List of Contributors

M. Altemus, Department of Psychiatry, Weill Medical College, New York, NY 10021, USA L. Arbogast, Department of Physiology, Southern Illinois University, Carbondale, IL 62901, USA B.W. Bluestein, Fuller Graduate School of Psychology, Pasadena, CA 91182, USA R.S. Bridges, Department of Biomedical Sciences, Tufts University, School of Veterinary Medicine, 200 Westboro Road, N. Grafton, MA 01536, USA I. Brockington, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham, B 15 2QZ, UK D.K. Buckwalter, Fuller Graduate School of Psychology, Pasadena, CA 91182, USA J.G. Buckwalter, Research and Evaluation, Kaiser Permanente Medical Group, Pasadena, CA 91101, USA A. Burlet, INSERM U308, Universit6 Henri Poincare, Nancy, France C.S. Carter, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL 60612, USA E Champagne, Developmental Neuroendocrinology Laboratory, Douglas Hospital Research Centre, Departments of Psychiatry and Neurology and Neurosurgery, McGill University, Montreal PQ H4H 1R3, Canada G.E Chrousos, Section on Pediatric Endocrinology, Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892, USA N. Coyle, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham, B15 2QZ, UK N. Craddock, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham, B 15 2QZ, UK A.J. Douglas, Division of Biomedical Sciences and Clinical Laboratory, Laboratory of Neuroendocrinology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK T.J. Garite, Department of Obstetrics and Gynecology, University of California at Irvine, College of Medicine, Irvine, CA 92697, USA B.S. Gingrich, Department of Psychiatry and Behavioral Sciences, Emory University, School of Medicine, Atlanta, GA 30322, USA A.R. Gintzler, Department of Biochemistry, Box 8, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn NY 11203, USA D.R. Grattan, Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, EO. Box 913, Dunedin, New Zealand A.E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, CB2 4AT, UK T. Higuchi, Department of Physiology, Fukui Medical University, Matsuoka, Fukui 9101193, Japan D.J. Hornsby, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada

vi C.D. Ingram, School of Neurosciences and Psychiatry, University of Newcastle, Leazes Wing, Royal Victoria Infirmary, Newcastle-upon-Tyne, NE1 4LP, UK R.R. Insel, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Yerkes Research Center, Atlanta, GA 30322, USA L.E. Johnstone, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK I. Jones, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham B 15 2QZ, UK Y.M. Kershaw, University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK E.B. Keverne, Sub-Department of Animal Behaviour, University of Cambridge, High Street, Madingley, Cambridge CB3 8AA, UK R.C. Kumar 1, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, UK Y. Lee, Department of Genetics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA C. Lendon, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham B15 2QZ, UK S.L. Lightman, University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK N.-J. Liu, Box 8, Department of Biochemistry, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn NY 11203, USA J.S. Lonstein, Center for Neuroendocrine Studies, Tobin Hall, University of Massachusetts, Amherst, MA 01003, USA K.-I. Maeda, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan P.E. Mann, Department of Biomedical Sciences, Tufts University, School of Veterinary Medicine, 200 Westboro Road, N. Grafton, MA 01536, USA M. Marks, Section of Perinatal Psychiatry, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, UK A.S. McNeilly, MRC Human Reproductive Sciences Unit, University of Edinburgh, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK M.J. Meaney, Douglas Hospital Research Center, 6875 LaSalle Boulevard, Montreal PQ H4H 1R3, Canada I.D. Neumann, Department of Zoology, University of Regensburg, D-93040 Regensburg, Germany D.A. Poulain, INSERM U378. Institut Francois Magendie, Universit6 Victor Segalen Bordeaux II, 1 rue Leo Saignat, F33076 Bordeaux CEDEX, France E. Robertson, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham B15 2QZ, UK J.A. Russell, Laboratory of Neuroendocrinology, Division of Biomedical Sciences and Clinical Laboratory, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK

*Deceased

vii C.A. Sandman, Department of Psychiatry and Human Behavior, Behavioral Perinatology Research Program, University of California Irvine, 3117 Gillespie Neuroscience Building, Zot Code 4260 Irvine, CA 92697, USA N. Shanks, University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK EZ. Stanczyk, Department of Obstetrics and Gynecology, University of Southern California, Los Angeles CA 90089, USA J.M. Stern, Department of Psychology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08903, USA I.E Stolerman, Section of Behavioural Pharmacology, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, UK A.J.S. Summerlee, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph ON N 1G 2W 1, Canada D.T. Theodosis, INSERM U378, Institut Francois Magendie, Universit6 Victor Segalen Bordeaux II, 1 rue L6o Saignat, F33076 Bordeaux CEDEX, France D.J. Toufexis, Department of Psychiatry, McGill University, Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun. Montreal PQ H4H 1R3, Canada H. Tsukamura, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan J.L. Voogt, Department of Molecular and Integrative Physiology, University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA P.D. Wadhwa, Department of Psychiatry and Human Behavior, Behavioral Perinatology Research Program, University of California Irvine, 3117 Gillespie Neuroscience Building, Zot Code 4260 Irvine CA 92697, USA C.-D. Walker, Department of Psychiatry, McGill University, Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun, Montreal PQ H4H 1R3, Canada B.C. Wilson, Department of Biology, Acadia University, Wolfville, NS, BOP1X0, Canada R.J. Windle, School of Nursing and Midwifery, University of Nottingham, Lincoln Education Centre, Lincoln County Hospital, Greetwell Road, Lincoln LN2 5QY, UK S.A. Wood, University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK S. Yang, Department of Molecular and Integrative Physiology, University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA L. Young, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA

Preface
The Maternal Brain conference was conceived in order to bring together people and information from research on the adaptation of the female brain during pregnancy, parturition and lactation. The adaptive changes within the central nervous system that prepare the body for the physiological requirements of pregnancy and motherhood are of major significance, and many scientists around the world are involved in elucidating these systems in humans and other mammals. The adaptive changes encompass diverse scientific disciplines, including neuroendocrinology, neuroscience and psychology; and failure of appropriate adaptation in mothers can lead to disorders that have profound and long-lasting consequences for individuals and for society. In the last decade or so much research has focussed on the neuroendocrine, emotional and psychological aspects of reproduction, and so the time was right to bring together research in these diverse disciplines to identify common perspectives and to plan for the future development of this crucial field. The conference was held in Bristol in July 1999, with the title The Maternal Brain: an International Meeting on Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum, and attracted over 100 delegates, with 37 symposium speakers and 53 poster communications. The conference was supported by Blackwell Science, the British Neuroendocrine Group, the Society for Endocrinology, the Ernst Schering Research Foundation GmbH., the Journals of Reproduction and Fertility Ltd., Organon Laboratories Ltd., Springer Verlag and Zeneca Pharmaceuticals. This was the first conference to address the maternal brain, and comprised wide ranging topics from molecular analysis of physiological systems using transgenic animals, through plasticity at the neurotransmitter and neuronal level, to the description of behavioural adaptation in terms of endocrinology, emotionality and its underlying causes, and analysis of psychosis; all in the peri-partum period. This volume contains review articles written by the symposium speakers, and thus brings together the major areas of research in the area of peri-partum plasticity. In the light of the success of the first conference, we are now looking forward to future developments and collaborations in the field of research into adaptive changes in motherhood, and to hearing about them at the next Maternal Brain conference, scheduled to be held in Montreal in 2003, and organised by Michael Meaney and Claire-Dominique Walker. J.A. Russell A.J. Douglas R.J. Windle C.D. Ingram (Editors)

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 1

Brain preparations for maternity - - adaptive changes in behavioral and neuroendocrine systems during pregnancy and lactation. An overview
John A. Russell 1,*, Alison J. Douglas 1 and Colin D. Ingram 2
1Laboratory of Neuroendocrinology, Section of Biomedical Sciences, Division of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK 2 School of Neurosciences and Psychiatry, University of Newcastle, Leazes Wing, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP, UK

Abstract: Pregnancy, parturition and lactation comprise a continuum of adaptive changes necessary for the development
and maintenance of the offspring. The endocrine changes that are driven by the conceptus and are essential for the maintenance of pregnancy and are involved in the preparations for motherhood are outlined. These changes include large increases in the secretion of sex steroid hormones, and the secretion of peptide hormones that are unique to pregnancy. The ability of these pregnancy hormones to alter several aspects of brain function in pregnancy is considered, and the adaptive importance of some of these changes is discussed, for example in metabolic and body fluid adjustments, and the induction of maternal behavior. The importance of sex steroids in determining the timing of the various adaptive changes in preparing for parturition and maternal behavior is emphasized, and the concept that the actions of prolactin and oxytocin, quintessential mammalian motherhood neuropeptides, can serve to coordinate a spectrum of adaptive changes is discussed. The part played by oxytocin neurons and their regulatory mechanisms is reviewed to illustrate how neural systems involved in maternity are prepared in pregnancy via changes in phenotype, synaptic organization and in the relative importance of their different inputs, to function optimally when needed. For oxytocin neurons secreting from the posterior pituitary, important in parturition and essential in lactation, these changes include mechanisms to restrain their premature activation, and adaptations to support synchronized burst firing for pulsatile oxytocin secretion in response to stimulation via afferents from the birth canal, olfactory system or suckled nipples. Within the brain, expression of oxytocin receptors permits centrally released oxytocin to facilitate the expression of maternal behavior. Changes in other neuroendocrine systems are similarly extensive, leading to lactation, suppression of ovulation, reduced stress responses and increased appetite; these changes in lactation are driven by the suckling stimulus. The possible link between these adaptations and changes in cognition and mood in pregnancy and post partum are considered, as well as the dysfunctions that lead to common problems of depression and puerperal psychoses.

The reproductive cycle: a continuum of adaptive changes

In mammals the reproductive cycle consists of distinct phases - - pregnancy, parturition and lactation - - each with their different demands on the

mother. These demands represent a conflict between the mother's need to maintain her own health and that of the offspring to maximize their potential. Thus, throughout this cycle it is essential that a continuum of adaptive changes occur in order that the maternal physiology satisfies both her needs and

* Corresponding author: John A. Russell, Laboratory of Neuroendocrinology, Division of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK. E-mail: j.a.russell@ed.ac.uk

the changing demands from the developing fetus or neonate. The aim of this chapter is to consider those changes that occur in the brain throughout pregnancy and post partum to allow the expression of neuroendocrine functions and behaviors that maximize the chances of a successful outcome from the pregnancy. This can be defined for the offspring as provision of an intrauterine environment with adequate nutrition and minimal exposure to damaging agents, a safe birth, post-natal maternal care and a sufficient lactation. For the mother reproductive success is measured in terms of the continuation of her genetic and epigenetic lineage, with the minimal expenditure, damage or discomfort of any sort from physical to mental, which for women at least is a sense of fulfillment or enjoyment. However to achieve this she will be required to undergo some of the most dramatic changes she will ever experience in the setting of physiological control mechanisms, with the preparation to perform new behaviors, to

suppress some behaviors but display others more vigorously. The adaptive changes of pregnancy are the consequence of signals from the conceptus, in large part as hormones, but partly arising from the physiological changes occurring in the pregnancy (Figs. 1 and 2), while post partum the signals result from the interactions between the behavior of the offspring and the mother's behavior and neuroendocrine mechanisms (Fig. 3). In this respect it is the developing offspring that determines, especially through the suckling stimulus, much of what happens in the mother's brain. The following sections will consider the major adaptive changes that occur at each of the distinct stages of the reproductive cycle, and how these are orchestrated. For the reproductive cycle to be successful it is essential that the complex sequence of adaptive events that occur during pregnancy and lactation are coordinated and correctly sequenced. An important factor that ensures harmony among the

Pregnancy
MOOD AND COGNITION

PLACENTAL/ CORPUS LUTEUM

HORMONES AND

NEUROSTEROIDS
act on neurotransmitter systems, e.g. GABAA receptor, opioids

PREPARATION ~ OFOXYTOCIN~) /~

NEURONES

. (~ DECREASED NEUROENDOCRINE STRESS RESPONSES

opioids/CRH/oxytocin/ prolactin

for parturition and lactation, sex steroids, opioids, NO, GABA

/ r ~ ~ ~ ~ ~ ~" ' ~N

INCREASED WATER

INTAKE
relaxin, altered smsensitivity/vaspressin /
SUPPRESSEI) FERTILITYf

4..1
/~
~J

I \ I(~) ~

r'f~ N " ~ ANALGESIA sex steroids/opioids, noradrenaline

prolactin, placental lactogens,sex steroids and opioids inhibit GnRH neurons

INCREASED FOOD INTAKE

progesterone

1~

Fig. 1. The maternal brain in pregnancy. The diagram shows the main physiological adaptations in pregnancy involving the brain/spinal cord, and the hormones and neurotransmitters that are involved. Anatomical regions: 1, limbic system, parvocellular PVN; 2, dorsal horn of the spinal cord; 3, arcuate nucleus, ventromedial nucleus, lateral hypothalamic nucleus, parvocellular PVN; 4, arcuate nucleus, preoptic area, medial amygdala; 5, OVLT, subfornical organ, magnocellular SON, PVN; 6, magnocellular SON, PVN; 7, limbic system, cerebral cortex.

Parturition
MATERNAL BEHAVIOR
onset by olfactory signals and cervical/vaginal stretch -

*eg3

DECREASED HPA AXIS ACTIVITY

opioids, CRH, ACTH

I I lPa U IXA./~..~ 1 5 I ~ t l d F l l / O

~t~fuiu~lOplOlU~,

__RTH

noradrenaline

Fig. 2. The maternal brain in parturition. The diagram shows the main physiological processes in parturition involving the brain/spinal cord, and neural stimuli, hormones and neurotransmitters that are involved. Anatomical regions: 1, spinal cord/brainstem neuronal pathways, olfactory bulb, medial preoptic area, limbic system, parvocellular PVN; 2, limbic system, parvocellular PVN; 3, dorsal horn of the spinal cord; 4, spinal cord/brainstem neuronal pathways, magnocellular SON, PVN, posterior pituitary.

changes as they unfold is the multiple parts played by each of the different endocrine signals (Carter and Altemus, 1997).
Pregnancy: an accommodation between mother and fetus

Uteroplacental signalling of pregnancy

The cycle of adaptive changes begins with the establishment and maintenance of the pregnancy. Implantation of the fertilized embryo involves initial local chemical signalling from the blastocyst to the endometrium to produce the reactions that allow anchoring and nourishment of the embryo. Soon thereafter the first hormonal signal from the embryo enters the mother's circulation to set off a series of endocrine changes in the mother that sustain the pregnancy and begin to prepare the mother to take care of the offspring once it or they are born. This first hormonal signal is chorionic go-

nadotrophin which acts on the corpus luteum to drive further the secretion of progesterone that has been continuing autonomously from ovulation long enough for the embryo to implant, but without which the endometrium will cease to succor the embryo and the pregnancy will abort. In most species, aside from leading to fertilization the process of mating plays little part in the adaptive responses of the mother. However, in some species, such as the laboratory rat, mating plays a key role as the initial support of the pregnancy is achieved by the stimulation of prolactin secretion from the mother's anterior pituitary as a reflex response to the copulation that led to fertilization (see Voogt et al., 2001, this volume, and see below). Here, prolactin acts with gonadotrophin on the corpora lutea to stimulate progesterone secretion; this mechanism is essential in this species because the corpora lutea otherwise rapidly regress after ovulation even before the embryos implant. Thereafter, the secretion of placental lactogen from the uteroplacental unit

Lactation
MATERNALBEHAVIOR MATERNAL DEFENCE/ CAREOF AGGRESSION YOUNG/NURSING CRH oxytocin,prolactin

MOOD AND DECREASED. ANXIETY BEHAVIOR NI ~ CRH, oxytocin, ~ ~ prolactin ~ p~LK#~ECTION~ (

"~ / ~ I ~ ~ / ~ /~ ~ ]~ / ~/ ~(/ ] DECREASED NEUROENDOCRINE

sPo s s

oxytoein,glutamate ~ ~ ~ and noradrenaline ~ ' ~ _ ~ . ' ' ~ mediateoxytocin ~.---~,.~..._.~ secretion

INCREASEDWATERv INTAKE ~)

~ prolactin, o.xytoein, ~ ~ ~ o r a d r e n a H n e

~'~1 ~

CRIt, vasopressin,

alteredosmosensltivity, vasopressin

(~

LACTOGENESIS/ GALACTOPOIESIS

dopamine,opioids mediatereflex prolactinsecretion

~ INCREASEDFOOD INTAKE prolactin,leptindecrease,NPY, % SUCKLING

SUPPRESSED FERTILITY

sucklingand prolaetininhibit
GnRH neurones

increasedmetabolicdemand

Fig. 3. The maternal brain in lactation. The diagram shows the main physiological adaptations and processes in lactation involving the brain/spinal cord, and the neural stimuli, hormones and neurotransmitters that are involved. Anatomical regions: l, olfactory bulb, limbic system, medial preoptic area, ventromedial nucleus, ventral tegmental area, periaqueductal gray, nucleus accumbens, parvocellular PVN; 2, brainstem, limbic system, parvocellular PVN; 3, spinal cord/brainstem neuronal pathways, arcuate nucleus, anterior pituitary; 4, arcuate nucleus, ventromedial nucleus, lateral hypothalamic nucleus, parvocellular PVN; 5, arcuate nucleus, medial preoptic area; 6, OVLT, subfornical organ, magnocellular SON, PVN; 7, spinal cord, brainstem, magnocellular SON, PVN, BNST, posterior pituitary; 8, limbic system, amygdala; 9, brainstem, limbic system, cerebral cortex, hippocampus, parvocellular PVN.

and gonadotrophin from the pituitary are important (Soares et al., 1998). If this is a first pregnancy, then two dramatic changes in the mother's endocrine experience have already taken place even at this early stage of gestation. First, the mother is experiencing increasing levels of the sex steroid progesterone in her circulation, to far exceed concentrations that her tissues, including the brain, have previously encountered. Secondly, there is a large and increasing concentration of chorionic gonadotrophin that is a stranger to her tissues, although this hormone shares a common c~-subunit with the anterior pituitary gonadotrophins, luteinizing hormone (LH) and follicle stimulating hormone (FSH). As the pregnancy progresses a cavalcade of such quantitative and qualitative changes in the hormones appears in the mother's circulation,

all emanating from each developing fetus, uteroplacental unit and the corpus luteum in the ovaries. The relative importance of each of these sources for the production of sex steroids varies among species, and according to the stage of the pregnancy, such that the fetus and placenta increase their endocrine autonomy as the pregnancy progresses. The quantities and range of hormones involved is impressive. The fetus and placenta certainly make a loud orchestrated endocrine statement that they have arrived and intend to stay. Throughout pregnancy the secretion of sex steroids (driven either by the mother or the fetus) can be considered to be the principle mediators of the adaptive changes that are taking place in the brain. This signalling involves both changes in concentration and in the relative ratios of the differ-

ent steroids. For instance maternal plasma progesterone concentration in a human pregnancy increases about 10-18-fold, and in women the proportions of 17[3-estradiol : estrone : estriol during peak secretion in the menstrual cycle are about 1 : 1 : 0 , but in pregnancy estriol concentration increases by up to 1000-fold, resulting in a ratio of 1 : 4 : 8. These steroids act, with different affinities, upon the classical estrogen receptor ~ and estrogen receptor and progesterone receptors which, in turn, modulate the transcription of secondary mediators (hormones, transmitters, enzymes, structural proteins, etc.) that lead to the adaptive changes. Estrogens and progestogens (or more importantly their metabolites) can also act at cell membranes, including on neurons, to evoke rapid non-genomic effects that play roles in the dynamic changes in pregnancy. As well as the larger amounts of sex steroid hormones in pregnancy, there are also increased amounts of certain peptide hormones in the maternal circulation. These are either novel hormones specific to pregnancy or those normally produced outside of pregnancy, but produced in greater quantities from the placenta and independently of the mechanisms that usually regulate their production from the mother's endocrine glands. The novel peptides are chorionic gonadotrophin and placental lactogen (chorionic somatomammotropin) from the placenta and relaxin from the corpus luteum. Each of these is homologous to a maternal hormone, chorionic gonadotrophin to the pituitary gonadotrophins as mentioned above, placental lactogen to anterior pituitary prolactin (see Grattan, 2001, this volume, and Mann and Bridges, 2001, this volume), and relaxin to insulin (see Hornsby et al., 2001, this volume). These peptides have functions related to the pregnancy, with chorionic gonadotrophin and placental lactogen acting through the receptors for the homologous maternal hormones (in the corpus luteum and mammary glands, respectively), but relaxin acting via a distinct, but still not characterized, receptor. In this respect relaxin is quite different from the other peptide hormones of pregnancy. Although they are peptides, and thus not expected to cross the blood-brain barrier, each is able to act directly on the brain, either as a consequence of transport via receptors in the choroid plexus (lactogens; see Grattan, 2001, this volume, and Mann

and Bridges, 2001, this volume) or by some other means (chorionic gonadotrophin accesses the hippocampus from the circulation; Lukacs et al., 1995), or by action on specific receptors in circumventricular organs (e.g. relaxin acts on the subfornical organ and anterior peri-third ventricular region). A further placental hormone with neuroendocrine homology is corticotrophin-releasing hormone (CRH), which in the human but not in the rat, is secreted by the placenta and is probably an important factor in regulating uterine contractility, and hence the time of onset of parturition (Majzoub et al., 1995; McLean et al., 1995; Wadhwa et al., 1998; Hillhouse and Grammatopoulos, 2001; King et al., 2001). But the potential cost to the mother is the takeover of control of adrenocorticotrophic hormone (ACTH) secretion from her anterior pituitary by this endocrine Trojan horse, the placenta. However, the mother mounts a defence, which is that her liver secretes a CRH binding protein that effectively protects the corticotrophs in the anterior pituitary from domination by placental CRH (Linton et al., 1990; King et al., 2001). This illustrates the fact that the mother does not necessarily accept passively all of the commands from the fetus and placenta, or from the infant once born, but a strategy emerges that accommodates the needs of both the mother and the offspring.

Adaptive changes in maternal physiology

There are remarkable physiological changes in pregnancy that are in part a direct consequence of the actions of the pregnancy hormones, and, in part, a consequence of their actions on the mother's own neuroendocrine control mechanisms, or responses to the physiological changes that are so wrought. Perhaps the most major changes are cessation of the signs of ovarian cycling, the change in metabolism that results in an increase in body weight, and the change in the activity of the hypothalamo-pituitaryadrenal axis. Suppression of fertility Once pregnancy is established the prevention of a further simultaneous pregnancy is clearly to the ad-

vantage of both mother and fetus(es) or offspring. There are several mechanisms to ensure that fertility is effectively suppressed. The progestogens secreted from the corpora lutea and/or placenta have a negative feedback effect on gonadotrophin-releasing hormone (GnRH) pulse generator activity, probably through the same gamma-aminobutyric acid (GABA)-ergic mechanisms that regulate GnRH secretion across the estrous cycle (e.g. in the sheep, Robinson and Kendrick, 1992). In the rat the mechanisms that maintain progesterone secretion illustrate how different adaptive changes may operate at different times in pregnancy. Mating reflexly triggers mechanisms that generate twice daily surges of prolactin, and these surges have a luteotrophic function through the first 10-12 days of gestation. The sensory stimuli from the vagina/uterine cervix associated with mating activate a neural circuit that includes the medial amygdala (MeA), preoptic area (POA), bed nucleus of the stria terminalis (BNST), ventromedial nucleus (VMH) and paraventricular nucleus (PVN; Tetel et al., 1993; Polston and Erskine, 1995; Yang et al., 1999), and studies using graded intromissions have shown that activation of the MeA is most closely associated with the surges of prolactin (Erskine, 1995; Polston and Erskine, 1995). The MeA acts via projections to the POA, which shows rhythmical activity that probably underlies the surges of prolactin (Lee et al., 1998). The POA acts by driving ~-endorphin-mediated suppression of the tuberoinfundibular dopaminergic (TIDA) neurons of the arcuate nucleus (Lee and Voogt, 1999b; Yang et al., 2000); these TIDA neurons release dopamine into the portal circulation to the anterior pituitary and tonically inhibit prolactin secretion. Furthermore, short-loop feedback effects of prolactin on enkephalin synthesis in TIDA neurons may contribute to further stimulation of prolactin release (Merchenthaler, 1994). So powerful are these mating-induced mechanisms that even in the absence of fertilization a pseudopregnancy with associated prolactin surges will persist for 10-12 days. However, as pregnancy progresses lactogens secreted from the developing placenta assume the luteotropic function and suppress prolactin secretion (Voogt et al., 1996). Placental lactogens can gain access to the brain through binding sites in the choroid plexus or ependymal cells near the arcuate nucleus (Pihoker et

al., 1993), and their action is believed to involve activation of the TIDA neurons (Lee and Voogt, 1999b), possibly as a result of a change in ~-endorphin synthesis (Mann et al., 1997) and reduced arcuate nucleus neuronal activity (Lee and Voogt, 1999a). This late gestation suppression of prolactin may prevent premature galactopoiesis, a role which prolactin will adopt following parturition. It is unclear whether prolactin or placental lactogens have other direct effects on the GnRH pulse generator that are independent of progesterone. The inhibition of the GnRH pulse generator by progesterone persists until luteolysis and the collapse in progesterone secretion is effected. This process triggers the sequence of events resulting in parturition and, in the rat, allows resumption of fertility and enables a post-partum estrus and mating to occur before the ovarian cycle is once again suppressed by lactation (see below, see McNeilly, 2001, this volume, and Tsukamura and Maeda, 2001, this volume). Changes in metabolism and fluid balance The increase in body weight is of course multi-factorial, comprising the uterus and its contents (fetus, placenta, amniotic fluid), increased body fluid and adipose tissue. The increase in body fluid is massive, and would be considered pathological outside of pregnancy, with blood volume increasing by some 40%, providing for the hemodynamic needs of the pregnant uterus. This is consequent on fluid retention and continued intake, such that extracellular fluid has a relatively increased water content and decreased osmolality. In this state, the secretion of vasopressin and drinking would be expected to be inhibited, but the thresholds for their stimulation are evidently lowered (Koehler et al., 1993). This is probably a consequence of central actions of relaxin to reset the regulatory mechanisms (see Hornsby et al., 2001, this volume). The metabolic demands of the growing fetus(es) are great (e.g. the human fetus near the end of pregnancy demands about 1 g glucose/h and 12 ml O2/min), and the growing and metabolically active placenta and developing mammary glands similarly strain the mother's metabolism. In addition, adipose stores are laid down in pregnancy to be drawn upon post-natally to support lactation. Clearly, as an increase in adipose tissue is supposed to reduce

appetite through leptin secretion from the fat acting centrally upon the hypothalamus there must be resetting of the central mechanisms regulating appetite and/or metabolism in pregnancy (see Johnstone and Higuchi, 2001, this volume). Hypothalamo-pituitary-adrenal axis activity and fetal neuroplasticity (programming) As a result of glucocorticoid secretion, the hypothalamo-pituitary-adrenal (HPA) axis plays widespread roles in regulating metabolism and immunocompetence, as well as transcription of a wide range of genes. Furthermore, HPA activation is one of the key elements of the physiological response to stressful stimuli. During pregnancy each of these functions is subject to modification and appropriate changes in HPA activity occur. It has been clear for several years that the HPA axis responses to stressors are reduced in lactation (see Lightman et al., 2001, this volume, and Walker et al., 2001a, this volume), and more recently that these responses are also reduced in pregnancy (see Neumann, 2001, this volume, and Douglas and Russell, 2001, this volume). The nature of the changes in the HPA axis is somewhat different between pregnancy and lactation in rats. In pregnancy the basal secretion of corticosterone is increased only in the last day or so, whereas in lactation there is sustained increased secretion of corticosterone (Atkinson and Waddell, 1995). ACTH secretion is primarily stimulated by CRH from the parvocellular (p)PVN CRH neurons, but vasopressin produced by the same neurons augments the actions of CRH. In the pPVN CRH neurons in pregnancy in the rat there is a continual decrease in the last week in the expression of CRH mRNA, with a similar decline in vasopressin mRNA expression (Johnstone et al., 2000). In contrast, in lactation, while there is also a decrease in CRH mRNA expression in pPVN neurons, there is a simultaneous increase in the expression of vasopressin mRNA (Shanks et al., 1999; Walker et al., 2001b). Furthermore, in late pregnancy intravenous injection of both CRH and vasopressin are less effective in stimulating ACTH secretion in pregnant rats than in virgins (Neumann et al., 1998; Russell et al., 2001), while in lactating rats the effectiveness of CRH also decreases

(Neumann et al., 1998; Toufexis et al., 1999), but that of vasopressin increases (Toufexis et al., 1999). Thus, it seems that in pregnancy the pPVN neurons decrease the production of both ACTH secretagogues, which are also less effective in stimulating ACTH secretion, but in lactation there is an increase in vasopressin production and effectiveness while CRH production remains decreased. However, reduced corticotrope sensitivity to secretagogues and reduced production of CRH or vasopressin by the pPVN neurons does not fully account for the decreased HPA axis responses to stressors in pregnancy or lactation. There is also reduced excitation in limbic brain regions that process stressors and regulate responses of pPVN neurons in pregnancy and lactation (Da Costa et al., 1996; see Walker et al., 2001a, this volume). These changes may themselves be a consequence of altered activity of pPVN neurons that project to limbic regions (see Walker et al., 2001a, this volume). Several explanations are proposed for the reduced responses in stressor processing circuits, including the pPVN CRH/vasopressin neurons, in pregnancy and lactation (see Walker et al., 2001a, this volume; Douglas and Russell, 2001, this volume), for instance the possibility that centrally released oxytocin attenuates activity in these circuits (see Neumann, 2001, this volume). This proposal comes from finding that in virgin rats oxytocin is released in limbic nuclei and the PVN during stress (a reflection of the excitation of magnocellular neurons by stressors in the rat; Nishioka et al., 1998), that central administration of oxytocin is also anxiolytic (Windle et al., 1997), and that intracerebroventricular injection of oxytocin antagonist increases HPA axis responses to stressors (Neumann et al., 2000a) involving oxytocin action within the PVN (Neumann et al., 2000a). Centrally administered oxytocin acts similarly in prairie voles (see Carter et al., 2001, this volume) and sheep (Cook, 1997). However, oxytocin antagonist is ineffective in modifying HPA axis responses in pregnant and lactating rats (see Neumann, 2001, this volume; Neumann et al., 2000b). Interestingly, there is a dissociation between HPA axis responses to stressors (reduced in pregnancy and lactation) and stress-induced anxiety-related behaviors. In pregnancy anxious behavior in a novel environment (exposure to the elevated plus maze) is increased but

the HPA axis stress response is reduced (Neumann et al., 1998). In lactation anxiety responses are reduced (see Walker et al., 2001a, this volume). The reduced anxiety in lactating rats depends upon recent suckling, which releases oxytocin centrally (Lambert et al., 1993b), and oxytocin antagonist given by intracerebroventricular injection has a pro-anxiety action in the presence of stressors in both pregnant and lactating (and also in virgin) rats. Overall, central actions of oxytocin do not account for the reduced HPA axis stress responses in pregnancy or lactation, but can attenuate the accompanying anxious behavior. Remarkably, parturition in the rat is not stressful in that ACTH and corticosterone secretion are not increased (Wigger et al., 1999), but the possible role of central oxytocin has not been studied.

HPA activity This will have major consequences for the health expectations of the offspring, and this programming most likely contributes to the fife-long adverse effects on the cardiovascular system, occurrence of adult onset diabetes, and other metabolic disorders that have been described for human populations (Barker, 1998).

Significance: prenatal programming. The functional significance of an attenuation of the HPA axis stress response throughout the period that the developing offspring are dependent on the mother may relate to the need to control exposure to excess glucocorticoids which can freely cross into milk and, despite the presence of placental l l~-hydroxysteroid dehydrogenase, can gain access to the late gestational fetus. One of the most important features of the mother-offspring relationship in pregnancy and lactation is the plasticity of the developing fetus, which can be programmed by the level of activity in the maternal neuroendocrine stress response system. In the rat, prenatal stress has programming effects that affect the offspring throughout their life. These effects include elevation of stress-induced HPA axis activity (Henry et al., 1994; McCormick et al., 1995), increased anxiety and behavioral reactivity/fearfulness (Alonso et al., 1991; Vallee et al., 1997; Caldji et al., 1998), decreased hippocampal glucocorticoid binding capacity (Henry et al., 1994; Maccari et al., 1995) and a decline in cognitive performance (Vallee et al., 1999). Studies in adrenalectomized corticosteroidreplaced animals have shown that this programming is due to stress-induced glucocorticoid secretion by the mother (Barbazanges et al., 1996) and could be mimicked by exogenous corticosterone administration (Bakker et al., 1995; Barbazanges et al., 1996). Furthermore, stress will even affect the development of the immune system (Sobrian et al., 1992; Bakker et al., 1995), possibly as a consequence of the altered

HPA axis in humans. In women, analysis of maternal HPA axis function in pregnancy is made difficult because the placenta produces ACTH and CRH. The effectiveness of CRH from the placenta on the maternal corticotrophs is strongly attenuated by the CRH binding protein produced by the liver (see Carter et al., 2001, this volume; Magiakou et al., 1996). Similarly, there is lack of an ACTH and cortisol secretory response to exogenous CRH in pregnant women (Schulte et al., 1990). The origin of the increased circulating ACTH level in pregnancy is thus not clear. It is possible that the increase in circulating cortisol feeds back to inhibit matemal pPVN neuron CRH (and vasopressin) production with decreased ACTH responses to stressors, as is seen in the pregnant rat (see above), but this is very difficult to study in humans. Nonetheless, the response to stressful stimuli also includes suppression of the sympathetic response and, for example, the hypertensive diastolic blood pressure response to a psychological challenge (serial subtraction) is reduced in the second trimester of pregnancy (Matthews and Rodin, 1992). In lactation, obviously with the confounding presence of the placenta removed, HPA axis responses to stressors are reduced in lactating, breast-feeding women compared with bottle-feeding women (see Carter et al., 2001, this volume; Altemus et al., 1995). It has been suggested that this may be a consequence of inhibitory actions of central oxytocin released during suckling (Uvnas-Moberg et al., 1990; see Carter et al., 2001, this volume). In rats, there is clearly increased central oxytocin release in pregnancy and lactation in response to parturition or suckling respectively, and in response to stressors. This central oxytocin is not responsible for the reduced HPA axis stress responses (Neumann et al., 2000b), but may be responsible for restraining the anxious behavior accompanying stress, and thus for enhancing the quality of maternal care (see Champagne and Meaney, 2001, this volume).

Stress, pain and parturition

HPA axis hormones and timing of delivery
Studies on women indicate that stress during pregnancy is a predictor for a sub-optimal outcome, including an increased risk of pre-term delivery (see Wadhwa et al., 2001, this volume; Wadhwa et al., 1998). Although, as discussed above, the maternal HPA axis and autonomic outflows are adapted in pregnancy to respond less to stressors, nonetheless there are still responses. A possible mechanism by which sustained maternal stress may advance the feto-placental mechanisms that determine the onset of parturition is through action on the placental production of CRH. Placental CRH relaxes the myometrium (Hillhouse and Grammatopoulos, 2001) and therefore offsets pre-term labor; however, the association between increased circulating CRH levels early in the third trimester and pre-term labor has led to the inference that CRH is responsible for pre-term labor (Wadhwa et al., 1998; Majzoub et al., 1999). The alternative explanation is that increased placental CRH secretion reflects a compensation to offset other factors predisposing to pre-term labor (Hillhouse and Grammatopoulos, 2001; King et al., 2001). Evidently, maternal stress hormones (ACTH, cortisol, catecholamines) can increase placental CRH production (Korebrits et al., 1998; see Wadhwa et al., 2001, this volume), which may be expected to delay labor, rather than advance it. How sustained maternal stress may affect the length of human pregnancy is clearly a complex problem (see Wadhwa et al., 2001, this volume; Hedegaard et al., 1993, 1996). In the rat the placenta does not produce CRH and we are not aware of studies that show an influence of stress in pregnancy on the duration of pregnancy. However, emotional stress during parturition interrupts births in rats and mice, but this is evidently via endogenous opioid-mediated inhibition of oxytocin secretion in rats (Leng et al., 1988) and via a [3-adrenergic mechanism in mice (Douglas et al., 2000a). The apparent paradox whereby oxytocin is a stress hormone in the rat yet inhibition of its secretion by stress in parturition explains the slowing of births, may be accounted for by the pulsatile pattern of oxytocin secretion (see below) that is optimal for stimulating uterine contractions and fetal expulsion,

whereas during stress there is a continuous secretion pattern and pulsatile secretion is inhibited (Leng et al., 1999). Like the reduced HPA axis responses to stress in pregnancy, oxytocin secretion in response to stressors is strongly inhibited by endogenous opioids, in this case driven by estrogen and progesterone (Douglas et al., 2000b; see Douglas and Russell, 2001, this volume). It is not known at which levels in the central stressor-processing circuitry opioids act, though there are reduced responses to stressors at multiple sites in these circuits in pregnancy (Da Costa et al., 1996).

Control of pain in parturition

One important action of sex steroids is to activate opioid-mediated antinociceptive mechanisms in the spinal cord, which is clearly important in reducing pain in parturition (see Gintzler and Liu, 2001, this volume). Pregnancy analgesia, revealed by increased threshold to noxious cutaneous stimulation, has been shown in rats and pigs to be mediated by endogenous opioid action as it is reversed by the opioid antagonist naloxone (see Gintzler and Liu, 2001, this volume; Jarvis et at., 1997). Such activation of central endogenous opioid mechanisms seems to be a feature of pregnancy and parturition, affecting the regulation of oxytocin and prolactin secretion for instance (Dondi et al., 1991; Douglas et al., 1993). The analgesia of pregnancy involves actions of opioids produced in the spinal cord, and acting via - and 3-opioid receptors in the dorsal horns (Dawson-Basoa and Gintzler, 1997, 1998). Vagino-cervical stimulation has a similar analgesic effect, involving spinal cord monoamines (Steinman et al., 1983; Gintzler and Komisaruk, 1991), which are also involved in pregnancy analgesia (Liu and Gintzler, 1999). 1713-Estradiol and progesterone actions underlie the analgesia of pregnancy (Medina et al., 1993a), evidently by activating dynorphin mechanisms, including its production (Medina et al., 1993b, 1995; Varshney et al., 1999).

Post-partum adaptations: the 'hormones of motherhood'

Female sex steroids, prolactin and oxytocin, which each have multiple co-ordinated actions in evoking maternal brain adaptations post partum, and can be termed the 'hormones of motherhood'. It is perhaps

10 not surprising that the sudden changes in the hormonal and physiological environment at the end of pregnancy can be associated with disturbances in mental state (see Kumar, 2001, this volume, and Jones et al., 2001, this volume; see below). It is remarkable that in the context of these sudden changes soon after the infant and placenta are delivered that the mother shows, or rather shows even during the birth process, the full blooming of maternal behavior. In the case of the laboratory rat or mouse this behavior will never have even been witnessed, let alone experienced, thus indicating the genetic basis of maternal behavior, evidently determined by paternal imprinted genes (see Keverne, 2001, this volume). However, it is clear that this rapid expression of maternal behavior, which can much more slowly be learned by rats that have not been pregnant, is dependent upon the changes in sex steroid secretion at the end of pregnancy, and involves actions of prolactin in the brain (see Mann and Bridges, 2001, this volume; Stern and Lonstein, 2001, this volume; and Grattan, 2001, this volume). Similarly, the triggering of lactation after birth involves the rapid and dramatic changes in hormone levels in the mother's circulation at the end of pregnancy, with removal of the stimulation by placental lactogens or prolactin of tubero-infundibular dopamine neurons, thus dis-inhibiting prolactin secretion (see Voogt et al., 2001, this volume, and Grattan, 2001, this volume). Thereafter the suckling stimulus maintains prolactin secretion (see Voogt et al., 2001, this volume, and Grattan, 2001, this volume), stimulates oxytocin secretion for milk transfer (see below), and prevents ovulation by inhibiting GnRH neurons (see McNeilly, 2001, this volume, and Tsukamura and Maeda, 2001, this volume), although the metabolic stress of lactation also contributes to GnRH suppression (see Tsukamura and Maeda, 2001, this volume). A striking feature of lactation is the suppression of HPA axis responses to stressors, although basal glucocorticoid secretion is increased (see above).
Maternal behavior and care of the neonate

without any longer a physical connection to the young, nurturance requires induction of the very strong motivation for maternity. Newborn pups are repulsive to virgin rats (Caldji et al., 1998), but in late pregnant and lactating rats they evoke a stereotypic pup-directed behavior. This involves carrying the pups and gathering them into a well-defined nest, pup-licking and grooming, and the adoption of the arched-back (kyphotic) posture to allow the pups to suckle (Numan, 1994; see Stern and Lonstein, 2001, this volume). In other species with more independent offspring, such as the sheep, bonding is induced that ensures that the mother will continue to accept and suckle her lamb (Keverne and Kendrick, 1994). Thus, the key features of maternal behavior are that internal signals prime the brain in order that appropriate behavior is expressed, and fear of the pups is suppressed, as soon as the neonate is born (induction/onset); and that signals from the neonates trigger maintenance of the behavior, perhaps with specific bonding to the individual offspring (maintenance). Furthermore, lactating rats become territorial and aggressive to intruding male or female rats. This inherent behavior has a primarily genetic basis, but can involve learning, and this is particularly important in humans (Miller et al., 1997).
Nature and nurture: determinants of maternal care

Changes in behavior even before parturition prepare for the arrival of the offspring, including nest-building, though this account focuses on post-partum behaviors. The young of many species are born wholly dependent on the parents (usually the mother) and,

Maternal behavior is not invariably expressed optimally; some new rodent mothers cannibalize or neglect their young. This could be a result of faulty priming, or of imbalance between fear and maternal drive, or have an inherited, genetic or epigenetic, basis. Although the genetic basis of maternal behavior is far from being understood, there is recent information about the source of the genes determining the neural substrate, and about specific genes essential for functioning of the neural circuitry. First, the key neural circuits for maternal behavior are in the limbic brain and hypothalamus, regions where paternal imprinted somatic genes are expressed (see Keverne, 2001, this volume). Disruption in mice of either of two such genes, mest or Peg1, leads to abnormal maternal behavior and growth retardation in the offspring (Lefebvre et al., 1998; Li et al., 1999d). Also, mice beating a knockout of the transcription factor genefosB show

11 a loss of maternal behavior (Brown et al., 1996). This and other transcription factor genes (c-fos in rats and sheep, zif/268 in sheep) are induced in neurons forming circuitry for maternal behavior by both sensory cues from the pups and maternal experience (Calamandrei and Keverne, 1994; Da Costa et al., 1997), and may be essential in tuning the network. Immediate early (transcription factor) gene expression studies have provided, together with longer established lesioning techniques, detailed information about the brain regions and the characteristics of neurons in the maternal behavior network. virgin than in lactating rats, suggesting that these areas may be actively involved in promoting defense responses and active avoidance of the pups (Sheehan et al., 2000). Interestingly, stress-induced c-fos mRNA expression in some of these areas is reduced in lactating rats (Da Costa et al., 1996) or in estrogen-primed rats treated with intracerebroventricular oxytocin, consistent with this role in avoidance. Involvement of the nucleus accumbens in motoric elements of maternal behavior is indicated by Fos induction by interaction with pups (Fleming and Walsh, 1994; Lonstein et al., 1998). Role of MPOA Although a leading role of the MPOA in the expression of maternal behavior is well-established, it is clear that the full repertoire of the behavior involves more extensive areas. Lesions of the MPOA block maternal responses and electrical stimulation of the MPOA can facilitate the onset of maternal responding in virgins (Morgan et al., 1999). However, as pointed out by Stern and Lonstein (2001, this volume), the effect of MPOA lesions on retrieval, which is the first event in the cascade of maternal responses, means that the normal sequence of the components of maternal behavior is anyway broken. Rather, maternal behavior depends on a diffuse circuitry in the hypothalamus as N-methyl-D-aspartate (NMDA) lesions of the ventral, dorsal or anterior hypothalamus stimulate rapid onset of maternal behavior in sex steroid-primed females (Bridges et al., 1999). Ibotenic acid lesions of the PVN increase maternal aggression in early lactation (Giovenardi et al., 1998), while kainic acid lesions to the lateral habenula but not hippocampus disrupt the normal pattern of pup retrieval and nest building (Matthews-Felton et al., 1995). The ventral BNST is strongly connected to many of the areas shown to be activated during maternal behavior and excitotoxic lesions of this region also disrupt pup retrieval in the post-partum rat (Numan and Numan, 1996). Role of periaqueductal gray Some limbic and hypothalamic areas (posterodorsal MeA, principal BNST, ventrolateral septum, dorsal premammillary nucleus, medial hypothalamus) are more activated by exposure to pups in non-maternal The ventrolateral caudal periaqueductal gray mediates sensorimotor integration of the arched-back nursing posture (kyphosis) (see Stern and Lonstein,

Neural circuitry of maternal behavior

Three main approaches have been employed to define the neural circuits that determine this complex behavior: lesions (electrolytic or neurochemical); either electrical stimulation or intracerebral administration of neuroregulators; and the immunocytochemical mapping of immediate early gene expression, typically after reuniting the pups with the mother following a period of separation. These studies show that discrete areas of the limbic system and hypothalamus and the periaqueductal gray are of key importance (Bridges et al., 1999). Thus, reuniting pups and mother after a separation increases Fos expression in these regions (in the limbic system, MeA, ventral BNST, lateral habenula, MPOA, anterior magnocellular PVN and periaqueductal gray; Lonstein et al., 1998; Numan et al., 1998; Stack and Numan, 2000). Some of these neurons are part of the mesotelencephalic dopamine system, and many contain estrogen receptor c~, providing a basis for genomic estrogen actions in the network (Lonstein et al., 2000). The expression of Fos and Fos B in the MPOA, ventral BNST and anterior magnocellular PVN is persistent in the lactating rat, indicating a continual role for these transcription factors in the activation of these maternal network neurons (Stack and Numan, 2000). Distributed functions within the maternal behavior neural network

12 2001, this volume). Fos expression is selectively stimulated here, and not in other brainstem regions, by suckling (Lonstein and Stem, 1997a,b). Lesions disrupt adoption of the kyphotic posture, also blocking sexual behavior and increasing aggression (Lonstein et al., 1998; Lonstein and Stem, 1998), suggesting that this area coordinates a number of related activities in the lactating rat. Role of ventral striatum/nucleus accumbens dopaminergic systems Dopaminergic neurons have a key role in the expression of maternal behavior. As well as regulating motor activity, central levels of dopamine have a powerful control over maternal motivation to express contact behavior (Stem and Keer, 1999). Inhibition of dopaminergic activity in the nucleus accumbens and striatum with a D2 receptor antagonist severely disrupts normal pup retrieval and grooming, but not kyphosis (Keer and Stem, 1999). Increased release of dopamine into the ventral striatum accompanies nursing during the reunion of a dam with her pups (Hansen et al., 1993). Although depletion of dopamine in the ventral striatum by the neurotoxin 6hydroxy-dopamine will also impair spontaneous pup retrieval, this deficit in motivation can be overridden by separation of the dam from the pups (Hansen, 1994). However, the dopaminergic system of the nucleus accumbens may not be specific for pup directed activity but generalized to social attachments, as shown by the effects that D2 receptor agonists and antagonist have on partner preference in prairie voles (Gingrich et al., 2000). In sheep the model of vagino-cervical stimulation of the female sex steroid-primed ewe, simulating the peripheral mechanism that evokes maternal behavior at parturition, activates circuitry involved in olfactory memory and behavior important for bonding with the lamb. This increases c-fos mRNA expression in somatosensory cortical regions and limbic areas (MeA, cingulate gyrus, hippocampus, BNST and lateral septum), mediodorsal thalamus and lateral habenula, and several hypothalamic areas (MPOA, mediobasal hypothalamus, periventricular area, PVN and SON; Da Costa et al., 1997). Infusion of antisense sequences to c-fos mRNA into the PVN significantly reduces parturition-induced oxytocin, CRH and c-fos mRNA expression, and, importantly, reduces the expression of some components of maternal behavior (low-pitched bleats and lamb sniffing; Da Costa et al., 1999), consistent with the involvement of immediate-early genes in the induction of this behavior (Brown et al., 1996). Estrogen and progesterone priming Although repeated exposure of virgin rats to pups can lead over several days to the expression of maternal behavior, a process termed concaveation (Sheehan et al., 2000), treatment of rats or prairie voles with estrogen and progesterone, to mimic the levels of secretion in pregnancy, leads to very rapid expression of parental behavior on exposure to pups (Bridges, 1984; Lonstein and De Vries, 2000). Similarly, the effectiveness of oxytocin and lactogenic hormones in evoking matemal behavior (see below) depends on prolonged prior estrogen and progestogen exposure. At least some of these sex steroid actions can be via cytoplasmic estrogen receptor and progesterone receptor as these are expressed, for instance, in the MPOA (Rosenblatt et al., 1994) where the number of neurons with progesterone receptors increases at the end of pregnancy (Numan et al., 1999). Many neurons of the mesotelencephalic dopamine system contain estrogen receptor c~ (Lonstein et al., 2000). However, non-genomic actions of estrogens and progesterone (via its metabolites) at neuronal cell membranes are also possible. Effects of sex steroids on expression of components of the transmitter mechanisms involved in the neural network underlying maternal behavior are evi-

Triggering the activation of maternal brain circuits

Sensory cues from the offspring The effective and sufficient stimuli from the pups are olfactory and auditory cues (Fleming and Walsh, 1994; Numan and Numan, 1995; Fleming and Korsmit, 1996; Walsh et al., 1996). In lactating rats, suckling activates no additional areas than contact with pups, except for the intercollicular periaqueductal gray (Fleming and Korsmit, 1996), which is a critical area for kyphosis (Lonstein and Stem, 1997a; see above).

13 dently important. For example, the expression of the oxytocin gene is up-regulated by estrogen and progesterone exposure followed by progesterone withdrawal (Broad et al., 1992, 1993; Crowley et al., 1995). Here, estrogen may act through estrogen receptor ~, which is expressed in oxytocin neurons (Hrabovszky et al., 1998), though this has not been established; and progesterone seems to act via modulation of GABAA receptors, presumably after metabolism to allopregnanolone (Thomas et al., 1999). Estrogen is also implicated in the up-regulation in pregnancy of oxytocin receptor expression in brain regions involved in maternal behavior (Young et al., 1997). Oxytocin promotes excitability of BNST neurons at the end of pregnancy, and these changes are enhanced by treatment of ovariectomized late pregnant rats with estrogen but not progesterone (Wakerley et al., 1998; Terenzi et al., 1999). Regarding central prolactin/lactogenic hormone actions, the expression of both estrogen receptor and prolactin receptor in the choroid plexus (Brooks et al., 1992; Pi and Grattan, 1998a; Hong-Goka and Chang, 1999) suggests a possible action of estrogen at this site of entry of lactogenic hormones into the brain. Expression of the long form of the prolactin receptor in the brain is found in the SON, PVN and choroid plexus (Pi and Grattan, 1998b), increases in pregnancy, and is up-regulated by progesterone, prolactin and growth hormone (Sugiyama et al., 1994); sex steroids also regulate prolactin receptor expression in neuroendocrine dopaminergic neurons (Lerant and Freeman, 1998). Thus, the behavioral potencies of rat prolactin and placental lactogen I are likely to be increased in pregnancy (Bridges et al., 1997). Prolactin and maternal behavior The role of lactogenic hormones in maternal behavior is well-established (Bridges and Mann, 1994; Dutt et al., 1994; Bridges et al., 1996, 1997; see Mann and Bridges, 2001, this volume; and Grattan, 2001, this volume). They act centrally through areas of the hypothalamus that express the prolactin receptor (Chiu and Wise, 1994). Maternal behavior may be induced by central actions of prolactin (Bridges and Mann, 1994; Bridges et al., 1997), and indeed the conceptus itself is involved in the generation of this behavior through the synthesis of placental lactogen from the placenta (Bridges et al., 1996). Infusions of lactogenic hormones (prolactin or placental lactogens) into the MPOA will accelerate the onset of maternal behavior in estrogen/progesterone-primed virgin rats (Bridges and Freemark, 1995; Bridges et al., 1997). Conversely suppression of prolactin secretion with haloperidol will reduce maternal behavior in post-partum rats (Bridges and Mann, 1994; Stern and Keer, 1999). Further support for a key role of lactogenic hormones comes from mice with a mutation of the prolactin receptor gene, which show deficits of maternal responses without effects on other behaviors (Lucas et al., 1998). There is evidence for increased expression of both the long and short forms of the prolactin receptor in the hypothalamus in lactation (Sugiyama et al., 1994; Pi and Grattan, 1999b). While, as discussed above, sex steroid priming actions involve effects on prolactin receptor expression, prolactin itself may promote prolactin receptor expression (Sugiyama et al., 1994; Sakaguchi et al., 1996). Furthermore, the induction of maternal behavior in virgin rats by repeated exposure to pups (concaveation) may depend on stimulation of expression of the prolactin receptor in the brain (Sugiyama et al., 1996). Oxytocin and maternal behavior The most powerful factors for the induction of maternal behavior are oxytocin, released within the brain (see below; Pedersen, 1997; Young et al., 1997), and prolactin or (placental lactogen), entering the brain from the circulation (Bridges et al., 1996) or perhaps produced in the brain (Devito et al., 1992). Thus, the conceptus begins directing maternal behaviors even before birth through placental lactogen or maternal prolactin, stimulated by estrogen, and the process of parturition triggers oxytocin release within the brain as well as into the circulation. As discussed above, the brain oxytocin and prolactin mechanisms are up-regulated by the actions of estrogens and progestogens in late pregnancy. Centrally released oxytocin facilitates the initial expression of maternal behavior, at least in rats and sheep (Keverne and Kendrick, 1992), species in which the oxytocin receptors in the brain are appropriately distributed (see Insel et al., 2001, this

14 volume). However, as oxytocin knockout mice show maternal behavior, oxytocin itself cannot be essential for maternal behavior in this species (Nishimori et al., 1996; see Insel et al., 2001, this volume). Interestingly, in mice with a mutation of the Peg3 gene, normally expressed in the hypothalamus but of paternal origin, there are deficits in maternal behavior and reduced numbers of PVN oxytocin neurons, and also deficient milk ejection (Li et al., 1999d; see Keverne, 2001, this volume). Earlier inconsistent findings conceming the effects of oxytocin on maternal behavior in rats led to the idea that it may act centrally to promote maternal behavior in conditions of stress (McCarthy et al., 1992) or inadequate stimulation by the pups (Pedersen et al., 1995). Furthermore, the density of oxytocin receptors in the BNST, POA and septum is greater in rats that display rapid induction of maternal behavior compared to the slow responders (Francis et al., 2000). The importance of olfactory cues from the newborn in activating the maternal behavior network (Walsh et al., 1996; Da Costa et al., 1997) has been mentioned above. Studies in sheep have shown an important action of oxytocin in the olfactory bulbs immediately post partum in the establishment of maternal behavior, and have characterized effects of oxytocin on the local release of neurotransmitters (acetylcholine, y-aminobutyric acid, glutamate and noradrenaline; Levy et al., 1995; Kendrick et al., 1997b; Kendrick, 2000). The importance of oxytocin action in the olfactory bulbs has been corroborated in studies on rats (Yu et al., 1996a,b). Oxytocin neurons in the PVN are the likely source of the oxytocin acting in the olfactory bulbs in the sheep (Da Costa et al., 1999), while lesioning experiments in rats indicate that this oxytocin reaches the olfactory bulbs in volume transmission mode, via the cerebrospinal fluid (Yu et al., 1996a). While central actions of prolactin and oxytocin are important for the induction of maternal behavior, any involvement in the maintenance of maternal behavior is far less certain. However, evidence has been provided for a continuing action of oxytocin in the olfactory bulbs in the maintenance of maternal behavior in the rat (Okere et al., 1999). Central cholecystokinin mechanisms may be involved in maternal behavior maintenance, as a cholecystokinin antagonist will decrease pup retrieval times (Mann et al., 1995). Furthermore, there are circuits that inhibit maternal behavior. Opiates, particularly ix-receptor agonists, will inhibit established maternal behavior in rats (Russell and Spears, 1984; Mann et al., 1990), but promote maternal behavior in the sheep (Keverne and Kendrick, 1994). Within the ventromedial hypothalamus NMDA-induced lesions promote the onset of maternal behavior (Bridges et al., 1999), while infusions of neuropeptide K into the ventromedial hypothalamus will delay the onset (Sheehan and Numan, 1997). Since the ventromedial hypothalamus receives a tachykinin input from the MeA, it is possible that this represents an inhibitory system for the suppression of maternal behavior. The maintenance of maternal behavior may involve the suppression of these inhibitory circuits.

Behavioral and cognitive changes in human pregnancy and lactation

The animal studies discussed above have revealed details of the nature, preparation and activation of the neural circuitry underlying the performance of maternal behavior, and these principles are presumed to apply to humans (Fleming et al., 1997; Stern, 1997). Human studies have revealed the changes in mood or emotion and cognition that accompany pregnancy or follow parturition, and which can be viewed as conducive to motherhood (Asher et al., 1995) although there are negative aspects also, as discussed below. While studies in animals show, for example, a marked increase in aggressive behavior in lactation to protect the young, and involving PVN oxytocin neurons (Giovenardi et al., 1998) and decreased anxious behavior in lactation (though not in pregnancy; Walker et al., 1995; Neumann et al., 1998), we are not aware of studies on changes in cognition in animals related to pregnancy. These might be expected in the light of the extensive literature on sex steroid effects on cognitive processes (e.g. McEwen et al., 1997), although central actions of progesterone metabolites or oxytocin may underlie the reduced anxiety behaviors in lactation (Freeman et al., 1993; Windle et al., 1997). In women, there are major changes during pregnancy and post partum in mood and behavior (Asher et al., 1995) and loss of reaction to stressful stimuli (Altemus et al., 1995; Carter and Altemus, 1997).

15 It is likely that the marked changes in steroid hormone levels play a major role in the mood changes (Buckwalter et al., 1999; see Buckwalter et al., 2001, this volume). Cognitive changes during pregnancy and post partum include reduced performance in certain mental tasks (e.g. Brindle et al., 1991; Eidelman et al., 1993; Morris et al., 1998; Buckwalter et al., 1999; see Buckwalter et al., 2001, this volume) but whether these confer an adaptive advantage for motherhood is not clear. Amnesic effects could be involved in forgetting the less pleasant aspects of childbirth, which may be important for continued procreation (Sharp et al., 1993; Keenan et al., 1998; Morris et al., 1998). However, it is also possible that these are simply consequences of the high sex steroid levels (see Buckwalter et al., 2001, this volume, and Herbison, 2001, this volume). Whether central oxytocin has any role in these changes, as has been proposed (Silber et al., 1990), is not known.
Lactation

Some of the arcuate nucleus neurons produce factors that directly regulate the secretion of anterior pituitary hormones after release into the hypothalamo-hypophysial portal system (e.g. dopamine acting as a prolactin-release-inhibiting factor; and growth hormone-releasing hormone), others produce inhibitory opioid peptides that act locally (e.g. enkephalins from TIDA neurons) or throughout the brain (~-endorphin from pro-opiomelanocortin (POMC) neurons, including projections to GnRH neurons), or peptides in projections to the PVN that are importantly involved in metabolic regulation (neuropeptide Y (NPY), agouti-related protein, c~-melanocyte stimulating hormone, (MSH another product of POMC); see Johnstone and Higuchi, 2001, this volume).

Regulation of prolactin
The stimulation of prolactin secretion by suckling involves inhibition of the TIDA neurons, which produce dopamine acting as a prolactin-release inhibiting factor, and perhaps stimulation of prolactinreleasing factor neurons (see Voogt et al., 2001, this volume). The suckling-induced suppression of dopaminergic activity is most likely mediated by the action of endogenous opioid peptides (Arbogast and Voogt, 1998; Hou and Voogt, 1999), perhaps through up-regulated enkephalin production in the TIDA neurons (Ciofi et al., 1993; Merchenthaler, 1994); or by [3-endorphin produced in nearby arcuate nucleus neurons, which are activated by suckling (Pape et al., 1996; Pape and Tramu, 1996). Studies on women indicate that the same neuroendocrine mechanisms are involved in stimulating prolactin secretion in lactation (Rossmanith et al., 1995). From mid-lactation onwards an important factor in the maintenance of lactation is the ability of the suckling stimulus to suppress the negative feedback effect of prolactin on TIDA neurons (Arbogast and Voogt, 1996). This short-loop feedback is absent in late pregnancy (Grattan and Averill, 1995; Flietstra and Voogt, 1997), but restrains prolactin secretion in early lactation (Flietstra and Voogt, 1997). Neurons in the arcuate nucleus and mediobasal hypothalamus express prolactin receptors (Chiu and Wise, 1994), and in vitro prolactin increases tyrosine hydroxytase activity, and hence dopamine synthesis (Arbogast and

The high levels of estrogen, progesterone, placental lactogen and then prolactin in pregnancy automatically prepare the mammary glands for lactation and prevent premature milk production. Consequently, following birth lactation is automatically initiated as the inhibitory feto-placental hormones are withdrawn, leaving the stimulatory action of maternal pituitary prolactin unopposed as its secretion increases. Then the continued secretion of prolactin and hence milk production is totally dependent on the suckling stimulus. The transfer of this milk to the young involves milk ejection during suckling, and for this oxytocin is essential (Nishimori et al., 1996). Clearly, the stimulation by suckling of prolactin and oxytocin secretion requires the expression of appropriate maternal behavior. The suckling-dependent neuroendocrine responses, which include suppression of the GnRH pulse generator and hence ovarian cycling, involve re-organization of neural circuitry. The metabolic and fluid demands of lactation are met by major changes in maternal regulatory mechanisms.

Arcuate nucleus
The arcuate nucleus has a pivotal role in the maintenance of milk production (galactopoiesis).

16 Voogt, 1997). However, as lactation progresses tyrosine hydroxylase activity in TIDA neurons decreases, and this is a result of prolactin action, but also perhaps as a result of inhibition by suckling-stimulated opioid mechanisms (Wang et al., 1993; Pape and Tramu, 1996; Li et al., 1999c). In lactation prolactin receptor expression increases in the tubero-infundibular hypothalamus (Pi and Grattan, 1999b,c; Pi and Voogt, 2000), but as far as we know changes in pregnancy are not described, although sex steroid administration to mimic pregnancy plasma levels increases serum prolactin and prolactin receptor in tyrosine hydroxylasepositive neurons (Lerant and Freeman, 1998). Without this suppression of prolactin negative feedback, dopaminergic activity induced by the high levels of prolactin would rapidly terminate sustained secretion and continued galactopoiesis. (Sauve and Woodside, 2000). However, a further important change in lactation is the suppression of circulating leptin levels, which will reduce the inhibition by leptin of arcuate nucleus NPY neurons and any stimulation of a-MSH production by POMC neurons (Brogan et al., 1999; see Johnstone and Higuchi, 2001, this volume).

Body fluid regulation

Drinking is closely coupled to eating to facilitate osmotic balance and solute excretion (Watts, 2000). However, in pregnancy the extracellular fluid osmotic and volume thresholds for drinking and vasopressin secretion are evidently reduced (Koehler et al., 1993, 1994), leading to expansion of fluid volume and a reduction in extracellular fluid osmolality (Atherton and Hutchinson, 1987). The enhancement by pregnenolone sulfate of NMDA-induced activity of vasopressin neurons (Richardson and Wakerley, 1998) may be involved in the evident decreased threshold for osmotic stimulation of vasopressin neurons in pregnancy. The coupling of the lamina terminalis osmoregulatory inputs to the magnocellular oxytocin neurons is weakened in pregnancy and osmotically stimulated secretion of oxytocin, a natriuretic hormone in the rat, is reduced, so accommodating sodium retention (Bull et al., 1994). These changes are evidently driven by relaxin from the corpora lutea, acting via circumventricular organs in the lamina terminalis (see Hornsby et al., 2001, this volume). In lactation there is no longer any relaxin, and the secretion of milk imposes a strain on the mother's hydromineral balance as milk has a lower salt content than maternal extracellular fluid. Consequently, there is incipient hyperosmotic drive to drinking and vasopressin secretion (Summy-Long et al., 1997). Water intake is greatly increased and the magnocellular PVN and SON neurons show increased vasopressin gene expression (Crowley et al., 1993) and increased connectivity to excitatory glutamatergic input (El Majdoubi et al., 1996).

Metabolic resetting
The metabolic demands of lactation are met by drawing on adipose tissue reserves laid down in pregnancy and by increased appetite and food intake. Food intake control mechanisms are clearly re-set (see Johnstone and Higuchi, 2001, this volume). This is of great interest in the light of the recent identification of several novel hypothalamic peptides that have powerful actions on appetite. Several of these peptides are produced in arcuate nucleus neurons, and there are marked changes in these neurons in lactation indicating roles in the changes in food intake (see Johnstone and Higuchi, 2001, this volume). Thus, in lactation in arcuate nucleus neurons projecting to the PVN there is increased expression of NPY, an appetite stimulant, and the co-localized agouti-related protein I (a stimulant through its antagonism at the melanocortin-4 (MC-4) receptor of the anorexic action of et-MSH; Ciofi et al., 1993; Pape and Tramu, 1996; Chen et al., 1999; Li et al., 1998, 1999c). The afferent input to these neurons excited by suckling comprises forebrain, limbic and brainstem regions (Li et al., 1999a). As central administration of prolactin increases food intake in virgin rats (Sauve and Woodside, 1996), it is possible that a central action of prolactin in lactation is important in re-setting the activity of the arcuate nucleus NPY neurons that stimulate appetite, but actions in the PVN and VMN are also likely

Suppression offertility in lactation

Post-partum infertility is a consequence of the suppressive effects of the suckling stimulus on GnRH

17 neuron activity (see McNeilly, 2001, this volume, and Tsukamura and Maeda, 2001, this volume). This provides an effective method of contraception (Kennedy et al., 1996), but requires a regular pattern of suckling, otherwise pulsatile GnRH, and consequently gonadotrophin secretion and ovulation resume (Tay et al., 1996; WHO, 1998a,b; see McNeilly, 2001, this volume). In the rat lactational infertility follows the postpartum estrus, and if mating and fertilization occur it features as delayed implantation. Three mechanisms have been considered as underlying the infertility of lactation. First is the possibility that the neural input from the suckling stimulus projects to, and inhibits, the GnRH neurons. Secondly, that the high levels of prolactin that are produced act centrally (and/or in the ovary). Thirdly, that the inhibition of GnRH neurons is a consequence of the metabolic strain of lactation. It is quite clear from studies in several species, including in women, that lactational infertility is primarily a consequence of suppressed gonadotrophin secretion (Tay et al., 1992). Although negative feedback sensitivity of gonadotrophin secretion to estrogen is enhanced (Illingworth et al., 1995), restoration of ovarian cycling by pulsatile GnRH administration indicates the primary importance of suppression of the GnRH pulse generator (Zinaman et al., 1995). Studies of the roles of dopamine and endogenous opioids show that in breastfeeding women pulsatile LH release is inhibited through a non-dopaminergic, non-opioidergic pathway (Tay et al., 1993). However, in rodents, the suckling-induced activation of POMC neurons in the rostral arcuate nucleus, detected as Fos expression (Pape et al., 1996; Pape and Tramu, 1996), and the inhibition of TIDA neurons (Wang et al., 1993; Li et al., 1999c) may have implications for the inhibition of GnRH neurons. In addition, as food restriction reduces fertility, there is the possibility that the mechanism of lactational infertility involves the increased metabolic demand from galactopoiesis, signalled as reduced energy availability. By acutely reducing glucose availability in the brain with intracerebroventricular infusion of 2-deoxyglucose, signalling via glucoreceptors in the brainstem and a noradrenergic projection to the pPVN, central CRH release inhibits pulsatile LH secretion (Tsukamura et al., 1994; Murahashi et al., 1996; Nagatani et al., 1996a; Nagatani et al., 1996b). Whether such a mechanism contributes to lactational infertility is not clear. Oxytocinergic neurons: a model for adaptive changes throughout pregnancy and lactation Here, we will explore changes in a maternal neuroendocrine system in pregnancy that has multiple functions in pregnancy and lactation, some of which have been discussed above, to illustrate some of the principles of adaptation, and to supplement the contributions that follow in this volume.

Importance of oxytocin to successful reproduction

Oxytocin is a distinctly mammalian hormone: a nine amino acid peptide closely related to vasopressin and more phylogenetically ancient similar peptides. Characterized in extracts of the posterior pituitary it was the first peptide hormone to be synthesized. The uterotonic, and later milk-ejecting, activity of posterior pituitary extracts led to the proposal that oxytocin secretion from the posterior pituitary played an important role in parturition and lactation. Subsequently, a mass of evidence in different species has given strong support to these proposals (Russell and Leng, 1998; Russell and Leng, 2000). The widespread use of synthetic oxytocin in clinical obstetric practice speaks to its effectiveness in initiating and sustaining parturition, and conversely the successful management of pre-term labor by administration of oxytocin antagonist indicates a role for endogenous oxytocin in parturition (Goodwin et al., 1994), as does the slowing of established parturition by oxytocin antagonist in rats (Antonijevic et al., 1995a). Increased secretion of oxytocin during parturition has been measured in several species, including in women, although this is difficult because of the rapid destruction of oxytocin by a circulating aminopeptidase from the human placenta (Fuchs et al., 1991). The importance of oxytocin in parturition is challenged by finding that mice with inactivation of the oxytocin gene deliver their offspring, but whether the process is normal is not clear (Nishimori et al., 1996; Young et al., 1996;

18 see Insel et al., 2001, this volume). These knockout mice indicate that there is redundancy in the mechanisms driving parturition, and not that oxytocin is not involved. Other mechanisms, particularly relating to the timing of the onset of parturition, involve local actions of placental hormones (e.g. CRH in women; McLean and Smith, 1999). In contrast, there is no redundancy with regard to the action of oxytocin in stimulating contraction of the myo-epithelial cells around the secretory alveoli in the mammary glands to cause milk ejection during suckling. In the oxytocin knockout mice, although the young are nursed they cannot obtain milk so they die, unless their mother is injected with oxytocin (Nishimori et al., 1996; Young et al., 1996). Oxytocin secreted from the posterior pituitary is released in the gland from axon terminals of neurons that have their cell-bodies in the supraoptic and paraventricular nuclei of the hypothalamus. There is indisputable evidence that these neurons in the maternal brain are activated during parturition. The content of oxytocin in the posterior pituitary decreases during parturition by about a third (Russell and Leng, 1998), and this is a result of increased firing of the cell bodies of the neurons in the magnocellular nuclei in the hypothalamus (Summerlee, 1981), while activation of the neurons is also indicated by the release of oxytocin from the dendrites of the neurons (Neumann et al., 1993), by the expression of the immediate early gene c-fos (Antonijevic et al., 1995b), and by stimulation of expression of the oxytocin gene in the neurons (Douglas et al., 1998). Taken together these different lines of evidence lead to the question of what mechanisms are involved in stimulating oxytocin neurons in parturition, and conversely what mechanisms prevent their premature activation? In addressing these questions in studies on rats, it has become evident that during pregnancy there are changes in these neurons and their inputs that prepare them for their role in parturition and beyond in lactation. The adaptations that occur in the oxytocin neurons and their input in pregnancy serve as a model for other neuron networks in the brain that are activated to perform functions related to the neuroendocrine mechanisms of lactation and maternal behavior.

Structural and functional plasticity of the oxytocin system

Oxytocin synthesis Although the amount of oxytocin stored in the posterior pituitary is increased by about 50% by the end of pregnancy, this may be a result of decreased release or breakdown rather than increased synthesis (Russell and Leng, 1998). There is disagreement in the literature concerning the timing of increased oxytocin gene expression in magnocellular neurons in pregnancy (Van Tol et al., 1988; Spinolo and Crowley, 1993; Horowitz et al., 1994; Douglas et al., 1998), with increased expression described consistently by some as occurring only after the fall in progesterone secretion (Crowley et al., 1995), or even only as a result of parturition (Douglas et al., 1998). In the former studies the increase in oxytocin gene expression is evidently a result of the withdrawal of the inhibitory action of progesterone (or allopregnanolone) acting via GABA receptors (Thomas et al., 1999). Thus, the withdrawal of the inhibitory action of progesterone on the electrical excitability of oxytocin neurons, allowing effective stimulation via the brainstem input driven by uterine contractions, coincidentally in the presence of estrogen, releases inhibitory restraint or otherwise stimulates oxytocin gene expression. This makes available more oxytocin for release in parturition, or in the subsequent lactation. In late human pregnancy (Lindow et al., 1996; Lindow et al., 2000) and in monkeys the nocturnal increase in uterine contractile activity seems to correlate with increased oxytocin secretion (Giussani et al., 1996). These rhythms may indicate a causal link between uterine contraction and oxytocin secretion and the onset of birth. In lactation, there is a consensus that the expression of oxytocin mRNA in the magnocellular neurons is increased after several days (see Douglas et al., 1998), evidently following a paradoxical decrease in the first few days of lactation (Crowley et al., 1993), although this may represent increased turnover. This stimulation of expression may reflect the continual drive to oxytocin secretion by suckling, which nonetheless results in depletion of the posterior pituitary content of oxytocin (Higuchi et al.,

19 1991). Prolactin has also been shown to have a role in regulating oxytocin gene expression (Ghosh and Sladek, 1995) while endothelin-1 expression in the SON changes in parallel with the oxytocin gene in the hypothalamus of the pregnant rat (Horowitz et al., 1994), and may also be a regulator of pregnancyrelated events. firing of oxytocin neurons have been on lactating rats. Three mechanisms seem to be important: closer contact between the cell bodies and dendrites of adjacent oxytocin neurons; an autoexcitatory action of oxytocin released from the dendrites and cell bodies of the oxytocin neurons; a change in the intrinsic mechanisms governing the excitability of the oxytocin neurons, such that the normal postspike hyperpolarization is not expressed (Brown and Moos, 1997; Russell and Leng, 2000). The contacts and synaptic connections of oxytocin neurons are reorganized in pregnancy, a fundamental change in the adult brain, and this is probably essential for the expression of bursting activity (see Theodosis and Poulain, 2001, this volume), although some of these changes accompany other states in which the magnocellular neurons are activated, such as dehydration (El Majdoubi et al., 2000). Retraction of glial processes separating adjacent neurons (Theodosis and Poulain, 1993), increased electrical coupling (Hatton et al., 1987) and changes in the dendritic tree of oxytocin neurons are involved (Stern and Armstrong, 1998). This plasticity is important for the expression of local excitatory glutamatergic (El Majdoubi et al., 1996; Boudaba et al., 1997; Moos et al., 1997) and inhibitory GABAergic (Gies and Theodosis, 1994; Boudaba et al., 1996; E1 Majdoubi et al., 1997) inputs to the SON and PVN which most likely underlie the bursting activity. These morphological changes, seen near the end of pregnancy, seem to depend on a local action of oxytocin in the presence of pregnancy levels of estrogen and progesterone (see Theodosis and Poulain, 2001, this volume). Indeed, in late pregnant rats suckling induces burst firing of oxytocin neurons, which resembles the suckling induced burst firing seen post partum more closely as term nears (Jiang and Wakerley, 1995). There are also changes in the noradrenergic synapses on oxytocin neurons (Michaloudi et al., 1997), that may enhance the effectiveness of the noradrenergic input (Bealer and Crowley, 1998), which acts via a local glutamatergic circuit (Parker and Crowley, 1993a,b; Daftary et al., 1998). However, a direct path from the brainstem conveying the suckling stimulus to oxytocin neurons has not been demonstrated, and there seems to be a diffuse

Burst firing: how pulses of oxytocin are generated in lactation

A particular aspect of the activity of oxytocin neurons in parturition and during suckling in lactation is that they fire in synchronized bursts a few minutes apart, so that oxytocin is secreted in pulses, and high concentration boluses of the peptide reach the receptors in the myometrium or mammary gland myo-epithelial cells (Poulain and Wakerley, 1982). This pattern of secretion evokes the optimal pattern of contractile activity in the targets: intermittent contractions and relaxation (Douglas et al., 2001), so the fetus(es) can continue the vital exchange of gases and metabolites with maternal blood in the placenta during myometrial relaxation, or the suckling young can receive milk to gulp intermittently during suckling. The effectiveness of this pattern of secretion is demonstrated by the restoration of births in rats in which oxytocin secretion has been inhibited by morphine, by pulsatile, but not continuous, low-dose oxytocin infusions (Luckman et al., 1993). Remarkably, so far as we know, the oxytocin neurons fire in bursts for the first time in the mother's life during parturition. Furthermore, it seems that the oxytocin neurons cannot fire in this bursting pattern until they have been modified or adapted in pregnancy to have the propensity to fire in bursts when appropriately stimulated in parturition or during suckling. Hence, a special question arises, what is the mechanism of burst-firing in oxytocin neurons, and how is this mechanism prepared in pregnancy? Although the burst-firing of oxytocin neurons is driven by input from the birth canal during parturition or from the nipples during suckling, the bursts are generated locally, as a result of interaction between intrinsic properties of the neurons and their local excitatory and inhibitory amino acid synapses (Jourdain et al., 1996, 1998). Most of the studies on the mechanisms of burst-

20 multi-synaptic path (Leng et al., 1999). A wealth of studies has indicated neurotransmitters in long projections that may be involved in mediating the driving of oxytocin neurons by suckling (see Fukuoka et al., 1984; Takano et al., 1992). Fos expression studies have not been helpful in delineating suckling-activated pathways to oxytocin neurons (see below). generated by each neuron over many minutes of suckling is calculated it is evident that this is not greater than their total activity over a similar period without suckling. GABA has a role in this gathering of action potentials into bursts with relative silence in between (Moos, 1995). The mechanism of the synchronization among the four magnocellular nuclei is not clear: among the nuclei there may be connections via contacts between dendrites allowing an internuclear synchronizing role for oxytocin (Neumann et al., 1995). Connections between the BNST and magnocellular oxytocin neurons are important in facilitating the milk ejection bursts, since oxytocin is released and acts here on up-regulated oxytocin receptors (Ingram et al., 1995). During suckling in the sheep oxytocin release in the BNST is increased (Kendrick et al., 1992). Electrophysiological studies show excitation of BNST and ventrolateral septum neurons correlated with milk-ejection bursts of magnocellular oxytocin neurons during suckling, and their excitation by oxytocin (Lambert et al., 1993a), although the effects of oxytocin antagonist are not reported. The capacity for burst firing, in response to suckling, emerges in late pregnancy, but the bursts increase in intensity post partum (Jiang and Wakerley, 1995). The effects of oxytocin on BNST neurons post parturn are optimal due to steroid priming and increased oxytocin receptor expression by the end of pregnancy (Ingram and Wakerley, 1993; Housham et al., 1997; Wakerley et al., 1998; Terenzi et al., 1999; Francis et al., 2000).

Burst triggering and synchrony: oxytocin, oxytocin, oxytocin

An essential feature of the burst firing of oxytocin neurons is that to generate a pulse of oxytocin secretion from the posterior pituitary, the bursts have to occur in all of the oxytocin neurons in the four magnocellular nuclei at the same time. Within the nuclei there are more close contacts between adjacent magnocellular neurons, as astrocytic glial processes retract in lactation, and there are more shared synaptic boutons (Hatton and Tweedle, 1982; see Theodosis and Poulain, 2001, this volume). Each burst of firing of oxytocin neurons during suckling, which occurs during an unchanging application of the suckling stimulus (see Stern and Lonstein, 2001, this volume), is triggered by local mechanisms in the magnocellular nuclei. The local release of oxytocin itself (Neumann et al., 1993) from the magnocellular neuron dendrites and cell bodies (Ludwig, 1998), is of key importance: intracerebroventricular injection of oxytocin can trigger a series of milk-ejection bursts in oxytocin neurons during suckling (Lambert et al., 1993a; Moos and Ingram, 1995), but not under other conditions, and oxytocin antagonist infusion stops reflex milk-ejection bursts (Lambert et al., 1993b; Neumann et al., 1994). The mechanism to explain this action of oxytocin may involve a presynaptic inhibitory action of oxytocin on excitatory amino acid-containing terminals on oxytocin neurons (Pittman et al., 2000). GABA input to oxytocin neurons has a predominant role in the regulation of their burst-firing during suckling (Moos, 1995; Voisin et al., 1995). It has been pointed out previously that although a characteristic of oxytocin neuron responses to suckling is short bursts of high frequency firing every few minutes, their activity between bursts is at a reduced level. Thus, if the total number of action potentials

Pathways to oxytocin neurons in parturition

Excitatory inputs It was proposed by Ferguson more than half a century ago that the secretion of oxytocin in parturition is stimulated reflexly by the distension of the birth canal with the passage of the fetus, a consequence of the uterine contractions induced by oxytocin (Ferguson, 1941). Clearly the Ferguson reflex is a positive feedback loop. This has been demonstrated by recording, in barbiturate-anesthetized late pregnant rats, the electrical activity of supraoptic nucleus oxytocin neurons during intermittent (10-min intervals) bolus infusions of oxytocin (10-20 mU) with

21 recording of intrauterine pressure. The firing rate of the oxytocin neurons increased progressively with the increase in intrauterine pressure following the injections of oxytocin (Douglas et al., 2001), although burst firing was not seen, perhaps because of the barbiturate anesthesia. In conscious or anesthetized late pregnant rats, repeated bolus injections of oxytocin also stimulate c-fos gene expression, detected immunocytochemically as Fos protein expression in the nuclei of supraoptic neurons (Antonijevic et al., 1995b; Douglas et al., 2001). The Fos technique has been used to study the inputs to oxytocin neurons that are activated in parturition. Brainstem Neural afferents from the uterus or birth canal are relayed via the nucleus tractus solitarius (NTS), while noradrenergic A2 neurons in the NTS have been shown to project to magnocellular oxytocin neurons (Raby and Renaud, 1989; see Theodosis and Poulain, 2001, this volume). Initial studies showed that NTS noradrenergic neurons (identified by their content of tyrosine hydroxylase, the enzyme essential for catecholamine synthesis) express Fos after births are complete, at a time indicating their activation in parturition (Luckman, 1995). Furthermore, bolus intravenous infusions of oxytocin resulted in similar stimulation of Fos expression in A2 neurons as well as in SON neurons (Antonijevic et al., 1995b). Taking this one step further, it has recently been shown that NTS A2 neurons identified as projecting to the SON, by retrograde labelling from microinjections of fluorescent latex microspheres into the SON some weeks previously, are activated in normal parturition (Meddle et al., 2000). However, only 60% of the activated (Fos-containing) NTS neurons projecting to the SON also contained tyrosine hydroxylase, so 40% were not noradrenergic. Their identity is still to be established. Measurement by high pressure liquid chromatography (HPLC) in microdialysis samples collected from the SON before and during parturition shows greater increase in release of noradrenaline than dopamine, serotonin or glutamate in parturition (Herbison et al., 1997), and pulsatile oxytocin infusion in anesthetized late pregnant rats stimulates release of occasional pulses of noradrenaline in the SON (Douglas et al., 2001). The stimulation of Fos expression in the SON by such intravenous pulses of oxytocin is prevented by the local infusion of an cq-adrenergic receptor antagonist, benoxathian, indicating the importance of noradrenergic neurons in this reflex activation of oxytocin neurons (Douglas et al., 2001). However, intracerebroventricular infusion of benoxathian does not affect spontaneous parturition (Douglas et al., 1999), so other transmitters must be involved, as indicated by the combined retrograde labelling, tyrosine hydroxylase and Fos expression studies in the NTS mentioned above. It seems therefore that, while noradrenaline is released to activate oxytocin neurons in parturition, there is redundancy; but the other transmitters involved are still to be identified. Olfactory bulbs Although emphasis has been on the most direct neural route from the birth canal to the oxytocin neurons, parturition in at least rodents and sheep is a major olfactory event. Indeed, as discussed above, olfactory stimuli from the newborn are essential in sheep, via the formation of olfactory memory at birth, for the establishment of maternal behavior and identification of the offspring (Kendrick et al., 1997a). While in rats, an important step in the rapid establishment of maternal behavior post partum is the suppression of the fear of pups that is mediated by olfactory stimuli (Lonstein et al., 1998; Winslow et al., 2000; see Champagne and Meaney, 2001, this volume). During parturition in the rat there is clearly abundant opportunity for olfactory signalling to the mother, who can be seen frequently to lick her perineum and the emerging pups, and to ingest the membranes, fluids and placenta (and remarkably infrequently to eat the attached pup). In the sheep activation of the olfactory bulbs is revealed by the release of neurotransmitters, in microdialysates (Kendrick et al., 1988), and in the rat by a substantial increase in the numbers of mitral cells (the second order neurons that project centrally) that express Fos. Some mitral cells are retrogradely labelled by fluorescent tracer micro-injected into the SON, and a substantial proportion of these neurons (~ 11%), greater than in virgin rats or in rats near the end of pregnancy, contain Fos (Meddle et al., 2000). Hence it is likely that mitral cells in the main olfac-

22 tory bulbs projecting to the SON, which are likely to be glutamatergic (Yang et al., 1995; Meddle et al., 2000), contribute to the stimulation of oxytocin neurons during parturition. Though this has not been critically tested, if correct, then the olfactory input to oxytocin neurons forms another positive feedback loop driven by stimuli arising during parturition. Although olfactory input seems likely to be the primary stimulus to activate the mitral cells, there is also the possibility that the noradrenergic projection to the olfactory bulbs from the brainstem (Meddle et al., 2000), activated during parturition as discussed above, also excites the mitral cells. Furthermore, oxytocin acts in the olfactory bulbs in parturition, evidently to promote the establishment of maternal behavior (see above; Levy et al., 1995). In the rat there is evidence that oxytocin released centrally from PVN neurons excites mitral cells in the olfactory bulbs (Yu et al., 1996a). Both the brainstem noradrenergic and paraventricular nucleus oxytocin projections to the mitral cells would also be parts of positive feedback loops in parturition. Other rostral inputs Progesterone and GABA Although there is extensive activation of rostral neurons as a result of parturition, evident as increased Fos protein expression, very few of these neurons project to the SON (Meddle et al., 2000), and are therefore unlikely to be involved, at least directly, in the stimulation of magnocellular oxytocin neurons in parturition. Rather, these neurons, for example those in the medial preoptic area, are likely to be involved in the establishment of maternal behavior (Numan, 1986). It is notable that other rostral inputs to the SON, in particular those concerned with osmoregulation arising in the lamina terminalis (organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus and subfornical organ), do not show activation at parturition, although OVLT cells are activated the day before parturition (Meddle et al., 2000). Local oxytocin A role for centrally released oxytocin in stimulating olfactory mechanisms in parturition is, as mentioned, well established. But, as in lactation, oxytocin has a So what prevents the premature activation of this positive feedback mechanism? The dramatic decrease in circulating progesterone level near the end of pregnancy in several species (though not in women) removes an inhibitory influence at several sites in the pathway from the uterus to the oxytocin neurons. First, progesterone has an inhibitory action on uterine contractile activity. This is exerted on the myometrial cells in the rat in part by reducing oxytocin binding to its receptor (Grazzini et al., 1998). Strikingly, there is a dramatic increase in oxytocin receptor expression in the myometrium over the day before parturition in the rat, following the decline in progesterone secretion (Fuchs et al., 1983). Hence, progesterone withdrawal greatly increases uterine excitability, particularly in response to oxytocin. Conversely, administration of progesterone when its secretion normally plummets delays parturition, rendering the uterus insensitive to oxytocin. In this condition intravenous bolus infusion of oxytocin is ineffective in inducing uterine contractions and parturition (Antonijevic et al., 2000). Furthermore, more direct role in the brain in the positive feedback stimulation of oxytocin neurons in parturition. It is released within the magnocellular SON and PVN during parturition (Neumann et al., 1993), probably from the dendrites and cell bodies of the oxytocin neurons (Ludwig, 1998) as synaptic terminals in the magnocellular nuclei containing oxytocin are quite sparse (Theodosis, 1983). This is evidently important in the excitation of oxytocin neurons as local infusion of an oxytocin antagonist, even unilaterally, slows parturition (Neumann et al., 1996). Further information about how oxytocin acts to control burstfiring comes from studies on this local mechanism in lactation (see above). There may be an additional action of oxytocin on indirect limbic system input, as oxytocin release increases during parturition in the BNST (sheep; Kendrick et al., 1992) and septum (rat), at least after naloxone (Neumann et al., 1991), and this region is where oxytocin facilitates burst-firing of oxytocin neurons during suckling (see above).

Restraint: inhibitory mechanisms

23 with the uterus insensitive to oxytocin as a result of sustained progesterone action, intravenous bolus oxytocin infusions at the end of pregnancy, at a time when they are normally effective, fail to induce Fos activation in either NTS or SON neurons (Antonijevic et al., 2000), indicating progesterone block of the Ferguson reflex. As this progesterone block wanes, so the effectiveness of intravenous bolus oxytocin infusion returns, or spontaneous parturition intervenes, about a day later than normal if progesterone is given the day before parturition normally occurs. However, it is evident from studying Fos expression in the NTS and SON in both spontaneous or oxytocin-induced parturition after progesterone-induced delay, that the progesterone block to activation of the Ferguson reflex pathway is withdrawn progressively. This is first at the uterus, next at the NTS, and finally at the SON (Antonijevic et al., 2000). The mechanism of progesterone action on the NTS or oxytocin neurons is unlikely to be via genomic mechanisms involving progesterone receptors within these neurons as we have not detected the receptor immunocytochemically in the SON. In the NTS there are fewer neurons expressing progesterone receptor at the end of pregnancy, with very low incidence of co-expression with tyrosine hydroxylase (Francis and Russell, unpublished observations). There is, however, convincing evidence for a direct inhibitory action of the progesterone metabolite allopregnanolone on oxytocin neurons, through allosteric modulation of the form of GABAA receptor expressed by oxytocin neurons in pregnancy (see Herbison, 2001, this volume). The switch in the subunit composition of GABAA receptors occurring in pregnancy, and reversing at parturition, represents one of the most basic changes in the neurobiology of the maternal brain (Brussaard et al., 1997; see Herbison, 2001, this volume; Follesa et al., 1998; Concas et al., 1998; Brussaard et al., 1999; Leng and Russell, 1999). A progesterone-induced increase in ~-subunit expression confers GABAA receptor sensitivity to the non-genomic inhibitory action of the progesterone metabolite, allopregnanolone, during pregnancy, allowing for suppression of burst firing of the oxytocin neurons until such time as steroid levels fall (Brussaard et al., 1997; Fenelon and Herbison, 2000). This mechanism can account for the more prolonged inhibition of the activation of SON neurons by exogenous oxytocin, described above, than of the myometrium, as the production or presence of allopregnanolone in the brain may outlast circulating progesterone. Whether progesterone acts to inhibit NTS neurons by a similar interaction with GABA mechanisms has not been studied, and whether progesterone has a similar blocking action at the oxytocin receptor in the brain to its reported action in the myometrium (Grazzini et al., 1998) is not clear. Clearly, the actions of allopregnanolone on oxytocin neurons require not only expression of the appropriate GABAA receptor subunits, but also an active GABA inhibitory input. Mechanisms regulating this GABAergic input are not known, but could involve genomic actions of estrogen, as local GABAergic neurons express estrogen receptor (Herbison, 1994). An important change, at least in lactation, is the increase in density of GABA synapses on oxytocin neurons (see Tbeodosis and Poulain, 2001, this volume). The activity of GABAergic synapses on oxytocin neurons may also be involved in the restraint by progesterone (or allopregnanolone) of oxytocin gene expression, which increases only after progesterone withdrawal at the end of pregnancy (see above; Amico et al., 1997; Douglas et al., 1998). Thus, the withdrawal of the inhibitory action of progesterone on the electrical excitability of oxytocin neurons, allowing effective stimulation via the brainstem input driven by uterine contractions, and coincidentally, in the presence of estrogen, releases inhibitory restraint of, or otherwise stimulates oxytocin gene expression. This makes available more oxytocin for release in parturition, or in the subsequent lactation. Nitric oxide A further inhibitory mechanism restraining oxytocin neurons is the production by oxytocin neurons themselves of nitric oxide (NO). The magnocellular PVN and SON neurons express neuronal nitric oxide synthase (nNOS) more strongly than other hypothalamic neurons, and expression increases with chronic stimulation (Kadowaki et al., 1994). NO produced by SON neurons acts both post-synaptically to inhibit electrical excitability, but also pre-synaptically to increase the frequency of GABA release (Stern and Ludwig, 2000). Near the end of pregnancy the activity of oxytocin neurons decreases (Summerlee, 1981), however,

24 the expression and activity of NOS in oxytocin neurons also changes (Woodside and Amir, 1996; Srisawat et al., 2000). Thus, it is not easy to explain the reduced activity of oxytocin neurons at the end of pregnancy in terms of increased NO production, although central administration of a NO donor in parturition slows births (Okere et al., 1999). So far, this account has described the removal of inhibitory mechanisms on oxytocin neurons at the end of pregnancy that will allow their stimulation by excitatory inputs arising in the contracting uterus or birth canal. However, such unrestrained stimulation may be expected, as a positive feedback loop is involved, to lead to runaway excitation of oxytocin secretion, whereas what is seen is a modest increase in oxytocin secretion (Higuchi et al., 1986), well below the secretory capacity of the neurons, with a superimposed intermittent pulsatile pattern of activity and release of boluses of oxytocin. The origin of the pulsatile pattern is enigmatic, but probably involves intrinsic properties of the neurons responsible for the high frequency synchronized firing followed by refractoriness to this behavior, as in lactation (Lincoln and Wakerley, 1974). Opioids In addition, there is increasing central endogenous opioid inhibitory tone on oxytocin neurons towards the end of pregnancy, so that administration of the opioid antagonist naloxone increases oxytocin secretion as a consequence of increasing the firing of the neurons (see Douglas and Russell, 2001, this volume). The effect of naloxone is greatest in parturition when it increases oxytocin secretion, but also accelerates the delivery of pups, and in the rat prevents the slowing of parturition and inhibition of oxytocin secretion as a consequence of environmental stress (Leng et al., 1988). Opioids inhibit oxytocin secretion in humans also in parturition, but endogenous opioid tone does not appear to develop (Lindow et al., 1992). It is not known whether naloxone, and hence endogenous opioids, alter the burst firing of oxytocin neurons in parturition. The source of the opioid peptide and activation by sex steroids are discussed elsewhere (see Douglas and Russell, 2001, this volume). It is possible that the endogenous opioid is co-produced in brainstem neurons projecting to the magnocellular neurons. The terminals of the A2 noradrenergic neurons projecting to the SON are sensitive to the inhibitory actions of the Ix-opioid agonist morphine (Onaka et al., 1995). There is evidence that the endogenous opioid mechanism restraining oxytocin neurons in pregnancy is activated by the combined action of estrogen and progesterone (Douglas et al., 2000b; see Douglas and Russell, 2001, this volume). It is striking that activation of central endogenous opioid mechanisms in pregnancy is important in the spinal analgesia of pregnancy (see Gintzler and Liu, 2001, this volume), and in the regulation of prolactin secretion (see Voogt et al., 2001, this volume), as well as in the control of oxytocin neurons. GnRH neurons are also inhibited by endogenous opioids during the estrous cycle (Bakker and Baum, 2000), but while opioid antagonists can disinhibit the GnRH pulse generator in women with hypothalamic ovarian failure, leading to pregnancy (Wildt et al., 1993), opioid inhibition does not apparently contribute to lactational amenorrhea (see McNeilly, 2001, this volume). After parturition, endogenous opioid restraint of oxytocin neurons is evidently not important in the regulation of oxytocin secretion in response to suckling (Bicknell et al., 1988). However, nNOS mRNA expression in the SON increases following parturition (Srisawat et al., 2000), perhaps as a result of the stimulation of the neurons during parturition, so NO evidently resumes importance as a regulator of oxytocin neurons (Liu et al., 1998; Okere et al., 1999).

Further roles of oxytocin

Thus, magnocellular oxytocin neurons are important in producing a pattern of oxytocin secretion from the posterior pituitary that efficiently and effectively stimulates the myometrium in parturition and the myo-epithelial cells in the mammary glands during lactation. The pattern of activity is importantly shaped by local positive feedback actions of oxytocin on the oxytocin neurons themselves. There are other central actions of oxytocin that promote successful motherhood, including facilitating expression of maternal behavior, at least in rats and sheep, species in which the oxytocin receptors in the brain are appropriately distributed (see Insel et al., 2001, this

25 volume). However, as oxytocin knockout mice show maternal behavior, oxytocin itself cannot be essential for maternal behavior in this species (see Insel et al., 2001, this volume). Interestingly, in mice with a mutation of the Peg3 gene, normally expressed in the hypothalamus but of paternal origin, there are deficits in maternal behavior and reduced numbers of PVN oxytocin neurons with deficient milk ejection (see Keverne, 2001, this volume). Earlier inconsistent findings concerning the effects of oxytocin on maternal behavior in rats led to the idea that it may act centrally to promote maternal behavior in conditions of stress (McCarthy et al., 1992). The question whether this generalizes to an anti-anxiety or antistress action of central oxytocin in motherhood has been considered in the discussion of altered stress responsiveness above. Pregnancy-related mental illness: a case of dysregulated neural plasticity? We have focused on one neural system, the oxytocin neurons, of particularly great importance in the success of motherhood. We have described substantial changes in these neurons and their connections in pregnancy and lactation, which are adaptations that optimize their performance in parturition and lactation. We have outlined changes in the level or type of neuropeptide expression in CRH pPVN neurons in pregnancy and lactation and the impact this has on pituitary-adrenal stress responses. Questions arise about other, perhaps adverse, consequences of the changes in the brain for motherhood that lead to the adaptations in these two neuroendocrine systems; about the regression of these changes following the two cataclysmic events of motherhood, first the birth and then the weaning of the offspring; and about the occurrence and regression of similar changes in intracellular and synaptic organization in other neural networks that are important in the expression of motherhood. The impact of the changes in the neural systems we have discussed is that motherhood does not merely involve activation, or inhibition, of preexisting neural circuits but involves major changes at multiple levels in these networks, including within neurons, the neurotransmitter phenotype, the cellular mechanisms governing excitability and patterns of electrical activity, and the connections between nearby and distant neurons. Changes in gene expression interacting with membrane actions of sex steroid metabolites are involved. Such changes occurring in networks other than those we have discussed may underlie and cause changes in other neuroendocrine functions, such as appetite and energy expenditure regulation, and changes in behavior and mood, including aggressiveness to protect the young (see Neumann, 2001, this volume). As pointed out by Herbison (2001, this volume) interaction of allopregnanolone with GABAA receptors in the hippocampus has an anti-seizure action, so there is predisposition to seizures post partum, as a result of progesterone withdrawal. Changes in GABAA receptor subunit expression in the cerebral cortex may contribute to the depressed mood post partum, while actions of progesterone, or metabolites, affect cognition (see Buckwalter et al., 2001, this volume). The high estrogen levels in pregnancy may have membrane or gene expression effects, with actions on the cerebral cortex also affecting cognition (see Buckwalter et al., 2001, this volume). Overall, there are deficits in memory in pregnancy, and this may outlast the pregnancy, although there seem to be stronger effects of the fluctuations in sex steroid levels on mood (see Buckwalter et al., 2001, this volume). The peri-partum period represents one of the greatest risks to the mental health of a woman. Whereas transient emotional lability post partum is common (>50%), some 10-15% of women experience post-partum depression, and 0.1% puerperal psychosis. The blues may be a response to the traumatic aspects of childbirth (see Kumar, 2001, this volume), but the greater risk of depression post partum seems likely to be a consequence of the withdrawal of the high levels of sex steroids, or their neurosteroid metabolites (Abou-Saleh et al., 1998; see Buckwalter et al., 2001, this volume), and similarly for post-partum psychosis (see Kumar, 2001, this volume) which also has a genetic basis, but the genes involved have not been identified (see Jones et al., 2001, this volume). The major disturbances in emotional state may be a consequence of withdrawal of the continual actions of progesterone and estrogen, or androgens, on limbic circuits, either as direct actions or secondary to the types of action described above. The adverse effects of such withdrawal could

26 be a consequence of the types of change outlined above (e.g. altered GABAA receptor subunit expression; Smith et al., 1998), altered phenotype or excitability of neurons, changes in the strength of specific synaptic inputs, either unbalanced by effects of the steroids, or reversing as a result of removal of the steroids responsible for the changes. One proposal is that the changes in the activity of the maternal HPA axis in pregnancy, involving altered activity of CRH neurons and responsiveness of corticotrophs, slowly reset after delivery and delay in this recovery may predispose to the blues or post-partum depression (see Carter et al., 2001, this volume). In view of the accepted role of CRH in the etiology of depressive illness (Arborelius et al., 1999), it is possible that post-partum depression arises from an inappropriate and persistent hyperactivity of the CRH system. This could arise from a rebound in the activity of central CRH mechanisms from an inhibited state in pregnancy, and relate to the withdrawal of estrogen, as well as progesterone (Gregoire et al., 1996).
Conclusion

DMH FSH GABA GnRH HPLC HPA LH MeA MPOA MC4 NMDA NPY NO nNOS NTS OVLT PAG PL POMC pPVN PVN SON TIDA VLS VMH

dorsomedial hypothalamus follicle-stimulating hormone gamma-aminobutyric acid gonadotrophin-releasing hormone high pressure liquid chromatography hypothalamo-pituitary-adrenal luteinizing hormone medial amygdala medial preoptic area melanocortin 4 receptor N-methyl-D-aspartate neuropeptide Y nitric oxide neuronal nitric oxide synthase nucleus of the tractus solitarius organum vasculosum of the lamina terminalis midbrain periaqueductal gray placental lactogen pro-opiomelanocortin parvocellular PVN paraventricular nucleus of the hypothalamus

The contemporary need to understand the mechanisms and importance of these changes relates to the attention that can now be given, at least in the countries where well-developed health care systems have minimized mortality and morbidity from conditions such as eclampsia, obstetric disasters and infection, to the more subtle and delayed impact of the quality of the internal environment in pregnancy, of the length of pregnancy (Knoches and Doyle, 1993) and of the interactions between mother and child post partum (Field, 1998; see Champagne and Meaney, 2001, this volume). The mother's mental health and the infant's potential for a long, happy and healthy life are importantly affected by these environmental factors (Francis et al., 1999).
Abbreviations

supraoptic nucleus tuberoinfundibular dopaminergic ventrolateral septum ventromedial nucleus of the hypothalamus

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Progressin BrainResearch, Vol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 2

Physiological roles for the neurosteroid allopregnanolone in the modulation of brain function during pregnancy and parturition
A l l a n E. H e r b i s o n *
Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, UK

Abstract: Allopregnanolone is a well-established allosteric modulator of the GABAA receptor but its physiological roles within the nervous system remain unclear. Derived principally from circulating progesterone, allopregnanolone achieves its highest concentrations within the nervous system during late pregnancy and recent studies have now begun to elucidate its roles at this time in the rat. At the molecular level it is clear that the regulation of GABAA receptor subunit gene expression by progesterone and its derivatives occurs in a subunit- and a neuron-specific manner and that both progesterone and allopregnanolone are involved. At the cellular level, the increasing concentrations of allopregnanolone with advancing pregnancy can be shown to have important physiological actions in repressing the electrical activity of specific neuronal phenotypes such as the magnocellular oxytocin neurons. The marked fall in progesterone and allopregnanolone concentrations prior to parturition equally appears to have a substantial impact upon GABAA receptor signaling in the hippocampus, frontal cortex and oxytocin neurons. Together, studies at a basic level suggest that the rise and fall in allopregnanolone concentrations during pregnancy are likely to exert a powerful regulatory influence upon neurotransmission in a variety of brain networks. The temporal correlation between these events and the observed cognitive, psychiatric and physiological changes associated with pregnancy and the peri-partum period in humans is striking and warrants close attention.

Introduction

Progesterone is one of the principal hormones secreted by the corpus luteum and placenta to ensure the survival and successful development of the embryo through to term and beyond. Accordingly, the gradually increasing concentrations of circulating progesterone over the course of pregnancy target a wide range of reproductive and non-reproductive tissues within the expectant mother. One such site of action is the nervous system where, not surprisingly, roles for progesterone in the regulation of * Correspondence to: Allan E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, UK. Tel.: +44-1223-496-528; Fax: +44-1223496-022, E-mail: allan.herbison@bbsrc.ac.uk

the neural networks controlling parturition and pain, as well as a spectrum of reproductive behaviors, have been described (Pfaff et al., 1994; Crowley et al., 1995; Dawson and Gintzler, 1998; Numan et al., 1999). Additionally, for many women, pregnancy and the peri-partum period represent a time of marked change in their cognitive and psychological well-being with reduced memory capability, altered mood and anxiety levels (Pugh et al., 1963; O'Hara, 1986; Buckwalter et al., 1998; Keenan et al., 1998). Some of these parameters, and particularly those of post-partum mood, have been positively associated with the absolute levels, or the peri-partum decline, of plasma progesterone concentrations (Nott et al., 1976; Harris et al., 1994; Buckwalter et al., 1998). While it seems reasonable to conclude that specific progesterone receptor-expressing neuronal pop-

40

Gonads, placenta
cholesterol

/
neurons

/-regnanolone glial cells

Fig. 1. Schematic representation of the production and action of allopregnanolone on GABAA receptors. Allopregnanolone is formed in the brain by glial cells either de novo from cholesterol, or principally, from circulating progesterone originating from the gonads and placenta. Progesteroneundergoes a two step reduction (enzymesin italics) within glial cells to become allopregnanolone,which then acts as an allosteric modulator at several different subtypes of GABAA receptors within the central and peripheral nervous system. Although the precise mechanismof action is not yet fully understood, it is clear that allopregnanolonemodulates the receptorto result in increased channel open times following activation by GABA. This is observed experimentally in voltage clamp recordings of individual neurons (inset) where allopregnanolone (+ allo) is found to have no effect upon opening of the channel (current amplitude measured in pA) but increases the decay kinetic (duration measured in ms) of inhibitory post-synapticcurrents.

ulations within limbic brain regions are responsible for some of these actions (Pfaff et al., 1994; Numan et al., 1999), they are unlikely to account for all of the known cognitive, psychological and anti-seizure effects of progesterone on the brain (Craig, 1966; Freeman et al., 1993; Baker et al., 1995; Herzog, 1995; Rupprecht, 1997). At present, the most attractive non-progesterone receptor-dependent mechanism of neuronal regulation by progesterone is that believed to occur through the progesterone derivative allopregnanolone (3~-hydroxy-5c~-pregnan-20one or 5c~-pregnan-3c~-ol-20-one). Allopregnanolone is one of a number of neuroactive 'neurosteroids' synthesized principally by glial cells within the brain (Fig. 1; Majewska, 1992; Paul and Purdy, 1992; Baulieu, 1998). Although the brain has the capacity to synthesize allopregnanolone de novo from cholesterol, the majority of allopregnanolone in the brain is derived from circulating progesterone (Corpechot et al., 1993; Cheney et al., 1995; Genazzani et al., 1998). Thus, during late pregnancy when progesterone levels reach their physiological maximum, it

has been shown that both circulating and brain levels of allopregnanolone also reach their peak; in the rat this amounts to a circulating concentration of ~ 4 0 ng/ml (>100 nM) and a brain concentration of ~ 1 0 ng/g (Paul and Purdy, 1992; Corpechot et al., 1993; Cheney et al., 1995; Concas et al., 1998; Fig. 2). It is now well established that allopregnanolone at low 'physiological' concentrations has a direct and highly selective allosteric modulatory action upon the "f-aminobutyric acid (GABA) type A ionotropic (GABAA) receptor (Majewska, 1992; Lambert et al., 1995; Rupprecht and Holsboer, 1999). Allopregnanolone exerts a pronounced effect upon neurons by enhancing GABAA receptor signaling, which is typically inhibitory in the adult brain. Allopregnanolone binds to the GABAA receptor in an as yet undetermined manner, to increase the mean channel open time of the receptor when activated by its ligand GABA (Fig. 1). This results in GABAA receptors remaining open longer and allowing more chloride ion flux in the presence of allopregnanolone. As chloride ion entry into a neuron normally hyperpolarizes the

41
oxytocin neurons

Prevention of premature firing ofoxytocin neuron Activation of oxytocin neuron at parturition

hippocampus

c~4 subunit: Increased anxiety and reduced seizure threshold

"[2 subunit

y2 subunit: unknown

cerebral cortex

? Pregnancy and post-partum changes in cognition and mood

advancing pregnancy birth

Fig. 2. Schematic representation of the fluctuating levels of circulating allopregnanolone (black) and progesterone (gray) concentrations over the course of pregnancy and parturition (left) and their effects upon the profile of specific GABAA receptor subunits expressed in oxytocin, hippocampal, and cerebral cortex neurons. The putative physiological implications of these changes are given for each region (right). Data for the c~4 subunit in the hippocampus are extrapolated from pseudopregnant animals and the full profile over pregnancy is not available (Smith et al., 1998b). The ct5 and y2 data are from Follesa et al. (1998) and the ctl results from Fenelon and Herbison (1996). While progesterone regulates subunit expression in oxytocin neurons, allopregnanolone influences subunit expression in the hippocampus and cerebral cortex.

cell, the net effect of allopregnanolone exposure is to enhance inhibitory GABA transmission. Although the mode of action of this progesterone derivative has been well established in several in vitro paradigms, many questions remain. In particular, because the pentameric GABAA receptor can be formed from among more than 15 different subunits (Barnard et al., 1998), it is not clear whether allopregnanolone acts at all GABAA receptor isoforms or only those receptors with a specific subunit stoichiometry (see Lambert et al., 1995). Furthermore, because of the lack of a specific antagonist for allopregnanolone's action at the GABAA receptor, and the inherent technical difficulties in examining neurons within their native environment, it is unclear what physiological roles allopregnanolone may play within the brain. In this respect, the time of late pregnancy, when the highest physiological levels of allopregnanolone are achieved in the brain, appears to be an ideal setting within which to explore this issue. Very recent studies have now hinted at the physiological roles of allopregnanolone in the control of the magnocellular oxytocin neurons, hippocampus

and cerebral cortex during pregnancy. While there are undoubtedly many more brain regions and neuronal phenotypes to be discovered which are targeted by allopregnanolone in the 'maternal brain', an examination of these three networks is likely to be instructive in uncovering the ways in which progesterone can influence the nervous system through its derivative allopregnanolone.

Aliopregnanolone, the magnocellular oxytocin neurons and parturition

The magnocellular oxytocin neurons reside within the supraoptic (SON) and paraventricular nuclei of the hypothalamus from where they project to the posterior pituitary and secrete oxytocin directly into the circulation. The electrical activity of these neurons undergoes substantial changes over the course of pregnancy and lactation (Summerlee, 1981; Leng et al., 1999), and progesterone is thought to have a particularly important role in synchronizing the activity of oxytocin neurons with other physiological processes during pregnancy (Negoro et al., 1973;

42 Crowley et al., 1995). The oxytocin neurons in the rat are known to exhibit a low level of firing during most of pregnancy, but then display an abrupt transition to synchronous bursting behavior, superimposed on elevated tonic firing, at the time of birth when the pulsatile release of oxytocin contracts the uterine myometrium to help expel the pups (Summerlee, 1981; Jiang and Wakerley, 1995). As the oxytocin neurons do not seem likely to possess progesterone receptors (Numan et al., 1999; Fenelon and Herbison, 2000), or respond directly in a non-genomic fashion to progesterone itself (Wang et al., 1995), it has not been clear how progesterone might influence this important neuronal phenotype. The oxytocin neurons receive a substantial GABAergic input, estimated to comprise approximately 40% of all synapses on these cells (Majdoubi et al., 1997), and express GABAA receptors comprised principally of ~1, 0t2, 132, 133 and y2 subunits (Fenelon et al., 1995). A potential role for progesterone in the regulation of GABAA receptors expressed by oxytocin neurons was first suggested by in situ hybridization studies which revealed that the expression of the c~l subunit transcript in oxytocin neurons fluctuated over the course of pregnancy in a highly selective manner (Fenelon and Herbison, 1996). The expression level of c~l subunit mRNA in putative oxytocin neurons was found to increase with advancing pregnancy and then fall by 30-40% over the last 2 days prior to birth (Fig. 2). This closely resembled the profile of progesterone secreted over pregnancy and parturition and recent studies have shown that progesterone does indeed enhance ctl subunit mRNA expression in the SON (Herbison et al., 1998; Fenelon and Herbison, 2000). This action of progesterone thus induces the expression in oxytocin neurons of a particular type of GABAA receptor that could be more sensitive to allopregnanolone. The most important observations have come from patch-clamp electrophysiological analyzes of oxytocin neurons within their native environment in an acute rat brain slice preparation (Brussaard et al., 1997, 1999; Brussaard and Herbison, 2000). These studies have shown that allopregnanolone is able to directly enhance oxytocin neuron GABAA receptor signaling in late pregnancy by slowing the decay kinetic of GABA-mediated inhibitory post-synaptic currents (IPSCs) and thus, engender a powerful inhibitory influence upon the neuron. Indeed, a substantial tonic inhibition of oxytocin secretion exists in late pregnancy in vivo (Brussaard et al., 1997) and this correlates perfectly with the peak of allopregnanolone concentrations found in the brain on day 19 of pregnancy in rats (Concas et al., 1998). Together, these observations have led us to suggest that allopregnanolone modulation of oxytocin neuron GABAA receptor activity represents an important physiological feedback mechanism through which progesterone represses oxytocin neuron activity in late pregnancy to prevent premature delivery (Brussaard and Herbison, 2000). More remarkably, however, we found that the sensitivity of the oxytocin neuron GABAA receptor to allopregnanolone declines dramatically over the last day of pregnancy so that it is reduced by over 10-fold on the day of parturition (Brussaard et al., 1997). As this decline in allopregnanolone facilitation of the GABAA receptor occurs in the face of unchanging GABA release upon the oxytocin neuron (Brussaard et al., 1999; Fenelon and Herbison, 2000), it seems likely to exert a powerful dis-inhibitory influence upon the electrical activity of the oxytocin neurons. Together with changes in other neurotransmitters such as glutamate, norepinephrine, and opioid peptides (Douglas et al., 1995; Herbison et al., 1997), the allopregnanolone dis-inhibition of oxytocin neurons in very late pregnancy is likely to be part of the mechanism enabling their transition to a synchronous bursting pattern of behavior necessary for parturition to proceed. Although a reasonable case may be made for a physiologically relevant role for allopregnanolone in the regulation of the oxytocin neurons, the precise mechanisms underlying the peri-partum changes in the allopregnanolone sensitivity of the GABAA receptors expressed by oxytocin neurons are unclear. On one hand, the clear correlation between declining allopregnanolone sensitivity and reduced ctl subunit mRNA expression makes it tempting to suggest that this subunit is particularly important for the allosteric modulation of the native GABAA receptor by allopregnanolone (Brussaard et al., 1997). While evidence from some in vitro heterologous recombinant studies would support this contention (Shingai et al., 1991), others would not (Lambert et al., 1999) and it remains unclear what relationship-specific subunits

43 may have on the allopregnanolone sensitivity of native GABAA receptors. An alternative explanation may be that changes in the phosphorylation state of the receptor underlie the altered efficacy of allopregnanolone (Leidenheimer and Chapell, 1997; Brussaard et al., 2000; Fancsik et al., 2000). Nevertheless, it remains intriguing that progesterone itself, rather than allopregnanolone, is likely to be responsible for increasing c~l subunit mRNA expression in oxytocin neurons (Fenelon and Herbison, 2000) and that this change may then possibly confer allopregnanolone sensitivity upon the GABAA receptor. If proven, this would represent a highly efficient mechanism through which progesterone could co-ordinate its own influence with that of the non-genomic actions of its derivative allopregnanolone. withdrawal of progesterone, or blockade of central allopregnanolone production, results in increased seizure susceptibility and anxiety. In parallel, they found a decrease in total GABAA receptor current within the hippocampus and the presence of GABAA receptors which were now relatively insensitive to benzodiazepines as well as allopregnanolone. All of these parameters correlated with a marked increase in the expression of the c~4 subunit of the GABAA receptor within the hippocampus (Fig. 2). Thus, Smith and colleagues have suggested that the fall in progesterone concentrations in late pregnancy or the luteal phase, results in lowered allopregnanolone concentrations within the brain and that this causes an increase in expression of the c~4 subunit within the hippocampus (Smith et al., 1998a,b). In turn, the subunit change produces GABAA receptors that exhibit relatively fast IPSCs, and consequently the inhibitory effect is shorter-lasting, while this form of the receptor is much less sensitive to enhancement by benzodiazepines and allopregnanolone. The combined result is that the progesterone withdrawal appears likely to result in hippocampal neurons with markedly reduced GABAA receptor 'tone' and the consequent elevated hippocampal excitability may, therefore, underlie the increased anxiety and reduced seizure threshold of the rats (Smith et al., 1998a,b). However, it remains to be determined whether this represents a truly cyclical phenomenon whereby the rising allopregnanolone concentrations of advancing pregnancy would have the opposite effects of suppressing ~4 subunit mRNA expression and reducing anxiety and increasing seizure threshold during pregnancy. Studies by others certainly suggest a more complex scenario in the hippocampus during mid to late pregnancy as progesterone and/or allopregnanolone administration to rats can also modulate the expression of the c~l and y2 subunits of the GABAA receptor within this structure (Weiland and Orchinik, 1995; Follesa et al., 1998). Although this, and other issues such as the hippocampal specificity and mechanism of allopregnanolone's influence upon c~4 gene expression are yet to be resolved, these studies are amongst the first to provide an insight into allopregnanolone action within the physiological context of fluctuating progesterone concentrations in pregnancy.

Allopregnanolone, the hippocampus, seizures and anxiety during pregnancy

Like the benzodiazepine compounds, progesterone can exert anti-seizure and anxiolytic effects in humans (Craig, 1966; Freeman et al., 1993; Herzog, 1995). Work in the rat now clearly suggests that these actions of progesterone are attributable to the direct allopregnanolone modulation of GABAA receptors within the brain (Gallo and Smith, 1993; Bitran et al., 1995; Frye and Bayon, 1998). During pregnancy, as well as the menstrual cycle, there is evidence for cyclical changes in seizure threshold and anxiety with adverse symptoms occurring mostly in the post-partum period and late-luteal, early-follicular phase of the cycle when circulating progesterone concentrations have just declined (Dennerstein et al., 1985; Herzog, 1995; Wang et al., 1996; Rupprecht and Holsboer, 1999). Accordingly, it has been suggested that the relatively acute withdrawal of progesterone and allopregnanolone at these times may have important implications for GABAA receptor functioning and thus neuronal excitability within the brain. Smith et al. (1998a,b) have very recently proposed a molecular mechanism within the hippocampus for understanding the increased anxiety and reduced seizure threshold of rats following progesterone withdrawal. Using either pseudopregnant or cyclical progesterone administration paradigms in rats, these investigators have shown that the acute

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Allopregnanolone and the cerebral cortex during pregnancy

The first indication that GABAA receptor functioning may be altered during pregnancy came from a study by Majewska et al. (1989) in which they demonstrated that whole forebrain GABAA receptor binding affinity was markedly increased in mid- to late-pregnancy before it fell to normal levels at parturition in the rat. At the time, they suggested that this may have resulted from the presence of high levels of GABAA receptor-facilitating neurosteroids in the brain of pregnant animals. More recently, Follesa et al. (1998) have reported that the abundance of y2 and ~5 subunit mRNA within the cerebral cortex declines with advancing pregnancy but then rapidly retums to control values over the last two days before birth (Fig. 2). They also demonstrated an identical temporal profile of changes in GABAA receptor-induced chloride ion uptake in the cerebral cortex over pregnancy and parturition, which suggested that the subunit changes were of functional significance. Interestingly, in a further paper, these authors showed that the change in y2 subunit mRNA expression was likely to be dependent upon allopregnanolone, rather than progesterone, exposure (Concas et al., 1998). Thus, as is thought to be the case for the c~4 subunit in the hippocampus (Smith et al., 1998a), allopregnanolone itself appears to be directly responsible for altering the expression of specific subunit mRNAs in a regionspecific manner (Fig. 2). The mechanism through which allopregnanolone modulates transcript stability and/or gene transcription in a subunit selective manner is presently unknown. In vitro studies have suggested that chronic allopregnanolone exposure will eventually uncouple GABAA receptor signaling at multiple levels (Yu et al., 1996). However, the almost perfect inverse relationship between subunit mRNA changes in the cerebral cortex and circulating allopregnanolone levels in these studies indicate a direct suppressive effect of allopregnanolone on subunit expression (Concas et al., 1998; Follesa et al., 1998). These new in vivo observations clearly suggest that the changing gonadal steroid levels of pregnancy are having a substantial impact upon GABAA receptor signaling within the cerebral cortex. The

direction of these changes in subunit mRNA expression and GABAA receptor agonist-induced chloride uptake are, however, opposite in direction to the GABAA ligand binding results reported initially by Majewska et al. (1989). As those workers examined the whole forebrain, while Follesa et al. (1998) analyzed the cerebral cortex, it seems reasonable to conclude that regional differences in the direction of GABAA receptor modulation must exist within the brain during pregnancy. Indeed, this is already clear from the opposite direction of subunit mRNA changes in the SON (Fenelon and Herbison, 1996) and cerebral cortex over pregnancy and parturition. The marked increase in GABAA receptor expression within the cerebral cortex of rats over the last 2 days of pregnancy is notable not only as a reversal of previous events but perhaps more importantly, for the rapidity with which it occurs. Two studies in women have clearly indicated that the degree of progesterone decline in the peri-partum period is the most important association with post-partum negative mood (Nott et al., 1976; Harris et al., 1994), and it is quite possible that this results from the rapid changes in GABAA receptor expression within the brain at this time. While post-natal depression and other neuropsychological events of pregnancy are very likely multi-factorial, and hormones such as estrogen may also be involved (Gregoire et al., 1996), the role of fluctuating allopregnanolone modulation and potentially widespread GABAA receptor changes deserves further attention.

Conclusions
There is clear evidence for a substantial influence of elevated progesterone and allopregnanolone concentrations during late pregnancy upon GABAA receptor functioning in the brain of the rat. However, as we already have evidence for both cell type- and GABAA receptor subunit-specific effects, this phenomenon is not simple or homogeneous in nature within the brain. While the elevated concentrations of allopregnanolone in late pregnancy selectively reduce a4/y2, and c~5/2, subunit expression in the hippocampus and cerebral cortex, respectively, progesterone enhances ~1 subunit mRNA expression in oxytocin neurons (Fig. 2). Thus, the response of any particular neuronal phenotype may be unique and reflect more

45 the underlying physiology of the neuronal network in which it operates. From an experimental view-point, it will be difficult to ascribe robust physiological meaning to the reported changes in the hippocampus and cortex, although clear hypotheses have been presented. In terms of the magnocellular oxytocin neurons, a reasonable physiological mechanism can be based upon the allopregnanolone-enhancement of inhibition in late pregnancy, when these neurons must be restrained, and the subsequent allopregnanolone- and GABAA receptor-mediated dis-inhibition of these cells, when they must be activated at parturition. However, the clinical relevance of these observations to the control of the timing of parturition in humans have yet to be determined. It is important to note that allopregnanolone is not the only neurosteroid derived from cholesterol and that many other related steroidal molecules have been discovered to have neuromodulatory actions. For example, pregnenolone, the immediate precursor to progesterone, can exert its own actions upon excitatory and inhibitory amino acid receptors once sulfated, and its dehydroepiandrosterone derivatives are also neuroactive (Lambert et al., 1995; Baulieu, 1998; Rupprecht and Holsboer, 1999). Thus, the state of elevated gonadal steroid synthesis in pregnancy is likely to expose the brain to a variety of different neurosteroids. Whether these neurosteroids are of physiological significance remains to be determined as, in general, high micromolar concentrations are usually required for them to be active in vitro (Lambert et al., 1995; Rupprecht and Holsboer, 1999). From another perspective, we already know that these other neurosteroids can act selectively; for example pregnanolone sulfate does not appear to influence oxytocin neurons (Richardson and Wakerley, 1998). While much is yet to be done to investigate each of these compounds, one of the future challenges will be to provide a coherent picture of co-ordinated neurosteroid action within defined neuronal networks. In terms of the cognitive and psychiatric changes associated with pregnancy and the post-partum period in humans, there would appear to be sufficient data in the rat to consider seriously the hypothesis that changes in both the GABAA receptor and allopregnanolone concentrations are at least partly involved. Although studies suggest roles for progesterone in certain facets of brain dysfunction during pregnancy, there is little hard data to distinguish between the actions of progesterone and allopregnanolone in the human. This situation seems unwarranted given the present state of basic science and potential impact of the substantial changes which occur peri-partum in the rat. In the laboratory, further work is clearly required to establish the precise nature of allopregnanolone's actions upon the GABAA receptor, as well as the physiological relevance of its actions during pregnancy in multiple neuronal networks. Ultimately, however, the highly regionand subunit-specific nature of the GABAA receptor changes over pregnancy will require the development of agents selective for specific GABAA receptor isoforms to enable any degree of specificity in the treatment of unwanted 'side-effects' of pregnancy on brain function.

Abbreviations
Allopregnanolone 3c~-hydroxy-5c~-pregnan-20-one or 5c~-pregnan-3c~-ol-20-one y-aminobutyric acid GABA GABAA receptor y-aminobutyric acid type A ionotropic receptor IPSCs inhibitory post-synaptic current SON supraoptic nucleus

Acknowledgements
Thanks to all those past and present in the Laboratory of Neuroendocrinology at Babraham who have participated in our contribution to the understanding of GABAergic regulation of the magnocellular oxytocin neurons. Thanks also to Arjen Brussaard at the Vrije University in Amsterdam for his many important contributions.

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Herbison, A.E., Voisin, D.L., Douglas, A.J. and Chapman, C. (1997) Profile of monoamine and excitatory amino acid release in rat supraoptic nucleus over parturition. Endocrinology, 138: 33-40. Herbison, A.E., Fenelon, V.S. and Brnssaard, A.B. (1998) Gonadal steroid regulation of GABAergic transmission. Eur. J. Neurosci., 10(Suppl. 10): 2. Herzog, A.G. (1995) Progesterone therapy in women with complex partial and secondary generalized seizures. Neurology, 45: 1660-1662. Jiang, Q.B. and Wakerley, J.B. (1995) Analysis of bursting responses of oxytocin neurones in the rat in late pregnancy, lactation and after weaning. J. Physiol., 486: 237-248. Keenan, P.A., Yaldoo, D.T., Stress, M.E., Fuerst, D.R. and Ginsburg, K.A. (1998) Explicit memory in pregnant women. Am. J. Obstet. Gynecol., 179: 731-737. Lambert, J.J., Belelli, D., Hill-Venning, C. and Peters, J.A. (1995) Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci., 16: 295-303. Lambert, J.J., Belelli, D., Callachan, A.C., Harney, S.A., Frenguelli, B. and Peters, J.A. (1999) The interaction of neurosteroids with recombinant and synaptic GABAA receptors. J. Physiol., Proceedings 518, 105. Leidenheimer, N.J. and Chapell, R. (1997) Effects of PKC activation and receptor desensitization on neurosteroid modulation of GABAA receptors. Mol. Brain Res., 52:173-181. Leng, G., Brown, C.H. and Russell, J.A. (1999) Physiological pathways regulating the activity of magnocellular neurosecretory cells. Prog. Neurobiol., 57: 625-655. Majdoubi, M.E., Poulain, D.A. and Theodosis, D.T. (1997) Lactation-induced plasticity in the supraoptic nucleus augments axodendritic and axosomatic GABAergic and glutamatergic synapses: an ultrastrnctural analysis using the disector method. .I. Neurosci., 80: 1137-1147. Majewska, M.D. (1992) Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog. Neurobiol., 38: 379-395. Majewska, M.D., Ford-Rice, F. and Falkay, G. (1989) Pregnancy-induced alterations of GABAA receptor sensitivity in maternal brain: an antecedent of post-partum 'blues'? Brain Res., 482: 397-401. Negoro, H., Visessuwan, S. and Holland, R.C. (1973) Unit activity in the paraventricular nucleus of female rats at different stages of the reproductive cycle and after ovariectomy, with or without oestrogen or progesterone treatment. J. Endocrinol., 59: 545-558. Nott, P.N., Franklin, M., Armitage, C. and Gelder, M.G. (1976) Hormonal changes and mood in the puerperium. Br. J. Psychiatry, 128: 379-383. Numan, M., Roach, J.K., Cruz, R., del Cerro, M., Guillamon, A., Segovia, S., Sheehan, T.P. and Numan, M.J. (1999) Expression of intracellular progesterone receptors in rat brain during different reproductive states, and involvement in maternal behavior. Brain Res., 830: 358-371.

O'Hara, M.W. (1986) Social support, life events, and depression during pregnancy and the puerperium. Arch. Gen. Psychiat~, 43: 569-573. Paul, S.M. and Purdy, R.H. (1992) Neuroactive steroids. FASEB J., 6:2311-2322. Pfaff, D.W., Schwartz-Giblin, S., McCarthy, M.M. and Kow, L.-M. (1994) Cellular and molecular mechanisms of female reproductive behaviors. In: E. Knobil and J.D. Neill (Eds.), The Physiology of Reproduction. Raven Press, New York. Pugh, T.F., Jerath, B.K., Smith, W.M. and Reed, R.B. (1963) Rates of mental disease related to childbearing. New Engl. J. Med., 268: 1224-1228. Richardson, C.M. and Wakerley, J.B. (1998) Supraoptic oxytocin and vasopressin neurones show differential sensitivity to the neurosteroid pregnenolone sulphate. J. Neuroendocrinol., 10: 829-837. Rupprecht, R. (1997) The neuropsychopharmacological potential of neuroactive steroids. J. Psychiatr. Res., 31: 297-314. Rupprecht, R. and Holsboer, F. (1999) Neuroactive steroid: mechanisms of action and neuropsychopharmacological perspectives. Trends Neurosci., 22: 410-416. Shingai, R., Sutherland, M.L. and Barnard, E.A. (1991) Effects of subunit types of the cloned GABAA receptor on response to a neurosteroid. Eur J. Pharmacol., 206: 77-80. Smith, S.S., Gong, Q.H., Hsu, EC., Markowitz, R.S., ffrenchMullen, J.M.H. and Li, X. (1998a) GABAA receptor c~4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature, 392: 926-929. Smith, S.S., Gong, Q.H., Li, X., Moran, M.H., Bitran, D., Frye, C.A. and Hsu, F.-C. (1998b) Withdrawal from 3c~-OH5c~-pregnan-20-one using a pseudopregnancy model alters the kinetics of hippocampal GABAA receptor c~4 subunit in association with increased anxiety. J. Neurosci., 18: 5275-5284. Summerlee, A.J.S. ( 1981 ) Extracellular recordings from oxytocin neurons during the expulsive phase of birth in unanesthetized rats. J. Physiol., 321: 1-9. Wang, H., Ward, A.R. and Morris, J.E (1995) Oestradiol acutely stimulates exocytosis of oxytocin and vasopressin from dendrites and somata of hypothalamic magnocellular neurons. Neuroscience, 68:1179-1188. Wang, M.D., Seippel, L., Purdy, R.H. and Backstrom, T. (1996) Relationship between symptom severity and steroid variation in women with premenstrual syndrome: study on serum pregnenolone, pregnenolone sulfate, 5 alpha-pregnane-3,30-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one. J. Clin. Endocrinol. Metab., 81: 1076-1082. Weiland, N.G. and Orchinik, M. (1995) Specific subunit mRNAs of the GABAA receptor are regulated by progesterone in subfields of the hippocampus. Mol. Brain Res., 32:271-278. Yu, R., Follesa, P. and Ticku, M.K. (1996) Down-regulation of the GABA receptor subunits mRNA levels in the mammalian cultured cortical neurons following chronic neurosteroid treatment. Mol. Brain Res., 41: 163-168.

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 3

Maternity leads to morphological synaptic plasticity in the oxytocin system

Dionysia T. Theodosis * and Dominique A. Poulain
INSERM U378, Institut Frangois Magendie, Universit~ Victor Segalen Bordeaux 11, 1 rue Ldo Saignat, F33076 Bordeaux Cedex, France

Abstract: The oxytocinergic system, which plays a major role in the control of different aspects of maternity, undergoes extensive synaptic and neuronal-glial remodelling during parturition and lactation and has thus become a remarkable example of activity-dependent morphological synaptic plasticity in the adult mammalian brain. The use of different comparative ultrastructural analyses on the rat supraoptic and paraventricular nuclei, together with identification of preand post-synaptic elements, has allowed us to show that there is a significant increase in the number of GABAergic, glutamatergic and noradrenergic synapses impinging on oxytocin neurons, concomitant with a reduction of glial coverage of the neurons. This synaptic plasticity involves axo-dendritic and axo-somatic contacts originating from terminals making one or several synaptic contacts in one plane of section. While noradrenergic afferents arise from medullary catecholaminergic neurons, our recent in vitro observations indicate that GABAergic and glutamatergic afferents derive, at least partly, from local intrahypothalamic neurons, in close proximity to oxytocin neurons. The cellular mechanisms underlying this morphological synaptic plasticity remain to be determined but it is highly likely that they depend on increased activity in both pre- and post-synaptic elements. Moreover, the oxytocin system continues to express 'embryonic' molecular features that may allow the morphological plasticity to occur. In particular, it expresses high levels of cell surface adhesion molecules currently thought to intervene in synaptic remodelling in the developing and lesioned central nervous system, including the weakly adhesive polysialylated isoform of the Neural Cell Adhesion Molecule, the axonal glycoprotein F3 and its ligand, the extracellular matrix glycoprotein, tenascin-C.

Introduction

During stimulation, magnocellular oxytocin-secreting neurons display significant changes in their electrical, biosynthetic and secretory activities (reviewed in Gainer and Wray, 1994 and Armstrong, 1995). In addition, they undergo morphological modifications that alter their relationship to adjacent glial cells and certain of their afferent inputs. Using quantitative ultrastructural analyses of immunoidentified neurosecretory profiles in the hypothalamic magnocellular * Corresponding author: D.T. Theodosis, INSERM U378, Institut Franqois Magendie, Universit~ Victor Segalen Bordeaux II, 1 rue Lro Saignat, F33076 Bordeaux Cedex, France. Tel.: +33 5 57 573731; Fax: +33 5 57 573750; E-mail: dionysia.theodosis @bordeaux.inserm.fr

nuclei of rats under various conditions of peptide release, we have established that at parturition and during lactation, conditions which greatly stimulate oxytocin release, there is an increased number of synaptic contacts on oxytocin neurons. At the same time, coverage of their somata and dendrites by astrocytic processes markedly diminishes and their surfaces become directly juxtaposed. It is noteworthy that similar changes are induced by chronic osmotic stimuli, which particularly stimulate vasopressin release, yet as in lactation, they affect the oxytocinergic system. The appearance of new synapses and neuronal surface juxtapositions is quite rapid, and they become obvious even a few hours after the onset of stimulation (for a review see Theodosis and Poulain, 1993). Since the synaptic and neuronal-glial changes are invariably associated, we have postulated that a

50 diminished glial coverage of neuronal surfaces is a prerequisite to allow the synaptic changes to occur. cal data would be in accord with light microscopic reconstruction of dendritic trees of identified SON neurons which indicated significant changes in dendritic arborization patterns in relation to lactation (Armstrong and Stern, 1998). It may be argued that an increased neuropil synaptic density is due to shrinking of the neuropil during lactation, especially in view of the neuronal-glial changes cited earlier, Also, a decrease of dendritic length and branching frequency was noted for oxytocin cells in lactating rats (Stern and Armstrong, 1998). Nevertheless, as clearly shown by several stereological analyses at the electron microscopic level, in spite of such dendritic and glial modifications, the overall volume of the SON increases significantly during lactation, which is due to hypertrophy of both neuronal somata and dendrites (Theodosis et al., 1986a; Salm et al., 1988; E1 Majdoubi et al., 1997). As for astrocytic processes, it is likely that rather than shrinking, there is actually only rearrangement. During stimulation, astrocytic coverage of magnocellular somata increases in absolute values (Chapman et al., 1986; Theodosis et al., 1986a; Modney and Hatton, 1989); but what is peculiar to hypertrophied oxytocin somata is that a significant proportion of their plasmalemma stays free of astrocytic processes (Chapman et al., 1986; Theodosis et al., 1986a; E1 Majdoubi et al., 1996). Additionally, we can discount major changes in the size of astrocytic cell bodies since very few occur in the SON itself, most being located along the base of the brain (Bonfanti et al., 1993).

Synaptic remodelling in the hypothalamic magnoceilular nuclei

The most visible manifestation of structural synaptic plasticity in the supraoptic nucleus (SON) and magnocellular portion of the paraventricular nucleus (PVN) is a changing incidence in axonal terminals that synapse onto two or more post-synaptic elements in the same plane of section (Theodosis et al., 1981; Hatton and Tweedle, 1982; Theodosis and Poulain, 1984; E1 Majdoubi et al., 1996, 1997). These 'shared synapses' couple adjacent somata, somata and dendrites or adjacent dendrites. It is not uncommon that the surfaces of the post-synaptic elements are not covered completely by astrocytic processes and are directly juxtaposed. In assessing the number of post-synaptic profiles coupled by such terminals, we found that the amplification of synaptic input takes place relatively quickly, within 24 hours (Montagnese et al., 1987). On the other hand, the rate at which synaptic input diminishes on cessation of stimulation depends on the length of time the system has remained stimulated (Theodosis and Poulain, 1984; Montagnese et al., 1987). Our recent analyses have revealed that synaptic plasticity in these nuclei is even more pronounced than we thought earlier, since the synaptic changes affect terminals making single as well as multiple synaptic contacts, on somata as well as on dendrites. Using different stereological analyses, and in particular, the unbiased 'disector' method to evaluate synaptic densities, we have seen that, in spite of the hypertrophy of magnocellular somata and dendrites consequent upon stimulation (Theodosis et al., 1986a; Modney and Hatton, 1989; E1 Majdoubi et al., 1996), the overall synaptic density in the SON of lactating rats does not diminish but remains similar to that recorded in virgin rats (Gies and Theodosis, 1994; E1 Majdoubi et al., 1997). Moreover, we can detect an increased numerical density of axo-somatic and axo-dendritic synapses when we evaluate the number of synapses in terms of the neuropil (samples of neuropil where the area occupied by soma profiles has been subtracted; Gies and Theodosis, 1994; E1 Majdoubi et al., 1997). These stereologi-

Synaptic remodelling affects different synaptic inputs to oxytocin neurons

Synapses in the magnocellular nuclei contain most classical neurotransmitters. Anatomical studies have thus visualized the amino acids, gamma aminobutyric acid (GABA; Van Den Pol, 1985; Theodosis et al., 1986c; Gies and Theodosis, 1994; E1 Majdoubi et al., 1997) and glutamate (Decavel and Van Den Pol, 1992; Meeker et al., 1993; E1 Majdoubi et al., 1996, 1997) and the catecholamines, noradrenaline (Sawchenko and Swanson, 1982; Buijs et al., 1984), dopamine (Decavel et al., 1987; Van Vulpen et al., 1999) and serotonin (Sawchenko et al., 1983; Shioda et al., 1989) in SON and PVN synapses.

51 Electrophysiological studies have indicated that the purines, adenosine triphosphate (ATP; Day et al., 1993; Hiruma and Bourque, 1995) and adenosine (Oliet and Poulain, 1999) are also present. in adjacent hypothalamic areas. However, because of the close proximity to their targets, visualization of these perinuclear afferents has been rendered difficult with the usual neuroanatomical tracing techniques. Nevertheless, GABAergic somata have been visualized in the perinuclear area of the SON in vivo (Theodosis et al., 1986c; Roland and Sawchenko, 1993). More recently, we examined organotypic slice cultures from postnatal rat hypothalamus enriched in magnocellular oxytocin neurons and found that intrahypothalamic GABA neurons do make anatomical and functional synaptic contacts onto the cells (Jourdain et al., 1996, 1998, 1999). There appear to be long-distance GABA afferents as well, however, since a direct GABAergic input from the diagonal band of Broca, at least to the SON, has been described (Jhamandas et al., 1989).

GABAergic afferents
From ultrastructural studies, it is obvious that GABA synapses are particularly numerous in both magnocellular nuclei. This has been confirmed by morphometric analyses. For example, estimates of synaptic densities in ultrathin sections of the rat SON immunolabelled for GABA revealed that of a mean overall synaptic density of 35 x 106 synapses/mm 3, between 30 and 50% are GABAergic, depending on the physiological condition of the animals (Gies and Theodosis, 1994; E1 Majdoubi et al., 1997). Moreover, analysis of sections in which both pre- and post-synaptic elements were identified clearly established that GABA afferents to the SON impinge both on oxytocin and vasopressin neurons; in unstimulated animals to approximately an equivalent degree (Theodosis et al., 1986c; Gies and Theodosis, 1994). GABAergic terminals form symmetrical synaptic contact on dendrites and somata; the density of axo-dendritic contacts is about twice that of axo-somatic contacts (Gies and Theodosis, 1994; E1 Majdoubi et al., 1997). Comparison of synaptic densities in the SON of virgin and lactating rats demonstrated a significant increase in the number of GABAergic synaptic contacts during lactation (Gies and Theodosis, 1994; E1 Majdoubi et al., 1997), but only on oxytocin neurons (Theodosis et al., 1986c; Gies and Theodosis, 1994). Moreover, these synaptic contacts derive from terminals making both simple (Gies and Theodosis, 1994; E1 Majdoubi et al., 1997) and multiple (Theodosis et al., 1986c; Gies and Theodosis, 1994; E1 Majdoubi et al., 1997) synaptic contact on oxytocin somata and dendrites. The origin of the GABAergic input to magnocellular neurons is uncertain. It is not intranuclear since GABAergic somata are not detected within the boundaries of the SON or PVN (Theodosis et al., 1986c; Decavel et al., 1989; Gies and Theodosis, 1994). Many early anatomical (Zaborszky et al., 1975; Tribollet et al., 1985) and electrophysiological (Boudaba et al., 1996, 1997) studies indicated that most afferents to the magnocellular nuclei originate

Glutamatergic afferents
Experimental approaches similar to those used to study GABAergic inputs permitted us to analyze putative glutamatergic synapses on identified magnocellular neurons (El Majdoubi et al., 1996, 1997). It should be pointed out, however, that immunocytochemical localization of glutamate is more problematic than that of GABA. Glutamate intervenes in the general metabolism of cells, which implies that it is ubiquitously distributed in nervous tissue and is therefore potentially visible in most cellular profiles by immunocytochemistry. Nevertheless, the concentration of glutamate varies in different cellular compartments and is particularly high in terminals utilizing it as neurotransmitter (Fonnum, 1984). By estimating quantitatively different levels of immunoreactivity, it is possible therefore to distinguish between the metabolic and neurotransmitter pools of the amino acid. Moreover, in glutamatergic terminals levels of immunolabel are significantly greater over synaptic vesicles and pre- and post-synaptic membrane densities. By using antibodies that recognize the fixation product of glutamate in tissues, together with sensitive post-embedding immunogold techniques that allow quantification and correct subcellular localization of antigens (see also (Merighi et al., 1989 and Ottersen, 1989), we were able to demonstrate that about 25% of all synapses in the rat SON are

52 glutamatergic. These contact both oxytocin and vasopressin neurons and twice as many synapse onto dendrites compared to the somata. In addition, our analyses made it clear that glutamatergic afferents on oxytocin somata and dendrites participate in lactation-induced synaptic plasticity (El Majdoubi et al., 1996, 1997). As strongly suggested by several electrophysiological observations, glutamatergic afferents appear to derive essentially from cells in hypothalamic areas adjacent to the magnocellular nuclei (Gribkoff and Dudek, 1990; Jourdain et al., 1996, 1998; Boudaba et al., 1997). We recently confirmed this from observations in our organotypic slice cultures in which glutamate neurons, with a different morphology and topology than those of intrahypothalamic GABAergic neurons, were seen to make contact on dendrites and somata of magnocellular oxytocin neurons (Jourdain et al., 1999). many areas of the adult central nervous system (Turner and Greenough, 1985; Desmond and Levy, 1986; Leedy et al., 1987; Murakami et al., 1987; Black et al., 1990; Brooks et al., 1991; Witkin et al., 1991; Shankaranarayana et al., 1999), but the neurochemical identity of the modified afferents in most cases has not been established. As shown by our own studies, immunocytochemical procedures, coupled to stereological analyses, are necessary for such identification and to date, have been successful in detecting sprouting of GABA synapses in different adult neuronal systems, including the red nucleus (Katsumaru et al., 1986), hippocampus (Morin et al., 1999) and arcuate nucleus (Parducz et al., 1993). At present, the cellular mechanisms underlying structural synaptic plasticity in these systems remain to be worked out but as illustrated by the synaptic remodelling in the magnocellular nuclei, it is highly likely that both pre-synaptic elements and post-synaptic elements are involved, as are adjacent glia. From developmental and lesion paradigms, it is obvious that formation and stabilization of synaptic contacts involves interaction with neighboring glial elements (for a review see Pfenninger et al., 1992). In the magnocellular nuclei, as in other hypothalamic systems (Olmos et al., 1989; Witkin et al., 1991), remodelling of synaptic inputs invariably occurs in concert with glial conformational changes, strongly suggesting that neuronal-glial interactions are a prerequisite for synaptic rearrangements, even under normal conditions. In its simplest context, decreased glial coverage would represent an increased neuronal surface for synapse formation. Assuming that this is the case, many questions still remain. One expects that an increase in synaptic numbers is preceded by neuronal sprouting while degeneration of boutons should accompany any decreases. Nevertheless, growth cones and degenerating boutons have not been detected, at least in the magnocellular nuclei. It is more likely, then, that boutons already in place produce and destroy synaptic specializations during different physiological conditions. The number of boutons would remain unchanged but sites of interaction with postsynaptic elements become operative or inoperative by the presence or absence of intervening glial processes. In this context, it is noteworthy that in the stimulated SON and PVN, two or more 'shared'

Noradrenergic afferents
In accord with earlier studies (Sawchenko and Swanson, 1981; Ginsberg et al., 1994) we have found that oxytocin and vasopressin neurons in both the SON and PVN are contacted directly by noradrenergic synapses (Michaloudi et al., 1997). These afferents arise from neurons in the A1 and A2 areas of the ventrolateral medulla and nucleus tractus solitarii, respectively (Sawchenko and Swanson, 1981; Cunningham and Sawchenko, 1988). Using our comparative stereological procedures, we established that noradrenaline terminals contact oxytocin and vasopressin somata to an equivalent degree under basal conditions of neurosecretion, and account for about 10% of all axo-somatic synapses in the SON and PVN (Michaloudi et al., 1997). Moreover, our analyses revealed that like GABAergic and glutamatergic afferents, these inputs also contribute to the activitydependent synaptic plasticity that characterizes the oxytocin system during lactation.

How does synaptic remodelling occur?

Cellular mechanisms
There are now numerous anatomical reports of stimulation-dependent increases in synaptic densities in

53 synapses are commonly seen within a cluster of oxytocin profiles whose surfaces are free of glial processes. Once neuronal surfaces are free of glia, there can be an increased number of synaptic contacts by enlargement, perforation and eventual splitting of already existing active zones. Since synapses are units, creating strong structural linkages between pre- and post-synaptic cells, the post-synaptic neuron must also intervene, if only to give rise to post-synaptic densities, which could be formed de novo or from the enlargement and perforation of those already in place (Carlin and Siekevitz, 1983; Dyson and Jones, 1984). Morphological remodelling of synapses represents, therefore, a coordinated process of change, bringing into play both pre- and post-synaptic elements and the intervening extracellular matrix of the synaptic cleft. The motor to drive such changes is very probably activity in pre-synaptic afferents and the corresponding responses in post-synaptic elements, although the inverse is also possible (see also Carlin and Siekevitz, 1983). The validity of these (or other) mechanisms still awaits rigorous identification. in these cells does not markedly vary in relation to physiological conditions (Theodosis et al., 1991; Bonfanti et al., 1992), which has led us to propose that polysialylation is a permanent feature, permitting them to undergo morphological remodelling when appropriate conditions are met. As we have shown recently, the carbohydrate PSA on NCAM is indeed an essential factor for the structural synaptic changes in the magnocellular nuclei, since specific enzymatic removal of the carbohydrate in vivo inhibited the synaptic changes associated with lactation and osmotic stimulation (Theodosis et al., 1999). Nevertheless, NCAM and its PSA cannot be considered solely responsible for the formation, maintenance and modification of cell contacts. As has been demonstrated in many in vitro models (for review see Keynes and Cook, 1990) these depend on a dynamic interplay of recognition events at the cell surface and the extracellular matrix which bring into play several molecules that can be classified grossly into categories displaying adhesive or repulsive characteristics. Here also, magnocellular neurons, their glia and synapses are no exception since they do express other molecules implicated in synaptogenesis. One is the F3 glycoprotein (Olive et al., 1995b; Pierre et al., 1998), a cell adhesion molecule structurally similar to NCAM. Unlike the expression of PSA-NCAM, that of F3, which in the hypothalamus is restricted to magnocellular neurons and is colocalized with the neurohormones, varies with the physiological condition of the animal (Pierre et al., 1998). Ligands for F3 include the extracellular matrix tenascins (Zisch et al., 1992) and axonal receptors, like L1 (Olive et al., 1995a), molecules which are also highly expressed in the magnocellular nuclei (Grant et al., 1992; Theodosis et al., 1994, 1997; Singleton and Salm, 1996). If molecules like those described above serve as permissive factors to allow synaptic remodelling in the magnocellular nuclei, then there must be particular stimuli to induce the changes. Oxytocin itself appears to be one such stimulus since intracerebroventricular infusion of oxytocin induced changes similar to those seen under physiological stimulation (Theodosis et al., 1986b; Montagnese et al., 1990). This indicates that the oxytocin released by dendrites and cells within the SON and PVN during parturition and lactation (Neumann et al., 1993) may play a ma-

Permissive and inductive factors

Like the cellular mechanisms underlying structural synaptic changes in the adult brain, the factors responsible are probably also shared by different neuronal systems capable of such plasticity. To identify these factors, we have been investigating the expression of molecules implicated in synaptogenesis during development, which include proteins on the cell surface and extracellular matrix as well as soluble and trophic factors. We have thus found that hypothalamic centers like the magnocellular nuclei that can undergo synaptic changes display certain molecular characteristics normally associated with axon growth and synaptogenesis. Adult magnocellular neurons and their glial cells thus continue to express the highly polysialylated, 'embryonic' isoform of the Neural Cell Adhesion Molecule (PSA-NCAM), which, as a negative regulator of cell adhesion, is considered an important regulator of most contact-dependent cell interactions (for review see Rutishauser, 1996). The distribution and extent of PSA-NCAM immunoreactivity

54 jor role in inducing the synaptic plasticity. However, oxytocin is not in itself sufficient, as central infusion of oxytocin induced plasticity only in animals undergoing prolonged diestrus or in castrated females that had been given progesterone and estrogen replacement therapy (Montagnese et al., 1990); i.e. in hormonal conditions similar to those of parturient and lactating female rats. tion potentials which occur synchronously throughout the population and result in a bolus release of hormone and the milk ejection reflex (reviewed in Wakerley et al., 1994). It may be then that strong inhibition, exerted by GABA, is necessary to maintain the neurons at a level of polarization which would facilitate their synchronous activation induced by glutamate. Another possibility is that oxytocin neurons require additional inhibition to prevent them from being stimulated by factors other than those necessary for lactation (Moos, 1995; Voisin et al., 1995). Indeed, reaction of the oxytocin system to osmotic (Hartman et al., 1987; Fenelon et al., 1993) and stressful (Lightman and Young, 1989) stimuli in lactating animals is greatly attenuated. Lastly, a diminished astrocytic coverage may also intervene. As discussed earlier, it is obvious that there must be glial retraction to allow formation of new synaptic contacts. However, it is possible that there are more direct effects, especially when one considers the role of astrocytes in the clearance of glutamate from the perisynaptic site (reviewed in Bergles et al., 1999). That many 'shared synapses' are glutamatergic and often found next to neuronal membranes that are not separated by astrocytes makes it highly likely that proliferation of glutamate synapses on oxytocin neurons is accompanied by a change in synaptic function, which would result in potentiation of glutamate action (Bergles et al., 1999) and therefore, further facilitation of their enhanced electrical activity.

Functional considerations
GABAergic, glutamatergic, and noradrenergic afferents constitute major synaptic inputs to oxytocin neurons. This is evident not only from morphological observations like those described in this review but also from numerous electrophysiological, biochemical and physiological data illustrating their importance on the electrical, and consequently, secretory activity of the cells. As in other neuronal systems, GABA inhibits the electrical activity of oxytocin neurons, essentially via GABAA receptors (Randle and Renaud, 1987; Wuarin and Dudek, 1993; Voisin et al., 1995; Jourdain et al., 1996). On the other hand, glutamate excites the cells (Gribkoff and Dudek, 1988, 1990; Van Den Pol et al., 1990) and stimulates oxytocin release in lactating rats (Parker and Crowley, 1993a,b), mainly via ionotropic non-N-methyl-D-aspartate (NMDA) receptors (Gribkoff and Dudek, 1990; Van Den Pol et al., 1990; Wuarin and Dudek, 1993; Jourdain et al., 1996, 1998). We have known for a long time that noradrenaline stimulates oxytocin secretion via 0q-adrenergic receptors (reviewed in Parker and Crowley, 1993b; Daftary et al., 1998), although recent studies have shown that this stimulation is not direct but via excitation of pre-synaptic glutamate neurons through cq-adrenergic mechanisms (Daftary et al., 1998). That both inhibitory and excitatory inputs are enhanced during lactation may be explained as a compensatory mechanism to meet the hypertrophy of oxytocin neurons induced by lactation, thus providing an equivalent degree of regulation as that acting in virgin rats. A more attractive possibility, however, is that an increased number of synaptic contacts play a role in the unique patterns of electrical activity that characterise these neurons. During lactation, they display intermittent high frequency discharges of ac-

Abbreviations
ATP F3 GABA NMDA PSA-NCAM PVN SON adenosine triphosphate F3 glycoprotein gamma amino butyric acid N-methyl-D-aspartate polysialylated Neural Cell Adhesion Molecule paraventricular nucleus supraoptic nucleus alpha adrenergic receptor

Acknowledgements
We thank G. Rougon, A. Faissner, G. Gennarini and H. Gainer for gifts of antibodies, M. Manning for gifts of oxytocin agonists and antagonists and R.

55

Bonhomme

for h e r c o n s t a n t s u p p o r t a n d e x c e l l e n t

technical assistance.

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Progress in Brain Research, Vol. 133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 4

Oxytocin: who needs it?

Thomas R. Insel 1,,, Brenden S. Gingrich 2 and Larry J. Young t
Center for Behavioral Neuroscience, Emory University, Atlanta, GA, 30322, USA 2 Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA

Abstract: The neuropeptide oxytocin has been implicated in the initiation of maternal behavior, based on studies in rats and sheep. Females in both of these species naturally avoid infants until parturition when they begin to show an intense interest in maternal care. Oxytocin pathways in the brain appear to be important for this transition from avoidance to approach of the young. Recent studies in mice with a null mutation of the oxytocin gene suggest a different scenario. These mice, which completely lack oxytocin, exhibit full maternal and reproductive behavior, except for a deficit in milk ejection. Apparently, oxytocin is not essential for maternal behavior in this species. Consistent with the role of oxytocin for the transition from avoidance to approach in rats and sheep, nulliparous mice show full maternal behavior and therefore do not require the peptide for the initiation of maternal care. The species differences in the behavioral effects of oxytocin are associated with profound species differences in the location of oxytocin receptors in the brain. Recent transgenic studies suggest that these species differences in the neuroanatomical distribution of oxytocin receptors may be a function of inter-species variation in the flanking region of the oxytocin receptor gene. So, who needs oxytocin? For maternal care, not mice and (possibly) other species, like primates, with promiscuous parental care. Most important, in considering the behavioral or cognitive functions of oxytocin, one cannot accurately extrapolate across species unless one knows the species have the same neuroanatomical location of oxytocin receptors.

Introduction

There are several excellent reasons to presume that the neuropeptide hormone, oxytocin, is important for maternal behavior. Oxytocin is found almost exclusively in mammals and it supports two critical mammalian maternal functions: uterine contraction during labor and milk ejection during lactation. Oxytocin is a nine amino acid neuropeptide comprising a ring structure and a tail, and is primarily synthesized and secreted from magnocellular supraoptic neurons and magnocellular and parvocellular paraventricular neurons in the hypothalamus. * Corresponding author: Thomas R. Insel, Center for Behavioral Neuroscience, Emory University, Atlanta, GA 30322, USA. Tel.: -I-1-404-727-8625; Fax: -I-1-404-727-8510; E-mail: insel@rmy.emory.edu

Oxytocin synthesis increases in the hypothalamus at parturition and during lactation. Oxytocin secretion from magnocellular neuron axon terminals in the posterior pituitary into the blood is co-ordinated with births and with nursing. In addition to serving as a hormone for peripheral tissues, oxytocin from parvocellular neurons projecting centrally can influence behavior by acting directly at a variety of sites in the brain via specific membranebound receptors that are coupled to intracellular signal transduction pathways. Given this evidence, one might hypothesize that oxytocin supports maternal behavior by acting as a neuromodulator or neurotransmitter within the brain, consistent with the hormone's effects on labor and nursing. In a sense, these presumed brain actions might be considered 'motivational' effects related to maternal interest that are critical for the peripheral actions of maternal care.

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Oxytocin and maternal behavior Studies of rat maternal behavior

Given the intuitive appeal of linking central and peripheral actions, it was gratifying that early studies reported that in estrogen-primed, virgin female rats, centrally administered oxytocin could induce maternal behavior within 30 min (Pedersen and Prange, 1979). It is important to recognize that virgin female rats are neophobic and will either avoid or attack pups. Oxytocin appeared to facilitate approach to the pups, followed by nest building, retrieval, and full maternal care. This effect was not observed with several unrelated neuropeptides and could not be elicited with oxytocin administered systemically. In addition to oxytocin, tocinoic acid (the ring structure of the oxytocin peptide) and to a more limited extent, vasopressin (nonapeptide differing by only 2 amino acids from the oxytocin sequence), could induce maternal behavior (Pedersen et al., 1982). While these effects were replicated in another lab (Fahrbach et al., 1984), other investigators (Rubin et al., 1983; Bolwerk and Swanson, 1984) had difficulty demonstrating an effect of oxytocin on the initiation of maternal behavior. Explanations that have been suggested for these discrepancies include the need to place the female in a novel cage for 2 h before testing (Fahrbach et al., 1986) and the possibility that olfactory deficits in some groups of rats could facilitate the oxytocin effects on maternal behavior (Wamboldt and Insel, 1987). In contrast to these conflicting reports with oxytocin administration in female rats, there have been several consistent demonstrations that decreases in oxytocin neurotransmission impair the onset of maternal behavior in the rat. Central administration of oxytocin antagonists, antisera, and anti-sense oligonucleotides, as well as lesions of oxytocin cells, inhibit maternal behavior (Fahrbach et al., 1985; Pedersen et al., 1985; van Leengoed et al., 1987; Insel and Harbaugh, 1989). These effects have been reported with the onset of maternal behavior in the course of a normal pregnancy or with hormone-induced, experimental models of maternal behavior. The critical point to remember is that the effects have been generally limited to the initiation of maternal behavior; reductions in oxytocin

function do not reduce maternal behavior once it is established (Giovenardi et al., 1998). Thus, in the rat, oxytocin appears to be necessary for the transition from pup avoidance to pup care, but it is not necessary once pup care has become established. More recent studies have focused on the neuroanatomical location for oxytocin's effects on maternal behavior in the rat. Although oxytocin receptor expression is found most densely in the ventral subiculum and ventromedial nucleus of the hypothalamus, site-specific injections of oxytocin antagonist have had the greatest effect in the ventral tegmental area and the medial preoptic area (Pedersen et al., 1994). Lesion studies previously implicated both of these regions in maternal behavior (Numan, 1994). Curiously, there are few if any oxytocin receptors evident in the ventral tegmental area and medial preoptic area using receptor autoradiographic techniques (Tribollet et al., 1988), but one homogenate binding study reported an increase in oxytocin receptors in both of these regions at parturition (Pedersen et al., 1994). In addition, given the possible importance of oxytocin effects on olfactory processing (Yu et al., 1996a), it is important to recognize that injections of an oxytocin antagonist directly into the bulb can block maternal behavior (Yu et al., 1996b).

Studies of sheep maternal behavior

These effects of oxytocin are not unique to rats. Several studies in sheep, which also exhibit maternal behavior for the first time at parturition, demonstrate an important role for oxytocin in maternal acceptance and recognition of the lamb, The ewe must bond to her lamb within 2-4 h of parturition or she will not become maternal. Both vagino-cervical stimulation, which releases oxytocin centrally (Kendrick et al., 1986), and administration of oxytocin into the lateral ventricle induce maternal behavior in non-parturient ewes or in post-partum ewes who are non-maternal due to receiving an epidural anesthetic (Keverne et al., 1983; Kendrick et al., 1987). An elegant series of in vivo microdialysis studies by Kendrick and colleagues have demonstrated the local release of oxytocin in the paraventricular nucleus (one location of oxytocin neurons), medial preoptic

61
Coordinat ion of OT Release by

Positive Feedback on Autoreceptors ~ BNST .~ j Motivati on

Recognition

Inhibit
Aggression/Avoidance

Postpartum Estrus
Fig. 1. A model of oxytocin effects on various components of maternal behavior, based on results from studies in rats and sheep. The release of oxytocin (OT) from the paraventricular nucleus of the hypothalamus (PVN) is regulated by dendritic autoreceptors as well as a short feedback loop to the bed nucleus of the stria terminalis (BNST). Targets in the olfactory bulb, medial preoptic area (MPOA), hypothalarnus, and noradrenaline (NA) and dopamine (DA) cells in the brainstem may mediate distinct components of maternal behavior.

area, medio-basal hypothalamus, and olfactory bulb concurrent with parturition (Kendrick et al., 1988). Administration of 1 IxM oxytocin (representing about 31 pmoles over 90 min) or tocinoic acid (but not vasopressin) directly into the paraventricular nucleus by retrodialysis appears to be as potent for inducing maternal behavior as an intracerebroventricular injection (10 nm) or vagino-cervical stimulation (Da Costa et al., 1996). This effect may co-ordinate oxytocin release at several other sites shown to be sensitive for inducing maternal behavior. Oxytocin release into the olfactory bulb at parturition facilitates lamb recognition (Levy et al., 1995), into the medial preoptic area decreases aggression toward the lamb (Kendrick et al., 1992), and into the medio-basal hypothalamus inhibits sexual receptivity (Kendrick et al., 1993). It is of note that all treatments that facilitate maternal behavior, including oxytocin administration, also increase noradrenaline release in the paraventricular nucleus (Da Costa et al., 1996). There have been no reports of oxytocin antagonist administration in lactating or steroid-primed ewes. Keverne and Kendrick (1992) have emphasized that oxytocin (or vagino-cervical stimulation) can induce all of the components of proceptive behavior leading to full maternal adoption of alien lambs, but these effects require steroid-priming, and even following estrogen and progesterone treatment, the quality and the intensity of the maternal be-

havior is not equivalent to what one observes with a post-partum ewe. Other factors, particularly opiate agonists, augment the effects of vagino-cervical stimulation and presumably amplify the effects of oxytocin to induce the full intensity of maternal behavior in the ewe (Keverne and Kendrick, 1992; see Fig. 1 for overview).

Maternal behavior in other species

What about other species? Although oxytocin has been reported to decrease infanticide in female house mice (Mus domesticus; McCarthy, 1990), there are no data implicating oxytocin in maternal behavior in other mammals beyond rats and sheep. Oxytocin has been implicated in several other aspects of rodent social behavior, including affiliation, social memory, and sexual receptivity (Insel et al., 1997). In the one report of oxytocin tested centrally in female primates, two nulliparous rhesus monkeys showed increased interest in unfamiliar infants (Holman and Goy, 1994).

Oxytocin knockout studies

To determine if oxytocin was necessary for maternal behavior, we investigated a mouse with a null mutation (knockout) of the first exon of the oxytocin gene. These mice were developed by homologous recom-

62 bination following germline incorporation of embryonic stem cells from ES/SV129 with a C57B6J background strain. The resulting mice lacked the oxytocin gene and peptide, but showed normal survival, unremarkable growth and development, and ostensibly an unaffected phenotype (Nishimori et al., 1996). There was no change in oxytocin receptor expression nor was there any alteration in vasopressin gene expression (Nishimori et al., 1996). The oxytocin-knockout females showed normal parturition with onset and duration indistinguishable from the wild-type controls. However, oxytocinknockout females were unable to nurse due to the absence of the milk ejection reflex. Administration of oxytocin reinstated milk ejection and permitted normal nursing (Nishimori et al., 1996). Maternal behavior, assessed by nest building, retrieval of pups, and time in the nest was identical between knockout and wild-type control mice (Nishimori et al., 1996). A similar result was reported with a different oxytocin-knockout mouse developed by deleting exon 3 of the gene (Young et ai., 1996). It seems possible that some other oxytocin receptor ligand, such as vasopressin, was compensating for the absence of oxytocin. To rule out this possibility, we administered the long-acting oxytocin receptor antagonist, d(CH2)5, [Tyr(Me)2, Thr 4, Orn 8, Tyr9-NH2]vasotocin, to females by chronic central Latency to pup retrieval
1800 1500 ,g, ~ 1200 120 ,., 100

(intracerebroventricular) infusion for 6 days. At the end of this period, oxytocin receptor binding was decreased by more than 50% in the lateral septum and ventromedial hypothalamus, but maternal behavior was remarkably unaffected (Fig. 2). These results demonstrate that oxytocin is not necessary for full maternal behavior in mice. Why the difference from previous reports in rats and sheep? Certainly a genetic knockout which removes the target hormone throughout development is not the same as administration of an antagonist to an adult. But administration of an oxytocin antagonist to adult mice was also without effect. No doubt, a key difference resides in the nature of maternal behavior in the strain of mice (C57B6J) that we studied. These mice are fully maternal in the virgin state and, unlike rats and sheep, do not show the sudden induction of maternal behavior at parturition. If oxytocin is essential for the initiation of maternal behavior, seen as a rapid transformation from avoidance to approach of the young in rat and sheep mothers, then one would not expect to find an oxytocin effect on maternal behavior in these mice. On the other hand, it is important to note that several other mouse strains with alternative gene knockouts have demonstrated profound deficits in maternal behavior. These include knockouts of thefos B (Brown et al., 1996), mesh (Li et al., 1999), and prolactin receptor genes

Time spent in nest

Receptor Binding
LS VMH
120 100 ,.~
e,,..

II

III

.~ 80
ID

8o ~. 6o ~
0~

900
,...1 600 300 0

60 40
20

4o ~
O

20 Q ~r
o

Fig. 2. Female mice with a null mutation of the oxytocin gene (knockout) lack oxytocin yet show full maternal behavior as measured by pup retrieval latency and time spent in nest. Even following 5 days of central infusion of the oxytocin antagonist d(CH2)5, [Tyr(Me) 2, Thr 4, Orn 8, Tyr9-NH2]vasotocin (OTA, II, or cerebrospinal fluid vehicle, CSF C3) to block other endogenous compounds (such as vasopressin) that could be compensating for the deficiency of oxytocin, mice exhibited full maternal behavior. The occupancy of the oxytocin receptors by the antagonist was observed by ex vivo binding studies and showed a reduction of [125I]oxytocin antagonist binding in lateral septum (LS) and ventromedial hypothalamus (VMH).

63 (Lucas et al., 1998). Presumably the protein products of these genes are critical for the expression and not the initiation of maternal behavior. Oxytocin-knockout mice show important deficits in other behaviors but these effects are found predominantly in males. Male oxytocin-knockout mice exhibit a profound deficit in social memory (Ferguson et al., 2000). Other forms of memory appear to be unaffected, but these males are unable to recognize conspecifics. This deficit is entirely rescued by central administration of oxytocin. found no effects on grooming or social interaction (Winslow and Insel, 1991). These differences in functional effects are associated with striking species differences in the distribution of oxytocin receptors in the brain (Fig. 3). Rats and mice, for instance, have completely different maps of oxytocin receptors. Indeed, comparative studies of laboratory mice, peromyscus, voles, rats, hamsters, sheep, and monkeys reveal a different map in every species, suggesting that, in each case, oxytocin has a unique set of central targets and therefore, unique functions (Young, 1999). What can we learn from these different maps? First, one should not generalize about oxytocin's central effects from one species to another. We have already seen how effects on maternal behavior are present in rats but not mice. Clearly, extrapolating from either mice or rats to humans is a misguided enterprise. Second, in species which share functional responses to oxytocin (e.g. rats and sheep), one might expect that receptor regions common to both species provide likely targets for oxytocin effects. For instance, oxytocin receptors are found in the olfactory bulb of both rats and sheep, suggest-

Species differences
The species differences in oxytocin function are interesting in and of themselves. Differences in the effects of central oxytocin have been previously reported: oxytocin increases social interest in prairie vole social behavior but in montane voles the same dose of oxytocin increases autogrooming (Winslow et al., 1993). Oxytocin decreases sexual receptivity in sheep whereas it increases receptivity in rats (reviewed in Insel et al., 1997). One study of central oxytocin administration in non-human primates

Mouse

Rat

Prairie Vole

Montane Vole

Fig. 3. Species variations in oxytocinreceptor density and distribution in the brain can be seen in anatomically matched brain sections from four differentrodents. Note the marked differencesin [125I]oxytocinantagonist binding in the bed nucleus of the stria terminalis (BNST) and the lateral septum (LS).

64 ing a likely target for oxytocin effects on maternal behavior. Unfortunately for this example, oxytocin receptors are also abundant in the mouse olfactory bulb (Insel et al., 1993). Nevertheless, this comparative approach can be useful for guiding site-specific injection studies to test out the relevant circuitry for oxytocin effects on behavior or physiology. Finally, these species differences raise an interesting question about mechanism. Why the differences in distribution of oxytocin receptors and function in closely related species, when most neuropeptide and neurotransmitter systems are conserved across broad taxa? behavior. In virgin mice that do not show pup avoidance, oxytocin is not necessary for full maternal behavior. There is no evidence that oxytocin is involved in maternal motivation in primates (including humans, see Carter et al., 2001, this volume), species in which non-pregnant females also show maternal behavior. The species differences in oxytocin effects on maternal behavior are associated with species differences in oxytocin receptor distribution in brain. These marked differences in receptor distribution demonstrate that oxytocin has different brain targets in each species studied thus far and, therefore, one would expect unique or species-specific behavioral effects. Given this information, it is important that we do not extrapolate from either rats or mice to humans without a full understanding of the oxytocin receptor distribution and without empirical studies of oxytocin function.

Molecular mechanisms of species differences

The differences in receptor distribution shown in Fig. 3 could represent structurally different receptors which all bind oxytocin. The oxytocin receptor gene has been cloned and sequenced in rodents (Rozen et al., 1995) and humans (Kimura et al., 1992). As sequence comparisons of the coding region of this gene demonstrate little sequence variation across species, it is clear that oxytocin is binding to the same receptor in mice, rats, voles, and humans. The species differences in the pattern of receptor expression probably result from variations in the sequences that flank the coding region. We have identified a tandem repeat sequence in the 5' flanking region that shows variable length across species (Young et al., 1997, 1998). This 5' flanking region may be an important determinant of the pattern of expression in the brain. When a segment of the prairie vole oxytocin receptor 5' flanking region was transfected into the mouse genome, the pattern of expression in the mouse brain resembled the pattern of the prairie vole oxytocin receptor (Young et al., 1997, 1998).

Abbreviations
BNST CSF DA LS MPOA NA PVN OT OTA VMH bed nucleus of the stria terminalis cerebrospinal fluid dopamine lateral septum medial preoptic area noradrenaline paraventricular nucleus oxytocin oxytocin antagonist (d(CH2)5, [Tyr(Me)2, Thr4, Orn 8, Tyr9-NH2]vasotocin) ventromedial hypothalamus

Acknowledgements
The authors wish to thank Katherine Murphey for technical assistance. This work has been supported by NIMH 56539, 56897, and the Whitehall Foundation.

Conclusion
Maternal behavior is a complex of related, motivated actions that result in the care and protection of offspring. In some species, such as laboratory rats and domestic sheep, virgin females avoid infants until the peri-partum period. In these species, oxytocin appears to be important for the onset (but not the maintenance) of maternal behavior, possibly by decreasing olfactory processing or reducing avoidance

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].A. Russell et al. (Eds.)

Progressin BrainResearch,Vol. 133 @ 200l Elsevier Science B.V. All rights reserved

CHAFFER 5

Endogenous opioid regulation of oxytocin and ACTH secretion during pregnancy and parturition
Alison J. Douglas * and John A. Russell
Laboratory of Neuroendocrinology, Department of Biomedical Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK

Abstract: Progress of parturition in the rat is optimal when there is increased oxytocin secretion, thus ensuring quick birth and otherwise risking adverse neonatal health. To ensure that the mechanisms for this are available, oxytocin neurons adapt in pregnancy and this includes development of a tonic inhibition by endogenous opioids. Endogenous opioid inhibition of oxytocin secretion increases in pregnancy, initially acting on the nerve terminals in the posterior pituitary and later on oxytocin cell bodies and their inputs. This inhibition enhances stores of oxytocin and enables restraint of oxytocin neuron responsiveness to selected excitatory inputs. The hypothalamic neurons which mediate stress also adapt in late pregnancy so that hypothalamo-pituitary-adrenal axis and oxytocin secretory responses to stressor exposure are attenuated. This is also partly due to endogenous opioid inhibition. Thus, in pregnancy oxytocin and hypothalamo-pituitary-adrenal axis secretion in response to stimulation is restrained, protecting the unborn fetus(es) from premature delivery and glucocorticoid exposure and preparing the oxytocin neurons for their important secretory role during parturition. In parturition itself, endogenous opioids continue to inhibit these neurons. Stress exposure during parturition delays births, probably due to endogenous opioid inhibition of pulsatile oxytocin secretion. On the other hand, basal ACTH and corticosterone secretion are reduced in parturition through inhibition by endogenous opioids. So, opioids continue to regulate the activity of oxytocin and hypothalamo-pituitary-adrenal mechanisms in labor; inhibition of oxytocin neurons at this time may control the spacing of pup births.

Introduction
Parturition is the culmination of a cascade of endocrine and neuroendocrine events that lead to the physiological and procreative outcomes: labor and birth of young. Oxytocin, which enhances uterine contractions, is secreted during birth from the posterior pituitary nerve terminals of oxytocin neurons, whose magnocellular cell bodies are located in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. Enhanced oxytocin neuron activity, demonstrated by an increase in firing rate * Corresponding author: Alison Douglas, Laboratory of Neuroendocrinology, Department of Biomedical Sciences, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK. Tel.: +44-131-650-3274; Fax: +44-131-650-2872; E-mail: alison.j.douglas@ed.ac.uk

(Summerlee, 1981) promotes oxytocin secretion at birth and there is also a rapid increase in the expression of the oxytocin gene in SON neurons (Douglas et al., 1998b) and immediate early genes in SON and PVN neurons, for example: Fos (Luckman et al., 1993). In contrast to the magnocellular oxytocin system, maternal hypothalamo-pituitary-adrenal (HPA) axis activity is suppressed during parturition (Wigger et al., 1999). The HPA axis involves the neuroendocrine hormones, corticotrophin-releasing hormone (CRH) and vasopressin, that are synthesised in the parvocellular PVN neurons and released at the median eminence into the portal system to regulate the synthesis and secretion of adrenocorticotrophic hormone (ACTH) from corticotrophs of the anterior pituitary, which in turn enhances the synthesis and secretion of corticosteroids from the adrenal cortex. This cascade is switched on when an animal

68 is exposed to a stressor, whether emotional (e.g. fear, novelty) or physical (e.g. forced swimming, restraint, pain), and leads to whole body and brain adaptation in terms of metabolism and future responses to stressor exposure. To perform optimally during birth, neuroendocrine neurons undergo adaptation during pregnancy and we have identified some of the mechanisms that are involved. During pregnancy both the oxytocin and the HPA axis systems are inhibited as part of this adaptation and, along with nitric oxide (Woodside and Amir, 1996) and GABA (Brussaard et al., 1999), endogenous opioid action in the hypothalamus contributes to the adaptation. The actions of opioids on oxytocin neuron activity and secretion, are inhibitory (Brown et al., 2000), but actions on CRH neurons, and thus on HPA axis activity, are less clear cut. Hormones such as the sex steroids, oestrogen and progesterone, are also important for the pre-parturition adaptation of the maternal brain, partly by regulating other inputs and factors, including the central endogenous opioids, and partly by controlling the neuroendocrine neurons directly. pressin neurons which co-express and secrete dynorphins from their nerve terminals in the posterior pituitary (Watson et al., 1982). These co-localised opioids are, additionally, simultaneously released from the magnocellular neuron dendrites in the hypothalamus (Ingrain et al., 1996). Selected inputs to the oxytocin neuron cell bodies, e.g. from the arcuate nucleus (Leng et al., 1988a) and brainstem (Raby and Renaud, 1989) also synthesise the endogenous opioids, 13-endorphin (Mann et al., 1997) and enkephalins (Sawchenko et al., 1990), respectively. The magnocellular cell bodies of oxytocin neurons in the SON and PVN have K- and Ix-opioid receptors (Sumner et al., 1992), but their nerve terminals in the posterior pituitary have only K-receptors (Herkenham et al., 1986). Although Met 5- and LeuS-enkephalins are not active at K-receptors, the extended forms Met-enkephalin-Arg6phe 7, and Met-enkephalin-Arg6GlyVLeu8 are; these are produced by oxytocin neurons, and like dynorphins can inhibit stimulated oxytocin secretion from the posterior pituitary (Panula and Lindberg, 1987). 3-receptors are not evident in the hypothalamo-neurohypophyseal system and g-acting opioids are not important in its regulation. Inputs to the magnocellular SON and PVN arising from other areas of the hypothalamus, the brainstem, circumventricular organs, bed nucleus of the stria terminalis (BNST), and olfactory bulb also express opioid receptor binding (Mansour et al., 1994), so opioids may inhibit such inputs. Thus opioids can mediate inhibition of oxytocin neurons at several sites of action: on inputs, the neuron cell bodies and at the terminals in the posterior pituitary; so oxytocin nerve terminal secretion into the blood can be regulated independently of neuronal firing rate. In the HPA axis endogenous opioids are co-synthesised by the PVN CRH neurons and the anterior pituitary corticotrophs, and have actions at the median eminence and the adrenal cortex to modulate responses to stress. In the PVN, the parvocellular CRH neurons synthesise enkephalins from the preproenkephalin A gene (Ceccatelli et al., 1989). Preproenkephalin A gene expression is rapidly stimulated in these neurons in response to stress (Lightman and Young, 1987). The corticotrophs in the anterior pituitary synthesise both ACTH and [3-endorphin

Endogenous opioids and opioid receptors in the neurohypophyseal system and the HPA axis
Opioids, such as morphine (produced by the opium poppy, Papaver somniferum), have been used for centuries to control pain, including during childbirth. The endogenous opioid peptides were identified in the early 1970s: the enkephalins (mainly from the Proenkephalin A gene) that bind to Ix- and 3-opioid receptors; 13-endorphin (from the proopiomelanocortin, POMC, gene) that binds to Ix- and ~c-receptors; and dynorphins (from the Prodynorphin A gene) that bind to K-receptors; see Kosterlitz (1985). Recently, an opioid peptide (endomorphin) selective for the Ix-receptor has also been identified in the brain (Zadina et al., 1997), although its precursor has not been described. In the oxytocin system, enkephalins and dynorphins are co-synthesised in magnocellular oxytocin neurons in the PVN and SON (Meister et al., 1990). Furthermore, they are co-localised in secretory vesicles with oxytocin and co-secreted, and thus can autoregulate oxytocin secretion into the blood. Endogenous opioids also come from the adjacent vaso-

69 from the POMC gene, and they are co-secreted into the peripheral circulation; however, the systemic role of ~-endorphin is uncertain. The adrenal cortex does not produce opioids in the rat, guinea pig, human or bovine, but the adjacent medulla produces and secretes enkephalins and endorphins that are reported to influence corticosteroid secretion (Pechnick, 1993). Neural inputs to the HPA axis that express endogenous opioids come from regions within the brain similar to those projecting to magnocellular oxytocin neurons, including the brainstem and arcuate nucleus (Sawchenko et al., 1990; Mann et al., 1997). Parvocellular PVN neurons express both Ix- and K-opioid receptors (Pechnick, 1993). There are opioid receptors in the median eminence, mediating local regulation at the level of the terminals of CRH/vasopressin parvocellular neurons and, since the blood-brain barrier is deficient in this region, opioids secreted peripherally (e.g. from anterior pituitary or adrenal medulla) could influence CRH release here too. Although some opioid binding has been reported in the anterior pituitary no direct effects on ACTH release from the corticotrophs have been reported (Calogero, 1996). Adrenal cortex secretion in the rat is influenced by both [3-endorphin and dynorphin. In the rat enkephalins and dynorphins are generally stimulatory to HPA axis secretion but in other species (e.g. sheep, pig, human) endogenous opioids are inhibitory. The opioids exert their major effects on stress responses by acting in the brain, but other mechanisms are also involved, e.g. naloxone increases adrenal corticosteroid secretion in the human, independently of a change in ACTH secretion. Neural inputs to the HPA axis, especially from limbic brain regions (amygdala, septum, BNST, and hippocampus) express opioid receptors and receive opioidergic inputs (Pechnick, 1993). The amygdala has g-opioid receptors in both rat and human; Ix-, 3and K-opioids all stimulate neurons in the amygdala in the guinea pig; the septum expresses Ix-opioid receptors. 3-Receptors are not found in the PVN, pituitary or adrenal cortex but may weakly contribute to regulating basal HPA secretion, perhaps via distant inputs. Thus, both brainstem and limbic system neurons involved in mediating activation of PVN CRH neurons by stressors either produce opioids or can be affected by them. Another peptide, nociceptin (orphanin FQ), related to dynorphin but from a different gene, has no affinity for opioid receptors and is not antagonised by naloxone, but acts on the ORL1 receptor (opioidreceptor-like 1). As for ~- and tx-opioids, nociceptin inhibits oxytocin neuron activity (Doi et al., 1998), and the SON and limbic regions that project to the PVN/HPA axis express nociceptin and ORLI. Its effects on the HPA axis are not described. Opioid receptors belong to the seven trans-membrane domain, G-protein-linked family; specifically they are linked to a Gi/o protein. The Ix-opioids act on magnocellular neurons via these receptors, causing activation of K + and inhibition of Ca 2+ conductances. Although K-opioid inhibition results from a similar effect on Ca 2+ current its effects on K + currents are less clear cut and K-receptor inhibition is not prevented by exposure to pertussis toxin (which irreversibly inactivates Gi/o protein; Brown et al., 2OOO).

Endogenous opioid control of oxytocin neuron activity and secretion

Endogenous opioid control of oxytocin neurons in pregnancy
Exogenously administered opioids strongly inhibit basal and stimulated oxytocin neuron firing rate and secretion in virgin rats (e.g. Pumford et al., 1993), acting on the opioid receptors on the cell bodies and nerve terminals. However, in conscious virgin animals endogenous opioids do not affect basal release of oxytocin since naloxone (an opioid antagonist) does not change plasma oxytocin concentration. On the other hand, when the oxytocin system is stimulated, e.g. by intravenous cholecystokinin (CCK, which acts on the vagus and, via the brainstem, stimulates oxytocin neurons, Leng et al., 1992), or electrical stimulation of rostral circumventricular organs (Bull et al., 1994), naloxone further increases oxytocin secretion in virgins. Since naloxone does not alter the firing rate of the neurons (Leng et al., 1992), this is due to reversal of the effect of the co-localised opioids which are co-released at the posterior pituitary and autoinhibit nerve terminal secretion (Bicknell and Leng, 1982).

70 In parturition, in response to uterine signals, the firing pattern of oxytocin neurons changes from a random continuous pattern to a bursting one (Summerlee, 1981), similar to the response to suckling, which results in pulsatile oxytocin secretion effecting the milk ejection reflex. There is a cascade of changes in the inputs and/or responsiveness of the oxytocin neurons in late pregnancy to facilitate this optimal firing pattern for parturition. In parallel there are mechanisms to restrain premature or excessive oxytocin neuron excitation. We have found that endogenous opioids strongly inhibit both basal and stimulated oxytocin neuron activity and secretion in late pregnancy and parturition in the rat. In the pregnant rat the magnitude and site of action of the endogenous opioid inhibition changes from mid-pregnancy onwards, since naloxone causes an increase in basal plasma oxytocin concentration, demonstrating an endogenous opioid inhibition of basal secretion not seen in virgins or in early pregnancy (Douglas et al., 1993b). At the posterior pituitary, opioid inhibition begins to increase in early- to mid-pregnancy: K-opioid antagonist (but not l~-opioid antagonist) enhances the oxytocin secretory response to CCK indicating an increase in endogenous opioid release and inhibition of oxytocin secretion (Leng et al., 1997). This is paralleled by an increase in neural lobe content of oxytocin (Fig. 1). Oxytocin gene expression may increase in pregnancy (Horowitz et al., 1994), but only after progesterone withdrawal (Crowley et al., 1995) or with acute stimulation in parturition itself (Douglas et al., 1998b). However, opioid inhibition of secretion may also contribute to the accumulation of stored oxytocin. These endogenous opioid influences at the posterior pituitary then decline in late pregnancy (Leng et al., 1997). Thus, naloxone is less effective at potentiating oxytocin release from electrically stimulated isolated pituitaries from late pregnant versus virgin rats (Douglas et al., 1993b), indicating a down regulation of endogenous opioid mechanisms at that site. This is partly due to a reduction in the sensitivity of the nerve terminals in the posterior pituitary to the action of opioids since a K-receptor agonist, U50488, is less effective at inhibiting the stimulated release (Douglas et al., 1993b) and K-receptor binding decreases (Sumner et al., 1992). There is also a

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Fig. 1. Oxytocinand dynorphincontents in the posteriorpituitary of pregnant rats. (a, b) Contentof oxytocinand dynorphinA1-8 respectively in the posterior pituitary of virgin (n = 5, 7) and late pregnant (n = 5, 8) rats (Douglas et al., 1993b). * P < 0.05 Students t-test vs. virgin control. (c) The dynorphin:oxytocin ratio calculated from data in (a) and (b), with a 70% reduction in pregnancy.The data indicate reduced capability of dynorphin action on the nerve terminals in the posteriorpituitary to inhibit oxytocinsecretionat the end of pregnancy. decrease in the posterior pituitary content of the endogenous opioid, dynorphin (Fig. 1), probably arising from prolonged release of the opioid; this could induce the observed receptor down regulation. Since the posterior pituitary content of oxytocin simultaneously increases, the ratio of dynorphin : oxytocin content falls steeply and the inhibitory power of the co-localised opioid falls and auto-inhibition decreases. This would unblock the nerve terminals from their opioid inhibition at the end of pregnancy and allow oxytocin secretion to follow closely the pattern of action potentials generated at the cell body in response to various inputs. In contrast there

71 is no effect of endogenous opioids on vasopressin release from the adjacent nerve terminals in the posterior pituitary as naloxone is ineffective, including throughout pregnancy (Douglas et al., 1993b). Simultaneously with the decrease in sensitivity to opioids at oxytocin nerve terminals there emerges an endogenous opioid inhibition centrally on oxytocin neuron activity, just before parturition. Naloxone increases basal immediate early gene (Fos) expression in SON neurons in late pregnancy, and this is evidently Ix-opioid mediated since the specific K-antagonist, nor-Binaltorphimine, is ineffective (Douglas et al., 1995). The central endogenous opioid inhibition also strongly restrains the neuron responses to stimulatory inputs. Naloxone enhances both the oxytocin neuron firing rate and secretory response to systemic CCK in late pregnancy (Douglas et al., 1995). The opioid action is input specific and appears to be focussed on inputs from the uterus, via the NTS in the brainstem, rather than inputs that mediate the oxytocin response to osmotic stimuli. Thus, naloxone is less effective at enhancing oxytocin secretory responses to stimulation of the rostral circumventricular organs in pregnancy than in virgin rats (Bull et al., 1994). The endogenous opioids are likely to act presynaptically to inhibit neurotransmitter release from the input from the brainstem, which partly comprises A2 noradrenergic neurons (Antonijevic et al., 1995). We have found that Ix-opioid receptor binding in the SON is reduced 5 days before the end of gestation suggesting action of released endogenous Ix-opioids; -opioid receptor binding is unaltered at this site (Sumner et al., 1992). Although others have differently reported that Ix-opioid binding capacity in the whole hypothalamus is enhanced in pregnancy (Dondi et al., 1991), change in Ix-receptor binding in the SON supports a role for endogenous IX- rather than ~:-opioid mechanisms in the inhibition of oxytocin neurons. The central inhibition of oxytocin neurons will restrain oxytocin neuron responsiveness to premature signals from the uterus and consequently prevent early secretion prior to parturition, which would deplete the oxytocin stores. In summary, the opioid inhibition of oxytocin neurons through mid to late pregnancy initially comprises inhibition of nerve terminal secretion and then shifts to inhibition of the oxytocin neuron cell bodies and their inputs from the brainstem.

Endogenous opioid control of oxytocin neurons in parturition

Even with the greatly enhanced activity of the oxytocin neurons in parturition, endogenous opioid inhibition remains strong. Naloxone causes an even larger increase in oxytocin secretion during birth than in pregnancy (Bicknell et al., 1988; Leng and Russell, 1989) and can speed up the birth process (Leng et al., 1985), at least in the rat, although apparently not in eady human labor (Lindow et al., 1992). It is clear that such opioids strongly inhibit oxytocin secretion and greatly prolong delivery time (Russell et al., 1989, 1991) and prevent SON neuron stimulation in rats (Luckman et al., 1993). Actions of opiates on the uterus do not explain these effects. Although pethidine inhibits uterine contractions in vitro in the rat, through its atropine-like and anti-prostaglandin actions on the uterus, any direct, naloxone-reversible, opioid action at this site is weak (Russell et al., 1991); morphine actions on the uterus have not been consistently reported, but may not be inhibitory (Russell et al., 1989). Simultaneous administration of naloxone with the opiate not only increases oxytocin secretion again in the rat (Russell et al., 1989, 1991) but also restores delivery time to normal showing that morphine acts via opioid receptors to inhibit oxytocin neuron activity. The site of Ix-opioid action is primarily central: intra-cerebroventricular morphine is strongly inhibitory and when given at the same dose intravenously is not effective; morphine does not act at the posterior pituitary due to the absence of Ix-receptors there. Lack of uterine contraction (and positive feedback to the hypothalamus, the Ferguson reflex) alone does not account for the reduction in oxytocin secretion since the suppression of SON neuron activity (Fos expression) in parturition by morphine is not restored by maintaining uterine contractile activity with simultaneous intravenous pulsatile oxytocin administration (Luckman et al., 1993). Opiates are often administered to women in labor for analgesia. Pethidine (meperidine, Demerol) and morphine (or heroin) have been extensively used in obstetric practice. Whilst being effective analgesics, these drugs may also inhibit oxytocin neurons (Lindow et al., 1992), although reports of their effect on

72 the progress of parturition in humans are few, and include evidence for both delay and acceleration of labor (Leng and Russell, 1989). Pethidine and morphine are both ~t-acting opioids, but a K-opioid (U50488) also inhibits oxytocin secretion and progress of parturition, and this is also reversible by naloxone or by intravenous oxytocin administration, and is not a result of direct uterine actions (Douglas et al., 1993a). Like the tx-opioids, U50488 not only slows births (Fig. 2a) but also inhibits SON neuron Fos expression during parturition (Fig. 2b), with reversal by a K-opioid (but not ~t-opioid) antagonist (Fig. 2b). Thus, oxytocin secretion in birth is inhibited by U50488 by actions at both the posterior pituitary and the oxytocin cell bodies. Oxytocin is released in the SON and PVN from magnocellular dendrites in parturition and this contributes positively to the pulsatile oxytocin neuron activity and secretion, and thus to birth (Neumann et al., 1996). In late pregnancy endogenous opioids inhibit this local oxytocin release in the SON (Douglas et al., 1995), presumably helping to restrain neuron activity. However, in parturition naloxone does not further increase oxytocin release in the SON and PVN (Neumann et al., 1993) suggesting loss of the inhibitory opioid control mechanism for dendritic secretion and predominance of excitation by oxytocin itself (Neumann et al., 1996). Interestingly, endogenous opioid control of oxytocin release in the septum is enhanced at the same time, in parturition (Neumann et al., 1991), and could contribute to regulation of maternal behaviour. In contrast to the acute effects of opioids, continuous administration of morphine, e.g. in women addicted to drugs such as heroin (an alkaloid related to morphine), does not apparently affect onset of parturition, and this can be replicated in rats (Yaksh et al., 1979), and may be explained by the development of tolerance to the opiate by the oxytocin neurons themselves (Russell et al., 1995). Relaxin, an insulin-like pregnancy hormone secreted from the corpus luteum, may play a role in induction of the endogenous opioid inhibition on oxytocin neurons in parturition: plasma concentration of relaxin increases during pregnancy with a pre-partum surge. Relaxin acts via the subfornical organ (Summeflee, 1989). A lack of relaxin fol\ + +

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Fig. 2. K- and Ix-opioid inhibition of parturition. At the birth of the second pup, parturient rats were injected intravenously with either vehicle (VEH) or nor-Binaltorphimine (NorBNI, a selective K-receptor antagonist, 0.5 mg/kg) and then vehicle (VEH + VEH, n = 6), U50488 (U50, K-agonist, 1 mg/kg; VEH + U50, n = 4, NorBNI + U50, n = 6), or morphine (MOR, Ix-agonist, 1 mg/kg; VEH + MOR, n = 4; NorBNI + MOR, n = 6). Pup birth times were noted and 90 min later the rats were killed and their brains removed, frozen and Fos immunocytochemistry was performed on cryostat sections containing SON. The data show: (a) cumulative times between the birth of pups 2 and 8, ANOVA P < 0.01, + P < 0.05 Student Newman Keuls post hoc test vs. VEH + VEH (modified from Douglas et al., 1993a); (b) the density of Fos-positive neurons in the SON. ANOVA P < 0.01, post hoc tests * P < 0.01 vs. VEH + VEH, + P < 0.05 vs. NorBNI + U50 in the same animals as in (a). The rate of pup births is fastest in the vehicle-treated parturient rats. Both U50,4988 and morphine slow births, and nor-Binaltorphimine prevents the slowing by U50488. In control rats, Fos is expressed in SON neurons during parturition (VEH + VEH). U50488 and morphine both strongly inhibit Fos expression; NorBNI partly prevented the inhibitory action of the K-agonist, U50488, but not the action of morphine.

lowing ovariectomy, with pregnancy maintained by oestrogen and progesterone, decreases oxytocin secretion and disrupts parturition (Way et al., 1993). In pregnancy relaxin has a dual role, activating an excitatory drive to oxytocin neurons (Way and Leng, 1992) while simultaneously inducing endogenous opioid inhibition.

73 Thus, endogenous opioids are inhibitory to oxytocin secretion in pregnancy and parturition. This provides a mechanism to control inputs/signals to oxytocin neurons, specifically related to parturition, but as a consequence, exogenous opiates given for analgesia may slow birth. Clearly the effects of exogenous and endogenous opioids on oxytocin secretion indicate that they are important regulators of defined neurons in the maternal brain in pregnancy, and have consequences for subsequent parturition and lactation and care of the offspring. Stress responses In the rat endocrine stress responses comprise increased secretion of HPA axis hormones, and other hormones including oxytocin. In other species oxytocin is generally not secreted and vasopressin is the posterior pituitary stress hormone. Although oxytocin may act directly on the adrenal cortex to acutely enhance basal corticosteroid secretion, chronic administration is reported to have other antistress effects, decreasing blood pressure and corticosterone secretion (Petersson et al., 1999), although its site and mode of action are unclear. Both HPA axis and oxytocin neuron responses to stress are regulated by endogenous opioids in the rat, sheep, pig, and human and these alter during pregnancy and parturition. 1998a; Neumann et al., 1998), and in lactation HPA responses in the rat are further reduced. Diminished HPA responses to a variety of stimuli in pregnancy have been reported in various species, including the human (Magiakou et al., 1996). The widespread phenomenon of reduced HPA axis responses in pregnancy is thought to be a protective mechanism for the unborn fetus(es), and there is accumulating evidence that excessive stress adversely affects the behavioral and endocrine development and long-term welfare of the offspring (Weinstock, 1997). We have investigated the underlying causes of the attenuated secretory responses in the rat and found that they are partly a result of reduced responsiveness of the anterior pituitary corticotrophs to CRH, with both a reduction in CRH receptor binding and an attenuated cAMP response to CRH stimulation (Neumann et al., 1998). However, these changes may follow altered central mechanisms. Stress-induced c-fos expression in parvocellular neurons and their inputs is reduced during late pregnancy and lactation (da Costa et al., 1996). Compared to virgins, there is reduced CRH mRNA expression in the PVN in late pregnancy (Douglas and Russell, 1994; Russell et al., 1999) and there is some evidence of altered stressor perception observed as changes in behavioral responses to stressors (Neumann et al., 1998).

Endogenous opioid control of HPA axis in pregnancy The hypothalamo-pituitary-adrenal axis in pregnancy
The rat is a good model to study changes in the maternal HPA axis in pregnancy because, unlike many species, the placenta does not produce CRH. In the rat from about day 15 of gestation onward HPA axis stress responses are attenuated, without change in basal plasma concentrations until later in pregnancy (Atkinson and Waddell, 1995; Neumann et al., 1998). Thus, stress-induced ACTH and corticosterone secretion is reduced in mid pregnancy, and strongly reduced in late pregnancy compared to virgins. This applies to emotional stressors such as placement on the elevated plus maze, a novel environment (Neumann et al., 1998), or following stressors such as restraint (Russell et al., 1999), or forced swimming in cold water for 90 s (Douglas et al., Endogenous opioids contribute to the regulation of HPA axis responses under a variety of physiological conditions. In the brain, opioids generally act to inhibit CRH secretion and hence ACTH secretion in most species. For example in the sheep, horse, pig and human endogenous opioids inhibit basal and stress stimulated HPA axis activity (Tsagarakis, 1997). On the other hand, in the rat the release of CRH is increased following central application of opioid in vivo or in vitro and this is blocked by naloxone; thus the net effect of opioids is stimulatory to the HPA axis in this species (Jezova et al., 1982). The effects of opioids have been extensively studied in the rat and they act centrally to stimulate or inhibit CRH release, via IX- or K-receptors, respectively (Cover and Buckingham, 1989; Calogero, 1996), so the overall effect depends

74

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The hypothalamo-pituitary-adrenal axis in parturition

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Fig. 3. Effect of naloxone on HPA axis and oxytocin secretory responses to forced swimming. Virgin and 21-day-pregnant rats were injected intravenously with either naloxone (NLX, 5 mg/kg) or vehicle (VEH) and forced to swim in 19C, deep water. The data are means 4- sem hormone concentrations in plasma from naloxone- and vehicle-treated (n = 7, 8) virgin rats, and naloxone- and vehicle-treated (n = 7, 8) pregnant rats at 5 min post-swim. Both oxytocin and ACTH plasma concentrations were significantly increased vs. pre-swim (not shown). (a) ACTH, t p < 0.05 vs. virgin vehicle-treated group. (b) Oxytocin: # P < 0.05 vs. virgin vehicle-treated, t p < 0.05 vs. all other groups. (Modified from Douglas et al., 1998a.) In virgin rats, naloxone revealed an endogenous opioid enhancement of the ACTH secretory response to swimming, and a weak endogenous opioid inhibition of oxytocin responses. Pregnancy attenuated the ACTH secretory responses to stress. In pregnancy, naloxone revealed loss of the opioid enhancement of the ACTH response as in virgins, but a large increase in inhibition of the oxytocin secretory response.

The action of endogenous opioids in regulating the HPA axis becomes stronger during parturition, parallel to the effects of opioids on oxytocin neurons. Parturition is considered to be 'stressful', as a consequence of the discomfort. However, in the rat, both ACTH and corticosterone basal plasma concentrations decrease just prior to parturition, remain low during birth and increase again afterwards (Wigger et al., 1999). Since a stress response is defined by the increased secretion of these hormones, parturition is thus not stressful to the rat. However, naloxone given during parturition greatly increases ACTH secretion, indicating that endogenous opioids are important for the reduced HPA secretion (Wigger et al., 1999). In rats that have abnormal parturition (excessive labor/straining without birth and/or long interval between births), HPA axis secretion is elevated without naloxone treatment, suggesting that dystocia stimulates ACTH and corticosterone secretion and thus is stressful. The stimulatory effect of naloxone in normal parturition may be a consequence of antagonising the endogenous opioid analgesic mechanisms in the spinal cord in pregnancy (Dawson-Basoa and Gintzler, 1993; see Gintzler and Liu, 2001, this volume). Stress and oxytocin secretion in pregnancy In the rat, oxytocin is secreted in response to various stressors including novel environment, restraint, forced swimming, social defeat, and pain (Lang et al., 1983), with peak secretion within 5-10 min after stress. The other neurohypophyseal hormone, vasopressin, is secreted in response to noxious stimuli in the rat. Whereas oxytocin neurons are prepared in pregnancy for activation at birth, the oxytocin secretory responses to stress are somewhat attenuated in late pregnancy (Neumann et al., 1998), conserving oxytocin stores in the posterior pituitary. Naloxone greatly increases the oxytocin secretory response to forced swimming in pregnant rats compared with virgins (Fig. 3b). These data suggest that endogenous opioids restrain a capacity for exaggerated oxytocin secretory responses to stress at this time (Douglas et al., 1998a). Although the endogenous opioids in-

upon the 'balance' of these actions under different stimulus conditions. In the presence of naloxone the stress responses are not inhibited in late pregnancy as they are in virgins (Douglas et al., 1998a), but there is a switch from inhibition by naloxone to weak enhancement (Fig. 3a), suggesting that endogenous opioids may now inhibit rather than excite HPA axis secretory responses. This opioid inhibition is most likely to be centrally on the parvocellular (CRH and vasopressin) PVN neurons, or their inputs.

75 hibit enhanced oxytocin secretory responses to CCK and stress in pregnancy and parturition (Douglas et al., 1995, 1998a), this does not extend to all stimuli (Bull and Russell, 1992). Thus, endogenous opioids probably act presynaptically on the inputs from brain areas involved in processing stressors, and the relays in the brainstem mediating stimuli from parturition and CCK. Endogenous opioids are not responsible for the lack of vasopressin secretion in response to stressors such as forced swimming or restraint in the rat, even in pregnancy (Douglas et al., 1998a). Stress and oxytocin secretion in parturition Despite the conclusion that oxytocin is a stress hormone in the rat, the stress of environmental disturbance in parturition delays births in rats, mice, pigs and humans (Newton et al., 1968; Leng et al., 1988b; Lawrence et al., 1992). In the rat and pig this evidently results from decrease in oxytocin secretion (Leng et al., 1988b; Lawrence et al., 1992). It seems likely that the pulsatile oxytocin secretion stops with consequent loss of coordinated uterine contractions; the lack of positive feedback from the uterus to the SON and/or PVN further preventing activation of oxytocin neurons. However, in such stressed rats or pigs, naloxone increases oxytocin secretion and restores births (Leng et al., 1988b; Lawrence et al., 1992). Whether the pulsatile pattern of oxytocin secretion is restored or not is unclear, and it may be that naloxone reveals the stimulation of oxytocin secretion by the stressor, as in pregnancy. However, the chronic stress of housing pigs in crates does not inhibit oxytocin secretion or the progress of parturition (Lawrence et al., 1995), indicating adaptation to the stressor. Immediately after parturition, and into lactation, endogenous opioids no longer appear to control basal oxytocin secretion since naloxone does not affect plasma oxytocin concentration (Bicknell et al., 1988), nor does it usually affect the milk ejection reflex, even though exogenous opioids are capable of inhibiting oxytocin neuron activity (Russell et al., 1995). However, oxytocin responses to stress are even more strongly attenuated than in late pregnancy (Neumann et al., 1998).

Sex steroids and endogenous opioid inhibition of oxytocin and the HPA axis in pregnancy and parturition
Oestrogen and progesterone may have both direct and indirect effects, via rapid membrane mechanisms or via the classical cytoplasmic receptors. Progesterone is the primary steroid hormone of pregnancy, being secreted from the corpus luteum of the ovary and/or the placenta throughout gestation in most mammals, and it penetrates throughout the body and brain. In general, progesterone acts on oestrogen-primed cells as oestrogen induces expression of progesterone receptors. Before the end of pregnancy there is an increase in the plasma concentration of oestrogens and/or a decrease in the plasma concentration of progesterone in several species (e.g. rat and mouse) resulting in an increase in the oestrogen:progesterone ratio (Bridges, 1984). In the SON and PVN oestrogen receptor ~3 is expressed (Shughrue et al., 1996), but not oestrogen receptor c~; a few magnocellular PVN but not SON neurons express progesterone receptor (Thomas et al., 1999). Additionally, there are neurosteroids, such as allopregnanolone, that are generated in brain tissue from progesterone, oestrogen and androgens and these act locally to rapidly change the responsiveness of receptors for neurotransmitters, including Ix-opioid (17[3-estradiol, Kelly et al., 1999) and GABA (allopregnanolone in the SON, Brussaard et al., 1999) receptors. Oestrogen can also rapidly stimulate dendritic peptide release in the SON (Wang et al., 1995). This discussion is confined to interactions of sex steroids with the endogenous opioids. Previously it has been shown that opioid inhibition of oxytocin secretory responses to stress is greater in female than in male rats (Carter et al., 1986), and that castration of males changes their response to resemble that of females. The development of the endogenous opioid inhibition in pregnancy, as described above, may be partly due to the concomitant increase in plasma oestrogen and progesterone. Sex steroid administration to virgin animals induces opioid synthesis in the brain. Progesterone increases the [3-endorphin content of the hypothalamus and enhances POMC expression in the arcuate nucleus (Bridges and Ronsheim, 1987) while the number of tx-opioid receptors in the brain is also increased

76

a)
=
O

30

~--~ 20 OE E 10 ~_ 0
J.

E+P-P Veh

E+P-P NIX

E+P NLX

b)._=

900
600 a00

Control

E+P

E+P-P

Fig. 4. Effects of sex steroids on opioid inhibition of oxytocin secretion. Virgin rats were subcutaneously implanted with silastic capsules containing estradiol and progesterone for 15-16 days (E + P), or received sham implants (Control). Progesterone capsules were left in place (+P) or removed ( - P , to mimic the pre-parturition fall in progesterone 1-2 days before experiment); all had a chronic jugular vein cannula implanted. The data are: (a) the change in basal plasma oxytocin concentration 20 min after naloxone (NLX) or vehicle (Veh) (E + P - P, vehicle: n = 6; E + P - P naloxone: n = 13; E + P naloxone, sham P withdrawal: n = 5). ANOVA for repeated measures P < 0.01, * P < 0.05 vs. pre-injection; (b) the change in plasma oxytocin concentration 5 min after forced swimming compared to pre-swimming (pre-swim = 100%; n = 5/6 per group; striped rectangle, after naloxone; open rectangle, after vehicle), 1 way ANOVA P < 0.0001; * P < 0.05 vs. vehicle controls and vs. non-steroid Control group. Note different scales in (a) and (b). (Data modified from Douglas et al., 2000.) Sex steroid administration induced weak endogenous opioid inhibition on basal oxytocin secretion (a), but stronger inhibition on oxytocin secretory responses to stress-exposure (b); progesterone withdrawal had no additional effect.

(Martini et al., 1989), similar to findings reported for pregnancy. On the other hand, oestrogens alone may inhibit [~-endorphin synthesis (Rosie et al., 1992). We implanted oestrogen- and progesterone-containing silastic capsules subcutaneously into virgin rats for 16-17 days, to increase sex steroid concentrations in plasma to levels similar to pregnancy (Bridges, 1984). Naloxone increased plasma oxytocin concentrations (Fig. 4a) in these sex-

steroid-treated rats, but did not increase Fos expression. Therefore it appears that opioid inhibition on oxytocin nerve terminals was increased by oestrogen/progesterone treatment but had no effect on the cell bodies of oxytocin neurons. Withdrawal of progesterone, by removing the subcutaneous capsules (to mimic the pre-partum fall) had no further effect. However, in these virgin rats there would be no reflex positive feedback stimulation from uterine contractions that can occur following oxytocin secretion at the end of pregnancy. Consequently, we studied the effect of oestrogen and progesterone administration on opioid inhibition of stimulated oxytocin neurons. Animals treated with the sex steroids in silastic capsules, as above, were given an intravenous naloxone or vehicle injection and were then exposed to a stressor (forced swimming). After naloxone, the sex-steroid-treated rats had a significantly greater oxytocin secretory response to the stressor compared to the non-steroid controls (Fig. 4b; Douglas et al., 2000), showing that sex-steroid treatment induced endogenous opioid inhibition on the oxytocin neurons by, at least partly, acting on their inputs. Again progesterone withdrawal had no additional effect from sex-steroid treatment without withdrawal, but 17~-estradiol treatment alone had a smaller but more prolonged effect on opioid inhibition of the oxytocin secretory response to stress (Douglas et al., 2000). In contrast, the ACTH and corticosterone secretory responses to the stress were not affected by the sexsteroid treatments (either with or without naloxone), so sex steroids may not be involved in the opioid regulation of the HPA axis (Douglas et al., 2000). Thus, it appears that exposure to changing oestrogen : progesterone levels as in pregnancy can induce endogenous opioid inhibition of oxytocin neuron activation in virgin rats. In pregnant rats the presence of the fetus(es) will additionally contribute to feedback signals from the uterus to the brain which may further enhance opioid mechanisms in late pregnancy and parturition.

Endogenous opioid expression in pregnancy

We have searched for pregnancy- and parturitionrelated changes in endogenous opioid gene expression in the SON and PVN and in regions

77 that project to these nuclei. We found no changes in Met enkephalin content of the posterior pituitary (Douglas et al., 1993b), or in the expression of Proenkephalin A mRNA in the PVN (Douglas and Russell, 1994), or dynorphin mRNA in the SON (Douglas et al., 1993b) in pregnancy, although Schriefer (1991) previously reported a decreased enkephalin content, decreased Proenkephalin A mRNA and increased dynorphin mRNA. Thus any role of co-localised opioids in the central regulation of oxytocin neuron activity in pregnancy is not clear. Inputs to the SON and PVN that express endogenous opioids may be a more likely source of the tonic opioid inhibition on these neurons in pregnancy and parturition than co-localised opioids. However among the inputs to the PVN, we observed no change in the Proenkephalin A mRNA content of the zona incerta with pregnancy, some of whose neurons project to synapse with nearby PVN neurons (Merchenthaler et al., 1986). However, these enkephalins are more selective for K- and Greceptors and the opioid inhibition on oxytocin neurons in late pregnancy is Ix-receptor mediated. ~-endorphin is a relatively Ix-selective endogenous opioid and it is synthesised from the POMC gene which is expressed in neurons of the arcuate nucleus and in the anterior and neurointermediate lobes of the pituitary. In pregnancy, there is increased ~-endorphin secretion into the blood in rats, humans and pigs (Bridges and Ronsheim, 1987; Chan and Smith, 1992; Aurich et al., 1993), which may be secreted in part from the placenta. Although it is uncertain where circulating [3-endorphin might act, as it does not cross the blood-brain barrier it could only influence nerve terminal secretion in the median eminence, posterior pituitary or at other sites where the barrier is leaky. However, the source of ~-endorphin that might inhibit neuroendocrine neurons is most likely to be the arcuate nucleus. ~-endorphin content of the hypothalamus increases in pregnancy in the rat (Wardlaw and Frantz, 1983) and the density of f3-endorphin-containing fibres in the SON and in the peri-SON region also increases in late pregnancy (Fig. 5). Also, POMC expression (Redmond et al., 1996) and number of neurons immuno-labelled with ~-endorphin increases in the arcuate nucleus in late pregnancy. However, despite the fact that arcuate neurons project to the SON and PVN, and inhibit
4 ./t

~o 2
z 0 SON PeriS O N region

Fig. 5. Density of 13-endorphin fibres in and around the SON. Virgin (n = 8) and 21-days-pregnant(n = 8) rats were perfused fixed and their brains removed and processed by immunocytochemistry for 13-endorphin. The density of [3-endorphin fibres in the SON and in the region adjacent to the SON (peri-SON) were counted using a stereological method. The data show an increase in both the SON and peri-SON region in the density of [3-endorphin fibres in pregnant rats (normalised to virgin density = 1). * P < 0.05 vs. virgin, Students t-test.

SON neuron firing rate, there is no electrophysiological evidence in virgin animals that this is mediated by opioids (Leng et al., 1988a). There is evidence that 13-endorphin can regulate the HPA axis; for example, it inhibits the PVN neuron firing rate (Pittman et al., 1980) and attenuates stress-induced CRH expression in the PVN (Suda et al., 1992). The newly discovered endogenous opioids, the endomorphins, are highly selective for the Ix-opioid receptor and are also expressed in the hypothalamus (Zadina et al., 1997), but changes with pregnancy are not known.

Summary
During late pregnancy and parturition endogenous opioid inhibition of oxytocin and the HPA axis increases. In the rat, opioid inhibition of oxytocin neurons switches from their nerve terminals in the posterior pituitary to their cell bodies and inputs. In the HPA axis opioid action switches from excitation to inhibition (Fig. 6). These opioid mechanisms prevent the oxytocin and HPA axes from responding inappropriately before and during parturition, thus isolating their responses to reproduction-related signals. The sex steroids of pregnancy, oestrogen and progesterone, appear to play a role in enhancing endogenous opioid regulation of oxytocin neuron activity, but other mechanisms may be responsible for opioid control of the HPA axis in late pregnancy.

78
;/h!!!; ....!i kii: i = i !!i)! ! iii~h/i ! iii

i!ii 2ii!

i ii!i!iii!i!ii! i !?

i i !~iii! ~ilili i!i i

~i !ii~i!il i~iii

PREGNANT UTERUS

ACTH

PITUITARY

Fig. 6. The maternal brain: changes in endogenous opioid inhibition of oxytocin neuron and HPA axis activity. At the end of pregnancy and in parturition endogenous opioid action: 0) switches from enhancement of HPA axis secretion (~) to inhibition: ((~) and switches from inhibition of oxytocin nerve terminals in the posterior pituitary to stronger inhibition of oxytocin cell bodies and/or their inputs. Both basal and stimulated (including stress and parturition) neuron activity and secretion are inhibited. Factors contributing to the changes in endogenous opioid action may include the sex steroids and activation of neural inputs by stressor exposure and/or uterine activity (or other fetal factors).

Abbreviations
beta delta kappa K p~ mu ACTH adrenocorticotrophic hormone BNST bed nucleus of the stria terminalis cholecystokinin CCK corticotrophin releasing hormone CRH hypothalamo-pituitary-adrenal HPA ORLI opioid-like receptor 1 POMC pro-opiomelanocortin paraventricular nucleus PVN supraoptic nucleus SON

Acknowledgements
The authors thank Ms. Paula Brunton, Mr. Ian Croy, Dr. Philip Bull, Dr. Karen Francis, Dr. Ruben Caron, Dr. Inga Neumann for discussions on the experiments and for technical analyses. Supported by the Royal Society, The Wellcome Trust, the BBSRC, and DAAD/ARC.

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Lindow, S.W., van der Spuy, Z.M., Hendricks, M.S., Rosselli, A.P., Lombard, C. and Leng, G. (1992) The effect of morphine and naloxone administration on plasma oxytocin concentrations in the first stage of labour. Clin. Endocrinol., 37: 349-353. Luckman, S.M., Antonijevic, I., Leng, G., Dye, S., Douglas, A.J., Russell, J.A. and Bicknell, R.J. (1993) The maintenance of normal parturition in the rat requires neurohypophysial oxytocin. J. Neuroendocrinol., 5: 7-12. Magiakou, M.A., Mastorakos, G., Rabin, D., Dubbert, B., Gold, P.W. and Chrousos, G.P. (1996) Hypothalamic corticotropinreleasing hormone suppression during the post partum period: implications for the increase in psychiatric manifestations at this time. J. Clin. Endocrinol. Metab., 81: 1912-1917. Mann, P.E., Rubin, B.S. and Bridges, R.S. (1997) Differential proopiomelanocortin gene expression in the medial basal hypothalamus of rats during pregnancy and lactation. Mol. Brain Res., 46: 9-16. Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H. and Watson, S.J. (1994) Mu, delta, and kappa opiold receptor mRNA expression in the rat CNS: An in situ hybridization study. J. Comp. Neurol., 350: 412-438. Martini, L., Dondi, D., Limonta, P., Maggi, R. and Piva, F. (1989) Modulation by sex steroids of brain opioid receptors: Implications for the control of gonadotropins and prolactin secretion. J. Steroid Biochem., 33: 673-681. Meister, B., Cortes, R., Villar, M.J., Schalling, M. and Hokfelt, T. (1990) Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels. Cell Tissue Res., 260: 279-297. Merchenthaler, I., Maderdrut, J.L., Altschuler, R.A. and Petrusz, P. (1986) Immunocytochemical localization of proenkephalinderived peptides in the central nervous system of the rat. Neuroscience, 17: 325-348. Neumann, I., Russell, J.A., Wolff, B. and Landgraf, R. (1991) Naloxone increases the release of oxytocin, but not vasopressin, within limbic brain areas of conscious parturient rats: a push-pull perfusion study. Neuroendocrinology, 54: 545551. Neumann, I., Russell, J.A. and Landgraf, R. (1993) Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: a microdialysis study. Neuroscience, 53: 65-75. Neumann, I., Douglas, A.J., Pittman, Q.J., Russell, J.A. and Landgraf, R. (1996) Oxytocin released within the supraoptic nucleus of the rat brain by positive feedback action is involved in parturition-related events. J. Neuroendocrinol., 8: 227-233. Neumann, I., Johnstone, H., Hatzinger, M., Liebsch, G., Russell, J.A., Shipston, M.J., Landgraf, R. and Douglas, A.J. (1998) Attenuated responses of the hypothalamo-pituitary-adrenal axis to stress in pregnant rats involve adenohypophysial changes. J. Physiol., 508: 289-300. Newton, N., Peeler, D. and Newton, M. (1968) Effect of disturbance on labor. An experiment with 100 mice with dated pregnancies. Am. J. Obstet. Gynecol., 101:1096-1102. Panula, E and Lindberg, I. (1987) Enkephalins in the rat pituitary

81

gland: immunohistochemical and biochemical observations. Endocrinology, 121: 48-58. Pechnick, R.N. (1993) Effects of opioids on the hypothalamopituitary-adrenal axis. Annu. Rev. PharmacoL Toxicol., 32: 353-382. Petersson, M., Hulting, A.-L. and Uvnas Moberg, K. (1999) Oxytocin causes a sustained decrease in plasma levels of corticosterone in rats. Neurosci. Lett., 264: 41-44. Pittman, Q.J., Hatton, J.D. and Bloom, EE. (1980) Morphine and opioid peptides reduce paraventricular neuronal activity: Studies on the rat hypothalamic slice preparation. Proc. Natl. Acad. Sci. USA, 77: 5527-5531. Pumford, K., Russell, J.A. and Leng, G. (1993) Effects of the selective kappa-opioid agonist U50488 upon the electrical activity of supraoptic neurones in morphine-tolerant and morphine-na;fve rats. Exp. Br. Res., 94: 237-246. Raby, W. and Renaud, L.P. (1989) Nucleus tractus solitarius innervation of supraoptic nucleus: anatomical and electrophysiological studies in the rat suggest differential innervation of oxytocin and vasopressin neurons. Prog. Br. Res., 81: 319327. Redmond, K., Douglas, A.J., Bicknell, R.J. and Russell, J.A. (1996) Evidence for plasticity in the 13-endorphin innervation of the supraoptic nucleus during pregnancy in the rat. J. Physiol., 495P: 13P. Rosie, R., Thomson, E., Blum, M., Roberts, L. and Fink, G. (1992) Oestrogen positive feedback reduces arcuate nucleus proopiomelanocortin messenger ribonucleic acid. J. Neuroendocrinol., 4: 625-630. Russell, J.A., Gosden, R.G., Humphreys, E.M., Cutting, R., Fitzsimons, N., Johnston, S., Liddle, S., Scott, S. and Stirland, J.A. (1989) Interruption of parturition in rats by morphine: A result of inhibition of oxytocin secretion. J. Endocrinol., 121: 521-536. Russell, J.A., Leng, G., Coombes, J.E., Crockett, S.A., Douglas, A.J., Murray, I. and Way, S. (1991) Pethidine (meperidine) inhibition of oxytocin secretion and action in parturient rats. Am. J. Physiol., 261: R358-R368. Russell, J.A., Leng, G. and Bicknell, R.J. (1995) Opioid tolerance and dependence in the magnocellular oxytocin system: A physiological mechanism? Exp. Physiol., 80: 307-340. Russell, J.A., Johnstone, H., Douglas, A.J., Landgraf, R., Wigger, A., Shipston, M.J., Seckl, J.R. and Neumann, I.D. (1999) Neuroendocrine stress mechanisms regulating ACTH and oxytocin in pregnancy. In: H. Yamashita, J.W. Funder, J.G. Verbalis, Y. Ueta and Y. Endo (Eds.), Control Mechanisms of Stress and Emotion: Neuroendocrine-Based Studies. Elsevier, Amsterdam, pp. 33-5 l. Sawchenko, P.E., Arias, C. and Bittencourt, J.C. (1990) Inhibin beta, somatostatin and enkephalin immunoreactivities coexist in caudal medullary neurons that project to the paraventricular nucleus of the hypothalamus. J. Comp. NeuroL, 291: 269-280. Schriefer, J.A. (1991) Diethylstilbesterol- and pregnancy-induced changes in rat neurointermediate lobe oxytocin, arginine vasopressin, methionine enkephalin and dynorphin. Neuroendocrinology, 54: 185-191. Shughrue, P.J., Komm, B. and Merchanthaler, I. (1996) The

distribution of estrogen receptor-13 mRNA in the rat hypothalamus. Steroids, 61: 678-681. Suda, T., Sato, Y., Sumitomo, T., Nakano, Y., Tozawa, F., Iwai, I., Yamada, M. and Demura, H. (1992) beta-Endorphin inhibits hypoglycemia-induced gene expression of corticotropin-releasing factor in the rat hypothalamus. Endocrinology, 130: 13251330. Summerlee, A.J.S. (1981) Extracellular recordings from oxytocin neurones during the expulsive phase of birth in unanaesthetized rats. J. Physiol., 321: 1-9. Summerlee, A.J.S. (1989) Relaxin, opioids and the timing of birth in rats. In: R.G. Dyer and R.J. Bicknell (Eds.), Brain Opioid Systems in Reproduction. Oxford University Press, Oxford, pp. 257-270. Sumner, B.E.H., Douglas, A.J. and Russell, J.A. (1992) Pregnancy alters the density of opioid binding sites in the supraoptic nucleus and posterior pituitary gland of rats. Neurosci. Lett., 137: 216-220. Thomas, A., Shughrue, P.J., Merchanthaler, I. and Amico, J.A. (1999) The effects of progesterone on oxytocin mRNA levels in the paraventricular nucleus of the female rat can be altered by the administration of diazepam or RU486. J. Neuroendocrinol., l 1: 137-144. Tsagarakis, S. (1997) Regulation of hypothalamic secretion of corticotropin releasing hormone - CRH. Rev. Clin. Pharmacol. Pharmacokin., 15: 35-40. Wang, H., Ward, A.R. and Morris, J.E (1995) Oestradiol acutely stimulates exocytosis of oxytocin and vasopressin from dendrites and somata of hypothalamic magnocellular neurons. Neuroscience, 68:1179-1188. Wardlaw, S.L. and Frantz, A.G. (1983) Brain beta-endorphin during pregnancy, parturition, and the postpartum period. Endocrinology, 113: 1664-1668. Watson, S.J., Akil, H., Fiscli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Griedanus, T.B. (1982) Dynorphin and vasopressin: common co-localization in magnocellular neurones. Science, 216: 85-87. Way, S.A. and Leng, G. (1992) Relaxin increases the firing rate of supraoptic neurones and increases oxytocin secretion in the rat. J. Endocrinol., 132: 149-158. Way, S.A., Douglas, A.J., Dye, S., Bicknell, R.J., Leng, G. and Russell, J.A. (1993) Endogenous opioid regulation of oxytocin release during parturition is reduced in ovariectomized rats. J. Endocrinol., 138: 13-22. Weinstock, M. (1997) Does prenatal stress impair coping and regulation of hypothalamic-pituitary-adrenal axis? Neurosci. Behav. Rev., 21: 1-10. Wigger, A., Lorscher, P., Oehler, I., Keck, M.E., Naruo, T. and Neumann, I.D. (1999) Nonresponsiveness of the rat hypothalamo-pituitary-adrenocortical axis to parturitionrelated events: inhibitory action of endogenous opioids. Endocrinology, 140: 2843-2867. Woodside, B. and Amir, S. (1996) Reproductive state changes NADPH-diaphorase staining in the paraventricular and supraoptic nuclei of female rats. Brain Res., 739: 339--342. Yaksh, T.L., Wilson, P.R., Kaiko, R.E and Inturrisi, C.E. (1979)

82 Analgesia produced by a spinal action of morphine and effects upon parturition in the rat. Anesthesiology, 51: 386-392. Zadina, J.E., Hackler, L., Ge, L.-J. and Kastin, A.J. (1997) A potent and selective endogenous agonis receptor. Nature, 386: 499-502.

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 6

The maternal spinal cord: biochemical and physiological correlates of steroid-activated antinociceptive processes
Alan R. Gintzler* and Nai-Jiang Liu
Department of Biochemistry, State University of New York Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA

Abstract: Physiological gestation, as well as the simulation of the associated changes in estrogen and progesterone, is
associated with significant elevations in nociceptive response thresholds. This is mediated by spinal cord K- and Gopioid systems. The predominant spinal ll-opioid system does not appear to participate. One hallmark of pregnancy- and hormonally-induced antinociception is the multiplicative interaction among its components. Approximately 40% results from spinal K/3 analgesic synergy on which is superimposed an additional increment (~60%) of synergy that results from the interaction between descending spinal c~2-noradrenergic and spinal K/3 activities. An intact hypogastric nerve is required for the spinal a2-noradrenergic component. This would explain the requirement for an intact hypogastric nerve in order for the antinociception of pregnancy and its hormonal simulation to be fully manifest. The predominant means by which spinal dynorphin-containing neurons adjust to increased demand is increased post-translational processing of dynorphin precursor intermediates which are present at ~ 10 the concentration of mature dynorphin peptides (1-17 and 1-8). This is indicated by the concomitant decline (~50%) in the spinal cord content of dynorphin precursors and increase (~87%) in the content of prohormone convertase 2, a processing enzyme sufficient to generate mature dynorphin peptides from prodynorphin. The presence of 'high gain' multiplicative spinal opioid antinociceptive pathways that can be activated by estrogen and progesterone has hyperalgesic implications as well, i.e. it could result in disproportionately increased pain responsiveness. This might explain, in part, findings that women are more prone to recurrent pain and pain of greater duration and intensity than men. The underlying mechanisms of gestational antinociception could point the way to pain pharmacotherapies that are gender-based.

Introduction
Hormonal milieu can be a critical determinant of neuronal plasticity (see Breedlove, 1992; ToranAllerand, 1995 for review). One manifestation of this is the sex dependence of responsiveness to environmental stressors, (Sternberg and Liebeskind, 1995; Sternberg et al., 1995; Mogil and Belknap, 1997), a critical determinant of which is the milieu of ovarian sex steroids (Kayser et al., 1996). For example,

the male phenotype-specific N-methyl-D-aspartate (NMDA) receptor-mediated antinociceptive pathway can be observed in females, but only in the absence of estrogen. In its presence, an alternative antinociceptive pathway is predominant (Mogil et al., 1993). Paradoxically, a propensity to develop hyperalgesia has also been attributed to ovarian sex steroids, e.g. they have also been shown to facilitate the development of tactile allodynia following partial sciatic nerve ligation (Coyle et al., 1996).

* Corresponding author: Alan R. Gintzler, Department of Biochemistry, State University of New York Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA. Tel.: +1 (718) 270-2129; Fax: +1 (718) 270 3316; E-mail: agintzler@netmail.hscbklyn.edu

Pregnancy-induced plasticity of spinal dynorphin neurons

Pregnancy is one naturally occurring physiological state associated with profound changes in circulating ovarian steroids. Not surprisingly then, gestation

84 TABLE 1 Influence of pregnancy and hormone simulated pregnancy on biochemical parameters of dynorphin metabolism in lumbar spinal cord PC2 a Pregnancy HSP 1" 1" Dyn 1-17b ~" 1" Dyn intermediates c 4, ? Dyn mRNAd ?

a Lumbar spinal content of prohormone convertase 2 (PC2) protein was quantified by Western blot analysis using an antibody generated against the COOH terminal 15 amino acids of this protein (courtesy of Dr. Christopher Rhodes, Harvard Medical School, Boston, MA; see Varshney et al., 1999). b Lumbar spinal content of dynorphin A (Dyn 1-17) was quantified by radioimmunoassay using an antibody highly specific for this peptide (courtesy of Dr. Eckard Weber, University of California, Irvine, CA; see Medina et al., 1993a,b). c Lumbar spinal content of dynorphin precursor intermediates (Dyn intermediates) was quantified by determining the amount of arginine6-1eucine enkephalin immunoreactivity that was generated by proteolytic (trypsin) treatment of lumbar extracts containing its precursor intermediates (see Medina et al., 1995). Arginine6-1eucineenkephalin immunoreactivity was determined by radioimmunoassay using an antibody highly specific for this C-terminal extended opioid (generously supplied by Drs. Lars Terenius and Engrid Nylander, Karolinska Institute). d A cDNA probe for rat prodynorphin mRNA (Dyn mRNA) was derived from a 1.7-kb fragment of the rat prodynorphin gene subcloned into pSP64 (generously provided by Dr. James Douglas). The 1.7-kb insert was removed with Pst/EcoR1 and made radioactive with 3zp-dCTP by random-primed labeling. Quantification of prodynorphin mRNA was accomplished by slot blot analysis and solution hybridization (see Franklin et al., 1991 for solution hybridization procedure). The increase in lumbar content of PC2 protein is consistent with decreased content of dynorphin precursor intermediates and increased content of mature dynorphin A (1-17) peptide. Collectively, these data suggest that augmented processing of dynorphin precursors, which contain approximately 10-fold more dynorphin A (1-17) than the spinal cord content of this peptide, is the primary means by which spinal dynorphin neurons adapt to the increased demands of pregnancy.

is associated with alterations in the functionality of spinal opioid antinociceptive systems. This is reflected by several neurochemical parameters of dynorphin and enkephalin neuronal function (see Table 1). During late pregnancy, the content of spinal dynorphin (1-17 and 1-8) is altered in a region specific fashion (Medina et al., 1993b). As a result, levels of dynorphin peptides are elevated, but only in the lumbar spinal region, the spinal area that receives pelvic afferents (Peters et al., 1987; Berkley et al., 1988; Robbins et al., 1990). In parturient animals, there is an additional increment in the lumbar content of dynorphin (1-17) (Medina et al., 1993b). The spinal content of methionine-enkephalin is also elevated during late gestation (Medina et al., 1993b). However, in contrast to dynorphin peptides, there is no interaction between condition and spinal level. Gestational antinociception is accompanied not only by an increase in the lumbar spinal cord content of mature dynorphin peptides (1-17 and 1-8) but also by a substantial ( ~ 5 0 % ) decrease in the content of dynorphin precursor intermediates (Medina et al., 1995). The magnitude of this decline vastly exceeds the magnitude of the increase in the content

of dynorphin peptides (1-17 and 1-8), a difference that would be consistent with the increased release of these peptides. Moreover, the content of messenger ribonucleic acid (mRNA) encoding preprodynorphin in the lumbar cord obtained from pregnant (day 22) versus non-pregnant rats does not differ (assessed using solution hybridization or slot blot analysis: Gintzler and Inturrisi; Gintzler and Rivera, respectively, unpublished observations, or Northern blot analysis, Draisci et al., 1994). Thus, the contribution of dynorphin genomic up-regulation to the increased content of spinal dynorphin during late pregnancy is, most likely, small at best. These observations suggest that regulation of the activity of the proteolytic enzyme(s) responsible for the conversion of dynorphin precursor intermediates to mature dynorphin peptides could be an essential part of the mechanism(s) by which spinal dynorphin neurons adapt to their increased activity during gestation.

Modulation of spinal cord prohormone convertase during pregnancy

Likely candidate enzymes include a superfamily of mammalian serine proteases (prohormone conver-

85 tase; PC) that has been identified in animal cells. These are dedicated to the excision of most peptide hormones and neurotransmitters from their respective precursors (Docherty and Steiner, 1982). Two of these mammalian enzymes, PC1 (also called PC3) and PC2 are expressed specifically in pancreatic islets, pituitary, adrenal medulla and many regions of the central nervous system (Seidah et al., 1991; Smeekens et al., 1991). Heterologous gene transfer experiments have demonstrated that they are capable of cleaving selectively at the dibasic amino acids Lys-Arg and Arg-Arg sites within precursor hormones (Benjannet et al., 1991, 1992; Thomas et al., 1991; Smeekens et al., 1992). The potential relevance of these processing enzymes to the adaptation of spinal cord dynorphin neurons to the increased demands of pregnancy is underscored by the presence of both PC1 and PC2, in relatively high amounts, in the superficial lamina of spinal tissue (Schafer et al., 1993). Moreover, using AtT20 cells in which PC1 and prodynorphin were co-expressed as well as in vitro incubation of prodynorphin with PC1, it has been demonstrated that PC1 can cleave prodynorphin at pairs of basic amino acids to generate 10 and 16 kDa high molecular weight intermediates. In addition, PC1 also cleaves prodynorphin at a single arginine residue to yield an 8-kDa product and C-peptide (Dupuy et al., 1994). More recently, it has been demonstrated that PC2, in the absence of PC1, can cleave prodynorphin at lysine-arginine residues to produce dynorphin A (1-17), dynorphin B (1-13) and c~-neoendorphin (Day et al., 1998). PC2 is also capable of cleaving at a single arginine residue of dynorphin A (117) and generates dynorphin A (1-8) and C-peptide. Recently obtained data suggest the relevance of PC2 to the adaptation of spinal cord dynorphin to the condition of pregnancy. During late pregnancy, the lumbar PC2 protein content, but not that of PC 1, is augmented (~87%). Interestingly, levels of PC2 protein in the thoracic cord were not altered at this time. In contrast to the elevated content of lumbar PC2 protein, levels of PC2 mRNA in the lumbar cord of pregnant rats, or rats with simulation of the hormonal conditions of pregnancy, were essentially unchanged. This indicates that increased transcriptional activity is not, necessarily, a prerequisite for increased PC2 protein content to be manifest.

Pregnancy-induced enhancement of spinal dynorphin precursor processing

There are several mechanisms that could underlie the increase in spinal cord content of dynorphin A (1-17). These include transcriptional up-regulation, reliance on a large pool of neuropeptide to buffer against demand-induced depletion and enhanced rates of post-translational processing of peptide precursor intermediates (see Morgan and Chubb, 1991 for review). The predominant importance of the latter to the pregnancy-induced increment in spinal dynorphin content is indicated by several observations. First, lumbar preprodynorphin mRNA levels are not elevated at this time. Second, the content of spinal dynorphin precursor intermediates is significantly (>50%) reduced and the lumbar spinal cord content of dynorphin A (1-17) that is contained in the form of precursor intermediates is at least 10-fold higher than the content of mature dynorphin peptides. This indicates that the pool of dynorphin precursor intermediates in the lumbar cord represents a large potential source of mature dynorphin peptides. These data strongly suggest that increased post-translational processing of dynorphin precursor intermediates is the primary mechanism responsible for the increased production of lumbar dynorphin during gestation. This is consonant with the presented findings that the content of PC2 protein, whose activity is sufficient to generate mature dynorphin peptides, e.g. dynorphin A (1-17) and dynorphin B (1-13) (Day et al., 1998), is increased in maternal spinal cord during late gestation. Moreover, the time of gestation (day 22) and spinal region (lumbar) in which this occurs parallels that of the decreased levels of dynorphin precursor intermediates, the absolute amount of which is approximately 3-fold greater in the lumbar cord than in cervical or thoracic areas (Medina et al., 1995). Thus, the up-regulation of PC2 protein content appears to be a component of the mechanism(s) by which maternal spinal dynorphin-containing neurons adjust to the increased demands of physiological pregnancy.

86

Ovarian steroid milieu of pregnancy is critical for increased lumbar content of dynorphin A (1-17) and PC2 enzyme protein
One critical peripheral component of the mechanisms that underlie gestational antinociception is the pregnancy blood concentration profile of 1713-estradiol (estrogen) and progesterone. Simulation of pregnancy ovarian sex steroid blood concentrations in non-pregnant rats (hormone-simulated pregnancy, HSP-accomplished via the subcutaneous implantation of Silastic tubing filled with either a solution of estrogen in sesame oil, or crystalline progesterone (Bridges, 1984) in ovariectomized females) also augments spinal cord content of dynorphin A (1-17). In contrast to the pregnant condition, the content of dynorphin A (1-8) is not affected (Medina et al., 1993a). Moreover, this regulation is time- (or dose-) dependent and is spinal region specific. Significant elevations in spinal levels of dynorphin (1-17) are observed during days 15-19 of steroid treatment, corresponding to the last week of actual gestation and parturition. Increased dynorphin (1-17) content is observed in only the lumbar spinal region. These changes are temporally and anatomically identical to those that occur during actual gestation and parturition (Medina et al., 1993b). Similarly, the increase in the content of lumbar PC2 protein observed during physiological pregnancy also occurs during sex steroid hormone simulation (Varshney et al., 1999). These observations indicate that changes in circulating estrogen and progesterone that occur as a natural consequence of gestation activate a dynorphin system in the lumbar spinal cord. Thus, the pattern of circulating ovarian sex steroids can be an important determinant of the activity of central opioid analgesic systems.

analgesia has been observed in rats and sows in response to somatic stimuli (Gintzler, 1980; Toniolo et al., 1987; Kristal et al., 1990; Jarvis et al., 1997), as well as visceral noxious stimuli (Iwasaki et al., 1991). Augmented response thresholds to nociceptive stimuli are not unique to laboratory animals. Pregnant women also manifest gestational antinociception (Cogan and Spinnato, 1986; Whipple et al., 1990). During the last 10 days of pregnancy, quantification of verbally reported discomfort thresholds revealed significantly greater tolerance of the progressive inflation of a blood pressure cuff than was observed in control, non-pregnant women, tested in parallel (Cogan and Spinnato, 1986).

Spinal versus supraspinal opioid contributions

In rats, the antinociception associated with pregnancy is comprised of multiple components. These include central (spinal and supraspinal) as well as peripheral constituents. Moreover, the opioid component, that represents the predominant central component and is the final mediator of gestational antinociception, is itself comprised of two antinociceptive systems (see below and Fig. 3 for summary). The ability of naltrexone (an opioid antagonist) to virtually abolish the entire pregnancy-associated increment in pain thresholds coupled with the lack of this effect in non-pregnant animals indicated that pregnancy-induced antinociception results from the activation of an opioid analgesic system(s) that is absent under basal physiological conditions (Gintzler, 1980). Additionally, anatomically discrete opioid receptor blockade indicated that activation of spinal but not supraspinal opioid antinociceptive systems were essential for the manifestation of pregnancy-related antinociception. This conclusion is based upon two observations. First, intracerebroventricular application of naltrexone does not attenuate gestational nociceptive response thresholds. On the contrary, at the earliest time point examined, intracerebroventricular naltrexone resulted in a paradoxical further elevation in pain thresholds (Steinman and Gintzler, unpublished observations). In contrast, blockade of spinal opioid receptors not only attenuates the analgesia of pregnancy (Sander and Gintzler, 1987) but abolishes it (Dawson-Basoa and Gintzler, 1997, 1998).

Behavioral correlates of augmented spinal dynorphin functionality

The condition of pregnancy is associated with a profound antinociception (see Fig. 1). This was initially demonstrated in rats in which nociceptive response thresholds to an electric foot shock progressively increased as gestation progressed, peaking just prior to parturition (Gintzler, 1980). Subsequently, the phenomenon of pregnancy-induced

87

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Fig. 1. Influence of pregnancy (A) or its hormonal simulation (HSP) (B) on nociceptive response thresholds. (A) Nociceptive response thresholds were determined on the indicated days in non-pregnant (hatched bars), pregnant (black bars) and post-partum rats, and pregnant rats given naltrexone (dotted bars). (B) Nociceptive response thresholds were determined over several days during estrogen/progesterone (HSP, black bars) or sham treatment (hatched bars) and in HSP rats given naltrexone (dotted bars). For HSP, each point represents the mean + SEM jump threshold that was observed during each hormone treatment period and is plotted as the midpoint of that period (n = 7 for each group). There was no difference in nociceptive response thresholds among pregnant/HSP animals receiving systemic naltrexone versus non-pregnant/vehicle-treated animals. During physiological gestation and HSP, there was increased activity of an endogenous opioid system(s) that is quiescent under normal physiological conditions but is activated by the pregnancy profile of estrogen and progesterone. As a consequence, thresholds for responsiveness to aversive stimuli are increased.

Opioid mediation of gestational analgesia is spinal opioid receptor type-selective

The central nervous system contains three distinct types of opioid receptor, termed ~, 3 and K. The relative contribution of each type to antinociceptive phenomena can be inferred from the effects, or lack thereof, of opioid receptor antagonists that can differentiate between ~, 3 and Ktypes of opioid receptor (see Figs. 2 and 3). The intrathecal administration of the K-opioid receptor selective antagonist nor-Binaltorphimine (nor-BNI; Takemori et al., 1988) essentially abolishes the pregnancy-related 75-100% increment in pain thresholds (Dawson-Basoa and Gintzler, 1998). A comparable reduction in nociceptive response thresholds is observed (Dawson-Basoa and Gintzler, 1997) following intrathecal application of 6-opioid receptor antagonist 7-Benzylidenenaltrexone (BNTX; Sofuoglu et al., 1993). In contrast, the

robust and predominant spinal g-opioid receptorcoupled analgesic system does not appear to contribute to gestational antinociception. This is indicated by the inability of the ~t-selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; Kramer et al., 1993) to diminish the pregnancy-induced enhancement in pain thresholds (Dawson-Basoa and Gintzler, 1996). Opioid mediation of antinociception is not observed in nonpregnant animals since blockade of spinal K- or 6-opioid receptors has no effect on nociceptive response thresholds. Therefore, these results demonstrate that activity of two of the three major types of opioid receptor is required for the manifestation of gestational antinociception. Furthermore, it results from the recruitment of relatively minor spinal opioid receptor (8 and K analgesic systems that are quiescent under basal (non-pregnant) conditions.

88
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Fig. 2. On day 20 of physiological gestation or day 19 of steroid treatment (HSP), jump thresholds were determined, after which the IX-, 6- or K-opioid receptor-selective antagonists CTAP (34 nmol), BNTX (10 pmol) or nor-Binaltorphimine (87 nmol), respectively, were intrathecally administered. Ten (for CTAP) and 5 rain (for BNTX), thereafter, jump thresholds were re-determined and compared with those obtained prior to the onset of pregnancy or ovarian steroid hormone treatment. The entire increment of pregnancy- and ovarian steroid-induced antinociception is abolished following the individual blockade of either spinal ~- or K-opioid receptors. In contrast, spinal IX-opioid receptor blockade (CTAP) was without any effect. These results not only indicate that spinal 8- and K-opioid receptors contribute to gestational and HSP antinociception but that their coincident activation is a prerequisite.

Activation of spinal K/8 analgesia of pregnancy is dependent on the milieu of ovarian steroids

As was observed for the increased lumbar content of dynorphin A (1-17) and PC2 enzyme protein, the pregnancy blood concentration profile of estrogen and progesterone is a critical peripheral component of the mechanisms that underlie gestational antinociception. This conclusion derives from experiments simulating pregnancy blood levels of estrogen and progesterone in non-pregnant rats either by inducing pseudopregnancy or by via the systemic administration of estrogen and progesterone. The pseudopregnant state, which lasts for "~9-14 days in the rat (deFeo, 1966), can be induced by brief intermittent mechanostimulation of the vaginal cervix during estrus. During this period, the pattern of change in circulating estrogen and progesterone parallel those that occur during physiological gestation (deFeo, 1966; Pepe and Rothchild, 1974). The mean jump thresholds of rats rendered pseu-

dopregnant are significantly elevated (mean increase of "--60%) relative to their thresholds observed in the period immediately preceding the onset of pseudopregnancy or subsequent to its offset (Gintzler and Bohan, 1990). Moreover, the entire pseudopregnant-induced increment in pain thresholds can be abolished by the systemic administration of naltrexone (Gintzler and Bohan, 1990), showing that endogenous opioids are responsible for the antinociception in this model, as for pregnancy. These results imply that the pregnancy blood profile of ovarian sex steroids activates endogenous opioid antinociceptive systems. This inference was confirmed by assessing the effects on pain thresholds of the simulation of the pregnancy blood concentration profile of circulating estrogen and progesterone in non-pregnant ovariectomized rats (HSP, see above). HSP resulted in a statistically significant elevation in pain thresholds (Dawson-Basoa and Gintzler, 1993), the temporal pattern and magnitude of which is strikingly similar to that which has been observed during

89 actual gestation. It should be noted that simulation of pregnancy levels of either estrogen or progesterone alone or estrogen with the delayed addition of progesterone (starting with hormone treatment day 15, the time in which the onset of analgesia is first manifest) is not sufficient to produce any increase in pain threshold. Therefore, the entire pregnancy profile of steroid hormones is responsible for the manifestation of analgesia. The types of opioid receptor that mediate HSP analgesia as well as their central nervous system localization parallels that observed for the analgesia of physiological pregnancy (see Fig. 2). Antagonism of spinal K- (Dawson-Basoa and Gintzler, 1996, 1998) or Gopioid receptors (Dawson-Basoa and Gintzler, 1997) abolishes the analgesia produced by ovarian hormones, as was observed for physiological pregnancy. Also, like the antinociception of physiological pregnancy, the antinociception of HSP is independent of spinal Ix-opioid receptor activity (DawsonBasoa and Gintzler, 1996). So HSP-induced K- and Gopioid mediated antinociception is also mediated via an endogenous opioid system(s). The similarity between the analgesia of HSP and actual gestation reaffirms that the profile of change in plasma estrogen and progesterone is a parameter of the pregnant condition essential for the manifestation of elevated pain thresholds. Additionally, these data underscore the existence of a spinal opioid analgesic system that is subject to a gender-specific hormonal modulation.

tine

nuclel~~
I Descending I NE pathways

Ascending I afferent I

hypog~trlc I

"~ .... .r~+

Fig. 3. Summary of known components of gestational antinocicepdon. The analgesia of pregnancy is comprised of both peripheral and central components. Peripheral components include changes in the blood concentration profile of ovarian sex steroids (estrogen and progesterone) and activity of the hypogastric nerve. Central components include augmented activity of spinal K- and ~-opioid and c~2-noradrenergic receptors in the spinal cord, the latter via augmented activity of descending spinal noradrenergic (NE) projection pathways from the pontine nuclei. Underlying augmented activity of spinal K-opioid neurotransmission is an increase in the content of the prohormone convertase 2 enzyme and post-translational processing of dynorphin precursor intermediates to mature dynorphin A (Dyn 1-17), the lumbar content of which is elevated during gestation and parturition. Interestingly, the spinal cord content of methionine-enkephalin is also elevated during pregnancy, consistent with the dependence of pregnancy-induced antinociception on spinal g-opioid receptor activity. A predominant characteristic of gestational and hormone simulation of pregnancy (HSP) antinociception is that it results from analgesic synergy between spinal ~- and -opioid receptor activity and an additional superimposed c~2-noradrenergic/opioid synergy. Spinal ~/K analgesic synergy is resistant to hypogastric neurectomy and thus could result from a direct spinal action of estrogen/progesterone. In contrast, since hypogastric neurectomy eliminates the c~2-noradrenergic component of HSR it is sug~ gested that HSP activates hypogastric neurotransmission, which in turn results in the increased activity of a spinal ascending pathway(s). This, directly or indirectly, results in increased activity of a descending noradrenergic pathway(s). The consequent augmented spinal c~2 activity would result in analgesic synergy with ongoing spinal K/~ neurotransmission. E2 = estrogen, P = progesterone.

Multiplicative spinal K/~ interactions during gestation and HSP

One very intriguing characteristic of the antinociception of gestation and its hormonal simulation is that blockade of either spinal K- or Gopioid receptors, individually, results in their abolishment. This indicates a special relationship between spinal K- and Gopioid receptors, the activity of which mediates gestational and HSP antinociception. Gestational and HSP antinociception could result from basically three types of interaction between spinal x- and ~-opioid antinociceptive systems: (1) the parallel activation of K and ~ types of opioid receptor, each of which, alone, is sufficient to mediate the full extent of the antinociception of each condition; (2) the parallel activation of spinal K-

90 and Z-opioid systems, each of which independently contributes a portion of the total analgesia; or (3) the coincident activation of spinal ~ and Z types of opioid receptor. In the latter scenario, activation of either type alone as a consequence of gestation or HSP, would not be sufficient to elevate maternal pain thresholds. The first putative mechanism requires that the analgesia of pregnancy not be antagonized unless more than one type of opiate receptor is blocked (Watkins et al., 1992) since the untargeted receptor type should remain fully functional and continue to mediate antinociception. This, however, is not consistent with the ability of intrathecal norBinaltorphimine, BNTX or Naltriben, individually at doses in which they interact with a single population of receptor, to not only attenuate but abolish the gestational- or HSP-induced increment in pain thresholds. Consequently, mediation of gestational or HSP antinociception via the first putative mechanism is not a viable possibility. Similarly, the second putative mechanism is not viable since it requires that individual blockade of a single opioid receptor type produce only a partial reduction in pregnancyor HSP-induced antinociception, i.e. the untargeted receptor should continue to mediate the portion of the total antinociception that results from its activity. More direct evidence that the antinociception of gestation and HSP derives from the coincident activation of spinal K- and Z-opioid pathways, i.e., ~/Z antinociceptive synergy, comes from a comparison of the effect on gestational or HSP jump thresholds of interrupting spinal K- and Z-opioid transmission individually versus concomitantly. The rationale for this approach is that if a combinatorial interaction underlies the analgesia of pregnancy and HSP, the magnitude of reduction in pain thresholds produced by concomitant elimination of spinal K- and Z-receptor activity should be greater than that produced by eliminating each individually. Alternatively, a requirement for coincident activation of more than one spinal opioid receptor type would be indicated if both perturbations produced an equivalent reduction in pain thresholds. During either physiological pregnancy (day 20) or HSP (day 19), the magnitude of reduction in jump thresholds produced by the intrathecal application of combinations of ~- and ~-antagonists, each at submaximal doses, is indistinguishable from that observed following their individual intrathecal application (Dawson-Basoa and Gintzler, 1998). This strongly suggests that gestational- and ovarian steroid-induced antinociception is not simply the sum of the independent analgesic effects of spinal - and Z-opioid systems but requires their coincident activation and resultant antinociceptive synergy. Heretofore, antinociception resulting from a multiplicative receptor interaction among component processes was demonstrated in response to the pharmacological application of multiple opioid agonists (Miaskowski et al., 1990; Sutters et al., 1990; Horan et al., 1992; Stewart et al., 1994). Activation of a single opioid receptor type by an exogenous opioid (e.g., sufentanil) results in substantial antinociception. This notwithstanding, the mechanism that subserves the antinociception of pregnancy or its hormonal simulation requires the coincident activation of multiple types of opioid receptor. This indicates that predominant mechanisms that underlie pharmacologically induced analgesia are not, necessarily, those utilized by endogenous opioid antinociceptive pathways. However, as the phenomenon of synergy between K- and Z-receptors is exhibited during normal gestation, the multiplicative interaction of opioid action appears to be relevant to physiological processes.

Evidence of spinal K/8-opioid receptor-mediated antinociceptive synergy

In addition to the endogenous opioid mechanisms described above, nociceptive response thresholds of pregnant or HSP animals can also be significantly reduced (~60%) following blockade of spinal cord a2(but not cq-) noradrenaline receptors (Liu and Gintzler, 1999). No effect of spinal c~2-noradrenaline receptor blockade can be observed in non-pregnant rats or ovariectomized rats treated with placebo Silastic implants. This indicates that spinal ct2-noradrenergic antinociceptive mechanisms do not contribute to basal nociceptive response thresholds but are activated by the (estrogen/progesterone profile of) pregnant condition. Spinal opioid receptor blockade has no effect on the antinociception produced by activation of spinal c~2-noradrenaline receptors (Ossipov et al., 1989) al-

91 though it abolishes antinociception associated with either pregnancy or HSP (Dawson-Basoa and Gintzler, 1997, 1998; Liu and Gintzler, 1999). Thus, it is unlikely that spinal noradrenergic systems would directly contribute to the antinociception associated with pregnancy or HSP. Instead, attenuation of the analgesia of these conditions following spinal noradrenergic c~2-receptor blockade, most likely, derives from the ongoing antinociceptive-coupled multiplicative consequence of concomitant activation of spinal c~2 and opioid receptors. Antinociceptive synergy following the concomitant activation of spinal ~2 (via clonidine) and opioid receptors has been extensively documented (Wang et al., 1980; Yaksh and Reddy, 1981; Hylden and Wilcox, 1983; Ossipov et al., 1989, 1990). The reduction in nociceptive response thresholds of gestation and HSP following blockade of spinal ~2-receptors suggests that an analogous analgesic synergy also occurs between their endogenous counterparts and subserves a significant component of the antinociception of gestation and HSP. Such a mechanism would be consistent with previous reports that spinal ~2-noradrenergic receptor activation is required for the full manifestation of the antinociception produced by the systemic, supraspinal (Camarata and Yaksh, 1985; Yaksh, 1979) or intrathecal (Ossipov et al., 1989) administration of morphine. Although spinal ~2-noradrenergic tone is a prerequisite for gestational and HSP antinociception to be fully manifest, it is not required for the spinal K/8 antinociceptive synergy that underlies, in part, this analgesia. The evidence for this is that following blockade of spinal c~2-noradrenaline receptors, the increment in pain thresholds that remains (440%) can be abolished by blockade of either spinal Kor 8-opioid receptors, individually. Thus, the component of ovarian steroid-induced antinociception that persists despite the absence of ~2-noradrenergic receptor activity results from K/~ antinociceptive synergy and not from their additive independent analgesic contributions. The absence of any dependence of the HSP-induced spinal K/8 analgesic synergy on spinal c~2-noradrenergic tone has significant mechanistic implications. It indicates that the attenuation of HSP and pregnancy nociceptive response thresholds following intrathecal yohimbine results from the removal of an additional level of antinociceptive synergy. Thus, multiple levels of analgesic synergy appear to underlie the totality of the antinociception associated with physiological gestation (and HSP). It results not only from the amplification that derives from analgesic synergy between spinal K/8 opioid antinociceptive pathways but from the superimposition of an additional synergy between endogenous opioid systems and descending noradrenergic pathways as well. Utilization of an opioid antinociception that results from endogenous analgesic synergies has decided benefits. It enables concentrations of spinal dynorphin and enkephalin that alone would produce minimal opioid receptor activation (and analgesia) to be analgesically effective in combination. Lowering the concentrations of endogenous opioids that are required to produce significant levels of antinociception would decrease the propensity for developing opioid tolerance and dependence. In other words, decreasing the amount of opioid that is required at its receptor will lessen the rate of tolerance development at that receptor for a given end point (analgesia) when the analgesia is achieved by multiple independent receptor systems (K, 3, c~2) that are activated concomitantly. This is well illustrated in primates where the analgesic potency of intrathecal morphine is substantially diminished over a period of 3-5 days. In contrast, antinociceptive responses to intrathecally administered combinations of morphine and the ~2 agonist, ST-91, do not manifest any apparent diminution over a 21-day period (Wang et al., 1980; Yaksh and Reddy, 1981).

The hypogastric nerve is required for activation of spinal c~2-noradrenergic antinociceptive mechanisms
The full manifestation of gestational and HSP antinociception is dependent on the presence of an intact hypogastric nerve (a major uterine afferent) (Gintzler et al., 1983). In HSP animals subjected to bilateral hypogastric neurectomy the magnitude of the increment in nociceptive response thresholds is reduced by ~60% (Liu and Gintzler, 1999) and is comparable to that observed following blockade of spinal ~2-noradrenergic receptors. Moreover, in HSP rats following bilateral hypogastric neurec-

92 tomy, spinal c~2-noradrenergic receptor blockade is devoid of any effect on HSP pain thresholds (Liu and Gintzler, 1999). These results suggest that the augmented spinal noradrenergic tone that occurs during pregnancy and its hormonal simulation requires hypogastric nerve activity. Conversely, the ability of hypogastric neurectomy to lower pain thresholds of pregnancy and HSP presumably results from the loss of augmented spinal a2-noradrenergic tone. well as central estrogen/progesterone loci of action. Regarding the former, there is precedent for ovarian steroid regulation of hypogastric nerve function. Functionality of the hypogastric nerve has been reported to vary across the estrous cycle (Robbins et al., 1992). For example, the minimum uterine pressure required to evoke multi-unit activity of the hypogastric nerve is highest in diestrus and significantly lower during both proestrus and estrus (Robbins et al., 1992). This suggests that the increasing concentrations of estrogen and progesterone that are characteristic of physiological pregnancy (and HSP) could similarly lower the threshold for hypogastric nerve impulse propagation, i.e. increase hypogastric nerve responsiveness. It is interesting to note that a profound uterine neuronal degeneration has been reported in the term pregnant rat (Haase et al., 1997). This would imply that the involvement of the hypogastric nerve in the antinociception of HSP and physiological pregnancy derives from the ability of estrogen/progesterone to directly augment hypogastric nerve activity. Since the yohimbine-resistant spinal /B antinociception of HSP persists after hypogastric neurectomy, its steroid activation would appear to be independent of hypogastric afferent activity and could be a consequence of a direct estrogen/progesterone action on spinal neurons. This explanation would be consistent with the recent finding that the c~-isoform of the estrogen receptor co-localizes with enkephalin in many neurons of the superficial lamina of the spinal dorsal horn (Amandusson et al., 1996). Moreover, it is also found in cells scattered among laminae I, II, VI and VII (Shughrue et al., 1997). Additionally, cells expressing the ~-isoform of the estrogen receptor have been identified in lamina lI of the dorsal horn (Shughrue et al., 1997). Thus, there is a biochemical basis for postulating that the increased content of lumbar dynorphin A (1-17) and PC2 protein as well as the antinociception associated with gestation results, in part, from a direct estrogen/progesterone action on spinal tissue. Peripherally administered sex steroid hormones have been shown to influence several parameters of central opioid activity. For example, opioid receptor binding density and t3-endorphin concentration increased approximately 52% in the pre-optic area of ovariectomized rats given pregnancy levels of estra-

Hypogastric nerve/spinal cl2-receptor link suggests relevance of supraspinal components to gestational and HSP antinociception
The involvement of spinal c~2-noradrenergic receptor activity in the antinociception of gestation and HSP is the first indication of a supraspinal contribution to these phenomena. Noradrenaline-containing cell bodies are located in the A5-A7 cell groups of the pons (locus coeruleus, subcoeruleus, the medial and lateral parabrachial and the Kolliker-Fuse nuclei as well as adjacent to the superior olivary nucleus; Westlund et al., 1983) and give rise to all noradrenergic spinal terminals. Consequently, the ability of hypogastric neurectomy to abolish the spinal noradrenergic component of gestational and HSP antinociception rules out the exclusive mediation via increased activity at spinal noradrenergic terminals and indicates the need for increased descending activity. Thus, it is likely that increased activity in descending noradrenergic pathways occurs during pregnancy and its hormonal simulation, resulting in an augmentation of spinal c~2-noradrenergic receptor activity. This underscores the relevance of pontine nuclei to the processing of pelvic afferent stimuli and the endogenous regulation of spinal K/3 antinociceptive systems.

Putative mechanisms underlying the antinociceptive consequences of the pregnancy blood profile of estrogen/progesterone
The mechanism(s) by which pregnancy levels of estrogen/progesterone enhance the lumbar spinal cord content of dynorphin A (1-17) and PC2 protein content and activate spinal ct2/opioid and /B antinociception is currently unknown. Collectively, the data suggest a peripheral (hypogastric nerve) as

93 diol and progesterone (Hammer and Bridges, 1987; Mateo et al., 1992). Additionally, estrogen has been shown to positively regulate proenkephalin mRNA expression in the ventrolateral aspect of the ventromedial hypothalamic nucleus within 1 h of its administration (Romano et al., 1989). Treatment with progesterone attenuates the rapid decline in hypothalamic levels of proenkephalin mRNA that occurs following the cessation of estrogen administration (Romano et al., 1988). It is tempting to speculate that an analogous ovarian steroid action occurs in the spinal cord of pregnant animals. tion of antinociceptive mechanisms that differ from those that underlie the analgesia that is characteristic of the gestational period. The onset of activation of each of these mechanisms would be temporally distinct but the analgesia that results would overlap and summate. Thus, during parturition, the analgesia that results from fetal stimulation of the uterine cervix and vagina would be in addition to the augmentation of descending spinal noradrenergic pathways and activation of spinal K/~ and c~2/opioid antinociceptive synergy that occurred in response to changes in circulating estrogen/progesterone. Following birth, ingestion of amniotic fluid/placenta potentiates opioid analgesia (Kristal et al., 1985, 1990), which would provide an additional increment of analgesia. Thus, as pregnancy progresses to parturition and birth there appears to be a crescendo of activated analgesic mechanisms.

Sequential activation and summation of multiple pain attenuating pathways

Pelvic afferent stimuli can also activate non-opioid antinociceptive processes. In non-pregnant rats and humans, vaginal and/or cervical mechanostimulation elevates pain thresholds (Komisaruk and Wallman, 1977; Cunningham et al., 1991; Komisaruk, 1991; Steinman et al., 1983) that is blocked by transection of the hypogastric and pelvic nerves (Cunningham et al., 1991). Moreover, direct mechanostimulation of the uterine cervix also produces analgesia. Uterocervical mechanostimulation, applied via a Silastic disc implanted in the uterus and abutted against the cervix, produced approximately a 2-fold increase in tail-flick latency that was naloxone-insensitive (Gintzler and Komisaruk, 1991). This antinociception, in contrast to that associated with pregnancy or HSP, was not altered by hypogastric neurectomy but could be abolished by transection of the pelvic nerve. Uterine mechanostimulation activates the hypogastric (Peters et al., 1987; Berkley et al., 1988; Robbins et al., 1990) but not the pelvic nerve (Peters et al., 1987) whereas vaginal mechanostimulation activates the pelvic (Komisaruk et al., 1972) but not the hypogastric nerve (Peters et al., 1987). Both of these nerves are activated by cervical stimulation (Komisaruk et al., 1972; Peters et al., 1987; Berkley et al., 1988). During late pregnancy the growing fetus(es) might stimulate the uterine horns more than the cervix and thus activate predominantly the hypogastric nerve. On the other hand, during labor (parturition) stimulation of the cervix/vagina, and therefore the pelvic nerve would predominate. Thus, the parturient period is also associated with activa-

Role of afferent input to nociceptive versus antinociceptive processes

Some of the above components that contribute to gestational and HSP antinociception highlight the dichotomous relationship between afferent input and nociceptive processing. In contrast to demonstrations that the hypogastric neuronal activity is critical to the antinociception of gestation and HSP (Gintzler et al., 1983; Liu and Gintzler, 1999) is the recent report that bilateral hypogastric neurectomy eliminates escape responses to uterine distention(Temple et al., 1999). These observations indicate that the hypogastric nerve activates nociceptive as well as antinociceptive mechanisms. This apparent inconsistency is mirrored by the fact that although neuropathic pain is relatively insensitive to opioids, (Arner and Meyerson, 1988; Portenoy et al., 1990; Dubner, 1991; Chemy et al., 1994) concomitant intrathecal administration of low doses of substance P and opioids significantly enhance antinociceptive consequences of the latter (Kream et al., 1993) and that spinal (K) opioids have been shown to have increased efficacy to attenuate hyperalgesia in rats subjected to chronic inflammation (Neil et al., 1986; Hylden et al., 1991). Collectively, these observations indicate that afferent input can play a dual role in the processing of nociceptive stimuli, the nature of which is determined by the type of afferent information transmitted. More-

94 over, the ability of the estrogen and progesterone to activate antinociception, dependent, in part, on hypogastric nerve activity, and the inability o f uterine distention to elicit escape responses in rats during proestrus and estrus (Bradshaw et al., 1999) demonstrate the importance of female-specific endocrine milieu to the influence of visceral (pelvic) afferent activity on nociceptive processing and its modulation by (endogenous) opioids. NTB P PC naltriben progesterone prohormone convertase

References
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Implications regarding gender-specific pain management

The finding that w o m e n are more likely than men to experience recurrent pain, more severe levels o f pain and pain of longer duration (Ellermeier and Westphal, 1995; Unruh, 1996; F i l l i g r a m et al., 1998), is seemingly inconsistent with the presence in female rats o f multiplicative spinal antinociceptive pathways that permit relatively small increases in the activity of one or more of its component processes to result in a disproportionately large decrease in pain sensitivity. It should be noted, however, that synergistic antinociceptive mechanisms also have reverse implications, i.e. a relatively minor impairment o f one or more contributing systems would result in an i n c o m m e n s u r a b l y large increase in pain perception. Thus, the relevance of gender-specific physiology to pain-coping mechanisms is underscored b y the phenomena o f gestational and H S P antinociception. Its underlying mechanisms could point the way to a rational understanding o f the gender specificity o f pain processing.

Abbreviations
alpha2-adrenergic receptor delta-opioid receptor k a p p a - o p i o i d receptor K m u - o p i o i d receptor Ix 7-benzylidenenaltrexone BNTX ix-receptor selective antagonist - - D-PheCTAP Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 estrogen E2 hormone-simulated pregnancy HSP mRNA messenger ribonucleic acid N M D A N-methyl-D-aspartate nor-BNI nor-Binaltorphimine (~2

95

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J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAFFER 7

Hypothalamic and limbic expression of CRF and vasopressin during lactation: implications for the control of ACTH secretion and stress hyporesponsiveness
Claire-Dominique Walker 1,,, Donna J. Toufexis 1 and Arlette Burlet 2
1 Douglas Hospital Research Center, Department of Psychiatry, McGill University, Montreal, Canada 2 INSERM U308, Universit~ Henri Poincare, Nancy, France

Abstract: Lactation is associated with physiological and behavioral changes that optimize conditions for development of the offspring. Although neuroendocrine and emotional stress responses are blunted, the central mechanisms involved are unclear. In addition to a reduction in stimulatory noradrenergic inputs to paraventricular nucleus (PVN) neurons, we demonstrate that lactation induces: (1) unique phenotypic changes in neuropeptide expression by hypothalamic PVN neurons (reduced expression of corticotropin-releasing factor (CRF) mRNA and increased expression of vasopressin mRNA in parvocellular PVN neurons); and (2) changes in pituitary sensitivity to CRF (reduced) and vasopressin (increased) as a consequence of differential CRF/vasopressin secretion into the hypophysial portal blood. Neurons in the bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA) that are implicated in the control of the hypothalamopituitary-adrenal axis also display changes in lactation: expression of CRF mRNA in the CeA is reduced, consistent with the diminished responsiveness to acoustic startle observed in nursing mothers. In contrast, expression of CRF mRNA is increased in the dorsolateral portion of the BNST, probably because of the tonic increases in endogenous glucocorticoid production during this period. Using immuno-targeted lesions of CRF or vasopressin in the PVN of virgin females, we have shown that CRF neurons of the PVN send inhibitory projections to the dorsolateral portion of the BNST and stimulatory inputs to CRF neurons in the CeA. Thus, it is possible that lactation-induced changes in the activity of parvocellular PVN neurons might also modulate the expression of neuropeptides and neurotransmitters in the BNST and the amygdala.

Introduction

In several species, lactation is associated with multiple changes in behavior (Numan, 1983), metabolic regulation (Stem and Levine, 1974), morphological plasticity (Montagnese et al., 1987) and neuroendocrine functions (Lightman, 1992) that are directed towards the maintenance of maternal behavior favoring optimal development of the young. In particular, *Corresponding author: Claire-Dominique Walker, Department of Psychiatry, McGill University, Douglas Hospital Research Center, 6875 Lasalle Blvd, Verdun, PQ H4H 1R3, Canada. Tel.: +1-514-761-6131, ext. 24934; Fax: + 1-514-762-3034; E-mail: waldom@douglas.mcgill.ca

lactation represents one of the few physiological conditions where neuroendocrine stress responses are blunted as demonstrated both in rodents and humans. Indeed studies described in this volume (see Carter et al., 2001, this volume) demonstrate that breast-feeding women exhibit decreased plasma corticotropin (ACTH), cortisol, vasopressin, epinephrine and glucose responses to treadmill stress when compared with bottle-feeding mothers (Altemus et al., 1995). Numerous studies in rodents have documented a decrease in stress-induced plasma oxytocin (Carter and Lightman, 1987; Higuchi et al., 1988; Neumann et al., 1993a), catecholamines, prolactin (Higuchi et al., 1989), ACTH and corticosterone (Stern et al., 1973; Walker et al., 1992; Windle et al., 1997) secretion during lactation. Similarly, a decrease in

100 stress-induced corticotropin releasing factor (CRF) (Lightman and Young, 1989) and c-fos (DaCosta et al., 1996) mRNA levels in neurons of the hypothalamic paraventricular nucleus (PVN) has been demonstrated during lactation, suggesting that CRF neurons controlling secretion of the pituitary-adrenocortical axis might be less responsive to stressful inputs. Reduced neuronal activation has also been demonstrated in extra-hypothalamic areas implicated in the integrated stress circuitry like the medial amygdala after restraint stress (see Lightman et al., 2001, this volume) and the hippocampus or cortex after N-methyl-D,L-aspartic acid (NMA) injection (Abbud et al., 1993, 1994). Behaviorally, this reduced response to stressors is manifested in a greater ambulation in an open field, reduced freezing responses to loud noise (Fleming and Leubke, 1981; Hard and Hansen, 1985) and lower startle responses (Toufexis et al., 1998). In fact, parturient and lactating females display an increased sensitivity and attraction to pup-related stimuli accompanied by reduced timidity and neophobia towards their pups (Fleming et al., 1989; Stem, 1989). Both the intensity and frequency of suckling regulate neuroendocrine and behavioral changes since litter removal restores most functions to normal with various time courses. For instance, following stress, CRF mRNA levels (Lightman, 1992) and ACTH secretion (Walker, 1995) appear to be normalized within 2-3 days after pup removal, whilst normalization of the fast component of glucocorticoid feedback appears to take up to 2 weeks (see Lightman et al., 2001, this volume). These different time courses of normalization are strongly suggestive of several different separate, or interacting mechanisms involved in maintenance of the stress hyporesponsiveness of lactation. At present several potential mechanisms have been considered including the role of central noradrenergic activity (Toufexis and Walker, 1996; Toufexis et al., 1998), central prolactin and oxytocin release (see Neumann, 2001, this volume), opioid inhibition (see Douglas and Russell, 2001, this volume), as well as changes in the efficacy of glucocorticoid feedback to reduce the activity of the hypothalamus-pituitary-adrenal (HPA) axis (Walker et al., 1992, 1995). Although much effort has been devoted to delineate afferent inputs modifying the activity of CRF neurons in the PVN during lactation, few studies have investigated whether intrinsic properties of these hypothalamic PVN neurons such as their phenotype are modified during this period and how these changes could relate to the control of pituitary ACTH secretion. In this chapter, we will focus our attention on lactation-induced changes in neuropeptide expression occurring within CRF neurons of the hypothalamic PVN, we will investigate CRF expression within parts of the limbic system implicated in the control of the HPA axis such as the bed nucleus of the stria terminalis (BNST) (Herman et al., 1994) and the amygdala (LeDoux, 1994), and attempt to relate these changes to the functionality of the HPA axis in lactation. One recent report demonstrated that diminished ACTH responses to stress in late pregnant females was associated with reduced pituitary responsiveness to CRF (Neumann et al., 1998). This observation suggests that pituitary modifications induced by changes in secretagogue release or glucocorticoid feedback during late pregnancy and lactation could also reduce ACTH secretion following a stressor. Indeed, we have found that the importance of vasopressin as a secretagogue is increased in lactating females (Toufexis et al., 1999a). Therefore we will discuss the consequences of phenotypic changes in hypothalamic neurons on pituitary ACTH secretion and responsiveness to secretagogues like CRF and vasopressin.

Experimentalapproaches
These studies compare Sprague-Dawley virgin females with lactating females in mid-lactation (days 10-14 after delivery). The synthetic capacity of hypothalamic and extra-hypothalamic neurons was investigated using both immunohistochemical detection of CRF and vasopressin and determination by in situ hybridization histochemistry of changes in mRNA levels. These procedures for CRF and vasopressin have been described in detail in our previous publications (Walker et al., 1997). In order to determine the functional role of specific neuronal populations within the PVN and their influence on other neighboring neurons or related structures, specific immuno-targeted lesions were used in the PVN as described previously (Walker et al., 1997). Briefly, monoclonal antibodies to CRF or vasopressin were associated non-covalently to purified ricin A chain

101 and monensin. These were then injected acutely over the PVN in virgin or lactating females in early lactation (day 2); CRF-ricin A is expected to enter CRF neurons and kill them, and similarly vasopressinricin A conjugate enters and destroys vasopressin neurons. CRF neurons will be damaged by the toxinvasopressin antibody conjugate if they express vasopressin, and their number will be reduced, and vice versa for vasopressin neurons expressing CRF and the toxin-CRF antibody. Control groups were injected with identical concentrations of ricin A and monensin mixed with non-specific rat immunoglobin type G (IgG). Rats were killed 12 days after the central lesion, i.e. on day 14 of lactation, and their brains processed for either in situ hybridization or immunohistochemical detection of neuropeptides in various brain regions, 24 h after central treatment with colchicine (Walker et al., 2001). To investigate pituitary responses to CRF and vasopressin, virgin or lactating females were fitted with jugular catheters. Two days later (on day 10-12 of lactation), they received an acute intravenous bolus injection of varying doses of CRF or vasopressin. Plasma ACTH concentrations were determined by specific radioimmunoassay at various times after neuropeptide injection as described previously (Toufexis et al., 1999b). duction during chronic stress also appear to be associated with an increased production and release of vasopressin from PVN neurons without concomitant changes in CRF secretion (De Goeij et al., 1991, 1992; Aguilera, 1994). During lactation, averaged dally glucocorticoid secretion is elevated above the levels seen in virgin females, mainly because nadir concentrations of corticosterone are elevated (Walker et al., 1992); this is analogous with many situations of chronic stress (Dallman et al., 1992). Consequently it has been shown that the expression of CRF in PVN neurons is reduced in mid-lactating females both in terms of the number of neurons expressing CRF (hybridized surface of sections) and of the synthetic capacity of each neuron (intensity of labeling) (Lightman and Young, 1989). CRF immunoreactivity within PVN neurons of lactating females under colchicine treatment is strongly diminished when compared to virgins, but the median eminence immunoreactivity is not altered. In contrast, parvocellular PVN (pPVN) expression of vasopressin is significantly increased in lactating females (Windle et al., 1997), suggesting that a greater number of neurons in this CRF-rich region express the vasopressin transcript and that each neuron is probably synthesizing more of the neuropeptide (increased labeling intensity) (Walker et al., 2001). Indeed, this was confirmed by our double immunohistochemical experiments showing that CRF and vasopressin colocalization in pPVN neurons, fibers and median eminence terminals is significantly enhanced in mid-lactating when compared with virgin females (Walker et al., 2001). At the level of the median eminence, the major difference between lactating and virgin females appeared in the number of vasopressin immunoreactive fibers in the external zone of the median eminence, which were numerous in lactating females, but always rare in virgins (Fig. 1). Terminals in the median eminence immunoreactive for CRF were not significantly modified between virgin and lactating females (data not shown). Phenotypic changes observed in pPVN neurons during lactation are unusual in that they demonstrate opposite regulation of CRF and vasopressin synthesis in the face of elevated basal glucocorticoid secretion and intact (Walker et al., 1992) or reduced feedback sensitivity (see Lightman et al., 2001, this

Phenotypic changes in PVN neurons during lactation

Neurons of the hypothalamic PVN are well known to synthesize several neuropeptides including CRF, vasopressin, oxytocin, neurotensin, enkephalins and cholecystokinin (Kiss et al., 1991; Watts, 1996). The magnitude of neuropeptide expression and colocalization within PVN neurons and in axon terminals in the median eminence depends on specific physiological conditions. For instance, following removal of endogenous glucocorticoids by surgical or pharmacological adrenalectomy, the production of vasopressin in parvocellular CRF neurons is increased (Antoni, 1993), the vasopressin: CRF ratio in the hypophysial portal circulation is augmented (Plotsky and Sawchenko, 1987) and more vasopressinimmunoreactive vesicles containing CRF are found in the external zone of the median eminence (Whitnall, 1993). Tonic elevations of glucocorticoid pro-

102

Fig. 1. Immunohistochemicaldetection of vasopressin in the median eminence of virgin (left) and lactating (right) females on day 14 of lactation. Brains were collected 24 h after colchicine treatment and fixed with 4% paraformaldehydeand picric acid. Lactating females display a greater density of immunoreactivevasopressin terminals in the external zone of the median eminence compared to virgins. Arrows also indicate a greater colocalizationof vasopressin within CRF terminals in nursing females. IT: infundibulartract; NT: neurohypophysialtract; V: third ventricle. volume). Thus, it appears that tonic elevation of glucocorticoid secretion in suckling females is efficient in reducing CRF expression, but does not prevent an increased expression of vasopressin in parvocellular neurons of the PVN. This result suggests that other central inputs that are activated by the suckling stimulus are able to supersede glucocorticoid feedback and specifically activate vasopressin expression in all PVN neurons (parvocellular and magnocellular). In fact, comparable changes in the relative expression of CRF and vasopressin heteronuclear RNA and mRNA was recently demonstrated in male rats following repeated restraint stress (Ma et al., 1997), emphasizing the extraordinary capacity of PVN neurons to adjust to varying physiological demands in specific ways. Interestingly, in addition to the changes described for the CRF/vasopressin system, other hypothalamic neuropeptides implicated in the control of the HPA axis also exhibit some plasticity during lactation. Examples of these include the well characterized modifications in the magnocellular oxytocin system described by Montagnese et al. (1987) and Theodosis and Poulain (2001, this volume), which allow for increased synaptic contacts and synchronization of firing activity. In addition, there is a suckling-induced expression of neuropeptide Y (NPY) mRNA in magnocellular neurons of the PVN (Burlet and Walker, unpublished) in addition to the well documented increase in expression in the arcuate nucleus (Smith, 1993). Thus, it is conceivable that suckling-induced changes in hormonal milieu and neuronal activity of brain afferents to the PVN favor the differential expression and release of several PVN neuropeptides implicated in the control of HPA activity and that normal activity seen in nonlactating females is reset in lactation. Examples of biochemical switching in hypothalamic circuits mediating stress responses have been documented with changes in steroid concentrations (glucocorticoids and sex steroids) and neural inputs to pPVN neurons (Swanson, 1991). As a consequence of differential hypothalamic hypophysial secretion and/or altered steroid secretion in lactating females, it is reasonable to postulate that changes in pituitary sensitivity to ACTH secretagogues might also contribute to the stress hyporesponsiveness of lactation.

Pituitary sensitivity to CRF and vasopressin is altered in lactating females

In rodents, CRF represents the major regulator of pituitary ACTH secretion (Antoni, 1993), and al-

103 Pituitary CRF challenge

2500 ~ Veh , CRF-R 60 ~ 2500

Pituitary AVP challenge

[---] Veh m AVP 0.5 ug Vlb-R 1500 ~"

2000
1500 1000

5.o ug :
'

40 o
20 ~

~ 2000
~ ~ < 1500

: , :
,

~5
1000

1000

500
0

j
V L

'
'

~ VL
0 ~

500
0

j
V L V L 500

Fig. 2. Changes in ACTH secretion in virgin (V) or lactating (L) females in mid-lactation (days 10-12) following an acute i.v. injection of vehicle, CRF (5.0 [zg/rat, left) or vasopressin (AVP; 0.5 txg/rat, right). Change (delta) in secretion was calculated as the difference between peak ACTH secretion at 5 min after injection and baseline concentrations. On the right of each graph, autoradiographic determination of pituitary receptor levels is shown. CRF-R: CRF-receptor; VIb-R: vasopressin lb-receptor. Redrawn from Toufexis et al., 1999b with permission. Values represent group sizes of 4-7. **P < 0.01 compared with virgins (Student's t-test).

though vasopressin is only a weak ACTH secretagogue it potentiates the stimulatory effect of CRF on pituitary corticotropes (Gillies et al., 1982). While secretion of both CRF and vasopressin from parvocellular neurons in the pPVN is required to elicit a full ACTH secretory response to an acute stressor (Linton et al., 1985), maintenance of tonically elevated activity within the HPA axis appears to require one or a combination of the following mechanisms: (a) an increased vasopressin:CRF ratio in the hypophysial portal circulation as described following adrenalectomy or chronic stress (Plotsky, 1991); and (b) changes in the number of CRF and/or vasopressin pituitary receptors as well as modifications in their coupling efficiency to second messenger systems (Wynn et al., 1985; Rabadan-Diehl et al., 1995). Concentrations of CRF and vasopressin in the hypophysial portal circulation have not been directly determined during lactation, but indirect evidence from our in situ hybridization and immunohistochemical studies suggests that the anterior pituitary of the lactating mother is exposed to a greater vasopressin : CRF ratio than under non-suckling conditions. This increase in vasopressin release might be specific to the anterior pituitary since earlier studies have failed to detect significant changes in peripheral plasma levels of vasopressin between lactating and non-lactating females (Fischer et al., 1995) and microdialysis studies conducted from the last 3 days of

gestation through day 10 of lactation failed to show any elevation in intranuclear vasopressin release in either the supraoptic or paraventricular nuclei (Neumann et al., 1993b). We recently demonstrated that potential changes in the vasopressin : CRF ratio during lactation induced a shift in pituitary sensitivity towards vasopressin and away from CRF (Toufexis et al., 1999b). When challenged acutely with exogenous CRF, lactating females displayed reduced ACTH responses, in particular with high, stress-level concentrations of CRF. Since pituitary CRF receptors were not altered in lactating females, we concluded that mechanisms distal to the receptor were likely to mediate the diminished pituitary responsiveness to CRF in these females (Fig. 2, left). In fact, these results are in complete agreement with a recent study conducted in late pregnant females, which showed that reduced pituitary responses to stress or CRF were accompanied by reductions in pituitary adenosine 3', 5'-cyclic monophosphate (cAMP) production (Neumann et al., 1998). In contrast to the blunted pituitary sensitivity to CRF, the pituitary ACTH response to a high dose of vasopressin was enhanced in lactating rats when compared with virgin females, again with no concomitant changes in pituitary vasopressin receptors (Fig. 2, right). Although hypophysial portal concentrations of endogenous vasopressin might be lower than those produced by systemic intravenous injection, the shift

104 to greater pituitary responsiveness to high doses of vasopressin in nursing compared with virgin females might represent a counter-regulatory mechanism ensuring that in the absence of adequate CRF secretion, nursing females could still exhibit robust responses to certain types of stressors. Finally, it is possible that in addition to vasopressin, increased secretion of oxytocin in the hypophysial portal circulation of lactating females would potentiate the effect of reduced CRF secretion and thus participate in the maintenance of adequate basal and stimulated ACTH secretion during lactation. have also been demonstrated recently by retrograde and anterograde tract tracing methods. In lactating females, several behavioral and biochemical experiments suggest that, in addition to changes in the activity of the neuroendocrine (HPA) stress axis, activation of neuronal stress circuits including the amygdala might be blunted. Indeed, using the startle reflex as an indicator of amygdalar recruitment (Davis, 1992), we previously demonstrated that lactating females showed reduced startle responses when compared with virgin females. In this paradigm, fear conditioning prior to the startle test was able to restore similar startle responses in the two groups, indicating that the fear component of emotional responses was unaltered by lactation (Toufexis et al., 1999b). Others have reported a decreased expression of c-fos mRNA in the PVN and amygdala following stress in lactating females (DaCosta et al., 1996; Woodside and Amir, 1997), suggesting that stress hyporesponsiveness might be associated with a reduction in neuronal activation of both structures. The central nucleus of the amygdala as well as the BNST are rich in CRF-containing neurons that have been suggested to play a significant role in mediating the behavioral stress responses (Gray, 1990). Our studies sought to determine whether lactation modified the basal expression of CRF in the BNST and in the CeA and whether specific lesions of CRF or vasopressin neurons of the PVN could produce discrete changes in the pattern of CRF expression in females. As shown in Fig. 3, CRF mRNA expression in the BNST was significantly enhanced in intact lactating compared to virgin females, but only in the dorsolateral portion of the nucleus. Expression of CRF mRNA in the ventral portion of this nucleus was not modified by lactation. In the dorsolateral region, increased surface of hybridization for CRF mRNA suggests that additional neurons were recruited to express CRF mRNA in lactating females and these neurons were slightly more active in CRF synthesis. Similarly, CRF immunoreactive neurons detected by immunohistochemistry in colchicinetreated females were more abundant in the BNST of lactating compared to virgin females (Fig. 4, left panel). In agreement with previous studies in male rats showing that high dose corticosterone injections for 2 weeks significantly increase CRF mRNA in the dorsolateral, but not ventral part of the BNST

Basal and reactive changes in limbic CRF neuron populations (BNST and CeA) during lactation
Undisputedly, CRF-containing neurons in the pPVN represent the primary convergent site of control for the activity of the HPA axis. However, these neurons receive numerous afferent inputs from brain stem neurons, limbic and cortical structures as well as intrahypothalamic and intranuclear neurons, which significantly modify their activity (Swanson, 1991). In particular, afferents arising in the telencephalon (medial prefrontal cortex, ventral parts of the hippocampus and the amygdala), which are primarily concerned with cognitive influences on the HPA axis, use the bed nucleus of the stria terminalis (BNST) as an important relay to pPVN neurons (Swanson, 1991). Various portions of the BNST can differentially affect CRF expression in the pPVN as elegantly demonstrated in earlier studies (Herman et al., 1994). The anterior/anterolateral portion of the BNST provides a tonic stimulatory input to pPVN CRF neurons. The lateral divisions of the BNST are functionally related to the central nucleus of the amygdala (CeA) and may relay the excitatory influence of this structure on pPVN CRF neurons (Beaulieu et al., 1986). In contrast, more dorsomedial regions of the BNST integrate excitatory signals from limbic structures and translate them into gamma-amino butyric acid (GABA)ergic inhibitory outputs to the PVN (Herman et al., 1996). Finally, the dorsolateral region appears to project multisynaptically to pPVN CRF neurons via neurons in close proximity to parvocellular neurons. Projections from the PVN to the BNST (CRF-ergic) (Champagne et al., 1998) and the amygdala (Palkovits et al., 1998)

105 CRF mRNA in the BNST

I ] Virgin m Lactating

~-,

18 15 =

] 2500 2000 1500 1000 "=-O 500

Dorsolat Ventral Dorsolat Ventral

"~ o No ~,--, 6 3 0

Region Fig. 3. Changes in CRF mRNA levels in the dorsolateral and ventral regions of the BNST between virgin (open bars) and lactating females in mid-lactation(dark bars). Hybridizedsurface of sections is depicted on the left panel and the intensity of labeling (grain counts) is depicted on the right panel. Values represent the mean i SEM of 24-28 determinationsper group using 4-6 animals per group. ***P < 0.001 comparedto virgins Student's t-test).

Makino et al., 1994), our results suggest that lactation-induced changes in the expression of CRF in the BNST might be mediated by tonic, long-term increases in endogenous glucocorticoid production. In contrast to the BNST, basal CRF mRNA expression in the CeA was reduced in mid-lactating females, both in terms of hybridized surface and grain intensity (Fig. 5). It is noteworthy that a lactation-induced decrease in CRF mRNA expression in this structure occurs despite tonically elevated glucocorticoid secretion, which has been reported to increase CRF mRNA in the CeA of male rats (Makino et al., 1994). This result suggests therefore that regulation of CRF expression in the CeA during lactation is not directly dependent upon glucocorticoid secretion, but might be regulated by changes in the activity of afferent inputs to this structure (Swanson and Petrovich, 1998). Interestingly, a large portion of afferent inputs originate from the main olfactory system and brainstem areas mediating autonomic regulation as well as sensory processing. Since olfactory and somatosensory inputs are so important to maintain endocrine and behavioral adaptations of lactation, it is possible that these specific inputs to the CeA play a critical role in reducing amygdalar

CRF production in nursing females. Although CRF mRNA was reduced in the CeA of lactating females, we did not observe significant modifications of CRF immunoreactivity in this structure between female groups (Fig. 4, right). Although the BNST sends direct projections to pPVN neurons, inputs from the amygdala are indirect and primarily relayed through the BNST. As there are projections from the PVN to the BNST (Champagne et al., 1998) and the amygdala (Palkovits et al., 1998), changes in the activity of pPVN neurons, such as these observed during lactation, could feedback on the BNST and amygdala to modify the expression of neuropeptides and neurotransmitters in these limbic regions. Most studies investigating the effects of PVN lesions have used large lesions, damaging numerous different cell types and thus preventing a more refined analysis of the source and nature of PVN inputs to the BNST and the amygdala. To circumvent this problem in our experiments, selective targeting of either CRF or vasopressin neurons in the PVN of virgin females was used to produce selective and restricted lesions of this nucleus (using ricin A-antibody conjugates, see above; Walker et al., 2001). Since virgin females do not exhibit a large degree of CRF and vasopressin colocalization compared to lactating females, we used this model to first identify a differential effect of CRF and vasopressin PVN populations on expression of CRF in the BNST and the amygdala. Twelve days after injection of the ricin A (Tx)-antibody conjugate to induce lesions in the PVN, we observed a large and significant increase in CRF mRNA levels in the dorsolateral, but not ventral portion of the BNST following lesion of PVN CRF neurons (CRFTx) compared to controls injected with non-specific immunoglobulins (IgG-Tx) (Fig. 6). Interestingly, in marked contrast with the CRF lesion, selective lesion of vasopressin neurons (vasopressin-Tx) of the PVN did not alter CRF expression in either region of the BNST. This differential effect of the lesions cannot be explained by changes in their efficacy since for both CRF and vasopressin lesions, reductions in CRF and vasopressin mRNA in the PVN was in the range of 20-50% for each targeted peptide (Walker et al., 2001). Our data suggest therefore that in virgin females, CRF neurons, but not vasopressin neurons of the PVN exert an inhibitory influence on the ac-

106

Fig. 4. Immunohistochemical detection of CRF in the BNST (left) and CeA (right) of virgin (top) or lactating (bottom) females in mid-lactation. Females were treated for 24 h with intra-cisternal colchicine prior to sacrifice and transcardiac perfusion with 4% paraformaldehyde. Note the increase in immunoreacfive CRF neurons in the BNST of lactating females compared to virgins.

107 CRF m R N A in the a m y g d a l a 16 _[----qVirgin Ii

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Lactating

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2

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Fig. 5. Changes in CRF mRNA levels in the central nucleus of the amygdala (CeA) between virgin (open bars) and lactating females in mid-lactation (dark bars). Hybridized surface is depicted on the left panel and the intensity of labeling (grain counts) is depicted on the right panel. Values represent the mean 4- SEM of 27-85 determinations per group using 4-8 animals per group. ***P < 0.001 compared to virgins (Student's t-test).

2ooldi d1
200 150 100 150 100 5O 0 50 0 Dorsolat Ventral Dorsolat Ventral Region CRF mRNA in the CeA Virgins IgG-Tx [ ~ AVP-TxI CRF-Tx 150 100

250

~"

.~

tivity of C R F containing neurons in the BNST. This inhibitory input appears to be restricted to the dorsolateral portion o f the BNST. In view of the increased expression of C R F in the B N S T o f lactating compared to virgin females (see above), it is interesting to speculate that this inhibitory CRF-ergic input to the dorsolateral B N S T might be reduced in lactation. However, further comparison o f the effects o f specific P V N lesions on the B N S T need to be performed in lactating females. In contrast to our observation in the BNST, lesion of C R F neurons in the P V N significantly reduced the expression of C R F in the C e A c o m p a r e d with animals receiving the sham lesion ( I g G - T x injection) (Fig. 7). In this structure, there was a trend towards a reduction in C R F expression also with the lesion targeted to vasopressin neurons. These results are opposite to the marked up-regulation of amygdalar C R F m R N A reported previously in male rats 6 weeks after complete bilateral lesion o f the P V N (Palkovits et al., 1998). It is possible that differences in the extent of the lesion, length of interval between lesion and tissue collection as well as hormonal factors might have contributed to these discrepant results. Clearly, more studies need to be performed to resolve these issues. However, our present results support an excitatory role of C R F neurons of the

Fig. 6. Expression of CRF mRNA in the dorsolateral and ventral region of the BNST in virgin females following immuno-targeted lesion of the PVN. Specific targeting was carried out against vasopressin neurons (by local injection of ricin A chainmonensin-vasopressin monoclonal antibody conjugate: AVP-Tx, hatched bars) or CRF neurons (ricin A chain-monensin-CRF monoclonal antibody conjugate: CRF-Tx, filled bars). Controls were injected with non-targeted immunoglobulin (IgG-Tx, open bars). Lesions were made 12 days prior to brain collection. Values represent the mean 4- SEM of 14-21 determinations per group using 4-6 animals per group. ***P < 0.001, **P < 0.01 compared to sham lesion with IgG-Tx (Student's t-test).

~'~ 150

lOO

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~q e'~

50

50

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0 Grains Surface Fig. 7. Expression of CRF mRNA in the central nucleus of the amygdala (CeA) in virgin females following immuno-targeted lesion of the PVN. Specific targeting (see Fig. 6) was carried out against vasopressin neurons (AVP-Tx, hatched bars) or CRF neurons (CRF-Tx, filled bars). Controls were injected with nontargeted immunoglobulin (IgG-Tx, open bars). Lesions were administered 12 days prior to brain collection. Values represent the mean 4- SEM of 24-29 determinations per group using 4-6 animals per group. ***P < 0.001, *P < 0.05 compared to sham lesion with IgG-Tx (Student's t-test). 0

108

PVN directed towards neurons of the CeA containing CRF. The comparable decline in CRF mRNA observed in lactating compared to virgin females suggests that the reduced activity of PVN CRF neurons in lactation contributes to limit the expression of CRF in the central nucleus of the amygdala. The relative importance of this feedback system in comparison with the feed forward input of the CeA to the PVN via the BNST remains to be determined.

cAMP CeA CRF GABA HPA IgG pPVN PVN Tx

adenosine 3', 5'-cyclic monophosphate central nucleus of the amygdala corticotropin-releasing factor gamma-amino butyric acid hypothalamus-pituitary-adrenal immunoglobin type G parvocellular PVN paraventricular nucleus ricin A

Conclusions
The stress hyporesponsiveness of the HPA axis in lactation is only one aspect of the many changes in neuronal systems and peripheral regulatory mechanisms that take place in females nursing their infants. However, modifications in this particular stress-response system probably prevent aberrant maternal behavior and ensure that relevant attention is dedicated to the young. As with other neuroendocrine systems, regulation of stress responsiveness is intimately linked to behavior, metabolism and perception of a threat by the individual. In this chapter, we have attempted to provide examples on how phenotypic plasticity in several key structures of the brain could lead to changes in secretory patterns and modify the responses of target glands (i.e. the pituitary). We have focused our discussion on the CRF/vasopressin system, but this is not to imply that other neuropeptides, endogenous opioids or even neurotransmitters do not have a critical role to play in the regulation of stress systems. As seen by the integration of the various chapters of this volume, many efforts are converging to unravel the specific mechanisms controlling changes in homeostasis occurring in pregnancy and lactation. One of the fascinating aspects of this research that we need to keep in mind is that all of these changes are adaptive and probably uniquely directed towards ensuring optimal and harmonious development of the young. This is an incredible expenditure of energy for a tremendously important outcome, one that should not suffer from the consequences of disturbing the nursing mother's physiology.

Acknowledgements
This work was supported by a grant from the Medical Research Council of Canada to C.D.W. and a collaborative grant from the INSERM-FRSQ to A.B. and C.D.W.

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during gestation, post-partum lactation and suckling-induced pseudogestation and lactation. J. Endocrinol., 115: 97-105. Neumann, I., Ludwig, M., Engelmann, M., Pittman, Q.J. and Landgraf, R. (1993a) Simultaneous microdialysis in blood and brain: oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat. Neuroendocrinology, 58: 637-645. Neumann, I., Russell, J.A. and Landgraf, R. (1993b) Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient, and lactating rats. Neuroscience, 53: 65-75. Neumann, I., Johnstone, H., Hatzinger, M., Liebsch, G., Shipston, M., Russell, J.A., Landgraf, R. and Douglas, A.J. (1998) Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J. Physiol., 508: 289-300. Numan, M. (1983) Brain mechanisms of maternal behavior in the rat. In: J. Balthazart, E. Prove and R. Gilles (Eds.), Hormones and Behavior in Higher Vertebrates. Springer-Verlag, Berlin, pp. 69-85. Palkovits, M., Young III, W.S., Kovacs, K., Toth, Z. and Makara, G.B. (1998) Alterations in corticotropin-releasing hormone gene expression of central amygdaloid neurons following long-term paraventricular lesions and adrenalectomy. Neuroscience, 85: 135-147. Plotsky, EM. (1991) Pathways to the secretion of adrenocorticotropin: a view from the portal. J. Neuroendocrinol., 3: 1-9. Plotsky, P.M. and Sawchenko, P.E. (1987) Hypophyseal-portal plasma levels, median eminence content, and immunohistochemical staining of corticotropin-releasing factor, arginine vasopressin, and oxytocin after pharmacological adrenalectomy. Endocrinology, 120: 1361-1369. Rabadan-Diehl, C., Lolait, S.J. and Aguilera, G. (1995) Regulation of pituitary vasopressin Vlb receptor mRNA during stress in rat. J. NeuroendocrinoL, 7: 903-910. Smith, S.M. (1993) Lactation alters neuropeptide Y and proopiomelanocortin gene expression in the arcuate nucleus of the rat. Endocrinology, 133: 1258-1265. Stern, J.M. (1989) Maternal behavior: sensory, hormonal and neural determinants. In: ER. Brush and S. Levine (Eds.), Psychoendocrinology. Academic Press, New York, pp. 105-226. Stern, J.M., Goldman, L. and Levine, S. (1973) Pituitary-adrenal responsiveness during lactation in rats. Neuroendocrinology, 12: 179-191. Stern, J.M. and Levine, S. (1974) Psychobiological aspects of lactation in rats. Prog. Brain Res., 41: 433-444. Swanson, L.W. (1991) Biochemical switching in hypothalamic circuits mediating responses to stress. Prog. Brain Res., 87: 181-200. Swanson, L.W. and Petrovich, G.D. (1998) What is the amygdala? Trends Neurosci., 21: 323-331. Theodosis, D.T. and Poulain, D.A. (2001) Maternity leads to morphological synaptic plasticity in the oxytocin system. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingram (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier,

Amsterdam, pp. 49-58. Toufexis, D.J. and Walker, C.-D. (1996) Noradrenergic facilitation of the adrenocorticotropin response to stress is absent during lactation in the rat. Brain Res., 737: 71-77. Toufexis, D.J., Thrivikraman, K.V., Plotsky, P.M., Morilak, D.A., Huang, N. and Walker, C.-D. (1998) Reduced noradrenergic tone to the hypothalamic paraventricular nucleus contributes to the stress hyporesponsiveness of lactation. J. Neuroendocrinol., 10: 417-427. Toufexis, D.J., Rochford, J. and Walker, C.-D. (1999a) Reduction in the acoustic startle response in lactating rats. Behav. Neurosci., 113: 176-184. Toufexis, D.J., Tesolin, S., Huang, N. and Walker, C.-D. (1999b) Altered pituitary sensitivity to corticotropin releasing factor and arginine vasopressin participates in the stress hyporesponsiveness of lactation. J. NeuroendocrinoL, 11 : 757-764. Watts, A. (1996) The impact of physiological stimuli on the expression of corticotropin-releasing hormone (CRH) and other neuropeptide genes. Front. Neuroendocrinol., 17: 281-326. Walker, C.-D. (1995) Modifications neuroendocriniennes relires l'activit6 de l'axe corticotrope au cours de la lactation chez le rat. Ann. Endocrinol. (Paris), 56: 169-172. Walker, C.-D., Lightman, S.L., Steele, M.K. and Dallman, M.F. (1992) Suckling is a persistent stimulus to the adrenocortical system of the rat. Endocrinology, 130: 115-125. Walker, C.-D., Trottier, G., Rochford, J. and Lavallre, D. (1995) Lactation-induced changes in behavioral and hormonal stress responses to the forced swim test in rats. J. Neuroendocrinol., 7: 615-622. Walker, C.-D., Tankosic, P., Tilders, F.J.H. and Burlet, A. (1997) Immunotargeted lesions of paraventricular CRF and vasopressin neurons in developing rats reveal the pattern of maturation of these systems and their functional importance. J. Neuroendocrinol., 9: 25-41. Walker, C.-D., Tilders, EJ.H. and Burlet, A. (2001) Increased colocalization of CRF and vasopressin in paraventricular neurons of the hypothalamus in lactating rats: evidence from immunotargeted lesions and immunohistochemistry. J. Neuroendocrinol., 13: 74-85. Whitnall, M.H. (1993) Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog. Neurobiol., 40: 573-629. Windle, R.J., Wood, S., Shanks, N., Perks, P., Condue, G.L., da Costa, A.P.C., Ingram, C.D. and Lightman, S.L. (1997) Endocrine and behavioural responses to noise stress: comparison of virgin and lactating female rats during non-disrupted maternal activity. J. Neuroendocrinol., 9: 407-414. Wynn, P.C., Harwood, J.P., Catt, K.J. and Aguilera, G. (1985) Regulation of corticotropin releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses. Endocrinology, 116: 1653-1659. Woodside, B. and Amir, S. (1997) Lactation reduces Fos induction in the paraventricular and supraoptic nuclei of the hypothalamus after urethane administration in rats. Brain Res., 752: 319-323.

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 8

Peripartum plasticity within the hypothalamo-pituitary-adrenal axis

Stafford L. Lightman

1 Richard J. Windle 1,3, Susan A. Wood 1 Yvonne M. Kershaw 1

Nola Shanks 1 and Colin D. Ingram 1,2,*

I University Research Centre for Neuroendocrinology, Dorothy Crowfoot Hodgkin Laboratories, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK 2 School of Neuroscience and Psychiatry, University of Newcastle, Leazes Wing, Royal Victoria Infirmary, Newcastle NE1 4LP, UK 3 School of Nursing and Midwifery, University of Nottingham, Lincoln Education Centre, Lincoln County Hospital, Greetwell Road, Lincoln LN2 5QY, UK

Abstract: The hypothalamo-pituitary-adrenal (HPA) axis plays important roles in the adaptive changes in physiology that occur during pregnancy and lactation. Although the axis still exhibits a pulsatile pattern of secretion, the normal diurnal rhythm of pulse amplitude is lost during lactation, such that mean basal levels remain constant throughout the day. In addition, the peripartum period is associated with a remarkable plasticity in stress-induced HPA activity, in that the increase of HPA activity normally seen in response to either physical or psychological stresses in the non-reproductive state become severely attenuated or absent in the lactating animal. This stabilization of both basal and stress-induced HPA activity may be important for maintaining a constant endocrine environment, thereby preventing any programming effects on the developing offspring. Attenuation of the stress response is initiated in late pregnancy and is temporally associated with luteolysis, indicating possible steroid hormone involvement. Indeed, mimicking the luteolytic changes in oestrogen and progesterone levels in non-pregnant animals induces a similar attenuation of the stress response. Furthermore down-regulation of the stress response is, at least in part, centrally mediated since in the period following luteolysis rats will show a decreased level of stress-induced neuronal activation of the PVN, as measured by the expression of either c-fos or CRH mRNAs. Persistence of this adapted state is dependent upon the continued suckling stimulus, as removal of the offspring litter rapidly leads to resumption of HPA responses to and the appearance of an exaggerated diurnal rhythm. The underlying mechanisms responsible for this stress hyporesponsiveness may include plasticity of noradrenergic and oxytocin pathways. In view of its role in other reproductive behaviors, a stress-inhibiting effect of oxytocin may reflect a more widespread co-ordinating role in the peripartum animal.

Introduction Adrenal corticosteroids play an important role in determining the activity of a wide range of homeostatic processes, including those regulating metabolic rate

* Corresponding author: Colin D. Ingrain, School of Neuroscience and Psychiatry, University of Newcastle, Leazes Wing, Royal Victoria Infirmary, Newcastle NE1 4LP, UK. Tel.: +44-191-282-5678; Fax: +44-191-227-5108; E-mail: c.d.ingram@ncl.ac.uk

and immunological reactivity. The synthesis and release of corticosteroids are largely under the control of the hypothalamo-pituitary-adrenal (HPA) axis which, through the synergistic actions of hypothalamic corticotropin-releasing hormone (CRH) and vasopressin released from the paraventricular nucleus (PVN), regulates the release of adrenocorticotropin (ACTH) from the pituitary gland. The activity of this neuroendocrine axis occurs both as diurnally regulated basal secretion and as part of the coordinated response to physical or psychological stimuli which are perceived as stressful. Various mechanisms exist

112 to ensure that both these modes of activity remain tightly regulated, since dysregulation of the HPA axis leads to widespread disruption of normal patterns of gene transcription, metabolism, renal, cardiovascular and immune function, and psychomotor activity. The HPA axis has a particular significance to pregnancy and lactation, times at which the metabolic demands placed on the mother are increased and when immune competence is challenged. Specific adaptations of both basal and stress-induced HPA activity occur during the reproductive cycle to fulfil the demands of the offspring. This chapter will review some of these adaptive changes which occur in HPA activity over the peripartum period, particularly focussing on the changes in the response to stressful stimuli. The potential mechanisms which underlie these adaptive changes will be considered, as well as the significance of the adaptive changes to ensure that the physiological and behavioral responses of the mother are appropriate for the successful care and growth of the offspring.

250]
2001 150 1 100]

?J
e~ ~D e~

2 E 8

0406001 Weane7 00 s
0
18 20 22 24 2

300 250 200 150 100 50 0

B. Lactating (Day 1O)

~12 14 16 18

4 6 8 10 Time of day (h)

Characteristics of HPA activity during pregnancy and lactation

Modulation of basal HPA activity

In both rat and man the basal activity of the HPA axis exhibits two principal temporal characteristics; ultradian (or pulsatile) secretory episodes and a diurual rhythm that has its acrophase shortly before the onset of locomotor activity (Fig. 1A; Veldhuis et al., 1989; Windle et al., 1998b), which in rats occurs at lights off. These two rhythms of secretion are linked in that, while ultradian pulses occur at more or less equal intervals throughout the diurnal cycle, variation in the amplitude of these episodes of HPA activity (but not their frequency or duration) generates the longer diurnal rhythm (Carnes et al., 1986; Jasper and Engeland, 1994; Windle et al., 1998a,b; Lightman et al., 1999). In order to resolve this dynamic pulsatile pattern of corticosterone secretion, we have utilized a computer-controlled sampling system which enables freely behaving animals to be sampled over extended periods of time at frequent intervals (usually 10 min) without operator intervention (Lightman et al., 1999). Using this sys-

Fig. 1. Examples of pulsatile corticosterone secretion in individual Sprague-Dawley rats before pregnancy (A), on day 10 of lactation (B), and 72 h after weaning following 10 days of lactation (C). Blood samples were obtained every 10 min over the 24 h period using an automated sampling system. All animals display a pulsatile pattern of secretion which comprises episodes of increased secretion lasting 10-20 min before levels declined at a rate similar to the clearance rate of the hormone. In the virgin animal the amplitude of these pulses was greatest at the start of the 10 h dark period (solid bar), and declined to a nadir around the time of lights on. This diurnal rhythm of pulse amplitude was lost in the lactating animal (B). However, 72 h after removal of the suckling pups a markedly exaggerated rhythm returned, with levels undetectable during the diurnal nadir.

tem it has been possible to study HPA activity under conditions in which the mother-offspring interactions are not disrupted and can be recorded by video camera during the sampling procedure (Windle et al., 1997c). Studies in rats have consistently shown that lactation is associated with a chronic hypercorticalism (Stern et al., 1973; Fischer et al., 1995; Windle et al., 1997c) which principally involves a flattening of the diurnal rhythm of secretion, such that there is a rise in the nadir levels of corticosterone and a decrease in the peak evening levels (Walker et al., 1992; Atkinson and Waddell, 1995). Studies using frequent, automated sampling of lactating rats has confirmed this change in the diurnal pattern (Fig. 1B), and clearly

113

200

A. Corticosterone (ng/ml)
* 100 ]

B. ACTH (pg/ml)

11 N
0-J

15C. PVN CRH mRNA

0.4-

D. PVN GR mRNA

5 0

*
E. CA1 GR mRNA

0.30.20.10F. CA1 MR mRNA

0.3 _ 0.2_1 ~ 4 0.1_ o_

Virgin Lactating

0.4 6t ~ 0.2 0

Virgin Lactating

Fig. 2. Mean levels of plasma corticosterone (A) and ACTH (B) in virgin (open bars) and lactating (hatched bars) SpragueDawley rats during the early light phase. Levels of both hormones are raised during lactation. However, the expression of CRH mRNA in the paraventricular nucleus (PVN) measured by in situ hybridization histochemistry (C) is significantly attenuated in the lactating animals (Data adapted from Shanks et al., 1999.) Glucocorticoid receptor (GR) mRNA did not differ in lactation in either the PVN (D) or CAI subfield of the dorsal hippocampus (E), but mineralocorticoid receptor (MR) mRNA expression was significantly decreased in the hippocampus (F). mRNA measurements are expressed in arbitrary optical density units. * P < 0.05 vs. virgin group.

demonstrates that this is not due to a loss of the pulsatile pattern of secretion, but rather pulse amplitude remains constant throughout the 24 h, in contrast to the virgin rat in which the pulse amplitude is low in the early light phase (Fig. 1A). This loss of diurnal rhythmicity appears not be due to a change in adrenal sensitivity to ACTH (Stem et al., 1973) and, since nadir levels of ACTH are also increased (Walker et al., 1992; Figs. 2B and 3B), a supra-pituitary change is implied. However, it should be noted that others have reported markedly depressed levels of ACTH in the face of elevated corticosterone (Fischer et al.,

1995), an observation for which the authors could offer no explanation. Although studies using a variety of paradigms in which the pups are separated and re-united with the dams have shown that the onset of suckling is ~stimulatory to HPA activity (e.g. Walker et al., 1992, 1993), this response is contingent upon pupgenerated cues (Smotherman et al., 1976) and does not occur naturally when the mother regulates the frequency and duration of the suckling bouts. Correlation of the individual pulses of secretory activity with the nursing behavior of the dam has shown that neither milk-ejection events nor the onset of individual suckling episodes show any relationship with the individual pulses of corticosterone secretion (unpublished data). Thus, while sensory stimuli from the pups are necessary to maintain HPA hypersecretion and separation leads to a rapid decline in hormone levels (Stem et al., 1973; Walker et al., 1992; Fischer et al., 1995), the primary mechanism underlying elevated basal HPA activity appears to involve a loss of circadian input to the endogenous pulse generator. This chronic elevation of HPA activity may subserve several roles in the lactating rat. Firstly, it may provide a more constant level of glucocorticoids that are necessary to cope with the high metabolic demands, including those associated with galactopoiesis. Secondly, since glucocorticoids can freely cross into maternal milk and influence the offspring, the stabilization of levels (including the absence of any stress-induced peaks described below) may prevent the brains of the developing neonates from being programmed by high or low hormone levels (see Champagne and Meaney, 2001, this volume). However, the consequence of this loss of the diurnal rhythm of corticosteroid secretion is the absence of the principal circadian signaling system that regulates diurnal changes in metabolism and gene transcription throughout the body. Despite the increased basal HPA activity, CRH messenger RNA (mRNA) expression is reduced in the PVN of lactating rats (Fig. 2; Lightman and Young III, 1989; Fischer et al., 1995; Windle et al., 1997a,c; Shanks et al., 1999). Since glucocorticoid negative feedback appears to be intact (see below), this depressed CRH expression is most likely a direct consequence of the raised nadir levels of

114 A. Corticosterone B. ACTH

250 -

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150
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50

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0 -110-90-70

-50-30-10

10

30

50

70

90

110

-3

21

Time (rain) relative to onset

Time (min) relative to onset

Fig. 3. Effect of noise stress (10 min 110 dB; shaded bar) on corticosterone (A, CORT) and ACTH (B) secretion in virgin (open symbols) and lactating (closed symbols). Note that basal levels of both hormones are markedly increased in lactating rats during the sampling period, which was performed during the nadir in the early light phase. However, there was no further increase in response to the noise, despite the fact that stress-induced ACTH levels in the virgin group exceeded the basal levels in the lactating group. This indicates that there is a mechanism for active suppression of the stress-induced response. Values represent mean 4- SEM of groups of 6-8 animals. * P < 0.05 vs. all equivalent values before noise, ANOVA and Tukey's post-hoc t-test. (Data in part B modified from Windle et al., 1997c.)

corticosterone. Although direct measures of CRH release have not been undertaken in lactating rats, it can be assumed that the decreased mRNA levels are reflected in decreased peptide release. This being so, the maintenance of ACTH secretion would appear to involve an increased contribution from other pituitary secretagogues, such as vasopressin. Although the number of neurons expressing vasopressin heteronuclear RNA transcripts appears not to be increased in lactation (Shanks et al., 1999), a small increase in vasopressin mRNA transcripts has been reported to occur in both parvocellular and magnocellular divisions of the PVN (Lightman and Young III, 1987; Fischer et al., 1995). It is possible that part of this increase is a response to the osmotic demand resulting from the production of milk, especially since a similar increase occurs in the supraoptic nucleus (Lightman and Young III, 1987). However, it has been recently reported that during lactation there is a marked increase in the co-localization of vasopressin with CRH, and an increased pituitary sensitivity to the stimulatory effect of vasopressin (Toufexis et al., 1999; Walker et al., 2001, this volume), suggesting that this may act to compensate for the decreased contribution from CRH, much in a similar way to that seen during chronic

stress-induced HPA activation (Harbuz et al., 1992; Chowdrey et al., 1995).

Attenuated HPA responses to stress in the peripartum period

A remarkable and consistent finding across many studies is that the HPA response to stress is markedly attenuated during the period of lactation. Since the original reports by Levine and colleagues that the HPA responses to footshock and ether stress are reduced in lactating rats (Thoman et al., 1970; Stern and Levine, 1972), it has been demonstrated that responses to a wide variety of stresses are attenuated. These include both psychological stresses, such as noise (Fig. 3; Windle et al., 1997c), conditioned footshock (Shanks et al., 1997), forced swimming (Walker et al., 1995; Toufexis and Walker, 1996; Toufexis et al., 1998), a combination of elevated plus maze and forced swimming (Neumann et al., 1998, 2000a), restraint (Banky et al., 1994; da Costa et al., 1996), and physical stresses, such as intraperitoneal injection of NaC1 (Lightman and Young III, 1989), ether vapor (Banky et al., 1994) and intraperitoneal injection of lipopolysaccharide (Shanks et al., 1999). This apparent lack of stimulus selectivity suggests

115 that at least part of the mechanism involved is common to the distinct neural pathways that mediate the afferent activation of the PVN in response to psychological (interoceptive) and physical (exteroceptive) stressors (Day et al., 1999b; Emmert and Herman, 1999). However, the magnitude of suppression varies between stimuli. In contrast to the complete absence of an HPA response to noise stress (Fig. 3) the endocrine response to immunological challenge with intraperitoneal lipopolysaccharide, though significantly attenuated, is not lost (Shanks et al., 1999). This lack of complete suppression is also reflected by the fact that the response of CRH mRNA remains intact. Whether this differential response to stresses merely reflects differences in stimulus intensity or is a function of the importance of particular stimuli to the survival of the animal (e.g. auditory stimulus vs. immunological function), remains to be determined. It is clear that this attenuation of responses to stress is not due to a refractoriness of the HPA axis to all afferent signals, since separation and reuniting of the litter with the dam will evoke HPA responses in animals which are otherwise insensitive to stressful stimuli (Walker et al., 1992, 1993). Furthermore, as described above, the basal ultradian pulses that are driven by hypothalamic output are not reduced but are of similar magnitude during the diurnal acrophase and increased during the nadir (Fig. 1). This profile of basal hypersecretion and stress-induced hyposecretion, is similar to that observed under other conditions of chronic HPA activation, such as experimental adjuvant-induced arthritis (Harbuz et al., 1999). This has been suggested to arise, in part, from the interaction between ultradian pulse frequency and activation by acute stress stimuli (Windle et al., 1998b), which results in reduced responses under conditions of increased pulse frequency. However, this appears not to account for the stress hyporesponsiveness in lactation as no difference in pulse frequency has been detected between virgin and lactating rats (unpublished data). An alternative condition which presents with this divergent profile of increased basal and reduced stress-induced secretion is that which occurs during severe mood disorders in humans (Gotthardt et al., 1995; Deuschle et al., 1997), and this has been attributed to changes in glucocorticoid negative feedback (see below). Measures of ACTH levels also show a loss of the stress-induced response (e.g. Fig. 3; Walker et al., 1995; Windle et al., 1997c), indicating that hyporesponsiveness is not due to adrenal insensitivity to ACTH. However, recent data have indicated that it may be due, in part, to an effect at the level of the pituitary corticotrophs. During lactation in vivo pituitary responsiveness to intravenous CRH is reduced (Toufexis et al., 1999), similar to that observed in late pregnancy when CRH-induced cAMP accumulation of pituitary fragments in vitro is also suppressed (Neumann et al., 1998). However, it is unclear whether this results from changes in pituitary CRH receptor expression (Neumann et al., 1998; Toufexis et al., 1999), and although this pituitary insensitivity combined with the reduced expression of CRH mRNA (Lightman and Young III, 1989; Fischer et al., 1995; Windle et al., 1997a,c; Shanks et al., 1999) would suggest a markedly attenuated CRH drive to the pituitary, this does not appear to be the sole mechanism underlying the lack of response to stress. In particular, data from measurement of stress-induced immediate early gene (da Costa et al., 1996; Wintrip et al., 1997; Woodside and Amir, 1997; Shanks et al., 1999) and CRH (Lightman and Young III, 1989) mRNA accumulation in the PVN suggest that central pathways are modulated. Indeed, further evidence that the adaptive plasticity of stress responses involves an action on the afferent stress pathways comes from the fact that a loss of stress responses is not selective for the HPA axis, since there is also a marked reduction in the stress-induced release of oxytocin (Carter and Lightman, 1987; Higuchi et al., 1988, 1991; Neumann et al., 1998) and prolactin (Higuchi et al., 1989). The potential mechanisms underlying this stress hyporesponsiveness are considered below.

Temporal profile of onset and recovery of stress hyporesponsiveness

The onset of the stress-hyporesponsive state of lactation is independent of signals related to either parturition or suckling, since it is already present in late gestation (da Costa et al., 1996; Neumann et al., 1998; Johnstone et al., 2000). Although pregnancy-associated hyporesponsiveness is described in detail elsewhere in this volume (see Douglas and

116
A. D a y 16 o f g e s t a t i o n 700 600 500 ~. 400 300

200
100 0 .o 700 600 .... , . . . . . . F;I', . . . . . . . . . . . .

B. D a y 19 o f g e s t a t i o n
F:;~.q

500
400 300 200 100 -110 -90 -70 -50 -30 -10

i:ii:i
F:.:.i

ii i::ii

10

30

50

70

90

110

Time relativeto onset of noise (rain)

Fig. 4. Effect of noise stress (10 min x 110 dB; shaded bar) on plasma corticosterone levels in pregnant female rats at day 16 of gestation before luteolysis (A) and day 19 of gestation after luteolysis (B). Note the decrease in basal level that occurs after luteolysis and the absence of a noise-induced response. Values are mean SEM. Noise was applied during the diurnal nadir three hours after lights on.

Russell, 2001), a brief mention here is valuable as the onset may provide evidence for possible underlying mechanisms that persist through into lactation. Using animals at different stages of the reproductive cycle we have tested the changing response to noise stress. On day 16 of gestation, despite greatly elevated basal levels of corticosterone, exposure to noise was able to evoke a further increase (Fig. 4A). This implies that the increased basal secretion of corticosterone seen in lactation is not the primary cause of the attenuated responses to stress. By contrast, on day 19 of gestation basal corticosterone levels had declined (although were still elevated compared to virgin animals; compare with Fig. 3A and Fig. 4B), and the response to noise stress was completely absent (Fig. 4B). This time frame of onset is similar to reports that the HPA responses to the combined effect of the novel anxiogenic environment of the elevated plus maze and a period of forced swimming are reduced between days 15 and 18 of gestation (Neumann et al., 1998).

In the rat, one of the main endocrine events that occurs over this late gestation period is luteolysis which leads to a dramatic fall in circulating levels of progesterone concomitant with an increase in the levels of estrogens (Yoshinaga et al., 1969; Garland et al., 1987). In order to investigate whether this dramatic change in the estrogen:progesterone ratio plays any role in the induction of stress hyporesponsiveness, ovariectomized virgin rats were implanted with capsules containing estradiol and received daily injections of progesterone to achieve levels comparable with those during mid-gestation. After 11 days of treatment, withdrawal of the progesterone administration three days prior to testing for responses to stress, resulted in a significant reduction in peak HPA activation compared to animals in which progesterone was continued (unpublished data). Although results obtained using a similar steroid paradigm showed no effects on the HPA response to forced swimming (Douglas et al., 2000), a variable period between progesterone withdrawal and testing could account for this difference and further studies will be required to evaluate the contribution of gonadal steroids to the onset of stress hyporesponsiveness. Importantly, gonadal steroid changes during late pregnancy have dramatic effect on central oxytocin systems (Crowley et al., 1995; Young et al., 1997; Wakerley et al., 1998; Douglas et al., 2000) which has implications for HPA activity (see below). While the onset of stress hyporesponsiveness occurs within a defined time window in late gestation, few studies have addressed the time course and mechanisms of recovery of the stress response. The HPA response to ether remains suppressed 24 h after removal of the litter (Stern et al., 1973), and the accumulation of CRH mRNA in the PVN following intraperitoneal injection of hypertonic NaC1 is still absent 18 h after weaning, only becoming fully restored 48-72 h after separation of the dam from the litter (Lightman and Young III, 1989). Likewise, lipopolysaccharide-induced c-fos mRNA expression in the PVN is restored within 12 days of removal of the litter (Shanks et al., 1999). Although the HPA response to noise stress is also restored 72 h after removal of the litter, frequent sampling shows that the dynamic profile of the response is very different: the peak levels did not differ from those in non-lactating animals, but the duration was twice

117 as long (Windle et al., 1997c). This suggests that the mechanisms responsible for the termination of HPA activity following acute activation are compromised. Furthermore, methodologically they illustrate important differences of interpretation depending on whether single or multiple samples are obtained. This differential time course of recovery of the basal, stress-induced and negative feedback components of HPA regulation, clearly indicates that peripartum plasticity involves the modulation of several separate mechanisms. diurnal negative feedback. Both during pregnancy (Nolten and Rueckert, 1981) and lactation (Owens et al., 1987) there is a reduction in the ability of dexamethasone to suppress circulating levels of cortisol. Plasma cortisol levels 17 h after a single oral dose of dexamethasone were significantly elevated over the first two weeks post-partum compared to non-pregnant controls. Furthermore, they displayed a negative correlation with the time post-partum, with levels falling over the first four weeks post-partum to reach levels similar to the non-pregnant controls by day 35 (Owens et al., 1987). This decreased suppression gradually developed over the course of gestation in parallel to the rise in plasma and urinary free cortisol levels (Nolten and Rueckert, 1981; Odagiri et al., 1988). In this respect it has been suggested that the attenuated negative feedback may be explained by elevated levels of corticosteroid binding globulin (CBG) providing a pool of dexamethasone-resistant cortisol (Owens et al., 1987). However, this has been challenged on the basis that non-pregnant women taking the combined oral contraceptive pill display elevated levels of CBG without any change in total cortisol levels (Scott et al., 1990). Furthermore, data from rats indicate that while the levels of CBG show a marked rise over the last few days of gestation (unpublished data), levels are significantly reduced during lactation (Walker et al., 1992; Shanks et al., 1997, 1999), suggesting that an alternative mechanism operates which may involve a resetting of the sensitivity of the HPA axis. The apparent loss of diurnal negative feedback may arise from changes in corticosteroid receptor expression, and this is supported by a report that the level of binding of [3H]dexamethasone in the soluble fraction prepared from the hippocampus is reduced in lactation (Meaney et al., 1989). To further investigate this receptor modulation, MR and GR mRNA expression have been compared in lactating and virgin rats. This has shown that MR mRNA expression is significantly reduced in the CA1 region of the hippocampus (Fig. 2F), while GR mRNA is unaffected in either CA1 or the PVN (Fig. 2D,E). It is possible that the elevated basal corticosteroid levels in lactation alter MR gene transcription (Chao et al., 1998), and that a reduction of MR-mediated feedback could underlie the loss of diurnal rhythmicity (Fig. 6). If this is the case it clearly differs from the situation

Glucocorticoid feedback regulation of basal and stress-induced HPA activity during pregnancy and lactation
The increased nadir levels of corticosterone (Fig. 1) and delayed termination of stress-induced HPA activity in weaned dams (Windle et al., 1997c) suggests that both the diurnal and rapid components of corticosteroid negative feedback may be modulated during lactation. However, it has been consistently reported that negative feedback is intact (Stern et al., 1973; Lightman and Young III, 1989; Walker et al., 1992) or even increased (Schlein et al., 1974) in lactating rats. The parallel time course and magnitude of CRH mRNA accumulation following adrenalectomy of virgin and lactating rats (Lightman and Young III, 1989), and the levels of ACTH following adrenalectomy and constant corticosterone replacement (Walker et al., 1992) both suggest that the sensitivity to negative feedback is not significantly altered. However, the clamping or removal of corticosterone levels used in these studies may itself lead to a normalization of feedback mechanisms which might be otherwise modulated by the high circulating corticosteroid levels during lactation. Furthermore, results of studies using adrenalectomy with or without corticosterone replacement do not distinguish between mineralocorticoid receptor (MR)-mediated diurnal negative feedback of PVN activity, which is mediated via the hippocampus, and the glucocorticoid receptor (GR)-mediated negative feedback of CRH and pituitary proopiomelanocortin mRNA levels, which is mediated via direct action on gene transcription. Contrary to the data from adrenalectomized rats, studies in humans support the possibility of reduced

118 prior to parturition, since MR mRNA is unaffected in late pregnant animals while GR mRNA is selectively increased in the dentate gyrus while remaining unaffected in the PVN and CA1 region (Johnstone et al., 2000). This recent paper also raises an interesting possibility in that there is a marked increase in 1 l~-hydroxysteroid dehydrogenase (type 1) activity in the PVN during late pregnancy (Johnstone et al., 2000). This increase might be expected to favor the conversion of inactive metabolites to active corticosterone, thereby raising local glucocorticoid levels and enhancing negative feedback. Whether this upregulation persists during lactation remains to be determined but, if so, could contribute to the decreased levels of CRH mRNA (Fig. 2C; Lightman and Young III, 1989; Fischer et al., 1995; Windle et al., 1997c; Shanks et al., 1999). Furthermore, it is possible that raised local corticosteroid action could also have an inhibitory effect on the electrical excitability of PVN neurons (Kasai et al., 1988; Kasai and Yamashita, 1988), thereby contributing to the reduction in activation following acute stressful stimuli. However, hyporesponsiveness does not appear to arise primarily from an increase in steroid negative feedback, since a reduction in stress-induced ACTH secretion is also seen in adrenalectomized dams (Walker et al., 1995), and while lactation is associated with complete suppression of the CRH mRNA response to intraperitoneal hypertonic NaC1 (Lightman and Young III, 1989), even very high levels of exogenous glucocorticoids cannot block this response in male animals (Harbuz et al., 1990). The following section will consider some of the other central mechanisms that may contribute to this suppression of stress responsiveness. in the PVN of lactating rats (Lightman and Young III, 1989), suggests that this cascade is disrupted in the peripartum period. This has been confirmed using quantitative measures of c-fos mRNA, that show the level of restraint-induced immediate early gene expression in the PVN is dramatically reduced in lactating rats (Fig. 5; da Costa et al., 1996). Furthermore, this reduction also occurred in animals tested in late pregnancy (days 19-21 of gestation; Fig. 5), in agreement with the pre-partum onset of this stresshyporesponsive state (Douglas et al., 1998; Neumann et al., 1998; Johnstone et al., 2000). This block of stress-induced c-fos mRNA not only demonstrates that the mechanisms underlying the hyporesponsive condition occur at a step earlier in the sequence of molecular responses than the induction of CRH mRNA (Imaki et al., 1992) but, since Fos is considered a marker of neuronal activity (Sagar et al., 1988), also suggests a reduction in either the afferent pathways or the signal transduction mechanisms within the PVN itself. Interestingly, separate analysis of c-fos mRNA expression in the parvocellular and magnocellular subdivisions of the PVN, revealed that while restraint-induced expression was significantly reduced in the parvocellular region throughout the period spanning late pregnancy, early lactation (days 3-4 post-partum) and mid-lactation (days 10-14 post-partum), gene expression in the magnocellular region, which presumably reflects the stress-induced release of oxytocin (Carter and Lightman, 1987; Higuchi et al., 1988; Miyata et al., 1995), was only reduced in the early lactation period (da Costa et al., 1996). Why stress-induced c-fos mRNA expression should be reduced in magnocellular neurons only in this immediate post-partum period, despite the fact that stress-induced oxytocin release is also significantly reduced in pregnancy (Neumann et al., 1998), remains to be determined. The attenuation of stress-induced c-fos mRNA expression in the PVN of lactating rats not only occurs in response to psychological stress (such as restraint), but also to physical stimuli such as intraperitoneal lipopolysaccharide (Shanks et al., 1999) or the osmotic stimulus of urethane administration (Woodside and Amir, 1997). Since psychological (interoceptive) and physical (exteroceptive) stimuli activate the HPA axis via distinct neural pathways (Day et al., 1999b; Emmert and Herman, 1999), the fact that

Potential mechanisms underlying stress hyporesponsiveness of lactation

Modulation of stress-activated circuits during lactation: immediate early gene mapping studies
Stress induces immediate early gene expression in the PVN, where it occurs in CRH-containing neurons as part of a cascade of receptor-activated molecular responses (Imaki et al., 1992; Kovacs and Sawchenko, 1996). The attenuated stress-induced increases of both CRH and proenkephalin mRNA

119

4...a

~.~

~..4

..)

0
V

II
LP

m
EL ML

Fig. 5. Stress-induced levels of c-fos mRNA in the PVN of virgin (V) Sprague-Dawley rats and of animals during late pregnancy (LP, days 19-21 of gestation), early lactation (EL, days 3-4 post-partum), or mid-lactation (ML, day 10-14 post-partum). The brains were collected immediately following 30 min of restraint in Plexiglas tubes. Note the marked reduction in immediate early gene expression in all pregnant and lactating groups of animals, indicating a reduction in the activation of PVN neurons. Analysis was not performed on unstressed animals as c-fos mRNA levels are undetectable in these animals. Photographs show representative autoradiograms indicating the distribution of hybridization signal in the PVN (arrowed). Histograms show mean optical density measures from autoradiograms (arbitrary units) measured in the parvocellular division of the PVN from groups of 5-9 animals/group; * P < 0.05 vs. virgin animals. (Figure adapted from da Costa et al., 1996.)

both are attenuated lends weight to the argument that the underlying mechanisms either involve a common afferent pathway or modulation of signal transduction within the PVN itself. One way to dissociate these possibilities has been to examine the regional patterns of immediate early gene expression outside the PVN. Restraint and other psychological stresses increase immediate early gene expression in a wide range of cortical, limbic, hypothalamic and brainstem regions, including the medial amygdala (MeA) and associated region of the ventrolateral septum (VLS) adjacent to the bed nuclei of the stria terminalis (Arnold et al., 1992; Imaki et al., 1992, 1993; Cullinan et al., 1995; Day et al., 1999b; Emmert and Herman, 1999). Studies conducted over the peripartum period have shown a pattern of changes in stress-induced c-fos mRNA in the MeA and VLS that closely follow that of the PVN, i.e. a decline during late pregnancy that reached a minimum in early lactation (da Costa et al., 1996). The fact that ibotenic acid lesions of the MeA block restraint-induced Fos expression in the PVN (Day et al., 1999b) suggests

that these structures may constitute a limbic circuit that determines the magnitude of neuroendocrine responses to stress and which could be the target for modulation during lactation. The cingulate cortex, another area activated by restraint (Imaki et al., 1992; Cullinan et al., 1995) and known to play a role in determining the magnitude of the HPA response to restraint (Diorio et al., 1993), also showed reduced gene expression but only in lactating and not pregnant animals (da Costa et al., 1996). These changes in stress-induced activation of the MeA, VLS and cingulate cortex were selective since no similar change occurred in other forebrain (piriform cortex and hippocampus) or brainstem (dorsal vagal complex and ventral tegmentum) regions. This dissociation between attenuated and unaffected regions, may form a neuroanatomical basis of the dissociation between the neuroendocrine and behavioral responses to forced swimming shown by lactating rats (Walker et al., 1995). The mechanisms underlying this regional reduction in c-fos mRNA induction may involve a modulation of the glutamatergic inputs to the PVN, since

120 expression of Fos-immunoreactivity evoked by injection of N-methyl-D-aspartate (NMDA) has been shown to be reduced in the lactating rat compared to non-lactating controls (Wintrip et al., 1997). Alternatively, it may involve a modulation of the central actions of CRH, which has been suggested to function to coordinate neural responses to stress (Dunn and Berridge, 1990; Herbert, 1993) and to exert a positive autoregulatory feedback effect on PVN function (Yamashita et al., 1991; Kiss et al., 1996). Intracerebroventricular (i.c.v.) administration of CRH in male and non-lactating female rats increases Fos and nerve growth factor inducible gene-B (NGFI-B) expression in a number of nuclei throughout the neuroaxis (Clark et al., 1991; Arnold et al., 1992; Andreae and Herbert, 1993; Imaki et al., 1993; Parkes et al., 1993; da Costa et al., 1997). However, in a circumscribed group of limbic nuclei, notably the PVN, arcuate nucleus, bed nuclei of the stria terminalis, and MeA, this increase does not occur in lactating rats (da Costa et al., 1997), possibly reflecting the attenuated CRH-induced neuroendocrine activity that occurs during lactation (Patel et al., 1991). This selectively reduced neuronal activation could arise from changes in the expression of central CRH receptors. In this respect, it is interesting that a reduction of [125I]CRH binding has been detected in the pituitary of late pregnant rats, coincident with a reduction in HPA responses to stress (Neumann et al., 1998), although others have detected no change in binding during lactation (Toufexis et al., 1999). Furthermore, although the levels of mRNA encoding CRH receptor type 1 have been found to be significantly lower in the PVN of pregnant, stressed rats compared to virgin controls, the levels are not lower (or are even increased) in lactating rats in which the stress-induced HPA response is still suppressed (da Costa et al., 2001). Indeed further evidence against a role of central CRH in determining HPA activity during lactation, comes from the fact that central blockade of the CRH receptor by intracerebroventricular (i.c.v.) injection of the antagonist cl-helical CRH (9-41) did not block the HPA response to separation and reunion with the pups (Walker et al., 1993). Thus, at least by inference from receptor expression in the PVN and pituitary, it would appear that changes in CRH receptor do not underlie the hyporesponsive state in lactation, although further studies will be required to specifically examine those limbic circuits which are suppressed. The mechanisms regulating the reduced CRH receptor expression in late pregnancy remain to be determined, but probably do not reflect the reduced level of corticosterone seen at this time as CRH receptor mRNA has been shown to vary independently of circulating glucocorticoids (Luo et al., 1995; Makino et al., 1995).

Modulation of noradrenergic activation of HPA axis during lactation

Noradrenaline is one of the principal transmitters that is activated by stress (Koob, 1999) and which contributes to afferent innervation of the CRH neurons in the PVN. Electrical stimulation of the ventral noradrenergic bundle causes an increase in CRH release into the portal system that can be blocked by C~l adrenergic receptor antagonists (Plotsky, 1987). alb receptors are located on CRH neurons of the PVN (Day et al., 1999a) and microinjections of noradrenaline close to the PVN will stimulate HPA activity and induce CRH mRNA transcription (Itoi et al., 1994). To what extent noradrenaline contributes to HPA regulation during lactation is not clear. However, microdialysis studies of lactating rats have shown that noradrenaline release into the PVN is decreased by a period of separation from the pups (Toufexis et al., 1998) and increased during a subsequent period of suckling (Bealer and Crowley, 1998) and it is possible that this contributes to the increase in ACTH that occurs during reunion with the litter (Walker et al., 1992, 1993). The expression of Cqb receptor mRNA in the PVN (but not hippocampus, amygdala or dorsal raphe) displays a strong negative correlation with circulating corticosteroids (Day et al., 1999a). Thus, the elevated basal levels of corticosterone during lactation may be expected to reduce the expression of this receptor and alter afferent input. Indeed a number of studies have suggested that the noradrenergic input to the HPA axis is altered during lactation. 6Hydroxydopamine lesions close to the PVN reduced the ACTH and corticosterone responses to swim stress in virgin animals, demonstrating the noradrenergic involvement (Toufexis and Walker, 1996). However, similar lesions did not have any effect on

121 the already blunted response of animals on day 911 of lactation (Toufexis and Walker, 1996), consistent with a reduced contribution of noradrenaline to stress-induced HPA activity during lactation. This has been tested more directly by i.c.v, injection of the Ctl agonist methoxamine. In virgin animals methoxamine evokes an acute elevation of plasma levels of oxytocin, ACTH and prolactin, and a long lasting increase in corticosterone levels (Patel et al., 1993; Windle et al., 1997a). However, similar administration in lactating rats results in much attenuated increases in both oxytocin and corticosterone (Patel et al., 1993; Windle et al., 1997a). Although this could be explained by reduced neuronal responses to cq activation, it should be noted that in the virgin but not lactating rats, methoxamine increased CRH but not vasopressin mRNA in the PVN (Windle et al., 1997a), suggesting that methoxamine selectively acts through CRH neurons. In view of the decrease in CRH peptide and pituitary CRH receptor expression previously described, the reduced HPA response to methoxamine could arise from changes downstream of cq receptor activation. To test this in vitro electrophysiological recordings were undertaken from PVN neurons in slices from proestrus virgin and lactating rats. This showed that both the proportion of neurons responding to a threshold dose of methoxamine (10 -5 M) and the magnitude of the methoxamine-induced excitation were significantly lower during lactation (Windle et al., 1997a), consistent with a reduced receptor efficacy. However, contrary to the predicted down-regulation of al receptors, binding studies have shown no change in [3H]prazosin binding in the PVN throughout lactation, but a significant decrease in [3H]rauwolscine binding between day 3 post-partum and day 10 post-partum, suggesting that (22 adrenergic but not ~1 receptor expression is modified (Toufexis et al., 1998). Furthermore, i.c.v, injection of the c~2 antagonist idazoxan markedly increased ACTH release in response to forced swimming in virgin, but not lactating rats, while the cq antagonist corynanthine failed to potentiate HPA activity in either group. Although this appears to suggest that ~2 receptors play the greater role in HPA regulation, the fact that idazoxan has anxiogenic properties (Handley and Mithani, 1984) means it will potentiate the effect of the forced swim in the virgin animals, while having no effect in the stress-hyporesponsive lactating animals. Thus, the relative importance of cq- and ct2-adrenoceptor function to the stress-hyporesponsive state remains to be clarified. Although the foregoing suggests that any noradrenergic involvement in the stress hyporesponsiveness may reflect a modulation of postsynaptic receptor transduction, there is some evidence that changes in activity of the noradrenergic neurons themselves may also occur. The induction of c-fos mRNA in the locus coeruleus in response to either stress (Cullinan et al., 1995; da Costa et al., 1996) or i.c.v, injection of CRH (Imaki et al., 1993; da Costa et al., 1997) is consistent with the involvement of the A6 cell group in neuroendocrine and behavioral responses. However, the degree of activation does not differ between virgin and lactating rats (da Costa et al., 1996, 1997), and the concentration of noradrenaline in the locus coeruleus is unaffected by chronic restraint stress in either virgin or late pregnant rats (Moyer et al., 1977). This may reflect the fact that certain stressand CRH-induced behaviors which involve the locus coeruleus, such as exploration and grooming, are unaffected during lactation (Almeida et al., 1994; Walker et al., 1995; Windle et al., 1997c). However, it is notable that in late pregnant rats levels of c-fos mRNA increase dramatically in the locus coeruleus (da Costa et al., 1996), there is a reduced content of noradrenaline (Moyer et al., 1977), and the spontaneous electrical activity of locus coeruleus neurons is reduced (Nakamura et al., 1988), and that this occurs at a time when the HPA responses to stress are attenuated. It is possible that variation in noradrenergic involvement in HPA activity could arise from the changing levels of gonadal steroids in the peripartum period. Gonadal steroids have marked effects on hypothalamic cq receptor function: estrogen elevates the density of Cqb receptors and potentiates cq-induced cAMP production (Etgen et al., 1992; Petitti et al., 1992; Karkanias et al., 1996). However, although this is an attractive mechanism, neither a simple steroid treatment of ovariectomy with or without estradiol or progesterone replacement for 14 days (Windle et al., 1997a), nor a combined estradiol and progesterone paradigm that mimics the steroid levels at the time of luteolysis (unpublished data),

122 are able to alter either the corticosterone or the CRH mRNA responses to i.c.v, injection of methoxamine. The recent demonstration that adrenalectomy increases Cqb mRNA in the PVN and that this is reversed by corticosteroid treatment (Day et al., 1999a) suggests that the raised levels of corticosterone during lactation may suppress noradrenergic inputs. Although this may underlie the different postsynaptic effects of methoxamine, it is unlikely to contribute to the stress-hyporesponsive state of lactation, as this persists in adrenalectomized animals (Walker et al., 1995). constant in the SON, suprachiasmatic nucleus or bed nuclei of the stria terminalis, it is increased early in pregnancy in the VLS and medial preoptic area, and at parturition in the central amygdala and ventromedial nucleus (Young et al., 1997). These changes in receptor expression are functionally reflected by changes in peptide-induced excitation measured in recordings from the VLS and bed nuclei of the stria terminalis (Ingram and Wakerley, 1993; Housham et al., 1997; Wakefley et al., 1998). Oxytocin is released in both hypothalamic and extra-hypothalamic sites in response to both stressful (particularly emotional) stimuli (Nishioka et al., 1998; Engelmann et al., 2000) and, in lactating rats, in response to the suckling stimulus (Neumann et al., 1993a,b). Although it is well established that this released peptide participates in the feedback facilitation of milk ejection and expression of maternal behavior, increasing evidence also supports a role in the control of anxiety behavior and HPA activity. This has been suggested from studies showing that peripheral injection of high doses of oxytocin have an anxiolytic effect on open field behavior (Uvn~is-Moberg et al., 1994) and that direct injection of oxytocin into the amygdala can attenuate conditioned fear responses (Roozendaal et al., 1992). Furthermore, we have demonstrated that constant infusion of low doses of oxytocin (1-100 ng/h) into the lateral cerebral ventricle of ovariectomized, estradiol-treated rats reduces the noise-induced HPA activity (Windle et al., 1997b), and caused a dosedependent inhibition of restraint-induced ACTH and corticosterone release and CRH mRNA transcription (unpublished data). Infusions also had an anxiolytic effect in tests employing the elevated plus maze (Windle et al., 1997b), similar to the report that either peripheral or i.c.v, administration of oxytocin to mice increased open arm entries on an elevated plus maze (McCarthy et al., 1996), an effect that was enhanced in ovariectomized mice by estradiol treatment that also caused an increase in the density of oxytocin binding sites in the septum. All these effects we have observed were highly selective for oxytocin over vasopressin, suggesting an action through specific receptors. From the above it is tempting to speculate that the raised oxytocin levels in lactating rats would have a similar effect on stress responses.

Coordinated actions of oxytocin in the peripartum rat: possible role in modulation of the HPA axis
As reviewed elsewhere in this volume, oxytocin plays a number of roles in the adaptive physiology and behavior in the peripartum period, including structural reorganization of the magnocellular neurons of the supraoptic nucleus (SON) and PVN, maintenance and facilitation of milk ejection, and the induction of pup-directed behavior, although not in the maintenance of this behavior in the lactating dam (Pedersen et al., 1982: van Leengoed et al., 1987; Insel and Harbaugh, 1989; Insel, 1992). It has been suggested that the expression of these multiple roles may be linked through the dynamic changes in the expression of oxytocin and its receptor that occur in the peripartum period (Insel, 1992; Ingram et al., 1995). Although up-regulated, the precise pattern of these changes in expression in the brain during lactation has not been clearly established. Hypothalamic oxytocin mRNA levels increase in late pregnancy and remain elevated through established lactation (Zingg and Lefebvre, 1988), although more detailed analysis of the time course of this response has shown a transient but marked decrease in the days immediately following birth, which may arise from an action of gonadal steroids (Crowley et al., 1993). This decline suggests that suckling is not the main stimulus for oxytocin mRNA transcription at this time, although removal of the litter for 24 h does result in a further decline in levels (Ghosh and Sladek, 1995). During the peripartum period oxytocin binding sites also increase in selective limbic sites, notably the VLS (Insel, 1990), and while oxytocin receptor mRNA has been shown to remain

123 A role of endogenous oxytocin in modulating anxiety and stress responses has been directly demonstrated by showing that administration of the selective oxytocin receptor antagonist desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4,O rnS]vasotocin either i.c.v, or by retrodialysis into the PVN increased both basal HPA activity and that induced by forced swim and exposure to the elevated plus maze (Neumann et al., 2000a,b). Furthermore, i.c.v. administration of the receptor antagonist prior to exposure to the elevated plus maze reduced exploratory behavior in pregnant and lactating rats, but not in virgin rats (Neumann et al., 2000a,b; see Neumann, 2001, this volume). This has been interpreted as due to an anxiolytic action resulting from the increased levels of oxytocin released into the limbic system during the peripartum period. Further recent data suggest a role for the dynamic pattern of gonadal steroid changes in late gestation in the induction of an oxytocin-dependent suppression of stress responses. A number of studies have shown that steroid treatments can have marked effects on the response of limbic neurons to oxytocin (Wakefley et al., 1998; Terenzi et al., 1999). Using the model of estradiol and progesterone treatment followed by progesterone withdrawal in virgin rats described earlier, it has been shown that progesterone withdrawal evokes a marked increase in hypothalamic oxytocin mRNA transcription (Crowley et al., 1995), and in the expression of oxytocin binding sites in the ventromedial nucleus, VLS and head of the caudate nucleus (unpublished data). This effect on binding site density was regionally selective, as no change occurred in either the medial and central amygdala or oval and principal bed nuclei of the stria terminalis. Importantly, animals receiving this steroid regime displayed significantly more entries into the open arm of the elevated plus maze, consistent with an anxiolytic-like effect, and the HPA response to noise stress was significantly attenuated. The role of oxytocin in mediating the effect on HPA activity was demonstrated by the fact that infusions of the oxytocin receptor antagonist, desGlyNH2,d(CH2)5 [Tyr(Me) 2,Thr4,O rn8]vasotocin, during the period of progesterone withdrawal restored the HPA response to that seen in control animals in which progesterone was continuously administered (unpublished data). From this it may be speculated that (1) the changes in gonadal steroids that occur at luteolysis trigger a sequence of events involving induction of oxytocin peptide synthesis and oxytocin receptor expression in selected limbic regions, and (2) that the action of oxytocin is to produce an anxiolytic effect and to suppress stress-induced activation of the HPA axis, possibly through gating of pathways running through the limbic system. As for maternal behavior, it is possible that, once initiated, this gating mechanism no longer requires oxytocin, but is maintained by sensory cues from the litter so that, once weaning takes place responses can be restored (Fig. 6). Conclusions The peripartum period is associated with marked adaptive changes in the HPA axis which subserve the metabolic demands of lactation, and provide a relatively stable level of steroid secretion that protects against any potentially deleterious programming effects on the late gestation fetus and suckling neonates. The most marked of these changes are the increase in basal secretion resulting from the loss of diurnal rhythmicity, and the marked attenuation of the neuroendocrine response to stress, including the release of oxytocin and prolactin as well as ACTH and corticosterone. Interestingly, the hormones that are normally released in response to stress, and which show suppressed responses during lactation (i.e. ACTH, corticosterone, oxytocin and prolactin), are the same hormones that are released in response to suckling and participate in various aspects of milk synthesis and milk ejection. Thus, it is possible that during lactation the neuroendocrine axes that are responsible for the release of these hormones have been subsumed for these alternative functions. Whether one single coordinating factor is responsible for determining these adaptive changes or whether they occur as a result of multiple interrelated mechanisms triggered in an appropriate sequence towards the end of gestation, remains unclear. Recent evidence strongly supports the participation of noradrenaline and oxytocin in the control of HPA activity in the peripartum rat. However, a number of other factors have been suggested to be involved in the modulation of stress-induced HPA activity (Lightman, 1992; Lightman et al.,

124

Fig. 6. Model for possible interacting mechanisms underlying adaptive changes in basal and stress-induced HPA activity during the peripartum period. In respect of basal secretion, reduced mineralocorticoid receptor (MR) expression in the hippocampus leads to a reduction in the diurnal (hippocampal) negative feedback control of the HPA axis. This results in raised corticosterone levels during the nadir and increased glucocorticoid receptor-mediated suppression of CRH mRNA transcription in the PVN and proopiomelanocortin (POMC) mRNA in the pituitary. Thus, despite the reduced levels of CRH mRNA (and presumably peptide), the increased activity of paraventricular nucleus (PVN) neurons due to the reduced mineralocorticoid-mediated feedback, combined with an increased synergistic effect of vasopressin, maintains HPA hypersecretion. In respect of stress-induced secretion, following luteolysis the change in progesterone to estrogen ratio induces increased expression of oxytocin and its receptor in the limbic system. The increased peptide activity acts to suppress the stress-activated afferents responsible for PVN activation. While oxytocin participates in the induction of this hyporesponsive state and probably participates in the induction of other behavioral and physiological adaptations, maintenance of stress hyporesponsiveness during lactation is dependent upon continued sensory signals from the offspring.

1998), including the opioid peptides (see Douglas and Russell, 2001, this volume) and prolactin (see Neumann, 2001, this volume), and future studies should be directed to determining their relative importance for determining the successful process of lactation.
Abbreviations

CBG CRH GR HPA

i.c.v.

~lb ~2

ACTH

~1 adrenergic receptor (/lb adrenergic receptor (/2 adrenergic receptor adrenocorticotropin

MeA NMDA MR mRNA NGFI-B PVN SON VLS

corticosteroid-binding globulin corticotropin-releasing hormone glucocorticoid receptor hypothalamo-pituitary-adrenal intracerebroventricular medial amygdala N-methyl-D-aspartate mineralocorticoid receptor messenger ribonucleic acid nerve growth factor inducible gene-B paraventricular hypothalamic nucleus supraoptic nucleus ventrolateral septum

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Acknowledgements The authors are grateful to the Wellcome Trust for their financial support, and to Dr. Ana da Costa, Marcella Brady, Thushitha Kunanandam, Fiona Dickinson and Marcus Andrews who have contributed to these studies. References
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J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 9

The neurobiology of stress in human pregnancy: implications for prematurity and development of the fetal central nervous system
Pathik D. Wadhwa

1,2,,,

Curt A. Sandman 1 and Thomas J. Garite 2

! Department of Psychiat~ and Human Behavior, University of California, Irvine, College of Medicine, 3117 Gillespie Neuroscience Building, lrvine, CA 92697, USA 2 Department of Obstetrics and Gynecology, University of California, Irvine, College of Medicine, 3117 Gille~pie Neuroscience Building, lrvine, CA 92697, USA

Abstract: Adverse early experience, including prenatal maternal psychosocial stress, has the potential to negatively influence developmental processes through both physiological and behavioral mechanisms. This in turn may have adverse consequences for the mental and physical health, well-being and aging of the individual throughout the entire life-span. We have initiated a program of research on humans to examine the consequences of maternal stress and related factors in pregnancy on the length of gestation, fetal growth, and brain development. We have also investigated the physiological mechanisms that are involved. In this chapter we outline the theoretical rationale for this work and give an overview of our findings to date. These findings support a significant and independent role for behavioral processes such as maternal prenatal stress in the etiology of prematurity-related outcomes, and suggest that these effects are mediated, in part, by the maternal-placental-fetal neuroendocrine axis; specifically by placental corticotropin-releasing hormone. Using a fetal challenge paradigm as a novel method for quantifying fetal neurologic maturity in utero, we have found that the maternal environment exerts a significant influence on the fetal autonomic nervous system and on central nervous system processes related to recognition, memory and habituation. Finally, our findings provide preliminary evidence to support the notion that the influence of prenatal stress and maternal-placental hormones on the developing fetus may persist after birth, as assessed by measures of temperament and behavioral reactivity in the first 3 years of postnatal life. The implications of these studies for life-span development and health are discussed.

Introduction Developmental processes involved in transforming a single-cell human embryo into a fully functioning organism whose cerebral cortex alone contains some 109 neurons, within a mere span of 40 weeks, are exceedingly complex and fascinating. It would be

* Corresponding author: Pathik D. Wadhwa, Behavioral Perinatology Research Program, University of California, Irvine, 3117 Gillespie Neuroscience Building, Zot Code 4260 Irvine, CA 92697, USA. Tel.: +1-949-824-8238; Fax: +1-949-824-8218; E-mail: pwadhwa@uci.edu

difficult to find another example in the physical or biological world that even begins to approximate the elegance of intrauterine development. The development of the fetus is characterized by proliferation and specialization of cells to form interconnected functional units. Whereas most of the structural and functional development of major organ systems is essentially complete by the end of the first trimester of gestation, the central nervous system (CNS) continues to grow and to develop throughout gestation and for the first 10-12 years of life after birth. CNS development may be characterized into various stages, overlapping in time. These include the proliferation and migration of neurons and glial cells, the

132 differentiation and maturation of neurons including axonal and dendritic development and synaptogenesis, myelination, and the development of neurotransmitter systems (noradrenergic, dopaminergic, serotonergic, cholinergic, y-aminobutyric acid (GABA), excitatory amino acid and peptidergic). During peak growth periods, neurons are generated at the rate of over 250,000 per minute, and by birth the brain contains over one hundred trillion connections among neurons (Cowan, 1979; Rakic, 1988; Institute of Medicine, 1992). Throughout the ages biologists have questioned whether the genetic material of the fertilized egg already contains a full set of building specifications for the human organism? Over the last few years, there has been a major paradigm-shift in developmental neuroscience regarding fundamental concepts of development and function. The answer to the above question is now believed to be an unequivocal 'no'. Genes and environment are no longer considered to exert separate influences, and development is viewed not as a gradual elaboration of an architectural plan pre-configured in the genes, but rather as a dynamic interdependency of genes and environment. This is characterized by a continuous process of interaction between these two factors in a place- and timespecific manner, and involves short- and long-term information storage, whereby genetic and epigenetic processes become represented in the evolving structural and functional design of the organism at every step of development (Hofer, 1988; Institute of Medicine, 1992; Smotherman and Robinson, 1995). According to this epigenetic view of development, events at one point in time have consequences that are manifested later in the developmental process, and the outcome of the interaction between genes and environment at any point in development has a profound influence on the developmental trajectory (Kolb, 1995). In other words, it appears that within the constraints imposed by the heritable germ line at conception, each developing organism plays an active role in its own construction. This dynamic process is effected by the emergence of various systems during embryonic and fetal life that acquire information about the nature of the environment, and use this information to guide development. In the context of this formulation, not only does environment play a necessary role for development to occur, but the nature of the environment may either play an advantageous role that promotes normal or optimal development, or may play a pernicious role which harms development (Bornstein, 1989). Clearly, if the nature of the environment is perceived to be stressful or hostile, it may promote developmental processes that result in deleterious short- and/or long-term consequences for health. For example, using animal models, several experimental studies have provided powerful evidence to support a causal role for prenatal stress in negatively influencing critical developmental and health outcomes. These negative effects are upon brain morphology and function (cognition, emotionality, and behavior), sexual differentiation, activity of the autonomic nervous, neuroendocrine, immune and reproductive systems, physical health, the time course of normal aging (rate of neuronal loss and cognitive decline), and longevity (Lauder et al., 1981; Fride and Weinstock, 1984, 1988; Peters, 1986, 1988, 1990; Rohde et al., 1989; Insel et al., 1990; Alonso et al., 1991; Ohkawa et al., 1991; Schneider et al., 1992; Sobrian et al., 1992; Takahashi et al., 1992; Schneider and Coe, 1993; Henry et al., 1994; Uno et al., 1994; Bakker et al., 1995; Barbazanges et al., 1996; Coe et al., 1996; Sanchez et al., 1996). These studies have offered valuable insights into putative physiological mechanisms that may be involved in mediating the effects of stressful environments on the developing organism. The ability to generalize these findings from animals to humans may be limited by the existence of inter-species differences in physiology, however. No single system exemplifies the magnitude of these inter-species differences as vividly as the reproductive system, even when compared between otherwise very closely related species such as humans and non-human primates (Smith, 1999). Our group has been working for the last few years on a program of research to examine the interface between biology and behavior in the context of human pregnancy and fetal development. Specifically, we have been interested in evaluating the effects of maternal stress during pregnancy on outcomes related to the length of gestation, fetal growth, and fetal brain development. We have begun to examine putative physiological mechanisms that may mediate the effects of the maternal environment on the devel-

133 oping fetus, with a particular emphasis on the role of the maternal-placental-fetal neuroendocrine axis. From this work we have articulated a neurobiological model of prenatal stress, which proposes that stress exerts a significant and negative influence on fetal developmental outcomes, in part by its effects on placental corticotropin-releasing hormone (CRH) production. This model, which is based on an understanding of the ontogeny of fetal development and the endocrinology of human pregnancy, further hypothesizes that the effects of prenatal stress are outcome-specific, and that they are modulated by the nature, timing and duration of stress (Wadhwa, 1998). The adoption of an epigenetic framework for early development, wherein the organism plays an active role in its own construction by evolving systems to acquire and use information about the nature of the environment to guide development, gives rise to two important questions. Firstly, how do the fetal and maternal compartments communicate with one another? Secondly, in light of the fact that the fetal nervous system is itself in a state of evolution and has yet to acquire its repertoire of structural and functional capabilities, what are the modalities available to the developing fetus to receive, process and act upon information acquired from the environment? There are no direct neural or vascular connections between the mother and her developing fetus, and bi-directional communication is mediated primarily via the exchange of blood-borne chemical signals produced by the endocrine and immune systems, for example (Meschia, 1994). One of the remarkable adaptations of pregnancy is the formation in early gestation of a transient organ of fetal origin - - the placenta. In addition to the long-recognized roles played by the placenta, this organ may also undertake functions that are usually ascribed to the central nervous system, i.e. the capability of receiving, processing and responding to certain classes of external stimuli. Indeed, we believe that a major role of the placenta is to act on behalf of the fetus as both a sensory and effector organ to facilitate the transduction of environmental information for its incorporation into the developmental process. The physiology of placental CRH illustrates this concept. This 41-amino acid neuropeptide is of predominantly hypothalamic origin and is one of the primary mediators through which the brain regulates the activity of the hypothalamo-pituitary-adrenal (HPA) axis and the physiological responses to stress and inflammation (Vale et al., 1981; Chrousos, 1992). However, during human pregnancy, the CRH gene and receptors are also richly expressed in the placenta, and this placental CRH is identical to hypothalamic CRH in structure, immunoreactivity, and bioactivity (Petraglia et al., 1996). The expression of CRH mRNA increases exponentially over the second half of gestation, effecting the production of CRH in the placenta and its release into the maternal and fetal compartments. We and others have proposed various crucial roles for placental CRH in regulating human pregnancy biology, including modulation of maternal and fetal pituitary-adrenal function, participation in fetal cellular differentiation, growth and maturation, and involvement in the physiology of parturition (Challis et al., 1995; Petraglia et al., 1996). Several convergent lines of evidence suggest that the activity of placental CRH is, in turn, regulated by characteristics of the maternal and intrauterine environment. For example, in vitro and in vivo studies have shown placental CRH output to be modulated in a positive, dose-related manner by the major biological effectors of stress responses, including cortisol, catecholamines, oxytocin, angiotensin-II, both forms of interleukin-1, and hypoxia (Petraglia et al., 1987, 1989, 1990; Korebrits et al., 1998a; Marinoni et al., 1998). Therefore we propose that the placenta plays an important role on behalf of the fetus as a sensory and effector organ, and more specifically that placental CRH may mediate the effects of the maternal environment on fetal development and related outcomes.

General methodology
Components of our neurobiological model of prenatal stress have been tested in prospective, longitudinal population-based cohort studies with a combined sample of approximately 375 women with singleton, intrauterine pregnancies, recruited during the second trimester of gestation and followed through delivery into the early postpartum period. Our recruitment strategy ensured heterogeneity in terms of sociodemographic and ethnic characteristics. Based on conventional measures of obstetric risk we included

134 approximately equal numbers of subjects at low- and high-risk for adverse perinatal outcomes. Structured interviews and questionnaires were administered at two or more time points in the second and third trimesters of gestation using standardized and validated instruments to assess maternal psychosocial constructs. These included various forms of prenatal stress, social support, personality, and several maternal behaviors, including diet and nutrition, physical activity, and smoking, alcohol and drug use. Maternal venous and umbilical cord blood (plasma) samples were collected during gestation and at delivery for bioassays of stress hormones, including CRH, adrenocorticotropin (ACTH), ~-endorphin, and cortisol. Obstetric and birth outcomes were abstracted from the medical record. All pregnancies were dated by best obstetric estimate using last menstrual period and early ultrasonographic confirmation. In a subsample of 156 pregnancies, fetal assessments were performed early in the third trimester of gestation. These assessments included fetal biometry, Doppler flow velocimetry of the uteroplacental circulation, and an experimental challenge paradigm to quantify indices of fetal arousal, reactivity, learning and habituation, assessed by fetal heart rate (FHR) responses to a series of vibroacoustic stimuli.
Results Maternal psychosocial processes and birth outcomes

To date, we have completed three studies that have each assessed and provided evidence to support the notion that maternal psychosocial processes significantly predict pregnancy outcomes related to the length of gestation and fetal growth, even after controlling for the effects of established sociodemographic and obstetric risk factors. In the first study, a sample of 90 pregnant women were prospectively assessed using measures of episodic and chronic stress, strain (response to stress), and pregnancy-related anxiety. Results, analyzed using multiple regression analyses, indicated that independent of obstetric risk, each unit increase of prenatal life event stress (from a possible sample range of 14.7 units of life event stress) was significantly associated with a 55 g decrease in infant birth weight and with a 32% increase in the relative risk of

low birth weight (<2500 g). Moreover, each unit increase of prenatal pregnancy-specific anxiety (from a possible sample range of 5 units of pregnancyspecific anxiety) was significantly associated with a 3-day decrease in gestational age at birth (Wadhwa et al., 1993). In the second study, a sample of 230 pregnant women was prospectively assessed using measures of prenatal psychosocial stress (state and pregnancyrelated anxiety), personal resources (mastery, selfesteem, optimism), and socio-cultural factors (income, education, ethnicity). Results, analyzed using structural equation modeling, revealed that women with stronger personal resources had higher birth weight babies (fl = 0.21), whereas those reporting more stress had shorter gestations (fl = -0.20), after controlling for the effects of parity, obstetric risk, sociodemographic and ethnic characteristics (X 2 = 141, P < 0.001; Cumulative Fit Index = 0.92; Killingsworth-Rini et al., 1999). In the third study, a sample of 192 women was prospectively assessed using measures of perceived social support. Structural equation modeling techniques were used to examine the relationship of a latent factor of perceived support to fetal growth (i.e. birth weight adjusted for length of gestation) after controlling for the effects of other established sociodemographic (marital status, socio-economic status, ethnicity) and obstetric factors (parity, medical risk, fetal sex) on birth weight. As predicted, women who perceived greater available support delivered babies with a significantly higher rate of fetal growth. Moreover, the magnitude (effect size) of the social support and fetal growth relationship was comparable and approximately equal to that between the other abovementioned factors and fetal growth (Feldman et al., 2000).
Maternal psychosocial processes and the neuroendocrine axis in human pregnancy

In non-pregnant humans psychological and social factors such as stress and social support exert significant influences on the HPA axis, the major endocrine stress response linkage between the brain and peripheral systems (Axelrod and Reisine, 1984; Chrousos and Gold, 1992; McEwen, 1998). However, this relationship may change in pregnancy, and thus alter

135 the pattern of physiological responses to exogenous stimuli or perturbations such as stress (Monga and Creasy, 1994; Wadhwa et al., 1996). Therefore we have carried out two studies to examine the nature of the relationship between matemal psychosocial processes and this neuroendocrine system during pregnancy. Results from both studies have supported our hypothesis that the maternal psychosocial environment during pregnancy impacts upon maternal physiology, as measured by the functioning of the maternal pituitary-adrenal axis. The first study, conducted in a sample of 54 women with a singleton, intrauterine pregnancy, examined the cross-sectional relationships between maternal measures of prenatal stress and social support and maternal plasma concentrations of pituitary-adrenal hormones (ACTH, [3-endorphin, cortisol) at 28 weeks' gestation. Factors known to influence neuropeptide and glucocorticoid hormone levels during pregnancy, including gestational age, diurnal rhythm and obstetric risk, were controlled for. Our results indicated that maternal stress was positively associated with maternal circulating ACTH and cortisol levels in addition to the pregnancy-associated elevations in baseline concentrations of maternal pituitary-adrenal hormones. In contrast, social support was negatively associated with the circulating concentrations of these hormones. Step-wise hierarchical multiple regression analyses, performed to examine the joint contribution of prenatal demographic and psychosocial factors to the maternal neuroendocrine parameters, indicated that our predictors accounted for 36% of the variance in maternal ACTH and 13% of the variance in maternal cortisol levels (Wadhwa et al., 1996). The second study involved administration of a standard, laboratory-based behavioral stressor - - a speech or mathematics task - - to a sample of 22 healthy, normotensive women (15 pregnant women ranging between 12 and 35 weeks' gestation, and 7 non-pregnant controls). This was used to determine whether mild behavioral stress could evoke a reliable physiological response during pregnancy, to compare the magnitude of responses between pregnant and non-pregnant women, and to examine whether gestational age at testing and baseline physiological states predicted the magnitude of physiological stress reactivity in pregnancy. Sympathetic-adrenalmedullary (SAM) responses were assessed in all subjects by quantifying changes in systolic and diastolic blood pressure and heart rate upon application of behavioral stress. Baseline HPA axis measures (plasma concentrations of ACTH and cortisol) were also obtained in all pregnant women. The possible effects of other factors on biological stress reactivity (i.e. high-risk obstetric conditions, endocrine disorders, circadian rhythms) were controlled for by the study design. The results indicated that each of the behavioral stressors elicited reliable SAM responses in both study groups. The magnitude of these responses was normally distributed across subjects, with sufficient variability for individual-difference analyses. SAM responsivity was significantly attenuated in pregnant subjects compared with non-pregnant controls. Moreover, among pregnant women there was a progressive increase in attenuation of the SAM response with advancing gestational age, accounted for, in part, by baseline levels of ACTH and cortisol (Wadhwa et al., 1997b).

Maternal pituitary-adrenal function and placental CRH in human pregnancy

To clarify the role of maternal pituitary-adrenal function in modulating placental CRH activity in vivo, we conducted a study in a sample of 260 adult women with a singleton, intrauterine pregnancy by examining the associations between maternal plasma CRH, ACTH, ~-endorphin and cortisol concentrations early in the third trimester of pregnancy (31-32 weeks' gestation). A second maternal plasma sample was obtained in a sub-sample of 158 women at 6 weeks postpartum. The results suggested that maternal ACTH and [3-endorphin levels during pregnancy and postpartum were highly correlated (P < 0.001), indicating their origin from a common precursorpro-opiomelanocortin. During pregnancy, CRH levels were significantly and positively correlated with ACTH (P < 0.01) and [3-endorphin (P < 0.001) levels. There was no direct association between CRH and cortisol in the present sample of subjects, however both plasma ACTH and [3-endorphin concentrations were significantly and positively correlated with cortisol concentration (P < 0.001). The time of day of blood sampling was significantly and nega-

136 tively correlated with maternal cortisol levels during pregnancy with higher levels in the morning and lower levels in the evening (r = -0.22, P < 0.001), indicating the presence of an intact circadian rhythm for cortisol. However, an analysis of covariance approach to control for the effects of time of day revealed no changes in the magnitude or significance levels of the above relationships. In contrast to the findings during pregnancy, maternal plasma CRH levels were not related with either ACTH, ~-endorphin, or cortisol during the post-partum period. Thus, these findings support the premise that in human pregnancy placental CRH activity is modulated, in part, in a positive manner by the maternal pituitary-adrenal axis (Wadhwa et al., 1997a). vation of the placental CRH system, as reflected by precocious elevation of maternal CRH levels, may be associated with earlier onset of spontaneous labor and resultant delivery. Secondly, placental CRH may be a marker of antepartum risk for preterm delivery, and therefore an indirect predictor of earlier delivery (Wadhwa et al., 1998a).

Biobehavioral studies of the human fetus

The developing human CNS may be more vulnerable to environmental perturbations than any other system because it develops over a much longer period of time (11-12 years); it has limited repair capabilities; its units have highly specific functional roles; the blood-brain barrier is not fully developed in utero; and the programming of neurotransmitter systems during critical developmental periods affects the organism's response to all subsequent experience (Rodier et al., 1994). However, the influence of the maternal and intrauterine environment on the developing human fetal brain is poorly understood. This is partly because the assessment and quantification of human fetal brain development represents many theoretical and methodological challenges (DiPietro et al., 1996a). We have performed three studies in order to quantify and examine the influence of the fetal environment on brain development. The first study was performed on a sample of 84 fetuses at 31-32 weeks' gestation to examine the ability of the fetus to learn and recall information. Three series of vibroacoustic stimuli were presented at pseudo-random intervals over the fetal head, and FHR responses to the first series of 15 stimuli (S1) were compared with responses to an identical second series of 15 stimuli (S1) separated from the first set by the administration of a single novel stimulus of different intensity and frequency ($2). A significant habituation pattern of responses was observed across trials for both series of stimuli, but this habituation pattern was attenuated for the series following the novel stimulus. These findings suggest that the 32-week-old human fetus may be capable of detecting, habituating and dishabituating to an external stimulus, and support the premise that areas of the human fetal CNS that are critical for some aspects of learning and memory have developed by early in the third trimester (Sandman

Placental CRH and prematurity

Endocrine and paracrine/autocrine roles have been proposed for placental CRH in the physiology of parturition and also in fetal growth and development. We have begun to explore these possibilities by investigating the prospective relationship between concentrations of CRH (derived from the placenta) in maternal plasma early in the third trimester of gestation and the risk for prematurity-related outcomes. This study involved a sample of 63 women with a singleton, intrauterine pregnancy. Maternal plasma was collected at 28-30 weeks' gestation, and CRH concentrations were determined by radioimmunoassay. The results indicated that maternal (placental) CRH levels at 28-30 weeks' gestation significantly and negatively predicted gestational length after adjusting for antepartum risk. Moreover, subjects who delivered preterm had significantly higher CRH levels early in the third trimester than those who delivered at term. In deliveries preceded by spontaneous onset of labor, maternal third-trimester CRH levels significantly and independently predicted earlier onset of labor and preterm labor, whereas in deliveries effected by induction of labor or cesarean section, maternal CRH levels were a marker of antepartum risk but not an independent predictor of gestational length. Thus these findings support the premise that placental CRH may be implicated in the timing of human delivery in at least two ways. Firstly, placental CRH may play a role in the physiology of parturition. Therefore premature or accelerated acti-

137 et al., 1997). In a sub-sample of 33 mother-fetus pairs from the above study, the relationship between maternal (placental) levels of CRH and this pattern of fetal habituation and dishabituation in response to external stimulation was examined. The results indicated that the fetuses of mothers with highly elevated CRH levels did not respond significantly to the presence of the novel stimulus, thereby providing preliminary support for the notion that abnormally elevated levels of placental CRH may play a role in impaired neurodevelopment, as assessed by the degree of dishabituation (Sandman et al., 1999). We have recently performed non-linear statistical analyses on our complete sample of 156 motherfetus pairs studied at 31-33 weeks' gestation. These analyses of FHR arousal and reactivity data, using a non-linear repeated-measures model with autocorrelated errors within subjects and independence across subjects, showed the following. Fetuses exhibited a significant, non-linear increase in FHR in response to the vibroacoustic stimulation protocol. After an initial response period, fetuses exhibited a FHR response decrement to subsequent stimuli, indicating habituation. Baseline FHR, presence of uterine contractions during trials, and characteristics of the challenge protocol such as inter-trial interval significantly influenced the magnitude of FHR responses. After accounting for these variables, maternal conditions related to psychological and physiological stress (i.e. psychosocial stress levels, placental CRH concentrations, umbilical blood flow, and presence of maternal medical risk conditions) were significantly associated with the pattern of FHR responses. Habituation rate, assessed in a two-parameter growth curve (power) model, accounted for approximately 70% of the variance in FHR response. Fetal sex and conditions related to maternal stress (i.e. maternal ACTH concentrations, presence of medical risk conditions) were significantly associated with the rate of habituation; thus these matemal factors are predictive for the differences between individuals in the patterns of fetal responses to external challenges (Wadhwa et al., 1999). This set of findings provides further support for the role of prenatal environment in modulating some aspects of human fetal brain development related to recognition, appraisal, response, memory and habituation.

Prenatal factors and infant developmental outcomes

To date, a very small number of human studies have been conducted to examine the effects of prenatal stress on subsequent infant development. Moreover, many of these studies suffer from methodological limitations such as retrospective design and inadequate control of covariates. Therefore we conducted preliminary studies to assess the relationship of maternal stress and stress hormones during pregnancy and delivery with indices of infant neuro-development. The study samples consisted of 47 motherinfant pairs 6 weeks after delivery, 24 mother-infant pairs 4 months after delivery, and 49 mother-infant pairs 3 years after delivery. All mothers had been participants in our above-described studies of prenatal stress, fetal development and birth outcomes. Questionnaires were administered to mothers of 6week-old infants to assess infant temperament and to mothers of the 3-year-old infants to assess behavioral characteristics of toddlers, including activity level, anger proneness, and social fearfulness. In collaboration with Kagan and colleagues, a laboratory-based behavioral assessment protocol (the Harvard Infant Behavioral Reactivity Protocol, Kagan and Snidman, 1991) was administered to the 4-month-old infants to measure infant motor and cry reactivity to a series of visual and auditory challenges. After adjusting for the effects of maternal influence, intra-partum compromise and prematurity, the overall pattern of results yielded support for the study hypotheses that higher levels of prenatal stress and stress hormones were significantly associated with an infant's temperamental difficulties at age 6 weeks, 4 months, and 3 years. Moreover, in utero measures of fetal arousal and reactivity also significantly predicted infant temperamental difficulties in these infants (Snidman et al., 1998; Wadhwa et al., 1998b).

Summary and conclusions

Our findings to date support a significant and independent role for behavioral processes such as prenatal stress in the etiology of prematurity-related outcomes, and suggest that these effects are mediated, in part, by the maternal-placental-fetal neuroendocrine axis. Our findings also suggest that the use of a fetal challenge paradigm offers a novel way to quantify

138 fetal neurologic maturity in utero, and that the maternal environment exerts a significant influence on the fetal autonomic nervous system as well as central brain processes related to recognition, memory and habituation. Finally, our findings provide preliminary evidence to support the notion that the influence of prenatal stress and maternal-placental hormones on the developing fetus may persist after birth, as assessed by measures of temperament and behavioral reactivity in the first 3 years of postnatal life. Our findings are consistent with our hypothesis and also with the work of several other research groups. In the past few years, several large, population-based epidemiological studies of prenatal stress and prematurity-related outcomes have suggested that high levels of maternal psychosocial stress are associated with a 1.5- to 2.5-fold increase in the adjusted risk for prematurity (Goldenberg et al., 1991; Cliver et al., 1992; Hedegaard et al., 1993, 1996; Pritchard and Teo, 1994; Copper et al., 1996; Nordentoft et al., 1996). Studies of physiological reactivity in pregnancy have reported attenuated responses to exogenous challenges. For instance, pregnancy has been associated with blunted autonomic and endocrine responses to a variety of physical and chemical challenges (Nisell et al., 1985a,b; Barron et al., 1986; Goland et al., 1990; Schulte et al., 1990; Matthews and Rodin, 1992). Longitudinal investigations of the functional development of the human fetal central nervous system over the course of gestation have indicated that chronic maternal psychological distress is significantly related to indices of fetal neurobehavioral maturation. Specifically, greater maternal stress was found to be associated with reduced fetal heart rate variability and reduced coupling between fetal heart rate and movement (DiPietro et al., 1996a,b). Other research groups have demonstrated that placental CRH is involved in the physiology of normal parturition and that elevated CRH concentrations significantly predict risk for spontaneous preterm birth (Campbell et al., 1987; Wolfe et al., 1988, Warren et al., 1992; McLean et al., 1995; Korebrits et al., 1998b; Hobel et al., 1999). Lastly, the small number of studies that have examined the effects of prenatal factors on infant development have reported significant associations of maternal prenatal stress with newborn and infant temperament (Zuckerman et al., 1990; Martin et al., 1997), with infant neurodevelopmental disorders (Mclntosh et al., 1995; Ward, 1990) and with infant psychopathology (Ward, 1991). Although this growing body of work by our group and others indicates that the prenatal environment may influence the development of the fetus and subsequently related outcomes, several questions remain. These questions relate to outcome-specificity, exposure-specificity, the timing of exposure during gestation, and the biological and behavioral mechanism(s) by which the prenatal environment may act upon the developing fetus. We are particularly interested in the importance of the timing of stress exposure during gestation, so we are serially assessing matemal and fetal responses to behavioral stress at three time points in early, mid and late gestation to determine whether there are specific periods of increased or heightened fetal vulnerability. We aim also to understand the biological mechanisms underlying the effects of stress on neural development. Maternal infection has been identified in the obstetric literature as being a particularly important risk factor in the etiology of extreme prematurity, and perhaps also in certain aspects of sub-optimal fetal brain development (Romero et al., 1994). We are exploring the hypothesis that maternal stress may negatively influence immune mechanisms, by altering the balance between Thl and Th2 lymphocyte responses, thus increasing maternal and fetal vulnerability to pathogen exposure. The importance of the types of research outlined above cannot be over estimated. Prematurity is the leading cause of infant mortality and morbidity in the non-anomalous fetus in the United States. The prevalence of prematurity is higher in the USA than in any other developed nation in the world and has not decreased significantly over the last 40 years. The etiology of prematurity is unknown in one-half to two-thirds of all cases and prevention programs to reduce the incidence have largely been unsuccessful (Creasy, 1994). Although the vast majority of premature newborns survive, studies of short-term outcomes find significantly higher rates of severe morbidity in the neonatal period, including asphyxia, meconium aspiration, hypoglycemia, polycythemia and respiratory distress syndrome. Furthermore, studies of long-term outcome have found higher rates of sensorineural impairments and dis-

139 abilities (e.g. cerebral palsy, and visual, auditory and intellectual impairments) and higher rates of complications affecting the respiratory, gastrointestinal and renal systems (Blair and Stanley, 1992; Low et al., t992; Knoches and Doyle, 1993). In the USA over 48 million people, or 15% of the population, suffer from some form of neurological disorder, not including mental disease. A large proportion of these disorders are believed to originate during prenatal life, and elucidating their etiology presents a perplexing neurological problem (Kolb, 1995). Another implication of this research relates to the risk of developing chronic degenerative disease in later life. Recent research suggests that 'set-points' for homeostatic feedback and control mechanisms are determined during intrauterine life and can thereafter alter the individual's response characteristics to her or his environment throughout life. From this work a model has been proposed by which human disease is programmed during fetal life. (Barker, 1998). Given that a fetus is most vulnerable during periods of rapid cell division, it has also been argued that humans are more vulnerable to the noxious effects of an adverse intrauterine environment due to the rapidity and complexity of human growth and development in utero. Epidemiological evidence from several recent population-based studies in Europe, North America, and Asia lends support for this hypothesis. It appears that pre- and perinatal processes, as indicated by measures of growth and size at birth and during early life, significantly predict the risk of chronic degenerative diseases such as hypertension, coronary artery disease, and adult-onset diabetes mellitus over and above those conferred by established physiological and behavioral risk factors, i.e. the magnitude of the effect equals or exceeds that of all other risk factors combined together (Barker, 1998; Nathanielsz, 1999). Some 60 years ago, the Fels study of early development, probably the first systematic investigation of factors that affect development before birth, suggested that "such factors as [maternal] emotional life and activity level during gestation may contribute to the shaping of physical status, behavioral patterns, and postnatal progress of the children they bear" (Sontag, 1941). Although we have come a long way since then, the continued investigation of the interface between biology and behavior in prenatal life promises to realize the full implications of this statement with respect to the nature of our origins and for our health and well-being.

Abbreviations
ACTH CNS CRH FHR GABA HPA mRNA SAM adrenocorticotropin central nervous system corticotropin-releasing hormone fetal heart rate gamma aminobutyric acid hypothalamo-pituitary-adrenal messenger ribonucleic acid sympathetic-adrenal-medullary

Acknowledgements
The authors would like to gratefully acknowledge the invaluable contributions of our collaborators and colleagues to this program of research: Aleksandra Chicz-DeMet, Ph.D., Christine Dunkel-Schetter, Ph.D., Laura Glynn, Ph.D., Kimberly Herbel, M.A., Calvin J. Hobel, M.D., and Manuel Porto, M.D. These studies were supported, in parts, by US PHS (NIH) Grants HD-33506, HD-28413, and HD-28202.

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Progressin BrainResearch,Vol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 10

Alterations in behavioral and neuroendocrine stress coping strategies in pregnant, parturient and lactating rats
Inga D. Neumann *
Department of Zoolog3; University of Regensburg, D-93040 Regensburg, Germany

Abstract: In the present chapter the behavioral and neuroendocrine alterations accompanying pregnancy and lactation will be discussed. It will be shown that many are dependent on the innate level of emotionality of the rats. In late pregnancy the level of anxiety, as measured on the elevated plus-maze is increased in rats with both high and low level of innate anxiety-related behavior, whereas lactating rats display less anxiety in such tests and higher degrees of aggressive behavior in tests for agonistic behavior. There is a dramatic reduction in the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis to various physical or emotional stimuli in both pregnant and lactating rats. This appears to be due to changes throughout the HPA axis. Oxytocin has been implicated in the control of the axis at this time, but the inhibitory action of central oxytocin on ACTH or corticosterone secretion seen in virgin female rats is not evident during pregnancy and lactation. However, central oxytocin is involved in the regulation of emotionality at this time. In addition to its anxiolytic effect, prolactin, acting at brain prolactin receptors, seems to exert an inhibitory effect on HPA axis responsiveness. At the time of parturition, the HPA axis is not stimulated by parturition-related stimuli and is under strong inhibition by endogenous opioids as revealed by the application of the opioid receptor antagonist naloxone.

Introduction

In the period around parturition, a variety of behavioral and neuroendocrine alterations have been reported in relation to stress responsiveness in several species including rats, sheep, and humans due to adaptations at almost all levels of the limbic-hypothalamo-pituitary-adrenal (HPA) axis. This may include changes in the process of stressor perception at limbic brain regions, i.e. the emotional evaluation of an external stimulus, resulting in: (1) a changed behavioral response to a given stressor (Walker et al., 1995; Windle et al., 1997a; Neumann et al., 1998a, see also Lightman et al., 2001, this volume); and (2) alterations of the excitatory

* Correspondence to: Inga D. Neumann, Department of Zoology, University of Regensburg, D-93040 Regensburg, Germany Tel.: +49-941-943-3055; Fax: +49-89-941-9433055; E-mail: inga.neumann@biologie.uni-regensburg.de

and inhibitory inputs to hypothalamic neurons of the paraventricular (PVN) and/or supraoptic nuclei (Toufexis and Walker, 1996; Douglas et al., 1998) synthesizing corticotropin releasing hormone (CRH), oxytocin or vasopressin. Consequently, the activity of CRH and vasopressin neurons which represent the major regulators of corticotropin (ACTH) synthesis and secretion at the level of the adenohypophysis of the HPA axis is expected to be changed. Down regulation, for example, of the CRH system including CRH synthesis in the PVN (Douglas and Russell, 1994) and CRH binding in the adenohypophysis (Neumann et al., 1998a) reported in late pregnancy can, at least in part, account for the reduced release of ACTH and consequently of corticosterone into blood in response to an external stimulus in both pregnancy and lactation (humans, Altemus et al., 1995; rats, Stern et al., 1973; Lightman and Young III, 1989; Walker et al., 1995; Windle et al., 1997a; Neumann et al., 1998a; sheep, Keller-Wood, 1994).

144 In addition to these changes in the feed-forward processing of received stimuli, alterations of negative feedback mechanisms of the HPA axis exerted by circulating glucocorticoids via mineralo- and glucocorticoid receptors at limbic, hypothalamic and adenohypophyseal levels may result in a shift of the excitatory set point of the stress response in the peripartum period (Johnstone et al., 1998, 2000). Furthermore, although highly activated in the peripartum period in order to guarantee reproductive functions, the response of the oxytocin system to stimuli not directly linked to reproductive events is attenuated as indicated by a reduced oxytocin secretion into blood in response to various stimuli (Carter and Lightman, 1987; Neumann et al., 1995, 1998a; Walker et al., 1995, see Douglas and Russell, 2001, this volume). The enhanced activity of the brain oxytocin system in the peripartum period leads to the hypothesis of an involvement of oxytocin in the behavioral and neuroendocrine adaptations seen at this time. This chapter will focus on: (1) alterations in behavioral stress coping strategies including anxietyrelated, agonistic and social behaviors of pregnant and lactating rats and the involvement of brain oxytocin in the regulation of these behaviors; (2) the basal and stress-induced activity of the HPA axis in late pregnancy and in lactation, and the possible involvement of intracerebral modulators of the HPA axis like oxytocin and prolactin; and (3) on the activity of the HPA axis to parturition-related stimuli during undisturbed delivery in rats. day 16 through to day 19 with maximal aggressive responses a few hours before parturition when rigorous and regular uterine contractions begin; this is seen also immediately postpartum in the presence of the litter (Rosenblatt et al., 1994). Maternal aggression is exemplified by an increased frequency of attacks or threats towards an intruder rat in the home cage (Mayer et al., 1987, Rosenblatt et al., 1994). An enhanced level of aggression, however, is expected to be related to a rather low level of anxietyrelated behavior. We have studied the anxiety-related behavior of virgin, pregnant and lactating rats by measuring the exploratory behavior of the open and exposed arms of the elevated plus-maze (Pellow et al., 1985). Beginning on day 15 of pregnancy, female Wistar rats showed a reduced percentage of time spent on the open arms, indicating an enhanced level of anxiety (Fig. I). However, only on day 18 was the level of anxiety found to be reduced and comparable to that seen in virgin and day 10 pregnant rats. These findings are of interest with respect to the distinction between home cage aggression/maternal aggression on the one hand (Rosenblatt et al., 1994) which ensures a safe place for the delivery process and protection of the well-being of mother and pups,

% entries 30 20 10 V % time 2o 10 D10 D15 D21

Behavioral adaptations in the peripartum period

Around parturition, the behavior of the female rat changes in order to ensure the offspring's survival. The most obvious behavior seen is the performance of maternal care, beginning immediately with the appearance of the pups during delivery which includes licking, grooming, nest-building, placentophagia and crouching over the pups. Another important aspect of maternal behavior is enhanced aggression and reduced fearfulness in the face of threat as a successful mechanism for the protection of the pups (Erskine et al., 1978; Hard and Hansen, 1984; for review see Rosenblatt et al., 1994). Thus, for example, aggressive behavior increases gradually from gestation

D10 D15

D21

Fig. 1. Anxiety-relatedbehaviorof virgin (V)and day 10, day 15 and day 21 pregnant rats on the elevatedplus-maze as indicated by the percentageof entries into and percentageof time spent on the open arms of the maze during a 5-min testing period. *P < 0.05, **P < 0.01 versus virgin (one-wayANOVA followed by Newman-Keuls post-hoc test; adapted from Neumann et al., 1998a).

145
I [] virgin
% time 30 20 10 30 20 10 pregnant I

% ;ntries
40

LAB

HAB

LAB

HAB

Fig. 2. Anxiety-related behavior on the elevated plus-maze of virgin (white columns) and day 18-21 pregnant rats (black columns) of the breeding lines with low (LAB) and high (HAB) innate level of anxiety as indicated by the percentage of entries into and percentage of time spent on the open arms of the maze during a 5-min testing period. #P < 0.05 versus virgin rats of the same breeding line; *P < 0.05 versus respective LAB rats (one-way ANOVA followed Tukey-Kramer procedure; adapted from Neumann et al., 1998b).

respectively, and risk assessment/anxiety-related behavior in an unknown and potentially dangerous environment on the other hand. Thus, an increased aggressive behavior in the home cage does not seem to be necessarily accompanied by a reduced level of anxiety-related behavior, as is evident with the progression of pregnancy. The increased anxiety-related behavior found at the end of pregnancy appears to be independent of the basal state of emotionality seen before pregnancy occurs (Fig. 2). We have compared the exploratory activity on the elevated plus-maze between two rat breeding lines, differing in their innate level of anxiety-related behavior (Liebsch et al., 1998), in late pregnancy. In both rat lines with high (HAB) and low (LAB) anxiety-related behavior, respectively, the exploration of the open arms of the maze was further reduced in both HAB and LAB rats between days 18 and 21 of pregnancy compared to the respective virgin group (Neumann et al., 1998b). Thus, irrespective of the initial anxiety level, pregnancy evidently increased anxiety-related behavior. In lactating rats, there is good evidence for increased maternal aggression towards intruders in the home cage as described above. However, it is not clear whether this aggressive behavior is only seen in the presence of the offspring. Furthermore, it is not clear whether, like the pregnant rat this aggression

is associated with an altered level of anxiety-related behavior on the elevated plus-maze. Therefore we have compared the aggressive behavior of virgin and lactating rats in a test for agonistic behavior in a 'neutral' cage as well as the anxiety-related behavior on the plus-maze. Lactating rats with litters adjusted to between 8 and 11 pups were tested between days 4 and 12 of lactation. The test for agonistic behavior is based upon the competition for a water bottle between two rats water-deprived for 48 h and differing in reproductive state (i.e. one lactating, one virgin). The two rats were placed in a neutral cage and the frequency and duration of drinking of both animals was monitored during a 10-min test period. Since in lactation the osmotic threshold is changed (Koehler et al., 1993) and thus a dehydration-induced osmotic challenge may not be comparable to that in virgin rats, we adapted this test by infusing angiotensin II (2 ng/5 Ixl) into the lateral cerebral ventricle of both the virgin and lactating rat just before the animals were introduced into the novel, neutral cage. Angiotensin II given via an intracerebroventricular (i.c.v.) route induces a robust and stereotype dipsogenic effect within 1-3 min after treatment. In this test we observed that lactating rats were more successful in competing for the water bottle, spending more time drinking during the first 5 min of the test period than the virgins (Fig. 3, lower panel). This indicates an enhanced level of aggressive behavior of lactating rats also in situations when the protection of the litter is not directly involved. In contrast to late pregnant rats, the enhanced level of home cage aggression and agonistic behavior in a neutral cage was accompanied by a reduced level of anxiety-related behavior on the elevated plus-maze. However, this was only evident when rats were transported to the neighboring testing room and separated from their pups immediately prior to testing (Fig. 3, upper panel), but not after the rats had been kept in the testing room for at least 24 h and separated from their pups 2 h before testing. Therefore, uninterruption may be important for reduced anxiety-related behavior in lactation; this may be a consequence of the anxiolytic effect of oxytocin released in the brain during nursing (Moos et al., 1989; Neumann and Landgraf, 1989; Kendrick and Keverne, 1992; Neumann et al., 1993).

146

Fig. 3. Anxiety-relatedbehavior, as indicated by the percentage of entries into and percentage of time spent on the open or closed arms of the maze and the latency to enter the open arms of the maze during a 5-min testing period (upper panel), and agonistic behavior following i.c.v, angiotensin II injection (2 ng/5 Izg; lower panel) of virgin (white columns) and mid-lactating rats (black columns). *P < 0.05, **P < 0.01 versus virgin (Mann-Whitney U-test).

Anxiolytic action of brain oxytocin in the peripartum period

There is a dramatic variation in plasma estrogen, progesterone and testosterone concentrations during pregnancy and lactation with a continuous rise in the three sex steroids with the progression of pregnancy and a rapid fall in plasma progesterone and testosterone between days 18 and 21 of pregnancy. These hormonal changes have been shown to form, at least partly, an essential foundation for some aspects of emotionality, i.e. aggressive and anxiety-related behaviors (Mayer and Rosenblatt, 1987; Rosenblatt et al., 1994; for review see Albert et al., 1992). Oxytocin, a neuropeptide closely linked to reproductive functions and maternal care (for review see Richard et al., 1991), is a candidate which has also to be considered in the regulation of these behav-

iors in the peripartum period. There are widespread intracerebral projections of oxytocinergic neurons, originating mainly in the hypothalamic PVN, within the limbic brain regions where a high density of oxytocin receptors has been reported (Brinton et al., 1984; Insel, 1990; Broad et al., 1993). Oxytocin is released within these regions in response to stressful and threatening stimuli in both male (Wotjak et al., 1998; Nishioka et al., 1998) and female (Wigger and Neumann, unpublished) rats, consistent with a role for oxytocin as a neurotransmitter/neuromodulator involved in stress coping strategies. Furthermore, anxiolytic and/or antidepressive effects have been described after oxytocin treatment in female mice and both male and female rats (Uvn~is-Moberg et al., 1994; McCarthy et al., 1996; Windle et al., 1997b). In order to evaluate the involvement of endogenous oxytocin in the regulation of anxiety-related

147

vehicle %

10XT-antagonist ] pregnant (6/7)

lactating (6/7)

entries time entries time entries time Fig. 4. Effects of blockade of brain oxytocin receptors by infusion of an oxytocinreceptor antagonist (0.75 Ixg/5 Ixl; black columns) or vehicle (gray columns) into the lateral ventricle on anxiety-relatedbehavior of virgin, day 19-21 pregnant and mid-lactating rats on the elevated plus-maze as indicated by the percentage of entries into and percentage of time spent on the open arms of the maze during a 5-min testing period. *P < 0.05 versus vehicle (two-wayANOVAfollowed by Newman-Keulspost-hoc test; adapted from Neumann et al., 1999a).

behavior and to compare the extent of oxytocin action at brain level between virgin, male, pregnant and lactating rats, a selective oxytocin receptor antagonist (0.75 tzg/5 Ixl, des Gly-NH2 d(CH2)5 [Tyr(Me)2,Thr4]OVT (ornithine vasotocin), Dr. M. Manning, Toledo, OH, USA) was given into a lateral cerebral ventricle 10 min prior to testing on the elevated plus-maze. To ensure stress-free conditions an extension line filled with vehicle or the oxytocin antagonist was connected to the i.c.v, guide cannula 2 h before infusion. Blockade of brain oxytocin receptor action significantly increased the exploration of open arms of the plus-maze in both pregnant and lactating rats (the latter were separated from their pups 2 h before test), indicating an anxiolytic action of intracerebral oxytocin in the peripartum period (Fig. 4; Neumann et al., 1999a). In contrast, a similar effect was not seen in either virgin female (Fig. 4) or male rats (Neumann et al., 1999b). Our preliminary results indicate that the central amygdala is a possible limbic site of this anxiolytic effect of oxytocin at least in pregnant rats (Neumann et al., 2000).

Reduced responsiveness of the HPA axis to non-reproduction-related stimuli: no effects of brain oxytocin
In contrast to the behavioral stress response it is well established that neuroendocrine responses to stressors including ACTH, corticosterone and/or oxytocin secretion into blood are reduced both at the

end of pregnancy and in lactation. A variety of stressors has been studied like immobilization, ether and osmotic stress, i.c.v. CRH administration, exposure to the elevated plus-maze, noise stress and forced swimming as well as treadmill exercise in humans all reveal attenuated HPA axis and/or oxytocin responses. These adaptations start to appear around day 15 of rat pregnancy and are probably due, at least in part, to a reduction in basal CRH synthesis in the PVN (Douglas and Russell, 1994), reduced CRH binding in the adenohypophysis and reduced CRH-stimulated ACTH secretion (Neumann et al., 1998a). Furthermore, there is a loss of excitatory inputs to the HPA axis, which has been described in virgin rats to involve endogenous opioids (Douglas et al., 1998) and noradrenaline (Toufexis and Walker, 1996). This is accompanied by a change in stress-induced expression of c-fos mRNA in limbic, hypothalamic and brainstem regions (Da Costa et al., 1996). Because of the increased activity of the brain oxytocinergic system beginning at the end of pregnancy and in lactation described above, oxytocin seems a likely candidate to have an inhibitory effect on HPA axis responsiveness at the brain level. In fact, chronic i.c.v. treatment of ovariectomized, virgin rats with synthetic oxytocin reduced the corticosterone response to noise stress (Windle et al., 1997b). To study the involvement of endogenous oxytocin in the attenuation of the HPA axis stress response in pregnant and lactating rats, we infused a selective oxytocin receptor antagonist (i.c.v., see section entitled Anxiolytic

148

[ ] vehicle
A. plus-maze

OXT-antagonist I
B. swim

--~ 600]

"8 0 0i'
virgin pregnant lactating virgin pregnant lactating
E ~

< 200]

6ooti i t
8t 200
.

-++-

ii
virgin pregnant lactating
virgin pregnant lactating

Fig. 5. Effects of blockade of brain oxytocin receptors by infusion of an oxytocin receptor antagonist (0.75 Itg/5 txl; black columns) or vehicle (white columns) into a lateral cerebral ventricle on the increase (delta) in plasma ACTH (upper panels) and corticosterone (lower panels) concentrations in response to exposure to the elevated plus-maze (left) or to 60 s of forced swimming (21C; right) of virgin, pregnant and lactating rats. *P < 0.05, **P < 0.05 versus vehicle; +P < 0.05, ++P < 0.01 versus virgin (two-way ANOVA followed by Newman-Keulspost-hoc test; adapted from Neumann et ai., 1999a). action of brain oxytocin in the peripartum period) 10 min prior to exposure to the plus-maze and swimming, respectively, on two consecutive days. While in virgin animals there was a significant dis-inhibition of stress-induced ACTH and corticosterone secretion by the antagonist independent of the stressor, this effect was absent in pregnant and lactating rats. Thus, the oxytocin antagonist did not affect the reduced ACTH and corticosterone secretory responses in pregnant and lactating rats after stress, compared to vehicle-treated virgin rats (Fig. 5). These results clearly indicate that endogenous intracerebral oxytocin does not significantly participate in the attenuation of the HPA axis responsiveness seen in late pregnancy and in lactation. However, stimuli relevant for the lactating animal like vocalization of the isolated pups or defense of the home cage against an intruder rat have not been studied with respect to neuroendocrine responses and oxytocin involvement.

Putative involvement of brain prolactin in behavioral and neuroendocrine adaptations

In addition to oxytocin, prolactin has also to be considered in the regulation of behavioral and neuroendocrine alterations seen in the peripartum period. In particular, prolactin has extensively been described to be involved in the regulation of maternal behavior (Bridges et al., 1990; Dutt et al., 1994; Lucas et al., 1998). Brain prolactin receptors, which belong to the superfamily of hematopoietin/cytokine/growth hormone-prolactin receptors, have been localized with highest density (Crumeyrolle-Arias et al., 1993; Pi and Grattan, 1998) and predominance of the long form (Chiu and Wise, 1994) in the choroid plexus, but also throughout the hypothalamus and in limbic brain regions. In this context it is of interest to note that there is a selective and active transport of plasma prolactin into the cerebrospinal fluid (Mangurian et al., 1992). In addition, prolactin immunoreactivity

149 has been described in the hypothalamic PVN, the medial preoptic area, septal nuclei and amygdala (Paut-Pagano et al., 1993) - - areas known to be involved in the regulation of maternal behavior, HPA axis activity and emotionality. Indeed, intracerebral infusion of ovine prolactin increased the open ann exploration of both virgin female and male rats on the elevated plus-maze (Torner and Neumann, 1998; Torner et al., 2001). This points toward the possibility that the brain prolactin system is activated in the peripartum period and is involved in the regulation of the complex behavioral changes including anxiety-related behavior seen at this time. Recently, increased prolactin receptor binding has been described in lactating rats (Pi and Grattan, 1999) supporting this hypothesis. Furthermore, co-localization of prolactin fragments and vasopressin/oxytocin in PVN and supraoptic neurons (Mejia et al., 1997) implies an involvement of brain prolactin in neuroendocrine functions. In fact, chronic intracerebral treatment with ovine prolactin (5 days, osmotic minipumps) increases basal ACTH and oxytocin secretion and attenuates the ACTH and oxytocin stress responses in virgin female rats (Torner et al., 1999, 2001). In the absence of a selective prolactin receptor antagonist we are currently using antisense oligodeoxynucleotides targeting the long (active) form of the prolactin receptor (Torner et al., 2001), in order to investigate whether the brain prolactin system plays a significant role in the reduction of the neuroendocrine stress response to non-reproduction related stimuli in the peripartum period (Torner et al., 1999). during the delivery process in chronically catheterized rats revealed a decline in HPA axis secretory activity after delivery of the second pup compared to pre-parturition values sampled within 3 h before appearance of the first pup (Wigger et al., 1999). The levels of circulating ACTH and corticosterone remained low throughout parturition and were found to be independent of the number of pups born in the 10-rain inter-sampling intervals (Fig. 6, left panel). At this time, the HPA axis activity is under efficient inhibition by endogenous opioids since treatment with the opioid receptor antagonist naloxone strikingly increases ACTH secretion (Fig. 6, fight panel). It is interesting to note that, in contrast, in virgin rats endogenous opioids exert a stimulatory action on the stress-induced release of ACTH, an effect which is absent in late pregnancy (Douglas et al., 1998, see Douglas and Russell, 2001, this volume). During parturition, however, the effects of endogenous opioids seem to be further reversed into a strong inhibition of the HPA axis; perhaps this protects the mother rat and/or the unborn pups from excessive metabolic changes due to high plasma glucocorticoids during birth. Whether brain oxytocin is involved in the efficient inhibition of the HPA axis reactivity to parturitionrelated stimuli is still to be investigated. It has been mentioned above that brain oxytocin exerts a tonic inhibitory effect on the secretion of ACTH and corticosterone under basal conditions and in response to stress only in male and virgin female, but not in pregnant and lactating rats. In these experiments, however, lactating rats were studied 2-4 h after separation from their litter and therefore in the absence of the suckling stimulus and intracerebral oxytocin release. During the parturition process an elevated release of oxytocin in a variety of brain regions (Landgraf et al., 1991 ; Kendrick and Keverne, 1992) has been reported including the PVN (Neumann et al., 1993) where CRH neuronal activity and, thus, HPA axis functions, might be regulated. In this context it is of interest to note that an inhibition of basal ACTH secretion by oxytocin could be localized to the PVN of both male and virgin female rats by bilateral retrodialysis of the oxytocin antagonist (Neumann et al., 1999b). In summary, a variety of behavioral and neuroendocrine adaptations are associated with preg-

Non-responsiveness of the HPA axis to parturition-related stimuli

The parturition process in the rat with the delivery of approximately 12-15 pups lasts about 90 rain and is characterized by significant behavioral activation of the dam. This includes nest-building before and during ongoing parturition, the experience of labor, licking of the newborn pups, placentophagia and intensive grooming. The enhanced activity is also reflected by an increase in plasma lactate measured at approximately 30 min after the onset of parturition as an indicator of general motor and metabolic activity (Wigger et al., 1999). However, monitoring plasma ACTH and corticosterone concentrations before and

150

_~

5OOT

V
,~t k'

2001

400 T'

g ,oo

Lvv

001 00r
. , 60 1 ~ 0

. _ /
0

\v
~ 40 60 min

. . . . .

basal 1./2.A+10 20 40 pu~L ~

basal 1./2.~+10 20 pul~ I

Fig. 6. Plasma ACTH concentrations during parturition of two representative rats treated with either vehicle or naloxone (NLX, 5 mg/kg, i.v.) after delivery of the 2nd pup. Arrows indicate the delivery of single pups.

nancy, parturition and lactation. Typical reproductive hormonal systems like the oxytocin and prolactin systems are activated at this time as reflected by enhanced secretion into blood in response to reproduction-related stimuli (birth process, lactation, milk ejection). Simultaneous release within various brain regions may directly (maternal behavior, autoregulation of oxytocin secretion) or indirectly (reduction of anxiety, attenuation of stress responsiveness) support these reproductive functions. Abbreviations
ACTH CRH HAB HPA LAB OVT PVN

References
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Acknowledgements I would like to thank Drs. A.J. Douglas and J.A. Russell for fruitful cooperation throughout many of the studies reported. Furthermore, I would like to acknowledge the contribution of my co-workers Alexandra Wigger, Luz Torner, Nicola Toschi, Agnes Pohlinger and Simone Krrmer. Supported by a Heisenberg stipend provided to IDN by the Deutsche Forschungsgemeinschaft.

151

aventricular nucleus during pregnancy and parturition in the rat. Gene Then, 1(Suppl.): $85. Douglas, A.J. and Russell, J.A. (2001) Endogenous opioid regulation of oxytocin and ACTH secretion during pregnancy and parturition. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingrain (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 67-82. Douglas, A.J., Johnstone, H.A., Wigger, A., Landgraf, R., Russell, J.A. and Neumann, I.D. (1998) The role of endogenous opioids in neurohypophysial and hypothalamo-pituitaryadrenal axis hormone secretory responses to stress in pregnant rats. J. Endocrinol., 158: 285-293. Dutt, A., Kaplitt, M.G., Kow, L.M. and Pfaff, D.W. (1994) Prolactin, central nervous system and behavior: a critical review. Neuroendocrinology, 59: 413-419. Erskine, M.S., Barfield, R.J. and Goldman, B.D. (1978) Intraspecific fighting during late pregnancy and lactation in rats and effects of litter removal. Behav. Biol., 23:206-213. Hard, E. and Hansen, S. (1984) Reduced fearfulness in the lactating rat. Physiol. Behav., 35: 641-643. Insel, T.R. (1990) Regional changes in brain OT receptors postpartum: time-course and relationship to maternal behaviour. J. Neuroendocrinol., 2: 539-545. Johnstone, H.A., Seckl, J.R. and Russell, J.A. (1998) Corticosteroid feedback on the hypothalamo-pituitary-adrenal axis in the late pregnant rat. J. Endocrinol., 156(Suppl.): P182. Johnstone, H.A., Wigger, A., Douglas, A.L, Neumann, I.D., Landgraf, R., Seckl, J.R. and Russell, J.A. (2000) Attenuation of hypothalamic-pituitary-adrenal axis stress responses in late pregnancy: changes in feed-forward and feedback mechanisms. J. Neuroendocrinol., 12:811-822. Kendrick, K.M. and Keverne, E.B. (1992) Control of synthesis and release of oxytocin in the sheep brain. Ann. New York Acad. Sci., 652: 102-121. Keller-Wood, M. (1994) Reflex regulation of hormonal responses during pregnancy. Clin. Exp. Pharmacol. Physiol., 22: 143151. Koehler, E.M., McLemore, G.L., Tang, W. and Summy-Long, Y.J. (1993) Osmoregulation of the magnocellular system during pregnancy and lactation. Am. J. Physiol., 264: R555R560. Landgraf, R., Neumann, I. and Pittman, Q.J. (1991) Septal and hippocampal release of vasopressin and oxytocin during late pregnancy and parturition in the rat. Neuroendocrinology, 54: 378-383. Liebsch, G., Linthorst, A., Neumann, I.D., Reul, J.M.H.M., Holsboer, F. and Landgraf, R. (1998) Behavioral, physiological, and neuroendocrine stress responses and differential sensitivity to diazepam in two Wistar rat lines bred for high and low anxiety-related behavior. Neuropsychopharmacology, 19: 381-396. Lightman, S.L. and Young III, W.S. (1989) Lactation inhibits stress-mediated secretion of corticosterone and oxytocin and hypothalamic accumulation of corticotropin-releasing factor

and enkephalin messenger ribonucleid acids. Endocrinology, 124: 2358-2364. Lightman, S.L., Windle, R.J., Wood, S.A., Kershaw, Y.M., Shank, N. and Ingram, C.D. (2001) Peripartum plasticity within the hypotbalamo-pituitary-adrenal axis. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingram (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 111-142. Lucas, B.K., Ormandy, C.J., Binart, N., Bridges, R.S. and Kelly, P.A. (1998) Null mutation of the prolactin receptor gene produces a defect in maternal behavior. Endocrinology, 139: 4102-4107. Mangurian, L.P., Walsh, R.J. and Posner, B.I. (1992) Prolactin enhancement of its own uptake at the choroid plexus. Endocrinology, 131: 698-702. Mayer, A.D. and Rosenblatt, J.S. (1987) Hormonal factors influence the onset of maternal aggression in laboratory rats. Aggress. Behav., 21: 253-267. Mayer, A.D., Reisbick, S., Siegel, H.I. and Rosenblatt, J.S. (1987) Maternal aggression in rats: changes over pregnancy and lactation in a Sprague-Dawley strain. Aggress. Beha~:, 13: 29-43. McCarthy, M.M., McDonald, C.H., Brooks, P.J. and Goldmann, D. (1996) An anxiolytic action of oxytocin is enhanced by estrogen in the mouse. Physiol. Behav., 60: 1209-1215. Mejia, S., Morales, M.A., Zetina, M.E., Martinez de la Escalera, G. and Clapp, C. (1997) Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei. Neuroendocrinology, 66: 151-159. Moos, E, Poulain, D.A., Rodriguez, E, Gueme, Y., Vincent, J.D. and Richard, P. (1989) Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats. Exp. Brain Res., 76: 593-602. Neumann, I. and Landgraf, R. (1989) Septal and hippocampal release of oxytocin, but not vasopressin, in the conscious lactating rat during suckling. J. Neuroendocrinol., 1: 305-308. Neumann, I., Russell, J.A. and Landgraf, R. (1993) Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: a microdialysis study. Neuroscience, 53: 65-75. Neumann, I., Landgraf, R., Bauce, L. and Pittman, Q.J. (1995) Osmotic responsiveness and crosstalk involving oxytocin, but not vasopressin or amino acids, between the supraoptic nuclei in virgin and lactating rats. J. Neurosci., 15: 3408-3417. Neumann, I.D., Johnstone, H.A., Hatzinger, M., Liebsch, G., Shipston, M., Russell, J.A., Landgraf, R. and Douglas, A.J. (1998a) Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes. J. Physiol., 508: 289-300. Neumann, I.D., Wigger, A., Liebsch, G., Holsboer, F. and Landgraf, R. (1998b) Increased basal activity of the hypothalamopituitary-adrenal axis during pregnancy in rats bred for high anxiety-related behavior. Psychoneuroendocrinology, 23: 449463. Neumann, I., Torner, L. and Wigger, A. (1999a) Brain oxytocin:

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differential inhibition of neuroendocrine stress responses and anxiety-related behavior in virgin, pregnant and lactating rats. Neuroscience, 95: 567-575. Neumann, I.D., Wigger, A., Torner, L., Holsboer, E and Landgraf, R. (1999b) Brain oxytocin inhibits basal and stress-induced activity of the hypothalamo-pituitary-adrenal axis in male and female rats: partial action within the paraventricular nucleus. J. Neuroendocrinol., 12: 235-243. Neumann, I.D., Kr6mer, S.A., Toschi, N., Ebner, K. (2000) Brain oxytocin inhibits the (re)activity of the hypothalamopituitary-adrenal axis in male rats: involvement of hypothalamic and limbic brain regions, Regul. Pept., 1: in press. Nishioka, T., Anselmofranci, J.A., Li, P., Callahan, M.E and Morris, M. (1998) Stress increases oxytocin release within the hypothalamic paraventricular nucleus. Brain Res., 781:57-61. Paut-Pagano, L., Roky, R., Valatx, J.L., Kitahama, K. and Jouvet, M. (1993) Anatomical distribution of prolactin-like immunoreactivity in the rat brain. Neuroendocrinology, 58: 682-695. Pellow, S., Chopin, P., File, S.E. and Briley, M. (1985) Validation of open:closed arms entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods, 14: 149167. Pi, X.J. and Grattan, D.R. (1998) Distribution of prolactin receptor immunoreactivity in the brain of estrogen-treated, ovariectomized rats. J. Comp. Neurol., 394: 462-474. Pi, X.J. and Grattan, D.R. (1999) Increased prolactin receptor immunoreactivity in the hypothalamus of lactating rats. J. Neuroendocrinol., 11: 693-705. Richard, P., Moos, E and Freund-Mercier, M.J. (1991) Central effects of oxytocin. PhysioL Rev., 71: 331-370. Rosenblatt, J.S., Factor, E. and Mayer, A.D. (1994) Relationship between maternal aggression and maternal care in the rat. Aggress. Behav., 20: 243-255. Stern, J.M., Goldman, L. and Levine, S. (1973) Pituitary-adrenal response during lactation in rats. Neuroendocrinology, 12: 179-191. Torner, L. and Neumann, I.D. (1998) Effects of intracerebral prolactin on anxiety-related behavior and HPA axis activity in rats. 28th Annual Meeting of the Society for Neuroscience, 371.19 (abstract).

Torner, L., Toschi, N., Pohlinger, A., Probst, C., Lrrscher, E and Neumann, I.D. (1999). Blockade of prolactin (PRL) receptors by antisense targeting and ICV infusion of PRL affect anxiety-related behavior and neuroendocrine responses to stress. 29th Annual Meeting of the Society for Neuroscience, 64.8 (abstract). Torner, L., Toschi, N., Pohlinger, A., Landgraf, R. and Neumann, I.D. (2001). Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of anitsense targeting of the prolactin receptor by molecular modeling. J. Neurosci., 21: 3207-3214. Toufexis, D.J. and Walker, C.D. (1996) Noradrenergic facilitation of the adrenocorticotropin response to stress is absent during lactation in the rat. Brain Res., 737: 71-77. Uvn~is-Moberg, K., Ahlenius, S., Hillegaart, V. and Alster, P. (1994) High doses of oxytocin cause sedation and low doses cause an anxiolytic-like effect in male rats. Pharmacol. Biochem. Behav., 49: 101-106. Walker, C.-D., Trottier, G., Rochford, J. and Lavallre, D. (1995) Dissociation between behavioral and hormonal responses to the forced swim stress in lactating rats. J. Neuroendocrinol., 7: 615-622. Wigger, A., Oehler, I., Lrrscher, P., Keck, M., Naruo, T. and Neumann, I.D. (1999) Non-responsiveness of the HPA axis to parturition-related events in the rat: involvement of endogenous opioids. Endocrinology, 140: 2843-2849. Windle, R.J., Wood, S., Shanks, N., Perks, P., Conde, G.L., da Costa, A.P.C., Ingram, C.D. and Lightman, S.L. (1997a) Endocrine and behavioral responses to noise stress: comparison of virgin and lactating female rats during non-disrupted maternal activity. J. Neuroendocrinol., 9: 407-414. Windle, R.J., Shanks, N., Lightman, S.L. and Ingram, C.D. (1997b) Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. Endocrinology, 138: 2829-2834. Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, E, Landgraf, R. and Engelmann, M. (1998) Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience, 85: 1209-1222.

J.A. Russell et al. (Eds.)

Progress in BrainResearch, Vol. 133

@ 2001 Elsevier Science B.V. All rights reserved

CHAPTER 11

The actions of prolactin in the brain during pregnancy and lactation 1

D a v i d R. Grattan *
Department of Anatomy and Structural Biology, School of Medical Sciences and Neuroscience Research Centre, Universi~ of Otago, Dunedin, New Zealand

Abstract: The vital role played by prolactin during pregnancy and lactation is emphasized by the physiological adaptations that occur in the mother to maintain a prolonged state of hyperprolactinemia. In many species the placenta provides a source of lactogenic hormones in the circulation, ensuring the continued presence of a hormone capable of activating the prolactin receptor throughout pregnancy. In addition, the tuberoinfundibular dopamine neurons, which normally maintain a tonic inhibitory influence over prolactin secretion, show a reduced ability to respond to prolactin during late pregnancy and lactation, allowing high levels of prolactin to be maintained unopposed by a regulatory feedback mechanisms. There is clear evidence that systemic prolactin gains access to the cerebrospinal fluid, from where it can diffuse to numerous brain regions. Prolactin receptors are expressed in several hypothalamic nuclei, including the medial preoptic and arcuate nuclei, and we have observed marked increases in expression of prolactin receptors in these nuclei during lactation. Moreover, a number of hypothalamic nuclei, including the paraventricular, supraoptic and ventromedial nuclei, in which prolactin receptors were not detected in diestrous rats, were found to express significant amounts of prolactin receptor during lactation. These observations have important implications for the variety of documented actions of prolactin on the brain. Prolactin has been reported to influence numerous brain functions, including maternal behavior, feeding and appetite, oxytocin secretion, and ACTH secretion in response to stress. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these effects of prolactin may be enhanced or exaggerated during lactation. Hence, prolactin may be a key player in the coordination of neuroendocrine and behavioral adaptations of the maternal brain.

Introduction Perhaps because of its name, the anterior pituitary hormone prolactin is usually associated with the stimulation of milk synthesis in the mammary gland during lactation. Such an association is not unreasonable because this is one of the major roles that

This chapter is dedicated to my friend and mentor, Professor Bob Averill (1929-1999). *Correspondence to: David Grattan, Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, P.O. Box 913, Dunedin, New Zealand. Tel.: +64-3-479-7442; Fax: -I-64-3-479-7254; E-mall: david.grattan@ stonebow.otago.ac.nz Homepage: www.otago.ac.nz/neuroendocrinology

prolactin plays in mammalian reproduction. However, among a vast number of other known functions of prolactin (Bole-Feysot et al., 1998) are numerous effects on the brain. This chapter will review these functions, and present evidence that there may be marked changes in the action of prolactin on the brain during pregnancy and lactation. In a remarkable example of conservation of signalling molecules, the same signal that the body uses to stimulate mammary gland development and function (i.e. prolactin) is apparently involved in fine-tuning aspects of hypothalamic function related to the state of lactation. Indeed, prolactin may be a key player in the organization and co-ordination of the neuroendocrine and behavioral adaptations of the maternal brain.

154

This review will focus primarily on research work using rats, but in keeping with the multidisciplinary nature of the volume, comparisons with other species will be made where appropriate. Patterns of lactogenic hormone secretion Patterns of secretion during pregnancy Pregnancy is a unique state with respect to prolactin action because, as well as the maternal pituitary gland, the placenta provides an additional source of lactogenic hormones in the circulation of many species. Fig. 1 shows the temporal relationships between the different lactogenic hormones during pregnancy in rats. In rodents, prolactin secretion during early pregnancy is characterized by surges of prolactin required for luteotrophic function (Gunnet and Freeman, 1983; Erskine, 1995). This unique neuroendocrine reflex is induced by the mating stimulus, after which prolactin surges continue twice-daily until approximately day 10 of pregnancy (mid-gestation), when the onset of placental lactogen secretion results in the inhibition of pituitary prolactin secretion (Voogt and de Greef, 1989; Arbogast et al., 1992; Voogt et al., 1996). Inhibition of prolactin secretion at mid-pregnancy occurs because placental lactogen is able to mimic the action of
Prolactin 1 O0
~

prolactin to stimulate hypothalamic dopamine secretion (Demarest et al., 1983a; Arbogast et al., 1992; Lee and Voogt, 1999), thereby inhibiting prolactin secretion by short-loop negative feedback. There are two forms of placental lactogen in rats. The first (rat placental lactogen I: rPL-I) is only present for a few days around mid-gestation (Robertson et al., 1982), but is likely to be the hormone that terminates the prolactin surges of early pregnancy (Voogt et al., 1996) and takes over the luteotrophic function of prolactin. The second (rat placental lactogen II: rPL-II) begins to be detected in the blood slightly after the appearance of rPL-I, and increases throughout the second half of pregnancy reaching peak levels approximately 1-2 days prior to parturition (Fig. 1; Robertson and Friesen, 1981). It is likely that this form of placental lactogen maintains an inhibitory control over prolactin secretion during the second half of pregnancy, as both rPL-I and rPL-II are able to bind to the prolactin receptor (Robertson et al., 1982), and prolactin secretion remains low through most of this time. During late pregnancy in rats, there is renewed prolactin secretion from the maternal anterior pituitary (Grattan and Averill, 1990, 1992). This takes the form of a large nocturnal surge of secretion (Grattan and Averill, 1990), and is of interest as it occurs during a time when placental lactogen is
Parturition Day 22

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on

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; I
I

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/ #

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~""

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I

-"

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l
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I I I I I I I I I I I l I I I I I I I I I I I

10

12

14

16 (Days)

18

20

22

Time during Pregnancy

Fig. 1. Diagrammatic representation of lactogenic hormone levels in the blood during pregnancy in the rat. Data are presented as a percentage of the maximum values present during gestation. The black and white bar along the x-axis represents the lights off and lights on periods of the light/dark cycle, respectively. Data for prolactin secretion are derived from Gunnet and Freeman (1983) and Grattan and Averill (1990). Data for placental lactogen secretion (PL-I and PL-II) are derived from Robertson and Friesen (1981) and Robertson et al. (1982).

155

still high. Neither human placental lactogen (hPL) (Grattan and Averill, 1991), ovine prolactin, nor rPL-I (Flietstra and Voogt, 1997) administered intracerebrally (all treatments known to inhibit the prolactin surges of early pregnancy) are able to inhibit this ante-partum surge of prolactin. Similarly, both intrahypothalamic anterior pituitary grafts which secrete prolactin (Grattan and Averill, 1995) and intraventricular transplants of placental tumor cells which secrete both rPL-I and rPL-II (Flietstra and Voogt, 1997) failed to affect prolactin secretion during late pregnancy. These data suggest that the autoregulatory mechanism by which lactogenic hormones inhibit pituitary prolactin secretion is not functional on the last day of pregnancy, and that the tuberoinfundibular dopamine neurons have become insensitive to prolactin. (Also see Voogt et al., 2001, this volume). In other rodents, such as mice and hamsters, placental lactogen secretion is almost identical to the rat, with two major forms of placental lactogen secreted at specific times during gestation (Ogren and Talamantes, 1988). Many other species, including non-human primates, cows, sheep and goats, show a gradual but continuous increase in placental lactogen in the blood starting relatively early in gestation, reaching a peak near parturition (Ogren and Talamantes, 1988). By contrast, there are some mammalian species, including pigs, rabbits and dogs, that apparently do not express a placental lactogen (Tala-

100
> 75

Prolactin

.........

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mantes et al., 1980). Interestingly, in these species, pituitary prolactin secretion is elevated throughout the latter half of gestation, especially immediately prior to parturition (Cowie et al., 1980). In humans, both prolactin (Tyson et al., 1972) and hPL (Braunstein et al., 1980) are present in increasing amounts with advancing gestation (Fig. 2). This is interesting, as one would expect that the placental hormone would feedback to induce an inhibition of prolactin secretion, as in other species. It is possible that the tuberoinfundibular dopamine neurons are insensitive to prolactin during human gestation, although this has not been studied. Alternatively, it is possible that hPL does not function at the prolactin receptors controlling dopamine activity in the human brain. In this respect, unlike placental lactogens of other species, hPL is actually more closely related to human growth hormone than human prolactin (Soares et al., 1998). The study of the actions of hPL has largely been undertaken in non-human systems, and it clearly exerts prolactin-like actions in some species. In the rat, for example, hPL clearly activates the rat prolactin receptor controlling dopamine activity (Demarest et al., 1983a). To my knowledge there have been no direct studies of the regulation of prolactin secretion by hPL in humans. The human decidua also produces significant amounts of a prolactin-like protein. The mature protein is identical to human pituitary prolactin, and is present in amniotic fluid by week 9 of pregnancy, in-

~E N ~ 25

8 10 ll2 lJ4 1'6 1'8 2'0 2'2 214 2'6 218 3'0 3'2 3'4 3'6 3~8 ; 0
Time during Pregnancy (Weeks)

Fig. 2. Diagrammaticrepresentationof lactogenic hormone levels in the blood during pregnancyin humans. Data axe presented as a percentage of the maximumvalues present during gestation.Data for prolactin secretionaxe derived from (Tysonet al., 1972) and data for placental lactogensecretion(hPL) are derivedfrom(Braunsteinet al., 1980).

156 creasing to peak levels around mid-gestation (Ogren and Talamantes, 1994). However, decidual prolactin does not get into the maternal circulation in significant concentrations (Riddick et al., 1979) and, hence, its contribution to lactogenic effects in the mother is minimal. It may, nevertheless, exert important effects in the fetal brain as discussed below. Regardless of the strategies utilized by different species, it is apparent that lactogenic hormones play a major role in pregnancy. The presence of both a pituitary and a placental source of lactogenic hormones bypasses the negative feedback regulatory control over the pituitary lactotroph, and ensures the continued presence of hormones capable of activating the prolactin receptor throughout pregnancy. During late pregnancy a further adaptation occurs within the hypothalamus to render the tuberoinfundibular dopamine neurons resistant to the stimulatory effects of prolactin, allowing increased pituitary prolactin secretion. Clearly, these adaptations are important for mammogenesis, where prolactin plays a critical role in lobulo-alveolar development of the mammary gland during pregnancy. In addition, the prolonged hyperprolactinemia resulting from these adaptations has important implications for effects of prolactin on the brain during pregnancy and lactation. could occur by two basic mechanisms: a decrease in inhibitory regulation by dopamine from the tuberoinfundibular dopaminergic neurons, and/or an increase in stimulatory regulation by one or more 'prolactinreleasing factors'. Certainly, there is good evidence for the former mechanism. Several investigators have reported a reduction in dopamine turnover (Selmanoff and Wise, 1981; Demarest et al., 1983b; Selmanoff and Gregerson, 1985) or release (Rondeel et al., 1988) in the median eminence in response to the suckling stimulus. Dopamine concentrations in the pituitary portal blood are reduced in lactating rats, but rise after prolonged separation of the mother from her pups (Ben-Jonathan et al., 1980). Levels of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, and its mRNA in the arcuate nucleus are also markedly reduced during lactation (Wang et al., 1993). Taken together, these data suggest that suckling suppresses tuberoinfundibular dopamine activity, which will certainly contribute to an increase in prolactin secretion. Despite the hyperprolactinemia induced by suckling, most indices of activity of the tuberoinfundibular dopaminergic neurons show low levels of activity during lactation (Ben-Jonathan et al., 1980; Demarest et al., 1983b; Wang et al., 1993). Treatment of lactating rats with physiological concentrations of ovine prolactin was ineffective in inhibiting the suckling-induced prolactin secretion (Whitworth et al., 1981), although higher doses of prolactin can attenuate but not abolish prolactin secretion after suckling (Whitworth et al., 1981; Selmanoff and Gregerson, 1984). Acute increases in prolactin concentrations markedly elevated tuberoinfundibular dopamine activity in diestrous rats, but had no effect on dopamine activity during lactation (Demarest et al., 1983b). Similarly, implantation of prolactin-secreting MMQ rat anterior pituitary adenoma cells into the third ventricle only partially activated dopamine neurons in early lactation and had no effect at mid-lactation (Arbogast and Voogt, 1996). These data suggest that the dopamine neurons may retain some ability to respond to prolactin during lactation, but that they are significantly less sensitive than in non-lactating rats. Hence, like during late pregnancy, short-loop negative feedback regulation of prolactin secretion is impaired during lactation, favoring the maintenance of a hyperprolactinemic state.

Patterns of secretion during lactation

Following parturition, prolactin secretion from the maternal pituitary again becomes the major source of lactogenic hormones in the maternal circulation. In all mammals, the concentrations present in the blood are dependent on the suckling stimulus, the most powerful natural stimulus to prolactin secretion. Suckling of the nipple by the young produces a signal in the somatosensory neuronal pathways innervating the gland. This neural signal is then relayed to the hypothalamus, where it is converted into a hormonal signal to regulate the anterior pituitary gland, inducing prolactin secretion (Neill and Nagy, 1994). The amount of prolactin released depends on the intensity and duration of suckling, and the time between suckling episodes. Details of the neuroendocrine mechanisms controlling the suckling-induced release of prolactin are covered detail elsewhere in this volume. Briefly, suckling-induced stimulation of prolactin secretion

157 There is also evidence that a stimulatory factor may be involved in the suckling-induced release of prolactin (for review see Neill and Nagy, 1994). Various studies have implicated thyrotropin-releasing hormone, oxytocin, and vasoactive intestinal polypeptide as possible releasing factors involved in stimulating prolactin secretion during lactation, while others have reported more equivocal findings (Neill and Nagy, 1994). An as yet unpurified factor from the intermediate lobe has also been implicated in the suckling-induced stimulation of prolactin secretion (Ben-Jonathan et al., 1991). The search for a physiological prolactin-releasing factor continues, with the latest putative candidate being the prolactinreleasing peptide identified in bovine hypothalamic extracts (Hinuma et al., 1998).
Access of prolactin to the central nervous system

As described above, pregnancy and lactation are physiological states characterized by the presence of hyperprolactinemia. But what amount of the lactogenic hormones present in the blood during pregnancy is able to get into the central nervous system? Being a relatively large polypeptide hormone (197199 amino acids), prolactin would be expected to be excluded from nervous tissue by the specialized tight junctions between vascular endothelial cells in brain which form the blood-brain barrier (Nilsson et al., 1992). There is clear evidence, however, that systemic prolactin does gain access to the cerebrospinal fluid (CSF), from where it can diffuse to numerous brain regions. Levels of prolactin in the CSF fluctuate in parallel with serum prolactin levels under a variety of physiological conditions (Login and MacLeod, 1977; Nicholson et al., 1980; Kalin et al., 1981; Kishi and Kobayashi, 1984; Simpkins, 1992). It appears that transportation of prolactin into the CSF involves a saturable, receptor mediated process (Walsh et al., 1987). It has been suggested that prolactin binding sites in the choroid plexus are involved in the transport of prolactin from the blood into the CSF (Nicholson et al., 1980; Silverman et al., 1986). The choroid plexus contains fenestrated capillaries, but maintains a blood-CSF barrier by means of continuous tight junctions between the choroid plexus epithelial cells (Nilsson et al., 1992). Hence, blood-borne prolactin would have ready ac-

cess to the choroid plexus epithelial cells, and could bind to receptors on these cells (Posner et al., 1983). There is now extensive evidence of prolactin and placental lactogen binding sites on the choroid plexus in a number of species (Posner et al., 1983; Muccioli et al., 1989; Walsh et al., 1990; Pihoker et al., 1993; Mangurian et al., 1994), including humans (Di Carlo et al., 1992; Lai et al., 1992). In light of the evidence of transport of systemic prolactin into the brain, and the fact that most prolactin-induced behaviors are accompanied by significant increases in plasma prolactin levels, the majority of the discussion in this chapter will be based on the assumption that the principal source of prolactin in the central nervous system is the systemic circulation. A review of the actions of prolactin on the brain would not be complete, however, without acknowledging at least two other potential sources of prolactin. First, it has been suggested that hypothalamic neurons might be exposed to very high concentrations of prolactin by retrograde diffusion in the pituitary portal vessels (Oliver et al., 1977). Prolactin from this source could theoretically enter the brain through the fenestrated capillaries of the median eminence. Second, there is considerable evidence of prolactin production by neurons. Immunoreactive prolactin of brain origin has been detected by radioimmunoassay (DeVito, 1988; Harlan et al., 1989) and immunohistochemistry (Shivers et al., 1989; Paut-Pagano et al., 1993). Prolactin mRNA has also been identified in brain tissue (DeVito et al., 1992a; Emanuele et al., 1992). Hence, it is possible that some of the behavioral effects ascribed to prolactin result from the activity of prolactin-containing peptidergic neurons. This possibility has been well reviewed by Dutt et al. (1994).
Actions of prolactin in the brain

Experimental studies have led to a wide range of reported actions of prolactin in the central nervous system that are summarized in Table 1. With the obvious exception of maternal behavior, most studies of prolactin effects on the brain have been carried out in non-pregnant animals. Clearly, maternal behavior is a prerequisite for successful reproduction in mammals, and prolactin appears to be a critical hormone in induction and regulation of this behavior.

158 TABLE 1 Prolactin effects on the brain in mammals Brain function Activity of tuberoinfundibular dopamine neurons Maternal behavior Appetite/food intake Fertility and gonadotropin secretion Effect of prolactin Increase Induce Increase Suppress Representative referencesa (Moore, 1987) (Bridges, 1994; Bridges and Mann, 1994; Bridges et al., 1996) (Moore et al., 1986; Gerardo-Gettens et al., 1989; Noel and Woodside, 1993; Sauv6 and Woodside, 1996; Heil, 1999; Sanv6 and Woodside, 2OOO) (McNeilly, 1980; Smith, 1980; Evans et al., 1982; Sarkar and Yen, 1985; Cohen-Becker et al., 1986; Fox et al., 1987; Curlewis and McNeilly, 1991; Koike et al., 1991; Park and Selmanoff, 1991; de Greef et al., 1995) (Doherty et al., 1981; Drago, 1984) (Harlan et al., 1983; Witcber and Freeman, 1985) (Drago, 1988) (Joseph et al., 1989; Gonzalez-Mora et al., 1990) (Drago et al., 1989; Cook, 1997; Torner et al., 2001) (Obal et al., 1994, 1997; Roky et al., 1995) (Roky et al., 1994) (Sarkar, 1989; Ghosh and Sladek, 1995) (Nicoletti et al., 1983a; Locatelli et al., 1985; Felman and Tappaz, 1989; Kolbinger et al., 1992) (Panerai et al., 1980; Nicoletti et al., 1983b; Sarkar and Yen, 1985; Pape and Tramu, 1996) (Weiland and Wise, 1989; Tong and Pelletier, 1992) (Drago and Amir, 1984) (Ramaswamy et al., 1983, 1989; Ramaswamy and Bapna, 1987; Shewade and Ramaswamy, 1995) (DeVito et al., 1992b, 1997)

Reproductive behavior Grooming behavior Motor activity Stress response REM sleep Sleep/wake cycle Oxytocin secretion GABAergic neuronal activity Opioidergic neuronal activity Body temperature Analgesia Astrocyte function

Suppress (males) Facilitate (females) Increase Stimulate Reduce Stimulate Alter Stimulate Stimulate Stimulate Inhibit Reduce Stimulate Promote mitogensis

a Where possible, reference to one or more reviews has been provided, or alternatively papers which contain a good coverage of the relevant literature. However, prolactin effects on other aspects of brain function may also play a key role in coordinating the neuroendocrine adaptations of the maternal brain. The fact that pregnancy and lactation are states characterized by a natural state of hyperprolactinemia suggests that the actions of prolactin on the brain may be more prevalent in these conditions. The following sections will present evidence suggesting that the responsiveness of the brain to prolactin changes during lactation. Increased expression of prolactin receptors in the hypothalamus during lactation is likely to enhance the expression of prolactin-mediated functions and behaviors. ceptors in specific neuronal populations. The actions of prolactin are mediated via its cognate receptor, a member of the class 1 cytokine receptor superfamily (Kelly et al., 1991; Bole-Feysot et al., 1998). In mice and rats, there are two isoforms of the receptor, a short form and a long form, produced by alternative splicing of a single gene. The two forms have identical extracellular portions, and hence are both able to bind prolactin, but they differ in their ability to activate intracellular signalling pathways (Jabbour and Kelly, 1997). In assays of milk protein transcription, only the long form will transduce the hormonal signal (Lesueur et al., 1991; O ' N e a l and Yu-Lee, 1994). Both forms, however, can mediate mitogenic actions of prolactin through an alternative signalling pathway (Das and Vonderhaar, 1995). Most other species, including humans, appear to have only a single form of the prolactin receptor, and this form is

Expression of prolactin receptors in the brain

One way of investigating the effects of prolactin in the brain is to examine the expression of prolactin re-

159 similar to the rodent long form (Jabbour and Kelly, 1997). Prolactin receptors are widely expressed in brain tissues of different vertebrate species including fish, amphibia, birds and mammals (Bole-Feysot et al., 1998). While early studies routinely detected prolactin binding in the choroid plexus (Walsh et al., 1978; Muccioli et al., 1989), receptors were not detected elsewhere in the brain, presumably due to a relative lack of abundance. Prolactin receptor mRNA is expressed in the epithelial cells of the choroid plexus, suggesting that the prolactin binding sites detected in these early studies are identical to prolactin receptors expressed elsewhere in the body (Brooks et al., 1992; Meister et al., 1992; Chiu and Wise, 1994). This has been confirmed by immunohistochemical studies identifying prolactin receptors in the choroid plexus using antibodies which were raised against the liver prolactin receptor (Roky et al., 1996; Pi and Grattan, 1998b). Both the short and long forms of the receptor mRNA have been identified in the choroid plexus (Nagano and Kelly, 1994; Pi and Grattan, 1998a). More recent studies have confirmed the presence of specific prolactin binding sites in the hypothalamus (Barton et al., 1989; Muccioli et al., 1990, 1991; Muccioli and Di Carlo, 1994), and the distribution of prolactin receptors has been further characterized by autoradiography (Barton et al., 1989; Muccioli et al., 1990, 1991; Crumeyrolle Arias et al., 1993) and immunohistochemistry (Roky et al., 1996; Pi and Grattan, 1998b). Similarly, the distribution of prolactin receptor mRNA expression in the hypothalamus has been investigated using in situ hybridization (Chiu and Wise, 1994; Bakowska and Morrell, 1997). Using microdissection and reverse transcription polymerase chain reaction (RT-PCR), we have demonstrated differential expression of short versus long form of the prolactin receptor mRNA in various hypothalamic nuclei, with the short form preferentially expressing the paraventricular and supraoptic nuclei, while the long form predominates in most other regions of the hypothalamus (Pi and Grattan, 1998a). In diestrous rats, we have observed prolactin receptor expression in the arcuate, periventricular and medial preoptic nuclei of the hypothalamus (Fig. 3) (Pi and Grattan, 1999c). In estrogen-treated ovariectomized rats, prolactin receptor expression was also observed in the supraoptic, suprachiasmatic, ventrolateral preoptic and ventromedial preoptic nuclei (Pi and Grattan, 1998b). Recent work has shown that many of the prolactin receptor containing neurons in the arcuate nucleus are also tyrosine hydroxylase positive, suggesting a direct action of prolactin on the tuberoinfundibular dopaminergic neurons of the hypothalamus (Fig. 4). Similar data have been obtained by other investigators using primary cultures of hypothalamic neurons (Arbogast and Voogt, 1997) or sections of adult female brains (Lerant and Freeman, 1998). The neurochemical identity of prolactin receptor-containing neurons in other brain regions is unknown at present.

Increased expression of prolactin receptors in the hypothalamus during pregnancy and lactation
As described above, pregnancy and lactation are states characterized by a physiological hyperprolactinemia. Hence, we were interested in whether expression of prolactin receptors in the brain changes during these times. An early study using RT-PCR to measure prolactin receptor mRNA in the whole brain demonstrated dramatic increases in expression of the long form mRNA during pregnancy (Sugiyama et al., 1994). Subsequently these authors reported that the major portion of this increase could be accounted for by changes in the choroid plexus (Sugiyama et al., 1996). We have recently reported dramatic increases in both short and long forms of the prolactin receptor mRNA in the choroid plexus during lactation (Pi and Grattan, 1999a,b) (Fig. 5). This observation is consistent with the hypothesis that there is increased transport of prolactin into the brain during conditions of elevated prolactin secretion. Using immunohistochemistry, we have compared expression of prolactin receptors in the brain of lactating rats with diestrous rats (Pi and Grattan, 1999c). The number of prolactin receptor-containing neurons in the medial preoptic and arcuate nuclei of lactating rats was significantly increased (2-3-fold) when compared with diestrous rats. Moreover, a number of hypothalamic nuclei, including the paraventricular, supraoptic and ventromedial hypothalamic nuclei in which prolactin receptors were not detected in diestrous rats, were found to express significant amounts of prolactin receptor during lactation. Fig. 6

160

Fig. 3. Examples of prolactin receptor expression in the brain of a diestrous female rat. (A) Rostral arcuate nucleus; note prolactin receptor immunoreactiveneurons in the dorsomedial aspect of this nucleus. (B) High power view of box in A, showing labelling of neuronal cell bodies and proximal processes. (C) Medial preoptic nucleus; note the few labelled cells in the periventricular regions of this nucleus, ox, optic chiasm. (D) High power view of box in C, showing labelling of neuronal cell bodies and also staining of neuronal processes (arrows). (E) Periventricularnucleus, with immunolabelledcells localized to the ventral regions around the base of the third ventricle, with a few cells more dorsally located along the sides of the ventricle. (F) High power view of box in E, showing immunolabelled neuronal cell bodies. (G) Choroid plexus. Note the punctate labelling of choroid plexus epithelial cells. Scale bars represent 100 txm (A, C, E) and 20 Ixm (B, D, F).

depicts the distribution of prolactin receptors in the hypothalamus during lactation. To complement these data, we have also examined expression of the two forms of prolactin receptor m R N A in these brain regions during lactation. A n increase in levels of

both the short and long forms of prolactin receptor m R N A was observed in numerous microdissected preoptic and hypothalamic nuclei of lactating rats (Fig. 5) (Pi and Grattan, 1999a,b). Similarly, in situ hybridization studies have demonstrated that levels

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Fig. 4. A and B show sequential sections of the mid-portion of the arcuate nucleus stained for tyrosine hydroxylaseand prolactin receptors, respectively. Note similar distribution of the immunolabelled cells. C-E shows prolactin receptor expression co-localized with tyrosine hydroxylasein the arcuate nucleus of the female rat using double-label immunohistochemistry.Tyrosine hydroxylase immunoreactivity(C) was visualizedusing anti-rabbit IgG-TexasRed (MolecularProbes), while prolactinreceptorimmunoreactivity(D) was developedusing biotinylatedanti-mouseIgG and Neutra Avidin-OregonGreen (MolecularProbes). E depicts a digital overlayof the two previousimages. Sections were examinedon a BioRad MRC600 Confocal Laser Scanning Microscopeusing 488 nm excitationfor prolactin receptorimmunoreactivityand 568 nm excitationfor tyrosinehydroxylase. of the long form prolactin receptor mRNA in the medial preoptic nucleus are significantly greater during late pregnancy than early pregnancy (Bakowska and Morrell, 1997). The mechanisms regulating increased expression of prolactin receptors in the brain of pregnant and lactating rats are not known. Estrogen, progesterone, growth hormone and prolactin itself have all been implicated (Sugiyama et al., 1996). The most likely possibility seems to be an agonist-induced up-regulation mediated by high levels of placental lactogen and/or prolactin during pregnancy. The hypothesis is supported by several observations: (1) experimental hyperprolactinemia up-regulates prolactin binding sites in the hypothalamus (Muccioli and Di Carlo, 1994); (2) high levels of expression of the long form mRNA in the choroid plexus of lactating rats requires the suckling stimulus (Sugiyama et al., 1996); and (3) in male rats that display maternal behavior, increased expression of long form prolactin receptor mRNA in the choroid plexus is observed concomitantly with elevated levels of serum prolactin (Sakaguchi et al., 1996). Much more work is required, however, to determine the precise mechanisms involved in stimulating prolactin receptor expression within specific brain regions during pregnancy and lactation.

Implications of changes in sensitivity for prolactin effects on the brain

The increased expression of prolactin receptors in specific hypothalamic nuclei during lactation has important implications for the variety of reported actions of prolactin on the brain (Table 1). Many of the specific hypothalamic functions described below are dealt with in greater detail elsewhere in this volume. This section will draw attention to the relatively

162

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Fig. 5. Increased expression of prolactin receptor mRNA during lactation in the rat. Bars represent the relative amount of mRNA of the long form (upper panel) and short form (lower panel) from different microdissected brain regions of diestrous (black bars) and lactating (gray bars) rats. Data redrawn from (Pi and Grattan, 1999a,b). For each brain regions, n = 6 for long form and n = 4 for short form. PaCTX, parietal cortex; CgCTX, cingulate cortex; ChP, choroid plexus; VMPO, ventromedial preoptic nucleus; VLPO, ventrolateral preoptic nucleus; MPO, medial preoptic nucleus; VLH, ventrolateral hypothalamic nucleus; LA, lateroanterior hypothalamic nucleus; SO, supraoptic nucleus; Pa, paraventricular nucleus; Arc, Arcuate nucleus; VMH, ventromedial hypothalamic nucleus; ME, median eminence. *P < 0.05 when compared with corresponding region in diestrous rats. t Data for choroid plexus presented from two independent experiments (ChP-I and ChP-2).

widespread influences of prolactin on hypothalamic function in the maternal brain, and present a case for prolactin as a physiologically important factor involved in organizing and coordinating the various neuroendocrine adaptations to pregnancy and lactation. Perhaps the most clearly defined behavioral change occurring during pregnancy and lactation is the onset and maintenance of maternal behavior. The mechanisms controlling maternal behavior are covered extensively in this volume (Mann and Bridges, 2001; Stem and Lonstein, 2001). There is clear evidence that prolactin is involved in stimulating the expression of this behavior (Bridges, 1994; Bridges and Mann, 1994; Bridges et al., 1996), and it appears to exert this effect by an action in the medial preoptic nucleus (Bridges et al., 1997). The increase in both prolactin receptor immunoreactiv-

ity (Pi and Grattan, 1999c) and prolactin receptor mRNA (Bakowska and Morrell, 1997; Pi and Grattan, 1999b) in this region during pregnancy and lactation is likely to contribute to the expression of matemal behavior. More recently, the ventromedial hypothalamic nucleus, another nucleus expressing prolactin receptors during lactation (Pi and Grattan, 1999c), has also been implicated in the regulation of maternal behavior (Bridges et al., 1999). Similarly, feeding and appetite changes in response to hyperprolactinemia (Moore et al., 1986; Gerardo-Gettens et al., 1989; Noel and Woodside, 1993; Sauv6 and Woodside, 1996; Heil, 1999) are likely to be mediated in the appetite centers of the brain. In birds, the hyperphagic effects of prolactin are clearly mediated in the ventromedial nucleus of the hypothalamus (Hnasko and Buntin, 1993; Li et al., 1995). In mammals, the only data available to

163

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Fig. 6. Distribution of prolactin receptors in the brain during lactation. Diagram represents sagittal sections through the medial (0.40 mm lateral to midline) and lateral (0.90 mm lateral to midline) hypothalamus, based on the atlas of Paxinos and Watson (1997). Dark gray shading represent major tracts of white matter (ac, anterior commissure; f, fomix; mt, mammillothalamic tract; ox, optic chiasm). Dotted lines represent approximate boundaries of various hypothalamic nuclei (AH, anterior hypothalamic nucleus; Arc, arcuate nucleus; DM, dorsomedial nucleus; LA, lateroanterior hypothalamic nucleus; MPA, medial preoptic area; MPO, medial preoptic nucleus; SCh, suprachiasmatic nucleus; SO, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus; VMPO, ventromedial preoptic nucleus). Pale gray shading represents nuclei in which the prolactin receptor is expressed during lactation (adapted from Pi and Grattan, 1999c).

date suggest that the paraventricular nucleus might be a site o f prolactin-mediated hyperphagia (Sauv6 and Woodside, 2000). Up-regulation of prolactin receptor expression in both of these nuclei in the hypothalamus of lactating rats, suggests that a lactat-

ing female m a y be significantly more sensitive to the hyperphagic effects of prolactin than a non-pregnant one. Indeed, prolactin m a y be a major factor mediating the hyperphagia associated with pregnancy and lactation (Moore and Brasel, 1984).

164 It has been suggested that prolactin secretion in response to stress has an adaptive function to regulate the magnitude of the stress response (Drago et al., 1989; Cook, 1997). The site of action of prolactin in mediating this action is not clear, but is likely to involve the hypothalamo-pituitary-adrenal axis, specifically, the paraventricular nucleus (Tomer et al., 2001). Elevated levels of prolactin during lactation, therefore, coupled with increased expression of prolactin receptors in the paraventricular nucleus, may result in prolactin contributing to the suppression of the stress response during lactation (for further details see Neumann, 2001, this volume). Oxytocin secretion is another hypothalamic function that changes dramatically during lactation (see Theodosis and Poulain, 2001, this volume). The major distribution of prolactin receptor-containing cells (Pi and Grattan, 1999c) and prolactin receptor mRNA (Bakowska and Morrell, 1997) in the supraoptic and paraventricular nuclei appeared to correlate with the distribution of oxytocin-containing cells although this has not been confirmed by double labelling techniques. Prolactin has been shown to influence both oxytocin mRNA expression (Ghosh and Sladek, 1995) and oxytocin secretion (Sarkar, 1989; Parker et al., 1991). Again, an increase in prolactin secretion during lactation coupled with increased expression of prolactin receptors in the magnocellular hypothalamic nuclei is consistent with a role for prolactin in influencing activity of oxytocin neurons during lactation. It is also tempting to speculate that prolactin might be involved in the suckling-induced suppression of fertility during lactation. There is no doubt that hyperprolactinemia inhibits gonadotropin secretion in a number of conditions (McNeilly, 1980; Smith, 1980; Evans et al., 1982; Sarkar and Yen, 1985; Cohen-Becker et al., 1986; Wise, 1986; Fox et al., 1987; Curlewis and McNeilly, 1991; Koike et al., 1991; Park and Selmanoff, 1991; de Greef et al., 1995). Such an effect might be mediated directly on gonadotropin-releasing hormone (GnRH) neurons, or indirectly by means of prolactin-sensitive afferent neurons. It is not known whether GnRH neurons contain prolactin receptors, although the immortalized GT1 cell line, derived from GnRH neurons, does appear to be directly inhibited by prolactin through a prolactin receptor mediated mechanism (Milenkovic et al., 1994). In lactating rats, we have observed increased expression of prolactin receptors in parts of the hypothalamus, such as the ventrolateral preoptic nucleus (Pi and Grattan, 1999b,c), where GnRH neurons would be found (Sagrillo et al., 1996). Hence, it is possible that prolactin acting in this region contributes to the suckling-induced suppression of fertility. Although this is an attractive hypothesis, there is evidence that prolactin may not be the primary mediator of lactational infertility, and that some other aspect of the suckling stimulus is critically involved (Maeda et al., 1990; McNeilly, 1994). Further information on the mechanisms of lactational infertility are discussed in McNeilly (2001, this volume) and Tsukamura and Maeda (2001, this volume). It seems likely that a number of the other described actions of prolactin on the brain (Table 1) will also occur during lactation. Whether prolactinmediated effects on rapid eye movement (REM) sleep (Obal et al., 1994, 1997; Roky et al., 1995), body temperature (Drago and Amir, 1984) or motor functions (Joseph et al., 1989; Gonzalez-Mora et al., 1990), for example, exert physiologically meaningful adaptations to support the lactating state, awaits further clarification. However, an element of caution must be retained in interpretation of the prolactin receptor data. An increased expression of prolactin receptors or receptor mRNA in a particular nucleus may not always be associated with increased responsiveness to prolactin. The effect of prolactin on tuberoinfundibular dopaminergic neurons during lactation is a very interesting example. As described earlier, there is good evidence that these neurons are less responsive to prolactin during late pregnancy and lactation than in non-pregnant animals. This prolactin-resistant state is likely to be essential for maintenance of lactational competency, as it allows the suckling stimulus to increase prolactin secretion unopposed by the activation of dopamine neurons. The mechanism of this important neuroendocrine adaptation during late pregnancy and lactation is not clear, but there does not appear to be a down-regulation of prolactin receptors on the tuberoinfundibular dopamine neurons. In fact, we have observed increased expression of prolactin receptors in the arcuate nucleus during lactation (Pi and Grattan, 1999c). Furthermore, using double-label immunohistochemistry, we

165 have identified prolactin receptors specifically on the tuberoinfundibular dopamine neurons in lactating rats (Grattan and Porteous, unpublished data). Hence, these neurons have receptors for prolactin, but apparently do not respond to prolactin with an increase in dopamine activity during lactation as they do at other times. It is possible that the intracellular response to prolactin has changed during pregnancy and/or lactation. For example, it has been suggested that prolactin stimulates expression of enkephalin in the tuberoinfundibular dopamine neurons during lactation (Merchenthaler, 1994; Merchenthaler et al., 1995), associated with a down-regulation of tyrosine hydroxylase expression. iologically important, factor involved in regulating hypothalamic function in the maternal brain. First, pregnancy and lactation are conditions characterized by a physiological hyperprolactinemia. Different species have developed different strategies to maintain high levels of lactogenic hormones during pregnancy, some involving placental lactogen production, others involving maintained pituitary prolactin secretion. During late pregnancy, and then into lactation, the state of hyperprolactinemia is maintained by the suppression of the ability of tuberoinfundibular dopamine neurons to be stimulated by prolactin. High levels of prolactin can then be maintained by the suckling stimulus, unopposed by a regulatory feedback mechanism. These high levels of systemic prolactin gain access to the central nervous system at the choroid plexus, providing high levels of prolactin in the cerebrospinal fluid. Second, there is a marked up-regulation of prolactin receptor expression in numerous hypothalamic nuclei during pregnancy and lactation, providing evidence of a relatively widespread action of prolactin throughout the hypothalamus. Finally, there are numerous studies documenting prolactin regulation of a variety of brain functions. Although many of these have been described in non-pregnant or male animals, they are likely to occur in lactating animals as well. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these functions may be enhanced or exaggerated in the maternal brain. The adaptations of the maternal brain allow the female to exhibit the appropriate behavior to feed and nurture her offspring, to adjust to the nutritional and metabolic demands of milk production, and to maintain appropriate hormone secretion to allow milk synthesis, secretion and ejection. The evidence that prolactin plays a key role in regulating hypothalamic function during lactation is compelling. Perhaps the overall role of prolactin is to organize and coordinate this wide range of behavioral and neuroendocrine adaptations during pregnancy and lactation. Further work will be required to determine the full extent of the part played by prolactin in mediating these adaptations. We also need to determine to what extent these actions of prolactin may occur in the male or non-pregnant female, to shed some

Prolactin effects in the fetal brain

This chapter has focused almost exclusively on prolactin actions in the maternal brain, but we should also consider the role of prolactin in the fetal and neonatal brain. Interestingly, the prolactin receptor is expressed at relatively high levels in the choroid plexus of fetal and neonatal rats (Royster et al., 1995). There is evidence that prolactin plays an important role in the development and maturation of the neonatal neuroendocrine system (Shah et al., 1988; Romero and Phelps, 1993; Phelps et al., 1995). Relatively high levels of prolactin receptor expression have also been observed in the olfactory bulb of perinatal rats from around day 18 of pregnancy until approximately postnatal day 5 (Freemark et al., 1996), suggesting that prolactin plays a role in the developing olfactory system. Prolactin is present in milk, and a significant portion of ingested prolactin is transferred intact into the neonatal circulation (Grosvenor and Whitworth, 1983). Furthermore, the prolactin-like protein from the decidua may reach the fetus. Hence, the presence of prolactin receptors in the developing brain suggests a novel mechanism by which lactogenic hormones of maternal origin could influence neonatal brain development and/or behavior.

Summary: prolactin effects on the brain during pregnancy and lactation

Three lines of evidence have been presented in this chapter suggesting that prolactin is a major, phys-

166

light on the mechanisms by which inappropriate hyperprolactinemia can influence emotions and mood (Sobrinho, 1993, 1998; Reavley et al., 1997). The recent development of prolactin antagonists (Kuo et al., 1998, Bridges et al., 2001), or the use of antisense oligonucleotides against the prolactin receptor (Torner et al., 2001) may be important tools in the future elucidation of the actions of prolactin on specific neuronal populations of the maternal brain. Abbreviations CSF GABA GnRH hPL MMQ REM rPL RT-PCR cerebrospinal fluid gamma aminobutyric acid gonadotropin-releasing hormone human placental lactogen rat anterior pituitary adenoma cell-line secreting prolactin rapid eye movement rat placental lactogen reverse transcription polymerase chain reaction

Acknowledgements The author's work in this field has been supported by the Health Research Council of New Zealand, by the New Zealand Lotteries Commission Lottery Health Grants, and by University of Otago Bequest Funds. References
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of messenger ribonucleic acid, immunoreactive protein, and ligand-binding activity and induction of expression in late gestation. Endocrinology, 136: 3892-3900. Sagrillo, C.A., Grattan, D.R., McCarthy, M.M. and Selmanoff, M. (1996) Hormonal and neurotransmitter regulation of GnRH gene expression and related reproductive behaviors. Behav. Gen., 26: 241-277. Sakaguchi, K., Tanaka, M., Ohkubo, T., Dohura, K., Fujikawa, T., Sudo, S. and Nakashima, K. (1996) Induction of brain prolactin receptor long-form mRNA expression and maternal behavior in pup-contacted male rats: Promotion by prolactin administration and suppression by female contact. Neuroendocrinology, 63: 559-568. Sarkar, D.K. (1989) Evidence for prolactin feedback actions on hypothalamic oxytocin, vasoactive intestinal peptide and dopamine secretion. Neuroendocrinology, 49: 520-524. Sarkar, D.K. and Yen, S.S. (1985) Hyperprolactinemia decreases the luteinizing hormone-releasing hormone concentration in pituitary portal plasma: a possible role for beta-endorphin as a mediator. Endocrinology, 116: 2080-2084. Sauv6, D. and Woodside, B. (1996) The effect of central administration of prolactin on food intake in virgin female rats is dose-dependent, occurs in the absence of ovarian hormones and the latency to onset varies with feeding regimen. Brain Res., 729: 75-81. Sauv6, D. and Woodside, B. (2000) Neuroanatomical specificity of prolactin-induced hyperphagia in virgin female rats. Brain Res., 868: 306-314. Selmanoff, M. and Gregerson, K.A. (1984) Autofeedback effects of prolactin on basal, suckling-induced, and proestrus secretion of prolactin. Proc. Soc. Exp. BioL Med., 175: 398-405. Selmanoff, M. and Gregerson, K.A. (1985) Suckling decreases dopamine turnover in both medial and lateral aspects of the median eminence in the rat. Neurosci. Lett., 57: 25-30. Selmanoff, M. and Wise, P.M. (1981) Decrease dopamine turnover in the median eminence in response to suckling in the lactating rat. Brain Res., 212:101-115. Shah, G.V., Shyr, S.W., Grosvenor, C.E. and Crowley, W.R. (1988) Hyperprolactinemia after neonatal prolactin (PRL) deficiency in rats: evidence for altered anterior pituitary regulation of PRL secretion. Endocrinology, 122:1883-1889. Shewade, D.G. and Ramaswamy, S. (1995) Prolactin induced analgesia is dependent on ATP sensitive potassium channels. Clin. Exp. Pharmacol. Physiol., 22: 635-636. Shivers, B.D., Harlan, R.E. and Pfaff, D.W. (1989) A subset of neurons containing immunoreactive prolactin is a target for estrogen regulation of gene expression in rat hypothalamus. Neuroendocrinology, 49: 23-27. Silverman, W.E, Walsh, R.J. and Posner, B.I. (1986) The ontogeny of specific prolactin binding sites in the rat choroid plexus. Brain Res., 389:11-19. Simpkins, J.W. (1992) Effects of haloperidol and prolactin secreting tumors on cerebrospinal fluid concentrations of prolactin in the female rat. Life Sci., 5l: 295-301. Smith, M.S. (1980) Role of prolactin in regulating gonadotropin secretion and gonad function in female rats. Fed. Proc., 39: 2571-2576.

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J.A. Russell et al. (Eds.)

Progress in Brain Research, gol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 12

Regulation of prolactin secretion during pregnancy and lactation

James L. Voogt *, Youngsoo Lee 1, Shuping Yang and Lydia Arbogast 2
Department of Molecular and Integrative Physiology, University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA

Abstract: Prolactin plays major roles in maintaining the corpora lutea of pregnancy and in the synthesis of milk during lactation. The hypothalamic mechanisms involved in these functions have been investigated. Mating leads to a surge of prolactin and programs daily surges during early pregnancy. The expression of Fos-immunoreactivity shows that mating activates several hypothalamic nuclei, particularly the arcuate nucleus and medial preoptic area. In the arcuate nucleus, mating is associated with Fos expression in ~-endorphin neurons, and infusion of naloxone blocks both mating-induced and diurnal prolactin surges. Tyrosine hydroxylase-immunoreactive dopamine neurons appear not to participate in surge generation. However, after day 10 of gestation the secretion of placental lactogens suppresses prolactin secretion via activation of dopamine neurons without involvement of ~-endorphin neurons. Intracerebroventricular implantation of placental lactogen-secreting cells will block pregnancy prolactin surges, increase Fos expression in dopamine neurons, and increase tyrosine hydroxylase activity. During lactation the mechanisms regulating dopamine and ~-endorphin neurons are further modified. In early lactation a prolactin-induced increase in tyrosine hydroxylase activity leads to negative feedback, but this effect is lost by mid-lactation. Overriding this negative feedback is the inhibitory effect that suckling has on dopaminergic activity. This may involve [3-endorphin-mediated inhibition of dopamine neurons, as naloxone causes a marked increase in tyrosine hydroxylase activity and suppression of circulating prolactin. However, removal of tonic dopamine inhibition is not sufficient to account for the high levels of prolactin attained during lactation, and additional releasing factors are probably involved. In situ hybrization histochemistry for the most recent candidate, prolactin-releasing peptide, suggests that this may involve brain stem neurons that co-localize noradrenaline. Thus, prolactin secretion during pregnancy and lactation involve complex interactions of regulatory factors and plasticity of neuronal responsiveness.

Introduction

Prolactin is a hormone of the anterior pituitary that is indispensable for successful pregnancy and lacta-

*Corresponding author: James L. Voogt, Department of Molecular and Integrative Physiology, University of Kansas School of Medicine, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA; Tel.: +1-913-588-7400; Fax: +1-913-588-7430; E-mail: jvoogt@kumc.edu Current address: Department of Genetics, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. 2Current address: Department of Physiology, Southern Illinois University, Physiology/CDALE, Carbondale, IL 62901, USA.

tion to occur in the rat. During pregnancy it acts on prolactin receptors in cells of the corpora lutea of the ovary to stimulate progesterone secretion, the essential hormone necessary to maintain pregnancy. During lactation, prolactin acts on epithelial cells of the m a m m a r y gland, stimulating synthesis of many components found in milk. Understanding how prolactin is regulated during these two reproductive states is very important to understanding reproduction itself. This chapter will review recent studies concerning the hypothalamic mechanisms regulating prolactin secretion, particularly focusing on the identification of the dopamine and [3-endorphin neurons of the arcuate nucleus involved in prolactin regulation, and the feedback effects of prolactin, placental lactogens,

174 opioid peptides, and prolactin-releasing peptides (see Tsukamura and Maeda, 2001, this volume, and Grattan, 2001, this volume, for further information on the regulation of prolactin secretion).

Immediate-early gene (Fos) mapping of mating-related circuits

In order to define the hypothalamic areas involved in the induction of mating-induced prolactin surges, an initial study was undertaken to examine patterns of Fos expression in several brain areas following mating, and prolactin was measured to provide an endocrine correlate of the neuronal activity. Mating of rats during the evening of proestrus resulted in a rapid (peak at 15 min) and short-lived increase in prolactin secretion, with a return to the levels seen in unmated controls within two hours. Ninety minutes after mating the brains of these animals were fixed and processed for immunocytochemical detection of Fos. Three different patterns of hypothalamic Fos expression were observed. Firstly, a striking increase in the number of Fos-positive neurons was detected in the mPOA and arcuate nucleus of mated animals undergoing intromission by comparison with either mounted (no intromissions) or control animals (Fig. 1). Secondly, the number of Fos-positive neurons in the VMH and medial amygdala was also significantly higher in mounted animals than in the control group, and mating increased this expression even further. Finally, exposure to either mounting or mating induced significant but similar increases in Fos expression compared to controls in the DMH and in the paraventricular nucleus. This finding that mating increased neuronal activity agrees with previous studies in which mating or manual vaginocervical stimulation increased Fos expression in the mPOA, medial amygdala and VMH (Tetel et al., 1993, 1994; Polston and Erskine, 1995). Importantly some of these brain areas containing neurons responsive to vaginocervical stimulation are similar to those shown to be important in regulating the twice daily prolactin surges of pregnancy. Of particular interest was the observation that mating caused a dramatic increase in Fos expression in the arcuate nucleus. This suggests that a neuronal network in this area of the brain may participate in conveying, integrating and redistributing the genitosensory stimulation. Several transmitters found in the arcuate nucleus are important in prolactin regulation. One of these, 13-endorphin, is a major stimulatory component in controlling prolactin secretion, including that induced by vaginocervical stimulation

Neural mechanisms underlying surge patterns of prolactin secretion during pregnancy

Vaginocervical stimulation that occurs during mating in the rat triggers twice-daily surges of prolactin that are present for the first ten days of pregnancy: a nocturnal prolactin surge occurs with a peak blood level of prolactin at 0200-0400 h and a diurnal surge occurs with a peak blood level of prolactin at 1800 h. These surges are responsible for the maintenance of the corpus luteum and progesterone secretion. Both of these surges cease by day 11, at which time the placenta secretes placental lactogens which replace prolactin as the luteotropic hormone that maintains the pregnancy. The neural mechanisms whereby coital and hormonal signals are integrated to induce mating-activated prolactin surges are not fully understood. Previous studies using brain-lesioning and estrogen-implantation techniques demonstrated the importance of medial preoptic area (mPOA), the ventromedial (VMH) and dorsomedial (DMH) nuclei of the hypothalamus (e.g. Freeman and Banks, 1980), and the corticomedial amygdala in mating-induced prolactin surges. Since these same areas also are activated by vaginocervical stimulation, they may be involved in processing the genitosensory stimulation and generating mating-induced prolactin surges. The utility of detecting the immediate-early gene protein Fos as an activity marker in neurons was first demonstrated by Hunt et al. (1987) and Sagar et al. (1988). Since these initial findings, numerous studies have assessed neuronal activity by measuring protein or mRNA levels of the Fos family. Fos is a transcription factor that forms a heterodimer with Jun which, in turn, is able to regulate gene expression through its interaction with the activator protein 1 site. Its immediate and transient expression in response to a stimulus allows measurement of activated or deactivated neurons simultaneously in several brain areas. We have applied this technique in studies to determine the neural substrates activated by mating and during daily prolactin surges.

175
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Fig. I. Comparison of mean number of Fos-positive cells (+ SEM) in several brain areas of control rats (homecage) or rats 90 min after receiving either intromissions (mated) or mounts without intromission. * P < 0.05 compared to control (homecage) or ** P < 0.05 compared to both the mounted and control groups. Areas examined were the medial preoptic area (mPOA), paraventricular nucleus of the hypothalamus (PVN), arcuate nucleus (ARC), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH) and medial amygdala (mAMYG). (Reproduced with permission from Yang et al., 1999.) (Sirinathsinghji and Audsley, 1985). Another factor is dopamine which is synthesized in the tuberoinfundibular neurons of the arcuate nucleus, and which acts to inhibit prolactin secretion in a chronic manner, working directly on lactotrophs. To further define the precise regions responsive to this stimulus, the arcuate nucleus was analyzed after dividing into three subdivisions - - rostral, middle and caudal. This was combined with immunocytochemistry to identify neurons containing ~-endorphin. Mating induced a significant increase in the proportion of 13-endorphin neurons that expressed Fos protein in all areas of the arcuate nucleus, especially in the caudal region (Fig. 2). However, neither mating with full intromissions nor mounting alone had any effect on the proportion of tyrosine hydroxylase-immunoreactive (dopamine-secreting) neurons that co-expressed Fos-related antigen in the arcuate nucleus. This occurred despite the fact that plasma prolactin increased during this time. Since [~-endorphin neurons in the arcuate nucleus were activated by mating, naloxone, a )t-opioid receptor antagonist, was infused intravenously (0.2 m g / 1 0 Ixl per minute) to determine the role of [3-endorphin on prolactin secretion (Hou and Voogt, 1999; Yang et al., 2000). As seen in Fig. 3 (upper panel), naloxone infusion during the period of mating completely blocked the mating-induced increase in prolactin. Furthermore, this naloxone infusion was able to block the subsequent diurnal prolactin surge three days post-mating (value measured at 1800 h), but did not significantly affect the nocturnal surge (value measured at 0200 h) (Fig. 3, lower panel). The results of these studies clearly demonstrate that mating induces an increase in neuronal activity in 13-endorphin neurons in the arcuate nu-

176

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Fig. 2. Comparison of mean (+ SEM) percentage of I3-endorphin (~-END) neurons colabeled with Fos in the rostral, middle and caudal regions of the arcuate nucleus in control (unmounted) proestrus rats or rats 60 min after receiving intromissions (mated) or mounts alone (mounted). ** P < 0.05 compared to mounted animals or controls. (Reproduced with permission from Yang et al., 2000.)

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cleus, and that blocking Ix-opioid receptors on which [3-endorphin may act (including those on arcuate nucleus neurons) results in the loss of mating-induced prolactin surges and subsequent diurnal prolactin surge during pregnancy, with no effect on the nocturnal surge of pregnancy. This suggests that [3-endorphin is important for processing the afferent genitosensory input relevant to the programming of the diurnal, but not the nocturnal, prolactin surge. However, it should be noted that other studies have suggested that [3-endorphin is involved in the nocturnal prolactin surge (Sagrillo and Voogt, 1991). It may be that each nocturnal surge requires direct stimulation by [3-endorphin, and that it has little to do with establishing the memory mechanisms responsible for the nocturnal surge. Given the important role that dopamine has in regulating prolactin secretion, it was surprising to find that tyrosine hydroxylase-containing neurons did not alter their expression of Fos in response to mating. Tyrosine hydroxylase is the rate-limiting enzyme for the synthesis of dopamine, and tuberoinfundibular dopamine neurons in the arcuate nucleus are primarily responsible for the production of dopamine which provides the tonic inhibition of pituitary lactotrophs. Previous studies (Arbogast and Voogt, 1991a) have shown that both nocturnal and diurnal prolactin

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Fig. 3. Naloxone (NAL) treatment during mating on the evening of proestrus inhibited the mating-induced increase in prolactin (PRL) (upper panel) and the diurnal prolactin surge three days post-mating (lower panel, 1800 h), but did not affect the nocturnal prolactin surge (lower panel, 0200 h). * Indicates significantly higher prolactin levels than before mating (time 0), and # indicates higher prolactin levels in saline (SAL)-treated animals than in naloxone-treated rats at the same time point (P < 0.05). Values are mean + SEM. The time of the naloxone or saline infusions during mating are indicated by the bar in the upper panel. (Reproduced with permission from Yang et al., 2000.)

surges coincide with declines in tyrosine hydroxylase synthetic activity, suggesting that a disinhibition of the lactotrophs may be at least a partial explanation for the generation of each surge. Because changes in the expression of Fos-related antigens in dopamine neurons closely mirror changes in dopaminergic activity in several physiological conditions (Hoffman et al., 1994), it has been widely used as a marker

177 for neuronal activity of dopamine neurons. In the present study expression of Fos was not suppressed in response to mating, suggesting that decreased dopamine activity during the surge times may not be directly induced by vaginocervical stimulation itself, and that dopamine neurons in the arcuate nucleus are likely not to be involved in integrating this somatosensory information. Hyperprolactinemia has been shown to induce an increase in tyrosine hydroxylase activity (Arbogast and Voogt, 1991b). Furthermore, the presence of prolactin receptors on dopamine neurons strengthens the hypothesis that prolactin acts directly on these neurons rather than through some other neuronal pathway (Arbogast and Voogt, 1997; Lerant and Freeman, 1998; Grattan, 2001, this volume). Placental lactogens from Rcho-1 cells completely eliminated prolactin surges of pregnancy, whereas control rats implanted with non-secretory rat chorioallantoic HRP-1 cells showed a normal pattern of two daily prolactin surges (Fig. 4A) (Lee and Voogt, 1999a). In the same animals the neuronal activity of tuberoinfundibular dopamine neurons was measured by the appearance of Fos-immunoreactivity in tyrosine hydroxylase-immunopositive neurons in the arcuate nucleus. Control animals bearing HRP-1 implants showed a pattern of neuronal activity in tyrosine hydroxylase-immunoreactive neurons which was the inverse of plasma prolactin levels (i.e. lowest levels during the surges at 1800 and 0200 h) at all levels of the arcuate nucleus (dorsomedial, ventrolateral and caudal) (Fig. 4B-D). In contrast, rats implanted with Rcho-1 cells which had significant amounts of placental lactogen in their cerebrospinal fluid, expressed greatly augmented neuronal activity in all areas of the arcuate nucleus at all three time points, with between 40 and 50% of tyrosine hydroxylase-positive neurons co-expressing Fos (Fig. 4). Overall, the level of tyrosine hydroxylase catalytic activity, as determined by HPLC-electrochemical detection of dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence, was elevated at both 0200 h and 1800 h on day 6 of pregnancy in animals implanted with Rcho-1 cells in the ventricles two days earlier (Lee and Voogt, 1999a). These results were correlated with very low prolactin levels during these times, indicating that the prolactin surges were most likely suppressed due to feedback effects of placental lactogens on tyrosine hydroxylase and consequent dopamine levels. Thus, placental lactogens (Rcho-I cells) administered via the lateral ventricle completely eliminated the prolactin surges of early pregnancy. However, when the activity of 13-endorphin neurons was determined by measuring the co-expression of ~-endorphin and Fos, the administration of Rcho-1 cells secreting

Regulation of prolactin surges during early pregnancy

Having established the activation of different hypothalamic areas during mating, a series of studies were undertaken to correlate the pattern of Fos expression with blood levels of prolactin throughout a 24-h period in pregnant rats. Prolactin levels in the blood demonstrated the expected twice daily surges, with peaks at 0200 h and 1800 h and a low value in the intersurge period at 1400 h. These prolactin levels correlated with the expression of Fos-immunoreactivity in both the mPOA and DMH, in that they showed the highest expression at 0200-0400 h and 1800 h and lowest at 1400 h. These results suggest that the two daily surges of prolactin during pregnancy may be temporally related to the neuronal activity in the mPOA and DMH. This pattern is lost after mid-pregnancy, suggesting that placental lactogens may interfere with this memory mechanism in the brain (Lee et al., 1997).

Role of placental lactogens in regulating activity of dopamine and [~-endorphin neurons

It has long been known that removal of the source of placental lactogens by hysterectomy results in the re-initiation of prolactin surges (Voogt, 1980). To determine the effect of raised levels of placental lactogen on the early establishment of prolactin surges, studies were performed in rats which had been implanted (either centrally or systemically) with rat choriocarcinoma cells (Rcho-1) that secrete placental lactogens. This procedure blocked the prolactin surges and did so, at least in part, by an action on tuberoinfundibular dopamine neurons (Arbogast et al., 1992). The neuronal activity of tuberoinfundibular dopamine neurons is partially regulated by the level of circulating prolactin or other lactogens.

178

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Fig. 4. Plasma prolactin (PRL) levels (panel A) and percentage of tyrosine hydroxylase (TH)-immunopositive neurons that also express Fos-immunoreactivity in three areas of the arcuate nucleus (dorsomedial, ventrolateral and caudal) in pregnant rats. Animals were implanted with either RCHO cells that secrete placental lactogen (open bars) or with control HRP-1 cells (solid bars) on day 4 of pregnancy. The neuronal activation of dopaminergic neurons is represented by the number of Fos/tyrosine hydroxylase-immunopositive neurons divided by the total number of tyrosine hydroxylase-immunopositive neurons. Values are mean -t- SEM, and bars with different letters are significantly different (P < 0.05). (Reproduced with permission from Lee and Voogt, 1999a.)

A) PRL (day 6 of pregnancy)

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B) rostral MBH
100 100] 80

C) caudal MBH

_~ 5O0

E
e-

~= 400

_[

[ ] 0200 h 1400 h []1800 h

a

~ 90
0

~ 8O
Q

Q" >u

70

7O
60

I
a

]
a/b

o
D,,

300 200 b

50 ~ " 40 U- o

E
el

"6~ 3o
*~e~_ 20 b
ImlUl

~.. 1oo

T
HRP

10 HRP RCHO HRP RCHO

RCHO

treatment

treatment

trealment

Fig. 5. Plasma prolactin (PRL) levels (A) and proportion of 13-endorphin-immunopositive cells that express Fos-immunoreactivity in the rostral (B) and caudal (C) mediobasal hypothalamus (MBH) on day 6 of pregnancy. Animals were injected in the cerebral ventricle with either control (HRP) or placental lactogen-secreting (RCHO) cells on day 4 of gestation and sacrificed on day 6 during the nocturnal (0200 h) or diurnal (1800 h) surges or the intersurge period (1400 h). Bars represent the mean value + SEM and bars with dissimilar letters are significantly different. An asterisk indicates significant differences between experimental groups. (Reproduced by permission from Lee and Voogt, 1999a.)

179 placental lactogens into the lateral cerebral ventricle did not change the daily pattern of activity in these neurons (Lee and Voogt, 1999b). Thus, in both control rats and those bearing Rcho-1 cells, the percentage of ~3-endorphin-immunoreactive neurons in the mediobasal hypothalamus that co-expressed Fos immunoreactivity was highest at 0200 h and 1800 h and low at 1400 h (Fig. 5). This suggests that although f3-endorphin neurons express a daily rhythm of activity, placental lactogens do not alter this rhythm.
Lactation studies Prolactin feedback on tuberoinfundibular dopamine neurons
PUP PUP PUP PUP EXPOSED DEPRIVED EXPOSED DEPRIVED
1000.

.EE
-I
s

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Control
MMQ-72h

U.I t,t}

4-1 +1

+1 '~

Following a successful gestation, the role of prolactin changes to that of the initiation and maintenance of lactation. Suckling causes an elevation in circulating prolactin levels that is essential for lactation. Each suckling episode results in prolactin release, which acts on the epithelial cells of the mammary gland to stimulate milk synthesis. Since this hyperprolactinemia of lactation lasts for several weeks, the question has been raised what role the tuberoinfundibular dopamine neurons have in this mechanism since these neurons are generally stimulated by prolactin. The fundamental hypothesis to be tested was whether the sensitivity to prolactin feedback on tuberoinfundibular dopamine neurons is absent or reduced in early pregnancy, but increases as lactation progresses. To study this, lactating rats on days 6 and 13 were used, and MMQ cells, which are rat anterior pituitary tumor cells known to secrete large amounts of prolactin, were injected into the lateral ventricle of the brain on lactation day 3 or 10 (Arbogast and Voogt, 1996). Dams were either exposed or deprived of pups, and prolactin levels, tyrosine hydroxylase activity in the stalk-median eminence and tyrosine hydroxylase mRNA levels in the arcuate nucleus were measured. The presence of MMQ cells inhibited plasma prolactin levels in dams on day 6 of lactation, but not on day 13 (Fig. 6). Similarly, either MMQ cells or treatment with subcutaneous injection of prolactin increased tyrosine hydroxylase activity (measured by DOPA accumulation) in the stalk-median eminence compared to controls on day 6, but not on day 13 (Fig. 7). Tyrosine hydroxylase

Fig. 6. Circulating prolactin (PRL) levels in pup-exposed or pupdeprived rats on days 6 and 13 of lactation. The pup-deprived dams had pups removed for 24 h. Prolactin-secreting MMQ cells were injected into the lateral ventricle 72 h earlier. This significantly suppressed pup-induced prolactin secretion on day 6 but not on day 13 of lactation. (Reproduced with permission from Arbogast and Voogt, 1996.)

mRNA was unaffected by implantation of MMQ cells, but did increase markedly in the absence of pups, as expected. To determine whether it was prolactin or suckling that was causing these changes, bromocriptine, a dopamine agonist, was used to inhibit prolactin secretion from the pituitary. This treatment decreased tyrosine hydroxylase activity in the stalk-median eminence in both day 5 and day 12 lactating rats (continuous suckling), but only in day 5 rats did ovine prolactin reverse the effects of bromocriptine (Arbogast and Voogt, 1996). The overall conclusion to this series of experiments is that prolactin negative feedback is present in early lactation, but is attenuated later. Although a positive effect of prolactin on tuberoinfundibular dopamine neurons was found in early lactation, this responsiveness is probably less than in non-lactating animals (Demarest et al., 1983; Arbogast et al., 1992). The increases in both tuberoinfundibular dopamine neuronal activity and tyrosine hydroxylase mRNA expression after 24 h pup removal suggests that the decrease in prolactin following removal of the suckling stimulus works primarily via changes in the activity of tuberoinfundibular dopamine neurons.

180
5O

[] []

Control MMQ-72h oPRL-36h

/
PUP EXPOSED

PUP DEPRIVED

PUP EXPOSED OVEX

30

PUP EXPOSED

PUP DEPRIVED

PUP EXPOSED OVEX

Fig. 7. In vivo DOPA accumulation in the stalk-median eminence of pup-exposed and pup-deprived lactating rats treated with prolactin during early (day 6) and mid- (day 13) lactation. A further group of pup-exposed animals were ovariectomized (OVEX). In each group animals were either implanted with prolactin-secreting MMQ cells in the lateral ventricle or treated with peripheral ovine prolactin (oPRL; 4 mg/kg, subcutaneous). Only in early lactation was central or peripheral prolactin capable of increasing tyrosine hydroxylase activity, whereas removal of the pups increased tyrosine hydroxylase activity at both stages of lactation. (Reproduced with permission from Arbogast and Voogt, 1996.)

Role of endogenous opioid peptides in prolactin release during lactation

The control of prolactin secretion in response to suckling not only involves a decrease in tuberoinfundibular dopamine neuronal activity (Selmonoff and Wise, 1981), but also an increase in prolactin releasing factors (Samson et al., 1986). However, the complex mechanisms involved in translating

the stimulus of suckling into prolactin secretion is not well understood, especially how it relates to tuberoinfundibular dopamine neurons. There are several reports that implicate endogenous opioid peptides as having a role in suckling-induced prolactin. Naloxone, as well as specific IX and K antagonists, have been shown to block prolactin in response to suckling (Selmonoff and Gregerson, 1986; Baumann and Rabii, 1991). The neuronal mechanism whereby opioid peptides exert their action at the hypothalamus to alter prolactin secretion is not understood, but may involve both a non-dopaminergic mechanism (Arita and Porter, 1984) as well as a mechanism involving tuberoinfundibular dopamine neurons (Deyo et al., 1979). Therefore, experiments were undertaken to test the hypothesis that suckling causes ~-endorphin release, which in turn inhibits tuberoinfundibular dopamine neuron activity, resulting in prolactin secretion (Arbogast and Voogt, 1998). The results indicate that naloxone infusion (60 mg/kg per hour for 12 h) significantly suppressed prolactin in constantly suckled rats and in rats that experienced only short-term (1 h) suckling periods. Measurement of tyrosine hydroxylase activity in the stalk-median eminence in both of these groups indicated that naloxone caused a major increase in DOPA accumulation. There also was a very large increase in tyrosine hydroxylase mRNA expression in the dopamine neurons in the arcuate nucleus as measured by in situ hybridization. There was no effect on tyrosine hydroxylase mRNA expression in the zona incerta, an area not thought to have any function in prolactin regulation. This study leads to the conclusion that endogenous opioid peptides are an integral part of the suckling-induced prolactin elevation during lactation. Furthermore, these peptides appear to act by decreasing tuberoinfundibular dopamine neuronal activity and tyrosine hydroxylase gene expression. Clearly opioid input is a major factor in normal prolactin secretion during lactation, and tuberoinfundibular dopamine neurons are an essential component of this action.

Prolactin-releasing peptide
In the absence of the hypothalamus, pituitary lactotrophs secrete prolactin at a very high rate. Only

181

pituitary gland

DM

reticular nucleus

(A)

(B)

(C)

Fig. 8. Representativephotomicrographsof the prolactin-releasingpeptide (PrRP) mRNA signals in the pituitary gland (left panel), dorsomedial nucleus of the hypothalamus (middle panel) and the reticular nucleus (right panel). Note there is no signal in the pituitary gland. In both the dorsomedial nucleus and reticular formation clear dark cell labelling is present (arrows). Abbreviations: 3V, third ventricle;AP, anteriorpituitary; DM, dorsomedial hypothalamus;IP intermediatelobe of the pituitary; PP, posteriorpituitary. (Reproducedwith permission from Lee et al., 2000.) when the hypothalamus is present and able to secrete dopamine is there tonic inhibition. However, during certain physiological states, such as pregnancy or lactation, the magnitude of prolactin release cannot be explained by a decline in dopamine alone, and this suggests the existence of prolactin-releasing factors. It is likely that prolactin release under these circumstances requires both an inhibition of dopamine and stimulation of releasing factors, acting simultaneously to cause a major and rapid increase in prolactin. A recent report by Hinuma et al. (1998) identified two new peptides, named prolactin-releasing peptides (PrRP) that directly stimulate prolactin release in vitro from lactotrophs. This report indicated that the potency of these peptides to release prolactin was equivalent to thyrotropin-releasing hormone. However, others have found that these peptides have little effect on prolactin (Samson et al., 1998). Intense investigation is underway to precisely locate these peptides and determine their physiological function. Using immunocytochemistry, several reports (Maruyama et al., 1999; Matsumoto et al., 1999) have indicated that PrRP-immunoreactive cell bodies are found primarily in the brain stem, specifically in the nucleus of the solitary tract and in the

182

183 reticular nucleus. In order to confirm these as sites of peptide synthesis we have employed a non-radioactive in situ hybridization method to demonstrate the location of PrRP mRNA in the rat brain. Following RNA extraction from the brain stem, cDNA was synthesized using reverse transcription polymerase chain reaction (RT-PCR), inserted into a vector, followed by plasmid purification. Digoxigenin-labeled riboprobes were synthesized by in vitro transcription of a full-length of prepro-PrRP cDNA. This was then used for in situ hybridization on brain sections of the rat (Lee et al., 2000). Positive PrRP mRNA signals were found in the hypothalamus and medulla oblongata (Figs. 8 and 9), and the pattern of distribution was the same in both males and females, and in females with differing levels of plasma prolactin (i.e. during the surge (0200 h) or trough (1400 h) of prolactin secretion on day 7 of pregnancy). In the hypothalamus, the DMH (particularly in the caudal ventral area) showed PrRP mRNA cells, but no other areas in the hypothalamus were positive. No signals were found in the pituitary gland. In the medulla oblongata, strong signals were found along the entire length of the nucleus of the solitary tract and in the reticular nucleus. These areas represent the location of cell bodies of A2 and A1 noradrenergic neurons, respectively. A recent report showed that PrRP is colocalized with tyrosine hydroxylase in both A1 and A2 neurons (Chen et al., 1999). Since A1 and A2 send nerve fibers to areas of the hypothalamus that are involved in prolactin regulation, it is possible that this colocalization in the medulla oblongata has some significance in prolactin regulation. This also is supported by the observation that A1 and A2 fibers terminate in the mPOA, the location of gonatotropin-releasing hormone cell bodies, providing a mechanism for a role in reproductive processes. Since PrRP nerve fibers have been reported in the posterior pituitary, it is possible that PrRP reaches the lactotrophs via the short portal vessels (Maruyama et al., 1999). Having defined this circuitry the challenge will be to determine precisely what function PrRP has, if any, in regulating either prolactin or reproductive processes. Conclusions The patterns of prolactin secretion show marked changes throughout the course of pregnancy and lactation. From the evidence presented here it can be seen that these changes result from differential participation of hypothalamic dopamine and ~-endorphin neurons. In particular, during early pregnancy activation of ~-endorphin neurons in the arcuate nucleus results in twice daily surges. However, as placental lactogens take over the luteotrophic activity, these surges are suppressed in mid-gestation by the action of placental lactogens activating dopaminergic neurons to tonically suppress lactotroph function. During lactation the interplay between inhibiting and releasing factors results in a high level of prolactin secretion which is maintained by a loss of negative feedback inhibition by prolactin. The challenge for the future will be to identify the precise neural mechanisms by which these interactions occur. Abbreviations DMH DOPA HRP- 1 MMQ dorsomedial hypothalamic nucleus dihydroxyphenlyalanine rat chorioallantoic placental cell-line rat anterior pituitary adenoma cell-line secreting prolactin mPOA medial preoptic area PrRP prolactin-releasing peptide RCHO rat choriocarcinoma RT-PCR reverse transcription polymerase chain reaction VMH ventromedial hypothalamic nucleus

Fig. 9. Representativephotomicrographsof the prolactin-releasingpeptide (PrRP) mRNA signals in the medulla oblongata, especially the nucleus of the solitarytract. Signals are restrictedmainlyin the nucleus of the medial part of the solitarytract that is representedby the A2 noradrenergicgroup. Arrowsindicate prolactin-releasingpeptide mRNA-positivesignals. Abbreviations: 10, dorsal motornucleus of the vagus nerve; 12, nucleus of the hypoglossalnerve; AP, area postrema; cc, central canal; Cu, cuneate nucleus; Gr, gracile nucleus. (Reproducedwith permissionfrom Lee et al., 2000.)

184

References
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Sekiguchi, M., Kurokawa, T., Nishimura, O., Onda, H. and Fujino, M. (1998) A prolactin releasing peptide in the brain. Nature, 393: 272-276. Hoffman, G.E., Le, W.W., Abbud, R., Lee, W.S. and Smith, S.M. (1994) Use of fos-related antigens (FRAs) as markers of neuronal activity: FRA changes in dopamine neurons during proestrus, pregnancy, and lactation. Brain Res., 654: 207-215. Hou, Y. and Voogt, J.L. (1999) Effects of naloxone infusion on nocturnal prolactin secretion and Fos/FRA expression in pregnant rats. Endocrine, 10: 145-152. Hunt, S.P., Pini, A. and Evan, G. (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature, 328: 632-634. Lee, Y. and Voogt, J.L. (1999a) Feedback effects of placental lactogens on prolactin levels and fos-related antigen immunoreactivity of tuberoinfundibular dopaminergic neurons in the arcuate nucleus during pregnancy in the rat. Endocrinology, 140: 2159-2166. Lee, Y. and Voogt, J.L. (1999b) Rhythmicity of 13-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat. Brain Res., 837: 152-160. Lee, Y., Arbogast, L.A. and Voogt, J.L. (1997) Semicircadian rhythms of c-Fos expression in several hypothalamic areas during pregnancy in the rat: relationship to prolactin secretion. Neuroendocrinology, 67: 83-93. Lee, Y., Yang, S.P., Stares, M.J. and Voogt, J.L. (2000) Distribution of prolactin-releasing peptide mRNA in the rat brain. Brain Res. Bull., 51: 171-176. Lerant, A. and Freeman, M.E. (1998) Ovarian steroids differentially regulate the expression of PRL-R in neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study. Brain Res., 802: 141-154. Maruyama, M., Matsumoto, H., Fujiwara, K., Kitada, C., Hunuma, S., Onda, H., Fujino, M. and Inoue, K. (1999) Immunocytochemical localization of prolactin-releasing peptide in the rat brain. Endocrinology, 140: 2326-2333. Matsumoto, H., Murakami, Y., Horikoshi, Y., Noguchi, J., Habata, Y., Kitada, C., Hunuma, S., Onda, H. and Fujino, M. (1999) Distribution and characterization of immunoreactive prolactin-releasing peptide (PrRP) in rat tissue and plasma. Biochem. Biophys. Res. Commun., 257: 264-268. Polston, E.K. and Erskine, M.S. (1995) Patterns of induction of the immediate-early genes c-fos and egr-I in the female brain following differential amounts of mating stimulation. Neuroendocrinology, 62: 370-384. Sagar, S.M., Sharp, ER. and Curran, T. (1988) Expression of c-fos proteins in brain: metabolic mapping at the cellular level. Science, 240: 1328-1331. Sagrillo, C.A. and Voogt, J.L. (1991) Endogenous opioids mediate the nocturnal prolactin surges in the pregnant rat. Endocrinology, 129: 925-930. Samson, W.K., Lumpkin, M.D. and McCann, S.M. (1986) Evidence for a physiological role for oxytocin in the control of prolactin secretion. Endocrinology, 119: 554-560. Samson, W.K., Resch, Z.T., Murphy, T.C. and Chang, J.K. (1998) Gender-biased activity of the novel prolactin releasing pep-

185

tides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects. Endocrine, 9: 289-291. Selmonoff, M. and Gregerson, K.A. (1986) Suckling-induced prolactin release is suppressed by naloxone and stimulated by f3-endorphin. Neuroendocrinology, 42: 255-259. Selmonoff, M. and Wise, P.M. (1981) Decreased dopamine turnover in the median eminence in response to suckling in the lactating rat. Brain Res., 212:101-115. Sirinathsinghji, D.J. and Audsley, A.R. (1985) Endogenous opioid peptides participate in the modulation of prolactin release in response to cervicovaginal stimulation in the female rat. Endocrinology, 117: 549-556. Tetel, M.J., Getzinger, M.J. and Blaustein, J.D. (1993) Fos expression in the rat brain following vaginal-cervical stimulation by mating and manual probing. J. NeuroendocrinoL, 5: 397404. Tetel, M.J., Celentano, D.C. and Blaustein, J.D. (1994) Intraneuronal convergence of tactile and hormonal stimuli associated with female reproduction in rats. J. Neuroendocrinol., 6 : 2 1 1 -

216. Tsukamura, H. and Maeda, K.-I. (2001) Non-metabolic and metabolic factors causing lactational anestrus: rat models uncovering the neuroendocrine mechanism underlying the suckling-induced changes in the mother. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingrain (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 187-205. Voogt, J.L. (1980) Regulation of nocturnal prolactin surges during pregnancy in the rat. Endocrinology, 106: 1670-1676. Yang, S., Lee, Y. and Voogt, J.L. (1999) Fos expression in the female rat brain during the proestrous surge and following mating. Neuroendocrinology, 69: 281-289. Yang, S., Lee, Y. and Voogt, J.L. (2000) Involvement of endogenous opioidergic neurons in modulation of prolactin secretion in response to mating in the female rat. Neuroendocrinology, 72: 20-28.

J.A. Russell et al. (Eds.)

Progressin BrainResearch, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 13

Non-metabolic and metabolic factors causing lactational anestrus: rat models uncovering the neuroendocrine mechanism underlying the suckling-induced changes in the mother
Hiroko Tsukamura * and Kei-ichiro Maeda
Graduate School of Bioagricultural Sciences, Nagoya Universi~, Nagoya, 464-8601, Japan

Abstract: Follicular development and ovulation are strongly inhibited during lactation. Administration of a high dose of estrogen induces luteinizing hormone (LH) surges in ovariectomized lactating rats, suggesting that brain mechanisms regulating cyclic LH release remain intact in lactating mothers. On the other hand, tonic LH release is profoundly suppressed in lactating rats. This suggests that lactational anestrus is mainly due to suppression of the mechanism regulating pulsatile gonadotropin-releasing hormone secretion in the hypothalamus, which is responsible for follicular development and steroid production. Both metabolic and non-metabolic factors are involved in suppressing pulsatile LH secretion throughout lactation in rats. During the first half of lactation, pulsatile LH secretion is strongly suppressed, even if milk production is attenuated by pharmacological blockade of prolactin secretion in ovariectomized lactating rats. Pulsatile LH release quickly recovers by removing pups or blocking neuronal input by hypothalamic deafferentation during the period. These data suggest that the suckling stimulus itself is responsible for suppression of LH release during the first half of lactation. During the second half of lactation, negative energy balance, which is caused by the milk production, appears to play a dominant role in suppressing LH secretion. Blockade of milk production by inhibiting prolactin release causes a gradual increase in LH release even if the vigorous suckling stimulus by foster pups remains. In conclusion, the suckling stimulus itself predominantly suppresses LH pulses during the first half of lactation and metabolic factors take over the role of the suckling stimulus during the second half of lactation.

Introduction

In most mammalian species, including humans, pregnancy does not occur during lactation. Being pregnant during lactation may endanger the mother's life as well as that of the offspring, because lactation demands that the mother expend a great amount of energy on the production of milk. There are two strategies for avoiding becoming pregnant during lactation. One is the absence of follicular development and subsequent ovulation throughout the period *Corresponding author: Hiroko Tsukamura, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan. E-mail: htsukamu @agr.nagoya-u.ac.jp

of lactation. The other is exhibited by animals showing postpartum ovulation, in which the embryo does not implant in the uterus during lactation. Either strategy ensures the survival of both the mother and offspring during lactation. This chapter describes results using the lactating and non-lactating rat models, including a fasted model, to explore the possibility of the energetic control of gonadal function. Since the lactating rat takes the second strategy, involving postpartum ovulation and subsequent delayed implantation, our efforts have been made toward developing a good rat model for the other species which display lactational anestrus, such as cows, sows and women. As a resuit, several endocrine manipulations on lactating rats have revealed physiologic mechanisms common

188 to most mammalian species, though the details of the mechanisms causing lactational anestrus still remain to be determined. implantation or embryonic diapause, and may also be caused by the suckling-mediated suppression of gonadotropin secretion and ovarian follicular development, and the subsequent inhibition of estrogen secretion from the ovary. In this regard, estrogen supplementation results in the immediate implantation of the blastocyst in the lactating rat with delayed implantation (Yoshinaga, 1961). In the tammar wallaby, the implantation of the embryo is also inhibited during lactational period (Renfree, 1979, 1993). The absence of the implantation is caused by the suckling-induced increase in prolactin secretion and subsequent inhibition of progesterone secretion from the corpora lutea present in the first half of lactation. Unlike the rat, in the tammar wallaby the increased prolactin secretion inhibits progesterone secretion from the lactational corpora luteum. In both types of blockade of pregnancy, the primary cause for the absence in the ovulation or implantation during lactation resides in suckling stimulus-induced hormonal or metabolic changes.

Two strategies to avoid pregnancy during lactation

Suckling-induced absence of ovulation

Most domestic animals and women adopt a strategy of being acyclic or anestrous to avoid becoming pregnant during lactation. In women, it has been shown that breastfeeding causes anovulation and amenorrhea in the postpartum period (Howie and McNeilly, 1982; Howie et al., 1982). For example, Hennart et al. (1985) reported that menstruation and luteinization of follicles reappeared earlier in lactating women who live in Europe than those living live in Africa (Zaire) who undertake higher frequency breastfeeding with long duration. In Japanese monkeys, ovarian cyclicity is inhibited during lactation even in the breeding season (Maeda et al., 1991). In dairy and beef cows, the inhibitory effect of suckling on postpartum return of estrus has also been well documented (Bluntzer et al., 1989). In addition, La Voie et al. (1981) reported that the interval between parturition and the first estrus was shorter in cows without calves than in those with calves. In the postpartum sow, reducing litter size before weaning resulted in earlier postweaning estrus (Stevenson and Davis, 1984) and suckling increases the interval of parturition (Grinwich and McKay, 1985). These facts suggest the importance of the intensity of the suckling stimulus in inhibiting the gonadal activities, such as ovulation, menstruation and estrous cyclicity.

Endocrine aspects of suppression of the hypothalamo-pituitary-gonadal axis

Suppression of the pulse mode of luteinizing hormone during lactation

The blockade or delay of ovulation in lactating mothers has been reported to largely be due to suppression of gonadotropin release, especially luteinizing hormone (LH). Suckled beef cows showed lower serum LH concentrations in the early postpartum period than non-suckled beef cows (Short et al., 1972; Randel et al., 1976; Carruthers et al., 1980). Similarly plasma concentrations of LH are lower in lactating rats than in non-lactating rats (Hammons et al., 1973; Taya and Sasamoto, 1980). Plasma LH concentrations in postpartum sows nursing 7-11 piglets for 4 weeks were significantly lower than those nursing only 2-4 piglets (Kunavongkrit, 1984), and in women, LH secretion in breastfeeding mothers is more strongly suppressed than that in bottle-feeding mothers (Glasier et al., 1983). On the other hand, follicle-stimulating hormone (FSH) secretion is not suppressed in lactating animals (Foxcroft et al., 1987; Schirar et al., 1990).

Suckling-induced absence of implantation: embryonic diapause

Several species, such as rats, mice and wallabies, have a postpartum ovulation, but the embryo does not implant in the uterus as long as the lactation persists. For instance, the rat ovulates within 30 h of parturition. When successful fertilization occurs at this postpartum ovulation, the embryo develops to a blastocyst and stays in the uterus until the weaning of the pups. This phenomenon is called delayed

189 Smith (1981a) reported that lactation differentially affects LH and FSH secretion, because estrogen-induced LH surges are decreased during lactation but FSH surges are not affected. Ovariectomy increases FSH secretion without any effect on LH secretion in lactating sows (Stevenson et al., 1981). Therefore, the suckling stimulus suppresses gonadal activity through controlling LH secretion, rather than FSH secretion. FSH release may be primarily controlled by other ovarian factors (e.g. inhibin) during lactation (Taya and Sasamoto, 1987). Ovulation is considered to be blocked during lactation through the suppression of follicular growth or pulse mode of LH secretion, but not by a direct inhibition of ovulation or the surge mode of LH secretion. This is because the estrogen-positive feedback mechanism controlling gonadotropinreleasing hormone (GnRH)/LH surge remains intact even when a mother is given a vigorous suckling stimulus by her pups. In our early study (Tsukamura et al., 1988), daily LH surges were induced by estrogen implants within 24 h, even in lactating rats receiving a vigorous suckling stimulus during the early half of lactation (Fig. 1). Although the amplitude of the LH surges in lactating mother rats declined more rapidly than non-lactating controls, the lactating mothers still showed clear LH peaks at a fixed time in the afternoon for a few days after the estradiol implantation. The more rapid decrease in LH peak levels in lactating rats than in non-lactating rats seems to be due to the exhaustion of LH in the anterior pituitary (Tsukamura et al., 1988), because LH synthesis is mainly maintained by tonic GnRH release (Ramey et al., 1987), which may be strongly suppressed in lactating mothers. These data clearly indicate that the suckling stimulus does not suppress the LH surge-generating mechanism, but inhibits basal or pulsatile mode of LH secretion which is necessary for the follicular development. Thus, the suckling stimulus appears to suppress estrous cyclicity through inhibiting tonic GnRH/LH releasing mechanism, which in turn causes delay of the follicular development and lowered plasma estrogen levels (Taya and Sasamoto, 1987), resulting in the absence of ovulation. The pulsatile pattern of LH secretion is well known to be due to the pulsatile release of GnRH into the hypophyseal portal circulation. Each LH
Lactating OVX

E2

E
~ I * .

., o.,.,.,.,

-E 5
~_4

Non-lactating OVX E2

2
1 =

~
. . . . lo . . . . . . . . .

o '

20

Days after parturition

Fig. 1. Daily LH surges in ovariectomized lactating and non-lactating rats induced by chronic estrogen treatment. Litter size was adjusted to eight (4 males and 4 females) on day 1 postpartum. Postparturient rats deprived of their litters on day 0 served as non-lactating controls. Lactating and non-lactating rats (n = 8 in each group) were ovariectomized on day 2 postpartum and implanted with Silastic tubing containing crystalline estradiol-17[3 on day 6 postpartum. Open and closed circles indicate plasma LH levels at 10.00 and 17.00 h, respectively. *P < 0.05, compared with non-lactating rats (Student's t-test). Values are means SEM. Arrows indicate the times of ovariectomy (OVX) and implantation of estradiol (E2).

pulse clearly corresponds to a GnRH pulse and monitoring the frequency of LH pulses is a bioassay of GnRH release (Moenter et al., 1992). In this context, LH pulses would be one of the most suitable indicators to investigate the activity of the brain mechanism regulating the reproductive axis. In the present chapter, lactational anestrus will be described in terms of the suppression of the pulsatile LH secretion. Again, the key factor inducing the dramatic changes in the mother's endocrine system is the suckling stimulus given by the pups. Undoubtedly, the suckling stimulus plays critical roles in causing the suppression of LH pulses in many mammalian species (see also McNeilly, 2001, this volume).

190
Non-metabolic and metabolic pathways to cause lactational anestrus

Lactational anestrus could be caused by a complex of several mechanisms. The suckling stimulus may directly cause the suppression of gonadotropin secretion. On the other hand, the suckling stimulus itself may not necessarily be directly involved in suppressing GnRH/LH secretion, because gonadal suppression is often found with infrequent suckling, i.e. milking twice a day may cause delay in the onset of postparturient ovarian cycles in milking cows (Butler et al., 1981). McClure (1994) suggested that there is a correlation between the level of nutrition and fertility during lactation: lower nutrition or an increase in milk yield often causes a decrease in pregnancy rate and increase in calving intervals in dairy cattle. The suckling stimulus, therefore, is considered to work in two ways to cause lactational anestrus or amenor-

rhea: one is non-metabolic (suckling direct) and the other is metabolic (suckling indirect) (Fig. 2). The suckling stimulus may first be involved in directly suppressing the activity of the brain mechanisms regulating GnRH/LH secretion. As described below, the suckling stimulus is conveyed to the hypothalamus to suppress the activity of the mechanism(s) controlling pulsatile GnRH/LH secretion probably through a specific neural pathway, and not through humoral factors, such as prolactin or ovarian steroids. Second, the suckling stimulus may cause a suppression of gonadotropin secretion by altering the metabolic status of the lactating mother, i.e. energy deficiency caused by the lactation. The suckling stimulus is indispensable to maintain milk production, because it stimulates the secretion of lactogenic hormones, such as prolactin, growth hormone and adrenocorticotropin (Cowie, 1984). As a result, the suckling stimulus forces the mother to expend a

e ~""]" I Directsuppression
(non-metabolic)

~,GnRH/LHrelease 1

Energydeficiency MilkPr~ductin t

Fig. 2. Schematic illustration of direct (non-metabolic)and indirect (metabolic) suppression of LH release by the suckling stimulus during lactation in the rat.

191 large amount of energy through the milk production. The energy metabolism in a suckling mother undergoes various drastic changes, such as the increase in bodyweight, food intake, the weight of gastrointestinal tract, and fat storage (Ota and Yokoyama, 1967; Tassanawat et al., 1990). The negative balance of energy metabolism caused by the suckling stimulus could, therefore, result in a suppression of LH secretion in the lactating mother.
Rat as a model for lactational anestrus

The ovariectomized lactating rat, therefore, would be a good model to study the activity of the GnRH/LH pulse-generating system during lactation.
Non-metabolic, direct action of the suckling stimulus on the LH release

Presence of the direct action of the suckling stimulus

Prolactin has been considered for years as one of the major factors causing suppression of LH release in lactating mothers throughout lactational period (Smith, 1980, 1981b), but previous studies, including our own, suggest that prolactin is not primarily responsible for the suppression of LH release, especially in the first half of lactation in rats (Lu et al., 1976; Maeda et al., 1990; Tsukamura et al., 1991: also see McNeilly, 2001, this volume). We have proposed an alternative hypothesis based on the non-metabolic and metabolic regulation of LH release during lactation from the following results. Pulsatile LH release is strongly inhibited during the first half of lactation in ovariectomized rats treated with the dopamine agonist 2-Br-c~-ergocryptine mesylate (CB-154) in which prolactin secretion is reduced in spite of the vigorous suckling stimulus given by pups fostered from other lactating rats (Figs. 4 and 5). On the other hand, non-lactating rats, which were deprived of their pups immediately after the parturition, display a gradual increase in plasma LH levels after the pup removal (Fig. 5b). This result suggests that, in early lactation, the sucking stimulus primarily causes suppression of LH release, in spite of the reduced prolactin secretion and consequent lack of milk production. The suckling stimulus itself, therefore, may be the major factor suppressing LH secretion in the first half of lactation. On the other hand, prolactin and/or other metabolic factors may contribute to the suppression of LH release in the second half of the lactation, because plasma LH concentrations in ovariectomized mothers receiving daily CB 154 administration were significantly higher than those in saline-injected controls from day 11 onward (Fig. 5a) (Maeda et al., 1990). During the second half of lactation, a large energy expenditure caused by the milk production would be a primary factor contributing to the suppression of LH secretion, because CB154-treated mothers which are

As described above, the rat ovulates immediately after parturition (generally within 30 h) and becomes fertilized when the female is mated with a male. In the lactating rat, lactational corpora lutea are formed after the postpartum ovulation and maintained throughout lactation by the high level of plasma prolactin which is stimulated by the suckling stimulus. In intact lactating rats, the high level of progesterone would partly contribute to this inhibition of gonadotropin secretion (Smith and Neill, 1977). Therefore the first model that we have investigated was to make a lactating rat model which lacks progesterone secretion. In intact rats, pulsatile LH release is profoundly inhibited throughout lactation. Our previous study revealed that the profound suppression of pulsatile LH release in the early lactation is still found in ovariectomized rats, which lack any lactational corpus lutea (Maeda et al., 1987, 1989). As shown in Fig. 3a, pulsatile LH secretion is strongly suppressed in ovariectomized mothers suckled by their litters at the first half of lactation. This suppression was withdrawn by removing pups from the ovariectomized lactating mother. In this case, LH pulses become obvious by 18-24 h after the pup removal with mean LH concentrations and the frequency and amplitude of LH pulses being significantly higher than in control lactating animals which were constantly suckled by their pups (Fig. 3b). When the pups were reintroduced to mothers that had been deprived of the pups for 24 h, LH pulses were inhibited within 47 h after the reattachment (Fig. 3c). This dynamic and reversible suppression obviously supports the concept that the suckling stimulus has an inhibitory role on gonadal activity through the suppression of GnRH pulses and consequent pulsatile LH release.

192

(a)
With pups 6h 12h

Time after pup removal 18h 24h 45h

2
e-

,..I

~2
"

00

30

(b)
1.0 Mean LH
c

2 30 Time (h)

30

30

E 1.0 ~'IAmplitude

b i

E
c-

~- 0.5
-J

-1"
.-,I

E u)
O.

a.

~ 0.5 ~
a I

ab

With 6 12 18 24 45 pups Hours after pup removal

(c)

With 6 12 18 24 45 pups Hours after pup removal

Frequency

With 6 pups

12 18 24 45

Hours after pup removal

Mean LH

Amplitude

I.

=,

-i- 0.5

t-"l" ,-I

i.
0 1 4 7 12 Hours resuckled

Q.

s 0 I

~ 12

Hours resuckled

0 1 4 7 12 Hours resuckled

Fig. 3. Changes in the pulsatile LH release after the removal of and subsequent resuckling by pups in ovariectomized lactating rats at day 8 postpartum. (a) Individual profiles of plasma LH concentrations in two representative animals, and (b) mean LH levels and mean pulse frequency and amplitude (mean 4- SEM) in lactating rats deprived of their pups for 6, 12, 18, 24, or 45 h. (c) LH pulse parameters in ovariectomized lactating rats resuckled by their pups for 1, 4, 7 or 12 h after a 24-h separation from pups. LH pulses were identified with the PULSAR computer program (Merriam and Wachter, 1982). Numbers in the column indicate the number of animals used. Values with different letters are significantly (P < 0.05) different from each other (Duncan's multiple-range test).

193 supplemented with ovine prolactin display an insufficient suppression of LH release (Fig. 5a) as well as an insufficient milk production, judging from the daily weight gain of the litters (Fig. 5c). Taken together with the results on pup removal and reattachment, these data suggest that, in rats, the suckling stimulus would be directly involved in suppressing LH secretion in the first half of lactation, and that humoral factors, such as prolactin or ovarian steroids, do not contribute to this early suppression. of lactation, intracerebroventricular injection of antagonists for c~-adrenergic, serotonergic, or gamma aminobutyric acid (GABA)ergic receptors does not block the inhibitory effect of the suckling stimulus on the LH pulses (unpublished observations). This may be because the direct action of the suckling stimulus would cooperate with other factors, such as the metabolic, to suppress LH pulses in the first half of lactation. In this context, postpartum CB154-treated rats lacking milk production and suckled by foster pups would serve as a good model to determine the neural mechanism mediating the direct involvement of the suckling stimulus on LH release.

Possible neural inputs mediating the direct action of the suckling stimulus
In early lactation, the suckling stimulus is conveyed to the hypothalamus by a neural pathway to suppress the activity of the GnRH/LH pulse-generating mechanism. This can be demonstrated by the fact that complete hypothalamic deafferentation, isolating the mediobasal hypothalamus from the rest of the brain, can restore pulsatile LH release in ovariectomized lactating mothers within 24 h of the deafferentation without any significant effect on the prolactin secretion (Fig. 6) (Tsukamura et al., 1990). In addition, anterior-lateral or roof deafferentation restored the suppressed LH pulses, similar to animals bearing complete deafferentation. Plasma prolactin levels in animals with anterior-lateral deafferentation also did not show any significant difference compared with levels in control lactating mothers with their pups. These results again suggest that the suckling stimulus itself, but not humoral factor(s), such as prolactin and gonadal steroids, may be directly involved in the inhibition of LH release in the first half of lactation. The deafferentation studies also suggest that neural inputs associated with the suckling stimulus enter the hypothalamus dorsally and directly result in the suppressed activity of the GnRH pulse-generating mechanism. The hypothalamic paraventricular nucleus (PVN) may not be involved in conveying the information on the suckling stimulus, because the electrolytic lesion of the PVN does not affect the suckling-induced suppression of LH pulses in the first half of lactation (Tsukamura et al., 1993), neither does central administration of an antagonist of corticotropin-releasing hormone (CRH), a treatment which does reverse fasting-induced LH suppression (see below). Furthermore, during this particular phase

Energetic regulation of LH release: implications for lactational anestrus

Implication of metabolic factors in the suppressed LH release in the lactating rat
Lactating mothers require an enormous amount of energy in order to maintain milk production. After the parturition, milk yield in lactating rats gradually increases and reaches a peak around day 15 of lactation (Maeda et al., 2000). Food consumption of a lactating mother rat also increases gradually up to day 16-17 postpartum and then dramatically over the last few days before weaning (Ota and Yokoyama, 1967). Blood glucose levels and plasma insulin levels are lower, but the glucose turnover rate is higher in lactating rats when compared with non-lactating animals (Burnol et al., 1983, 1986). Thus, lactating mothers adapt their energy metabolism to the increased energy demand, and this may be another factor leading to the lactational anestrus. In the CB 154-treated mother rat, prolactin supplementation does not restore the LH secretion to the levels found in the ovariectomized lactating rat during the late lactation (Fig. 5b). Interestingly, the pup weight gain also remains at a low level in prolactinreplaced rats (Fig. 5c). This indicates that the dose of prolactin replaced was insufficient to maintain a normal milk yield. The prolactin-treated mothers may not suffer an extreme energy deficiency because of this reduced milk yield. These results raise a possibility that energy expenditure caused by the milk production could become a major factor suppressing LH secretion in the latter half of the lactation of the

194

(a)

Lactating
Saline

CB-154

CB-154+oPRL

3 E I0 ..,J E O9 _~3
EL
=---..........................

Non-lactating Saline _~3 E

-J

CB- 154

CB-154+oPRL

"1- 0 E O9 --~3 n
O

(b)
Lactating Mean LH 1 (ng/ml)

Time (h) Frequency 5 t (pulse~3h) Amplitude

Non-lactating 2 MA~n I H

Frequency

Amnlih IdA

0 -0 Saline CB154 CB154 Saline CB154 CB154 +oPRL +oPRL

Saline CB154 CB154 +oPRL

195

rat. Thus, in the rat, the direct action of the suckling stimulus on LH release becomes relatively weaker, but metabolic factors become more important in late lactation. At present we have no direct evidence for the metabolic regulation of LH secretion in the late lactation of the rat, but recent data concerning the metabolic regulation of LH secretion in the fasted rat model may have an important bearing.

Lactating
44
"1" ...I

(a)
Saline (n=9)

CB154 (n=9) --o-- CB154+oPRL (n=6)

.=, _, I *~..~

~ 2 E
,
-_ __. . . . . . . .

10 12

14 16 18 20

Fasted rat model for the study on the energetic regulation of LH release
Undernutrition or fasting has been well established to suppress gonadal activity through inhibiting LH secretion (Pirke and Spyra, 1981; Dyer et al., 1985; Foster and Olster, 1985; McClure and Saunders, 1985; Bronson, 1988; Wade and Schneider, 1992; Maeda and Tsukamura, 1996). When female rats are subjected to 48 h of fasting starting on the day of estrus, the next ovulation is blocked (Cagampang, 1992), apparently due to the suppression of pulsatile LH secretion (Cagampang et al., 1990). This suppression is dependent on the steroidal milieu, because fasting-induced suppression of LH pulses is enhanced by a low level of estrogen treatment mimicking the level found at diestrus (Cagampang et al., 1991). Thus, estrogen-primed ovariectomized rats serve as a good model to investigate the neuroendocrine pathway mediating nutritional regulation of gonadal function.

Non-lactating
CB154 (n=7) --o-- CB154+oPRL (n=7)
{7)

(b)
~ ~ ,dr~ T M ,,If" /

"1--I

t~

E (t}

E_ 2 4 6 8 10 12 14 16 18 20

---m-- Rotated (n=20) ---o-- CB154+oPRL (n=6) --m.- OVX lactating (n=6)

(c)

._~30
~- 20
~

10 4 6 8 10 12 14 16 18 20 Days after parturition

_,J Fig. 4. Lack of effect of daily injection of a dopamine agonist with or without prolactin replacement on pulsatile LH release in lactating or non-lactating ovariectomized rats at day 8 postpartum. (a) Individual profiles of plasma LH concentrations in two representative animals from each treatment group, and (b) LH pulse parameters (mean 4- SEM) in each group. Animals were daily injected with saline or 2-Br-c~-ergocryptine mesylate (CB154, 0.6 mg/day) from day 2 postpartum. Half of the CB154-injected rats were infused with ovine prolactin (oPRL) solution (0.3 mg/day) via a mini-osmotic pump. Litters in the lactating group were rotated every day among four kinds of foster mother rats consisting of a CB154-treated group, two untreated groups and a saline-injected group, so that the mothers received the similar strength of the suckling stimulus. No statistical significance was found in each LH pulse parameters compared with corresponding saline-treated animals (Student's t-test). Numbers in or on the column indicate the number of animals used.

$ 0 ,=2_

Fig. 5. Effect of daily injection of a dopamine agonist with or without prolactin replacement on LH release in lactating (a) or non-lactating (b) ovariectomized rats, and (c) daily weight gains of litters of ovariectomized lactating rats. Plasma samples were taken daily from days 3 to 20 postpartum. The closed and open circles indicate the LH concentrations in rats received daily CB154 treatment alone and daily CB154 with ovine prolactin (oPRL), respectively. See Fig. 4 for additional details for a and b. (c) The open squares show the weight gain of litters that were rotated every day among a set of CB154-treated, a set of saline-treated and two sets of untreated lactating rats. Open circles show the weight gain of litters that were treated with CB154 daily and infused with oPRL. The solid squares show the weight gain of litters nursed by ovariectomized lactating mothers. Values are mean 4- SEM. *P < 0.05, **P < 0.01 vs. saline-treated controls. *P < 0.05 vs. CB154-treated rats with PRL replacement. #P < 0.05 vs. ovariectomized lactating group. P < 0.05 vs. rotated group.

196

(a) 3
SD
PRL: 63.8 17.5 ng/ml

AD
MPOA ~ , .ALIA

62.2 31.7 ng/ml

-. UMId

PD
MPOA a-..

64.4 18.2 ng/ml

,."
..td-lA

'"~

--- . t~UMI-I

1 E
7)

ev

-i- 0
_1

CD

74.4 11.4 ng/ml A L D

67.7 29.7 ng/ml

RD
M~OA .AH

20.1 1.0 ng/ml DMH

E 09
13.

M P O A ALIA,.--DMH

0
0
(b)

30

1 2 Time (h)

30

Mean LH (ng/ml)

10 Frequency (pulses/3 h) 8

Amplitude (ng/ml)

*"

SD CD AD ALD PD RD

SD CD AD ALD PD RD
Treatments

SD CD AD ALD PD RD

llii

Fig. 6. Effect of various hypothalamic deafferentations on the suppression of pulsatile LH release in ovariectomized lactating rats at day 8 postpartum. (a) LH profiles in a representative animal for each group. Right upper numbers inside each panel indicate plasma prolactin (PRL) levels (in ng/ml, mean -4- SEM) in each group. (b) LH pulse parameters (mean 4- SEM) in ovariectomized lactating rats with various hypothalamic deafferentations. Numbers in or on the column indicate the number of animals used. *P < 0.05, **P < 0.01 compared with SD (Mann-Whitney U test). SD, sham deafferentation; CD, complete deafferentation; AD, anterior deafferentation; ALD, anterior-lateral deafferentation; PD, posterior deafferentation; RD, roof deafferentation. AHA, anterior hypothalamic area; ARC, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus; MPOA, medial preoptic area; OC, optic chiasm; VMH, ventromedial hypothalamic nucleus.

197

PVN

GnRH neuron

neuron "~Gastdc branchof vagus nerve

k
LH suppression
raet

Fig. 7. Neuroendocrine pathway mediating fasting-induced suppression of GnRH/LH release in female rats. Informationon the fasting reaches the nucleus of the solitary tract (NTS) and activates noradrenergic neurons in the A2 region via afferent gastric vagus nerve originating in the upper digestive tract. Noradrenaline released in the paraventricular nucleus (PVN) activates CRH neurons to release CRH. CRH inhibits GnRH release from neurons in the preoptic area (POA) via the actions of opioidergic intemeurons. This suppresses pulsatile LH secretion.

Neuroendocrine pathway mediatingfasting-induced suppression of LH release

Using the estrogen-treated ovariectomized rats subjected to 48 h fasting, injections of various neurotransmitters/neuropeptides antagonists or the physical denervation of vagus nerve have revealed the pathway mediating the fasting-induced suppression of G n R H / L H release (Cagampang and Maeda, 1991; Cagampang et al., 1991, 1992a,b). Fig. 7 shows a schematic illustration describing this neural pathway. The information originating in fasting signals is partly received by peripheral organs, such as the upper digestive tract, because acute transection of the vagus nerve immediately restores fasting-induced suppression of LH release (Cagampang et al., 1992a). The effect was reproduced by dissecting the gastric branch of vagus nerve, but not by either hepatic or celiac vagotomy. The involvement of

noradrenergic neurons in this LH inhibition has been also suggested by a series of experiments (Cagampang et al., 1992b; Maeda et al., 1994). The suppression of pulsatile LH release during fasting was blocked by a local injection of a-methyl-p-tyrosine, a catecholaminergic synthesis inhibitor, into the PVN. Furthermore, fasting-induced inhibition of LH pulses was immediately reversed when an c~2-adrenergic receptor antagonist, but not cq- or ~-adrenergic antagonists, were injected into the third ventricle. These results clearly suggest that noradrenergic neurons projecting to the PVN play a key role in suppressing LH secretion in fasted animals via c~2-adrenergic receptors. Furthermore, the A2 region of the nucleus of the solitary tract (NTS) is one of the major brain regions where noradrenergic cell bodies projecting to the PVN are located (Sawchenko and Swanson, 1983). The NTS receives neural inputs from visceral organs via the vagus nerve. Thus, it is possible that the visceral information originating in the upper di-

198
Unfasted
Saline

Fasted

Saline

E
"'~ 0 Z .d 5
I I I I I I I f

c~-helieal CRF

s-helical CRF

E u} _m
a.

Hours

Fig. 8. Effect of a CRH antagonist on fasting-induced suppression of LH pulses in estrogen-primedovariectomizedrats. The CRH antagonist (a-helical CRF) or saline was injected into the third cerebroventricle 1 h after the onset of blood sampling (arrows). Each panel shows the profileof plasma LH concentrations in a representativeanimal in 48-h-fasted or unfasted rats. Intracerebroventricularinjection of a-helical CRF immediately restored suppressed LH pulses in the fasted rat, but did not affect LH pulses in the unfasted animal. gestive tracts is relayed to the NTS via the gastric vagus nerve to activate the ascending noradrenergic pathway. In addition, the administration of a CRH antagonist in the third ventricle reversed the LH suppression during fasting (Fig. 8), suggesting that CRH mediates the fasting-induced inhibition of LH pulses (Maeda et al., 1994). This result is consistent with the notion that noradrenaline stimulates CRH release from the PVN, and in this respect catecholaminergic terminals have been shown to have a close relationship with CRH neuronal elements (Alonso et al., 1986; Kitazawa et al., 1987; Hornby and Piekut, 1989; Liposits and Paull, 1989; Mezey and Palkovits, 1991). Furthermore, third ventricle injection of a tx-opioid receptor antagonist also blocked the LH inhibition (Cagampang and Maeda, 1991). From these results it is possible to speculate that the noradrenergic neurons originating in the lower brainstem and projecting to the PVN induce CRH release, and this subsequently leads to release of endogenous opioids, resulting in the suppression of GnRH/LH release during fasting (Fig. 7). The PVN has been shown to be a center for the physiological responses when an animal is subjected to a stressor(s), and CRH plays a key role

in mediating the stress responses. For instance, suppression of gonadal function or LH secretion by experimental stress, such as immobilization or electrical foot shock stress, has been well documented (Rivier and Vale, 1984; Rivier et al., 1986; Maeda et al., 2000). The stress-induced inhibition of LH release is mediated by the hypothalamic CRH and opioid peptides (Vale and Spiess, 1981; Gindoff and Ferin, 1987; Petraglia et al., 1987). Therefore, the results obtained from our fasting model suggest that fasting shares the common neural pathway with the other stress responses to suppress LH release. Interestingly, the PVN has also been known as a center regulating feeding responses (Sawchenko et al., 1981; Leibowitz, 1988). Noradrenergic inputs to and c~2-adrenergic receptors in the PVN play the major role in inducing feeding behavior in rats. Thus, the PVN could coordinate two important functions, reproductive functions and food intake, to preserve the energy under conditions of malnutritional.

Role of estrogen in modulating the activity of neuroendocrine pathway

Estrogen has been shown to have diverse effects on brain functions, such as neuronal plasticity and regulation of neuroendocrine system and behaviors (Naftolin et al., 1993, Naftolin et al., 1996). Classical estrogen receptor a (ERc0 are distributed in various brain regions, such as preoptic area, hypothalamic ventromedial nucleus and arcuate nucleus (Sar and Parikh, 1986; Shughrue et al., 1997). As already shown above, 48 h fasting suppresses pulsatile LH release in an estrogen-dependent manner, suggesting that estrogen may modulate the activity of the neuroendocrine pathways involved in the fasting-induced inhibition of GnRH/LH release. Ovariectomized animals bearing local estradiol implants in the PVN or A2 region in the NTS show clear suppression of pulsatile LH release after 48 h fasting (Fig. 9), while the suppression is not found in fed animals (Nagatani et al., 1994). In addition, animals with estradiol implants in the preoptic area, arcuate nucleus or A1 region do not show any significant changes in LH release under both fasted and unfasted condition (Fig. 9). Thus, the PVN and/or A2 region appear to be the major estrogen feedback sites related to the fasting-induced suppression of

199

PVN

L?I

NTS

Vagus nerve I

Fasting information

POA ARC Changes in number of ERa immunoreactive cells after 48-h fasting Changes in LH pulses after local implantation of E2 in 48-h fasted OVX rats ~ b

PVN t
/

PeVN VMH l .

LC ne

A1 ne

NTS (A2) l
/

t,

ne

ne

P
5

: no change

l :increase

~ : decrease

ne: not examined

Fig. 9. How and where estrogen acts in the brain to enhance the fasting-inducedsuppressionof LH pulses. Schematic illustrationshowing that fasting information increases number of estrogen receptor c~ (ERc0-containingcells in the nucleus of the solitary tract (NTS) via the vagus nerve, and in the hypothalamic paraventricular nucleus (PVN). Estrogen in the peripheral circulation acts on the estrogen receptors in these brain nuclei to activate the neural pathway mediating fasting-inducedsuppression of GnRH/LH release. Summary of our previous results indicating the sites of action of estrogen revealed by estrogen implant studies and the fasting-inducedchanges in the number of ER-immunoreactivecells in various brain nuclei. POA, preoptic area; ARC, arcuate nucleus; PeVN, periventricularnucleus; VMH, ventromedialhypothalamicnucleus; LC, locus coeruleus.

LH release. It should be noted that these two brain regions (PVN and A2) are the nuclei where noradrenergic neurons are projecting to and originating from, respectively. Interestingly, acute infusion of estrogen into the PVN, but not into the A2 region, in 48-h fasted, ovariectomized rats suppresses pulsatile

LH release within an hour (Nagatani et al., 1996a). This result suggests that estrogen may modulate these two nuclei by temporally distinct mechanisms. Furthermore, the local injection of noradrenaline into the PVN suppresses pulsatile LH release strongly in the presence of estrogen, and this inhibition is re-

200 stored by central administration of a CRH antagonist (Tsukamura et al., 1994). On the contrary, the same noradrenaline treatment shows only transient suppression of LH pulses in the absence of estrogen. Therefore, estrogen may either enhance the CRH expression in the PVN and then ensures the strong suppression of GnRH/LH release, or increase neuronal responsiveness to noradrenaline. The PVN and NTS are not the major nuclei containing ERa, while this receptor is consistently expressed in areas like the preoptic area and arcuate nucleus. Actually, little ERc~-immunoreactivity is found in the PVN and NTS under normal-fed conditions. However, 48 h fasting significantly increases the numbers of ERc~ immunoreactive cells both in the PVN and NTS (Fig. 9) (Estacio et al., 1996b). The increase in the number of the ERc~-containing cells was blocked by vagotomy (Estacio et al., 1996a), suggesting that fasting increases ERc~ expression in the PVN and NTS through the vagus nerve. Interestingly, the number of ERc~-immunoreactive cells is also increased by 1 h of immobilization stress. Fasting and other experimental stresses induce very similar responses of ERc~ expression in these two nuclei. Recent studies have shown that fasting does not affect estrogen receptor [3 (ER[3)-immunoreactivity in the PVN (unpublished observation), which is one of the major nuclei expressing ER[3 (Shughrue et al., 1997). Thus, environmental cues (e.g. fasting or other stresses) can regulate the reproductive axis by stimulating ERc~ expression in the PVN and NTS. Consequently estrogen acts on these nuclei to enhance the activity of neural pathway via inducing protein synthesis or modifying neuronal plasticity. In the fasted rat model, estrogen may enhance the expression of adrenergic receptors or CRH in the NTS and PVN, respectively (Fig. 9). 1985; Bronson, 1988; Wade and Schneider, 1992; Wade et al., 1996). Energy substrates, such as glucose and free fatty acids, are candidate signals which are sensed by the brain or peripheral organs to monitor the nutritional condition. Schneider and Wade (1989) first demonstrated that pharmacological glucoprivation with 2-deoxyglucose (2DG), a glucose antagonist, impairs estrous cyclicity in the hamster, and that lesions of the area postrema block the effect of 2DG (Schneider and Zhu, 1994). This clearly indicated that the brain monitors glucose availability to regulate the gonadal function. Actually, the presence of the brain glucose-sensing mechanism has been well documented in feeding studies in which central 2DG injection increases food intake (Ritter et al., 1992). Our studies support this idea of a glucose sensor, because central as well as peripheral administration of 2DG suppresses pulsatile LH release in the rat (Murahashi et al., 1996; Nagatani et al., 1996b). The neural pathway mediating the suppression of LH pulses induced by pharmacological blockade of the glycolysis, is very similar to that mediating fasting-induced suppression of LH pulses, because 2DG administration increases noradrenaline release in the PVN and the blockade of catecholamine synthesis in the PVN nullifies the effect of 2DG (Nagatani et al., 1996c), as was demonstrated in the fasted model. Furthermore, central administration of CRH antagonist blocks the 2DG-induced suppression of pulsatile LH release in estrogen-treated ovariectomized rats (Tsukahara et al., 1998). It is reasonable to consider the presence of brain glucose sensing system at least in the lower brainstem, because 2DG infusion into the fourth ventricle, with slow enough flow rate to avoid the backward flow to the rostral direction, inhibits LH pulses in male rats (Murahashi et al., 1996). This result is consistent with a previous result indicating the presence of a glucosensor in the hindbrain involved in the induction of feeding behavior (Ritter et al., 1981). The question on the location of brain glucose sensor could be answered by considering the glucosesensing mechanisms of peripheral organs. One of the most well known organs monitoring the circulating glucose level is the pancreas. Pancreatic B-cells are responsible for insulin secretion to maintain the homeostatic control of blood glucose levels. Thus, this type of cells always monitors the glucose avail-

Signal(s) and sensor(s) mediating energetic regulation of LH release

It is apparent that nutritional cues are critically important for regulating reproductive functions, and the next question would be what signal(s) conveys the metabolic condition and how the brain recognizes it. Of various nutrients, energy level has been suggested by many studies to play a critical role in regulating the activity of reproductive axis (Foster and Olster,

201

10

15

20

Days after parturition

Fig. 10. Relative contribution of the factors involvedin the suppression of LH secretion in lactating rats. The suckling stimulus itself strongly suppresses GnRH/LH pulses in the first half of lactation through a neural pathway but this inhibitoryinfluence gets weaker towards the end of lactation. Metabolic factors, such as energy deficiency,may take over the role in suppressing LH secretionafter day 10 postpartum. Ovarian steroids may play a role in the suppression, particularlyby modulatingresponsesto metabolicfactors. ability in the general circulation. Combination of glucokinase (hexokinase IV) and glucose transporter (GLUT) 2 with high Km values in the pancreatic B-cell has been considered to compose the glucose sensor that regulates insulin secretion. High Km values of these functional proteins permits pancreatic B-cells to monitor changes in blood glucose level at around 5.5 mM. Our recent immunohistochemical study revealed that immunoreactive pancreatictype glucokinase but not hepatic-type glucokinase is present in some discrete brain areas (Maekawa et al., 2000). Interestingly, the immunoreactivity is present in the cytoplasm of ependymocytes lining most of the cerebral ventricles and in serotonergic neurons in the raphe nuclei located in the midbrain and medulla oblongata. The ependymocytes containing the glucokinase have also GLUT1 and GLUT2 in its cilia, and GLUT4 in the cytoplasm (Maekawa et al., 2000). This result may imply that the brain takes up glucose from the cerebrospinal fluid by those glucose transporters and monitors the glucose availability in the cerebrospinal fluid with the glucokinase. On the other hand, glucose availability would not be the only signal representing the energetic or metabolic condition of the animals. Lipoprivation with a [3-oxidation blocker drastically suppressed pulsatile LH release (unpublished data), as well as inducing the feeding behavior (Ritter et al., 1992). This result suggests that not only glucose but also free fatty acids may serve as signals and are probably sensed by both peripheral and central organs. Total energy availability may alter the reproductive function through regulating tonic GnRH/LH release.
Non-metabolic and metabolic cues leading to lactational anestrus

The evidence presented in the foregoing sections suggest that three major factors, ovarian steroids, suckling stimulus and metabolic factors, may be involved in suppressing LH secretion, and the complex of these factors ensure the infertility in lactating animals (Fig. 10). In lactating rats, progesterone released by the lactational corpora lutea may be effective throughout the lactation. Estrogen, the plasma level of which gradually increases in the last half of lactation (Taya and Sasamoto, 1987), may also exert an inhibitory effect on GnRH/LH secretion. The suckling stimulus itself strongly suppresses GnRH/LH pulses in the first half of lactation through a neural pathway, but this inhibitory influence gets weaker towards the end of lactation (Fig. 10). Metabolic factors may take over the role in suppressing LH secretion after day 10 of lacta-

202 tion onward. Ovarian steroids may participate in LH suppression along with the negative energy balance in the last half of lactation to secure the metabolic suppression of LH release. In this respect it should be noted that a metabolic deficiency causes LH suppression in the presence of estrogen, as shown in our fasted model. This idea is in accordance with our previous result showing that central administration of Ix-opioid receptor antagonist did not affect suppressed LH release during early lactation, but blocked the suppression in late lactation in the intact rat (Tsukamura et al., 1992). The mechanism mediating the suppression of LH release in late lactation involves metabolic cues and shares a common mechanism with the fasted model, because both models include endogenous opioid peptide-dependent pathway in the presence of ovarian steroids. Recently, Smith and colleagues reported that gene expression or the immunoreactive levels of several brain peptides, such as neuropeptide Y, leptin, and agouti-related protein, are altered by the suckling stimulus (Li et al., 1998, 1999; Brogan et al., 1999; Chen et al., 1999). These peptides are well known to affect both feeding behavior and reproductive function, suggesting that energetic deficiency alters the dynamics of such peptides in the brain to regulate both food intake and reproductive performance during lactation. Several more peptides, such as motilin and melanin-concentrating hormone, have recently been found to induce feeding and suppress LH secretion in female rats (Tsukamura et al., 2000a,b), and these may also be involved in the hormonal changes during lactation. The data presented here suggest that the lactating rat serves as a good model for the study of the non-metabolic and metabolic factors regulating LH secretion during lactation, providing their endocrine status is properly understood and manipulated.
Abbreviations

GnRH GLUT LH NTS PVN

gonadotropin-releasing hormone glucose transporter luteinizing hormone nucleus of the solitary tract paraventricular hypothalamic nucleus

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Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 14

Neuroendocrine changes and fertility in breast-feeding women

Alan S. M c N e i l l y *
MRC Human Reproductive Sciences Unit, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK

Abstract: Breast-feeding through the suckling stimulus suppresses fertility for a variable time after birth. Initially there is a period of pituitary gonadotroph recovery from the suppressive effects of the high steroid levels of pregnancy, followed by a period of suppressed ovarian activity associated with limited follicle growth. During this period of breast-feeding-induced amenorrhea, the pulsatile secretion of luteinizing hormone (LH), which reflects hypothalamic gonadotropin-releasing hormone (GnRH) release, is erratic and much slower than the one pulse per hour required in the normal follicular phase of the menstrual cycle to drive follicle growth. At some time the suckling stimulus drops below a threshold resulting in a resumption of reasonably organized pulsatile LH secretion, which is associated with development of follicles and some steroid secretion. However, positive feedback of estradiol which triggers the preovulatory LH surge and ovulation appears to be blocked by continued suckling, until suckling is reduced further and positive feedback and ovulation resumes. Often while women continue to breast-feed the first few ovulations and menses are associated with inadequate corpus luteum function, which would probably not support a pregnancy. Eventually normal menstrual cycles resume when suckling declines further. The duration of amenorrhea and subsequent period of inadequate luteal function varies greatly between mother-baby combinations, and in different societies. Exactly how the suckling stimulus reduces pulsatile secretion of GnRH/LH is not clear, although clinical studies do not support a role for opioids or dopamine. The role of prolactin remains uncertain since suckling releases both prolactin and suppresses GnRH release. Regardless of the precise mechanism, it is clear that breast-feeding in women can suppress fertility for prolonged periods, and women may proceed from pregnancy through lactation to another pregnancy and lactation with no menstrual period for several years.

Introduction

In spite of overwhelming demographic, epidemiological and physiological evidence (McNeilly, 1994, 1997; McNeilly et al., 1993; Short, 1993; WHO, 1998a,b) many people remain unconvinced that lactation in women can successfully inhibit fertility. However, women, in c o m m o n with almost all other

* Correspondence to: Alan S. McNeilly, MRC Human Reproductive Sciences Unit, University of Edinburgh Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK. Tel.: -t-44-131-229-2575; Fax: -1-t-44-131-228-5571; E-mail: a.mcneilly@hrsu.mrc.ac.uk

mammals, do experience a period of infertility associated with breast-feeding. However, there is enormous variation in the duration of this infertility which can be related to the complex interactions between each m o t h e r - b a b y unit, the suckling pattern established between this pairing, and the strictures of society, which can impose irrational and uninformed prejudices to breast-feeding, and indirectly undermine the effectiveness of the suckling of the baby in suppressing fertility. While a detailed description of the potential mechanisms controlling fertility in animal models is presented elsewhere in this volume (Tsukamura and Maeda, 2001), this brief review will highlight the major factors regulating the suppression of fertil-

208 ity by breast-feeding in women, and the major areas that still need to be addressed if there is going to be universal acceptance of breast-feeding as a reliable regulator of fertility. it is able to grow and develop normally. There is evidence that the shorter the interbirth interval the greater the risk of both morbidity and mortality of the first child (Thapa et al., 1988; Short, 1993). Thus, there are sound biological reasons for breast-feeding to have evolved a dual role of feeding the baby and suppressing fertility. However, a major hurdle for the application of breast-feeding as a reliable contraceptive is the relative lack of signs of the imminent return of fertility. A consensus statement (Kennedy et al., 1989), subsequently modified after confirmation (Kennedy et al., 1996), has proposed that women remain at a very low risk of pregnancy if they remain amenorrheic, and this is applicable up to at least 6 months postpartum. Large scale trials of this Lactational Amenorrhea Method have confirmed the efficacy of using the natural contraceptive effects of breast-feeding and adopting it in contraceptive provision, but there is still much resistance to a universal adoption of this method. In many studies it has been difficult to determine which component of breast-feeding is responsible for the suppression of fertility. Often this is because the relevant observations on the pattern of breast-feeding and supplementary food provision were not adequately recorded. However, in most studies in which adequate data are available there is a recognition that the major factor is the strength of the suckling stimulus, both in terms of frequency and duration (McNeilly, 1993, 1994, 1997). However, the influence of the different components of the suckling pattern that results in suppression of fertility in different societies is not clear. Hence, in some situations it is normal to
BIRTH

Suckling and the resumption of fertility

In the absence of breast-feeding menstrual cycles resume 4 - 6 weeks after birth, but these cycles are usually associated with reduced or inadequate corpus luteum function, and probably would not sustain a pregnancy (McNeilly et al., 1982). The second menstrual cycle is usually normal (McNeilly, 1993). Thus, it takes up to 8 weeks for the hypothalamicpituitary-ovarian axis to return to normal menstrual cycles. In contrast, breast-feeding can delay the resumption of menstrual cycles for longer than 4 years. Indeed some women become pregnant during this period of lactational amenorrhea without experiencing a menstrual period at all (Fig. 1; McNeilly et al., 1983a). Even in the Western World, it has been known for some women to have two children, breastfeeding both, and not having had a menstrual period for 6 years. This highlights the potential that the biology of women has evolved to avoid menstruation, emphasizing the importance of maintaining a period of infertility during breast-feeding, and has two important implications. The first is that the baby that is suckling requires breast milk for a reasonable time and suppressing fertility allows the mother's metabolic resources to be directed to this activity. The second is that the suckling of the baby is delaying the arrival of its sibling until such time as
BIRTH

INTERBIRTH INTERVAL CONCEPTION

PREGNANCY

RECOVERY FROM PREGNANCY 11

AMENORRHOEA ~

OVULATORY CYCLES

PREGNANCY

BREAST FEEDING

"::>

Fig. 1. The componentsof the interbirth interval. After pregnancythere is a 4-8-week period of recoveryof the hypothalamo-pituitary axis from the suppressive effects of pregnancy steroids followedby an indefinite period of amenorrheadetermined by the pattern of suckling. The menstrual cyclesthat return may have inadequate corpus luteum functionwhich is associated with reduced fertilitybefore conceptionand pregnancy.

209 suckle very frequently for short periods, whereas in other societies infrequent periods of suckling each of much longer duration occur (Short, 1993). Since the frequency of suckling is determined by many factors it is difficult to define in precise terms exactly which parameter is most important. Nevertheless, it is clear that suckling in many different patterns results in the suppression of reproductive function. Before considering the changes in pituitary and ovarian function in breast-feeding, the normal control of the menstrual cycle will be briefly described. The central control is the pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus which stimulates the secretion of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. The output of both LH and FSH is regulated by the frequency of GnRH release, controlled by feedback of estradiol from the ovarian follicle, and progesterone from the corpus luteum in the luteal phase of the cycle. At menses, plasma concentrations of FSH increase and stimulate the growth of follicles which secrete inhibin B to suppress FSH secretion. At the same time LH pulse frequency increases and this stimulates estradiol secretion from these developing follicles. Around day 5 of the follicular phase, a single follicle is selected and this continues to grow into the dominant follicle which will ovulate. All other follicles stop growing due mainly to the suppression of FSH, which occurs as a result of the negative feedback of inhibin B and estradiol directly at the pituitary. The maintenance of estradiol secretion is dependent on a high pulse frequency of LH release, controlled by the pulsatile release of GnRH. When the follicle reaches a diameter of 15-20 mm, estradiol will normally have reached the threshold required to trigger a major release of GnRH from the hypothalamus, resulting in the release of the preovulatory LH surge from the pituitary. This surge causes rupture of the preovulatory follicle, release of the egg, and formation of the corpus luteum which produces progesterone, estradiol and inhibin A. Thus, for a normal ovulatory menstrual cycle all components of this axis must function in the correct sequence. There is little, if any, role of prolactin in regulating the menstrual cycle (McNeilly, 1985). The changes in reproductive activity during breast-feeding resulting in suppression of fertility, and hence regulation of the interbirth interval, are summarized in Fig. 1. After a period of recovery from the suppressive effects of placental steroids on the hypothalamo-pituitary axis lasting probably 4-6 weeks, there is a period of amenorrhea during which time menstrual activity is suppressed. During this time ovarian activity may resume, and ultrasound has now revealed that medium to large follicles may be present, although they do not necessarily produce normal amounts of estrogen (Flynn et al., 1991). More recently it has been shown that these follicles do produce inhibin B (H. Burger, personal communication; Peerhentupa et al., 2000). As suckling declines so menstrual cycles resume, with apparently normal follicle growth in terms of estradiol secretion. However, these often result in the formation of an inadequate corpus luteum, either in terms of duration of function, or of progesterone production, or both (McNeilly et al., 1982). Several menstrual cycles may have inadequate luteal function before a normal menstrual cycle occurs with return of fertility (McNeilly et al., 1993). The following sections will examine the effect of suckling on the different components of the reproductive system.
GnRH and the secretion of LH and FSH

During pregnancy the high levels of placental steroids suppress plasma levels of both LH and FSH, presumably by suppressing GnRH output from the hypothalamus. At term, pituitary content of LH is less that 1% of normal (De la Lastra and Llados, 1977). Within 4 weeks of delivery plasma concentrations of FSH return to levels equivalent to those in the menstrual cycle (Glasier et al., 1984). However, ovarian activity remains suppressed both in terms of follicle growth, and steroid and inhibin B production (Fig. 2). Indeed, with the low level of negative feedback from the ovaries, equivalent to that in menopausal women, it is clear that FSH levels are inappropriately low, and thus are effectively suppressed. Pulsatile LH secretion, reflecting GnRH output, is also severely suppressed. Although very low amplitude, high frequency pulses of LH have been recorded (Nunley et al., 1991) normal pulsatile release is severely compromised, and no normal pulses of LH are observed until around 5-6

210

Fig. 2. The relationship between pulsatile LH secretion over a 24-h period and the resumption of ovarian activity during and after breast-feeding. In the early postpartum period LH levels are low with no pulsatile activity. By 6-8 weeks postpartum low frequency sporadic pulses of LH occur and this restores some estradiol secretion from the developing follicles. Once the frequency is restored to approximately one pulse per hour ovulation resumes with consequent progesterone secretion and menstruation.

~,,~tlUlt

.:

~ t ~ l M l l l o ~

L H m ~ m

Fig. 3. The control of secretion and feedback modulation of prolactin (left panel), FSH (middle panel) and LH (right panel) in breast-feeding women. Prolactin is required for milk production but appears to have little influence on reproductive function directly. FSH release requires some GnRH input from the hypothalamus hut the major control of release is via a direct negative feedback by follicular estradiol and inhibin B at the pituitary, although withdrawal of GnRH will also reduce secretion. LH output is regulated exclusively by GnRH pulses which are controlled by the negative feedback effects of estradiol in the follicular phase, before stimulation of the preovulatory GnRH surge by elevated levels of estradiol secreted by the preovulatory follicle. During breast-feeding the hypothalamus shows increased sensitivity to the negative feedback effects of estradiol resulting in an inability to sustain normal frequency of pulsatile GnRH output. The suckling-induced enhanced sensitivity of the hypothalamus to the negative feedback effects of estradiol maintains a control over the normal release of GnRH preventing normal patterns of FSH and LH release. Ovulatory menstrual cycles resume when the suckling stimulus is reduced below a threshold with return of normal sensitivity of the hypothalamus to steroid negative feedback.

211 weeks postpartum (Glasier et al., 1984; Tay et al., 1992). During this time the capacity of the pituitary to release LH and FSH has returned since exogenous GnRH will evoke a normal release of LH and FSH (Tay et al., 1993). Thus, the failure of a return to normal pulsatile LH release and lower than expected FSH release, both appear to depend on the reduced release of GnRH during this period. Between 6 and 8 weeks the pulsatile release of LH returns, although at a low frequency, and with a sporadic interpulse interval (Fig. 2), and the pattern is quite different to that observed during puberty (Wu et al., 1991). The return of some pulsatile secretion of GnRH also enhances FSH secretion (Tay et al., 1992) and may stimulate follicular growth. The resumption of some LH release is associated with a stimulation of estradiol release from these follicles, but the infrequent pattern of GnRH/LH release is insufficient to maintain normal estradiol release (Fig. 2). The infrequent pattern of GnRH/LH release is sustained for a variable time during breast-feeding. The resumption of menstrual cycles, which is preceded by ovulation, only appears to occur when the pulse frequency of LH release approaches one pulse per hour typical of the follicular phase of the menstrual cycle (Fig. 2). Treatment of amenorrheic, breast-feeding women with GnRH delivered every 60-90 min by a pulsatile infusion pump to mimic the normal follicular pattern of GnRH release resuited in the induction of normal follicle growth, ovulation and the formation of a functional corpus luteum (Glasier et al., 1986; Zinaman et al., 1995). This supports the contention that suckling maintains infertility by altering and slowing the pattern of pulsatile release of GnRH. Only when the suckling input declines below a threshold can GnRH pulsatile secretion return to a normal pattern of release every hour, and control of pulse frequency return to the normal negative feedback regulation by gonadal steroids. breast-feeding women which would allow a proper evaluation of the mechanisms involved, since any drug that might be used can pass into the baby through the breast milk. Nevertheless some insights have been gained. The preovulatory LH surge is generated in response to the increase in estradiol secretion by the developing and mature preovulatory follicle which induces a major discharge of GnRH. Breast-feeding suppresses the ability of estradiol to induce an LH surge (Baird et al., 1979). Furthermore, there is also an increase in the sensitivity to the negative feedback action of estradiol on the secretion of both LH and FSH (Baird et al., 1979). This has been confirmed in recent studies in which both LH and FSH secretion has been dramatically reduced by low levels of estradiol delivered from 50 Ixg hormone replacement patches (Illingworth et al., 1995; Peerhentupa et al., 2000). These low levels of estradiol do not affect gonadotrophin secretion in non-breast-feeding women. However, in breastfeeding women they result in the suppression of FSH secretion with resultant suppression of inhibin B secretion (Peerhentupa et al., 2000), indicating a reduction in the competence of any small follicles present, and a complete suppression of the pulsatile release of LH (Illingworth et al., 1995). Since GnRH induced a normal release of LH and FSH it can be concluded that the suppression of gonadotrophin secretion was due to a suppression of GnRH release from the hypothalamus. Thus, suckling had induced a major increase in the sensitivity of the hypothalamus to the negative feedback effects of estradiol (Fig. 3). Dopamine does not appear to be involved in the suppression of GnRH pulsatility during breastfeeding. While treatment of breast-feeding women with the dopamine antagonist metoclopramide can cause a massive release of prolactin, there is no effect on the basal or pulsatile release of either FSH or LH (Tay et al., 1993). Furthermore, treatment with the opioid blocker naloxone also failed to affect LH and FSH release (Tay et al., 1993; Illingworth et al., 1995). Interestingly naloxone did enhance the release of LH in response to GnRH, but this only occurred in women in whom estradiol levels were slightly raised above basal levels normally occurring in early lactation (Illingworth et al., 1995). This suggests that opioids are not

Relative contribution of steroid sensitivity, dopamine and opioids

While suckling clearly suppresses GnRH secretion, the mechanisms whereby the suckling stimulus affects GnRH pulsatile secretion are not clearly understood. It is difficult to undertake many of studies in

212 involved in the suppression of GnRH pulsatile secretion during the period of breast-feeding when ovarian activity is completely suppressed. However, when ovarian activity resumes with some secretion of estradiol then opioids may become involved, but apparently by affecting the sensitivity of the pituitary to GnRH. This requires further investigation since this result is unexpected, particularly as studies in other species would suggest that opioids act at the hypothalamus to modulate GnRH secretion.
Prolactin Conclusions

The major role of prolactin is to stimulate the production of milk. Suckling causes the release of prolactin and high mean plasma levels of prolactin are sustained during breast-feeding (Fig. 3). The changes in the pattern of prolactin release are determined entirely by the pattern of suckling. Low frequency suckling results in discrete episodes of prolactin release, while high frequency suckling will maintain high basal levels of prolactin with indistinct increases in response to suckling (Tay et al., 1996). However, the release of prolactin is directly related to the tactile stimulus of the nipple during suckling, and is not associated with non-tactile stimulation (McNeilly et al., 1983b). While the high levels of prolactin have been associated with the suppression of fertility during breast-feeding, there is no compelling evidence to support this contention. Altered forms of prolactin may be released during lactation (Campino et al., 1999), but there is no evidence that these forms of prolactin have any direct or indirect effect on the reproductive axis. The high levels of prolactin may simply reflect the strength and frequency of suckling, and be acting as a monitor of suckling strength, rather than being a causal agent in the suppression of fertility (McNeilly, 1994, 1997). However, there appears to be a variation in both basal and lactation-related plasma prolactin levels associated with both patterns of breast-feeding, and other behavioral factors (Stallings et al., 1998). Thus there is no doubt that the role of prolactin in the suppression of fertility will remain a matter of conjecture - - for further evidence see Grattan (2001, this volume) and Tsukamura and Maeda (2001, this volume).

There can be no doubt that suckling during breastfeeding has a profound suppressive effect on the resumption of fertility postpartum. Although prolactin is elevated there is little good evidence to support a role for prolactin in the suppression of fertility in human (Fig. 3). It is clear that suckling disrupts the normal pattern of pulsatile GnRH secretion and replacement of a normal input of pulsatile GnRH results in a resumption of normal ovarian activity. Furthermore, the increase in sensitivity to estradiol associated with suckling may be the mechanism whereby prolonged periods of amenorrhea can be maintained without a complete cessation of GnRH pulsatile secretion. The return of erratic pulsatile GnRH release allows a return of a more normal release of FSH which, in turn, induces follicle growth and inhibin B secretion. The reduced pulsatile LH release will stimulate the developing follicles to produce some estradiol, which will have two negative effects. Firstly, this estradiol acting with inhibin B will suppress FSH release by a direct action at the pituitary, withdrawing support for continued follicle growth (Fig. 3). Secondly, because of the enhanced sensitivity to negative feedback, the estradiol will switch off further pulsatile GnRH and LH release (Fig. 3). Thus, all gonadotrophic support for the follicle that had been growing will be withdrawn and it will die. This cycle could be repeated for a prolonged period providing an adequate suckling input is maintained (McNeilly, 1993; McNeilly et al., 1994). As soon as suckling declines below some undefined threshold there would be loss of enhanced sensitivity to negative feedback and a normal pattern of pulsatile GnRH release will resume, with the return of sustained follicle growth and ovulation. Whether subsequent corpus luteum function will be normal remains uncertain. In many instances inadequate corpus luteum function may occur and infertility remains, even though menstrual cycles have resumed. The mechanisms causing inadequate corpus luteum function remain unknown. Furthermore, exactly how suckling disrupts the puisatile secretion of GnRH and enhances the sensitivity of the GnRH pulse generator to estradiol remains unknown. The task ahead is to devise safe and ethical studies to allow the elucidation of these

213

mechanisms in order to provide potential ways of enhancing the natural biological suppression of fertility both for breast-feeding and non-breast-feeding women. Abbreviations FSH GnRH LH follicle-stimulating hormone gonadotropin-releasing hormone luteinizing hormone

References
Baird, D.T., McNeilly, A.S., Sawers, R.S. and Sharpe, R.M. (1979) Failure of estrogen-induced discharge of luteinizing hormone in lactating women. J. Clin. Endocrinol. Metab., 49: 500-506. Campino, C., Torres, C., Ampuero, S., Diaz, S., Gonzalez, G.B. and Seron-Ferre, M. (1999) Bioactivity of prolactin isoforms: lactation and recovery of menses in nursing women. Hum. Reprod., 14: 898-905. De la Lastra, M. and Llados, C. (1977) Luteinizing hormone content of the pituitary gland in pregnant and nonpregnant women. J. Clin. Endocrinol. Metab., 44: 921-923. Flynn, A., Docker, M., Brown, J.B. and Kennedy, K.I. (1991) Ultrasonographic patterns of ovarian activity during breastfeeding. Am. J. Obstet. Gynecol., 165: 2027-2031. Glasier, A.F., McNeilly, A.S. and Howie, P.W. (1984) Pulsatile secretion of LH in relation to the resumption of ovarian activity post partum. Clin. Endocrinol., 20: 415-426. Glasier, A.E, McNeilly, A.S. and Baird, D.T. (1986) Induction of ovarian activity by pulsatile infusion of LHRH in women with lactational amenorrhoea. Clin. Endocrinol., 24: 243-252. Grattan, D.R. (2001) The actions of prolactin in the brain during pregnancy and lactation. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingram (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 153-171. Illingworth, EJ., Seaton, J.E.V., McKinlay, C., Reid-Thomas, V. and McNeilly, A.S. (1995) Low dose transdermal oestradiol suppresses gonadotrophin secretion in breastfeeding women. Hum. Reprod., 10: 1671-1677. Kennedy, K.I., Rivero, R. and McNeilly, A.S. (1989) Consensus statement on the use of breastfeeding as a family planning method. Contraception, 39: 477-496. Kennedy, K.I., Labbok, M.H. and Van Look, EEA. (1996) Consensus statement - - lactational amenorrhoea method for family planning. Int. J. Gynecol. Obstet., 54: 55-57. McNeilly, A.S. (1985) Prolactin and the corpus luteum. In: S.L. Jeffcoate (Ed.), The Luteal Phase. Current Topics in Reproductive Endocrinology, Vol. 4. John Wiley, Chichester, pp. 71-87. McNeilly, A.S. (1993) Breastfeeding and fertility. In: R.H. Gray, H. Leridon and. A Spira (Eds.), Biomedical and Demographic

Determinants of Reproduction. Clarenden Press, Oxford, pp. 391-412. McNeilly, A.S. (1994) Suckling and the control of gonadotropin secretion. In: E. Knobil and J. Neill (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 1179-1212. McNeilly, A.S. (1997) Lactation and fertility. J. Mammary Gland Biol. Neoplasia, 2: 291-298. McNeilly, A.S., Howie, P.W., Houston, M.J., Cook, A. and Boyle, H. (1982) Fertility after childbirth: adequacy of postpartum luteal phases. Clin. EndocrinoL, 17: 609-615. McNeilly, A.S., Glasier, A.E, Howie, P.W., Houston, M.J., Cook, A. and Boyle, H. (1983a) Fertility after childbirth: pregnancy associated with breast feeding. Clin. Endocrinol., 18: 167173. McNeilly, A.S., Robinson, 1.C.A.F., Houston, M.J. and Howie, P.W. (1983b) Release of oxytocin and prolactin in response to suckling. Br. Med. J., 286: 257-259. McNeilly, A.S., Forsyth, I.A. and McNeilly, J.R. (1993) Regulation of postpartum fertility in lactating mammals. In: G.E. Lamming (Ed.), Marshall's Physiology of Reproduction, 4th edn. Chapman and Hall, London, pp. 1037-1101. McNeilly, A.S., Tay, C.C.K. and Glasier, A. (1994) Physiological mechanisms underlying lactational amenorrhoea. Ann. New York Acad. Sci., 709: 145-155. Nunley, W.C., Urban, R.J. and Evans, W.S. (1991) Preservation of pulsatile luteinizing hormone release during postpartum lactational amenorrhoea../. Clin. Endocrinol. Metab., 73: 629636. Peerhentupa, A., Chritchley, H.O.D., Illingworth, P.J. and McNeilly, A.S. (2000). Enhanced sensitivity to steroid negative feedback during breast-feeding: Low dose estradiol (Transdermal Estradiol Supplementation) suppresses gonadotropins and ovarian activity assessed by inhibin B. J. Clin. Endocrinol. Metab., 85: 4280-4286. Thapa, S., Short, R.V. and Potts, M. (1988) Breast feeding, birth spacing and their effects on child survival. Nature, 335: 679682. Tsukamura, H. and Maeda, K.-I. (2001) Non-metabolic and metabolic factors causing lactational anestrus: rat models uncovering the neuroendocrine mechanism underlying the suckling-induced changes in the mother. In: J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingrain (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 187-205. Short, R.V. (1993) Lactational infertility in family planning. Ann. Med., 25: 175-180. Stallings, J.F., Worthman, C.M. and Panter-Brick, C. (1998) Biological and behavioural factors influence group differences in prolactin levels among breastfeeding Nepali women. Am. J. Hum. Biol., 10: 191-210. Tay, C.C.K., Glasier, A.E and McNeilly, A.S. (1992) The twentyfour hour pattern of pulsatile luteinizing hormone, follicle stimulating hormone and prolactin release during the first eight weeks of lactational amenorrhoea in breastfeeding women. Hum. Reprod., 7: 951-958. Tay, C.C.K., Glasier, A.E and McNeilly, A.S. (1993) Effect

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of antagonists of dopamine and opiates on the basal and GnRH-induced secretion of luteinizing hormone, follicle stimulating hormone and prolactin during lactational amenorrhoea in breastfeeding women. Hum. Reprod., 8: 532-539. Tay, C.C.K., Glasier, A.E and McNeilly, A.S. (1996) Twentyfour hour patterns of prolactin secretion during lactation and the relation to suckling and the resumption of fertility in breastfeeding women. Hum. Reprod., 11: 950-955. WHO (1998a) The World Health Organization multinational study of breasffeeding and lactational amenorrhoea. I. Description of infant feeding patterns and of the return of menses. Fertil. Steril., 70: 448-460. WHO (1998b) The World Health Organization multinational study of breastfeeding and lactational amenorrhoea. II. Factors

associated with the length of amenorrhoea. Fertil. Steril., 70: 461-471. Wu, EC.W., Butler, G.E., Kelnar, C.H.J., Stirling, H.F. and Huhtaniemi, I. (1991) Patterns of pulsatile luteinizing and follicle stimulating hormone secretion in prepubertal (midchildhood) boys and girls and patients with idiopathic hypogonadotrophic hypogonadism (Kallman's syndrome): a study using an ultrasensitive time-resolved immunofluorometric assay. J. Clin. Endocrinol. Metab., 72: 1229-1237. Zinaman, M.J., Cartledge, T., Tomai, T., Tippett, P. and Merriam, G.R. (1995) Pulsatile GnRH stimulates normal cycle ovarian function in amenorrhoeic lactating postpartum women. J. Clin. EndocrinoL Metab., 80: 2088-2093.

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 15

Food intake and leptin during pregnancy and lactation

Louise E. Johnstone 1 and Takashi Higuchi 2,*
l Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK 2 Department of Physiology, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan

Abstract: Successful reproduction requires the accumulation of energy reserves. Although acute and chronic food deprivation disrupts reproduction, surprisingly, an over-abundance of energy reserves can also result in infertility. The infertility of obese, ob/ob mice can be reversed by the reintroduction of leptin, the protein product of the ob gene. In rats, energy reserves are increased during pregnancy by fat accumulation and during lactation by hyperphagia. We have therefore investigated the interactions of leptin and food intake during late pregnancy and lactation in rats. Cycling rats consume their daily food intake during the dark phase and this is accompanied by a subsequent increase in plasma leptin concentration compared to light levels. During late pregnancy, rats increase their food intake during the dark phase and this is accompanied by a nocturnal increase in plasma leptin level. However, the nocturnal increase is not seen on the day prior to parturition, and is absent throughout lactation. Surprisingly, despite the massive increase in food intake during lactation plasma leptin levels continue to fall, suggesting that leptin release in response to food intake is suppressed during lactation. Furthermore, central leptin administration is less effective in reducing food intake in late pregnant and early lactating rats compared to cycling rats which suggests that these rats are insensitive to leptin. This may result from downregulation of brain leptin receptors. Decreased leptin production and action during late pregnancy and lactation will result in a decreased satiety effect, with up-regulation of orexigenic factors that produce hyperphagia, so allowing adequate energy intake for successful rearing of offspring.

Introduction: energy reserves and reproduction Reproduction can be considered to have two essential parts: first, the capability to become pregnant, and second the ability to maintain development of the conceptus through pregnancy and lactation to produce viable offspring. Adequate energy reserves are essential, in all mammals, for each component, with the amount of additional energy required for successful reproduction being related to body size. As a result, rodents require a relatively higher energy intake for reproduction compared to larger m a m mals. Therefore it is not surprising that modest dis-

* Corresponding author: Takashi Higuchi, Department of Physiology, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan. Fax: +81-776-61-8134; E-mall: higuchit @fmsrsa.fukui-med.ac.jp

ruptions in food intake can affect rodent fertility and the success of rearing offspring. An acute lack of energy intake through food deprivation disrupts the estrous cycle, pregnancy or lactation in rodents whereas human reproduction is relatively unaffected (Wade and Schneider, 1992). Chronic deprivation of food leads to a delay in puberty, amenorrhea or persistent anestrus in humans and rodents, respectively (Wade and Schneider, 1992). Surprisingly, an over-abundance of energy stores, as in obesity, as well as a lack of energy reserves can result in infertility (Barash et al., 1996; Chehab et al., 1996). Taken together these findings imply that there is an essential signal that conveys the level of energy reserves to the reproductive axis, which then enables reproduction to occur. Obesity arises from the lack of some humoral inhibitory factor to reduce food intake (Coleman, 1978), and this blood borne factor has been recently identi-

216 fled through the cloning of the obese gene (Zhang et al., 1994) as its protein product, leptin (Lonnqvist et al., 1995). Subsequently, obese, infertile, ob/ob mice have been shown to be deficient in leptin (Zhang et al., 1994) and db/db mice (Lee et al., 1996) andfa/fa (Chua et al., 1996) rats to be resistant to leptin due to a mutation in the leptin receptor. Leptin is a 146-amino acid peptide that is secreted by adipocytes (Zhang et al., 1994), and its receptor (Ob-R) is a member of the cytokine receptor family (Tartaglia et al., 1995). Many different forms of the receptor have been isolated (Tartaglia, 1997) although only one form, Ob-Rb, the long receptor, contains an intracellular domain (Tartaglia et al., 1995) which is coupled to the JAK-STAT intracellular pathway (Tartaglia, 1997) and so is thought to mediate the actions of leptin. However, the short forms of Ob-R (a and c) may be coupled to the MAP kinase pathway (Yamashita et al., 1997) and so mediate cell signaling. Leptin is thought to bind to a soluble leptin receptor, Ob-Re, in the plasma (Tartaglia, 1997). The relationship between leptin and the reproductive axis is under intensive investigation. We will discuss the current evidence for a role of leptin in energy regulation during reproduction. The role of ieptin in energy regulation Leptin is synthesized and secreted from fat cells in adipose tissue in response to an improvement in metabolic status. Leptin not only decreases excessive appetite that can result in obesity (Halaas et al., 1995), but also increases metabolic rate, locomotor activity and therefore energy utilization (Campfield et al., 1995; Halaas et al., 1995; Collins et al., 1996). Leptin thus has a major effect on energy balance in rodents. Leptin as a satiety factor An increase in food intake results in an increase in plasma leptin concentration (Pickavance et al., 1998), which is thought to act centrally as leptin receptor (Ob-R) mRNA has been localized in the choroid plexus, piriform cortex, thalamus, hippocampus, cerebellum and hypothalamus (Tartaglia et al., 1995; Guan et al., 1997). There is a high density of Ob-R mRNA in the arcuate nucleus, and to a lesser extent in the ventro- and dorsomedial hypothalamic (VMH, DMH, respectively) nuclei (Schwartz et al., 1996a): areas that have long been implicated in the control of energy homeostasis. The widespread distribution of leptin receptor forms in the brain (Guan et al., 1997; Hakansson et al., 1998) demonstrates the large number of potential sites and pathways that may be influenced by leptin. However, peripherally produced leptin cannot pass through the blood-brain barrier but may enter the brain by binding to the choroid plexus (Devos et al., 1996) before being transported into the cerebrospinal fluid (CSF) by a saturable mechanism (Banks et al., 1996; Caro et al., 1996). The arcuate nucleus contains the densest population of neuropeptide Y (NPY) containing neurons in the hypothalamus, and these project to the paraventricular nucleus (PVN) and the DMH (Bai et al., 1985). Central NPY is a powerful orexigenic peptide and over-expression of NPY leads to obesity (Frankish et al., 1995). Acting centrally, NPY has also been shown to decrease heat production and locomotor activity to produce a net energy gain (Billington et al., 1991). Leptin has been proposed to inhibit the actions of NPY as Ob-Rb mRNA is colocalized with NPY in neurons in the arcuate nucleus (Mercer et al., 1996), and intracerebroventricular (i.c.v.) administration of leptin decreases NPY mRNA expression in the arcuate nucleus (Schwartz et al., 1996b). Potentially leptin may act through its receptors in the arcuate nucleus, DMH and the lateral hypothalamus to directly inhibit the expression of other orexigenic peptides such as dynorphin, ~-endorphin, galanin, orexin A and B and melanin-concentrating hormone (Hakansson et al., 1998; Kalra et al., 1999) thereby counteracting the signals for increasing food intake. As yet the action of leptin on the production of these orexigenic peptides is unknown. Leptin has also been shown to augment the actions of other satiety factors as it potentiates the satiety effect of cholecystokinin (CCK) (Emond et al., 1999) and also upregulates POMC mRNA expression in the arcuate nucleus that produces melanocortin (Thornton et al., 1997) whose dysfunctional MC-4 receptor is linked to obesity (Fan et al., 1997). It has been suggested that melanocortin may act in the lateral hypothalamic and perifornical areas to inhibit the production

217 of the orexigenic neuropeptides, orexin and melaninconcentrating hormone (Schwartz et al., 2000). It is clear that leptin can either directly or indirectly inhibit production of orexigenic neuropeptides.

Food intake during the reproductive cycle in the rat

The estrous cycle

Normal estrous cycling is dependent on an adequate energy intake, as food deprivation delays the onset of the first estrus in prepubertal rats (Bronson, 1988), suppresses plasma LH concentration in adult female rats (Howland, 1972) and disrupts the estrous cycle by extending diestrus (Wade and Schneider, 1992). Refeeding results in an almost immediate return to normal cyclicity. Conversely, during the estrous cycle, food intake is suppressed by estrogen and stimulated by progesterone (Wade and Schneider, 1992). Under controlled experimental conditions rats have an ad libitum food supply, but despite this free access they do not feed randomly throughout a 24-h period. Both male and female rats display a significant diurnal variation in their food intake, consuming 75-80% of their daily food intake in the dark period, when rats are active, and 20-25% during the day when they are inactive (Kimura et al., 1970; Tartellin and Gorski, 1971). This pattern of food intake is associated with a daily rhythm in the arcuate nucleus expression levels of NPY, POMC and galanin mRNAs, which are increased during the light phase (Brady et al., 1990; Xu et al., 1999). It may be the release of these orexigenic peptides at the onset of the dark phase that induces feeding. During the dark phase, when food intake is maximal, plasma leptin levels and adipose tissue ob mRNA expression are significantly elevated compared to the light phase (Pickavance et al., 1998) (Fig. 1). Feeding during the estrous cycle therefore appears to be governed by an accumulation of orexigenic factors in the hypothalamus during the light phase, which then act at the onset of the dark phase to increase feeding which in turn indirectly induces increased secretion of leptin.

Leptin and the reproductive axis

Reversal of obesity in ob/ob mice by leptin administration occurs through its action as a satiety factor. In addition, their infertility is also reversed by continuous leptin administration and pregnancy and parturition then occur successfully (Chehab et al., 1996; Mounzih et al., 1998). This reversal of infertility is not simply a result of the decrease in food intake, but rather depends on the presence of leptin. Thus, food restriction does not restore fertility in ob/ob mice (Mounzih et al., 1997), and withdrawal of leptin treatment reverts the leptin treated ob/ob mice to an infertile state (Chehab et al., 1996). In particular, leptin treatment of infertile adult ob/ob mice induces maturation of the non-functional immature gonads, and as a result the formerly infertile mice produce viable sperm and eggs (Barash et al., 1996). However, leptin has also been shown to inhibit ovarian estradiol production (Zachow and Magoffin, 1997). Although leptin induces puberty in normal rats (Cheung et al., 1997) and mice (Chehab et al., 1997), the timing of puberty in rhesus monkeys is unaffected (Plant and Durrant, 1997). This suggests that leptin may only be acting as a permissive factor in reproductive maturation rather than having a direct action (Cheung et al., 1997). Some studies have shown that leptin can induce luteinizing hormone (LH) secretion in female rats and follicle stimulating hormone (FSH) secretion in male rats (Ahima et al., 1996; Barash et al., 1996), and may regulate gonadotropin releasing hormone (GnRH) secretion in fasted rats (Nagatani et al., 1998). The administration of leptin anti-serum reduces LH pulsatility and halts the estrous cycle in normal rats (Carro et al., 1997). However, leptin administration to fasted rats only partially restores circulating LH levels (Kalra et al., 1998). Thus availability of systemic leptin is evidently a requirement for a functional reproductive axis, and may act as a signal between peripheral energy reserves and the central mechanisms that directly control the ability to reproduce.

Pregnancy and lactation

Pregnancy and lactation are states of physiological hyperphagia and as a result present a good model for the investigation of mechanisms of feeding without experimental manipulation. This hyperphagia is

218 1984) which is thought to be due to the increase in progesterone secretion (Hervey and Hervey, 1967). Loss of body fat only occurs during the suckling period (Naismith et al., 1982). This mobilization of fat is under hormonal rather than dietary control, as lactating dams fed either a high or low protein diet utilize the same amount of body fat to produce their milk (Naismith et al., 1982). Food deprivation in early pregnancy results in decreased litter size though to a lesser extent than that seen with food deprivation in lactation (Wade and Schneider, 1992) and can result in an extended gestation in rats (Woodside et al., 1987). In comparison to the effects of short-term food deprivation on the estrous cycle and lactation, late pregnant rats are resistant to these adverse effects due to the presence of the placental hormones (Wade and Schneider, 1992). Food restriction (50% of ad libitum intake) during the first 2 weeks of lactation results in a further week of post-weaning infertility (Woodside, 1991). Here, food restriction decreases LH secretion via decreased GnRH release (Walker et al., 1995) in a suckling independent manner (Woodside et al., 1998). In the rat, food consumption usually occurs in the dark phase only (Fig. 1) and it increases in pregnancy: peaking in mid-gestation (Cripps and Williams, 1975; Morgan and Winick, 1981) before significantly decreasing on the day prior to parturition (day 21: Cripps and Williams, 1975; Johnstone et al., 1997). During lactation though, food intake increases with a characteristic change in the pattern of feeding. Food intake during the first week postpartum doubles as a result of increased meal size during night feeding only, then it almost triples during the second week by an increase in the frequency of meals with a change to both day and night feeding (Strubbe and Gorrisen, 1980) (Fig. 1). However, after weaning, food intake rapidly falls to pre-pregnancy values. This changing pattern of food intake depends upon the continual suckling stimulus throughout lactation; furthermore, the patterned increases in food are abolished by lesioning of the ventromedial hypothalamus (Becker and Kissileff, 1974). In addition, the hypothalamic content of serotonin, a known suppressor of food intake, is negatively correlated whereas that of norepinephrine, a stimulator of food intake, is positively correlated with increased food

22 20 18 16 14

14

21

14

20

DI

Day of Pregnancy

Day of Lactation

Fig. 1. Food intake (g, mean 4- SEM) during the light phase (07.00-19.00 h, open bars) and dark phase (19.00-07.00 h, solid bars) during diestrus (DI), pregnancy (days 7, 14, 21) and lactation (days 7, 14, 20) in Wistar rats (n = 40). Food intake during the dark period was significantly greater than in the light phase in diestrus and pregnancy *P < 0.05 vs. same day, paired t-test. Food intake during the dark was significantly greater in mid pregnancy, d14, compared to days 7 and 21: #P < 0.05 ANOVA on ranks, Kruskal-Wallis. In lactation food intake increased during the light phase, becoming similar to nocturnal intake, which was greater by L20 than in all other groups: f~P < 0.05.

only maintained for the duration of pregnancy and lactation and its extent is a function of the relative body size of the species and the litter size, such that with larger litter sizes there is a greater proportional increase in food intake. For the rat, food intake initially decreases then increases through pregnancy by approximately 10-20% (Fig. 1, Morgan and Winick, 1981) but an increase of 100% has also been reported (Cripps and Williams, 1975) and during lactation intake increases to a massive 180-450% of nonpregnant levels (Cripps and Williams, 1975; Morgan and Winick, 1981). In contrast, women only increase food intake during pregnancy by 10-15% and by 20-25% during lactation; however, this is dependent on the availability of food (Wade and Schneider, 1992). Pregnancy in the rat is associated with an increase in maternal fat and body weight (Shirley,

219 intake during pregnancy and lactation (Morgan and Winick, 1981). The highly ordered patterning of feeding in lactation in the rat maximizes food intake with the increasing energy demands from the pups as lactation progresses.
5.0 4.5 4.0 3.5

Plasma leptin concentrations during pregnancy and lactation

Plasma leptin concentrations have been found to be higher during late pregnancy compared to the estrous cycle in rats (Chien et al., 1997; Kawai et al., 1997) and mice (Tomimatsu et al., 1997). However, circulating leptin levels have been reported to fall significantly on the day prior to parturition compared to late pregnancy values (Chien et al., 1997; Kawai et al., 1997; Terada et al., 1998) and by day one of lactation plasma leptin levels are not significantly different from those seen in non-pregnant rats (Chien et al., 1997; Tomimatsu et al., 1997). The time points used for plasma collection in the studies described above were all in the light phase. Age-matched rats do not demonstrate an increase in leptin levels during late pregnancy (Terada et al., 1998) and when a greater number of samples were obtained from time points during both the light and dark phases, significant increases in plasma leptin were evident during the dark phase (the period of increased food intake) in diestrus and in late pregnancy, days 16 and 20, with no differences between these stages of pregnancy and non-pregnant rats (Fig. 2: Johnstone et al., 1998). This significant nocturnal increase in plasma leptin concentration, in response to the increased food intake during the dark phase, has been reported previously during the estrous cycle (Pickavance et al., 1998). While we found a small increase in plasma leptin concentration during the day in pregnancy (Fig. 2), the dramatic change was abolition of the nocturnal increase on day 21, the day before parturition (Fig. 2; Johnstone et al., 1998). Plasma leptin levels are also significantly less during parturition itself (after delivery of pup 2) compared to the dark phase (Johnstone et al., 1998) and light phase levels during late pregnancy days 16-21 (Kawai et al., 1997; Johnstone et al., 1998). Furthermore, during lactation, no increases of leptin levels occurred in the dark phase (Fig. 2; Pickavance et al., 1998) and the light and dark levels

J= i,o

3.0 2.5 2.0

u~ D

1.5

1.0
0.5

0.0

DI

P16 P20 P21

L2

L20

Reproductive Day

Fig. 2. Plasma leptin concentrations, measured by radioimmunoassay, in diestrus (DI), late pregnant (P16, P20, P21) and lactating rats (L2, L20) at 10.00 and 22.00 h. The rats were kept in a 12-h light : 12-h dark cycle (lights on at 07.00 h and off at 19.00 h). Jugular vein blood samples were taken from indwelling cannulae, implanted under ether anesthesia, and obtained on the same day from the same group of rats (n = 5 rats per group). Values are mean + SEM *P < 0.05 vs. same day 10.00-h sample, paired t-test; #P < 0.05 vs. DI, P16, P20 22.00-h sample, unpaired t-test. ~P < 0.05 vs. all other 10.00 h samples.

for days 2-15 were comparable to light phase levels during the estrous cycle (Fig. 2; Pickavance et al., 1998). Therefore, during lactation increases in leptin levels do not occur in the dark phase as they do in pregnancy (except at the end of pregnancy). As lactation progresses both light and dark phase plasma leptin levels fall greatly to significantly lower levels on lactation day 20 than on lactation day 2 (Fig. 2; Johnstone et al., 1998), and are then even lower than those seen during the estrous cycle (Fig. 2; Kawai et al., 1997). The low levels of leptin seen during lactation can be further suppressed by acute (48 h) fasting (Woodside et al., 1998). The profile of plasma leptin concentrations in the rat is similar to that seen in human pregnancy (Masuzaki et al., 1997). In women, plasma leptin concentration is increased during the first trimester compared to non-pregnant levels, and it remains

220 elevated through to lactation when leptin levels fall (Sivan et al., 1998). nesis, increased lipolysis results in elevated levels of non-esterified fatty acids in the serum which are inversely proportional to the levels of circulating leptin (Pickavance et al., 1998). Importantly, in pregnancy, the placenta is a potential source of leptin, as human placental cells secrete leptin into the matemal circulation and amniotic fluid (Masuzaki et al., 1997) and express ob mRNA at a significantly higher level than adipose tissue (Hassink et al., 1997). However, rat placental ob mRNA is expressed at a low level on days 12 and 19 (Kawai et al., 1997), but at a high level on day 21 of pregnancy (Amico et al., 1998). An upregulation of short-form leptin receptors (Ob-Ra and Ob-Rc) has been shown in the myometrium of pregnant rats (Chien et al., 1997) and mice (Hoggard et al., 1997). In contrast, leptin receptor expression in human placenta declines with approaching term (Henson et al., 1998). The role of leptin production by the placenta and of uterine leptin receptors is as yet unknown.

Relationship of plasma leptin to food intake during pregnancy and lactation

During the estrous cycle, food intake during the dark phase is followed by a significant increase in plasma leptin levels (Pickavance et al., 1998). This relationship persists through pregnancy, until day 21 when nocturnal plasma leptin concentration decreases although nocturnal feeding still predominates (Figs. 1 and 2). Thus, from the day prior to parturition, circulating leptin concentration no longer increases at night, despite the continued pattern of nocturnal feeding (Fig. 2), and this dissociation persists for the first few days of lactation while nocturnal feeding is still predominant. Thereafter, as feeding increases both at night and during the day as lactation progresses, both night and day circulating concentrations of leptin decrease. Thus, in the third week of lactation the total daily food intake is substantially greater than in the first 2 weeks, and intake during the day is as great as during the night, yet both day and night circulating leptin concentrations are at their lowest (Fig. 2). Therefore during late pregnancy and throughout lactation plasma leptin levels do not increase with increased food intake, but show an inverse relationship. The increases in plasma leptin in response to food intake, as observed during the estrous cycle and through most of pregnancy, are abolished or actively suppressed during lactation. The changes observed in circulating leptin concentrations may result from changes in the levels of ob mRNA expression in the adipose tissue. The accumulation of stored fat in late pregnancy is accompanied by a 2.5-fold increase in ob mRNA content per unit mass and leptin production in the adipose tissue compared to virgins, in both rats and mice (Kawai et al., 1997; Tomimatsu et al., 1997), which has been suggested to underlie the increased day-time plasma leptin levels found in these studies. This increased ob mRNA expression rapidly decreases early in lactation (Kawai et al., 1997; Tomimatsu et al., 1997). In particular, ob mRNA expression in parametrial adipose tissue is positively correlated with circulating leptin concentration during pregnancy and lactation (Terada et al., 1998). In addition to decreased lipoge-

Causes of reduced blood leptin concentration in lactation

This decreased production of leptin in lactation may simply follow the utilization of the fat reserves laid down in pregnancy. In addition, the average adipose cell volume is significantly decreased by one-third in lactating rats compared to virgin rats (Pickavance et al., 1998). Overall, circulating leptin levels may decrease as lactation progresses due to the decreased leptin production that results from decreased ob mRNA expression in the decreasing fat mass. Leptin is also produced by human mammary epithelial cells (Smith-Kirwin et al., 1998) and is present in human milk, at a higher concentration than that found in maternal serum in all reproductive states (Houseknecht et al., 1997; Smith-Kirwin et al., 1998). Ob mRNA expression in mouse mammary gland was found to decrease following parturition and to be maintained at a low level during lactation (Aoki et al., 1999). Increased ob gene expression then occurred following weaning (Kawai et al., 1997). Therefore there are considerable differences in patterns of leptin expression in the mammary glands in lactation between humans and rodents, and hence potential differences in physiological roles. Lactation-dependent down regulation in this tissue in

221 rodents may occur through local autocrine/paracrine mechanisms. Brogan et al. (1999) have suggested that leptin secretion may be inhibited as a result of milk production rather than as a direct response to the suckling stimulus. Ovarian hormones do not seem to be responsible for the reduced leptin production in lactation as ovariectomized lactating dams have a significant reduction in plasma leptin levels similar to that in intact dams (Brogan et al., 1999).

18o

-~ 17o

~ 160
n, 150 140 ,,o 0

d ~ O

d6

Leptin administration in late pregnant rats

To determine whether or not the increasing food intake during late pregnancy results from a decrease in the satiety action of leptin, we administered 3.5 txg leptin (in 3.5 ~1; mouse recombinant leptin: Linco Research, USA) or vehicle (artificial cerebrospinal fluid: aCSF) by intracerebroventricular (i.c.v.) infusion once per day to late pregnant rats at 17.00 h on days 18, 19, 20, 21 and 22, the day of parturition. Non-pregnant rats were similarly infused with either leptin (3.5 btg/3.51tl) or vehicle (3.5 I-tl) once per day for 5 consecutive days. Rat body weight (g) and food intake (g) were measured for all groups: on the day prior to the infusions (dO), day of the third infusion (d3) and on the day following the infusions (d6) (Fig. 3). In comparison to non-pregnant rats (Fig. 3A,B) the same dose of leptin was less effective in decreasing food intake and body weight in late pregnant rats (Fig. 3C,D). Thus, i.c.v, leptin had no effect on the body weight changes in pregnant rats, but decreased body weight in virgins (Fig. 3), while food intake was significantly decreased within 3 days in virgin rats, but only after 3 days in pregnant rats (Fig. 3). The presence of pups did not mask a dam weight decrease in response to leptin treatment as dam weights following parturition (d6) were not significantly different from vehicle treated dams (Fig. 3C). Thus, in pregnancy there is evidently an initial central resistance to satiety actions of leptin that may result from the decreased expression of the long form of the leptin receptor in the hypothalamus during pregnancy (Garcia et al., 2000). When a relatively large dose of leptin, 50 Ixg/g was administered, via an intraperitoneal injection, daily to ob/ob pregnant mice substantial increases in body weight were also attenuated (Mounzih et al., 1998). Furthermore, 5 days of i.c.v, leptin administration to

D 16

14
200 ~ 12

n,' 160 140

oo oo

O 20~ ' ~ L ~
oo oo

Fig. 3. Non-pregnant (A,B) and day 16 pregnant (C,D) rats were implanted with an intracerebroventricular (i.c.v.) cannula under ether anesthesia 48 h prior to the experiment. Rats received an i.c.v, infusion of either leptin (3.5 I*g/3.5 Ixl; solid bars) or 3.5 Itl vehicle (open bars) (artificial cerebrospinal fluid) once per day, at 17.00 h, for 5 consecutive days: for pregnant rats days 18-22 of pregnancy. Rat body weights, A, C (g, mean 4- SEM) and food intake, B, D (g, mean 4- SEM) were measured at 09.00 h prior to (dO), during (d3) and for 1 day following (d6) the i.c.v, infusions. (Note the pregnant rats gave birth on day 5 of treatment). Statistical differences were determined by repeated measures, two-way ANOVA, followed by Student-NewmanKeuls test, * P < 0.05 vs. same-day vehicle treated controls.

mid (L6) and late (L13) lactating rats did not induce a significant fall in body weight or food intake until the 6th day (Johnstone et al., 1998) which suggests that leptin has a reduced satiety effect in lactating rats. This could be a result of downregulation of leptin receptors in the brain at this time. Together with the dramatic decrease in leptin secretion in lactation, this serves to permit the greatly increased energy intake for successful lactation. These changes in leptin production and action are already evident by the end of pregnancy. The reduction in leptin production during lactation is thus evidently important as it will permit the increased food intake by decreasing satiety fac-

222 tor signaling to the brain. However, leptin secretion is not downregulated during most of pregnancy, although there is an evident need for an increased food intake, or increased efficiency of utilization, to support the accumulation of fat stores as well as growth of the fetuses and placentae. Recent investigations have shown that ob/ob mice treated with leptin that became pregnant, showed normal gestation with leptin treatment lasting for 6 days or more, but a prolonged gestation and parturition if leptin treatment was given only once post-copulation (Mounzih et al., 1998). However, the pups did not survive as lactation failed, although the mothers nursed (Mounzih et al., 1998). These results suggest that although leptin is not critical for implantation, fetal growth, gestation and parturition it is required for successful lactation. Although, as we have described here, circulating levels of leptin in lactation are greatly reduced compared to pregnancy and the estrous cycle, it seems that the residual secretion of leptin in lactation is nonetheless important. Taken together, the above studies indicate that the overriding requirement for massive energy transfer to the suckling young during lactation is accommodated by the removal of a satiety factor, i.e. leptin. However, during lactation hypothalamic NPY content as well as NPY mRNA expression increase (Smith, 1993; Pickavance et al., 1996; Wilding et al., 1997; Li et al., 1998), which may be a consequence of an overriding effect of reduced inhibition of NPY neurons by leptin. Leptin has been shown to act in the hypothalamus to inhibit NPY synthesis and secretion (Stephens et al., 1995; Schwartz et al., 1996b), so the increased production of NPY in lactation could be a consequence of the reduced circulating levels of leptin. In particular, there is an increase in NPY mRNA expression in the medial preoptic area and arcuate nucleus during lactation (Pickavance et al., 1996), and NPY peptide content in the median eminence compared to estrous cycle levels (Smith, 1993). The increased expression of NPY mRNA is localized to a specific region of the arcuate nucleus at the level of the dorsomedial hypothalamic (DMH) nucleus (Smith, 1993). Such increased activity of NPY neurons may account for increased food intake. This increased NPY production, permitted by reduced leptin permitted by reduced leptin production and action, is expected, through the orexigenic actions of NPY, to meet the huge metabolic demands of lactation. This interpretation is consistent with the finding that food deprivation, like lactation, induces an upregulation of NPY expression in the arcuate nucleus, specifically in a population of neurons that project to the PVN (Sahu et al., 1988; Brady et al., 1990) and the DMH (Kalra et al., 1999). Whether the increased NPY expression reflects increased NPY release is not clear, as NPY receptors (type Y1) in the hypothalamus are not down regulated during lactation, which is expected as a consequence of increased NPY release (Pickavance et al., 1999). However, upregulation of NPY-immunoreactivity in the PVN indicates increased transport to this site (Ciofi et al., 1991). Other changes in central mechanisms regulated by leptin can also be expected. For example, lactation, like food restriction, decreases POMC expression in the arcuate nucleus (Brady et al., 1990).

Possible central consequences of reduced leptin secretion in lactation

Whether the progressive decrease in the circulating leptin concentration is alone sufficient to induce the massive increase in food intake and altered feeding pattern in lactation is not clear. In addition, the central actions of leptin that normally stimulate the utilization of adipose tissue energy stores (Schwartz et al., 2000) are expected to be decreased in lactation. Leptin receptors are located in the arcuate nucleus, PVN and DMH, which are areas important in the regulation of food intake (Mercer et al., 1996; Schwartz et al., 1996a), so these areas are presumably responding in lactation to the reduced leptin availability. With regard to the regulation of food intake in lactation, one mechanism may be reduced inhibition by leptin of orexigenic mechanisms (Fig. 4). Lactation is an estrogen-deficient state (Fox and Smith, 1984) and this lack of estrogen may be expected to decrease NPY mRNA expression (Smith, 1993).

Lactational infertility
Lactation is characterized by an inhibition of ovarian cyclicity, probably resulting from a suppression

223

A
I Food intake

B
I Food intake

I Leptin secretion

1 Leptin secretion

INPYexpression

|aMSH

INPYexpression

|orexin

|MCH

|orexin

IMCtt

/
I Food intake

/
I Food intake

Fig. 4. The interaction between food intake and leptin secretion in (A) estrous cycling and (B) lactating rats. (A) Increased food intake during the estrous cycle results in leptin secretion which inhibits NPY and POMC neurons in the arcuate nucleus thereby decreasing appetite via inhibition of orexigenic factors in the lateral hypothalamus and ventromedial hypothalamus. (B) In lactation the increased food intake does not result in an increase in leptin secretion, through an as yet unknown mechanism(?). This allows increased NPY and decreased POMC expression in the hypothalamus,which in turn results in an upregulation of orexigenic factors in the appetite centers to further increase food intake. of pulsatile LH secretion, which is independent of feedback actions of sex steroids (Fox and Smith, 1984; Lee et al., 1989). Food deprivation in lactating rats increases the duration of lactational infertility (Wade and Schneider, 1992) which is reversed by leptin administration (Woodside et al., 1998); however, chronic leptin administration to normal lactating rats does not result in an early resumption of fertile LH levels (Woodside et al., 1998). As leptin absence has been shown to lead to infertility, it could be that the lack of leptin in lactation is involved in the infertility of lactation, including delayed implantation if mating occurs at the post-partum estrus. Lactational infertility defends the mother's metabolic status from the additional demands of a further set of fetuses, while giving metabolic advantage to the suckling offspring. Mating at the post-partum estrus will lead to a further pregnancy (Gilbert, 1984), and in such lactating-pregnant rats delayed implantation extends beyond the weaning of the first litter (Koiter et al., 1999). Here there is an inherent competition between the normal laying down of fat during pregnancy and the mobilization of fat stores during lactation. This natural condition may give an insight into the roles of leptin in regulating food intake, but also into the mechanisms regulating its production in late pregnancy and early lactation. With a concurrent pregnancy, there is a reduction in lactational performance that is attributable to the relatively lower food intake of pregnancy (Koiter et al., 1999); this suggests that the energy demands of pregnancy override those of lactation, and this is independent of maternal behavior. Increased estrogen concentrations seen over the second half of gestation (Bridges, 1984) may be responsible for the reduced lactational performance (Fleming, 1976). However, plasma leptin concentrations are not significantly different between pregnant and non-pregnant lactating rats, so it is unlikely that leptin alone is responsible for the differences in their lactational performance (Koiter et al., 1999).

Conclusion
The profile of leptin secretion shows characteristic changes according to stage of pregnancy and

224 with lactation. Through much of pregnancy there is a nocturnal increase that accompanies the increased food intake at night; near the end o f pregnancy, the nocturnal increase in leptin secretion is lost. There m a y be a mechanism suppressing leptin production at this time, perhaps a consequence of the collapse in progesterone secretion. The state of reduced leptin production, particularly at night, persists in lactation, while the circulating levels o f leptin during the day and at night progressively decrease. F o o d intake is increased both in pregnancy and lactation, permitting the laying down o f fat stores in pregnancy as well as providing for the immediate needs of the developing fetuses, but the increase in food intake is much greater in lactation, while the fat reserves now are also depleted, The reduced production of leptin in lactation will, by removing this centrally acting satiety signal, permit increased food intake, partly through reduced inhibition o f orexigenic mechanisms in the hypothalamus.

References
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Abbreviations
CCK CSF DMH FSH GnRH i.c.v. LH MC-4 NPY Ob-R Ob-Re POMC PVN VMH cholecystokinin cerebrospinal fluid dorso-medial hypothalamic nucleus follicle stimulating hormone gonadotropin releasing hormone intracerebroventricular luteinizing hormone melanocortin receptor-4 neuropeptide Y leptin receptor soluble leptin receptor pro-opiomelanocortin paraventricular nucleus ventromedial hypothalamic nucleus

Acknowledgements
L.E.J. was in receipt of a Fellowship from the Japanese Society for the Promotion o f Science. The authors are very grateful to Professor John A. Russell for his constructive advice and criticism during the preparation of this manuscript.

225

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J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 16

Relaxin and drinking in pregnant rats

David J. Hornsby

1, Brian C. Wilson 2 and Alastair J.S. Summerlee 1,*

1 Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, N1G 2W1, Canada 2 Department of Biology, Acadia Universi~, Wolfville, NS, BOPIXO, Canada

Abstract: Work reported in this chapter describes the potential role of relaxin in resetting cardiovascular thresholds in pregnant rats. Relaxin, a polypeptide produced primarily by the ovary in pregnant animals in many species, is also produced in the brain. Exogenous administration of relaxin into the brain causes a profound drinking response which is negated by pretreatment with a specific monoclonal antibody to rat relaxin when the antibody is injected into the brain. Neutralizing the action of endogenous brain relaxin in pregnant rats also blocks the normal increase in drinking that is observed in rats at night during the second half of pregnancy. Relaxin acts through the forebrain angiotensin system at the level of the subfomical organ (an important interface between the blood, the brain and the cerebrospinal fluid) as blockade of the angiotensin II receptor action negates several central actions of relaxin. Expression of angiotensin II AT1 receptors in the subfornical organ increases in parallel with the increase in circulating relaxin seen in the second half of pregnancy. Neutralizing the effects of endogenous brain relaxin, using central injections of the monoclonal antibody, blocks this increase in the expression of angiotensin II AT~ receptors in subfornical organ. These data imply that relaxin in the brain may act to affect central cardiovascular thresholds in rats and this may be important for the normal physiology of pregnancy.

Introduction

Cardiovascular changes in pregnancy

There are several significant changes in cardiovascular control during pregnancy that help the mother adapt to the growing presence of the fetus(es). In the rat, these changes are most marked in the second half of pregnancy: there is a gradual expansion of plasma volume but, at the same time, blood pressure falls, plasma osmolality decreases, and glomerular filtration rate increases (Atherton et al., 1982; Ahokas et al., 1989). Normally these cardiovascular parameters are carefully controlled within a narrow margin by a set of inter-related central mechanisms (De

Swiet, 1988). Therefore the changes that occur in pregnancy imply that there is a major shift in the central thresholds for cardiovascular control. Several different factors have been implicated in resetting the central thresholds but no clear link between the endocrine changes in the reproductive tissue and the cardiovascular controls has been established. This chapter explores the possibility that relaxin, a peptide which has recently been shown to have a number of effects outside the reproductive system, may be involved in setting cardiovascular thresholds in the rat during pregnancy. It also highlights the potential importance of endogenous relaxin in the brain to modify cardiovascular thresholds in the pregnant rat versus the role of relaxin in the peripheral circulation.

Relaxin
* Corresponding author: A.J.S. Summerlee, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada. Tel.: +I-519-824-4120, ext. 3846; Fax: +1-519-767-1693; E-mail: alastair @exec.uoguelph.ca Relaxin was originally named as the factor that caused relaxation of the pelvic ligaments of the guinea pig (Hisaw, 1926). It has been shown to have a important role in preparing the mother for birth by

230 allowing relaxation of the cervix and elements of the birth canal (see review: Sherwood, 1994). However, there are species differences in the biological activity of relaxin, its site of production and circulating profiles. For example, in the rat, pig and mouse where relaxin is primarily produced in the corpora lutea of pregnancy, relaxin concentration is elevated in the second half of pregnancy (O'Byrne and Steinetez, 1976). Hormone levels rise and peak shortly before birth. There is a precipitous fall in relaxin immediately before delivery (Sherwood et al., 1980) and it has been suggested that relaxin may have a role in the timing of birth in rats (Jones and Summerlee, 1986a,b; Summerlee et al., 1998b). In contrast, in the human, relaxin is produced primarily from the placenta. The circulating levels of relaxin rise to a peak at the end of the first trimester of pregnancy, then fall and remain at a plateau for the remainder of pregnancy (Bell et al., 1987). The role(s) of relaxin in human pregnancy remain to be elucidated. The molecular structure of relaxin has been determined in many species (Bryant-Greenwood and Schwabe, 1994; Tregear and Wade, 1995). There is a remarkable lack of structural homology between these relaxins. Although the peptide is comprised of two chains linked together with disulfide bridges, the number of amino acids in different relaxins varies between 43 and 64. There is no more than 50% homology between relaxins in different species (Tregear and Wade, 1995) and these differences may be related to the biological activity of relaxin. Some species do not possess a functional relaxin gene (Roche et al., 1993; Hartung et al., 1995). However, these species possess a relaxin-like factor (Leydig cell insulin-like peptide) in ovarian cells (Bullesbach, 1999; Ivell et al., 1999). It is possible that relaxinlike factor may have important roles in preparing the mother for birth in ruminants (Ivell et al., 1999) or may have completely different functions in species where there is also a functional relaxin gene (Neff and Parada, 1999). Our knowledge of the physiological actions of relaxin is incomplete. The relaxin receptor has not been identified, isolated and characterized. This is largely due to technical difficulties encountered in labeling relaxin and the relatively low numbers of relaxin binding sites (Sherwood, 1994). Binding sites for relaxin have been identified in tissues of the reproductive tract including the uterus (Tan et al., 1998), cervix (Weiss and Bryant-Greenwood, 1982; Kuenzi and Sherwood, 1995), mammary gland and nipple (Kuenzi and Sherwood, 1995; Kuenzi et al., 1995; Min and Sherwood, 1996). In addition, binding sites have also been located in the cardiac atrium and brain (Osheroff and Phillips, 1991; Osheroff et al., 1992; Osheroff and Ho, 1993; Tan et al., 1998).
Relaxin and pregnancy

Circulating relaxin is essential for successful birth and lactation. Endogenous relaxin promotes growth and softening of the cervix (Hwang and Sherwood, 1988; Hwang et al., 1989) and growth of the vagina (Zhao et al., 1996) to allow for delivery of the young. Uterine contractions are reduced by the actions of relaxin during the second half of pregnancy until shortly before birth (Downing and Sherwood, 1985) protecting fetuses against premature delivery. Finally, relaxin stimulates development of mammary nipples (Hwang et al., 1991; Kuenzi and Sherwood, 1992) so the mother rat can nurse her young successfully. Much of the data on the role of relaxin in pregnancy and birth is based on information obtained in the rat and to some extent in the pig (Sherwood, 1994). Recently, a relaxin-deficient mouse model was developed using gene targeting (Zhao et al., 1999). Contrary to expectation from the evidence in rats, the relaxin-deficient mice were able to produce healthy litters. However, their mammary development was deficient and they were unable to suckle their young. The nipples did not enlarge during pregnancy and mammary parenchyma was poorly developed at the start of lactation. Further investigation showed that the pelvic ligament did not relax in the deficient mice although no evidence of dystocia was revealed. There was, however, a difference in plasma osmolarity in the relaxin-deficient mice compared with wild-type controls which may indicate a potential role for relaxin in cardiovascular control in pregnancy in mice (see later).
Relaxin and non-reproductive tissues

In addition to pregnancy-related actions, relaxin also affects a number of non-reproductive tissues includ-

231 ing the heart and brain. Exogenous administration of relaxin affects the force and frequency of contraction of the isolated heart (Kakouris et al., 1992; Ward et al., 1992) and may also have a direct action on the heart in intact animal preparations (Parry et al., 1990; Yang et al., 1995). There are also a number of actions of relaxin reported in the central nervous system (Geddes and Summerlee, 1995). The central actions include: the suppression of reflex milk-ejection (Summerlee et al., 1984; Way and Leng, 1992); a profound pressor response (Parry et al., 1990; Parry and Summerlee, 1991; Geddes et al., 1994; Parry et al., 1994; Yang et al., 1995), and the release of a number of hypothalamic and pituitary peptides, for example, oxytocin and vasopressin (Geddes et al., 1994; Parry et al., 1994; Summerlee et al., 1998a; Way and Leng, 1992), luteinizing hormone (Summerlee et al., 1991) and prolactin (Betbea et al., 1989; Sortino et al., 1989). Recently there have been reports that centrally administered relaxin is dipsogenic (Summerlee and Robertson, 1995; Thornton and Fitzsimons, 1995; Sinnayah et al., 1999) and there is a possibility that this dipsogenic action is physiologically relevant to pregnancy.
20E

~ 15~=1o8

.~.

~=

Treatment (iev)

Fig. 1. Stimulation of water consumption in 60 min after intracerebroventricular injection of relaxin (RXN) or angiotensin II (AII) in female rats. Values are means -t- SEM. Significant (P < 0.05) increases compared with injection of vehicle (0.9% NaC1) are shown by the asterisks. Data redrawn from Summerlee et al., 1998c with permission. specific to injection of bioactive relaxin peptide. Neither treatment with denatured relaxin, insulin nor one of the insulin-like factors (Summerlee and Robertson, 1995) causes drinking and pre-treatment of rats with a specific monoclonal antibody to rat relaxin blocks the dipsogenic effect of relaxin treatment. Thornton and Fitzsimons (1995) observed a comparatively slower and smaller dipsogenic response to injection of porcine relaxin into the third ventricle of the rat brain compared with injection into the lateral ventricle (Summerlee and Robertson, 1995). This atA 7.5--I

Role of relaxin and drinking

Acute injection of exogenous relaxin causes drinking
Dose-dependent dipsogenic effect of exogenous relaxin Intravenous infusion of human relaxin (Sinnayah et al., 1999) and intracerebroventricular (i.c.v.) injection of porcine relaxin (Summerlee and Robertson, 1995; Thornton and Fitzsimons, 1995) causes a marked drinking in water-replete rats. The response to central injection is remarkably sensitive to low doses of hormone and is dose-dependent (Fig. 1). Injection of 10 ng exogenous relaxin causes female rats to drink approximately 4 ml of water in 15 min, and most of the water is consumed in the first 5 min after treatment (Fig. 2). Increasing the dose to 50 ng increases the amount of water consumed to ca. 10 ml. This represents about 2 - 5 % of body weight consumed in 15 min. After treatment, rats appear to compensate for the increase in water intake by reducing water intake for 24 h. The dipsogenic response is

E E 5.0O o

~2.5E
O O

> 0.0-

10

15

20

25

30

time after treatment (min)

Fig. 2. Time course of stimulation of water consumption in 5-min time bins after intracerebroventricularinjection of 50 ng relaxin in female rats. Values are means 4- SEM. Data redrawn from Summerleeet al., 1998c with permission.

232 tenuated response may be due to differences in the access to relaxin sensitive structures from these different sites of injection. O'Byrne et al. (1987) and Mumford et al. (1989) reported differences in the pressor response and inhibition of milk-ejection to injection of relaxin into different sites in the ventricular system: slower and smaller pressor responses were seen after injection into the third and fourth ventricles compared with injection into the lateral ventricles. These workers suggested that the different responses were due to the pattern of flow of cerebrospinal fluid within the ventricular system and the site of relaxin-binding sites. Thus, injection of relaxin into the third ventricle was ineffective because it is 'downstream' of relaxin binding sites. Subsequently, Osheroff and colleagues (Osheroff et al., 1990; Osheroff and Phillips, 1991; Osheroff and Ho, 1993) reported an abundance of relaxin binding sites in the subfornical organ. These receptors would be 'downstream' from the lateral ventricle but 'upstream' from the third ventricle. Intravenous treatment with relaxin also induces water drinking. Infusions of human gene 2 relaxin at doses of 25-80 txg/kg/h for 1 h induced a dosedependent water intake in both male and female rats (Sinnayah et al., 1999). Most of the water consumed occurred in the first 30 min after the infusion was started. Blockade of central angiotensin II receptors negates the dipsogenic response to central and peripheral injection of relaxin. Initial work by Summerlee and Robertson (1995) showed that the response to central relaxin was completely blocked using saralasin (a general angiotensin II antagonist), but the specificity of the angiotensin receptor-type has been further established. Sinnayah et al. (1999) showed that losartan (an angiotensin II AT1 receptor antagonist) reduced the dipsogenic response to intravenous infusion of human hormone, and Summerlee et al. (2001) reported that the central effects of relaxin were completely blocked by losartan. Sinnayah et al. (1999) demonstrated combining the infusion of relaxin at (40 l~g/kg/h) with a nondipsogenic dose of angiotensin II (0.5 Ixg/h) almost tripled the relaxin-induced water intake. The authors suggested that angiotensin and relaxin might act in concert to induce drinking. They further commented that this potential synergy might be important in hypovolemic conditions in pregnancy when the circulatory function of both the mother and fetus may be impaired. In contrast, however, they reported that the dipsogenic response to an infusion dose of relaxin was not affected by simultaneous infusion of hypertonic NaC1 solution. As plasma tonicity is a major physiological stimulus for water drinking (Fitzsimons and Simons, 1969; Fitzsimons, 1998) this lack of interaction between relaxin and hypertonicity indicates that there is some degree of specificity in the interaction of relaxin with angiotensin II in potentiating drinking. The site of relaxin action in the brain has been localized to two of the forebrain circumventricular organs. Specific binding sites for relaxin are present in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (Osheroff et al., 1990; Osheroff and Phillips, 1991; Osheroff and Ho, 1993). These structures are located, respectively, in the ventral and dorsal limits of the lamina terminalis, the rostral wall of the third ventricle, and are in close contact with the cerebrospinal fluid flowing from the lateral ventricles. However, with respect to actions of circulating relaxin, this is not expected to freely enter the brain, being excluded by the blood-brain barrier as it is a peptide of approximately 55 amino acid residues and a molecular weight of approximately 6 kDa (Bryant-Greenwood and Schwabe, 1994). Unless there are active transport mechanism for relaxin into the brain, which seems unlikely, relaxin in the circulation could only have an action in the brain through regions that lack a blood-brain barrier. Both the subfornical organ and OVLT lack a blood-brain barrier (Pardridge, 1988; McKinley et al., 1990) and they are known as part of the central pathways involved in modulating drinking behavior in response to dipsogenic stimuli such as elevated endogenous or exogenous angiotensin II or changes in plasma tonicity (Simpson et al., 1978; McKinley et al., 1990, 1998, 1999). The median preoptic nucleus (also known as the nucleus medianus) is a group of neurons in the lamina terminalis with connections with the OVLT and subfornical organ. Ablation of the median preoptic nucleus blocks the dipsogenic effects of relaxin (Geddes et al., 2001), so it is likely that the median preoptic nucleus represents an intermediate pathway in the central actions of relaxin.

233 It is therefore proposed that relaxin acts on one or both of the circumventricular organs to stimulate the neural pathways involved in thirst. It is plausible that both endogenous relaxin in the peripheral circulation and/or relaxin in the brain could act at these sites. Furthermore, it has been clearly shown that angiotensin II acts at the same sites to subserve thirst mechanisms and it is possible that an interaction of relaxin and angiotensin on the same neurons might explain the potentiation of dipsogenesis observed with injection of both peptides. It is also plausible that relaxin may be acting to stimulate the central angiotensin system and therefore promote thirst and other changes. Effect of photoperiod on the dipsogenic response to relaxin Rats are essentially nocturnal animals and demonstrate a diurnal rhythm of drinking behavior: rats consume more water at night when they are active compared with drinking during daylight hours. There is a well established link between central angiotensin II, thirst and sodium appetite (Fitzsimons, 1998) and evidence that angiotensin II-induced dipsogenesis is affected by the light : dark cycle (Gardiner and Stricker, 1985). Therefore to investigate the possibility that the dipsogenic response to relaxin might vary with time of day, female rats were treated with porcine relaxin (i.c.v. injection) in the subjective morning (08.00-10.00 h), in the afternoon (14.0016.00 h) and in the night (22.00-24.00 h) to compare the effects on drinking behavior (lighting regimen: 10 h light: 12 h dark - - lights on at 08.00 h). Water consumption in response to relaxin varied with the time of injection (Fig. 3). Less water was consumed (ca. 3.5 ml at 50 ng) and the drinking response least sensitive (minimal effective dose at 25 ng) in the afternoon. In contrast, the response was maximal (ca. 17.5 ml at 50 ng) and most sensitive (minimal effective dose at 5 ng) at night. The latency to the drinking response, however, remained at about 1 min and did not vary significantly with either the time or the dose of injection.

Possible physiological role of relaxin and drinking in pregnancy

Neutralization of circulating (peripheral) relaxin on drinking behavior in pregnancy Using monoclonal antibodies to neutralize the effects of endogenous relaxin in the periphery, Zhao et al. (1995) demonstrated that endogenous circulating relaxin had marginal effects on drinking during pregnancy: relaxin-neutralized rats consumed less water during the day compared with intact controls. Recently Omi et al. (1997) expanded these observations by showing that peripheral injection of exogenous relaxin promotes only moderate increases in water intake during late pregnancy in rats and does not affect drinking during the night. These authors suggested it was possible that circulating relaxin might enter the cerebrospinal fluid and have a central action on drinking. However, they did comment that it was possible that relaxin produced within the brain could have a direct, local action on drinking behavior. There is evidence that relaxin is synthesized within the brain (Osheroff and Ho, 1993; Gunnersen et al., 1997). Furthermore, data from our laboratory suggests that passive neutralization of relaxin in the brain may reveal roles for relaxin which are different from those observed for relaxin in the systemic circulation (Summerlee et al., 1998b).

25-

~
v "0 u) c 0

20-

E 1510" "4 _ / ' " Z oi " ---m-. A f t e r n o o n

E =.0 go 6'0 8'0 16o

Dose rat relaxin iev (ng)

Fig. 3. Diurnal variation in the effects of intracerebroventricular injection of relaxin on drinking behavior in rats. Water consumed is shown at different doses of relaxin given in the morning between 08.00 and 10.00 h, in the afternoon between 14.00 and 16.00 h, or at night between 22.00 and 24.00 h. Values are means -4- SEM. Significant (P < 0.05) differences are shown by the asterisks. Data redrawn from Summerlee et al., 1998c with permission.

234 Neutralization of brain (central) relaxin on drinking behavior in pregnancy To examine whether central, as opposed to peripheral relaxin might have a physiological role in drinking behavior during pregnancy, Summerlee et al. (1998c) examined the effects of central administration of monoclonal antibodies specific to rat relaxin on drinking behavior of pregnant rats. Pregnant rats were injected daily with a specific monoclonal antibody raised against rat relaxin from day 12 to day 22 of pregnancy through a chronically implanted i.c.v. cannula. Drinking and eating behavior was monitored every 12 h during pregnancy and the data compared with control animals treated with saline alone. There was a significant decrease in water consumed at night in the relaxin-neutralized versus the relaxin-intact animals (Fig. 4). In contrast, there was no effect on drinking during the day. The relaxin-neutralized animals also showed a decrease in weight gain during pregnancy. These data indicate that neutralizing the action of relaxin inside the ventricular system of the brain disrupts the increase in drinking that occurs in the second half of pregnancy in rats (Omi et al., 1997) and implies that relaxin may have a physiological role in water balance in late pregnancy. When compared with data on the effects of systemic relaxin on water balance in pregnancy (see above), the data suggest that the actions of relaxin in the brain may be separate and different from the actions in the periphery. There is, however, some evidence that relaxin may also affect blood vessel tone in the periphery in normotensive and hypertensive rats (Massicotte et al., 1987). Original work done on the effects of relaxin on the timing of birth (Jones and Summerlee, 1986a,b) was based on the premise that very high levels of relaxin seen in the circulation at the end of pregnancy (Sherwood et al., 1980) might 'spill' over into the brain and have a central effect. However, there is a growing body of evidence to support the contention that relaxin is produced in the brain (Osheroff and Ho, 1993; Gunnersen et al., 1997) and acts locally (Summerlee et al., 2001). The potential involvement of relaxin in water balance in pregnancy raises the possibility that relaxin might also be involved in water balance at other stages of the reproductive cycle but this remains to be investigated.
_

Control

50lk .

- , t - MCA-3

-n wA v

40-

30o >

20 12

1'4

1'6

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2~.

days of pregnancy

u (~ f.. >,

201816-

~.c_ ~

141210 ,

E=

12

14

16

I'8

20

22

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Fig. 4. Effects of intracerebroventricularinjection of monoclonal antibody to rat relaxin (MCA-3) on water consumption in pregnant rats during the 12-h dark period (upper panel) or the 12-h light period (lower panel). Values are means SEM. Intact rats, B; rats treated with MCA-3, A. Significant (P < 0.05) differences are shown by the asterisks. Data redrawn from Summerlee et al., 1998c with permission.

Mechanisms of physiological action of central relaxin

Circumventricular organs and the central action of relaxin There are several lines of evidence to suggest that the dipsogenic actions of relaxin are mediated through the circumventricular organs: lesion of the subfornical organ blocks the action of relaxin on blood pressure (Mumford et al., 1989), milk-ejection (O'Byrne et al., 1987) and birth (Summerlee and Wilson, 1994); there are relaxin-binding sites in the subfornical organ and OVLT (Osheroff and Phillips,

235 1991; Osheroff and Ho, 1993); intracerebroventricular treatment with relaxin is followed by an increased expression of c-fos in the subfomical organ (McKinley et al., 1997); exogenous relaxin activates subfornical organ neurons (B.C. Wilson, personal observations; McKinley and Oldfield, 1998; McKinley et al., 1998) and finally osmoreceptors for the regulation of thirst and vasopressin secretion are present in the circumventricular organs (Miselis, 1981; McKinley et al., 1982; Thrasher and Keil, 1987). Relaxin-activation of the forebrain angiotensin II system results in the release of vasopressin and oxytocin (Geddes et al., 1994) in addition to the reported effects on drinking behavior. The release of vasopressin, but not oxytocin, appears to be mediated through the cell bodies of the median preoptic nucleus as destruction of neuronal cell-bodies in this region block the increase in vasopressin secretion in response to exogenous relaxin (Geddes et al., 2001). This is not a direct action of relaxin on the median preoptic nucleus as these neurons do not respond to topical relaxin in vitro (B.C. Wilson, personal observation). Geddes et al. (2001) have shown that an intact median preoptic nucleus is required for the relaxin-induced dipsogenesis in conscious rats but the role and importance of the neurons in this nucleus remain to be established. Angiotensin II and the central actions of relaxin The pathways that emanate from the subfomical organ and OVLT are involved as part of the forebrain angiotensin II system (Saavedra, 1992). It has been suggested that angiotensin II mediates the central action of relaxin (Geddes and Summerlee, 1995). Evidence in support of this contention comes from the demonstration that prior treatment with a peptidic angiotensin II receptor antagonist saralasin (which blocks ATl and AT2 receptors) inhibits relaxin-stimulated drinking (Summerlee and Robertson, 1995). It also blocks the relaxin-induced release of oxytocin and vasopressin and the pressor response to relaxin (Parry and Summerlee, 1991; Geddes et al., 1994; Parry et al., 1994). More recently it has been shown that the specific AT1 receptor antagonist, losartan, blocks the dipsogenic action of centrally administered relaxin (Summerlee et al., 2001). It is unlikely that the effects of losartan represent a non-specific depressive effect on drinking behavior because i.c.v. losartan does not inhibit water intake after dehydration, or salt intake after sodium depletion in rats (Weisinger et al., 1997). Angiotensin II receptor expression and the central actions of relaxin To accommodate the changes in drinking responses seen in the second half of pregnancy in rats, the central mechanisms controlling cardiovascular parameters, in particular drinking behavior, must change in pregnancy. Circulating relaxin levels increase during the second half of pregnancy and evidence suggests that relaxin may be implicated in the control of drinking behavior in pregnancy through the forebrain angiotensin II system. Therefore, experiments were done to examine the possibility that differential responsiveness of angiotensin II (AT1) receptors in the subfornical organ may be responsible for the changing responsiveness to relaxin during pregnancy. The expression of angiotensin II (AT1) receptor mRNA in subfornical organ neurons was examined in pregnant rats (Summerlee et al., 2001). Expression of ATI receptor mRNA increased from day 12 and reached a peak at day 16 of pregnancy in control rats treated with an antibody to fluoresce (Fig. 5). In contrast, expression of AT1 receptor mRNA was unaffected throughout pregnancy in animals in which the effects of central relaxin was neutralized by treatment with a specific monoclonal antibody to rat relaxin. These data imply that brain relaxin may be responsible for changing AT1 receptor expression during pregnancy.

Possible relationship between relaxin, angiotensin and steroids

In addition to AT1 receptors in the subfornical organ (Mendelsohn et al., 1984; Yamada and Mendelsohn, 1989; Tsutsumi and Saavedra, 1991; Bunneman et al., 1992: Song et al., 1992; Phillips et al., 1993) which may be co-localized with relaxin receptor, there is evidence that subfornical organ neurons contain estrogen receptors. Pfaff and Keiner (1973) reported the presence of estrogen concentrating neurons in the region of the subfomical organ and it has been shown that subfornical organ neurons contain

236 d a y s post coitum

10

12

14

16

18

20

22

+2

+10

D
MW 1 2 3 4 5 6 7 8 9 10 11

Fig. 5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of AT1 gene expression in the subfornical organ of pregnant female rats. Panel A shows data from animals treated with monoclonal antibody to fluorescein (control), and panel C shows data from animals treated with monoclonal antibody to rat relaxin. In the intact animals (A) the positive signal for the ATI mRNA is highest between days 12 and 18 of pregnancy (lanes 3-6). No gene transcripts were observed before day 12 post coitum (lanes 1 and 2), and after day 18 (lanes 7-10) or if water replaced the cDNA template in the PCR reaction (lane I 1, *). The pattern of positive ATI mRNA signal is absent in animals treated with antibody (C) although there was a slight positive signal on day 2 of lactation. All samples showed strong positive signals for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (B,D), demonstrating equivalent quality of reverse transcription reactions.

m R N A for estrogen and androgen receptors (Simerly et al., 1990). M o r e recently Rosas-Arellano et al. (1999) reported coexistence of estrogen and AT1 receptors in subfornical organ neurons especially those closely associated with blood vessels and the ventricular lining. This raises the intriguing possibility that there could be a relationship between relaxin, estrogen and angiotensin II at the level o f the subfornical organ, and this m a y be the site for cardiovascular

control and water balance not only pregnancy but also in the menstrual and estrous cycles.

Conclusions
Central relaxin appears to have a critical role in the control of drinking behavior in the second half of pregnancy in the rat and to be involved in changing angiotensin II receptor activity in the subfornical

237 organ in pregnancy. There are clinical conditions recognized in pregnant w o m e n where the usual changes in cardiovascular thresholds are disrupted. In particular, there is a clinical condition known as pregnancy-induced hypertension which occurs in approximately 10% of primiparous w o m e n (Pipkin, 1988). This can have a deleterious effect on the mother and baby. In both rats and women there are changes in the hemodynamic axis in pregnancy which include: decreased plasma osmolality, increased blood volume; an increase in fluid intake; decreased threshold for vasopressin secretion (Lindheimer et al., 1989) and blunted pressor response to systemic administration of angiotensin II (Paller, 1984). The onset and course of these changes parallel changes in plasma relaxin levels and it is tempting to speculate that there may be a causal relationship: disruption of this relationship might result in pregnancy-induced hypertension. and normotensivepregnant rats. J. Obstet. Gynecol., 161: 618622. Atherton, J.C., Dark, J.M., Garland, H.O., Morgan, M.R.A., Pidgeon, J. and Soni, S. (1982) Changes in water and electrolyte balance, plasma volume and composition during pregnancy in the rat. J. Physiol., 330: 81-93. Bell, R.J., Eddie, L.W., Lester, A.R., Wood, E.C., Johnston, P.D. and Niall, H.D. (1987) Relaxin in human pregnancy serum measured with an homologous radioimmunoassay.Obstet. Gynecol., 69: 585-589. Bethea, C.L., Cronin, M.J., Haluska, G.J. and Novy, M.J. (1989) The effect of relaxin infusion on prolactin and growth hormone secretion in monkeys. J. Clin. Endocrinol. Metab., 69: 956-962. Bryant-Greenwood, G.D. and Schwabe, C. (1994) Human relaxins: chemistry and biology. Endocr. Rev., 15: 5-26. Bullesbach, E. (1999) The relaxin like factor is a hormone. Endocrine, 10: 167-169. Bunneman, B., Iwai, N., Metzger, R., Fuxe, K., lnagami, T. and Ganten, D. (1992) The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain. Neurosci. Lett., 142: 155-158. De Swiet, M. (1988) The physiology of normal pregnancy. In: P.C. Rubin (Ed.), Handbook of Hypertension, Hypertension in Pregnancy, Vol. 10. Elsevier, New York, pp. 1-9. Downing, S.J. and Sherwood, O.D. (1985) The physiological role of relaxin in pregnant rats. II. The influenceof relaxin on uterine contractile activity.Endocrinology, 116: 1206-1214. Fitzsimons, J.T. (1998) Angiotensin, thirst and sodium appetite. Physiol. Rev., 78: 583-686. Fitzsimons, J.T. and Simons, B.J. (1969) The effect of drinking in the rat of intravenous infusion of angiotensin, given alone or in combinationwith other stimuli of thirst. J. Physiol., 203: 45-57. Gardiner, T.W. and Stricker, E.M. (1985) Impaired drinking in rats with lesions of nucleus medianus: circadian dependence. Am. J. Physiol., 248: R224-R230. Geddes, B.J. and Summerlee, A.J.S. (1995) The emerging concept of relaxin as a centrally acting peptide hormone with hemodynamic actions. J. Neuroendocrinol., 7:411-417. Geddes, B.J., Parry, L.J. and Summerlee, A.J.S. (1994) Brain angiotensin-IIpartially mediates the effects of relaxin on vasopressin and oxytocin release in anaesthetizedrats. Endocrinology, 134:1188-1192. Geddes, B.J., Hornsby, D.J., Poterski, R.S. and Summerlee, A.J.S. (2001) Role of the median preoptic nucleus in the central cardiovascular actions of relaxin. Can. J. Physiol. Pharmacol., in press. Gunnersen, J.M., Crawford, R.J. and Tregear, G.W. (1997) Expression of the relaxin gene in rat tissues. MoL Cell. Endocrinol., 110: 55-64. Hartung, S., Kondo, S., Abend, N., Hunt, N., Rust, W., Balvers, M., Bryant-Greenwood, G. and Ivell, R. (1995) The search for ruminant relaxin. In: A. MacLennan, G.W. Tregear and G.D. Bryant-Greenwood(Eds.), Progress in Relaxin Research. World ScientificPublishing,Singapore,pp. 439-456.

Abbreviations
AII AT1 or AT2 GADPH MCA OVLT RT-PCR RXN angiotensin II angiotensin receptor type 1 or 2 glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody organum vasculosum of the lamina terminalis reverse transcription polymerase chain reaction relaxin

Acknowledgements
This paper is dedicated to the memory of David G. Porter who was the stimulus for this work. It is also dedicated to the rats used in the experiments and serves as a reminder for all of the members of the laboratory of the times we have all spent involved in the experiments on drinking behavior. Supported by Natural Science and Engineering Research Council (NSERC) Canada.

References
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238

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Progressin BrainResearch, Vol. 133 2001 Elsevier Science B.V. All rights reserved

CHAFFER 17

Neuroendocrine and emotional changes in the post-partum period

C. Sue Carter

1,,,

Margaret Altemus 2 and George E Chrousos 3

1 Department of Psychiatry, University of Illinois at Chicago, Chicago, IL 60612, USA 2 Department of Psychiatry, Weill Medical College, New York, NY 10021, USA 3 Section on Pediatric Endocrinology, Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892, USA

Abstract: As well as having widespread effects on many aspects of mammalian physiology, the hormones of both the
reproductive and stress axes can directly and indirectly influence behavior. Here we review possible mechanisms through which centrally active hormones of the female reproductive system and the hypothalamo-pituitary-adrenal stress axis may interact to influence behavior and mood states during the post-partum period. We will focus primarily on the behavioral effects of selected neuropeptide hormones, in particular oxytocin, vasopressin and corticotrophin-releasing hormone. The literature documenting central behavioral effects of these neuropeptides arises almost exclusively from research in experimental animals. In particular, it has been reported that during lactation in rats there are high blood and brain levels of oxytocin. At the same time there is a reduction in corticotrophin-releasing hormone in the brain and in its secretion in response to stress. These changes may contribute to optimal maternal care of the offspring. Correlational studies of peptides and behavior in the post-partum period also support the hypothesis that neuropeptides may influence human physiology and behavior. Studies of post-partum women reveal powerful regulatory effects of lactation on the reactivity of the hypothalamo-pituitary-adrenal axis and of autonomic and immune systems, especially in the face of challenge. The integrative function of neural systems that influence both reproduction and the hypothalamo-pituitary-adrenal axis suggests one central mechanism for mediating the effects of environmental challenges.

Introduction: neuropeptides coordinate physiological and behavioral interactions

Many factors, including neuroendocrine peptide hormones, regulate the physiology of pregnancy, parturition and the subsequent post-partum period. Best known among these are corticotrophin-releasing hormone (CRH), oxytocin and vasopressin; and they play a pivotal role in the integration of physiology and behavior. CRH is the primary hypothalamic signal comprising the hypothalamic-pituitary-adrenal (HPA) * Corresponding author: C.S. Carter, Department of Biology, University of Maryland, College Park, MD 20742, USA. Tel.: +1-301-405-6940; Fax: +1-301-314-9358; E-mail: ccl 1@umail.umd.edu

axis (reviewed by Chrousos et al., 1998; Gold and Chrousos, 1998). CRH is a 41 amino acid peptide that is synthesized in the parvocellular paraventricular nucleus (PVN) of the hypothalamus and is secreted at the median eminence into the hypothalamo-hypophysial portal system, through which it reaches the anterior pituitary. There, CRH stimulates synthesis and secretion of adrenocorticotrophic hormone (ACTH) by the corticotrophs. CRH synthesis and release are dynamically regulated by other endocrine factors including the catecholamines, opioids, and cytokines, and interacts with vasopressin and other peptides in the regulation of the HPA axis. CRH also is synthesized in neurons of the bed nucleus of the stria terminalis and the amygdala (Watts and Sanchez-Watts, 1995). Neurons from the parvocellular PVN, bed nucleus

242 of the stria terminalis and amygdala project to parts of the brainstem that regulate arousal and autonomic function, and CRH has been shown to activate the noradrenergic systems of the locus ceruleus and the nucleus of the tractus solitarius. Activation of the amygdala also leads to increased activity in the HPA axis; for example, fear responses are mediated by the amygdala. Thus, CRH also has important central effects on stressor processing centers in the brain. As a consequence of HPA activation, glucocorticoids are secreted from the adrenal gland and mediate negative feedback to the cerebral cortex, hippocampus and hypothalamus in the brain and to the anterior pituitary. These regions express glucocorticoid receptors, which have been implicated in the inhibition of the activated HPA axis (Diorio et al., 1993). Conversely, the production of CRH in some brain regions, including the central nucleus of the amygdala, is glucocorticoid-dependent (Makino et al., 1994) and glucocorticoid action in the amygdala probably plays a role in the potentiation of fear responses (Corodimas et al., 1994). In particular, the involvement of the cerebral cortex in this system permits an integration of cognitive, endocrine and autonomic processes (Garcia et al., 1999). HPA axis hormones are also produced in the placenta during pregnancy, including CRH and circadian ACTH, and corticosteroids from the placenta/fetus are involved in timing of the onset birth in humans. Oxytocin and vasopressin are nine amino acid hormones, each configured as a six amino acid ring with a three amino acid tail, and are identical with the exception of one amino acid in the ring and one in the tail of the molecules. Oxytocin and vasopressin are synthesized primarily within magnocellular neurons in the supraoptic nucleus (SON) and PVN, and are released into the systemic circulation from the magnocellular neuron nerve terminals in the posterior pituitary gland. Parvocellular neurons, located in the PVN, and cells in other brain areas including the bed nucleus of the stria terminalis and amygdala, also synthesize and release oxytocin and vasopressin from their centrally projecting axons/terminals. Additionally, oxytocin and vasopressin are synthesized in peripheral parts of the body (oxytocin from the ovary, uterus and thymus and vasopressin from the thymus) and have local paracrine effects; for example in enhancing local production of prostaglandins. Oxytocin plays a central role in the integration of the behavioral and physiological processes characterizing female reproductive physiology (Russell and Leng, 1998). Cervical stimulation during parturition and nipple stimulation during nursing are proximate stimuli for the release of oxytocin into the blood and brain. Social signals, including tactile and olfactory cues that may be present in the mother-infant interaction, also play a major role in the release of oxytocin (Uvnas-Moberg, 1998; Turner et al., 1999). Vasopressin is released into the blood and brain by various stressors, including dehydration, and its release, especially within the central nervous system, is also sensitive to social experiences. With both of these neuropeptides, release within the central nervous system and from the posterior pituitary into the blood can occur independently, so plasma concentrations of the hormones do not always reflect central levels; although central and peripheral release patterns also may be coordinated with each other (Kendrick et al., 1986; Neumann and Landgraf, 1989; Engelmann et al., 1996). Thus we cannot reliably infer the effects of factors or neuropeptides in the human brain from their blood profiles. Within the central nervous system, oxytocin and vasopressin receptors are found in the olfactory system, limbic-hypothalamic system, brainstem and spinal cord areas that regulate reproductive and autonomic functions (Tribollet et al., 1998). The distributions of the receptors for these peptides within the central nervous system vary across development and among mammalian species. Steroid hormones, including estrogens, progestagens, androgens and glucocorticoids (Insel, 1997; Liberzon and Young, 1997) can also influence the density and pattern of distribution of binding for oxytocin and vasopressin.
Pregnancy and hormones of the HPA axis

In human pregnancy, plasma concentrations of glucocorticoid (cortisol) increase, reaching their highest level in the third trimester (Okamoto et al., 1988; Goland et al., 1992; Lockwood et al., 1996). The elevated levels of cortisol in late pregnancy are similar to those observed during strenuous exercise or in severe depression. Plasma ACTH levels also rise in pregnancy. Although hypothalamic CRH expression cannot directly be measured in humans, it may be

243 suppressed during late pregnancy, as in rats (Johnstone et al., 2000). In the human, CRH of placental origin becomes elevated in plasma in late pregnancy, peaks during labor and falls to low or undetectable levels after parturition (Goland et al., 1992). CRHbinding protein is thought to limit the bioavailability of CRH and its concentration in blood declines during late gestation, possibly allowing higher levels of 'free' CRH and augmenting the hypercortisolism of late pregnancy (Magiakou et al., 1996a; Florio et al., 1997). Analysis of blood samples taken at 30-min intervals in women revealed the expected circadian rhythms of ACTH and cortisol, but not CRH. This lack of correlation between CRH and ACTH/cortisol is not surprising as the CRH is unlikely to originate from the hypothalamus, but to come from the placenta and to have little action on the corticotrophs because it is bound to CRH-binding protein. However, another secretagogue, possibly vasopressin, released at the median eminence from terminals of parvocellular PVN neurons, may be important in regulating circadian ACTH secretion during this period. During stress the HPA axis exerts profound, primarily inhibitory, effects on the reproductive axis (Chrousos et al., 1998). For example, in rats CRH can inhibit hypothalamic gonadotrophin-releasing hormone secretion, and glucocorticoids can inhibit pituitary luteinizing hormone and ovarian estrogen and progesterone secretion. Glucocorticoids also can render estrogen target tissues, such as the endometrium, resistant to the gonadal steroids. Therefore stress-induced activation of the HPA axis during pregnancy can be expected to have serious, negative implications for fetal development. ated suppression of hypothalamic CRH during the post-partum period. To test this hypothesis, HPA axis activity was examined in a prospective study of 17 healthy women (Magiakou et al., 1996b). Psychometric testing was performed and CRH stimulation tests were given at 3, 6 and 12 weeks post-partum. During the postpartum period 7 of the 17 women developed the 'blues' and one developed post-natal depression. The ACTH response to exogenous ovine CRH (oCRH) was blunted in these women at 3 and 6 weeks post-partum, but returned to normal at 12 weeks, compared to before parturition. At all three time points, cortisol levels were in the upper-third of the normal range, and responded to oCRH. When the data were analyzed according to the mood states of the women (non-depressed or blues/depressed), the cortisol responses were identical in both groups, while ACTH responses were lower in those women reporting symptoms of 'blues' or depression. Normal cortisol responses in the face of reduced ACTH responses suggests that the hypertrophy/hyperactivity of the adrenal cortex which develops during pregnancy has a delayed recovery in depressed women. These findings are also compatible with the hypothesis that any suppressed hypothalamic CRH activity, which recovers slowly during the post-partum period as adrenal cortical hypertrophy resolves, is associated with the mood changes experienced by some women during this period. Whether this group difference represented a pre-existing condition or a differential response to pregnancy, parturition and/or the post-partum period is not known.

Lactation and reactivity to stressful experiences

Lactation is the defining property of Mammalia, and until modern times was essential to human reproduction. Successful lactation involves the oxytocinmediated milk ejection reflex and neuroendocrine adaptations of the HPA and gonadal axes. Based on previous research on animals we have hypothesized that oxytocin, vasopressin, and CRH may regulate dynamic behavioral states in breast-feeding women, including the capacity of an individual to respond to both social and physical challenges. It has been found that lactating women interact more positively with their babies, directing more

The post-partum period and the HPA axis

At the time of parturition, the placental source of CRH is lost, and the maternal HPA axis may readjust. The resetting of the HPA axis and post-parturn recovery of the CRH activity remains poorly characterized, and varies among women. Because hypofunctioning of the CRH system has been implicated in the symptomatology of individuals with atypical depression (Gold and Chrousos, 1998), it is possible that the development of 'post-partum blues' or depression is related to prolonged or exagger-

244 touching and smiling toward their infants than do bottle-feeding mothers (Dunn and Richards, 1977). It also is reported that nursing mothers versus bottlefeeding mothers are more likely to describe positive mood states (reviewed by Carter and Altemus, 1997) and less anxiety (Berg-Cross et al., 1979; Virden, 1988; Heck and deCastro, 1993; Klein et al., 1995). Lactation also inhibits reactivity to stressful experiences. It has been known since the early 1970s that lactating female rodents showed reduced adrenal reactivity, often indexed by reduced glucocorticoid (corticosterone) secretion, following exposure to stressors such as ether, surgical trauma and electric shock (Thoman et al., 1970; Higuchi et al., 1989; Lightman and Young, 1989; Walker et al., 1992). In rats, injections of hypertonic saline normally are considered stressful and are expected to enhance CRH and enkephalin synthesis in the parvocellular PVN and cause increased secretion of corticosterone. However, during lactation there is a selective inhibition of these normal hypothalamic stress responses (Lightman and Young, 1989). In lactating women, Wiesenfeld et al. (1985) measured reduced autonomic reactivity (skin conductance and heart rate) in response to infant cries, showing lower levels of sympathetic arousal in lactating versus non-lactating mothers. The reduced responsivity to stressful experiences associated with lactation may be viewed as an adaptive response that protects a nursing female from overreacting to stressful stimuli and promotes successful lactation. Altemus et al. (1995) have examined the effects of physical stress in lactating versus recently delivered, bottle-feeding women, with the two groups matched in age. In that study, women underwent 20 min of strenuous treadmill exercise to 90% of their VO2max. The peak blood lactate level, a measure of exercise intensity, was similar in both groups, and lactating and non-lactating subjects had similar basal circulating levels of ACTH and cortisol. Blood concentrations of ACTH, cortisol and vasopressin increased following exercise in bottle-feeding women, as would be expected in normal controls. However, the magnitude of the increase in ACTH, cortisol and vasopressin in response to exercise stress was blunted in the lactating women. Thus, lactating women show a marked inhibition of stress hormone secretion in response to exercise. In another study of responses to psychological stressors, bottle-feeding women had higher systolic blood pressure and heart rate and lower cardiac vagal tone than breast-feeding or non-stressed women did (Altemus et al., 2001). Taken together these studies suggest that for humans, as for other mammals, lactation reduces physiological reactivity to various stressors. The reduced responsivity to stressful experiences associated with lactation may be viewed as an adaptive response that protects a nursing female from overreacting to stressful stimuli and promotes successful lactation. Animal research also implicates central action of a variety of neuropeptides or neurotransmitters in the reduced stress responses. Central actions of oxytocin have been implicated as a cause of the down-regulation of the HPA axis reactivity as well as behavioral changes that may be associated with the post-partum period. The activity of other neural systems in the central part of the stress axis also change in lactation. For example, catecholamine release in response to stress is reduced in lactating rats (Higuchi et al., 1989). Suckling also increases central production of the inhibitory amino acid transmitter, gamma aminobutyric acid (GABA), in rats and sheep (Qureshi et al., 1987; Kendrick et al., 1992). GABA is known to suppress the locus ceruleus/noradrenaline system, and play an important role in the regulation of anxiety and behavioral reactivity. Additionally, lactating female rats do not show the expected activation in cerebral cortical neurons following exposure to a glutamatergic agonist, NMDA (Abbud et al., 1993), suggesting that the functional modifications associated with lactation extend beyond the hypothalamus and brainstem to include cortical functions. Effects of vasopressin and oxytocin on the HPA axis Vasopressin synergizes with CRH at the anterior pituitary to enhance the secretion of ACTH. However, the regulatory effects of CRH and vasopressin and their interactions differ as a function of the types of stressors and other physiological conditions at the time a stressor is experienced. It is possible that differential patterns of the release of CRH and vasopressin allow social versus physical stressors to have

245 different consequences. For example, the effects of physical exercise, such as running on a treadmill, are very different from the consequences of social deprivation or the death of a child, although each of these may be considered 'stressful' experiences and might be associated with the release of hormones in the HPA axis. It has long been known that stress and hormonal products of the HPA axis can influence the release and actions of oxytocin. Both milk ejection and labor are inhibited by stressful experiences (Newton, 1973), suggesting that oxytocin secretion in women could also be inhibited by stress. The converse effects of oxytocin on the HPA axis are complex and species specific. For example, in rats there are reports that oxytocin can both increase and decrease the activity of the HPA axis, as measured by glucocorticoid levels. Uvnas-Moberg and her associates (reviewed 1998) have discovered a biphasic effect of oxytocin on corticosterone release in rats. In this species oxytocin initially stimulates the HPA axis. However, over a matter of hours following treatment or after repeated injections of oxytocin, animals show an inhibition in HPA axis activity and reductions in blood pressure. The half-life of circulating oxytocin is 1-2 rain, but the effects of chronic oxytocin may last for days or weeks. In fact, animals that are exposed to extra oxytocin during early life may show a permanent reduction in the reactivity of the HPA axis and lower levels of blood pressure (Uvnas-Moberg et al., 1998). Oxytocin production and release are increased during lactation and in women both ACTH and cortisol levels decrease during a bout of breastfeeding (Chiodera et al., 1991; Amico et al., 1994), while there is evidence that peripheral injections of oxytocin can inhibit ACTH and cortisol release in both men and women (Legros et al., 1984). In addition, there are reports that oxytocin administration and lactation inhibit the release of ACTH and/or cortisol which normally follow stressful experiences, treatment with vasopressin plus CRH, or exercise (Coiro et al., 1988). However, oxytocin circulating at physiological concentrations is unlikely to cross the blood-brain barrier in sufficient amounts to act centrally. Oxytocin released within the brain may have actions on the HPA axis. In highly social mammals, including prairie voles (Microtus ochrogaster), intracerebroventricular treatment with oxytocin is inhibitory to the HPA axis, producing an approximately 50% decline in plasma corticosterone within 30-60 min following administration (Carter, 1998). A similar decline in corticosterone is observed when reproductively naive prairie voles are paired with a member of the opposite sex (DeVries et al., 1996). In both cases treatment with a selective antagonist for the oxytocin receptor prevents the decline in corticosterone. Dynamic interactions among vasopressin, oxytocin and CRH also have the potential to affect behavior. CRH and vasopressin released in the brain are anxiogenic and high levels of brain CRH and vasopressin have been measured in anxiety disorders (Altemus et al., 1992; Bremner et al., 1997; Baker et al., 1999). In contrast, oxytocin tends to have anxiolytic actions, and may serve as a natural antagonist to vasopressin (McCarthy and Altemus, 1997; Carter, 1998).

Lactation and post-partum depression

Thus, the physiology of lactation may buffer women from stressors, and has the potential to reduce vulnerability to depression. So, although there is a high risk of a psychotic episode within the first three weeks post-partum (Kendell et al., 1987) for women with a personal or family history of bipolar disorder, this complication occurs in less than 0.1-0.4% of mothers. Controlled studies indicate that the incidence of the much more common non-psychotic major depression is not elevated in the first year post-partum (Cooper et al., 1988; O'Hara et al., 1990). As a group, women do experience more minor depressive symptoms post-partum, which seems to be primarily due to sleep disruption (Swain et al., 1997). On the other hand, as with a history of bipolar disorder, women with a history of major depression do have an increased rate of relapse during the post-partum period (Whiffen, 1988; O'Hara et al., 1991; see also Kumar, 2001 (this volume) and Jones et al., 2001 (this volume)). Because gonadal steroids are important modulators of both the HPA axis and the autonomic nervous system (Burgess and Handa, 1992; Lindheim et al., 1992; Kirschbaum et al., t996; 1999; Komesaroff et al., 1999), the large shifts in gonadal steroid levels that occur post-partum may contribute to a relapse of major depression

246 in these women. Impaired homeostatic regulation of these two stress response systems is thought to be a key vulnerability factor for major depression. Suppression of excessive reactivity of the HPA axis and autonomic reactivity during lactation may help to prevent development of depression in vulnerable women. Animal research would also seem to predict that breast-feeding might protect women from post-partum depression. However, studies attempting to relate lactation to post-partum depression have produced conflicting results. Several studies have reported no association between breast-feeding and post-partum depression (O'Hara and Swain, 1996; Wisner and Stowe, 1997; Hendrick et al., 1998). There also are reports that breast-feeding women may have a higher incidence of post-partum depression (Adler and Cox, 1983). Others have reported that breast-feeding women have fewer symptoms of depression (Abou-Saleh et al., 1998). Thus, it seems that post-partum depression is not limited to bottle-feeding mothers but is experienced by lactating women also. Case studies of women with major depression have associated the onset of depression with weaning (Susman and Katz, 1988). On the other hand, in one study of women who were suffering from major depression it was reported that the majority of the women (83%) became depressed before the cessation of breast-feeding, while only 17% became depressed following the cessation of breast-feeding (Misri et al., 1997). Although studies of lactating versus bottle-feeding women have yielded inconsistent conclusions about correlation to mood, several studies have reported relationships between prolactin and mood states including depression and anxiety. Prolactin secretion is high in lactating women and transiently increases with each bout of nursing. Among lactating women, higher levels of circulating prolactin have been associated with hypoanxiety (Asher et al., 1995) and lower levels with the occurrence of depression (Abou-Saleh et al., 1998). Prolactin secretion from the anterior pituitary is mainly regulated by the release of dopamine from hypothalamic tuberoinfundibular neurons, along with various other neuroendocrine factors and hormones, and so the above reported findings do not necessarily directly implicate direct action of prolactin or lactation on behavior. Still, because prolactin secretion is markedly elevated in lactation, and appears to be actively transported into the brain the correlation between lactation and post-partum mood states is consistent with a beneficial role for breast-feeding. Abou-Saleh et al. (1998) hypothesize that "breast-feeding started shortly after childbirth restores the 'milieu interieur' that prevailed during pregnancy with the high circulating levels of prolactin and averts the onset of a prolactin 'withdrawal' state which may be conducive to mood states." Conclusion The neuropeptides oxytocin and vasopressin participate peripherally in important reproductive functions and homeostatic responses, including modulation of the HPA axis. Recent evidence also implicates these hormones centrally, in the regulation of social behaviors. Thus, natural conditions associated with high levels of oxytocin may facilitate both social contact and selective social interactions associated with social attachment and pair bonding and reduced HPA reactivity. Vasopressin, in contrast, is associated with behaviors that might be broadly classified as 'defensive' including enhanced arousal, attention or vigilance, increased aggressive behavior, and a general increase in HPA reactivity. Based on the literature about the functions of these hormones and our own recent findings, we propose that dynamic interactions between oxytocin and vasopressin are components of a larger system which integrates neuroendocrine and autonomic changes associated with mammalian social behaviors and the concurrent regulation of the stress axis. In addition, studies of lactating females provide a valuable model for understanding the more general neuroendocrinology of the stress axis. Abbreviations ACTH CRH GABA HPA NMDA oCRH PVN adrenocorticotrophic hormone corticotrophin-releasing hormone gamma-aminobutyric acid hypothalamic-pituitary-adrenal N-methyl-D-aspartate ovine CRH paraventricular nucleus

247

SON supraoptic nucleus VO2max body maximum oxygen utilization

Acknowledgements
Support from the Department of Defense, National Science Foundation (BNS 7925713, 8506727, 8719748), the National Institutes of Health, including the Institute of Child Health and Human Development (HD 16679) and the National Institute of Mental Health (MH 45836), NARSAD and the New York Community Trust was essential to this research. We are especially grateful to our many collaborators who have contributed to the studies described here.

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Progress in BrainResearch, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 18

Lactogenic hormone regulation of maternal behavior

Phyllis E. Mann * and Robert S. Bridges
Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, 200 Westboro Road, N. Grafton, MA 01536, USA

Abstract: Biological factors can profoundly affect a mother's response to her young. For example, it is well known that the hormones of pregnancy act on the maternal brain to stimulate the spontaneous onset of maternal behavior at parturition. Studies in the rat have provided an excellent model to investigate maternal behavior in mammals, since maternal behavior in rats is easily observable and readily quantifiable and it is well-documented that the endocrine state of gestation helps to bring about the onset of maternal behavior around the time of birth. The same response in virgin animals requires a number of days of constant exposure to pups before maternal-like behaviors emerge. To date, research has established that the steroid hormones, estradiol and progesterone, and the lactogenic hormones, prolactin and the placental lactogens, act in concert to stimulate maternal behavior in the pregnant female. Treatment of adult, virgin rats with these hormones can stimulate a rapid onset of maternal care. In the present chapter experiments are described that demonstrate key roles for prolactin and placental lactogens in the onset of maternal behavior. Central sites of action of prolactin and placental lactogens, including the medial preoptic area, appear to be involved in stimulating the onset of maternal care. Other studies are discussed which support the involvement of the prolactin receptor in the endocrine regulation of maternal behavior using prolactin receptor antagonist and 'knock-out' models in rats and mice, respectively. Overall, these studies indicate that during pregnancy the endocrine system primes the mother's brain so that the new mother displays appropriate and successful behaviors toward her newborn at parturition.

Introduction

Female mammals display a wide variety of behaviors in order to nurture their offspring. In rodents, these maternal behaviors are readily observable and quantifiable. Pregnant rats will begin displaying maternal behavior within hours of parturition or immediately following the birth of the young (Slotnick et al., 1973; Mayer and Rosenblatt, 1984). At parturition, the female rat helps in the delivery of the pups, cleans the pups of all fluid and ingests the placenta. During the post-partum period, the female will retrieve pups to the nest (see Fig. 1A), exhibit anogenital licking (to allow for urination and defecation) and crouch over

* Corresponding author: EE. Mann, Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, 200 Westboro Road, N. Grafton, MA 01536, USA. Tel.: +1-508-887-4911; Fax: +1-508-839-7091; E-mail: phyllis.mann @tufts.edu

the pups in a nursing posture (see Fig. 1B). Additional behaviors include nest-building and defense of the young against intruders. Unlike post-partum females, virgin, inexperienced rats will either ignore, avoid or cannibalize pups when they are first placed in the cage. Constant exposure to pups for 5 to 10 days, however, will 'sensitize' the female, stimulating her to display the full range of maternal behaviors (Rosenblatt, 1967). The capacity to induce maternal behavior in inexperienced females demonstrates that the neural substrate for the behavior is already in place. In fact, prepubertal rats (between 21 and 30 days of age) have short-latency (1-2 days) maternal behavior, retrieving and crouching over the pups, sometimes immediately after exposure (Bridges et al., 1974b; Mayer and Rosenblatt, 1978, 1979; Mayer et al., 1979; Brunelli et al., 1985, 1987; Brunelli and Hofer, 1990). The developmental profile of maternal responsiveness alternates between fast responders for juvenile rats, to slow responders for adult virgin rats, returning to a very fast response around parturition (Fig. 2).

252

Fig. 1. Examples of maternal behavior in lactating rats. (A) Lactating female retrieving a pup back to the nest. (B) Lactating female crouching over the pups. The changes in the endocrine system during pregnancy are thought to underlie the display of maternal behavior around or at parturition. The following section will describe the pregnancy profiles of the hormones involved with the onset of maternal behavior. Subsequent sections will describe studies that investigated the role of the lactogenic hormones, prolactin and the placental lactogens, in the onset of maternal behavior. In addition, the role of the medial preoptic area in lactogenic mediation of maternal behavior will be described. The final section will describe studies on the onset of maternal behavior after prolactin receptor antagonist administration and in a transgenic mouse strain that lacks the prolactin receptor. The endocrine state of pregnancy The steroid hormones (estrogen and progesterone) and the lactogenic hormones (prolactin and the placental lactogens), which increase in the serum during pregnancy (Figs. 3 and 4), are involved in the onset of maternal behavior at parturition in the rat. After conception the rat ovary is the primary source of

253
Impregnation

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1 / / Birth Lactation Ends

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50

60

70 80 Age (Days)

90 100 110

120

Fig. 2. Developmentalprofileof the latencies in days to respond maternallyto foster pups in nulliparous() and primiparous(0) female rats. (Adapted from Bridges, 1990.) estradiol and progesterone in the serum. Estradiol concentration steadily increases during pregnancy, reaching a peak just before parturition (Rosenblatt and Siegel, 1981; Bridges, 1984). Progesterone concentration, on the other hand, rapidly increases following mating and reaches a plateau during early gestation on days 7 to 10 and increases to another plateau later, between days 15 and 20 of pregnancy (Morishige et al., 1973; Rosenblatt and Siegel, 1981). Near term, progesterone levels sharply decline allowing parturition to occur and maternal behavior to be displayed (Bridges et al., 1978). Dur-

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Fig. 3. Pregnancyprofilesofsernm progesterone(O)and es~ogen(U) concentrationsin~malerats.(Adapted ~om Bridges, 1984.)

254

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-

400

200

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12

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18

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Fig. 4. Pregnancy profiles of serum prolactin (0) and placental lactogen (ll) concentrations in female rats. (Adapted from Bridges, 1990.)

ing the first half of pregnancy, prolactin secretion is rhythmic, exhibiting both diurnal and nocturnal surges (Butcher et al., 1972; Bridges et al., 1993). In mid-pregnancy, when the conceptus starts secreting placental lactogens I and II (Robertson and Friesen, 1981), the dam's prolactin secretion is suppressed and remains at low concentrations until the day before parturition. Closely related in structure to prolactin (Nicoll, 1982), the placental lactogens circulate in the dam's serum during the second half of gestation, with placental lactogen I peaking around days 10-13 and placental lactogen II at high titers from day 12 to parturition (Pihoker et al., 1993). Placental lactogens are also found in the cerebrospinal fluid of late-pregnant women and rats (Peake et al., 1983; Bridges et al., 1996), and bind to lactogenic receptors in the maternal choroid plexus and hypothalamus (Pihoker et al., 1993; Freemark et al., 1994). Together, these studies allow us to conclude that the conceptus exposes the pregnant female to a changed hormonal milieu during much of pregnancy, which in turn may affect her behavior at parturition (Bridges et al., 1996). Before parturition there

is a shift in the ratio of estradiol to progesterone, with progesterone declining and estradiol increasing. This causes the dramatic increase in serum prolactin at parturition (Linkie and Niswender, 1972; Bridges and Goldman, 1975). Studies investigating the role of prolactin and the placental lactogens in the onset of maternal behavior will be described below.
I n d u c t i o n o f m a t e r n a l b e h a v i o r with p r e g n a n c y hormones

Research into the involvement of hormones in the onset of maternal behavior began as early as the 1930s. Riddle et al. (1935) demonstrated that prolactin when injected into virgin rats stimulated pup retrieval behavior. Unfortunately, it was not until the 1970s that this work was replicated and expanded upon. In the 1960s, previous work had failed to reproduce the early findings (Lott and Fuchs, 1962; Beach and Wilson, 1963). Then in 1970, Moltz and his colleagues demonstrated that a combination of estradiol, progesterone and prolactin stimulated maternal behavior in ovariectomized, virgin rats (Moltz

255 et al., 1970). Throughout the next decade, convincing evidence began accumulating that estrogen was a key factor in the onset of maternal behavior at parturition. Rosenblatt and colleagues demonstrated that estrogen stimulates a rapid onset of maternal behavior in both ovariectomized and hysterectomized virgin rats (Rosenblatt and Siegel, 1975; Siegel and Rosenblatt, 1975a,b, 1978) and in pregnancy-terminated rats, following hysterectomy (Rosenblatt and Siegel, 1975). In addition, estrogen infused into the medial preoptic area of the brain stimulated maternal behavior in pregnancy-terminated and virgin rats (Numan et al., 1977; Fahrbach and Pfaff, 1986; Rosenblatt et al., 1994). In contrast, the central actions of progesterone in the onset of maternal behavior are less clearly understood, but appear to be somewhat inhibitory. When circulating levels of progesterone at the end of pregnancy are maintained at high levels the display of maternal behavior is delayed (Siegel and Rosenblatt, 1975b; Bridges et al., 1978; Numan, 1978; Siegel and Rosenblatt, 1978). A recent study by Numan et al. (1999) showed that the inhibitory effect of progesterone on maternal behavior in estrogen-treated rats can be blocked by RU486, a progesterone receptor antagonist.
Lactogenic hormones

Studies which showed that the anterior pituitary hormone, prolactin, is clearly involved in the onset of maternal behavior occurred in the mid-1980s (Bridges et al., 1985a,b; Loundes and Bridges, 1986; Bridges and Dunckel, 1987). Fig. 5 is a diagrammatic representation of the steroid and hormonal protocols for the experiments on maternal behavior described in this chapter. Earlier work had shown that a steroid regimen that delivers near-pregnancy levels of estradiol and progesterone (enclosed in silastic capsules implanted subcutaneously for 17-22 days), stimulated a rapid onset of maternal behavior in ovariectomized virgin rats (Bridges, 1984). When the steroid regimen was administered to hypophysectomized, virgin rats no behavioral stimulation was found. Moreover, the latencies to respond maternally were the same in experimental and control rats that did not receive the steroid hormones (Fig. 6). This suggests that a pituitary hormone is important for the induction of maternal behavior. In addition, when circulating prolactin levels were reinstated in these animals by either exogenous administration of ovine prolactin or ectopic pituitary grafts that secrete large amounts of prolactin, short-latency maternal behavior could be re-

Bromocriptine (2 mg/kg x ?./day,s.c.)

Figs. 9,10 [ & 11

(2 mg/kg x 2/day, s.c.)

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11 12 ......................

Fig. 7

~ F i g . 6,7,8,9 10 & 11

17/22

Fig. 5. Diagram representing the protocol of administration of steroid hormones and drugs in each maternal behavior experiment described in Figs. 6-11. Seven days after ovariectomy,rats were implanted subcutaneouslywith three 30-mm progesterone-filledsilastic capsules. Between 0900 and 1000 on day 11 the progesterone capsules were removed and one 2-mm estradiol capsule was implanted. MPOA = medial preoptic area; s.c. = subcutaneously injected; i.c.v. = intracerebroventricularlyinjected. Maternal behaviortesting was performed on days indicated by the arrows.

256

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Intact

Hypophysectomized

Fig. 6. Latencies in days to respond maternally in hypophysectomized, ovariectomized virgin rats after steroid-treatment (m). * Significantly different from control group (D), P < 0.05. Numbers of rats per group are shown in brackets. (Adapted from Bridges et al., 1985.)

stored (Bridges et al., 1985a,b; Bridges and Dunckel, 1987; Fig. 7), strongly indicating that prolactin is important in the onset of maternal behavior. Since h y p o p h y s e c t o m i z e d rats are not the ideal choice as experimental animals, a model which involves pharmacologically suppressing endogenous prolactin release was used. Twice-daily injections o f bromocriptine, a dopamine agonist, has been shown 10

to inhibit the release of prolactin from the pituitary (Ben-Jonathan et al., 1989). W h e n ovariectomized steroid-treated virgin rats were given bromocriptine twice a day and exposed to foster pups, the display of maternal behavior was inhibited (Bridges and Ronsheim, 1990; Fig. 8), and short-latency maternal behavior could then be restored with concurrent treatment with ovine prolactin (Fig. 8).

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Prolactin Replacement

Fig. 7. Latencies in days to respond maternally in hypophysectomized, ovariectomized virgin rats after steroid- (m) or control-treatment (n) in rats given a graft replacement or ovine prolactin infusions (s.c.). * Significantly different from control group, P < 0.05. Numbers of rats per group are shown in brackets. (Adapted from Bridges et al., 1985 and Bridges and Dunckel, 1987.)

257

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3

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Full Maternal Behavior

Fig. 8. Latencies in days to respond maternally in bromocriptine-treated (U), CB-154 + prolactin-treated ( ) or control (D) ovariectomized virgin rats after steroid-priming, expressed as group median. * Significantly different from control group, P < 0.05. * Significantly different from CB-154 alone group, P < 0.05. The number of rats in each group was 12. (Adapted from Bridges and Ronsheim, 1990.)

Even though these experiments demonstrated that prolactin is involved in the onset of maternal behavior in virgin rats, they do not reveal the site of action of prolactin. The central nervous system

is the most likely candidate as its site of action for the following reasons: (1) prolactin is actively transported across the blood-brain barrier through an active transport mechanism (Martenez and Her-

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Fig. 9. Percentage of bromocriptine-treated, ovariectomized virgin animals responding maternally after ovine prolactin (10 Ixg, , n = 11; 50 Ixg, n = 10) or vehicle (D, n = 22) infusions into the lateral ventricle. * Significantly different from vehicle group, P < 0.05. (Adapted from Bridges et al., 1990.)

258 bert, 1982; Walsh et al., 1987); (2) elevated levels of prolactin are found in the cerebrospinal fluid when plasma prolactin levels are high (Rubin and Bridges, 1989; Felicio and Bridges, 1992); and (3) prolactin receptors are present in the choroid plexus and the brain (Barton et al., 1989; Fechner and Buntin, 1989; Michel and Parson, 1989; Bakowska and Morrell, 1997; Pi and Grattan, 1998). Behavioral studies found that when ovine prolactin is infused into the lateral ventricle of ovariectomized, steroid-treated, bromocriptine-treated females, latencies to respond maternally were reduced from approximately 6 to 2 days (Bridges et al., 1990; Fig. 9). In addition, bilateral infusions of ovine or rat prolactin directly into the medial preoptic area, a site that is critically involved in the regulation of maternal behavior (Numan, 1994), stimulates a rapid onset of maternal responsiveness (Bridges et al., 1990; Bridges and Mann, 1994). Significantly more animals responded maternally by the second test day (60% vs. 0%) following prolactin infusions (data not shown). Under normal physiological conditions during the first half of pregnancy the pregnant female rat is exposed to high levels of plasma prolactin. After the prolactin surges subside, the pregnant female is exposed to elevated lactogenic hormones originating from the placenta, which are high throughout the second half of pregnancy (Robertson and Friesen, 1981; see Fig. 4). In addition to high titers of placental lactogens circulating in the maternal bloodstream, rat placental lactogens I and II are found in the cerebrospinal fluid during gestation. Moreover, when rat placental lactogens I and II were infused into the medial preoptic area of ovariectomized, steroid-treated rats, the latencies to display maternal behavior were reduced (Bridges et al., 1996; Fig. 10). The placental lactogens presumably act at the prolactin receptor to stimulate maternal behavior, but there is no direct evidence for this. As well as the medial preoptic area, the ventromedial nucleus of the hypothalamus is also involved in reproductive behavior (Pfaff and Schwartz-Giblin, 1988), although it has not until recently been considered to be important in maternal responsiveness (Numan, 1978; Rubin and Bridges, 1984). We examined maternal behavior in steroid-primed, bromocriptinetreated virgin rats after ventromedial nucleus infusions of rat prolactin (Bridges and Mann, 1994; Bridges et al., 1999). Surprisingly, both rat prolactin and vehicle infusions stimulated fast onset maternal behavior in these females (Bridges and Mann, 1994; Fig. 11A). In addition, infusions of the neurotoxin, N-methyl-D-aspartic acid into the ventromedial nucleus, which destroys cell bodies but not fibers of

4 o ~d -~ 3

0 Retrieval Crouching Full Maternal Behavior Fig. 10. Latencies in days to respond maternally following medial preoptic area infusions of rat placental lactogen I (rPL-I II, n = 12), rPL-II ( i , n = 11) or vehicle (ff], n = 13) in steroid-primed, virgin rats, expressed as group median. * Significantly different from vehicle group, P < 0.02. ** Significantly different from vehicle group, P < 0.05. (Adapted from Bridges et al., 1996.)

259

~" 5
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Vehicle rPRL
(8) (9)

Vehicle rPRL
(9) (8)

NMA NMA (9) Vehicle

MPOA (12) VMH Fig. 11. Latencies in days to respond maternally in steroid-primed, bromocriptine-treatedvirgin rats: (A) following infusions into the medial preoptic area (MPOA) and ventromedial nucleus (VMH) of rat prolactin (rPRL) or vehicle (adapted from Bridges and Mann, 1994); (B) following N-methyl-D-asparticacid lesions (NMA) and sham (NMA vehicle) of the ventromedial nucleus. Numbers of rats per group are shown in brackets. Data are expressed as group median. (A) * Significantly different from medial preoptic area vehicle, P < 0.05. (B) * Significantly different from NMA vehicle. (Adapted from Bridges et al., 1999.)

passage, stimulated short-latency maternal behavior in steroid-primed virgin rats (Bridges et al., 1999; Fig. 11B). Taken together, these results indicate that the ventromedial nucleus may have an inhibitory role in the regulation of the onset of maternal behavior. Overall, the above findings indicate that prolactin and the placental lactogens can act directly on the central nervous system to stimulate maternal behavior. However, the initial priming with estrogen and progesterone is a critical factor in the stimulation, as infusion of prolactin into the lateral ventricle of nonsteroid-primed virgin female rats does not stimulate the onset of maternal behavior (Bridges et al., 1990).
Inhibition of maternal behavior

the females also received twice-daily bromocriptine injections and 5 infusions of rat prolactin with either the prolactin receptor antagonist, rbPRLR-ECD, or the antagonist vehicle (0.1% bovine serum albumin) over a 3-day period. The day after they received the estradiol capsule (day 12) maternal behavior testing begin. The prolactin receptor antagonist significantly blocked the stimulation of pup retrieval behavior by prolactin over the first two test days (P < 0.05, data not shown). These results further prove the role of prolactin in the onset of maternal behavior. Further studies employing other prolactin receptor antagonists are ongoing in our laboratory.

Gene knockout ( - / - ) mice

Using gene-targeting techniques, a new model was developed to study the role of prolactin in maternal behavior. The prolactin receptor gene was removed from the set of expressed genes in mice carrying a germ line null mutation (Lucas et al., 1998). Virgin, female, homozygous and heterozygous mutant mice were exposed to foster pups every day in order to induce maternal behavior. Fig. 12A demonstrates that both the homozygous and heterozygous mutant mice had longer latencies to become fully maternal than the wild-type mice. The homozygous mice

Prolactin receptor antagonists

Recently, in collaboration with Dr. Arieh Gertler, a prolactin receptor antagonist, raised against the extracellular domain of the rabbit prolactin receptor, was used to examine the role of the prolactin receptor in maternal behavior. Adult, nulliparous, female rats were stereotaxically implanted with bilateral cannulae in the medial preoptic area 1 week prior to starting the steroid regimen, of 11 days of progesterone followed by estradiol. Starting on day 11

260

A
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.
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~ 900 600 ~ ~ 300

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Full Maternal

First pup

Second pup

r - I pup Third

Full Maternal Behavior

Behavior

. . . . . . . . . . Retrieval . . . . . . . . . .

Fig. 12. Median latencies to respond maternally. (A) 6-8-week-old nulliparous female mutant mice (heterozygous II, n : 6; homozygous II, n = 10) and corresponding wild-type mice (D, n = 7) to display pup retrieval and crouching behaviors, expressed in days. * Significantly different from wild-type, P < 0.05. (B) 6-8-week-old post-partum female mice to retrieve and crouch over pups, expressed in seconds. * Significantly different from wild-type, P < 0.05. (Adapted from Lucas et al., 1998.)

had median latencies of 6 days to become maternal, while the heterozygous mice had intermediate latencies of 4-5 days to display the behavior, compared to latencies of 1-2 days for the wild-type mice. Additionally, when transgenic mice heterozygous for the prolactin receptor were tested post partum, the maternal behavior of the heterozygous mutant mice was found to be disrupted (Lucas et al., 1998; Fig. 12B). In the absence of the prolactin receptor, lactogenic hormones produced by the placenta were unable to stimulate maternal behavior. Interestingly, some heterozygous mutants had response latencies similar to wild-type mice, while others were more severely affected. Taken together, these two studies support a role for the endogenous prolactin receptor, in the onset of maternal behavior.
Conclusion

ering which hormones are involved in the onset of maternal behaviors in the rat should lead to a better general understanding of the hormonal basis of parental behavior.
Abbreviations

i.c.v, MPOA NMA rPL RPRL s.c. VMH

intracerebroventricular medial preoptic area N-methyl-D-aspartic acid rat placental lactogen (I and II) rat prolactin subcutaneous ventromedial nucleus

Acknowledgements

The results described above demonstrate the important role of hormones, especially lactogenic hormones, in the onset of maternal behavior in female rats. Once maternal behavior is established, however, the endocrine system is not thought to be crucially involved (Bridges, 1990). Since the hormonal changes of pregnancy in rats are similar to those in other mammals, including primates, discov-

This work was supported by grants awarded to R.S.B. by the NIH (R01 HD19789 and K05 MH01374).
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262 Robertson, M.C. and Friesen, H.G. (1981) Two forms of placental lactogen revealed by radioimmunoassay. Endocrinology, 108: 2388-2390. Robertson, M.C., Gillespie, B. and Friesen, H.G. (1982) Characterization of the two forms of rat placental lactogen (rPL): rPL-I and rPL-II. Endocrinology, 111: 1862-1866. Roky, R., Paut-Pagano, L., Goffin, V., Kitahama, K., Valaix, J.-L., Kelly, P.A. and Jouvet, M. (1996) Distribution of prolactin receptors in the rat forebrain. Neuroendocrinology, 63: 422-429. Rosenblatt, J.S. (1967) Nonhormonal basis of maternal behavior in the rat. Science, 156: 1512-1514. Rosenblatt, J.S. and Siegel, H.I. (1975) Hysterectomy-induced maternal behavior during pregnancy in the rat. J. Comp. Physiol. Psychol., 89: 685-700. Rosenblatt, J.S. and Siegel, H.I. (1981) Factors governing the onset and maintenance of maternal behavior among nonprimate mammals. The role of hormonal and nonhormonal factors. In: D.J. Gubernick and EH. Klopfer (Eds.), Parental Care in Mammals. Plenum Press, New York, pp. 13-76. Rosenblatt, J.S., Mayer, A.D. and Giordano, A.L. (1988) Hormonal basis during pregnancy for the onset of maternal behavior in the rat. Psychoneuroendocrinology, 13: 29-46. Rosenblatt, J.S., Wagner, C.K. and Morrell, J.I. (1994) Hormonal priming and triggering of maternal behavior in the rat with special reference to the relations between estrogen receptor binding and ER mRNA in specific brain regions. Psychoneuroendocrinology, 19: 543-552. Rubin, B.S. and Bridges, R.S. (1984) Disruption of ongoing maternal responsiveness in rats by central administration of morphine sulfate. Brain Res. 307: 91-97. Rubin, B.S. and Bridges, R.S. (1989) Immunoreactive prolactin in the cerebrospinal fluid of estrogen-treated and lactating rats as determined by push-pull perfusion of the lateral ventricles. J. Neuroendocrinol., 1: 345-349. Siegel, H.I. and Rosenblatt, J.S. (1975a) Hormonal basis of hysterectomy-induced maternal behavior during pregnancy in the rat. Horm. Behav., 6:211-222. Siegel, H.I. and Rosenblatt, J.S. (1975b) Progesterone inhibition of estrogen-induced maternal behavior in hysterectomizedovariectomized virgin rats. Horm. Behav., 6: 223-230. Siegel, H.I. and Rosenblatt, J.S. (1978) Duration of estrogen stimulation and progesterone inhibition of maternal behavior in pregnancy-terminated rats. Horm. Behav., 11: 12-19. Slotnick, B.M., Carpenter, M.L. and Fusco, R. (1973) Initiation of maternal behavior in pregnant nulliparous rats. Horm. Behav., 4: 53-59. Walsh, R.J., Slaby, F.J. and Posner, B.I. (1987) A receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid. Endocrinology, 120: 1846-1850.

behavior in the laboratory rat: early origins in 18- to 27-dayold young. Dev. Psychobiol., 12: 407-424. Mayer, A.D. and Rosenblatt, J.S. (1984) Prepartum changes in maternal responsiveness and nest defense in Rattus norvegicus. J. Comp. Psychol., 98: 177-188. Mayer, A.D., Freeman, N.C.G. and Rosenblatt, J.S. (1979) Ontogeny of maternal behavior in the laboratory rat: factors underlying changes in responsiveness from 30 to 90 days. Dev. Psychobiol., 12: 425-439. Michel, E. and Parson, J. (1989) Histochemical demonstration of prolactin binding sites. J. Histochem. Cytochem., 37: 773-779. Moltz, H., Lubin, M., Leon, M. and Numan, M. (1970) Hormonal induction of maternal behavior in the ovariectomized nulliparous rat. Physiol. Behav., 5: 1373-1377. Morishige, W.K., Pepe, G.J. and Rothchild, I. (1973) Serum luteinizing hormone, prolactin and progesterone levels during pregnancy in the rat. Endocrinology, 92: 1527-1530. Nicoll, C.S. (1982) Prolactin and growth hormone: specialists on one hand and mutual mimics on the other. Perspect. Biol. Med., 25: 369-381. Numan, M. (1978) Progesterone inhibition of maternal behavior in the rat. Horm. Behav., 11: 209-231. Numan, M. (1994) Maternal Behavior. In: E. Knobil and J.D. Neill (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 221-301. Numan, M., Rosenblatt, J.S. and Komisaruk, B.R. (1977) Medial preoptic area and onset of maternal behavior in the rat. J. Comp. Physiol. PsychoL, 91: 146-164. Numan, M., Roach, J.K., Del Cerro, M.C.R., Guillam6n, A., Segovia, S., Sheehan, T.P. and Numan, M.J. (1999) Expression of intracellular progesterone receptors in rat brain during different reproductive states, and involvement in maternal behavior. Brain Res., 830: 358-371. Peake, G.T., Buckman, M.T., Davis, L.E. and Standefer, J. (1983) Pituitary and placentally derived hormones in cerebrospinal fluid during normal human pregnancy. J. Clin. Endocrinol. Metab., 56: 46-52. Pfaff, D.W. and Schwartz-Giblin, S. (1988) Cellular mechanisms of female reproductive behaviors. In: E. Knobil et al., (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 1487-1568. Pi, X.J. and Grattan, D.R. (1998) Distribution of prolactin receptor immunoreactivity in the brain of estrogen-treated, ovariectomized rats. J. Comp. Neurol., 394: 462-474. Pihoker, C., Robertson, M.C. and Freemark, M. (1993) Rat placental lactogen-I binds to the choroid plexus and hypothalamus of the pregnant rat. J. Endocrinol., 139: 235-242. Riddle, O., Lahr, E.L. and Bates, R.W. (1935) Maternal behavior induced in virgin rats by prolactin. Proc. Soc. Exp. Biol. Med., 32: 730-734.

J.A. Russell et al. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 19

Neural mediation of nursing and related maternal behaviors

Judith M. Stern 1,, and Joseph S. Lonstein 2
1 Department of Psychology, Rutgers - The State University o f New Jersey, New Brunswick, NJ 08903, USA : Center for Neuroendocrine Studies, Tobin Hall, University of Massachusetts, Amherst, MA 01003, USA

Abstract: Nursing is the behavioral concomitant of lactation and the most generalizable maternal behavior across mammals. In lactating rats nursing often occurs in the kyphotic (upright crouched) posture; like the neuroendocrine determinants of milk synthesis and release, kyphosis requires suckling by the young. The dam's active pronurturant behaviors, such as retrieval and licking of pups, requires perioral somatosensory stimulation, which is often a precursor of kyphosis as well, and is inhibited by suckling. The sequential nature of maternal behaviors and the dissociations in their somatosensory regulation are critical to understanding their neural mediation, as exemplified by our recent work in lactating rats. We found that the caudal lateral and ventrolateral midbrain periaqueductal gray (cPAGI.vl) is a sensorimotor integration site for the kyphotic nursing posture. Destruction of the cPAGl.vb or increased activity of the inhibitory neurotransmitter GABA within it, severely reduced kyphosis, increased nursing in more atypical postures, and had little or no effect on pronurturance. Various forebrain sites are known to mediate retrieval and licking of pups. Inhibition of dopaminergic activity in the nucleus accumbens of dams via microinfusions of a mixed D1/D2 dopamine receptor antagonist, cis-flupenthixol (FLU), dose-dependently reduced these active behaviors, while increasing nursing duration. Retrieval was inhibited, however, only by infusions of FLU that included the nucleus accumbens shell, which is reciprocally connected with other sites implicated in retrieval of pups. Thus, maternal behavior is not a unitary process but rather a complex category consisting of sequential behavioral components that have their own sensory and neural determinants.

Introduction

The goal of a m a m m a l i a n mother is to ensure the survival of her offspring; in addition to providing nourishment, this usually entails temperature regulation, cleaning, protection, and instruction. There are wide species differences in the display of various aspects of maternal care, but nursing is the most generalizable and functionally significant component among m a m m a l s and the defining behavior of the class (Stern, 1989). Because a major theme of this volume is the neurobiological adaptations of lactation, it is fitting that the present chapter on the neural deter-

* Corresponding author: Judith M. Stern, Department of Psychology, Rutgers - The State University of New Jersey, New Brunswick, NJ 08903, USA. Fax: -t-1-732-4452263; E-mail: jmstern@rci.rutgers.edu

minants of maternal behavior emphasizes the behavioral concomitant of lactation - - nursing - - and its relationship to other aspects of maternal care. Further, given the complexity of neural circuitry underlying mammalian behaviors, the focus on a relatively simple component makes sense, as demonstrated by decades of fruitful study of the sexually receptive lordosis posture of female rats (Pfaff et al., 1994). What does the mother do to ensure a successful nursing episode? First, she is not only tolerant of contact with the young, meaning neither killing nor avoiding them, but she also makes contact with them regularly. Second, once in proximity, she provides the young access to her teats which enables them to suckle. Third, during nursing there is typically an inhibition of all active behaviors and, in some species, adoption of a unique posture. We begin with a description of the sensory regulation of maternal behavior in laboratory Norway rats, the m a m m a l that

264 is best studied with respect to the physiological bases of maternal behavior (Stern, 1989, 1996a, 1997a; Numan, 1994). attach to, and suck on a teat, the young at all ages play an active role in being nursed (Stern, 1997a). Whereas trigeminal stimuli that the dam receives from contacting the young with her mouth elicit retrieval and licking of pups (Stem and Kolunie, 1989, 1991; Stern, 1996a,b, 1997b), suckling stimuli elicit the state and postural changes of nursing (Stern and Johnson, 1990; Stern et al., 1992; Stern and Lonstein, 1996), followed by neuroendocrine secretions and milk-ejection (see Russell et al., 2001, this volume). When the rat dam's perioral somatosensations are blocked acutely, nursing of neonates is unlikely because she needs this stimulation to position her ventrum over the pups, but older, more mobile pups can bypass the dam's perioral attentions by stimulating nursing directly (Stem and Johnson, 1989), which is a routine occurrence later post parturn (Rosenblatt and Lehrman, 1963). Once a sufficient number of pups begin to suck, the previously active dam becomes quiescent and adopts a reflexive crouching posture that is unique to nursing, termed kyphosis (Stem, 1996a). This posture occurs while the dam is upright, bilaterally symmetrical, with rigid limb support and legs splayed to accommodate a large litter mass, and with spinal ventroflexion that results in a dorsal (or kyphotic) arch, culminating in depression of the head and rump when the arch is high. Pups that are too cool or too warm do not suck or even attach to a nipple, indicating that only a small deviation from nest temperature (~34C) impairs mobility and prevents nipple attachment. Litters that are small for a rat (which has 12 teats), such as two or four pups, are inefficient in eliciting their dam's quiescent kyphotic nursing, so that the latency to begin nursing is increased substantially. In the absence of sufficient sucking or its perception, sustained kyphosis will be absent or greatly reduced in duration. When kyphosis is not elicited, the maternally motivated dam continues to display active behaviors, both pup-oriented and not (Stern and Johnson, 1990; Stern et al., 1992; Stern and Lonstein, 1996). Maternal aggression toward an unfamiliar intrnder is related to both perioral and ventral trunk somatosensory regulation (Stem, 1996a). Whereas trigeminal stimulation elicits biting and attacking (Kolunie and Stem, 1990; Stem and Kolunie, 1991), continual ventral trunk contact with the litter is nec-

Perioral and ventral trunk somatosensory determinants of rat nursing behavior

Upon reunion with her litter, the rat mother goes through a typical sequence that constitutes a stimulus-response chain (Fig. 1). Distal cues - sight, sound, and smell - - from pups presumably serve to arouse and orient the dam, so that contact with the pups is achieved. No one of these distal cues, however, is essential for the induction of maternal behavior in naive virgins by cohabitation with pups, for its natural post-partum onset, or for its maintenance (Stem, 1989, 1990, 1997a), suggesting that they are not sufficient to activate the neural circuitry necessary for the elicitation of specific components of maternal behavior. In support of this interpretation, in lactating rats the activation of an immediate-early gene, c-fos, which occurs readily in many parts of the brain in response to maternal stimulation involving physical contact, does not occur in response to only distal stimulation from pups (Fig. 2; Lonstein et al., 1998a; see also Numan and Numan, 1995). Indeed, in non-maternal rats the odor of pups apparently mediates the inhibition of maternal responsiveness via well-known olfactory processing circuits (Numan, 1994). The hormones of pregnancy effect a change from aversion to preference of odors associated with birth fluids and neonates, as demonstrated in sheep (LEvy et al., 1996). Once the dam is willing to make and maintain physical contact with pups, sensory cues other than the sight, sound, and smell of the young are necessary for the dam's performance of particular maternal behaviors; these critical sensory inputs are tactile. Rats visit their maternal burrow frequently, staying long enough to nurse if stimulated to do so (Jans and Leon, 1983). Once there, the dam retrieves to the nest any pups that have strayed or that were displaced by her if she left the nest precipitously while the pups were still attached to a nipple. Mostly, the dam hovers over the gathered pups, while licking and rearranging (mouthing) them, thereby providing them with access to her nipples. Because mammalian young are equipped with reflexes to find by rooting,

265

SENSORY REGULATION OF RAT MATERNAL BEHAVIOR

MOTHER Distal Cues Stimulate Contact Seeking Perioral Contact Stimulates PronurturanceWhile Hovering Over I

PUPS SIGHT,SOUND AND SMELL

I<

..1 (MOTHER PUPS) TOUCH

TOUCH P (MOTHER4-- PUPS)

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Rooting And Suckling < Stimulate Quiescence And Kyphosis

=1 MILK-EJECTIONS

Fig. 1. The sequence of mother-litter interactions that culminate in a nursing bout in laboratory Norway rats. Although licking of pups

takes up a substantial amount of time before the onset of quiescent nursing, maternal snout but not actual tongue contact is required for hovering over pups (Stern and Johnson, 1989). Older pups can directly provoke kyphosis by suckling from their dam while she is supine or prone, thereby bypassing the dam's receipt of perioral stimulation while hovering over the litter. If pups are incapable of sucking, the dam does not become quiescent and continues a variety of behaviors, including licking, self-grooming, nest repair, and excursions from the nest. Modified from Stern (1989), with permission from Academic Press. essary to maintain the propensity to protect the litter (Ferreira and Hansen, 1986; Stern and Kolunie, 1993). In mice, suckling stimulation per se is essential for the onset and maintenance of maternal aggression (Svare, 1981). pup-directed behaviors that typically precede nursing - - that pertain to our understanding o f their neurobiological determinants. These include both a reevaluation of past studies and a need for well-informed research designs in the future. 'Maternal behavior' is a motivational category, not a unitary phenomenon Natural, motivated behaviors in vertebrates share many organizational characteristics, among them being a site in or near the hypothalamus that is sensitive to and integrates responses to relevant stimuli, both

Relevance of somatosensory regulation of nursing to its neural mediation

There are important theoretical and methodological implications of our findings on the separate somatosensory regulation of nursing and o f pronurturant behaviors (Stern, 1996a) - - motorically active,

266

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267 internal, such as circulating hormones, and external, such as those from the incentive; lesions of these sites typically result in profound alterations in the given behavior. A m o n g post-partum rats, large lesions of the medial preoptic area (MPOA) virtually eliminated maternal retrieval of pups and nest building and greatly reduced nursing (Numan, 1974). These findings have led to an implicit assumption that maternal motivation in rats is a unitary phenomenon in the sense that a disruptive manipulation in a key region affects all of its major components. As is true of other motivated behaviors, however, maternal behavior is a complex category consisting of several elements, each with its own function, sensorimotor characteristics, and other determinants. Actually, assessment of the entire literature on the effects of damage to the M P O A or its connections reveals that only retrieval of pups is consistently, severely, and permanently impaired (see below). A failure to correctly quantify other components, such as pup licking and nursing, creates a biased impression of the importance of a neural site such as the M P O A in all aspects of maternal behavior. relatively slight cooling disrupted nursing (Stern and Johnson, 1990). If a neurobiological insult causes an impairment in a dam's pronurturance, then the stimulus conditions for eliciting nursing will be absent as well - - unless there are a sufficient number of healthy, vigorous, and mobile pups that are able to initiate suckling contact with the dam. Because nursing occurs in bouts, a dam-litter separation of a few hours is needed before assessing maternal behavior, including nursing. The typical failure in many studies to impose such a separation is based on the implicit assumption that the entire sequence should be elicited normally at any time, as if deprivation is unrelated to this motivated behavior unlike others, such as sexual and ingestive behaviors. In contrast, the satiety or hunger of the pups greatly influences nursing behavior (Keer and Stern, 1996). Unfortunately, brief spot checks of nursing are still common, yet observations that are sufficiently long and continuous are needed to distinguish between the mother hovering over the pups while she is motorically active and the sustained, quiescent kyphotic nursing posture. Furthermore, the success of nursing should be verified by weighing the litter before and after a nursing bout to estimate milk transfer. In sum, the prevalence of inadequate pup stimuli and experimental designs that do not carefully assess nursing behavior raise serious doubts about the validity of earlier conclusions on the neurobiological bases of nursing behavior.

Sustained quiescent nursing requires more stringent stimulus conditions than do retrieval and licking, and appropriate observational criteria
Monkey and chimpanzee mothers are known to carry about their dead infant for hours or days before finally abandoning it. Similarly, rat pups that are dead, anesthetized, or at an immobilizing temperature elicit retrieval and licking, yet the gathered pups are incapable of stimulating quiescent nursing (Stern and Johnson, 1990; Stern and Lonstein, 1996). Following chilling of the pups, only very low skin temperatures inhibited the dam's retrieval and licking, whereas

Forebrain regulation: excitatory for pronurturance and inhibitory for nursing?

The role of various brain regions in the maternal behavior of rats has been studied for decades, mostly with lesions (Stern, 1989; Numan, 1994). In recent

Fig. 2. Interactions with pups and neuronal Fos expression. Effects of interaction with suckling or non-suckling pups for 60 rain or control conditions - - pups-in-box, empty box (Box), or no stimulus (No Stim.) - - on the number of Fos-IR nuclei (mean number 4SE) expressed relative to the no stimulus condition, the mean of which is set to 100%. (A) Medial preoptic area (MPOA) and primary somatosensory cortex (SI) representation of the face and trunk; * P < 0.001, suckled and non-suckled > other groups; in addition, in MPOA and SI-face, Pups-in-box and Box levels are significantly higher than those in No Stim. (B) Nucleus accumbens (NA), lateral septum (LS), lateral habenula (LHb), paraventricular thalamic nucleus (PVT), and bed nucleus of the stria terminalis (BNST); P < 0.001, suckled and non-suckled > other groups. (C) Rostral and caudal periaqueductal gray (rPAG and cPAG), peripeduncular nucleus (PP), and pontine nucleus (PN); different letters above the standard error bar indicate that the means are significantly different, P < 0.05. (A-B) Modified from Lonstein et al. (1998a), with permission from Pergamon. (C) Modified from Lonstein and Stern (1997a), with permission from the Society for Neuroscience.

268 years, the activation of an immediate-early gene, such as c-fos, assessed by the immunocytochemical labeling of its product, the nuclear protein Fos, has enabled researchers to simultaneously visualize brain sites that are selectively excited by stimulation. This has enabled the identification of neurons in the maternal rat brain that are excited by interaction with pups. Using this method, our data (Fig. 2A,B; Lonstein et al., 1998a) support an excitatory role in rat maternal behavior of several forebrain sites identified previously. These sites include the MPOA and both the face and trunk representation areas of the primary somatosensory cortex (SI) (Fig. 2A; Xerri et al., 1994), nucleus accumbens (NA) (see below), lateral septum (LS) (Kor~inyi et al., 1988), lateral habenula (LHb) (Matthews-Felton et al., 1995), and bed nucleus of the stria terminalis (BNST) (Numan and Numan, 1996). Another such site, the paraventricular thalamic nucleus (PVT), not previously known to be involved in maternal behavior of rats, receives dense innervation from the MPOA and ventral BNST, projects to the nucleus accumbens, and is implicated in sensory gating and regulation of behavior (Su and Bentivoglio, 1990; Otake and Ruggiero, 1995). Fos is also expressed selectively during parental behavior in the lateral septum and BNST in prairie voles (Rood et al., 1999) and in the MPOA in female mice (Calamandrei and Keveme, 1994). Most germane to the present discussion, by using pups that were either capable of suckling or not due to perioral anesthesia, we showed that nursing behavior did not elicit greater activation of c-fos in these forebrain sites than did maternal care without nursing. Also, similar numbers of Fos-labeled neurons were found in the MPOA in post-partum dams that were lactating and presumably suckled or that were not suckled due to pre-partum nipple removal (Numan and Numan, 1995; Walsh et al., 1996). In contrast, the only site of over twenty-five that were quantified in which suckling elicited higher levels of Fos than non-suckling interactions with pups was in the midbrain periaqueductal gray (PAG; Fig. 2C). These findings, and the requirement that active maternal behaviors are necessarily inhibited during the display of quiescent nursing, led to our conjecture that nursing behavior may be largely independent of excitatory forebrain influences that mediate pronurturance (Lonstein et al., 1998a). We did not quantify all possible forebrain sites, and cannot exclude the possibility that the study of a different immediateearly gene would reveal forebrain sites responsive to nursing behavior. Furthermore, we realize that not all sites involved in a given behavior show immediate-early gene activation. Nonetheless, our present understanding of the somatosensory regulation of nursing casts doubt on many claims of forebrain lesion-induced disruptions in this behavior.

Methodological flaws of many lesion studies with respect to nursing behavior

The absence of appropriate stimulus conditions for nursing has occurred in many experiments in which a neurobiological insult disrupts retrieval and grouping of pups; this is true not only acutely during a particular observation of maternal behavior, but chronically as well. A maternal behavior test is typically initiated by placing the pups opposite the nest site to assess retrieval. If the mother does not gather the pups by any means, and they are not gathered by the experimenter, then they are unable to initiate contact with the mother. Consequently, her entire maternal responsiveness will inevitably decline over days due to a continuous lack of sufficient contact with pups (Rosenblatt and Lehrman, 1963). Indeed, even a oneday separation from pups after an insult exacerbates its debilitating effects (Stem, 1997b). Thus, disparate findings with respect to nursing behavior after similar brain insults are likely due to differences in the stimuli from the pups, and the resulting acute and chronic effects on the mother. These disparities are most evident with respect to effects of MPOA lesions on nursing behavior, reported in numerous studies. Although the willingness to retrieve pups is permanently impaired by well-placed electrolytic or cell-body lesions of the MPOA or transections of its dorsolateral connections (Numan, 1994), the impairment in nursing behavior varies both between and within studies (table 6 in Stem, 1989). When retrieval is assessed all day, instead of in a brief test, by dividing the cage into quadrants with barriers that are too high for young pups to climb over, then of course, in such non-retrieving mothers nursing behavior can not occur. In contrast, the retention or recovery of nursing behavior was demonstrated in studies that utilize a litter that increases in age

269 and mobility during the post-surgical testing interval and thus permits free access between the dam and her litter (Terkel et al., 1979; Jacobson et al., 1980; Franz et al., 1986; Numan, 1990). Likewise, in postpartum cats, large lesions of the MPOA-anterior hypothalamic continuum caused a moderate reduction in maternal licking of kittens and in retrieval or effort to join a separated kitten as compared with sham-lesioned mothers, but nonetheless permitted the successful rearing of robust litters (Voith, 1982). Forebrain sites other than the MPOA have received much less attention with respect to maternal behavior. For example, in a single study on the effects of pre-mating lesions of the dorsal hippocampus, maternal nest building and retrieval were found to be inferior to controls, nursing time greatly reduced, and, therefore not surprisingly, there was a high rate of pup mortality (Kimble et al., 1967). But parturition was not observed, there was no replacement of dead pups, and subjects were not disturbed until post-partum day 6. Inadequate maternal behavior during parturition itself leads to a high rate of pup mortality and morbidity (Stem, 1996b). Thus, we cannot know from this research design what the nursing behavior of the lesioned dams would have been like if they were tested from early post partum with a sufficient number of healthy pups. more accessible and quiescence during nursing have been reported for rabbits, domestic swine, ewes, pony mares, bushbabies (see Stern and Johnson, 1990) and guinea pigs (Hennessy and Jenkins, 1994). The PAG - - a critical component of the Emotional (or Limbic) Motor System (Holstege, 1991) - is well-known as an integrative region vital for survival and reproduction, including analgesia, defense, vocalizations, female sexual behavior, and cardiovascular, respiratory, and visceral autonomic reflex regulation. These functions of the PAG are accomplished by processing somatosensory inputs from the trigeminal nerve and spinal cord, interconnections with other components of the limbic system, some direct projections to the spinal cord, and extensive projections to autonomic and somatic pre-motor nuclei of the ports and medulla (Depaulis and Bandler, 1991; Holstege, 1991; Beitz, 1995; Cameron et al., 1995a,b). These characteristics make the PAG a likely site for the sensorimotor integration of kyphosis.

Caudal lateral-ventrolateral periaqueductal gray (cPAGI,vl) responds to suckling

Our first evidence for a function of the PAG in suckling-induced kyphotic nursing is that lactating dams physically interacting with suckling pups had 50% more Fos-labeled neurons in the cPAGl,vl than dams interacting with pups rendered unable to suckle due to anesthetization of their mystacial pads (Fig. 2C; Lonstein and Stem, 1997a), in contrast to our findings with forebrain sites (Fig. 2A,B). Also, the degree of Fos-labeling in this area was highly correlated with the duration of time that dams spent in kyphosis. Maximal levels of Fos in the cPAGl,vl following maternal behavior occur in response to suckling but not other types of somatosensory stimulation of the ventral trunk from pups, such as rooting. Further, the selective increase in response to suckling was not found throughout the PAG of suckled dams, but instead was restricted to the cPAGl,vl at the level of the trochlear nucleus (Lonstein and Stem, 1997b).

Neural mediation of kyphotic nursing behavior

In rats, nursing often occurs via the kyphotic posture when the pups are neonates and at any stage post partum when the dam initiates a nursing episode by returning to the nest. On day 7 post-partum kyphotic nursing accounts for close to 100% of nursing time in a 60-min observation following a 3-h dam-litter separation, versus 30% of nursing time, lasting maximally for about 20 min continuously, during an undisturbed 24-h videotaped observation. This is the most efficient nursing posture for elicitation of milk ejections; most of the remainder of nursing is in the supine, or cat-like, posture (Lonstein et al., 1998b). Although this specific posture is not generalizable to all mammals, kyphosis has been described in detail in prairie voles (Lonstein and De Vries, 1999), and is probably characteristic of other small rodents with altricial young. Both postural adjustments in response to tactile stimulation from offspring that render the teats

cPAGt.vt mediates kyphosis and lordosis and inhibits maternal aggression

Bilateral electrolytic lesions of the cPAGl,vl performed either pre partum or post partum, revealed

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the function of this area of the midbrain in maternal behavior (Lonstein and Stern, 1997a, 1998; Lonstein et al., 1998b). Lesions delayed the onset of suckling-induced quiescence, reduced total nursing duration somewhat, and reduced the display of kyphosis in lactating dams by over 85%, while pronurturant maternal behaviors, such as retrieval, licking, and nest building, were virtually unaffected. The effect is site-specific since lesions of the dorsal rostral PAG had no effect on kyphosis (Fig. 3A). The reduction in kyphotic nursing, however, was largely compensated by nursing in other postures, such as the aberrant prone posture, in which the dam lays on top of the litter with little or no limb support. The functional significance of kyphosis was evident by the 1015% reduction in daily litter weight gains in litters interacting with cPAQvl-lesioned dams, which is understandable since few milk ejections occur during prone nursing. Because other nursing postures are not unique to nursing and do not seem to be reflexive in nature, there may not be a site akin to the cPAGl,vl that mediates them. The many parallels between feminine sexual and maternal behaviors (Stern, 1990, 1996a) include mediation of lordosis, as well as kyphosis, by the cPAGl,vl, and not the dorsal, rostral PAG (Fig. 3B; Lonstein and Stern, 1998; Lonstein et al., 1998b). A difference in the effects of these lesions, however, is that proceptivity (hopping and darting movements that solicit the male's attention) also was inhibited, while pronurturance was spared. The rapid alternation between solicitation behaviors and lordosis may account for their tighter linkage compared to the active and quiescent aspects of maternal behavior. Finally, the maternal aggression of lactating rats was doubled by bilateral lesions of the cPAGl,vl (Fig. 3C) (Lonstein and Stern, 1997a, 1998; Lonstein et al., 1998b), and reduced fearfulness of dams, measured in an elevated plus-maze, was reduced still further (Lonstein et al., 1998b). These results suggest

that this region usually is inhibitory to aggression, which fits with the incompatibility of this behavior with nursing. Also, stimulation of the lateral PAG is known to elicit defensive behavior (Depaulis and Vergnes, 1986). Possibly, ventral contact with the litter, usually including suckling, brings about a neurochemical change in the cPAG~,vl that lasts a few hours and that maintains the propensity to defend the litter and nest site by disinhibition of the lateral PAG; lesions of the cPAGl,vl would exaggerate the hypothesized neurochemical change.

GABAergic neurons in cPAGvl regulate kyphosis and lordosis, but not maternal aggression
The activity of most neurons within the PAG is tonically inhibited by the inhibitory neurotransmitter gamma aminobutyric acid (GABA) (Behbehani et al., 1990) and suckling may alleviate this tonic inhibition to allow for the display of kyphosis. This hypothesis is supported by the effects of bilateral microinfusions of GABAergic drugs into the caudal (c) PAG of lactating rats after a dam-litter separation of 4 h compared with controls only infused with saline (Salzberg et al., 1999). When dams were provided with non-suckling pups that normally do not elicit kyphosis, infusions of the GABAA receptor antagonist bicuculline (15 ng/0.25 txl per side) into the ventrolateral, but not the dorsolateral, cPAG resulted in prolonged periods of kyphosis in response to the pups' rooting. Conversely, when dams were provided with suckling pups, infusions of the GABAA receptor agonist muscimol (125 ng/0.25 Ixl per side) into the ventrolateral, but not the dorsolateral, cPAG substantially reduced the duration of kyphosis compared with controls infused with saline, while nursing in the prone posture increased. Normal pronurturance was found in all these tests. Similar effects on proceptivity and lordosis were found post weaning after infusions in the cPAG,,j,

Fig. 3. Effects of pre-partum bilateral electrolytic lesions of rostral or caudal periaqueductal gray (rPAG-x, cPAG-x), or sham lesions (Sham) on the behavior of lactating rats on days 1-6 post partum. (A) Duration of kyphotic nursing in 45 min observations after a 3-h dam-litter separation. (B) Lordosis quotient during the post-partum estrus. (A and B) * P < 0.0001, cPAG-x < other groups. (C) Frequency of maternal attacks toward an unfamiliar adult male rat on days 2 and 5 post partum just after the maternal behavior observation, without the pups present, and on day 8 post partum, 24 h after litter removal; * P < 0.01, cPAG-x > other groups, days 2 and 5 only. Values are means ::k SE. Modified from Lonstein and Stern (1998), with permission from Elsevier Science Ltd.

272 i.e., facilitation with bicuculline after sub-threshold ovarian hormone treatment and inhibition with muscimol after optimal ovarian hormone treatment. In contrast, maternal aggression does not appear to be under tonic GABAergic inhibition. This behavior was absent or at low levels when tested after mothers were placed with non-suckling pups, and therefore after 4.5 h without nursing, and bicuculline did not increase it or decrease it further. Robust maternal aggression, reinstated by 0.5 h with suckling pups, was not altered by muscimol infusions. stimulation alone does not activate axial musculature necessary for the control of the analogous lordosis posture (Cottingham et al., 1987). In support of our hypothesis that dis-inhibition of lower brainstem premotor neurons mediates kyphosis, more than half of the cPAG neurons that show increased c-fos activity in response to suckling themselves synthesize the inhibitory neurotransmitter GABA (Lonstein and De Vries, 2000). Of the many cPAGl,vl efferents to the medulla, the most likely to mediate kyphosis are those to the nucleus retroambiguus, a rostrocaudally oriented column of pre-motor neurons in the ventrolateral caudal medulla that projects to abdominal and pre-vertebral muscles responsible for flexion and bending of the trunk and head, respectively (Holstege, 1991; Holstege et al., 1997). Other PAG projections to the rostral medulla may contribute to kyphosis as well or may mediate suckling-induced physiological changes such as hypotension, analgesia, and slow-wave sleep.

The kyphosis circuit: possible afferents to and efferentsfrom the cPAGi.vt

Suckling inputs critical for oxytocin secretion leading to milk ejection travel through the spinal cord via the dorsolateral funiculus, with contralateral predominance (Fukuoka et al., 1984). Similarly, this column, but not the dorsal column, carries the suckling inputs necessary for kyphotic nursing as well (Stern et al., 1993). Dams with dorsolateral funiculus lesions recovered retrieval and licking of pups, and allowed the young to suckle, but they did so in a hunched position, without reflexive adjustments to the tactile stimulation from the young. Of the various tracts that ascend in the dorsolateral funiculus, the most likely to carry the sucking-induced information to the PAG is the spino-mesencephalic tract, which originates in the superficial to deep laminae of the dorsal horn throughout the spinal cord, and terminates in various midbrain sites, including the cPAG (Menttrey et al., 1982; Keay et al., 1997a). Further, the spino-mesencephalic tract utilizes the excitatory amino acid neurotransmitter, glutamate, in a majority of its neurons (Yezierski et al., 1993). The latter finding is consistent with our Fos results (Lonstein and Stern, 1997a,b), since neuronal firing activates c-los, and with the behavioral quiescence and hyporeactivity elicited by excitatory amino acid application to the ventrolateral PAG of male rats (Depaulis et al., 1994). Via excitatory neurochemicals, suckling may alleviate or overcome the tonic inhibition of cPAGl,vj neurons that is present when dams are not being suckled. The cPAG probably activates kyphosis not by direct projections to spinal cord interneurons or motoneurons, but rather indirectly via pre-motor neurons of the medulla; for example, because PAG

Suckling-induced mediation of quiescence

Prolonged inhibition of motorically active behaviors is a necessary concomitant of kyphosis. Yet our cPAG1,vl lesions that severely reduced kyphosis only delayed quiescence and slightly reduced its total duration. This suggests that additional inputs from suckling - - in a wider region of the cPAG, in the adjacent tegmentum (Keay et al., 1997b), or in another region - - are responsible for quiescence. One possible mechanism entails reciprocal connections between the MPOA and the cPAG~,v~ (Rizvi et al., 1992), some of which are inhibitory (MacLeod and Mayer, 1980). Another involves regulation of mesotelencephalic dopamine by inhibition of its source neurons in the ventral tegmental area, mediated by dense projections from the PAG to the ventral tegmental area (Beitz, 1995; Cameron et al., 1995a); the evidence for a function of mesotelencephalic dopamine in suckling-induced quiescence will be reviewed in the next section.

Mesotelencephalic dopamine: dissociation between pronurturance and nursing

Dopamine is well-known to influence incentive motivation, such as maternal behavior, via mesolimbic

273 pathways, including the nucleus accumbens (Robbins and Everitt, 1996), which serves as an interface between limbic structures and the motor system (Groenewegen et al., 1996). In prairie voles, the establishment of the pair-bond in this monogamous species is dependent upon dopamine in the nucleus accumbens (Gingrich et al., 2000). In rats, basal levels of dopamine underlie the dam's motivation to contact the pups (Stern and Keer, 1999), while higher levels of dopamine, released in response to active interactions with the pups (Hansen et al., 1993), are needed to display nest building, retrieval and licking (Giordano et al., 1990; Hansen et al., 1991a,b; Stern and Taylor, 1991). In contrast, when movement initiation was severely impaired following a high systemic dosage of haloperidol, a dopamine receptor blocker, kyphotic nursing behavior was facilitated (Stern and Taylor, 1991). When non-maternal virgin female or male rats were administered a cataleptic dosage of haloperidol, kyphosis was elicited by ventrum stimulation from a litter of hungry, rooting pups, but not by anesthetized pups (Stern, 1991). Thus, whereas dopamine is necessary for the motorically active components of maternal behavior, suckling-induced inhibition of dopamine may be required for kyphosis. The roles of dopamine in the active and inactive phases of maternal behavior are paralleled by its effects on feminine sexual behavior, in which dopamine is necessary for proceptivity, but its inhibition facilitates lordosis (Caggiula et al., 1979). reduced dose-dependently by nucleus accumbens infusions of FLU, but this effect was less site-specific than the FLU effects on retrieval (Fig. 4B). An enhancement of kyphotic nursing was also observed and occurred only after FLU infusions in the nucleus accumbens, whether or not there was also a pup retrieval deficit (Fig. 4C). Finally, among dams with nucleus accumbens infusions there was a relationship between alterations in maternal behavior and induction of mild catalepsy (Fig. 4D). One implication of the above findings is that the dopaminergic neurons of the dorsomedial nucleus accumbens shell, but not the nucleus accumbens core, are part of a neural circuit that is critical for retrieval of pups. Indeed, there are reciprocal connections of the shell - - but not core - - with several sites such as the lateral hypothalamus, MPOA, BNST, ventral tegmental area, and rostral periaqueductal gray (Heimer et al., 1991; Brog et al., 1993) that are implicated in pup retrieval (Numan, 1994; Numan and Numan, 1996; Lonstein and Stern, 1998). FLU-induced reductions in retrieval and licking of pups are also consistent with the role of the nucleus accumbens in numerous oral behaviors (Prinssen et al., 1994; Cools et al., 1995; Stratford and Kelley, 1997), including hoarding of food (Kelley and Stinus, 1985), which is similar in topography to retrieval of pups. A further implication of the above findings is that the induction of mild catalepsy by dopamine receptor blockade in the striatum, especially in the nucleus accumbens, inhibits active maternal behaviors such as pup-licking, while it facilitates the kyphotic nursing posture. Because licking was not eliminated in any dam, however, perhaps dopamine receptor blockade in the lateral striatum (Dells and Kelley, 1990), or at more than one site, is necessary to block this behavior more completely. Catalepsy is induced by dopamine receptor antagonism in the nucleus accumbens of rats (Ossowska et al., 1990), mediated by its projections to the substantia innominata-ventral pallidum complex (Van den Bos and Cools, 1989). This adaptive state subserves the readiness to stand still by promoting "stable, static equilibrium" (De Ryck et al., 1980), thereby forming an appropriate precursor for sustained kyphosis by reducing motoric responsiveness to behaviorally activating stimuli and by providing the necessary

Nucleus accumbens: mediation of transition from pronurturance to kyphosis ?

To obtain more site-specific information on the effects of different degrees of dopamine receptor blockade on pup retrieval, as well as on pup licking and nursing behavior, microinfusions of the mixed dopamine receptor types D~/D2 antagonist cis-flupenthixol (FLU) were carried out in lactating rats (Keer and Stern, 1999). FLU dose-dependently reduced retrieval of pups when placed in the nucleus accumbens, but not in the dorsomedial striatum (DMS) or lateral ventricle; this reduction occurred only if the infusions included the shell region of the nucleus accumbens (Fig. 4A). Pup-licking also was

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Fig. 4. Dopamine mechanisms and maternal behavior. (A-D) Effects of bilateral microinfusions of 5, 10, or 20 Ixg/Ixl cis-flupenthixol, a mixed dopamine DI/D2 receptor antagonist, within the nucleus accumbens (NA) or dorsomedial striatum (DMS), or unilateral microinfusions of 0, 20, or 40 Ixg/Ixl FLU within the lateral ventricle (LV; n = 5) on days 7-9 post partum, or saline (0) on day 6. A-C show the effects, after a 4-h dam-litter separation, on maternal behavior for 30 min after suckling onset. The insets shows the nucleus accumbens results divided between the retrieving (Ret+) and non-retrieving (Ret-; n = 5) dams after 20 txg FLU. There are statistically significant differences between the means with different letters in the nucleus accumbens group. (A) Number of pups retrieved in the first 5 min. The infusions of only the retrieval-impaired dams included the shell region. Inset: * P < 0.02. (B) Duration of pup licking from reunion to 30 min after onset of suckling by >6 pups. The pups of retrieval-impaired dams were placed in the nest after 5 min. * In the DMS group, licking was reduced after 10 or 20 I~g FLU, P < 0.04. (C) Duration of kyphotic nursing in the 30 min after onset of suckling by >6 pups. (D) Duration of clinging to bars, tested soon after the end of the maternal behavior observation. * P < 0.001, FLU versus saline. Modified from Keer and Stern (1999), with permission from Elsevier Science Ltd.

postural f o u n d a t i o n 1991).
Conclusions

for q u i e s c e n t nursing

(Stern,

Progress in e l u c i d a t i n g the neural circuitry o f m a ternal b e h a v i o r is facilitated by u n d e r s t a n d i n g the sensory r e g u l a t i o n o f its various c o m p o n e n t s . In rats, a useful dissociation can be m a d e b e t w e e n pronurturance, or the m o t o r i c a l l y active b e h a v i o r s

that p r e c e d e nursing such as retrieval and licking, and q u i e s c e n t nursing, often in the k y p h o t i c posture. R e s e a r c h r e v i e w e d shows that there are differences in the n e u r o b i o l o g i c a l d e t e r m i n a n t s o f retrieval, licking, and k y p h o t i c nursing, e m p h a s i z i n g the fact that m a t e r n a l b e h a v i o r is far f r o m a unitary process. O n l y w e l l - i n f o r m e d e x p e r i m e n t a l designs and careful observation o f m a t e r n a l b e h a v i o r s after neural insults will a l l o w us to p r o p e r l y e x a m i n e the intricacies o f this c o m p l e x m a m m a l i a n behavior.

275

Abbreviations
BNST bed nucleus of the stria terminalis central (c) midbrain periaqueductal gray cPAG cPAGI,vj caudal lateral and ventrolateral midbrain periaqueductal gray cis-flupenthix ol FLU gamma aminobutyric acid GABA lateral habenula LHb lateral septum LS medial preoptic area MPOA nucleus accumbens NA midbrain periaqueductal gray PAG paraventricular thalamic nucleus PVT

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Progress in Brain Research, Vol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 20

Genomic imprinting and the maternal brain

E. Barry Keverne *
Sub-Department of Animal Behaviour, University of Cambridge, High Street, Madingley, Cambridge CB3 8AA, UK

Abstract: Those parts of the genome that contain imprinted genes are relatively small (between 100 and 150 genes predicted) but their impact on mammalian development and evolution is substantial. Most of the imprinted genes that have been studied are regulatory: transcription factors, alternative splicers, oncogenes, tumor suppressors, growth factors, or are involved in complex signalling pathways such as the tumor necrosis factor (TNF) and ubiquitin pathways. This review considers the effects of imprinted genes on brain development by examining the distribution of androgenetic and parthenogenetic cells in the brains of chimeric mice using in situ markers. At birth, cells that are disomic for the paternal genome (androgenetic) contribute substantially to the hypothalamus, septum, preoptic area and bed nuclei of the stria terminalis and fail to survive in the developing neocortex and striatum. In contrast, cells that are disomic for the maternal genome (parthenogenetic) proliferate in the cortex and striatum but are excluded from the diencephalic structures. Growth of the brain is enhanced by the presence of parthenogenetic cells and hence increased maternal gene dosage, whereas the brains of androgenetic chimeras are smaller. Mest and Peg3, two imprinted genes that are paternally expressed, have been disrupted by gene targeting and show high levels of expression in regions where androgenetic cells accumulated, namely the hypothalamus, preoptic area and septum. Although of different structural classes and located on different chromosomes, both of these paternally expressed genes influence placental growth and maternal behavior. The implications of these findings for brain evolution and maternal behavior are discussed.

Introduction

Genomic imprinting, which results in the monoallelic expression of certain genes according to parent of origin, represents a form of gene regulation which plays an important role in mammalian development (Barlow, 1995). Hence the Mendelian law that the two copies of autosomal genes are functionally equivalent does not hold true for the functionally haploid genes that are imprinted in mammals. The realization that male and female autosomal genes do not contribute equally to mammalian development derives from experiments in mice where normal development was found to require the presence of

* Correspondence to: E.B. Keverne, Sub-Department of Animal Behaviour, University of Cambridge, High Street, Madingley, Cambridge CB3 8AA, UK. E-mail: ebkl0@cus.cam.ac.uk

both a maternal and paternal genome (McGrath and Solter, 1984; Surani et al., 1984). In these experiments both parthenogenetic embryos (diploid with two sets of maternal chromosomes) failed to show normal development, and survival extended for only a few days beyond implantation. Parthenogenetic conceptuses developed embryos up to the 25 somite stage but the extra embryonic tissues which form the placenta were rudimentary. Androgenetic conceptuses (diploid with two sets of paternal chromosomes) possessed over-developed placental tissue, but embryonic development rarely progressed beyond the 6-8 somite stage. Hence placental development appeared to depend on the expression of paternally inherited alleles, and embryonic growth on alleles of maternal origin, and these differing phenotypes resulted from the disruption of parent of origin imprinted genes. These early observations have been substantiated by the identification of certain imprinted genes and by inducing null mutations

280 that experimentally produce some features of the placental/embryonic phenotype (De Chiara et al., 1991). The principles of gene dosage control through imprinting has been integral to the evolutionary development of the placenta. Haig and Graham (1991) have proposed that in the development of the placenta a conflict of interest between the maternal and paternal imprinted genes has arisen. There is molecular genetic evidence for this with the paternally expressed insulin-like growth factor 2 gene and the maternally expressed insulin-like growth factor 2 receptor which binds the ligand but fails to transduce the signal to the cell (Haig and Graham, 1991). This theory of parental conflict in the context of genomic imprinting provides a conceptual framework that explains paternally expressed genes enhancing placental growth and maternally expressed genes restricting this growth. Since the mother provides the placental nourishment, it is in her interests to ensure survival of all her generations of offspring, while the interests of each father is to maximize the extraction of maternal resources for his own offspring at the expense of those from future fathers. A question of some relevance to our understanding of the 'maternal brain' is whether or not Haig's theory of 'genomic conflict', derived from imprinting in the placenta, can be applied to brain development and maternal behavior. precise location of the parthenogenetic and androgenetic cells in chimeras can be determined using in situ markers (lacZ or multiples copies of the B-globulin gene). Using these techniques, a clear and distinct patterning in the distribution of these cells emerges during brain development. At birth, cells that are disomic for the paternal genome (i.e. both alleles are from the father) contribute substantially to those parts of the brain that are important for primary motivated behavior (e.g. the hypothalamus, septum, preoptic area and bed nuclei of stria terminalis) and are excluded from the developing neocortex and striatum. At the earliest stages of mouse brain development (days 9-10), androgenetic cells are present in all neural tissues and, as gestation progresses, they proliferate extensively in the medio-basal forebrain. At parturition, however, they are virtually absent from telencephalic structures. By contrast, parthenogenetic cells (i.e. both alleles inherited from the mother) are excluded from these medio-basal forebrain areas, but accumulate selectively in those regions where androgenetic cells are excluded, especially the neocortex and striatum. Furthermore, growth of the brain of parthenogenetic chimeras is enhanced by this increased maternally expressed gene dosage, whereas the brains of androgenetic chimeras are smaller, both in absolute size and especially relative to body weight. Not only is it surprising that parthenogenetic cells seem to proliferate at the expense of normal cells and to produce a larger telencephalon in chimeras, but this enlarged brain appears anatomically and functionally normal. This is particularly surprising because a large number of genes have been silenced in these cells (i.e. all the imprinted genes that are paternally expressed), and others that are maternally expressed have been duplicated. Such a finding emphasizes the importance of maternally expressed alleles in telencephalic development and the lack of importance of paternally inherited imprinted genes in these regions. The distinct patterning in the distribution of parthenogenetic and androgenetic cells and their differential effects on brain growth suggest genomic imprinting may have been important in forebrain evolution (Keverue et al., 1997). Allometric scaling of the parts of the brain to which matemally or paternally expressed genes differentially

Genomic imprinting and brain development

The first step in identifying whether or not imprinted genes impact on the brain was obtained by investigating the cumulative effects of the imprinted genome. Since both androgenetic and parthenogenetic embryos die long before brain development occurs, then such studies require the construction of chimeric embryos containing androgenetic plus wild-type cells or parthenogenetic plus wild-type cells (Allen et al., 1995; Keverne et al., 1996). The androgenetic cells are XY and differ from wild-type cells in possessing a duplicated paternal genome which increases the dosage of paternally expressed imprinted genes and effectively silences the maternally expressed imprinted genes. The converse applies to parthenogenetic cells which are XX and differ from wild-type in possessing a duplicated maternal genome. The

281 contribute reveals that a remodelling of the brain has occurred during mammalian evolution. On moving across phylogenies from insectivorous mammals to prosimian and then simian primates, it can be seen that the neocortex and striatum have increased significantly in size relative to the rest of the brain and body, while the hypothalamus, medial preoptic area and septum have relatively decreased in size. Genomic imprinting may thus have facilitated a rapid, nonlinear expansion of the brain (especially the neocortex and striatum) relative to body size during its development over an evolutionary timescale. Imprinted genes that are paternally expressed and influence maternal behavior Two imprinted genes that are paternally expressed have been disrupted by gene targeting in embryonic stem cells. Mest (also known as Pegl) is only expressed from the paternal allele and in adult animals is expressed almost exclusively in the central nervous system (Lefebvre et al., 1998). Particularly high levels of expression are found in the ventral forebrain, including the hypothalamus, amygdala, medial preoptic area and also in olfactory processing structures. Females carrying the targeted mutation have a normal pregnancy rate, deliver at term, but have few surviving progeny, even when mated with normal males. All pups from such matings are phenotypically wild-type, since they only express the Mest allele inherited from their normal father. Progeny from these matings when cross-fostered to wild-type mothers show the normal rate of survival. Hence the low survival rate seen with mutant mothers is not attributable to a phenotype of their pups, but reflects a decreased maternal fitness of the mothers. Investigation of maternal behavior shortly after birth has revealed that mutant Mest mothers do not respond to their pups, fail to suckle them, and do not ingest the placentae. Furthermore, pup induced maternal behavior in non-parturient females shows severe deficits in all aspects of maternal behavior (e.g. retrieval, nest building, crouching) in mutants compared with wild-type females. Sniffing of pups and location of buried food is not significantly different from wild-type females, suggesting the impaired maternal behavior is not primarily from an olfactory deficit. In addition to the maternal deficit in Mest mutant females, both male and female mutant offspring from normal mothers (i.e. inheriting the mutated allele from + / - fathers) are smaller and lighter than wild-type littermates (i.e. inheriting the normal allele from + / - fathers). Mutant embryos and placental weights are approximately 87% of normal and their postnatal growth is slower (65% of normal) at the first week postpartum and when weaned, while adult animals are still only 62% of the weight of normal littermates. Moreover, their survival rate is lower with less than 50% of the expected number of mutant animals surviving to weaning. This finding, that the paternally expressed Mest gene acts as a positive regulator of embryonic growth is consistent with Haig's evolutionary model of genomic imprinting based on the conflicting interests of the parental genomes in mammals. Another paternally expressed gene, Peg3, is also expressed in embryonic placental tissues and in the adult hypothalamus (Li et al., 1999). To study the function of Peg3 in vivo, a targeted mutation of this gene was achieved by insertion of a ~-geo selection cassette in the Y-coding exon. A distinct behavioral phenotype became evident from interbreeding of heterozygotes. Very few first litters (8%) of mutant mothers ( + / - ) grew to weaning age compared to those nursed by wild-type mothers. Again, this occurred when the pups of the mutant mother were normal (paternal transmission from + / + fathers), placing the cause for postnatal fatalities as being maternal. Observations of maternal behavior with mutant, primiparous mothers revealed a complete deficit in all aspects of maternal behavior (retrieving, nest-building and crouching) during a 30-min period. To validate these observations the response of postpartum females towards cross-fostered newborn pups was also tested. Mutant mothers took 11 times longer to retrieve the pups and eight times longer to build a nest than wild-type females, and were never observed to crouch over these pups. An inability to find the pups was not a factor for the impaired maternal response, as the mutant mothers sniffed the pups as quickly as wild-type mothers. Further observations on multiparous and virgin females for induced maternal behavior by pup exposure revealed significant impairments in all aspects of maternal responding compared with matched wild-type control females.

282 Hence, the mutation would appear to affect maternal behavior independently of the hormonal influences of pregnancy and parturition. High levels of Peg3 expression are present in those structures of the hypothalamus that are known to regulate maternal behavior and can be shown to be active following pup exposure. The exposure of virgin females to pups causes a rapid activation offos B, an immediate-early gene, in the medial preoptic area, a neural region strongly implicated in maternal behavior (Brown et al., 1996). A null mutation of fos B also impairs maternal behavior, but mutant Peg3 females showed a normalfos B response to pup exposure, suggesting Peg3 expression is downstream or independent of this gene. Despite the small litter size of Peg3 females, the surviving (non-mutant) progeny gains less weight during the first 3 weeks after birth, although their weight did catch up after weaning. The preweaning deficiency in weight gain could result either from impaired maternal response, and/or a defect in lactation in the mutant females. To test this we measured both matemal behavior and the weight gain by the pups following 2 h separation from mother. The mutant mothers were slower in adopting the crouching posture compared to wild-type mothers (15.3 vs. 8.7 min, n = 6, P < 0.004) but the pups of both groups attached to the nipples after 1 h. Pup's weight increased by 1.8 4- 0.5 mg and 3.2 4- 0.25 mg after 6 and 24 h, respectively, with normal mothers. By contrast, the pups suckled by the mutant mothers gained no weight after 6 h and only 0.98 + 0.2 mg after 24 h (P < 0.01). The reduced weight gain in the latter suggested a defect in lactation in the mutant mothers. Examination of the mammary glands of the mutant mothers revealed that they were histologically normal both at prepartum and postpartum. Milk ejection is controlled by oxytocin released from the hypothalamic paraventricular and supraoptic nuclei in response to the suckling stimulus. We found that postpartum mutant mothers had reduced oxytocinpositive neurons compared to the wild-type females. There were a total of 2984 -4- 209 oxytocin-positive neurons in the mutant hypothalamus compared to 4496 4- 252 in the wild-type hypothalamus (n = 5, P < 0.02). Even allowing for the difference in body weight and paraventricular nucleus size between controls and mutant females, the number of oxytocin neurons was still significantly smaller in the latter. The reduced weight gain of pups could therefore be explained by an insufficient oxytocin surge, since a mutation of the oxytocin gene in mice abolished milk ejection (Nishimori et al., 1996; Young et al., 1996). In rodents, central oxytocin synthesis increases at parturition (Caldwell et al., 1987), and central infusions of this hormone stimulate a rapid maternal behavior while the hormone antiserum or antagonists inhibit maternal behavior (Insel, 1992; Pedersen et al., 1992). Thus, the behavioral and neuroendocrine responses in this study have oxytocin as a common component for both the peripheral and central events that are impaired in the Peg3 mutants. The reduced number of oxytocin-producing neurons would not eliminate oxytocin secretion but may have impaired the neuronal coupling and synchrony, which is required for a bolus of oxytocin release at postpartum to achieve milk let-down (Hatton et al., 1987; see also Theodosis, 2001 (this volume)). The involvement of Peg3 in the tumor necrosis factor (TNF) signalling pathway affecting the balance of apoptosis and cell proliferation (Relaix et al., 1998) could account for these decreases in oxytocin neurons in the paraventricular nucleus and other hypothalamic nuclei. Since Peg3 mutant mothers conceived and gave birth, and they had normal development of mammary glands during lactation, multiple endocrine dysfunction is an unlikely cause of this behavioral phenotype. Why

imprinting?

The evolutionary consequences and impact of genomic imprinting on mammalian development does not in itself explain why such a mechanism is required. A wide variety of theories have been advocated for genomic imprinting (Hurst, 1997), including conflict over parental investment (Moore and Haig, 1991), or a defence mechanism against parthenogenesis (Varmuza and Mann, 1994), invasive retroviral DNA (Barlow, 1993), and chromosome gain or loss (Thomas, 1995), while other theories relate to the regulation of gene dosage (Solter, 1988). No single theory succeeds in completely explaining genomic imprinting, and since the functions of relatively few imprinted genes are known, these the-

283 ories tend to address ultimate causation (Haig and Trivers, 1995). From a mechanistic viewpoint genomic imprinting has much in common with allelic exclusion in that only a single allele is expressed to encode RNA and protein, but in allelic exclusion the parent-of-origin for the expressed allele is random. This monoallelic expression ensures that only a single receptor type is expressed from a family of receptors and is exemplified in phylogenetically old systems, such as the olfactory receptor genes, immunoglobulin genes, natural killer cell receptor genes and T-cell receptor genes (Chess, 1998). Such allelic exclusion has enabled expansion of receptor gene families while at the same time maintaining each cell's specificity. Likewise, expansion of gene control mechanisms has required cooperative binding of transcription factors regulated by a variety of signalling pathways. When a number of these factors must be present for any of them to bind, this may lead to all-or-none transcription, as is the case for the interleukin-2 gene which has been recently shown to exhibit monoallelic expression (Hollander et al., 1998). Dosage regulation for interleukin-2 is crucial since overproduction results in suppression of T-cell function and autoimmunity, while immunodeficiency occurs with decreased production. Interleukin-2 expression is, therefore, tightly controlled by multiple signalling pathways. It is not just the cytokines like interleukin-2 which regulate biological effects in a dose-dependent mode, since during development localized gradients of chemokines specify cellular migration, and growth factors are regulated according to a discriminating threshold. These events would also benefit from allelic exclusion. Control of cellular differentiation during development is more tightly regulated with single allele expression during mitosis when segregation and the pairing of chromatids produces daughter cells of differing configuration. This occurs when a stem cell gives rise to another stem cell and a differentiated lineage (Holliday, 1990). It is, therefore, well established that allelic exclusion can tightly control the cellular events that are crucial to development. Genomic imprinting may be viewed as a variation on this theme with even tighter regulation on gene dosage control when the receptor and ligand are reciprocally imprinted. Although gene silencing is not uniquely mammalian, its inheritance through specific imprinted genes according to parent of origin does appear to be a mammalian trait (Efstratiadis, 1994). Imprinted genes influence embryonic growth and, since a large proportion of those identified are expressed in the placenta, this suggests that an important role for genetic imprinting lies in the control of intrauterine growth and development. From an evolutionary standpoint, it is clear that in order for internal fertilization and placentation to become established, it would have been essential that the embryonic growth program were free of errors. Any errors late in mammalian development would not only constitute a cost and risk to the mother, who had committed a large portion of her resources to fetal development, but they would also put at risk sibling embryos of the same and possibly future generations. Unlike the egg laying reptiles and amphibia where vast numbers of eggs are produced, and where small losses from developmental dysfunction are of no great consequence to the mother's health and reproductive potential, mammals can tolerate no loss of embryos once placentation is firmly established. There is, therefore, an additional need in mammals to either ensure that the developmental program is perfect, and if not, then to reject the embryo at the earliest possible stage. By imprinting key regulatory genes that may be implicated in implantation/placentation as well as embryonic growth and brain development, a fail-safe mechanism may have been achieved.
Conclusions

There are now two imprinted genes, Peg3 and Mest, of different structural classes implicated in the behavioral function of the central nervous system. This suggests that maternal behavior may be a specifically selected function for the imprinting of neurally expressed genes. The most widely accepted hypothesis for genomic imprinting is that of 'parental conflict' in which the parental genomes compete to regulate intra-uterine embryonic growth through different sets of imprinted genes (Moore and Haig, 1991). However, the impaired behavior of the mother with respect to paternally expressed genes does not, from an evolutionary viewpoint, occur until the following generation. Since this deficit would equally affect transmission of both grand paternal and grand maternal genes, it does not fit appropriately with the

284

conflict theory. However, fixation of the gene in the population may have been boosted by the evolutionary advantage of its effects on maternal behavior, milk let-down and postnatal growth. In order to understand the evolutionary development of the maternal brain, it is important to expand our thinking beyond neurobiology. A larger brain requires a larger skull and changes in the female pelvic morphology to permit parturition. Brain tissue has one of the highest demands for oxygen and glucose in the body, metabolic needs that can only be met by an adequate vascular supply to the placenta, a feature of development that is also regulated by imprinted genes. Moreover, large brained mammals such as primates produce fewer offspring, usually in a neotanous state and often requiring long periods of lactation to reach weaning, while parental care may continue long beyond weaning. Hence the lifetime reproductive success of large brained primates is dependent on extended matrilines to sustain care giving. Not only are the sisters and daughters necessary for sharing the care giving, but they, themselves, gain mothering experience before they reach reproductive age (Keverne et al., 1997). Hence a number of genes with differing functions require a co-ordinated regulation to enable synchronization of evolutionary events at multiple levels.

Acknowledgements
The work reported in this review was undertaken in collaboration with Professor Azim Surani. The targeted mutations in Peg1 and Peg3 were done by Dr. Louis Lefebvre and Dr. Li-Lan Lee, and Sheila Barton pioneered development of the mouse chimeras.

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Progressin BrainResearch,Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAFFER 21

Like mother, like daughter: evidence for non-genomic transmission of parental behavior and stress responsivity
Frances Champagne and Michael J. Meaney *
Developmental Neuroendocrinology Laboratory, Douglas Hospital Research Center, Departments of Psychiatry, and Neurology and Neurosurgery, McGill University, Montreal PQ H4H 1R3, Canada

Abstract: Considerable evidence demonstrates that the quality of the early environment influences patterns of development that, in turn, determine the health and productivity of the individual throughout their life span. However, the processes through which early life influences health are not clearly understood. Through the activation of the hypothalamo-pituitaryadrenal (HPA) axis and corticotropin-releasing hormone (CRH) pathways, prolonged or exaggerated responses to stress have profound effects on physiological and cognitive functions. Early maternal separation or handling of neonatal rats can program widespread and lifelong changes in various transmitter systems that regulate the HPA and CRH systems. Our studies show that a high level of maternal licking/grooming, and arched-back nursing correlates with reduced CRH mRNA expression and enhanced glucocorticoid negative feedback, and lower stress responses in the adult. This behavior is stably transmitted between generations and cross-fostering studies show that the offspring inherit the behavior from the nursing mother and not the biological mother. Such intergenerational transmission of maternal behavior is seen in rodents, primates and humans, and may underlie adaptive changes in the HPA axis. The neural basis of this inheritance pattern appears to reside in the central oxytocin system which determines features of maternal behavior. Through these various adaptive neural mechanisms the environmental demand on the mother is reflected in the quality of maternal care to her offspring. This, in turn, programs stress reactivity and maternal behavior patterns of the offspring. This not only determines certain health outcomes but also establishes the relationships between mother and offspring in the next generation. These findings suggest that for neurobiologists, the function of the family is an important level of analysis and the critical question is that of how environmental events regulate neural systems that mediate the expression of parental care.

Parental care and the health of offspring

Considerable evidence has accumulated to show that the quality of family life can have a major influence on the development of individual differences in vulnerability to illness throughout later life (Taylor and Seeman, 1999). As adults, victims of childhood physical or sexual abuse are at considerably greater risk for mental illness, as well as for diabetes and

* Corresponding author: Michael J. Meaney, Douglas Hospital Research Center, 6875 LaSalle Boulevard, Montreal, PQ H4H 1R3, Canada. Tel.: + 1-514-762-3048; Fax: +1-514-762-3034; E-mail: mdmm@musica.mcgill.ca

heart disease (e.g. Bifulco et al., 1991; Brown and Anderson, 1993; Felitti et al., 1998). Children need not be beaten to be compromised. Persistent emotional neglect or conditions of harsh, inconsistent discipline serve to increase the risk of depression and anxiety disorders to a level comparable to that observed in cases of abuse (Holmes and Robins, 1988). Indeed, for certain outcomes, the consequences of persistent neglect exceed those of abuse (Trickett and McBride-Chang, 1995; A m m e r m a n et al., 1986). However, more subtle relationships also exist. Low scores on parental bonding scales, reflecting cold, distant parent-child relationships, significantly increase the risk of depression and anxiety in later life (e.g. Parker, 1981; Canetti et al., 1997). Furthermore,

288 the risk is not unique to mental health. Russek and Schwartz (1997) found that by mid-life those individuals who, as undergraduate students, rated their relationship with parents as cold and detached, had a four-fold greater risk of chronic illness, including depression and alcoholism, as well as heart disease and diabetes. Parental factors also serve to mediate the effects of environmental adversity on development. For example, the effects of poverty on emotional and cognitive development are mediated by parental factors, to the extent that if such factors are controlled there is no discernible effect of poverty on child development (Conger et al., 1994; Mclloyd, 1998). Moreover, successful treatment outcomes associated with early intervention programs are routinely correlated with changes in parental behavior, and in cases where parental behavior proves resistant to change, treatment outcomes are seriously limited. These findings suggest that variations in parental care mediate, in part at least, the effects of environmental adversity on child development. However, the sword cuts both ways, and family life can also serve as a source of resilience in the face of chronic stress (Rutter, 1979). Thus, warm, nurturing families tend to promote resistance to stress and to diminish vulnerability to stress-induced illness (Smith and Prior, 1995). The critical question to be addressed in this review concerns the nature of these parental influences on the health of the offspring. What are the factors that mediate such enduring effects? We have argued that the relationship between early life events and health in adulthood is mediated by parental influences on the development of neural systems which underlie the expression of behavioral and endocrine responses to stress (Francis and Meaney, 1999). There are two critical assumptions here: first, that prolonged activation of neural and hormonal responses to stress can promote illness and, second, that early environmental events influence the development of these responses. The follow sections will address the evidence favoring these assumptions. the development of such divergent forms of illness involve the same hormones that ensure survival during a period of stress (McEwen and Stellar, 1993; McEwen, 1998). These effects can, in part, be understood in terms of the responses elicited by stressors (Dallman et al., 1993; De Kloet et al., 1998). The increased adrenal release of catecholamines, adrenaline and noradrenaline, as well as the glucocorticoids, orchestrate a move to catabolism, increasing lipolysis and mobilizing glucose reserves, and insulin antagonism. These actions serve to increase the availability and distribution of energy substrates. At the same time the increase in circulating levels of catecholamines and glucocorticoids is associated with increased cardiovascular tone. Prolonged activation of these pathways provides an obvious risk for decreased sensitivity to insulin and a risk of steroid-induced diabetes, hypertension, hyperlipidemia, hypercholesterolemia, abdominal fat deposition, and arterial wear and tear (Brindley and Rolland, 1989), all of which are associated with an increased risk for heart disease (Seeman et al., 1997). There are also cognitive responses to stressors that include systems which mediate attentional processes as well as learning and memory (Arnsten, 1998). During stress individuals become hypervigilant; the level of attention directed to the surrounding environment is increased at the expense of our ability to concentrate on a focused set of tasks that are not essential for survival. As a function of these changes in attentional processes, as well as the effects of glucocorticoids on brain structures such as the hippocampus, episodic memory is less effective during periods of stress (Lupien et al., 1998; Newcomer et al., 1999). At the same time, glucocorticoids act on areas of the brain, such as the amygdala, to enhance learning and memory for emotional stimuli (Davis, 1992; LeDoux, 1992; Quirarte et al., 1997). These changes in psychological arousal are also associated with altered emotional states: feelings of apprehension and fear predominate during a stressful experience. While these responses are highly adaptive, chronic activation of these systems can promote the emergence of specific forms of cognitive impairments, states of anxiety and dysphoria, and sleep disorders (Arborelius et al., 1999). Herein lies the dilemma: the same stress hormones that permit survival during stress, can ul-

Stress and its relationship to illness

Stress is a risk factor for a variety of illnesses, ranging from auto-immune disorders to mental illness. The pathways by which stressful events can promote

289 timately lead to disease. In human and nonhuman populations individuals that show exaggerated hypothalamic-pituitary-adrenal (HPA) responses to stress are at increased risk for a variety of disorders including heart disease, diabetes, as well as anxiety, depression and drug addiction. The following sections will address the neurobiological basis for this relationship between HPA activity and vulnerability to illness. 1991). The amygdaloid CRH projection to the locus coeruleus (Moga and Gray, 1989; Koegler-Muly et al., 1993; Van Bockstaele et al., 1996) is also critical for the expression of behavioral responses to stress. Microinjections of the CRH receptor antagonist, c~-helical-CRH(9-41), into the locus coeruleus attenuate fear-related behaviors (Butler et al., 1990; Swiergiel et al., 1993; Rosen and Schulkin, 1998). Hence, the CRH neurons in the PVN and the central amygdala serve as important mediators of both behavioral and endocrine responses to stress. Not surprisingly, increased CRH levels have been associated with serious mood disorders (Arborelius et al., 1999). These findings have provided a basis for understanding how stress can influence health. Yet the influence of stress can only really be fully appreciated when we factor into the equation some appreciation of the individual's response to stress. A major research theme on the development of psychopathology focuses on the role of early life events in determining individual differences in vulnerability to stress. This hypothesis rests on the assumption that chronic activation of central and endocrine stress responses can promote illness (see above). Thus, early life events which increase stress reactivity result in a greater vulnerability for stress-induced illness over the life-span.

Corticotropin-releasing hormone
In large measure the physiological and cognitive reactions to a stressful environment are governed by the activity of central corticotropin-releasing hormone (CRH) systems which coordinate behavioral, emotional, autonomic and endocrine responses to stressors (e.g. Gray, 1993; Koob and Heinrichs, 1999). There are two major CRH pathways regulating the expression of stress responses. First, there is a CRH pathway from the parvocellular regions of the paraventricular nucleus of the hypothalamus (PVN) to the hypophysial-portal system of the anterior pituitary, a system which serves as a principal network for the transduction of neural signals into a pituitary-adrenal response (Plotsky, 1991). In responses to stressors, CRH, as well as co-secretagogues such as arginine vasopressin, is released from PVN neurons into the portal blood supply of the anterior pituitary where it provokes the synthesis and release of adrenocorticotropic hormone (ACTH). Pituitary ACTH, in turn, causes the release of glucocorticoids from the adrenal gland. This is the classical hypothalamo-pituitary-adrenal axis. The second CRH pathway involves CRH neurons within other parts of the limbic system. CRH neurons in the central nucleus of the amygdala project to the locus coeruleus and increase the firing rate of locus coeruleus neurons, resulting in increased noradrenaline release in the highly distributed terminal fields of this ascending noradrenergic system (Gray et al., 1989; Lavicky and Dunn, 1993; Valentino et al., 1998). One of the principal targets for this noradrenergic system is actually the CRH neurons of the PVN, thereby leading to a potential feed-forward mechanism. Noradrenaline is the major known source of drive to CRH release from PVN neurons during stress (Plotsky et al., 1989; Plotsky,

Environmental regulation of HPA and behavioral responses to stress

One of the strongest models for environmental regulation of the development of responses to stress is that of postnatal handling or maternal separation of rodents. This handling involves daily periods of separation of the pup from the mother. In the rat and mouse, brief (i.e. 3-15 min) episodes of handling in the postnatal period decreases the magnitude of behavioral and endocrine responses to stress in adulthood (Levine, 1957, 1962; Levine et al., 1967; Ader and Grota, 1969; Hess et al., 1969; Zarrow et al., 1972; Meaney et al., 1989, 1996; Viau et al., 1993; Maccari et al., 1995). In contrast, longer periods (i.e. 3-6 h) of daily separation from the mother increase behavioral and endocrine responses to stress (Plotsky and Meaney, 1993; Ladd et al., 1996; Liu et al., 2000). These effects are pro-

290 grammed to persist through the life of the animal (Meaney et al., 1988) and are associated with health outcomes, such as changes in the susceptibility to allergic encephalomyelitis (Laban et al., 1995). The central CRH systems are critical targets for the effects of neonatal handling procedures (Francis and Meaney, 1999). Predictably, handling decreases and maternal separation increases CRH gene expression in the PVN and the central amygdala in the adult. Moreover, there are also potent effects on systems which are known to regulate CRH gene expression in the PVN and the central amygdala. These include both the glucocorticoid receptor system which serves to inhibit CRH synthesis in and release from PVN neurons, and the y-aminobutyric acid (GABA)/central benzodiazepine systems which regulate amygdaloid CRH activity as well as having effects at the level of the noradrenergic neurons of the locus coeruleus and nucleus of the solitary tract (Caldji et al., 2000). Predictably, stress-induced activation of ascending noradrenergic systems in adult animals is enhanced by maternal separation and decreased by handling in early life (Liu et al., 1997). Thus, environmental manipulations can alter the expression of behavioral and endocrine responses to stress by altering the development of central CRH systems and the mechanisms which regulate them. In addition, maternal separation in early life alters the development of ascending serotonergic systems in both monkey (Higley et al., 1991) and rat (Ladd et al., 1996). Kraemer et al. (1989) have shown that repeated periods of maternal separation in early life increase cerebrospinal fluid measures of central noradrenaline and 5-hydroxy-tryptamine (5-HT) responses to stress in the rhesus monkey. Considering the importance of the ascending noradrenaline and 5-HT systems in the aetiology of depression, these findings suggest a mechanism whereby early life events might predispose an individual to depression in later life. The decreased mother-infant contact resulting from long periods of maternal separation seems likely to be a critical variable in understanding how this procedure increases behavioral and HPA responses to stress. But does this imply that, under normal conditions, maternal care actively contributes to the development of neural systems that mediate stress responses, or simply that the absence of the mother is so disruptive to pup physiology that it affects the development of these systems? If maternal care is relevant, then what are the relevant features of mother-pup interactions, and how do they influence neural development?

Critical features of early life events

Handling, although a brief interlude in the routine of mother-pup interactions, does alter the behavior of the mother towards the offspring (Bell et al., 1971; Lee and Williams, 1974). Overall, mothers of handled pups spend the same amount of time with their litters as mothers of non-handled pups; however, mothers of handled litters spend significantly more time licking/grooming their pups (Lee and Williams, 1974; Liu et al., 1997). A question, then, is whether this altered pattern of maternal behavior serves as a critical stimulus for the environmental effects on the development of endocrine and behavioral responses to stress. Interestingly, there are substantial, naturally occurring variations in maternal licking/grooming in rat dams. Maternal licking/grooming of pups occurs most frequently prior to or during periods where the mother nurses her young in the arched-back position. As you might imagine, the frequency of these two behaviors is closely correlated (r = +0.91; Liu et al., 1997) across mothers. Thus, it is feasible to characterize mothers as either High or Low on licking/grooming and arched-back nursing (LGABN). Such naturally occurring variations were first described by Myers and colleagues (Myers et al., 1989) using behavioral observations of mothers with their pups in the home cages. Interestingly, these individuals differences are stable across multiple litters (Francis et al., 1999). In one series of studies, mothers were divided into two groups, High LG-ABN or Low LG-ABN, on the basis of behavioral observations performed over the first 10 days of life (6-8 h of observation per day). It is important to note that there were no differences between these groups in the overall amount of time in contact with pups (Liu et al., 1997; Caldji et al., 1998; Francis et al., 2000a). It was hypothesized that if the handling-induced differences in licking/grooming or arched-back nursing are relevant for the effects of handling on

291 HPA development, then the offspring of High LGABN mothers should resemble the handled animals. This is exactly what was found (Liu et al., 1997). As adults, the offspring of High LG-ABN mothers showed reduced plasma ACTH and corticosterone responses to restraint stress. These animals also showed significantly increased hippocampal glucocorticoid receptor messenger ribonucleic acid (mRNA) expression, enhanced glucocorticoid negative feedback sensitivity and decreased hypothalamic CRH mRNA levels. Moreover, the magnitude of the corticosterone response to acute stress was significantly correlated with the frequency of both maternal licking/grooming (r = -0.61) and archedback nursing (r = -0.64) during the first 10 days of life, as was the level of hippocampal glucocorticoid receptor mRNA and hypothalamic CRH mRNA expression (all r values > 0.70; Liu et al., 1997). In addition, we found that the adult offspring of Low LG-ABN mothers showed significantly increased noradrenergic responses to stress at the level of the PVN (Caldji et al., 1999). These studies suggest that the critical feature for the handling effect on HPA development involves the increase in maternal licking/grooming. The offspring of the High and Low LG-ABN mothers also differed in behavioral responses to novelty (Caldji et al., 1998). As adults, the offspring of the Low LG-ABN, showed increased startle responses, decreased open-field exploration, and longer latencies to eat food provided in a novel environment. These animals also showed increased CRH receptor levels in the locus coeruleus and decreased central benzodiazepine receptor levels in the basolateral and central nuclei of the amygdala, as well as in the locus coeruleus (Caldji et al., 1998) and increased CRH mRNA expression in the central amygdala (D.D. Francis, J. Diorio and M.J. Meaney, unpublished data). These differences map perfectly onto the differences in handled and non-handled animals, and provide support for the idea that the effects of handling are mediated by changes in maternal behavior. It may be surprising that apparently subtle variations in maternal behavior have such profound impact on infant development. However, for a rat pup, the first weeks of life do not hold a great deal of stimulus diversity. Stability is the theme of the burrow, and the social environment in the first days of life is defined by the mother and littermates. The mother, then, serves as a primary link between the environment and the developing animal. It seems reasonable that variations in mother-pup interaction would serve to carry so much importance for development.
Transmission of individual differences in maternal care to the offspring

Interestingly, individual differences in maternal behavior show intergenerational transmission. The female offspring of High LG-ABN mothers showed significantly more licking/grooming and archedback nursing than did the female offspring of Low LG-ABN mothers (Francis et al., 1999). This intergenerational transmission of parental behavior has also been reported in primates. In rhesus monkeys there is clear evidence for family lineages expressing child abuse (Maestripieri et al., 1997). There is also evidence for transmission of individual differences in parental styles falling within the normal range. In studies of vervet monkeys, Fairbanks (1996) found that daughters who were reared by mothers which consistently spent a higher amount of time in physical contact with their offspring, became mothers who were similarly more attentive to their offspring. In rhesus monkeys, Berman (1990) found that the rate of rejecting the infant by mothers was correlated with the rejection rate of their mothers. In primates, such individual differences in maternal behavior may also be revealed in juvenile, nulliparous females. Thus, amongst juvenile female vervet monkeys, time spent in proximity to non-related infants was associated with the maternal behavior of their mothers (Meaney et al., 1991). In all cases these findings were independent of the social rank of the mother. Equally impressive findings exist in humans, where Miller et al. (1997) found that scores on measures of parental bonding between a mother and her daughter were highly correlated with the same measures of bonding between the daughter and her child. These findings suggest perhaps a common process of intergenerational transmission of maternal behavior. The critical question then concerns the mechanism underlying this intergenerational transmission of individual differences in behavior.

292 While genomic factors are important for some aspects of maternal behavior (see Keverne, 2001, this volume), we have provided evidence for a non-genomic transmission of individual differences in maternal behavior (Francis et al., 1999). In one study, we performed reciprocal cross-fostering of the offspring of Low LG-ABN and High LG-ABN mothers. A major concern in undertaking this was that the wholesale fostering of litters between mothers is known to affect maternal behavior (Maccari et al., 1995). In order to avert this problem and maintain the original character of the host litter, no more than two of twelve pups were fostered into or from any one litter (McCarty and Lee, 1996). In interpreting this study the critical groups of interest were the biological offspring of Low LG-ABN mothers fostered onto High LG-ABN dams, and vice versa. The control groups included: (1) the offspring of Low LG-ABN fostered onto other Low LG-ABN, as well as offspring of High LG-ABN dams fostered onto other High LG-ABN mothers; (2) sham-adoption animals, which were simply removed from the nest and fostered back to their biological mothers; and (3) unmanipulated pups. The limited cross-fostering design did not result in any effect on maternal differences in maternal behavior, i.e. the frequency of pup licking/grooming and arched-back nursing across all groups of High LG-ABN mothers was significantly higher than that for any of the Low LG-ABN dams regardless of litter composition. The results of the behavioral studies are consistent with the idea that the variations in maternal care are causally related to individual differences in the behavior of the offspring. The biological offspring of Low LG-ABN dams reared by High LG-ABN mothers were significantly less fearful under conditions of novelty than were any of the offspring reared by Low LG-ABN mothers, including the biological offspring of High LG-ABN mothers. A separate group of female offspring were then mated, allowed to give birth and observed for differences in maternal behavior. The effect on maternal behavior followed the same pattern as that for differences in fearfulness. As adults, the female offspring of Low LG-ABN dams reared by High LG-ABN mothers did not differ from normal High LG-ABN offspring in their frequency of pup licking/grooming or arched-back nursing. The frequency of licking/grooming and arched-back nursing in animals reared by High LG-ABN mothers was significantly higher than in any of the Low LGABN groups and, again, this included female pups originally born to High LG-ABN mothers, but reared by Low LG-ABN dams. Thus, individual differences in both fearfulness and maternal behavior mapped onto those of the mother who reared them, rather than the biological mother. A second series of studies was designed to examine the intergenerational effects of an 'early intervention' program. As already stated, handling increases maternal licking/grooming and arched-back nursing. Handling of pups, in fact, turns Low LG-ABN dams into High LG-ABN mothers (Francis et al., 1999). As adults, the handled offspring of Low LG-ABN mothers resemble the untouched offspring of High LG-ABN mothers, a finding that is consistent with the non-genomic transmission hypothesis. We then studied the F2 generation, focusing on the Handled and Non-Handled offspring of Low LG-ABN mothers. These mothers were completely unmanipulated. Note that these mothers are referred to as Low LGABN because they are derived from Low LG-ABN mothers themselves. However, the Low LG-ABN mothers with handled pups behave in manner that is indistinguishable from High LG-ABN dams. Importantly, their female offspring (F2 animals) also behave as High LG-ABN. As shown by Francis et al. (1999), the F2 offspring of handled/Low LG-ABN mothers resemble the offspring of High LG-ABN dams on measures of hypothalamic CRH and hippocampal glucocorticoid receptor mRNA expression, as well as central benzodiazepine receptor binding. These findings suggest that individual differences in gene expression in brain regions that regulate behavioral and endocrine responses to stress can also be transmitted across generations via a non-genomic mechanism. These findings are consistent with the results of studies using the cross-fostering technique as a test for maternal-mediation hypotheses. For example, the spontaneously hypertensive rat (SHR) is a strain bred for hypertension which appears in adolescence. While the selective breeding suggests a genetic background to this condition, the expression of the hypertensive trait is also influenced by epigenetic factors (McCarty et al., 1992). Pups of spontaneously hypertensive dams reared by wild-type (Wistar Kyoto

293 - WKY) mothers do exhibit hypertension to the extent of kin reared by spontaneously hypertensive dams. However, when borderline hypertensive rats, a hybrid formed by S H R - W K Y matings, are reared by WKY mothers, they do not express the spontaneous hypertensive phenotype. The potential effects of maternal behavior is also seen on the development of behavioral and endocrine responses to stress in BALB/c mice. The BALB/c is normally a strain that is very fearful and shows elevated HPA responses to stress compared to the C57 strain. However, BALB/c mice cross-fostered to C57 mothers are significantly less fearful, with lower HPA responses to stress (Zaharia et al., 1996). Importantly, C57 mothers normally lick and groom their pups about twice as frequently as BALB/c mothers (Anisman et al., 1998). Comparable findings have emerged with rat strains. Typically Fisher 344 rats are more responsive to novelty and have increased HPA responses to acute stress by comparison to Long-Evans rats. Moore and Lux (1998) reported that Long-Evans dams tick/groom their offspring significantly more often than do Fisher 344 mothers. Under normal circumstances, of course, BALB/c mice are reared by BALB/c mothers. In this case the genetic and environmental factors conspire to produce an excessively fearful animal. This is usually the reality of nature and nurture. Genetic and environmental factors work in concert, and are often correlated (see Scarr and McCartney, 1983). Because parents provide both the genes and the environment for their biological offspring, the offspring's environment is, in part, correlated with their genes. The genes of the offspring are correlated with those of the parents, and the genes of the parents influence the environment they provide for the offspring. The reason why many epidemiological studies based on linear regression models often find that epigenetic factors, such as parental care, do not add predictive value above that of genetic inheritance is because of this correlation. The environment the parent provides commonly serves to enhance the genetic differences; in a sense they are redundant mechanisms. In the case of BALB/c mice, knowledge of its pedigree is sufficient to predict a high level of timidity in adulthood. Additional information on maternal care would statistically add little to the predictability as the two factors work in the same direction. But this is clearly different from concluding that the maternal care is not relevant, and the results of the crossfostering studies attest to the importance of such epigenetic influences. The value of this inheritance process is that it can provide for variation. If the genetically determined trajectory is not adaptive for the animal, then development can move in the direction of the current environmental signal (which would be of adaptive value). Hence, environmental events can alter the path of the genetically established trajectory in favor of more adaptive outcomes. This, of course, is the adaptive value of plasticity. In our minds, these are adaptive processes. Children inherit not only genes from their parents, but also an environment (Eisenberg, 1990): Englishmen inherit England, as Francis Galton remarked. We believe that the transmission of individual differences in stress reactivity from mother to offspring can provide an adaptive level of 'preparedness' for the offspring. Under conditions of increased environmental demand, it is commonly in the animal's interest to enhance behavioral (e.g. vigilance, fearfulness) and endocrine (HPA and metabolic/cardiovascular) responsivity to stress. These responses promote the detection of any potential threat, the learning of avoidance, and the mobilization of energy reserves that are essential under the increased demands of the stressor. Since the offspring usually inhabit a niche that is similar to their parents, the adaptive transmission of these traits from parent to offspring could serve to be adaptive. In this context it is understandable that parents inhabiting a very demanding environmental niche might 'transmit' a high level of stress reactivity to their offspring.

Maternal responsivity in High and Low LG-ABN mothers

Two important questions concerning this non-genomic inheritance are, what is the neural basis for these individual differences in maternal behavior, and what mechanisms underlie this apparent transmission of parental behavior from one generation to the next? We believe that these questions can, to some degree, be addressed in non-human populations and the focus of our work lies in the findings of Allison Fleming (Fleming et al., 1998) showing a

294 direct relationship between fearfulness and maternal behavior. In the rat, maternal behavior emerges as a resolution of an interesting conflict between attraction and repulsion of pups (Rosenblatt, 1994). Female rats, unless they are in late pregnancy or lactating, exhibit a fearful, neophobic reaction to pups. Habituation through continuous exposure to pups renders females more likely to exhibit maternal behavior (a process sometimes called concaveation). In the classic behavioral test for 'maternal responsivity' virgin females are exposed continuously to pups of 3-6 days of age (Bridges, 1994; Stern, 1997). Following a number of days most females begin to show active care of the pups including crouching over the pups in a nursing posture and licking/grooming. Thus habituation through continuous exposure to pups renders females less neophobic, and more likely to exhibit maternal behavior. In general, procedures that reduce fearfulness, including amygdaloid lesions, enhance maternal responsivity, reducing the amount of time taken for females to exhibit maternal behavior (Fleming et al., 1989). Such findings may apply to the human condition. Fleming and Corter (1988) reported that many factors contribute to the quality of the mother's attitude towards her newborn, but none are correlated more highly than the women's level of anxiety. More anxious or depressed mothers are, not surprisingly, less positive towards their baby (also see Field, 1998). Behaviorally, more fearful mothers, such as the Low LG-ABN dams, appear to be less maternally responsive towards their offspring. Considering the differences in fearfulness in the female offspring of High and Low LG-ABN, we expected to see differences in the maternal responsivity test in these animals. This was exactly what occurred (E Champagne and M.J. Meaney, unpublished data). The virgin female offspring of High LG-ABN mothers exhibited the full pattern of maternal behavior in about one-half the exposure time compared to the offspring of Low LG-ABN (4.4 vs 8.9 days exposure). These findings suggest that naturally occurring variations in maternal care are reflected in differences in the maternal responsivity test. Moreover, variations in maternal responsivity in the female offspring of High and Low LG-ABN mothers are apparent even in nulliparous animals. If naturally occurring variations in maternal care are associated with differences in matemal responsivity, then we should be able to screen a population of nulliparous females with the pup sensitization paradigm and use the data on individual differences in the latency to express maternal behavior to predict variations in actual matemal care. This admittedly obvious hypothesis has, to the best of our knowledge, never actually been tested. This seems surprising considering the degree to which our knowledge of the neural basis of maternal behavior rests on the use of the pup-sensitization paradigm. Results of this study showed that the frequency of licking/grooming over the first 10 days post-partum in primiparous females was highly correlated to the latency in which females exhibited maternal behavior in the maternal responsivity test (Champagne and Meaney, unpublished). Thus, interindividual differences in neural mechanisms regulating maternal behavior can be detected either with natural or induced behaviors. Neural basis for individual differences in maternal behavior The onset of maternal care in the rat is mediated by hormonal events prior to and during parturition (Bridges, 1994; Fleming et al., 1998), including critical variations in circulating levels of progesterone and estrogen. Estrogen acts at the level of the medial preoptic area to enhance maternal behavior (Rosenblatt, 1994). The medial preoptic area is also a site of action for the effects placental lactogens, including prolactin, on maternal behavior (Bridges, 1994). The influence of ovarian hormones on maternal behavior in the rat is mediated, in part, by effects on central oxytocin systems (Pedersen, 1995). Estrogen induces oxytocin receptor gene expression De Kloet et al., 1986; Krrmarik et al., 1995; Young et al., 1997), and intracerebroventricular administration of oxytocin rapidly stimulates maternal behavior in virgin rats (Pedersen and Prange, 1979; Fahrbach et al., 1985). The effect of oxytocin is abolished by ovariectomy and reinstated with estrogen treatment. Moreover, treatment with oxytocin-antisera or receptor antagonists blocks the effects of ovarian steroid treatments on maternal behavior (Pedersen et al., 1985; Pedersen, 1995).

295 Oxytocin receptor levels are enriched in sites such as the medial preoptic area, the ventral tegmental area and the central amygdala, and increase following parturition in each of these regions (Insel, 1992; Young et al., 1997). Oxytocin infusion into the medial preoptic area or the ventral tegmental area increases the expression of maternal behavior (Pedersen and Prange, 1979; Fahrbach et al., 1985; Pedersen et al., 1985; Pedersen, 1995). Oxytocin neurons which project to the ventral tegmental area have been located in the ventral bed nucleus of the stria terminalis-lateral preoptic area as well as the PVN (Pedersen, 1995) and lesions of these areas inhibit maternal behavior (Numan, 1994). The ventral tegmental area is, of course, the source for the mesocorticolimbic dopamine system, and dopamine receptor blockers suppress the expression of pup licking/grooming (Stern and Taylor, 1991). Functionally, the onset of maternal behavior emerges from the decreased fearful response of the female to pups and an increase in the attraction of the mother to her pups (see Rosenblatt, 1994, Fleming and Corter, 1988 and Stern, 1997 for reviews). The positive cues associated with pups emerge from tactile, gustatory and auditory stimuli (Stern, 1997). Thus, pup stimuli can either be aversive, eliciting withdrawal, or positive, eliciting approach. The onset of maternal behavior clearly depends on decreasing the negative-withdrawal tendency, and increasing the positive-approach responses. For virgin females, pups elicit withdrawal and avoidance associated with odor cues transduced via both the vomeronasal and accessory olfactory bulb projections to the medial preoptic area. The vomeronasal projections arise via the amygdala. Thus, anosmic females are more readily maternal (Fleming, 1998) and lesions of the amygdala enhance maternal responsiveness in virgin females (Fleming et al., 1980; Numan, 1994). These findings suggest that the cues which elicit withdrawal are transmitted through the amygdala. Morgan et al. (1975) found that amygdaloid kindling, which enhances fearfulness in the rat, increases neophobia and decreases approaches to pup-related stimuli in virgin females. Additionally, oxytocin projections to the olfactory bulb may mediate a decrease in odor-induced fear responses to pups (Yu et al., 1996). What is particularly interesting to consider is the possibility that neural systems involved in the expression of fearfulness, notably the CRH systems, can directly influence maternal behavior. Pedersen et al. (1991) reported that central CRH infusions disrupt maternal behavior in the rat. Such effects of CRH could explain, at least in part, differences in the maternal behavior of High and Low LG-ABN mothers which differ in the expression of CRH mRNA (Francis et al., 2000a). In addition, there are differences between High and Low LG-ABN mothers in neural systems that have been found to mediate the expression of maternal behavior. We found significantly reduced oxytocin receptor levels in the central amygdala of Low LG-ABN mothers, as well as increased CRH receptor in this same region (Francis et al., 2000b). Such findings are apparent even in virgin animals and underscore the relationship between neural systems mediating fear and those involved in maternal behavior. We also found differences between High and Low LG-ABN mothers in oxytocin receptors in the medial preoptic area and the bed nuclei of the stria terminalis that were evident only during lactation (Francis et al., 2000b). These brain regions have been implicated in a limbic circuitry that activates maternal behavior in the rat (Insel, 1992; Numan, 1994). Individual differences in maternal care could, therefore, be derived from early environmental effects on the development of neural systems mediating fearfulness as well as those involved in mediating the attraction of females towards pups. Both may be, in part, associated with differences in oxytocin receptor gene expression. The net effect are differences in maternal responsivity between High and Low LGABN mothers. These effects, in turn, provide the basis for stable individual differences in stress reactivity and maternal behavior in the offspring. This hypothesis could account, at least in part, for the stable transmission of individual differences in maternal behavior in the rat.

Environmental regulation of maternal behavior

A critical issue here is the relationship between the environment of the mother and the nature of her behavior towards here offspring. We propose that such individual differences are, in turn, functionally

296

Low levels of resources High predation Social turmoil, isolation, etc.

Environmental conditions

Maternal Care

Developmental signal F1

Neural Development

1" CRH geneexpression $ GR levels

~" Oxytocin receptor levels ? Dopamine

,I, GABAA/CBZ receptor levels

Developmental consequence for F1 and developmental signal for F2

endocrinereactivity to stress

Increased behavioral /

Maternal behavior of F1

II
Neural development of next generation

Developmental consequence for F2

Fig. 1. A schematic representing the potential outcomes of the proposed relationship between environmentaladversity and infant care. The key feature of this formulation is the hypothesized relationship between fearfulness (i.e. reactivity to stress) and maternal behavior. Thus, variations in maternal care affect the developmentof neural systems that mediate stress reactivity, which may then influence the development of the subsequent generation and thus provide a basis for the transmissionof individualdifferences in stress reactivity from one generation to the next. F1, first generation;F2, second generation.

related to the level of environmental demand which confronts the animal. Under natural conditions, and the sanctity of the burrow, rat pups have little direct experience with the environment. Instead, conditions such as the scarcity of food, social instability, low

dominance status, etc., directly affect the status of the mother and, thus, the quality of maternal care. The effects of these environmental challenges on the development of the pups are then mediated by alterations in maternal care which serves to transduce

297 an environmental signal to the pups (Fig. 1). The environmentally driven alterations in maternal care then influence the development of neural systems that mediate behavioral and HPA responses to stress. These effects can, thus, serve to increase or decrease stress reactivity in the offspring. We suggest that more fearful, anxious animals (such as the Low LG-ABN mothers) are, therefore, more neophobic and lower in maternal responsivity to pups than are less fearful animals. Hence, these effects serve as the basis for comparable patterns of maternal behavior in the offspring, and for the transmission of these traits to the subsequent generation. A critical assumption in this hypothesis is that variations in parental behavior are related to the level of environmental demand. Human research suggests that the social, emotional and socioeconomic context are overriding determinants of the quality of the relationship between parent and child (Eisenberg, 1990). Human parental care is disturbed under conditions of chronic stress. Conditions which most commonly characterize abusive and neglectful homes involve economic hardship, martial strife and a lack of social and emotional support (Eisenberg, 1990). Such homes, in turn, breed neglectful parents, such that individual differences in parental behavior are reliably transmitted across generations. While this analysis may seem to be a parental indictment it is important to note that these same environments are also associated with considerable anxiety and depression. It is important to note that under a high level of environmental demand, increased fearfulness and hypervigilance might well be considered as adaptive. Of course, increased stress reactivity is also associated with enhanced vulnerability to stress-induced illness. Since, individual differences in parental care can influence the development of stress reactivity and, therefore, vulnerability for chronic illness in later life, vulnerability for chronic illness is also transmitted across generations. The assumption here is that variations in parental behavior reflect environmental demand. Perhaps the most compelling evidence for this process emerges from the studies of Rosenblum et al. (1994). Bonnet macaque mother-infant dyads were maintained under one of three foraging conditions: Low Foraging Demand, where food was readily available; High Foraging Demand, where ample food was available, but required long periods of searching; and Variable Foraging Demand, a mixture of the two conditions on a schedule that did not allow for predictability. At the time that these conditions were imposed, there were no differences in the nature of mother-infant interactions. However, following a number of months of these conditions there were highly significant differences in mother-infant interactions. The Variable Foraging Demand condition was clearly the most disruptive, and mother-infant conflict increased in this condition. Infants of mothers housed under these conditions were significantly more timid and fearful. These infants showed signs of depression commonly observed in maternally separated macaque infants, remarkably even while the infants were in contact with their mothers. As adolescents, the infants reared in the Variable Foraging Demand conditions were more fearful, submissive and showed less social play behavior. More recent studies have demonstrated the effects of these conditions on the development of neurobiological systems that mediate the organisms behavioral and endocrine/metabolic response to stress. Coplan et al. (1996, 1998) showed that, as adults, monkeys reared under Variable Foraging Demand conditions showed increased cerebrospinal fluid levels of CRH. Increased central CRH drive would suggest altered noradrenergic and serotonergic responses to stress, and this is exactly what was seen in adolescent animals reared under Variable Foraging Demand (Coplan et al., 1998). Predictably, these animals show increased fearfulness. We would predict that if the environmental conditions remained stable these differences would, in turn, be transmitted to the offspring (Fig. 1). These studies underscore two critically important points. First, variations in maternal care falling within the normal range of the species can have a profound influence on offspring development. One does not need to appeal to the more extreme conditions of abuse and neglect to see evidence for the importance of parental care. Second, environmental demands can alter parental care, and thus infant development. Indeed, we hypothesize that environmentally induced alterations in maternal care mediate the effect of variations in the early postnatal environment on the development of specific neural systems that mediate the development of fearfulness.

298 Such individual differences in fearfulness, in turn, influence the parental care of the offspring, providing a neurobiological basis for the intergenerational transmission of specific behavioral traits.
Conclusions

CRH GABA HPA LG-ABN mRNA PVN SHR WKY

Biologists have defined how parental behavior, as a component of reproduction, emerges as an adaptation to habitat. In large measure the variations studied here are those occurring between species. In each case we have come to expect that the pattern of parental care of a species can be understood, in part, by the demands placed upon the animal by its environmental context. And yet only in anthropology and sociology do we find a similar level of understanding applied to intra-species variations in parental behavior. There are notable exceptions (e.g. Altmann, 1980) but, in general, we have little understanding of how environmental factors, including socioeconomic forces, influence child care. It is clear from the studies of Conger et al. (1994) and Mclloyd (1998) that the effects of poverty on child development are mediated through changes in parenting: poverty influences parental style which, in turn, affects development. Anthropologists and evolutionary biologists have shown that variations in family organization and maternal behavior can be meaningfully understood in terms of prevailing economic factors (Reiter, 1975; Draper and Cashdan, 1988; Fairbanks, 1995; Blaffer Hrdy, 1999). It is clear that maternal behavior is not the inevitable, invariant outcome of pregnancy and birth (Altmann, 1980; Blaffer Hrdy, 1999). Maternal behavior occurs within a context, and involves a constant balance between the immediate needs of the parent and those of the offspring. The socioeconomic environment greatly determines the tenor of this balance. The studies of Rosenblum and colleagues have shown that the relationship between socioeconomic factors and parental behavior can be examined in other species, and such studies provide a remarkably rich and socially important topic of study for neurobiology.
Abbreviations

corticotropin releasing hormone gamma aminobutyric acid hypothalamo-pituitary-adrenal licking/grooming and arched back nursing messenger ribonucleic acid paraventricular hypothalamic nucleus spontaneously hypertensive rat Wistar Kyoto wild type rats

References
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Progressin BrainResearch, Vol. 133 2091 Elsevier Science B.V. All rights reserved

CHAPTER 22

Pregnancy and post partum: changes in cognition and mood

J. Galen Buckwalter 1,,, Deborah K. Buckwalter 2, Brendon W. Bluestein 2 and Frank Z. Stanczyk 3
1 Andrus Gerontology Center, University of Southern California, Los Angeles, CA 90089, USA 2 Fuller Graduate School of Psychology, Pasadena, CA 91182, USA 3 Department of Obstetrics and Gynecology, University of Southern California, Los Angeles, CA 90089, USA

Abstract: Steroidal hormones are increasingly recognized as highly relevant in multiple aspects of brain functioning. While basic science has actively worked to advance understanding of fundamental steroid mechanisms within the brain, investigation of the neurobehavioral outcomes of reproductive hormone actions on the human brain has received less attention. We argue that the dramatic steroidal hormone changes seen in human reproduction must be systematically studied and may provide novel explanations of cognitive and mood disorders associated with reproductive events. This chapter provides a review of current literature establishing a role for a variety of steroids on neuroactivity, and evidence from a variety of observational and experimental paradigms linking hormones and clinical aspects of cognition and mood in humans. The specific hormonal changes of pregnancy are described and discussed in relation to concomitant alterations in cognition and mood across the peri-natal period. A review of studies that have systematically observed cognitive and affective changes both during pregnancy and the post-partum period is presented, as well as new data that follow a small cohort of women for an extended period of time after delivery. We conclude that women may show specific areas of cognitive changes during and after pregnancy, notably deficits in verbal learning and memory. Mood appears to be impacted as well. While steroidal hormones show a pattern of associations with mood during and after pregnancy, no such pattern is evident for cognition. The embryonic state of our knowledge regarding reproductive hormones and neurobehavioral functioning is evident, as are the scientific and public health reasons to redress this lacuna.

Introduction Interest in the effects of a variety of reproductive states on brain functioning has dramatically increased in recent years. Much of this work has arisen from findings suggesting that steroidal hormones have not only organizational effects (Hutchison and Beyer, 1994) on the brain during early development but also activational effects (Arnold and Gorski, 1984) across the lifespan. Most salient among such

* Corresponding author: J.G. Buckwalter, Kaiser Permanente Medical Group, Research and Evaluation, 100 S. Los Robles, 2nd Floor, Pasadena, CA 91101, USA. Tel.: +1-626-564-5535; Fax: +1-626-564-3430; E-mail: galen.x.buckwalter @kp.org

studies are those suggesting that the use of estrogen replacement therapy (ERT) in the post-menopause may lessen the risk of Alzheimer's disease (Paganini-Hill et al., 1993; Henderson et al., 1994). Given such possibly profound effects of steroids on brain functioning, even in advanced age, the study of the effects of hormonal exposures across the lifespan becomes highly relevant. This chapter will (1) review the current literature establishing a role for a variety of steroids on brain functioning, (2) link these effects to clinically relevant aspects of cognition and mood in humans, (3) discuss the pattern of changes in steroid levels during pregnancy and the post-partum period, (4) review studies that have evaluated changes in cognition and mood across this time period, and (5) discuss the possible role of steroids in such changes. While the present chapter focuses on

304 the role of steroids, this reflects the interests of the authors and is not suggested to be a comprehensive discussion of all factors (e.g., peptides, growth factors) that may be relevant to the understanding of pregnancy-induced changes in cognition and mood. ing neural circuitry of the cerebral cortex (McEwen and Brinton, 1987) as well as in lateralization of functioning (McManus and Bryden, 1991). McEwen (1992) describes effects of steroid hormones on brain development and function at two levels. Intracellular steroid hormone receptors, which are DNA-binding proteins, are active at the level of regulation of gene transcription. Other effects occur at the membrane level by means of cell surface receptors that generate rapid effects on electrical activity and secondary messenger systems (McEwen, 1992). The synthesis of specific neurosteroids in many sites in the brain suggests that steroid hormones may have diverse functions, including modulation of affect and cognition via influence on neurotransmitter mechanisms (Halbreich et al., 1992). Substantial evidence associates steroids and neuroactivity (neuronal transmission or trophism) in the rodent brain. Neurotransmission in critical brain areas related to behavior and cognitive functioning has been demonstrated to correspond with changes in availability of estradiol (Gould et al., 1991), testosterone (Teyler et al., 1980), progesterone (Jussofie, 1993) and DHEA (Baulieu and Robel, 1996). The actions of several neurotransmitters are enhanced or preserved in the presence of estradiol, including dopamine in the hippocampus (Joseph et al., 1989), norepinephrine in the hypothalamus (Best et al., 1992) and acetylcholine in the frontal cortex and hippocampus (Menard and Dohanich, 1994; Eichenbaum, 1996). Effects on acetylcholine are particularly relevant to the study of cognition, given its clear role in memory. DHEA and DHEA-S also appear to have a critical role in neurotransmission. The inhibitory neurotransmitter y-aminobutyric acid (GABA), which is widely distributed through out the brain, has roles in behavior and learning. DHEA, like other 3f3-hydroxysteroids, modulates GABA neurotransmission (Demirgoren et al., 1991) in areas related to memory, particularly in the hippocampus (Steffensen, 1995), where it increases activity in the CA1 subfield (Meyer and Gruol, 1994) and in the dentate gyrus (Yoo et al., 1996). Evidence also supports neurotrophic effects (dendritic growth and/or increased synaptic density) of these neuroactive steroids. Axonal sprouting (Loy and Milner, 1980; Morse et al., 1992) and dendritic growth, as well as synaptogenesis (Matsumoto and

Steroid hormones and brain functioning

Steroid hormones have a basic 4-ring chemical structure and are classified according to the number of carbon atoms they possess. Androgens have 19-carbons, while estrogens are 18-carbon steroids (Speroff et al., 1994; Stanczyk, 1997). All steroid hormones are synthesized from pregnenolone that has been converted from the precursor cholesterol in the mitochondria. The conversion of cholesterol to pregnenolone is stimulated by anterior pituitary hormones, specifically luteinizing hormone in the ovary and testis, and adrenocorticotropic hormone in the adrenal cortex (Findling et al., 1997). Androgens and estrogens are synthesized mainly in endocrine glands, specifically the gonads and adrenals and by the fetoplacental unit. They are also synthesized in much smaller quantities in the nervous system and other non-endocrine (peripheral) tissues, primarily the liver, kidney, adipose tissue and skin (Lingappa and Mellon, 1997). The human fetus and placenta act jointly to produce estrogens. Although the placenta is able to synthesize substantial amounts of progesterone from pregnenolone, it cannot form androgens, the precursors of estrogens. Also, the fetus cannot convert androgens into estrogens. Rather, both maternal and fetal adrenals produce dehydroepiandrosterone sulfate (DHEA-S) from which estrone and estradiol are formed in the placenta. DHEA-S is first converted to dehydroepiandrosterone (DHEA) and then convetted to androstenedione, which undergoes placental aromatization, forming estrone, which can be converted to estradiol. Another estrogen, estriol, is also formed in the placenta from 16c~-hydroxy-DHEA-S, which originates primarily in the fetal liver (Taylor and Martin, 1997). Steroid hormones clearly impact the brain with respect to both neural organization and the functioning of neural substrates (McEwen, 1992). During fetal and early childhood development, gonadal hormones are involved in the critical processes organiz-

305 Arai, 1981; Gould et al., 1991), have been observed in the hippocampus and hypothalamus in the presence of endogenous or exogenous steroids. Specifically, axonal sprouting in the hippocampal dentate gyms occurs in the presence of estradiol and testosterone (Loy and Milner, 1980; Morse et al., 1992). Estradiol and progesterone act in the hippocampal CA1 subfield to increase dendritic growth (Gould et al., t990) and synaptic density (Gould et al., 1991). The hypothalamic arcuate nucleus and the ventromedial nucleus appear to be highly reactive to the presence of estradiol (Matsumoto and Arai, 1981). Estradiol markedly increases the numbers of dendritic shafts and spine synapses in the arcuate nucleus and restores structural density in rodents with brain lesions. Estradiol also promotes synaptogenesis in the ventromedial nucleus of the hypothalamus (Jones et al., 1985), an effect that is augmented by the presence of progesterone (Meisel and Luttrell, 1990). Steroidal hormones interact with monoamines that have been widely associated with affect regulation (Carlson, 1994) including serotonin (5-HT) (Cone et al., 1981; Sumner and Fink, 1995), dopamine (McEwen and Parsons, 1982), epinephrine and norepinephrine (McEwen and Parsons, 1982; Halbreich, 1990). Cone et al. (1981) reported that administration of estradiol and sequential administration of estradiol and progesterone to rat brain increases serotonin levels and accumulation rates. Steroidal hormones also exert effects on amino acid neurotransmitter functions, primarily on GABA (Paul and Purdy, 1992; Bixo et al., 1995; see Herbison, 2001, this volume). Such relationships are important to consider in view of the recognized role of GABA in the modulation of emotions and behavior and in the etiology of some affective disorders (Berretini and Post, 1984). GABA's function in depressive illness is indicated by observations that GABA levels are low in the cerebrospinal fluid of depressed patients (Majewska, 1992) suggesting that depression may be associated with decreased GABA-ergic neurotransmission. GABA agonists have been utilized in the successful treatment of both depression (Hollister et al., 1980) and anxiety (Gasior et al., 1999). Progesterone has a wide range of metabolic effects such as generating changes in protein, carbohydrate and lipid metabolism (Goldfien and Monroe, 1997). Progesterone also has neural effects, acting in conjunction with estrogen in expanding dendritic spines (Woolley and Gould, 1989) and in conjunction with DHEA in regulating the balance between excitatory and inhibitory systems (Paul and Purdy, 1992). In addition, the possible effects of progesterone, or its metabolites, on mood-related neuronal structures and processes have been demonstrated in its binding to membrane receptors for neurotransmitters (Chadwick and Widdows, 1990), and in particular potentiating inhibitory effects of GABA, by enhancing its binding capability (Majewska, 1992; Paul and Purdy, 1992). While the effects of androgens on neural functioning have received less attention than estrogen, animal studies have found that androgen receptors generally colocalize with estrogen receptors in the brain (Simerly et al., 1990). Androgen activity is involved in sexual differentiation of the brain (McEwen, 1992), as well as modulating neuronal activity that may influence behavior including cognitive abilities (Berenbaum et al., 1995), aggression (Uzych, 1992) and libido (Coope, 1996). Testosterone and its receptors are found concentrated in those areas of the brain involved in sex and emotions (McEwen, 1984). Utilizing animal studies, Morse et al. (1986) demonstrated a role of testosterone in synaptic growth in the hippocampus. Like DHEA, testosterone can be converted to estrogens, including in the brain (Stanczyk, 1997). Testosterone has been shown to have inhibitory effects on monoamine oxidase, a class of enzymes that destroys dopamine, norepinephrine and serotonin (Klaiber et al., 1967).
Evidence for a link between hormones, cognition and mood

Evidence from a variety of observational and experimental paradigms suggests that sex steroid actions on the brain can result in clinically relevant changes. Such paradigms include the study of gender, conditions in women associated with endogenous fluctuations in steroids, e.g., menstruation and menopause, and exogenous administration of hormones. As with rodents, estradiol, progesterone, testosterone and DHEA/DHEA-S appear to have

306 specific and often dose-dependent effects on cognition and mood. Higher levels of estradiol in women are associated with enhanced verbal abilities (Nyborg, 1984), including verbally based learning and memory (Phillips and Sherwin, 1992; Robinson et al., 1994), attention (Schmidt et al., 1998), verbal fluency (Barrett-Conner and Kritz-Silverstein, 1993) and articulation (Hampson, 1990a). Well-practiced tasks (Broverman et al., 1981) and increased motor response repetition (Hampson, 1990b) are also enhanced, along with creativity and divergent thinking (Krug et al., 1994). However, a rise in circulating estradiol concentrations also corresponds with poorer visual-spatial abilities (Hampson, 1990a). Increased blood concentration of progesterone is related to dysexecutive effects, including disinhibition (Keenan et al., 1992) and decreased processing of non-verbal and verbal information (Freeman et al., 1993). Although possibly an artifact of progesterone's anxiolytic properties, psychomotor speed is lowered with increasing progesterone concentrations (Freeman et al., 1993). On the other hand, progesterone has been associated with enhanced visual memory (Phillips and Sherwin, 1992). Testosterone concentration in blood is positively associated with spatial advantages in both men and women (Nass and Baker, 1991; Van Goozen et al., 1994). Enhanced mathematical ability is apparent in women with high testosterone levels (Gouchie and Kimura, 1991). Testosterone level is also associated with decrements in verbal memory (Van Goozen et al., 1994). Limited studies on cognition and DHEA/DHEA-S demonstrate that this steroid appears to enhance information processing speed, visual-spatial abilities, verbal memory (Wolkowitz et al., 1997), attention and category fluency (Buckwalter et al., 1998). DHEA and DHEA-S may enhance these domains, as well as providing a protective effect against organic brain disorders, secondary to conversion in the brain to androgens and estrogens (Leblhuber et al., 1992, 1995). Gender differences in some affective conditions, most notably depression, provide support for the relevance of hormonal involvement in mood states. A biological susceptibility hypothesis has been offered to explain the sex ratio in mood disturbances based on strong evidence for an association/interaction between the hypothalamic-pituitary-gonadal axis, female sex hormones and affective disorders (Steiner, 1992; Toren et al., 1996). According to Steiner (1992), the extremely complex, delicately integrated neuroendocrine system, related to the female reproductive cycle, is vulnerable to changes, not only physiological, but also psychosocial and environmental. Toren et al. (1996) add that a dysregulation of the hypothalamic-pituitary-ovarian axis activity, which may arise from any number of sources (e.g., hormonal changes), may play a significant role in mood regulation. This intricate balance may come into play as early as puberty for girls. Warren and Brooks-Gunn (1989) found a significant curvilinear trend for depressive affect during times of rapid increases in sex hormone levels of adolescent girls. Brace and McCauley (1997) identified a subgroup of women, at risk for negative psychological symptoms during periods of hormonal change that responded to stabilization of hormone levels by exhibiting improved psychological well being. Estrogen has been described as psychoprotectant in that its supplementation has been found to have both mood-elevating and anti-depressant properties (Klaiber et al., 1979; Studd, 1993). Sands and Studd (1995) discuss the necessity of balanced estrogen and testosterone for psychological well being. Montgomery et al. (1987) randomized peri- and post-menopausal women to receive placebo, estrogen alone, or an estrogen/testosterone combination. Although results were not significant, after 2 months, patients in the combined treatment group scored better on a self-rating scale of distress than the group receiving estrogen alone. In another controlled study, Sherwin (1988) found that surgically menopausal women who received both estradiol and testosterone reported more positive mood than both an untreated control group and women who were treated with estradiol alone. Testosterone in women has been traditionally investigated in its relationship to libido. However, a study by Baisher et al. (1993) found significantly higher plasma levels of testosterone in women diagnosed with major depression. Contradictions in the literature may indicate that the pattern of exposure to testosterone in women is not obvious and that more complex interactions and/or factors may be involved in testosterone's effect on mood.

307 Sex hormones have been associated with mood fluctuations during menarche (Bemstein et al., 1991), ovulation and menstruation (Bancroft, 1993), the special case of premenstrual syndrome (Backstrom et al., 1983; Halbreich et al., 1986) and the climacteric (Gerdes et al., 1982).
Steroid hormones and pregnancy

The hormonal changes of pregnancy are unprecedented among reproductive events in women. Rather than the expected decline in serum levels of estrogens and progesterone that usually occurs 10 to 12 days after ovulation in the normal menstrual cycle, with conception and embryonic implantation these hormone levels do not decrease (Taylor and Martin, 1997). Instead, production of these hormones is maintained and slightly increased for the first few weeks of pregnancy by the corpus luteum (Parker, 1993; Stanczyk, 1997). In conjunction with a variety of other hormonal changes, estrogen and progesterone increase dramatically, after the ninth week of gestation, when the placenta begins to secrete substantial amounts of these hormones. Serum levels of estradiol increase from 0.5 to 1 ng/ml in the first weeks of pregnancy (Parker, 1993), to an overall mean of 16 ng/ml at the end of pregnancy (Stanczyk, 1997). After the first few weeks of pregnancy, the main source of estrogen is that produced by interactions among the fetoplacental unit and adrenal steroidogenesis of the mother (Parker, 1993). As stated earlier, the fetal adrenal provides DHEA-S as a precursor for placental production of estrogen (Speroff et al., 1994). Just prior to parturition, estrogen secretion again increases (Buster and Abraham, 1975; Speroff et al., 1994), and contributes to enhancement of rhythmic uterine contractions, and the increased uterine sensitivity to oxytocin prior to commencement of labor (Speroff et al., 1994). Progesterone is produced in pregnancy by the ovary, specifically the corpus luteum, and the placenta (Speroff et al., 1994; Stanczyk, 1997). During the first 9 weeks, the corpus luteum predominates resulting in plasma progesterone levels ranging from 10 to 35 ng/ml (Stanczyk, 1997). At approximately the 10th week after ovulation, the placenta takes over the major responsibility for progesterone secretion resulting in a steady rise, until term plasma

levels ranging from 125 to 190 ng/ml are reached (Speroff et al., 1994; Stanczyk, 1997). Following parturition, serum levels of estrogen and progesterone are dramatically reduced with concentrations of estradiol close to menopausal levels (Petrakis et al., 1987). Nott et al. (1976) found that in a group of 27 pregnant women total estrogen decreased dramatically from a mean of 13.8 (4-5.6) ng/ml plasma in the 2 weeks prior to delivery to 0.6 (4-0.4) ng/ml on the first post-partum day, 0.21 (4-0.15) ng/ml on day 3 and 0.13 (4-0.07) ng/ml on day 5. Estrogen levels continued to decrease during the 2 weeks following parturition to a low level of 0.06 (4-0.03) ng/ml. Buckwalter et al. (1999a) reported levels of estradiol with a mean of 25.1 (4-8.2) ng/ml during the last trimester of pregnancy and a mean of 0.02 (4-0.01) ng/ml after delivery. Mean values for progesterone were 174.1 (4-33.6) ng/ml during pregnancy and 0.50 (4-0.88) ng/ml after delivery. Estrogen levels return to normal within a month after delivery if lactation does not occur. However, with lactation, estrogen levels remain low for as long as the mother is the sole source of nourishment for the infant (Petrakis et al., 1987). In normal pregnancy, circulating testosterone concentration increases from mid-menstrual cycle levels of approximately 30 ng/dl plasma to approximately 200 ng/dl (Boots, 1993), at least 5- to 10-fold higher (Stanczyk, 1997). Despite this dramatic increase in testosterone production during pregnancy, there is a notable absence of virilization in pregnant women (Demisch et al., 1968). Circulating sex-hormonebinding globulin (SHBG) concentrations increase concomitantly during pregnancy, which is likely to reduce bioavailability of testosterone (Pearlman and Crepy, 1967; Speroff et al., 1994). Bamman et al. (1980) also found that, although total testosterone levels increase throughout pregnancy, free circulating testosterone concentration is unchanged until week 28, when it is significantly elevated. Serum concentrations of testosterone remain at pregnancy levels immediately following delivery (Rivarola et al., 1968), but decrease by one-half between the 4th and 6th post-partum day (Demisch et al., 1968). In contrast to estrogens, progesterone and testosterone, maternal serum levels of DHEA and DHEA-S in pregnancy actually decrease (Rivarola et al., 1968) to about 30% to 50% of normal men-

308 strual cycle values (Stanczyk, 1997). Peter et al. (1994) report a decrease in DHEA-S level from 3.25 (+0.38) Ixg/ml in early gestation to a minimum of 1.50 (4-0.16) Ixg/ml at week 38. This decline is due to rapid metabolism by the placenta and fetal liver (Stanczyk, 1997) to produce estrogens (Rivarola et al., 1968). In comparison to maternal production, the fetal adrenal produces more than 200 mg of DHEA-S daily, about 10 times more than the mother (Speroff et al., 1994). Buckwalter et al. (1999a) reported circulating testosterone levels of 57.5 (4-23.1, s.d.) ng/dl during the last trimester of pregnancy and 24.6 (4-10.8) ng/dl after delivery. DHEA levels during pregnancy and following delivery were 4.5 (4-1.7) ng/ml and 2.5 (4-1.0) ng/ml, respectively. While post-partum levels of androgens were approximately half of those during the last trimester of pregnancy, the decreases in circulating androgen concentrations were not nearly as dramatic as the decreases in estradiol or progesterone following parturition (Buckwalter et al., 1999a). learning 2 and recall. A similar pattern was found for pregnant women in all trimesters of pregnancy. Eidelman et al. (1993) found that women in the third month of a high risk pregnancy performed worse than non-pregnant controls on the ability to recall a short passage of prose, but not on a visual memory task. In a separate group of women with normal pregnancies, deficits in both passage recall and visual memory were reported during the first post-partum day, but not during the second or third day. In a longitudinal study, Silber et al. (1990) found no cognitive differences between pregnant women and controls during pregnancy, in the immediate post partum or at the three-month testing. However, the women who had been pregnant showed significant increases in performance on word list learning and reaction 6 and 12 months post parturn. The authors interpret this as indicative of a peri-partal cognitive impairment. Similarly, Keenan et al. (1998) performed a longitudinal study with 10 pregnant women matched with 10 non-pregnant controls for age, education and estimated intelligence. Two tests of general cognitive ability were included to obtain estimates of IQ for matching. Paragraphs of narrative material were chosen from both the original and revised Wechsler Memory Scales and the California Discourse Memory Test to assess explicit, contextually mediated memory. A word stem completion task was administered to test implicit memory. The Zung Anxiety Scale and the Beck Depression Inventory assessed degree of anxiety and depression, respectively. A Likert scale 'Subjective Memory Questionnaire' was administered to evaluate perceived memory ability. Subjects were tested during the first, second and third trimester, and post partum. Circulating estradiol and progesterone levels were assayed at all four testing sessions. Whereas both immediate and delayed recall on the explicit memory task were worse in the pregnant group than in the control subjects, this difference was significant in the third trimester only. Similar results were not evident on the implicit memory task.

Pregnancy and cognition

Subjective reports of cognitive dysfunction during pregnancy have long been noted. Attention, memory and concentration problems have been reported in the early puerperium (Kane et al., 1968). Poser et al. (1986) analyzed the results of 66 women who completed a symptom checklist in each trimester of their full term pregnancy. Symptoms such as forgetfulness, disorientation, confusion and reading difficulties were common among pregnant professional women. Subjective memory complaints during pregnancy have also been associated with impairments in implicit memory 1 (Brindle et al., 1991). Only a few studies have systematically observed these cognitive changes during pregnancy. Sharp et al. (1993) revealed deficits in the recall of word lists in both primigravid and multigravid pregnant women, when compared to non-pregnant women. This deficit was greater for incidental versus explicit

l Implicit memory: procedural memory, or unconscious knowledge that is expressed in performance.

2Incidental learning: acquisition of information without effortful attention. Explicit learning: conscious and intentional acquisition of information.

309 The simultaneous evaluation of cognitive functioning and mood, which is essential to determine if cognitive changes are reflective of changes in mood, has not been conducted until quite recently. Morris et al. (1998) found that subjective reports of cognitive failures in the workplace were related significantly to mood, but not to pregnancy status. Buckwalter et al. (1999a) examined the effects of pregnancy on both cognition and mood. Nineteen women, average age 33, were tested with a comprehensive neuropsychological battery during their last month of pregnancy and again two to six weeks after delivery. Blood samples were obtained from all subjects and were assayed for a variety of steroids implicated in cognitive and mood functioning. Participants also completed several standardized selfreport measures of mood. The women demonstrated significantly more impairment, relative to normative groups, in aspects of verbal memory primarily during the last trimester of pregnancy, but also after delivery. During pregnancy, this group of highly educated women (average years of education = 16) performed at expected levels on tests of intelligence and on tests of simple attention. However, they performed well below expected normative levels on tests of psychomotor speed and, most notably, on aspects of verbal learning and memory. The average normative performance on aspects of verbal memory was below the 10th percentile for this group of generally high functioning women. Verbal learning was characterized by an inefficient, haphazard style, suggesting difficulty in processing large amounts of information. Deficits were also apparent on tasks requiring the ability to simultaneously track more than one piece of information. Performance in these areas, including semantic memory 3, was improved after delivery, but remained below normative values for age. Cognitive deficits were not explained by mood disturbances. No hormones assayed (estradiol, progesterone, DHEA and testosterone) consistently related to cognitive performance during pregnancy; however, positive associations between circulating estradiol and DHEA levels and aspects of cognitive performance emerged after delivery. These resuits strongly suggest a peri-partum memory deficit, which cannot be explained by the dramatic rise in the circulating hormones measured. The authors speculate that the pattern of cognitive deficits observed is consistent with a disruption of cholinergic functioning, suggested to mediate the processing of new information, in the brain (Hasselmo et al., 1996; Sarter and Bruno, 1997). However, research addressing possible cholinergic disruption during pregnancy is in its infancy. A study by Holdcroft (as cited in Moore, 1997) poses the plausibility of dramatic changes in brain plasticity associated with pregnancy. Ten women with normal pregnancy received a magnetic resonance image brain scan in the last 2 weeks of pregnancy, at 6 to 8 weeks post partum and 6 months post-delivery. Comparing ante-natal to the first postpartum scans revealed a significant increase in brain size in all women with additional enlargement noted between the second and third scans. Global changes in brain size, present in both cerebral hemispheres and also in the medulla and brainstem were found. The authors conclude that, while it is possible that the brain increases from its normal size after pregnancy, it is more feasible that the brain reduces in size in association with pregnancy. Such changes in the brain may have implications for fluctuations in mood in the peri-natal period, particularly if areas typically associated with mood (such as the prefrontal cortex and amygdala) are altered.

Pregnancy and mood

Mood during pregnancy has been investigated primarily in relation to post-natal blues and depression. Of investigators who have assessed mood during pregnancy, some found ante-natal depressed and anxious mood to predict post-partum affect (Cox et al., 1982; Saks et al., 1985), whereas others found little continuity from ante-natal to post-natal maternal affective conditions (Kumar and Robson, 1984; Watson et al., 1989). O'Hara et al. (1991) report that women who had higher levels of depressive symptoms during pregnancy and/or who had reported previous premenstrual depression, were at increased risk for post-partum negative affect. Watson et al. (1984) report that 27% of the 128 women in their pregnancy study experienced one or more periods

3 Semantic memory: the ability to recall overlearned, habituated information (e.g. the alphabet or common names).

310 of 'clinical disorder' during pregnancy and the postnatal period. While the majority of these disorders constituted depressed affect, there was one case of alcoholism and two cases of agoraphobia. The post-partum period is widely reported to be a time of increased risk for negative mood ranging from milder forms of dysphoria (Pritchard and Harris, 1996) to clinical depression and puerperal psychosis necessitating hospitalization and/or other treatment for a relatively small number of women (Cox et al., 1982; Gotlib et al., 1989; Harris, 1994; Pritchard and Harris, 1996). Mild to moderate depression is common among women through the first 3 months after childbirth (Pitt, 1973; Saks et al., 1985), with a prevalence rate of 30% to 70%, depending on the study and methodology (Pritchard and Harris, 1996), and a rate of onset approximately three times higher in the month following delivery than in non-gestational controls (O'Hara et al., 1990). Several studies report variable onset of depression both during pregnancy and following delivery. Estimates of negative symptomatology during pregnancy range from 28% (O'Hara et al., 1984) to 34% (Rees and Lutkins, 1971), whereas post-partum rates range from 22% (Hayworth et al., 1980) to 30% (Rees and Lutkins, 1971). While investigators have attempted to differentiate between post-partum negative mood as a continuation of ante-natal versus specific postnatal onset, results are inconsistent, possibly due to differences in assessment time frames (e.g., earlier vs. later in the post-partum period) and the use of different measures of depressed mood (Watson et al., 1984; Gotlib et al., 1989). The prospective study by Glover (1992) of pregnancy-related mood changes found different times of onset in both pregnancy and post partum. However, Nott et al. (1976) found that women who reported more mood disturbance before delivery also reported more post partum. While negative affective state has been investigated in relation to a number of peri-natal variables, post-natal depressed mood is commonly attributed to hormonal changes related to the peri-natal period. Nott et al. (1976) report a correlational trend for greater ante-natal irritability with higher circulating estrogen levels and a greater likelihood for subjects with the greatest decrease in progesterone level to rate themselves as depressed within 10 days postnatally. O'Hara et al. (1991) examined progesterone and mood from the second trimester of pregnancy to the 9th post-partum week but failed to find a similar correlation. However, this group found some support for the proposal that estrogen withdrawal is a cause of post-partum blues. Another prospective study of primiparous women, which assessed mood concurrently with circulating progesterone and cortisol concentrations 2 weeks prior to delivery and in the month following parturition, found that women with maternity blues had significantly higher ante-natal and lower post-natal progesterone concentrations than women without blues (Harris et al., 1994). In contrast, Heidrich et al. (1994) report no significant difference in estradiol or progesterone levels in women with and without post-partum blues. Nonetheless, estrogen has been used to successfully treat women who have experienced recurrent postpartum depression (Sichel et al., 1995; Gregoire et al., 1996). While studies investigating possible relationships between estrogen and progesterone levels and mood during and following pregnancy have been completed, there is a dearth of information regarding androgens and mood during pregnancy and post parturn. The study by Buckwalter et al. (1999a) reports correlations between circulating estradiol, progesterone, DHEA and testosterone and mood. During pregnancy, levels of progesterone were associated with greater mood disturbances while higher levels of DHEA corresponded with better mood. After delivery, testosterone concentration was strongly and consistently associated with greater reported mood disturbances, while a decline in DHEA after delivery was associated with an increase in mood disturbances. Certain aspects of mood, particularly negative thought pattems, were found to be more frequent during pregnancy than post partum. It appears that the dramatically different hormonal milieus of pregnancy and post partum have very different implications for mood. While a negative role for progesterone during pregnancy is consistent with previous literature, a positive role for DHEA during pregnancy and a strong negative role for testosterone in the post-partum period suggest that future research on peri-partal mood disturbances must include consideration of the possible role of androgens.

311
Long-term implications of pregnancy on cognition and m o o d

Long-term effects of pregnancy on mood and cognition have not been reported. In an effort to examine this issue, we have recently completed follow-up testing on a sub-sample (n = 11, average age = 34 years) of the women who participated in the Buckwalter et al. (1999a) study. None had experienced an intervening pregnancy. Evaluation procedures were identical to those used in the original study and were completed, on average, 2 years after delivery. All tests were conducted on the second or third day of the woman's menstrual cycle. In comparing neuropsychological results from testing 2 years after delivery (T3) with results during the last month of pregnancy (T1), performance at T3 evidenced improvements in simple attention and psychomotor speed. Improvements in verbal learning and memory were also observed; however, performance continued to be below expected normative values (Fig. 1). Women at T3 demonstrated a more complex learning strategy, i.e., they encoded words by utilizing semantic information. Semantic memory (the ability to recall over-learned information) was greatly improved from T1 to T3. From the post-partum period to 2 years later, there were iraPregnancy and Cognition 80 70 60 50

provements in complex attention and working memory 4. While the current results appear to support a long-term post-partum cognitive recovery of attention, working memory, psychomotor speed, semantic memory, episodic memory and the use of a more complex learning strategy, the improvements were not statistically independent of mood. In the original Buckwalter et al. (1999a) study, cognitive variation from pregnancy to the post partum was independent of mood. From the immediate post-partum period to two years post partum, improvement in cognition was not independent of an improvement in mood. This suggests that the mechanism for improvement in cognition from pregnancy (T1) to post partum (T2) may involve a neurotransmitter system that is not highly related to mood, while the long-term improvements seen from T1 and T2 to T3 may involve a system that is associated with mood. Changes in mood from Tl to T3 were dramatic. There were significant improvements in reported

4 Working memory: the temporary, limited capacity information manipulation and retrieval system. Requires attention as well the ability to manipulate the information stored in the short-term retrieval system.

Visual Perception 40 30 20 0 Verbal Learning Verbal Memory --I1-- Processing Speed

10
t i i

Pregnancy

1 mo postpartum

2 yr post partum

Fig. 1. Pregnancyand cognition.(Data from Buckwalteret al., 1999a; and from unpublished data.)

312
Pregnancy and Mood
70

60
5O
=m o

40
i

--O- Obsessive Compulsive -I:]-- Depression --k-Anxiety

Hostility --O-- Global

~ 3o
2O
10
! i i

Pregnancy

1 mo postpartum

2 yr postpartum

Fig. 2. Pregnancy and mood. (Data from Buckwalter et al., 1999a,b.)

levels of depression, somaticization, obsessive-compulsiveness, anxiety and a global index of mood disturbances (Fig. 2). From T2 to T3 there were significant improvements in depression, confusionbewilderment and hostility. The pattern of associations between circulating sex steroid levels and mood is particularly informative at T3. DHEA was negatively associated with depression, fatigue, somaticization, anxiety, hostility and psychoticism. There was a significant negative relationship of testosterone with fatigue. There were no significant associations for either estradiol or progesterone with any measures of mood. These findings suggest that pregnancy and the post-partum period have a unique impact on cognitive and affective attributes in the lives of women. Cognitive disturbances greatly improve 2 years after delivery; however, verbal episodic memory continues to be below expected normative levels. Negative mood exhibited during pregnancy and the post-parturn period appears to improve over time. Longitudinal clinical studies of mood extending beyond the immediate post-partum period are lacking. The sole long-term study (e.g., longer than 1 year postnatally) of emotional disorders related to pregnancy was accomplished by Kumar and Robson (1984). A prospective study of 119 primiparous mothers in-

corporated interviews at fixed intervals during their pregnancy and 1 year after delivery. A subset was followed when their children were 4 years of age. Although 'many' who had experienced first-time depression during or after pregnancy continued to experience psychological problems up to 4 years after birth, the study's 4-year follow-up was still in progress at the time of this writing and it is unclear how many of the original sample participated in the long-term follow-up. In contrast to our findings that mood improves over time following pregnancy, Kumar and Robson (1984) suggest that long-term emotional problems consequent on pregnancy may occur for a subset of women. These different conclusions are possibly accounted for by differences in time frame and/or the methods of assessment.
Conclusions

The role of steroid hormones in the cognitive and affective characteristics of pregnancy continues to be unclear, yet evocative. There is no consistent suggestion that steroid hormones impact upon cognition during pregnancy and minimal evidence that they play a large role post partum. It is possible that the very high levels of sex steroids during pregnancy have a negative effect on aspects of brain function-

313 ing that underlie cognitive performance. However, Buckwalter et al. (1999a) found no negative associations between circulating levels of these hormones and cognitive performance. This does not preclude the possibility of some threshold level for a specific hormone(s) above which the effects of the steroid(s) on the brain is uniformly negative. There is more consistent evidence that steroids play a role in the mood disturbances associated with pregnancy and the post-partum period. During pregnancy there appears to be a clear negative role for progesterone and a positive role for DHEA. In the post-partum period testosterone may play a strong role in negative mood states. Interestingly, while DHEA was not reported to have an association with mood post partum (Buckwalter et al., 1999a), the preliminary data we report here suggest that a positive role reemerges after the menstrual cycle has resumed. That DHEA levels are reduced post partum may indicate that these must rise above a certain value for the positive effect to be apparent. The mechanisms by which DHEA might affect mood are unknown. No receptor for DHEA has been discovered in the brain. This hormone does not seem to act like the bioactive androgen, namely testosterone. Thus, its effect is not likely to be mediated by the androgen receptor. Mechanistic possibilities include the prospect that a DHEA receptor is yet to be discovered. Another possibility is that the conversion of DHEA into other metabolites, such as estrogen and testosterone, account for its effect on mood (Schwarz, 1990; Wolkowitz et al., 1997). However, as previously stated, we did not find estradiol or testosterone associations with mood. That DHEA does not have an effect similar to either testosterone or estradiol also argues against DHEA being effective through conversion to its most immediate metabolite, androstenediol. Androstenediol is of particular interest because of its ability to bind to both androgen and estrogen receptors. Thus, conversion of DHEA to androstenediol would be expected to yield a pattern of associations for DHEA like those of testosterone or estradiol, which was not the case. Rather, we found testosterone to be strongly and consistently associated with negative mood only in the post-partum period, at which time levels of testosterone and other hormones are at significantly decreased levels. A final possibility is that the DHEA molecule itself is able to bind to other receptors and to interact with neurotransmitter systems. Such a possibility supports the dopaminergic and serotonergic actions of DHEA (Abadie et al., 1993; Porter et al., 1995) as well as its antagonistic behavior in relation to GABA (Majewska, 1992; Steffensen, 1995). Our findings suggest that the effect of DHEA is immediate, yet it may not have a positive effect if levels drop below a threshold level, as during the post-partum period. It is also possible that, if levels rise above certain values, such as around ovulation, there is no positive effect (Eriksson et al., 1992). The general consistency and strength of our findings suggests the need for further investigation of the effects of DHEA on mood in women, particularly during the course of reproductive events and also raises the possibility that DHEA supplementation may be effective in the treatment of mood disturbances. The mechanisms of progesterone action with regard to its effect on mood are unknown. Specific receptors for progesterone have been found in various areas of the brain (Maxson, 1987) and have been demonstrated to impact on mood-related neuronal structures through its binding to membrane receptors for neurotransmitters (Chadwick and Widdows, 1990). As stated previously, progesterone and its metabolites potentiate the major inhibitory neurotransmitter, GABA, in brain by binding stereoselectively and with high affinity, to specific GABA receptors (Paul and Purdy, 1992). The typical effect of such neuroaction is augmentation of GABA-activated chloride ion currents in a manner similar to that of anesthetic barbiturates. It may be that this effect varies with the serum concentrations of progesterone. As previously noted, fluctuations in progesterone and estradiol secretion have been linked to premenstrual symptomatology (Backstrom et al., 1983; Steiner, 1992; Reid, 1993). Several investigators have suggested that the interaction of cyclic changes in estradiol and progesterone production with the serotonergic system may be important in the pathogenesis of negative cyclic mood (Rapkin, 1992; Endicott, 1993; Mortola, 1998). Despite the temporal association of negative mood symptoms with both rising and falling serum progesterone concentrations, particularly during the luteal phase of

314 the menstrual cycle, neither progesterone augmentation nor treatment with progesterone agonists result in substantial improvement of symptoms (Mortola, 1998). Both the effects of progesterone on affective functioning and the mechanisms by which such effects transpire remain open questions. Our study joins those who have postulated negative impact on mood with rising levels of progesterone. Contradictions in the literature suggest that the pattern of exposure to progesterone that leads to negative mood is not clear-cut. It is possible that no simple or singular effect exists (e.g., that higher progesterone equals worse mood), but that mood effects result from more complex ratios of progesterone with other steroid hormones and interactions with neurotransmitters. Our finding regarding testosterone and negative mood post partum also has intriguing implications. That testosterone plays a role only in the post-partum period, when testosterone levels are lower than during pregnancy and the menstrual cycle, suggests that it is not levels of testosterone alone that are relevant. Rather, it suggests that testosterone is a negative factor in mood only in the specific hormonal milieu that exists during the post-partum period. This milieu is characterized by very low levels of estrogen and progesterone. These findings suggest that future research should consider the interactions among these steroid hormones. Future research of the role of steroid hormones also should consider the binding proteins. For example, DHEA is bound primarily to albumin, therefore the majority of DHEA should be bioavailable given the relatively weak bond between DHEA and albumin (Findling et al., 1997). However, in pregnancy, more than 85-95% of estradiol and testosterone is bound with high affinity to SHBG and approximately 40% of progesterone is bound to corticosteroid-binding globulin (CBG; Speroff et al., 1994). The increased estrogen production in pregnancy induces hepatic biosynthesis of SHBG and CBG, resuiting in approximately 5-fold and 2-fold increases in circulating levels of SHBG and CBG, respectively. It is unclear if the total amount of a hormone serves as an effective marker of the bioavailable portion of that hormone. Although we have observed clear patterns of association of circulating concentrations of total steroid with affect, measurement of the concentrations of unbound hormones during and after pregnancy and correlation with mood levels remains to be done. Another intriguing possibility is raised by the presence of SHBG receptors in brain tissue (Joseph et al., 1991, 1997). While SHBG has primarily been considered as facilitating steroid transport, a direct role in brain functioning is possible. Given the momentous and monumental experience of pregnancy and childbirth, it is not surprising that there is an impact on cognitive and affective function in mothers. Yet we are not prepared to conclude that the negative cognitive and mood states experienced by women during these reproductive events is attributable solely and/or primarily to psychological, social and cultural aspects of the transition to parenthood, or the adding of another child to the family. Rather, we argue that the major changes in sex steroid hormone production may provide novel explanations of the cognitive and mood disorders associated with these reproductive events. Such explanations will in turn expand understanding for all neuroscientists. Further investigations of the effects of hormones on cognition and mood related to pregnancy are clearly needed. An ideal study would incorporate longitudinal examination of primiparous women, with hormonal and neuropsychological assessment commencing prior to conception and continuing across the trimesters of pregnancy and for an extended period of time following parturition. Abbreviations 5-HT CBG DHEA DHEA-S ERT GABA SHBG References Abadie, J.M., Wright, B., Correa, G., Browne, E.S., Porter, J.R. and Svek, E (1993) Effect of dehydroepiandrosteroneon neurotransmitter levels and appetite regulation of the obese Zucker rat. Diabetes, 42: 662-669. serotonin corticosteroid-binding globulin dehydroepiandrosterone dehydroepiandrosterone sulfate estrogen replacement therapy y-aminobutyric acid sex-hormone-binding globulin

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J.A. Russell et al. (Eds.)

Progressin BrainResearch,Vol.

133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 23

Molecular genetic approaches to puerperal psychosis

Ian Jones *, Corinne Lendon, Natasha Coyle, Emma Robertson, Ian Brockington and Nick Craddock
Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham B15 2QZ, UK

Abstract: Puerperal psychosis, an episode of mania or psychosis precipitated by childbirth follows approximately one in 1000 deliveries. The evidence of clinical, outcome and genetic studies supports the hypothesis that the majority of puerperal psychotic episodes are manifestations of an affective disorder diathesis with a puerperal trigger. Furthermore the available evidence supports the hypothesis that genes are involved in susceptibility to both diathesis and trigger. For complex genetic disorders such as affective illness there are marked benefits in focussing on a homogenous subtype which allows a subset of hypotheses to be tested. Molecular genetic studies of puerperal psychosis provide an excellent example of this strategy, allowing a hierarchy of hypotheses concerning the involvement of neurosteroid pathways in pathophysiology to be tested. Puerperal psychosis results in considerable suffering to a woman and her family. Elucidating the pathophysiological basis of this disorder will lead to better prevention and treatment and, it is anticipated, inform research on affective disorders more generally.

Introduction "Women have the fight to the enjoyment of the highest attainable standard of physical and mental health."
The Beijing PlaO~orm for Action - - adopted at the Fourth World Conference on Women in 1995 (quoted in the Lancet 1997:349 (suppl. I): 1-26).

Childbirth is a period of rapid and significant change at the biological, psychological and social levels. Perhaps more than any other event it provokes a wide range of psychiatric disorders, from the brief and common experience of the baby blues to some of the most severe episodes of psychotic illness seen in psychiatric practice. From the wide variety of psychiatric disorders that occur in the puerperium (see Brockington, 1996a for review; see also Kumar, 2001, this volume) attention is often

* Corresponding author: Ian Jones, Division of Neurosciences, Department of Psychiatry, University of Birmingham, Birmingham B15 2QZ, UK. E-mail: i.r.jones @bham.ac.uk.

focussed on the trio of baby blues, post-natal depression and puerperal psychosis (Table 1). Over 50% of women experience transient episodes of emotional lability following childbirth (Brockington, 1996a). Symptoms of this syndrome, referred to as the baby or maternity blues, include crying, irritability and depressed mood but last only a few days and do not require intervention (Kennerley and Garth, 1996). Post-natal depression, on the other hand, if untreated may last for many months and cause significant disruption to the woman and her family. Many studies in a variety of settings and employing differing definitions of post-natal depression have been conducted. Summarising this extensive literature, Brockington (1996a) concluded that although 10-20% of mothers are depressed at some time during the first post-natal year a much lower figure (2-4%) receive treatment from primary care services. The symptoms are those of a major depressive illness generally (see Table 1), with negative thoughts often focussed on the baby and the mother's ability to cope. The focus of our research is puerperal psychosis, a psychotic episode precipitated by birth. It is the rarest but most severe psychiatric disturbance fol-

322 TABLE 1 The clinical features of puerperal psychosis, post-natal depression and the baby blues 'Baby blues' Incidence Typical onset after delivery Duration Symptoms include ~50% around days 3-5 few days depressed mood irritability lability of mood crying Post-natal depression ~5-15% within 6 months weeks to months depressed mood lack of pleasure poor sleep/appetite suicidal thoughts self blame/guilt Puerperal psychosis ~0.1% first two weeks weeks to months Elated, irritable or depressed mood lability of mood confusion/perplexity psychotic symptoms, including delusions and hallucinations rapidly changing clinical picture Severe but prognosis of recovery from puerperal episode good. Remains at risk of further puerperal and non-puerperal affective episodes. Anti-psychotic medication, anti-depressant medication, mood stabilizers (e.g. lithium), support and counselling. Often requires admission, jointly with baby if possible.

Prognosis

Transient. Increased risk of subsequent post-natal depression. Requires no intervention

May be severe and long lasting without treatment. At risk of further puerperal and non-puerperal affective episodes. Anti-depressant medication, support and counselling. Most often may be treated at home but severe cases may need admission, jointly with baby if possible.

Treatment

lowing childbirth with an episode following approximately one in 1000 deliveries. Parturition is the most potent known precipitant of severe psychiatric disorder with a 22-fold increased risk of affective psychosis in the 4 weeks following childbirth (Kendell et al., 1987). The symptoms of puerperal psychosis include a wide variety of psychotic phenomena such as delusions and hallucinations, the content of which is often related to the new child (Brockington et al., 1981). Affective (mood) symptoms, both elation and depression, are prominent as is a disturbance of consciousness marked by an apparent confusion, bewilderment or perplexity. The clinical picture often changes rapidly with wide fluctuations in the intensity of symptoms and severe swings of mood. Puerperal psychosis is relatively rare when compared to conditions such as schizophrenia and bipolar disorder more generally, but the importance of studying this disorder should not be underestimated. The occurrence of the disorder at such an important time in a woman's life results in considerable suffering and may put a strain on relationships with her child, her partner and her family. In addition to alleviat-

ing this distress, understanding the pathophysiology of puerperal psychosis may yield important insights into the aetiology of other post-natal disorders and affective disorders more generally. The association between madness and childbirth has been noted for hundreds if not thousands of years. Despite this long history there has been little consensus regarding the nosological status of episodes of psychotic illness in the puerperium. Opinion has varied from those who have argued that puerperal psychosis is a condition in its own right to those who have regarded childbirth as a non-specific stressor like any other life event, acting to trigger a wide variety of psychotic illness (see Brockington, 1996b for an extensive review of this debate). There are two themes, however, that emerge from a study of the literature on puerperal psychosis: (i) the close relationship to bipolar disorder, and (ii) the probable role of biological aetiological factors. In this chapter we will first review the evidence that leads to these conclusions before considering molecular genetic approaches to puerperal psychosis which we hope will result in major advances in our understanding of its pathophysiology.

323 TABLE 2 DSMIV diagnostic criteria for (1) major depressive episode and (2) manic episode; presence of all of A, B, C, D and E is required Major depressive episode Manic episode (A) Abnormallyand persistentlyelevated, expansiveor irritable mood lasting at least one week (any duration if hospitalized). (A) Five (or more) of the following symptomspresent during the same two week period and represent a change from normal functioning:at least one of (1) depressed mood or (2) loss of interest or pleasure. (1) Depressed mood (2) Loss of interest or pleasure (3) Change in appetite and or weight (4) Sleep change (5) Psychomotor agitation or retardation (6) Loss of energy (7) Feelings of worthlessness or excessive or inappropriateguilt (8) Diminished concentration (9) Recurrent thoughts of death/suicidal ideation (B) The symptomsdo not meet the criteria for a mixed episode. (C) The symptoms cause clinically significantdistress or impairmentin social, occupational,or other important areas of functioning. (D) The symptoms are not due to the direct physiologicaleffects of a substance (e.g. a drug of abuse, a medication or other treatment) or a general medical condition. (E) The symptoms are not better accounted for by bereavement. (B) Three or more of the following symptoms (four if mood only irritable) present to a significantdegree. (1) Increased self esteem or grandiosity (2) Decreased need for sleep (3) More talkative than usual or pressure to keep talking (4) Flight of ideas or racing thoughts (5) Distractibility (6) Increase in goal directed activity or psychomotor agitation (7) Excessive involvementin pleasurable activitiesthat have a high potential for painful consequences

(C) The symptomsdo not meet the criteria for a mixed episode. (D) The mood disturbanceis sufficientlysevere to cause marked impairmentin occupationalfunctioning or in usual social activities or relationshipswith others or to necessitatehospitalizationto prevent harm to self or others, or there are psychotic features. (E) The symptomsare not due to the direct physiologicaleffects of a substance (e.g. a drug of abuse, a medicationor other treatment) or a general medical condition.

The link to bipolar disorder

The core feature of bipolar disorder is a pathological disturbance of mood (affect) ranging from extreme elation (mania) to severe depression usually accompanied by disturbances in thinking and behaviour, which may include psychotic symptoms such as delusions and hallucinations. The occurrence of an episode of mania during the course of illness distinguishes bipolar disorder from unipolar disorder, a more c o m m o n form of mood disorder in which patients only suffer episodes of depression (WHO, 1993; American Psychiatric Association, 1994). Table 2 summarizes the diagnostic criteria for unipolar and bipolar disorder according to the most recent version of the American Psychiatric Associations Diagnostic and Statistical Manual - - DSMIV (American Psychiatric Association, 1994). Bipolar disorder has a lifetime prevalence of approximately 1% with no evidence that rates differ widely between pop-

ulations. Rates of illness are similar in males and females and the mean age of onset is around 21 years (Goodwin and Jamison, 1990). Despite the variety of clinical features seen in episodes of puerperal psychosis there is strong evidence from clinical, outcome and genetic studies for a close relationship with bipolar disorder.

Clinical studies
Despite variations in inclusion criteria and definitions of puerperal period, studies consistently demonstrate that the majority of puerperal psychosis episodes are affective with mania particularly c o m m o n in the 2 weeks following childbirth (Protheroe, 1969; Thuwe, 1974; Dean and Kendell, 1981; Meltzer and Kumar, 1985; S c h r p f et al., 1985; McNeil, 1986; Kendell et al., 1987; Dean et al., 1989). The study of Brockington and colleagues in Manchester examined the symptoms ex-

324 perienced in 58 puerperal episodes compared to 52 episodes of non-puerperal psychotic illness occurring in women of childbearing age (Brockington et al., 1981). It found systematization of delusions, persecutory ideas, auditory hallucinations, odd affect and social withdrawal were less common in the puerperal patients whereas manic symptoms - elation, rambling speech, flight of ideas, lability of mood, distractibility, euphoria and excessive activity - - were all more frequent and severe. In the puerperium, it would seem, episodes of mania and other affective disorders are relatively common whereas the onset of a schizophrenic episode is rare.
Outcome studies

Further evidence for a close link to bipolar disorder comes from studies examining the natural history of puerperal psychotic episodes. The excellent prognosis associated with this condition has been noted for hundreds of years. Hunter, in the late eighteenth century, wrote "in general, this species of madness cures itself" (Hunter, 1771). Women who have suffered an episode of puerperal psychosis, however, remain at high risk of developing further puerperal and non-puerperal affective episodes (Davidson and Robertson, 1985). A number of studies have examined the risk of puerperal recurrences in women with bipolar disorder or affective psychosis and demonstrate that puerperal episodes of illness follow 20 to 30% of births to women in this group (Bratfos and Haug, 1966; Reich and Winokur, 1970; Kendell et al., 1987). We have examined the occurrence of puerperal episodes in 152 parous bipolar women who have taken part in our ongoing molecular genetic studies of bipolar disorder. Using a definition of puerperal psychosis that included all episodes of mania or psychotic illness within 6 weeks of delivery we found that such an episode occurred after 26% of deliveries to these women (Jones and Craddock, 2001a). Blehar and colleagues reported that in the sample of 139 women with bipolar disorder ascertained as part of the National Institute of Mental Health (USA) genetics initiative study 45.3% of parous women reported experiencing "severe emotional problems" during pregnancy or within one month of childbirth (Blehar et al., 1998). However, not one of 51

women with severe emotional disturbance following pregnancy reported a manic episode in the puerperium. This stands in contrast to other published results examining the occurrence of puerperal manic episodes in women with bipolar disorder and we can only conclude that this discrepancy must arise from methodological differences between this and previous studies. Further support for the particular relationship between bipolar disorder and puerperal psychosis comes from the recent study of Terp and Mortenson (1998) linking the Danish birth and psychiatric admission registers and assessing over one million deliveries. This important and in some respects unique study found that the highest relative risk (RR = 6.82) was obtained for first episode bipolar manic depressive psychosis 2-28 days following delivery and obtained an even higher figure if only the first two weeks of the puerperium are considered (Terp, 1999). In contrast, the relative risk for schizophrenia for the same time period after delivery was less than one.
Genetic studies

Family, twin and adoption studies provide compelling evidence that genes influence susceptibility to bipolar disorder (Craddock and Jones, 1999). The mode of inheritance is complex and the (epistatic) interaction of several susceptibility genes represents a plausible model for transmission (Craddock et al., 1995). Esquirol in the early 19th century first noted the tendency for puerperal psychosis to run in families (Esquirol, 1845). Formal family studies of puerperal psychosis have consistently demonstrated familial aggregation of psychiatric (particularly affective) disorder, with morbidity risks for first degree relatives in the range 10% to 50% (Protheroe, 1969; Thuwe, 1974; Kadramas et al., 1979; Whalley et al., 1982; Schtpf et al., 1985; Platz and Kendell, 1988; Dean et al., 1989). No differences were found in rates of illness in male and female relatives and a wide range of diagnoses were observed in the families of puerperal probands - - including unipolar depression, bipolar disorder and atypical psychoses. There are two ways of conducting family studies: the family history method in which the proband is interviewed and asked about the occurrence of

325 illness in her relatives and the methodologically superior family study method in which first-degree relatives are interviewed directly. The study of Dean et al. (1989) using direct interview of relatives provides evidence that suggests that puerperal psychosis may be a more genetic subtype of bipolar disorder. It demonstrated a substantial (50% versus 30%) and significant (P = 0.0018) increase in affective disorder morbidity in first-degree relatives of subjects who had experienced at least one episode of puerperal psychosis when compared to relatives of bipolar women who had never experienced a puerperal psychosis episode. Analysis of families recruited for our bipolar family study provides independent support for the increased familiality of mood disorder for bipolar subjects having puerperal psychosis episodes compared to those without such episodes (P = 0.012) (Jones and Craddock, unpublished data). Two further studies which compared women with psychosis limited to the puerperium with those who also had non-puerperal episodes found lower rates of illness in first-degree relatives of those with pure puerperal disease (Kadramas et al., 1979; Scht~pf et al., 1985). In both studies, however, the pure puerperal probands had a milder condition in that they had predominantly only experienced one episode of illness. Reich and Winokur (1970) found a non-significant trend for morbid risk to first degree relatives to be lower for bipolar probands with puerperal episodes compared to bipolar patients more generally and also reported no significant family loading for post-partum illness. Numbers were small, however, with only 8 bipolar women with a puerperal episode and 12 parous non-puerperal bipolar probands. In summary, the family data suggest a major overlap in the genetic factors predisposing to puerperal psychosis and bipolar disorder. There are no twin or adoption studies of puerperal psychosis but the importance of genetic factors in the puerperal trigger is further supported by a number of reports of identical twin pairs concordant for puerperal psychosis (Kallman, 1938; Kane, 1968). Moreover, we have previously reported a case in which familial clustering of puerperal psychosis was associated with consanguinity, raising the possibility of a recessive gene contributing to susceptibility (Craddock et al., 1994).

What is the evidence concerning the inheritance of the post-partum trigger in bipolar subjects ?
The key comparison is the rate of puerperal psychosis in relatives of puerperal psychosis probands compared to the rate in relatives of non-puerperal bipolar probands. Data are provided by the study of Dean et al. (1989) which found a rate of 22/1000 affected pregnancies in relatives of patients with pure puerperal disease, 12/1000 in combined disease, and 5/1000 in non-puerperal bipolar disorder (Fisher's exact test, P = 0.05, non-puerperal disease versus puerperal psychosis (Brockington, 1996b)). This suggests that a vulnerability to puerperal episodes themselves may aggregate in families but in order to provide further evidence in support of this hypothe~ sis we have conducted a study of the occurrence of episodes of puerperal psychosis in families multiply affected with bipolar disorder participating in our ongoing sib-pair study of this condition (Jones and Craddock, 2001a). Our results conclusively demonstrate that familial factors are implicated in susceptibility to the puerperal trigger with puerperal episodes shown to cluster in families (x -----0.67, P = 0.001). Dividing our sample of parous bipolar women into those with a first-degree relative with an episode of puerperal psychosis and those without such a relative reveals a significant difference in the rates of puerperal psychosis episodes following deliveries to women in the two groups (57% of deliveries in bipolar women with a family history of puerperal psychosis (n = 49), 20% in bipolar women without such a relative (n = 264, P < 0.000001) (Jones and Craddock, 2001a). While these results provide compelling evidence of familiality it is important to point out that we have not proved that genes are involved. However, given what is known about bipolar disorder and puerperal psychosis, genes provide by far the most plausible mechanism for this striking familiality.

Evidence for biological aetiological factors

There is no evidence that the psychosocial context in which a delivery occurs influences the susceptibility to puerperal psychosis (McNeil, 1988; Marks et al., 1991). Consideration of the abrupt onset during a

326 time of major physiological change (e.g. hormonal fluctuations) convinces most researchers that biological factors are of fundamental importance. The possible role of several hormones (e.g. prolactin, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) has been considered but evidence for involvement of estrogen is most compelling (Cookson, 1982; Wieck et al., 1991). Estrogen levels drop precipitously following parturition (Speroff et al., 1994), estrogen can be anti-depressant (Henderson et al., 1991) and affects expression of several neuronally relevant genes including those encoding dopamine receptors (Wieck et al., 1991; Guivarch et al., 1995), 5-hydroxytryptamine type 2A (5HT2A) receptor (Fink and Sumner, 1996), monoamine oxidaseA (Ma et al., 1995) and tyrosine hydroxylase (Raab et al., 1995). Aetiological theories based on estrogen's interaction with serotonin systems have been forwarded (Fink and Sumner, 1996) and its interaction with dopamine systems forms the basis of a theory first put forward by Cookson (1982). He suggested that the rapid fall in estrogen levels after delivery exposes dopamine receptors that have been rendered supersensitive by high levels of estrogen during pregnancy. This theory formed the basis for the work of Wieck and colleagues (1991). They measured growth hormone response to the injection of the dopamine agonist apomorphine on day 4 after delivery in 15 'high risk' women (history of puerperal psychosis or non-puerperal mania) and 15 normal post-partum controls. The finding of a significantly greater average growth hormone response in the 8 women who went on to develop puerperal episodes provides support for the supersensitive dopamine receptor hypothesis. Meakin and colleagues (Meakin et al., 1995), however, were unable to replicate this result in a sample of 10 high-risk women, three of whom went on to develop puerperal mania and 34 controls.

peral trigger. Furthermore the evidence supports the hypothesis that genes are involved in susceptibility to both diathesis and trigger. Interestingly, 10% of females have transient subclinical mania within days of childbirth (Glover et al., 1994) which may represent the operation of the trigger in the absence of the diathesis. The relationship between genes for diathesis and trigger is not known. It is possible that genes for the diathesis and trigger are distinct with a gene that increases vulnerability to the puerperal trigger having no influence on susceptibility to bipolar disorder generally. It is also possible that a certain gene may both increase vulnerability to bipolar disorder and increase susceptibility to episodes of illness in the puerperium.

Molecular genetic approaches

The diathesis-trigger model outlined above forms the basis of the approach our group is currently taking in the investigation of puerperal psychosis. Molecular genetic studies, using the complementary approaches of linkage and association studies offer powerful paradigms for the genetic dissection of psychiatric disorders such as bipolar disorder and puerperal psychosis (Craddock and Owen, 1996a). As more genes are identified and sequenced, candidate gene association studies, particularly those using polymorphisms (Variations) that Affect Protein Structure or Expression (VAPSEs) (Sobell et al., 1992), represent an increasingly attractive paradigm for the identification of genes of relatively modest effect (Craddock and Owen, 1996b). Candidate gene studies depend critically on the availability of good candidates. Given our knowledge of the likely pathophysiology of puerperal psychosis, plausible candidates include polymorphisms in both estrogen receptor genes (ERa and 13) genes involved in estrogen pathways and neuronally relevant genes whose product is influenced by estrogen. As knowledge develops of the complex interplay between sex steroid hormones and neuronally relevant systems it will be important to test novel candidates as they become available. Molecular genetic studies of bipolar disorder are ongoing in several centres, promising findings are emerging but no gene has yet been identified (Risch and Botstein, 1996; Craddock and Jones, 1999). It

Hypothesis
Although questions about the nosological status of puerperal psychosis remain, the clinical and genetic evidence outlined above supports the hypothesis that the majority of post-natal psychotic episodes are manifestations of a bipolar diathesis with a puer-

327 is likely that susceptibility genes for bipolar disorder will be discovered in the early part of the new century, a task that will greatly be facilitated by the completion of the Human Genome Project. As outlined above, there is evidence that puerperal psychosis may be under greater genetic influence than bipolar disorder more generally. For complex genetic disorders there are marked benefits in focussing on a homogenous subtype which allows a subset of hypotheses to be tested (Lander and Schork, 1994). Molecular genetic studies of puerperal psychosis provide an excellent example of this strategy, allowing a hierarchy of hypotheses concerning the involvement of neurosteroid pathways in pathophysiology to be tested. Our strategy for identifying susceptibility genes involved in puerperal psychosis takes two broad approaches. (1) We are investigating the involvement in puerperal psychosis of genes implicated by positional or candidate studies of bipolar disorder (Reviewed in Jones and Craddock, 2001b, and Cradock and Jones, 2001). (2) We are testing the involvement of polymorphisms in a set of candidates involved in neurosteroid pathways: (i) neurosteroid receptor genes (e.g. E R a and 6); (ii) genes involved in neurosteroid metabolism (e.g. the cytochrome system); (iii) genes involved in the action of neurosteroids (e.g. heat shock proteins); and importantly (iv) neutonally relevant genes containing hormone response elements (e.g. tyrosine hydroxylase). The involvement of genes in this hierarchy is being tested by polymorphism detection (e.g. Denaturing High Performance Liquid Chromatography (O'Donovan et al., 1998)) followed by association studies in samples of puerperal psychosis probands and appropriate comparison groups. We are currently recruiting a large sample of probands with puerperal psychosis and have ascertained over 400 women from around the UK who have suffered an episode of this disorder of which over 200 have already been interviewed. Genotyping is currently ongoing in our puerperal psychosis sample for polymorphisms in such genes as ESR a and b (Jones et al., 2000), catechol-o-methyl transferase, and a number of genes involved in the serotonin system (5HT). We have recently investigated the hypothesis that variation at the 'variable number tandem repeats' (VNTR) polymorphism in intron 2 of the serotonin transporter gene (SERT/5-HTT) influences susceptibility to puerperal psychosis (Coyle et al., 2000). This is an attractive candidate polymorphism because (a) 5-HTT expression is influenced by estrogen (McQueen et al., 1997), (b) variation at the VNTR can influence expression of 5-HTT (McKenzie and Quinn, 1999), and (c) several previous studies have found an increase in the STin2.12 allele of the VNTR in bipolar probands compared with controls (e.g. Collier et al., 1996; Rees et al., 1997). We compared genotype and allele distributions in two United Kingdom-born Caucasian female samples: (a) 97 women who had experienced at least one episode of bipolar affective puerperal psychosis with onset within 14 days of childbirth, and (b) 72 attendees at a general practitioner for non-psychiatric reasons. We found significant evidence (P < 0.003) that variation at 5-HTT exerts a substantial (odds ratio -----4) and important (population attributable fraction = 69%) influence on susceptibility to such episodes. It is possible that the previously inconsistent findings with the 5-HTT VNTR in bipolar disorder may in part be related to variation in the proportion of puerperal cases within the samples and it will be important for researchers to re-analyse their data taking this into account. Functional studies Evidence to support the pathogenic relevance of polymorphisms identified in the mutation detection and association studies is being sought through functional studies addressing the demanding question of how might gene variants predispose to or modify disease. Relevant assays are designed according to the known or presumed function and expression of the gene. For genes involved in steroid hormone systems assays these include: (1) Studies of the influence of steroid hormone receptor binding to steroid hormone response elements. Electrophoretic mobility shift assays and supershift assays using specific antibodies (Driscoll et al., 1998), provide a sensitive method for the investigation of the influence of polymorphisms on sequence specific DNA-protein interactions

328 Identificationof genespotentiallyinvolved in psychiatricdisorderswhichare potentially hormonaUyregulated Genesimplicatedby positionalor candidate studiesof bipolar disorder Hierarchyof candidates involvedin neurosteroid pathways

Computeranalysisof genetic Lad Prioritiseby likely sequencefor potentialHRE's ] ~ rolein ~

"

Idebyn~ fYHp~Ue nseq~u::c~ a~t ~

SequenKn: VwnaCant ~
YES

Associationand genotype:phenotypic correlations

I Replication I
+

If functionknown

+
Functionalstudies

Designappropriatespecific assay, comparevariant versuswild-type

T If suggested or unknown function

Studyof functionand furtheranalysisof I effectof genevariants,comparisonversus wildtype

Fig. 1. Summaryflowdiagramof process involvedin the identificationof polymorphicvariationspotentiallyinvolvedin predisposition to puerperalpsychosis.HRE = hormoneresponseelement.

(2) Studies of the effect of the polymorphism on modulation of expression of neuronally relevant genes. Relevant cell lines and primary cultures are

transfected with constructs bearing candidate polymorphisms in a reporter construct. This technique is being used to examine differential modulation

329 of promoter activity following the administration of steroid hormone. Our approach to the identification of polymorphisms potentially predisposing to puerperal psychosis and subsequent functional studies is summarized in Fig. 1.

Acknowledgements
Ian Jones is a Wellcome Trust Training Fellow. Nick Craddock is a Wellcome Trust Senior Research Fellow in Clinical Sciences. Natasha Coyle is a Wellcome Trust Prize PhD student. E m m a Robertson is a West Midlands Regional Health Authority New Blood Training Fellow. We would like to thank the Wellcome Trust, South Birmingham Mental Health Trust, the West Midlands Regional Health Authority and the Women's Mental Health Trust for the support that made this work possible. We are grateful to Ms Jackie Benjamin for advice and support, and to members of Action on Puerperal Psychosis and sufferers who have helped in this work.

Summary and conclusions

The status of puerperal psychosis has been debated for generations but three themes have emerged: (1) the close relationship to bipolar disorder, (2) the probable role for biological factors in aetiology, and (3) the importance of genetic factors in increasing vulnerability to puerperal psychosis. These strands of evidence lead to the hypothesis that forms the basis of our work in puerperal psychosis - - that the majority of episodes of puerperal psychosis represent a bipolar disorder diathesis acted on by puerperal trigger and that genes influence susceptibility to both diathesis and trigger. Molecular genetic approaches now provide the tools needed to uncover susceptibility genes for both diathesis and trigger leading to an improved understanding of the biological factors involved in this condition. Elucidation of the pathophysiological basis of the puerperal trigger will lead to major benefits in treatment and prevention of puerperal psychosis and is likely to inform research on a wide range of affective disorders, particularly those occurring in relation to other hormonal triggers such as menstruation and steroid therapy.

References
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Abbreviations
DSMIV ER ct and [3 5HT 5HT2A receptor RR SERT/5-HTT VAPSEs Diagnostic and Statistical Manual IV estrogen receptor a and [3 serotonin system 5-hydroxytryptamine type 2A receptor relative risk serotonin transporter gene Variations (polymorphisms) that Affect Protein Structure or Expression variable number tandem repeats

VNTR

330

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Kallman, EJ. (1938) The Genetics of Schizophrenia. Augustin, New York. Kane Jr., EJ. (1968) Postpartum psychosis in identical twins. Psychosomatics, 9: 278-281. Kendell, R.E., Chalmer, J.C. and Platz, C. (1987) Epidemiology of puerperal psychoses. Br. J. Psychiatry, 150: 662-673. Kennerley, H. and Garth, D. (1996) Maternity blues reassessed. Psychiatr. Dev., 1: 1-17. Knmar, R.C. (2001) The maternal brain as a model for investigating mental illness. In: J,A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingrain (Eds.), The Maternal Brain. Neurobiological and Neuroendocrine Adaptation and Disorders in Pregnancy and Post Partum. Progress in Brain Research, Vol. 133. Elsevier, Amsterdam, pp. 333-338. Lander, E.S. and Schork, N.J. (1994) Genetic dissection of complex traits. Science, 265: 2037-2048. Ma, Z.Q., Violani, E., Villa, E, Picotti, G. and Maggi, A. (1995) Estrogenic control of monoamine oxidase A activity in human neuroblastoma cells expressing physiological concentrations of estrogen receptor. Eur. J. Pharmacol., 284: 171-176. Marks, M.N., Wieck, A., Checkley, S.A. and Kumar, R. (1991) Life stress and postpartum psychosis: a preliminary report. Br. J. Psychiatry, 158: 45-49. McKenzie, A. and Quinn, J. (1999) A serotonin transporter gene intron 2 polymorphic region, correlated with affective disorders, has allele-dependent differential enhancer-like properties in the mouse embryo. Proc. Natl. Acad. Sci. USA, 96: 1525115255. McNeil, T.F. (1986) A prospective study of postpartum psychoses in a high risk group, I. Clinical characteristics of the current postpartum episodes. Acta Psychiatr. Scand., 74: 205216. McNeil, T.E (1988) A prospective study of postpartum psychoses in a high risk group, 3. Relationship to mental health characteristics during pregnancy. Acta Psychiatr. Scand., 77: 604-610. McQueen, J.K., Wilson, H. and Fink, G. (1997) Estradiol-17[~ increases serotonin transporter (SERT) mRNA levels and the density of SERT-binding sites in female rat brain. Mol. Brain Res., 45: 13-23. Meakin, C.J., Brockington, I.E, Lynch, S. and Jones, S.R. (1995) Dopamine supersensitivity and hormonal status in puerperal psychosis. Br J. Psychiatry, 166: 73-79. Meltzer, E.S. and Kumar, R. (1985) Puerperal mental illness. Clinical features and classification: a study of 142 mother-andbaby admissions. Br. J. Psychiatry, 147: 647-654. O'Donovan, M.C., Oefner, P.J., Roberts, S.C., Austin, J., Hoogenddorn, B., Guy, C., Speight, G., Upadhaya, M, Sommer, S.S. and McGuffin, P. (1998) Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics, 52: 44-49. Platz, C. and Kendell, R.E. (1988) A matched-control follow-up and family study of 'puerperal psychosis'. Br. J. Psychiatry, 153: 90-94. Protheroe, C. (1969) Puerperal psychoses: a long term study 1927-1961. Br. J. Psychiatry, 115: 9-30. Raab, H., Pilgrim, C. and Reisert, I. (1995) Effects of sex and es-

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J.A. Russell et at. (Eds.)

Progress in Brain Research, Vol. 133

2001 Elsevier Science B.V. All rights reserved

CHAPTER 24

The maternal brain as a model for investigating mental illness

R. Channi Kumar t
Department of Perinatal Psychiatry, Institute of Psychiatry, King's College London, De Crespigny Park, London, SE5 8AE UK

Abstract: The idea that a particular type of severe mental illness, puerperal or post-partum psychosis, is a disease entity and that its causes lie in some kind of physiological disturbance of the reproductive process, can be traced back to antiquity. Epidemiological studies provide strong support for such an hypothesis, but, despite the powerful methodological attractions of using childbirth as a model for research, there has been surprisingly little neuroendocrine research into this subject. There have been preliminary reports of prospective research into women with histories of affective psychosis who are pregnant and who are at particularly high risk of recurrence of illness. This work suggests that it may be very fruitful to investigate interactions between the massive changes that occur in sex hormones around parturition and the activity of selected neurotransmitter systems. Because of the prospective research paradigm, it becomes possible to test whether the measures can predict who will become ill and who will stay well.

Introduction and historical background

The debate about whether, or how, childbearing plays a part in the etiology of mental illness can be traced at least as far back as 400 BC to Hippocrates. He crystallized his clinical observations into the following aphorisms: "It is a sign of madness when blood congeals about a woman's nipples" and "Those women who have white lochia in the puerperium and in whom cessation of the discharge is attended with pain, deafness and fever, go into a fatal dementia" (translation by Chadwick and Mann, 1950). The first aphorism suggests that a serious abnormality of the lactational process is associated with madness and the second implies that 'bad' lochial humours which are unable to escape from the uterus, flow 'upwards' and thus cause the fatal dementia. Although almost all the cases described by Hippocrates probably had developed toxic, and eventually fatal, psychoses as a consequence of puerperal infections, the hypotheses

+Deceased.

that were contained in his aphorisms were remarkably prescient, even though they lay dormant during the next two millennia. Sporadic case reports of puerperal mental illness appeared in the medical literature of the 16th, 17th and 18th centuries and the first systematic description of a series of cases was initially reported in 1818 by the great French psychiatrist E. Esquirol, and was republished in his classic text book Des Maladies Mentales (Esquirol, 1838). Echoing Hippocrates, he commented "There is a lacteal diathesis which modifies all secretions of the female and imposes on them its own character". Twenty years later, Esquirol's student L.V. Marc6 (1858) published the first book devoted entirely to puerperal and lactational psychoses and he additionally drew links between onsets of illnesses and the return of the menses. He too was impressed by possible connections between disorders of the uterus and related organs and disorders of the mind. Bearing in mind that hormones had not yet been discovered it is remarkable how astute were these 19th century French clinicians. Their colleagues across the channel in

334 England were of like mind. Thus Henry Maudsley (1899) in his authoritative text book, The Pathology of Mind, wrote as follows: "Affectations of the uterus and its appendages afford notable examples of a powerful sympathetic action on the brain, and not infrequently play an important part in the production of insanity, especially of melancholia". It is sobering to reflect that despite advances such as the identification of hormones, of mechanisms and sites of their secretion, regulation and action, of interactions with neurotransmitters, that we do not seem since 400 BC to have made any significant advances on the concepts propounded by Hippocrates. In fact an explicit contemporary restatement of the original Hippocratic hypotheses is contained in the Infanticide Act (1938) of England and Wales, which uniquely recognizes post-natal mental illness as grounds for diminished responsibility, traceable back to physiological rather than psychological or social causes. An offence which would otherwise be regarded as murder is reduced to manslaughter if "at the time of the act or omission the balance of her mind was disturbed by reason of her not having fully recovered from the effects of giving birth to the child or by reason of the effect of lactation consequent upon the birth of the child." The law rests upon two fundamental assumptions, first that there are mental disorders that are peculiar to childbearing and second, that their origins are rooted in some kind of physiological dysfunction arising out of the reproductive process. How firm is the medical and scientific evidence in support for, or against either assumption?
Puerperal mental illnesses as disease entities

Three kinds of mental disturbance are traditionally linked with childbirth - - maternity 'blues', postnatal depression and post-partum psychosis. The maternity blues are mild, transient episodes of mood disturbance that are almost universally prevalent and while some cases may progress to depression, the blues are not in themselves a disorder. The main interest has been to try and find possible physiological correlates of the blues and then to see whether such research may provide clues to mechanisms underlying the more severe disorders (see Wieck, 1996; Harris, 1996). Post-natal depression (non-psy-

chotic depression) is present in 10-15% of mothers (O'Hara and Swain, 1996) and the general consensus is that although there may be a few women in whom the onset of depression predominantly reflects genetically determined vulnerability specifically related to childbirth, in the majority of cases it is the psychological and social aspects of parenthood that, in the context of acquired vulnerabilities and concurrent life stress, are predominant in the causal pathways into depression. The third of these conditions, puerperal or post-partum psychosis is the one which has figured most prominently in the debate about whether there is a disease entity associated with childbirth. Most of the early literature was restricted to studies of hospitalized and hence very severely ill patients and it is in relation to puerperal psychosis that opinions became most sharply polarized. One way of distinguishing a disease or syndrome is by means of unique clinical features, as one does, for example, in discriminating between Parkinson's disease and Sydenham's chorea. Esquirol (1838) believed that there were clinical features peculiar to puerperal illnesses but Marc6 (1858) was less certain, commenting that the symptoms found in puerperal psychoses could also be seen in illnesses not associated with childbearing. He was certain, nevertheless, of an underlying morbid process that was rooted in the reproductive organs. If there were pathognomonic symptoms or signs, what might they be? Many authors drew attention to the presence of perplexity, patchy and transient disorientation quite unlike the confusion and disorientation seen in organic illnesses (see Brockington, 1996) but perplexity is also often found in the acute early stages of schizophrenic and manic breakdowns. Other authors from Savage (1875) to Hamilton (1962, 1992) have commented on the variety and changing nature of the predominant symptoms of the illness. Savage wrote of his patients "We cannot classify them with any degree of precision into mania, melancholia or dementia. We shall note typical cases of each of these varieties, but I must premise by saying that it is common for one to pass through all these forms." Present day studies, using operational criteria (Brockington et al., 1981; Dean and Kendell, 1981) confirm that there are no specific distinguishing clinical characteristics of puerperal psychoses and they also confirm that the clinical picture has

335 remained largely unchanged over a century (Rehman et al., 1990). From a clinical, diagnostic standpoint (Kraepelin, 1906) was probably fight when he wrote "People often speak of 'puerperal mania' in the sense of a particular form of insanity produced exclusively by the puerperium . . . . The puerperium cannot be regarded as the cause, but as the last impulse to the outbreak of the disease (manic-depressive insanity)." The opinions of earlier sceptics (e.g. ChrichtonBrowne, 1896) were thus given added weight; he had criticized "those who were rather too apt to seize upon the puerperium and to connect it with insanity." But they did not refer to measles as 'puerperal measles' or pleurisy occurring during that time as 'puerperal pleurisy'. Several years later, two influential articles by North American authors seemed to nail down the lid of the coffin containing the concept of a specific puerperal mental illness. "Old names die hard . . . . All others who have studied this problem are unanimous in the belief that there is no psychosis which may be diagnosed puerperal" (Stecker and Ebaugh, 1926). Foundeur et al. (1957) added "Since those patients whose mental illness was apparently precipitated by childbirth did not differ substantially from control groups as regards diagnosis, previous mental illness or prognosis, the conclusion seems justified that the post-partum illness is not a psychiatric entity." Thus at a time when clinical diagnostic systems were themselves in a state of chaos (see Kendell, 1975), apparently authoritative opinions based on suspect clinical data, were challenging the etiological assumptions handed down since antiquity - - if there was no disease entity how could there be a disease process? International classification systems followed suit, but in a curiously ambivalent and inconsistent manner. Thus, in 1968 (General Register Office, 1968), British psychiatrists using the 8th Revision of the International Classification of Diseases (ICD) of the World Health Organization were strongly advised not to classify cases as puerperal psychosis but rather to classify them under other headings: schizophrenia, affective psychosis, paranoid states or 'other' psychoses. They were, nevertheless, left the option of classifying them in the sub-category psychoses associated with childbirth (onset within 6 weeks following delivery) if they could not classify them as instructed. It seemed that most psychiatrists ignored these instructions, in South-East England anyway (see Meltzer and Kumar, 1985) until the 9th revision (ICD-9, World Health Organization, 1978) came along, deleting any reference to puerperal psychosis in an attempt to avoid etiological assumptions, yet the same glossary retained categories such as psychogenic psychosis! The upshot was chaos (see Meltzer and Kumar, 1985) and there were two serious consequences. First, except in places with both obstetric and psychiatric case registers, it became impossible accurately to identify subjects for research and second, conditions which were deemed not to exist did not easily attract resources. The 10th Revision (ICD-10, World Health Organization, 1992) has effectively reverted to the position held in ICD-8, i.e. facing both ways, recommending classification under other headings as before and only using the category associated with the puerperium if insufficient information is available or if special additional clinical features are present (but without indicating what these might be). The North American Classification System (American Psychiatric Association), has been similarly ambivalent, either not mentioning puerperal illness or echoing ICD-8 (Diagnostic and Statistical Manual (DSM)-III; American Psychiatric Association, 1980). DSM-IV (American Psychiatric Association, 1994) does take a major step forward in incorporating the opportunity to add a post-parturn onset specifier (within 4 weeks of delivery) to mood disorders or to brief psychotic disorders. The problem here is an unwarranted assumption that schizophreniform and schizo-affective disorders do not merit a post-partum onset specifier (see e.g. Dean and Kendell, 1981; Meltzer and Kumar, 1985; Davies et al., 1995); thus rendering it very difficult to include such subjects in research into putative links between childbirth and mental illness.

Puerperal psychosis and the epidemiological evidence

Incidence rates: it is remarkable that estimates of the incidence of this condition have remained constant at about 1/1000 live births during the past 150 years and they appear to be independent of culture or race (see Kumar, 1994). Meanwhile in developed countries, rates of maternal mortality have fallen

336 massively, thus the etiology of puerperal psychosis is independent of all the changes behind the improvements in expectation of life and health, i.e. changes that have occurred in society, in public health and education, in womens' roles and also in medicine, including obstetrics. These facts, allied to the consistently observed short interval between birth and onset of illness (see Brockington et al., 1982) and the at least 200-fold increase in occurrence rates of psychosis following birth in women with histories of previous episodes of affective psychotic illness, irrespective of whether they were puerperal or were unrelated to childbirth (Brockington et al., 1982; Marks et al., 1992), point very clearly to a physiological vulnerability, which, in the context of childbearing, explodes into psychosis. The epidemiological studies of Paffenbarger (1964) and of Kendell et al. (1987) showing near 20-fold increases of relative risk of occurrence of illness in the month post-partum in comparison with similar time periods prior to conception or during pregnancy are also compelling evidence for some kind of specific disease process. Terp and Mortensen (1998), who compared rates of illness in women who were childbearing with those who were not, found a much smaller, but nevertheless increased relative risk. nant. Given that the nature and clinical course of illness is very similar in first episodes and in recurrences, it is probable that the underlying disease processes are the same. This type of research design carries with it the added advantage that potential subjects can be identified relatively easily by screening obstetric populations, pregnant women are less likely to be taking drugs and as they are typically in remission, women with histories of illness are able to give informed consent. Very importantly, measures can be standardized on a time-scale in relation to childbirth, thus greatly facilitating comparisons if, for example, endocrine measures are compared post-natally. Finally, in terms of research methodology, this kind of protocol is almost unique in that it is possible to calendar date (through knowledge of the expected date of delivery) the time of likely onset of illness within a margin of 1 or 2 weeks. It is surprising that so little research has exploited the advantages of this model. It is possible that potential investigators have been put off by the sterile debate about whether or not there is a condition to investigate and have overlooked the powerful epidemiological evidence in support. Up to now there are only three published studies known to the author which have explored putative neuroendocrine-neurotransmitter dysfunctions in a prospective manner in childbearing women at risk of post-partum recurrence of mental illness. Wieck et al. (1991) tested a hypothesis originally put forward by Cookson (1985) suggesting that estrogen 'withdrawal' post-partum might trigger psychosis through an action on the central dopamine system. They tested all their subjects on the 4th day postpartum, i.e. before the likely onset of illness, by measuring the growth hormone response to a 'challenge' dose of a dopamine (D2) receptor agonist, apomorphine. Elevated growth hormone responses were observed in women who experienced a recurrence of mood disorder in comparison with those who remained well, but it was not clear whether the increased receptor sensitivity was related to affectire psychotic relapse or to onset of non psychotic depression and anxiety. With the small number of subjects (n = 15 in all), it was also not possible meaningfully to test for trait-versus-state differences. Meakin et al. (1995) were unable to replicate these preliminary findings in ten women at risk of re-

Childbirth as a model for investigating puerperal psychosis

There are limits to the amounts of information that can be obtained from epidemiological or clinical studies and ultimately the confirmation of a disease entity rests upon identification of causes. In general, studies of putative etiological mechanisms in populations of women who have become ill are confounded by the presence of illness, by treatments and by bias arising out of knowledge of the presence and nature of illness. Thus the most rigorous studies should be prospective and longitudinal, but they are not feasible when in this case the expectation is that only one woman in a thousand will become ill soon after delivery. There is, however, an opportunity to conduct prospective research, not into first episodes, but into recurrence of illness in women at high risk - - that is in women with histories of affective and schizoaffective psychosis, possibly also schizophreniform psychosis, who are in remission and who are preg-

337 currence of psychosis, three o f w h o m subsequently developed manic relapse. Variations in dose regime - - intravenous or subcutaneous, absolute dose or m g / k g , the small number o f w o m e n who relapsed and the lack of information about clinical measures, e.g. o f non-psychotic affective disorder during the 42-day period following delivery and other procedural differences discussed by Meakin et al., might account for the discrepancy between these two small studies. A third study by M c I v o r et al. (1996) used the same protocol as W i e c k et al. (1991) but tested women with histories of major depression. Recurrence of depression and anxiety disorder was associated with an augmented response to apomorphine, i.e. the measure was predicting recurrence o f depression.
Future studies References

The kind o f research which is described above can be extended in various ways and a project is nearing completion (Kumar et al., 1997, 1998) with larger numbers of subjects, simultaneously examining two measures o f d o p a m i n e receptor responsiveness to apomorphine (growth hormone and prolactin secretion) as well as possible associations between measures of sex hormones and the neurotransmitter system 'resting' or ' b a s e l i n e ' levels as well as responsiveness to agonist challenge. These measures are being tested as trait markers - - contrasting childbearing women with histories of affective or schizoaffective psychosis, subjects with histories o f major depression and healthy controls. Assessments of clinical outcome in the months following delivery will permit analyses of the predictive value of the neuroendocrine-neurotransmitter measures, i.e. of impending m o o d disorder - - affective psychotic illness and non-psychotic depression and anxiety. Depending on the findings it m a y be fruitful to extend such research to explore other neurotransmitter systems.
Abbreviations

DSM ICD

Diagnostic and Statistical Manual (American Psychiatric Association, APA) International Classification o f Diseases (World Health Organization, W H O )

American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders, 3rd edn. Washington, APA. American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edn. APA, Washington. Brockington, I.E (1996) Motherhood and Mental Health. Oxford University Press, Oxford. Brockington, I.F., Cernik, K.E, Schofield, E.M., Downing, A.R., Francis, A.E and Keelan, C. (1981) Puerperal psychosis. Phenomena and diagnosis. Arch. Gen. Psychiatry, 38: 829-833. Brockington, I.E, Winokur, G. and Dean, C. (1982) Puerperal psychosis. In: I.E Brockington and R. Kumar (Eds.), Motherhood and Mental Illness. Academic Press, London, pp. 3769. Chrichton-Browne, J. (1896) Meeting of Medical Society of London. Prevention and treatment of insanity of pregnancy and puerperal period. Lancet, 1: 164-165. Cookson, J.C. (1985) The neuroendocrinology of mania. J. Affect. Disord., 8: 233-241. Davies, A., Mclvor, R.J. and Kumar, R. (1995) Impact of childbirth on a series of schizophrenic mothers: a comment on the possible influence of oestrogen on schizophrenia. Schizophr. Res., 16: 25-31. Dean, C. and Kendell, R.E. (1981) The symptomatology of puerpal illness. Br. J. Psychiatry, 139: 128-133. Esquirol, E. (1838) Des Maladies Mentales, Tome 1. Bailliere, Paris. Foundeur, M., Fixsen, C., Triebel, W.A. and White, M.A. (1957) Postpartum mental illness, a controlled study. Arch. Neurol. Psychiatry, 77: 503-512. General Register Office (1968) Studies on Medical and Population Subjects, No. 22. A Glossary of Mental Disorders. HMSO, London. Hamilton, J.A. (1962) Postpartum Psychiatric Problems. Mosby, St. Louis. Hamilton, J.A. (1992) Patterns of postpartum illness. In J.A. Hamilton and EN. Hargerger (Eds.), Postpartum Psychiatric Illness, University of Pennsylvania Press, Philadelphia, PA, pp. 5-32. Harris, B. (1996) Hormonal aspects of postnatal depression, lnt. Rev. Psychiatry, 8: 27-36. Hippocrates (c. 400 BC) The Medical Works of Hippocrates. Translation by J. Chadwick, and W.N. Mann (1950). Blackwell, Oxford. Kendell, R.E. (1975) The Role of Diagnosis in Psychiatry. Blackwell, Oxford. Kendell, R.E., Chalmers, J.C. and Platz, C. (1987) Epidemiology of puerperal psychoses. Br. J. Psychiatry., 150: 662-673. Kraepelin, E. (1906). In: T. Johnstone (Rev. and Ed.), Lectures on Clinical Psychiatry, 2nd edn. Bailliere, Tindall and Cox, London. Kumar, R. (1994) Postnatal mental illness: a transcultural perspective. Soc. Psychiatry Psychiatr. Epidemiol., 29: 250-264. Kumar, R., Marks, M.N., Wieck, A., Davies, R.A., McIvor, R., Brown, N., Papadopoulos, A., Campbell, I.C. and Checkley,

338 O'Hara, M.W. and Swain, A.M. (1996) Rates and risk of postpartum depression - - a meta-analysis. Int. Rev. Psychiatry, 8: 37-54. Paffenbarger Jr., R.S. (1964) Epidemiological aspects of parapartum mental illness. Br. J. Soc. Prey. Med., 18: 189-195. Rehman, A.U., St. Clair, D. and Platz, C. (1990) Puerperal insanity in the 19th and 20th centuries. Br. J. Psychiatry, 156: 861-865. Savage, G.H. (1875) Observations on the insanity of pregnancy and childbirth. Guy's Hosp. Rep., 20:83-117. Stecker, E.A. and Ebaugh, EG. (1926) Psychoses occurring during the puerperium. Arch. NeuroL Psychiatry, 15: 239-252. Terp, I.M. and Mortensen, P.B. (1998) Postpartum psychoses. Clinical diagnoses and relative risk of admission after parturition. Br. J. Psychiatry, 172: 521-526. Wieck, A. (1996) Ovarian hormones, mood and neurotransmitters. Int. Rev. Psychiatry, 8: 17-25. Wieck, A., Kumar, R., Hirst, A.D, Marks, M.N., Campbell, I.C. and Checkley, S.A. (1991) Increased sensitivity of dopamine receptors and recurrence of affective psychosis after childbirth. Br. Med. J., 303: 613-616. World Health Organization (1978) Mental Disorders: Glossary and Guide to their Classification in Accordance with the Ninth Revision of the International Classification of Diseases. WHO, Geneva. World Health Organization (1992) The 1CD-IO Classification of Mental and Behavioural Disorders, Vol. 1. WHO, Geneva, pp. 311-387.

S.A. (1997) Neuroendocrine mechanisms in postpartum psychosis and postnatal depression. Biol. Psychiatry, 42: 130S131S. Kumar, R., Marks, M.N., Wieck, A., Davies, R.A., Mclvor, R., Brown, N., Papadopoulos, A., Campbell, I.C. and Checkley, S.A. (1998) Neuroendocrine treatments for postpartum psychosis and postnatal depression. Int. J. Neuropsychopharmacol., l(Suppl. 1): $20. Marcr, L.V. (1858) Traitd de la Folie des Femmes Enceintes, des Nouvelles Accouches et des Nourrices. Bailliere et ills, Paris. Marks, M.N., Wieck, A., Checkley, S.A. and Kumar, R. (1992) Contribution of psychological and social factors to psychotic and non-psychotic relapse after childbirth in women with previous histories of affective disorder. J. Affect. Disord., 29: 253-264. Maudsley, H. (1899) The Pathology of Mind. Appleton, New York. Meakin, C.J., Brockington, I.F., Lynch, S.E. and Jones, S.R. (1995) Dopamine supersensitivity and hormonal status in puerperal psychosis. Br. J. Psychiatry, 166: 73-79. Mclvor, R.J., Davies, R.A., Wieck, A., Marks, M.N., Brown, N., Campbell, I.C., Checkley, S.A. and Kumar, R. (1996) The growth hormone response to apomorphine at 4 days postpartum in women with a history of major depression. J. Affect. Disord., 40: 131-136. Meltzer, E.S. and Kumar, R. (1985) Puerperal mental illness, clinical features and classification: a study of 142 mother-andbaby admissions. Br. J. Psychiatry, 147: 647-654.

J.A. Russell et al. (Eds.)

Progressin Brain Research, Vol. 133 2001 Elsevier Science B.V. All rights reserved

CHAPTER 25

In memoriam

An appreciation of Professor Ramesh Kumar t (1938-2000)

Maureen Marks 1 and Ian E Stolerman 2,*
1 Section of Perinatal Psychiatry, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, UK 2 Section of Behavioural Pharmacology, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AE UK

Ramesh (Channi) Kumar was born in India in 1938. At age thirteen he came to England and spent the following five years at Epsom College in Surrey. He was successful in obtaining a place to study medicine at Corpus Christi College in Cambridge where he also received a degree in experimental psychology. He went on to complete a Ph.D. in pharmacology at University College London and trained in psychiatry at The Maudsley. After winning an academic post at the Institute of Psychiatry he founded its Section of Behavioural Pharmacology but already, as a trainee psychiatrist his interest in postnatal mental illness had been emerging; this eventually became the main focus of his work. In 1981 he was appointed Consultant to The Maudsley's newly opened Mother and Baby Unit and he also founded the Section of Perinatal Psychiatry at the Institute of Psychiatry. Some years later he was awarded the title of Professor of Perinatal Psychiatry. He had attained international standing in two distinct research areas,

* Corresponding author: I.E Stolerman, Section of Behavioural Pharmacology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK. Tel.: +44-207848-0370; Fax: +44-20-7848-0579; E-mail: i.stolerman @iop.kcl.ac.uk

had established an important clinical facility at the linked Bethlem Royal Hospital, and had also found time to start a successful international society. These continuing activities are appropriate memorials to his bright, creative, generous and well-intentioned nature. Channi's work in behavioral pharmacology began with support from a Beit Memorial Fellowship. Having presented his Ph.D. thesis on the psychopharmacology of curiosity, learned fear and anxiety in rat models, he turned to studies of drug dependence in collaboration with Ian Stolerman and Hannah Steinberg. In the late 1960s they started to publish a series of papers that helped to mould the perception of addiction as learned behavior resulting from defined interactions of drugs with environmental circumstances, rather than as simply a reflection of drug withdrawal syndromes (Kumar et al., 1968). This research highlighted the critical role of learning factors in the development of drug dependence and brought out the limitations of theories based solely upon the consequences of bathing nerve cells in drugs. These studies demonstrated that drug-seeking behavior was powerfully influenced by conditioned reinforcers, stimuli associated with the repetitive administration of morphine (Kumar and Stolerman, 1972). In several respects the research paralleled and supported

340 the work in progress at prominent laboratories in the United States and helped to establish an international reputation for Channi at an early stage in his career. Having spent five years immersed in fundamental psychopharmacology, Channi, with encouragement from Malcolm Lader, returned to clinical medicine in 1969 by undertaking a full training in psychiatry at the Maudsley Hospital, London. He never lost sight of the basic science that underpinned the use of drugs in psychiatry and thus he trained several psychopharmacology researchers who now hold senior positions in diverse countries. Especially notable were the investigations of nicotine dependence carried out by his postgraduate student Paul Clarke who subsequently became a prominent investigator in that area; these observations significantly advanced understanding of the adaptations of the nervous system that occur during daily exposure to nicotine and which contribute to the development of tobacco addiction (Clarke and Kumar, 1983). The work on nicotine continued in collaboration with Ian Stolerman who eventually took over as head of the group, enabling Channi to concentrate on his interest in peri-natal psychiatry. Channi then worked consistently to develop our understanding of post-natal mental illness and to improve services and treatment for mentally ill women and their babies. The innovative development of the in-patient Mother and Baby Unit allowed mothers who were mentally ill to be admitted together with their babies rather than being separated. Working closely with midwives at King's College Hospital he developed a multidisciplinary approach to care, combining psychiatric and obstetric resources, for pregnant and post-natal women. This emphasizes looking after prospective mothers' minds at the same time as their bodies and is now a model of its kind. Over the years his work attracted the interest of clinicians and researchers from many different countries. Some came and worked with him at The Maudsley and took back his enthusiasm and ideas for improving psychiatric services for parturient women and their infants to their own countries. His scientific work was wide ranging and included studying the biochemical aspects of postpartum mental disorder, the extent to which medication taken by the mother to help her mental condition can be passed through her breastmilk to her baby, new methods for treating mothers with post-natal depression, and the impact of mothers' post-natal depression on the child's development. In a series of studies he followed up his interest in physiological factors that may be involved in the etiology of post-partum psychosis, in particular that estrogenrelated augmented dopaminergic activity in the brain may be implicated. His research demonstrated that women with histories of bipolar disorder, who are at high risk of post-partum psychosis, indeed have elevated dopaminergic activity (assessed by measuring the growth hormone secretory response to an apomorphine challenge at 4 days postpartum), but that this is a trait effect only and does not predict post-partum psychotic relapse. In contrast, post-partum depressive relapse, in both bipolar and unipolar women, is predicted by augmented dopaminergic activity (Kumar et al., 1997, 1998). These findings were effectively translated into clinical practice in a randomized controlled trial which showed that transdermal estrogen is an effective treatment for post-natal depression (Gregoire et al., 1996). His most recent study, reported in an as yet unpublished paper, which he was completing at the time of his death, was an open clinical trial of the efficacy of transdermal estrogen in preventing recurrence of affective psychosis after childbirth in women at high risk. The study showed that estrogen did not prevent relapse but women receiving the highest starting dose (800 Ixg/day) needed less medication and had shorter admissions than those receiving lower starting doses (400 and 200 txg per day), suggesting that while estrogen administration may not prevent recurrence of psychosis, it may potentiate the therapeutic response to anti-psychotic drugs. While his research into the causes, impact and treatment of post-natal illness has made a highly significant contribution, his overarching achievement has been that of raising levels of awareness, in the public as well as the medical and academic arenas, about post-natal mental illness and its impact not only on the women who suffer from it but also their new infants and other family members. Indeed he has been one of the key figures in the development of Perinatal Psychiatry as a speciality, not only in the UK but internationally. He also took a leading role in the formation of the Marc6 Society, together with

341

Professor Ian Brockington, Dr Jim Hamilton and others. He was the first vice-president of the Society and then President for 1984-1986, after which he remained actively involved for many years. Channi loved his work. Before he became ill he would be one of the first to be at his desk in the morning and the last to leave at night. Even when his illness necessitated some periods of time in King's College Hospital he continued to work. He had tremendous energy, enthusiasm and determination and was always open to new experiences and challenges. He was a charismatic public speaker and particularly good at explaining his work to nonspecialist audiences. His ability to motivate others was remarkable. He was courteous and charming and people felt at ease with him. He had a way of making colleagues and patients alike feel they were respected, trusted and valued by him. It was these personal characteristics as much as his considerable abilities as a clinician and scientist that contributed to the success of his many national and international projects.

Selected Publications
Clarke, EB.S. and Kumar, R. (1983) The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br. J. Pharmacol., 78: 329-337. Gregoire, A.J.E, Kumar, R., Everitt, B., Henderson, A.E and Studd, J.W.W. (1996) Transdermal oestrogen for severe postnatal depression. The Lancet, 347: 930-933. Kumar, R., Steinberg, H. and Stolerman, I.P. (1968) Inducing a preference for morphine in rats without premedication. Nature, 218: 564-565. Kumar, R. and Stolerman, I.E (1972) Resumption of morphine
self-administration by rats after periods of enforced abstinence: an attempt to modify tendencies to relapse. J. Comp.

Physiol. Psychol., 78: 457-465. Kumar, R., Marks, M.N., Wieck, A., Davies, R.A., Mcivor, R., Brown, N., Papadopoulos, A., Campbell, I.C. and Checkley, S.A. (1997) Neuroendocrine mechanisms in postpartum psychosis and postnatal depression. Biol. Psychiat~, 42(1S): 130-131. Kumar, R., Marks, M.N., Wieck, A., Davies, R.A., Mcivor, R., Brown, A., Papadopoulos, A, Campbell, I.C. and Checkley, S.A. (1998) Neuroendocrine treatments for postpartum psychosis and postnatal depression. Int. J. Neuropsychopharmacol., I(S)1 :Abstract from the XXlst CINP Congress.

343

Subject Index

3c~-hydroxy-5c~-pregnan-20-one s e e allopregnanolone 5c~-pregnan-3c~-ol-20-one s e e allopregnanolone Adipose tissue s e e body fat Adrenal medulla, endogenous opioid synthesis, 69 Adrenocorticotropic hormone (ACTH) control by corticotropin-releasing hormone (CRH), 5, 7, 102-103 diurnal rhythms, 243 levels during parturition, 74, 149 maternal levels in pregnancy, 135-136 placental CRH production and, 9 pregnancy levels, 8, 242 secretion during lactation, 100, 114 stress and, 289, 291 stress hyporesponsiveness, 115 Affiliation bonding in prairie voles, 273 bonding in sheep, 10 oxytocin and, 61 parental bonding and alcoholism, 288 parental bonding and anxiety, 287-288 Aggression s e e maternal aggression Agouti-related protein, effect of suckling, 202 Alcoholism, parental bonding and, 288 Allelic exclusion, 283 s e e genomic imprinting Allopregnanolone action on GABAA receptors, 40-45 anxiolytic effects, 43 cerebral cortex control, 41, 44 hippocampus control, 41, 43 neuronal regulation, 23, 40, 41-43 oxytocin neuron control, 23, 41-43 pregnancy levels, 40 seizures and, 43 synthesis, 40 Alzheimer's disease, estrogen replacement therapy, 303 Amenorrhoea s e e a l s o fertility; fertility suppression; infertility; menstrual cycle dopamine suppression of fertility, 211

energy expenditure and LH secretion, 191-193, 200-201 food deprivation and, 195, 215 induction by breastfeeding, 188, 207-213 lactational amenorrhoea and GnRH, 24, 208, 211 Amnesia s e e a l s o cognition; memory pregnancy and, 15 sex steroids and, 15 Amygdala central nucleus and lactation, 104-108 corticomedial nucleus and prolactin surges, 174 fear responses and, 242 maternal behavior and, 295 medial nucleus and prolactin surges, 6 medial nucleus and stress, 119 M e s t gene expression, 281 opioid receptors, 69 stress and, 119, 289, 290, 291 Analgesia s e e antinociception; pain thresholds Analgesics s e e morphine; pethidine Androgenetic embryos, 279 Androgens s e e a l s o sex steroids; testosterone mood and, 310 sexual differentiation of brain, 305 Angiotensin II receptors, relaxin and, 232, 235-236 Anovulation, s e e amenorrhoea; fertility suppression; menstrual cycle Antidiuretic hormone (ADH) s e e vasopressin Antinociception s e e a l s o pain thresholds c~2-noradrenaline receptors, 90-92 8-opioid receptors, 87, 89-92 dynorphins and, 84, 86, 91 enkephalins and, 91 estrogen receptor and, 92 estrogens and, 86, 92-93 exogenous opioids, 90 gestational, 83-94 hormone-simulated pregnancy (HSP), 86, 89-90, 91-92 hypogastric nerve and, 91-92 ~-opioid receptors, 87, 89-92

344 N-methyl-D-aspartate, 83 non-opioid, 93 opioids and, 9, 24, 86 parturition, 93 pregnancy, 9, 24, 86, 87, 89-92 progesterone and, 83, 92-93 pseudopregnancy, 88 sex differences, 94 sex steroids and, 9, 83 yohimbine-resistant, 92 Anxiety s e e a l s o fearfulness; stress allopregnanolone and, 43 anxiogenic effects of CRH, 245 estrogen and, 146 GABA receptors and, 43 hippocampus and, 43 lactation and, 7-8, 14, 145 maternal behavior and, 294 oxytocin and, 7-8, 146-147, 295 parental bonding and, 287-288 pregnancy and, 7, 144 progesterone and, 43, 146 prolactin and, 148-149 rats, 144-149 reduction during suckling, 7 stress and, 289 testosterone and, 146 Appetite s e e a l s o food intake increase by prolactin, 158, 162-163 lactation and, 16 reduction by leptin, 6-7,216 regulation in pregnancy, 6-7 Arcuate nucleus ~-endorphin neurons and mating, 175-176 galactopoiesis and, 15 lactation and, 15, 16, 216 neuropeptide Y neurons, 216 prolactin receptors, 159, 164 Auditory cues, maternal behavior, 12 [3-endorphin s e e a l s o opioids increase in pregnancy, 77 inhibition of synthesis by estrogens, 76 maternal levels in pregnancy, 135-136 mating-induced prolactin surges and, 175-176 stimulation by progesterone, 75 stimulation of prolactin secretion, 174-176 synthesis, 68-69 ~-endorphin neurons activation by mating, 175-176 daily activity rhythm, 177, 179 regulation by placental lactogens, 177-179 Baby blues s e e maternity blues Bed nucleus of the stria terminalis (BNST) lactation and, 104-108 maternal behavior and, 268, 295 milk ejection and, 20 Behavioral adaptations, peripartum, 144-145, 148149 Behavioral changes, human pregnancy, 14-15 Benzodiazepines, sensitivity of GABAA receptors, 43 Bipolar disorder s e e a l s o depression diagnostic criteria, 323 heritability, 324 prevalence, 323 puerperal psychosis and, 245, 323-325, 326-327, 329 symptoms, 323 Birds, hyperphagic effects of prolactin, 162 Birth, relaxin and, 229-230, 234 Birth canal, relaxation by relaxin, 229-230 Birth weight, maternal factors and, 134 Blood pressure fall in pregnancy, 229 pregnancy-induced hypertension, 237 Blood-brain barrier, prolactin transport, 257 Blues s e e maternity blues Body fat increase in pregnancy, 6-7, 218, 223 lactation and, 16 loss during suckling, 218, 220, 223 Body fluids increase in pregnancy, 6, 16 regulation during lactation, 16 Body temperature, effect of prolactin, 158, 164 Body weight, increase in pregnancy, 6 Bottle feeding maternal stress reaction, 8, 244 mood and, 244, 246 mother-infant interaction and, 243-244 Brain s e e a l s o central nervous system; fetal brain development and genomic imprinting, 280-281 development and steroid hormones, 304 evolutionary remodelling, 280-281 sexual differentiation and androgens, 305 size in pregnancy, 309

345 Brain dysfunction in pregnancy, progesterone and, 45 Brain functioning, sex steroids and, 304-305 Breastfeeding s e e a l s o bottle feeding; lactation; milk ejection; suckling fertility suppression, 207-213 induction of anovulation and amenorrhoea, t88 maternal stress reaction, 8, 244 menstruation and, 188 mood and, 244, 246 mother-infant interaction and, 243-244 post-natal depression and, 246 Bromocriptine, inhibition of prolactin release, 256
c - f o s s e e Fos expression Cannibalization of young, rodents, 10 Cardiovascular changes, pregnancy, 229, 237 Catecholamines, placental CRH production and, 9 stress hyporesponsiveness, 244 Cattle s e e cows Central nervous system (CNS) development in human, 131-132, 136-137 developmental stages, 131-132 growth rate, 132 imprinted genes, 281-282, 283 Cerebral cortex allopregnanolone control of, 41, 44 GABAA receptor expression, 44 number of neurons in human, 131 Cerebrospinal fluid (CSF), prolactin levels, 157, 165 Cervical mechanostimulation induction of maternal behavior, 60, 61 pain thresholds and, 93 prolactin surges, 3, 74 Cervix, relaxation by relaxin, 229-230 Child abuse, intergenerational transmission, 291 Child development, parental care and, 287-288 Cholecystokinin (CCK) maternal behavior and, 14 satiety factor, 216 Chorionic gonadotrophin action on brain, 5 action on corpus luteum, 3 implantation and, 3 increase in pregnancy, 3-5 structural similarity to other hormones, 4 Chorionic somatomammotropin s e e placental lactogens

Choroid plexus prolactin binding, 157 prolactin receptors, 159, 165, 258 Chronic degenerative diseases, prenatal origins and, 139 Circumventricular organs, relaxin action and, 232233, 234-235 Cognition s e e a l s o memory changes in pregnancy, 15, 303-309, 311-314 changes post partum, 14-15 dehydroepiandrosterone (DHEA) and, 306 estrogens and, 25 long-term effects of pregnancy, 311-312 mood and, 309, 311 post partum, 14-15, 311 sex steroids and, 305-307, 312-313 Cognitive development, parental factors, 288 Concaveation s e e a l s o maternal behavior rat, 12, 294 Contraception, Lactational Amenorrhoea Method, 208 Corpus luteum chorionic gonadotrophin action, 3 maintenance by prolactin, 191 progesterone secretion, 3, 6 resumption of function after birth, 208, 212 Corticosteroid receptors, stress hyporesponsiveness and, 117-118 Corticosteroid-binding globulin (CBG), lactation and pregnancy and, 117 Corticosteroids, control by hypothalamo-pituitaryadrenal (HPA) axis, 111-112 Corticosterone s e e a l s o cortisol; glucocorticoids lactation and, 7, 112-114 parturition and, 74, 149 pregnancy and, 7 pulsatile secretion pattern, 112-113 Corticotropin s e e adrenocorticotropic hormone (ACTH) Corticotropin-releasing factor (CRF) s e e corticotropin-releasing hormone (CRH) Corticotropin-releasing hormone (CRH) s e e a l s o paraventricular nucleus activation of noradrenergic systems, 242 anxiogenic effects, 245 control of adrenocorticotropic hormone (ACTH), 5, 7, 102-103 depressive illness and, 26

346 expression in paraventricular nucleus (PVN) neurons, 101-102 fearfulness and, 295 hypothalamo-pituitary-adrenal (HPA) axis and, 26, 241-242 inhibition by endogenous opioids, 68, 73 lactational hyporesponsiveness to stress, 115 luteinizing hormone (LH) suppression in fasting animals, 198-200 maternal behavior and, 295 maternal separation and, 290 mood and, 289 mRNA levels in lactation, 100, 104-105, 107-108, 113-114 neurons, endogenous opioid synthesis and, 68 parvocellular PVN neuron secretion, 103 pituitary sensitivity in pregnancy and lactation, 100-101,102-104 placental, 9, 133, 135-136 post-natal depression and, 25-26 post-partum activity, 243 postnatal handling and, 290, 291 pre-term delivery and, 9 pregnancy levels, 135-136, 243 reduced response to in pregnancy, 73 regulation of uterine contractility, 5 stress and, 289-290, 291 synthesis, 241 Cortisol s e e a l s o corticosterone; glucocorticoids diurnal rhythms, 243 placental CRH production and, 9 pregnancy levels, 135-136, 242 Cows, inhibition of return of estrus by suckling, 188 ~-opioid receptors, gestational antinociception and, 87, 89-92 Decidual prolactin-like protein, humans, 155-156, 165 Dehydroepiandrosterone (DHEA) s e e a l s o androgens; testosterone cognitive effects, 306 mood and, 313 neuroactive derivatives, 45 pregnancy levels, 307-308 Delivery s e e parturition; pre-term delivery; prematurity 2-Deoxyglucose, suppression of luteinizing hormone (LH) secretion, 200 Depression s e e a l s o bipolar disorder; mood; mood changes; post-natal depression adolescent girls, 306 corticotrophin-releasing hormone (CRH) and, 26 estrogens and, 306 gamma-aminobutyric acid (GABA) and, 305 parental bonding and, 287-288 post-natal, 25, 309-312 pregnancy, 309-310 serotonergic system development and, 290 stress and, 289 testosterone in women and, 306 weaning and, 246 Development control of cellular events by allelic exclusion, 283 effects of stress, 132 role of environment, 132 Diabetes quality of parental care and, 287-288 stress and, 289, 297 Dipsogenic response s e e drinking Diurnal rhythms adrenocorticotropic hormone (ACTH), 243 ~-endorphin neurons, 177, 179 cortisol, 243 drinking patterns, 233 feeding patterns, 217 glucocorticoid receptors (GR) and, 117-118 hypothalamo-pituitary-adrenal (HPA) axis, 112113, 117-118 leptin in pregnancy, 219, 220, 223-224 mineralocorticoid receptors (MR) and, 117-118 Dopamine s e e a l s o dopamine neurons female sexual behavior and, 273 fertility suppression and, 211 maternal behavior and, 273 nursing and, 272-274 prolactin secretion and, 175-176, 177, 181,256 pronurturance and, 272-274 Dopamine neurons s e e a l s o dopamine maternal behavior and, 12 mating and, 176-177 prolactin receptors on, 177 regulation by placental lactogens, 177-179 Dopamine systems, puerperal psychosis and, 326 Dorsolateral funiculus, kyphosis and, 272 Dorsomedial nucleus (DMH) mating-induced prolactin surges and, 174, 177

347 prolactin-releasing peptides (PrRP) in, 183 Drinking s e e a l s o body fluids diurnal rhythm, 233 induction by relaxin, 231-233 lactation and, 16 pregnancy and, 229-237 Drug addiction, stress and, 289 Dynorphins, s e e a l s o opioids antinociception in pregnancy, 84, 86, 91 cleavage of prodynorphin by prohormone convertases (PC), 85 effects on hypothalamo-pituitary-adrenal (HPA) axis, 69 pituitary oxytocin and, 70-71 pregnancy levels, 84, 86, 92 regulation of oxytocin secretion, 68 precursor intermediates in lumbar spinal cord, 85 Embryonic diapause, 188; s e e a l s o implantation Embryonic growth imprinted genes and, 10, 283 regulation by M e s t gene, 281 Emotional development, parental factors, 288 Emotional motor system s e e periaqueductal gray Endogenous opioids s e e dynorphins; [3-endorphin; enkephalins; opioids Endorphin s e e [3-endorphin Energy expenditure, suppression of luteinizing hormone (LH) secretion, 191-193, 200-201 Energy regulation, leptin and, 216 Energy requirements, lactation, 193 Enkephalins antinociception in pregnancy, 91 effects on hypothalamo-pituitary-adrenal (HPA) axis, 69 regulation of oxytocin secretion, 68 synthesis, 68 Environment regulation of maternal behavior, 295-298 role in development, 132 Estradiol (17[3-estradiol) s e e a l s o estrogens, sex steroids fertility suppression and, 211 maternal behavior and, 255 menstrual cycle control, 209 neurotrophic effects, 304-305 pre-menstrnal stress and, 313-314 pregnancy levels, 253, 307 suppression of follicle-stimulating hormone (FSH) secretion, 211 suppression of luteinizing hormone (LH) secretion, 211 verbal ability and, 306 Estrogen receptor genes, puerperal psychosis and, 326, 327 Estrogen receptors antinociception and, 92 subfornical organ, 235-236 Estrogen replacement therapy, Alzheimer's disease, 303 Estrogens s e e a l s o estradiol; sex steroids anti-depressant properties, 306 anxiety-related behavior and, 146 cognition and, 25 embryonic diapause and, 188 gestational antinociception and, 86, 88-89, 92-93 implantation and, 188 inhibition of 13-endorphin synthesis, 76 lactation and, 15, 222, 307 luteinizing hormone (LH) suppression in fasting animals, 198-200, 201-202 maternal aggression and, 146 maternal behavior and, 12-13, 252, 254-255, 259, 294 maternity blues and, 310 mood and, 306, 310 oxytocin response to stress, 9 post-natal depression and, 310, 340 post-partum levels, 307 pregnancy levels, 252-253, 307 puerperal psychosis and, 326 relaxin and, 235-236 stress hyporesponsiveness and, 116 synthesis, 304 Estrous cycle food intake and, 217, 220 leptin and, 219-220 F3 glycoprotein, synaptic remodelling, 53 Fasting s e e a l s o food intake inhibition of ovulation, 195 luteinizing hormone (LH) secretion inhibition, 195-200 suppression of gonadotropin-releasing hormone (GnRH) release, 197-198, 201

348 Fatty acid deficiency, suppression of luteinizing hormone (LH) secretion, 201 Fearfulness see also anxiety amygdala and, 242 BALB/c mouse, 293 corticotropin-releasing hormone (CRH) and, 295 glucocorticoids and, 242 maternal responsivity and, 294 non-genomic transmission, 292 Feeding patterns see also food intake orexigenic peptides and, 217 Female sexual behavior see mating Ferguson reflex, parturition, 20-21 Fertility food intake and, 17, 215-216 leptin and, 216, 217, 222-223 suppression by prolactin, 158, 164 Fertility suppression see also amenorrhoea; infertility; lactational amenorrhoea; lactational anestrus; menstrual cycle breast feeding, 207-213 dopamine and, 211 estradiol and, 211 follicle-stimulating hormone (FSH) and, 209-211, 212 gonadotropin-releasing hormone (GnRH) and, 6, 16-17, 164, 209-212 lactation, 16-17, 187-202, 207-213, 222-223 luteinizing hormone (LH) and, 209-211,212 obesity and, 215, 217 opioids and, 211-212 pregnancy, 5-6 progestogens, 6 prolactin and, 17, 212 suckling, 16-17, 207-213 Fetal behavior, human, 136-137 Fetal brain, prolactin and, 165 Fetal development, maternal stress and, 132-133 Fetal growth, maternal social support and, 134 Fetal habituation, human development, 136-137 Fetal neuroplasticity, hypothalamo-pituitary-adrenal (HPA) axis and, 7-8 Fluid balance, pregnancy, 6-7, 16 Follicle-stimulating hormone (FSH) gonadotropin-releasing hormone (GnRH) secretion and, 209-211 induction by leptin, 217 inhibin B secretion and, 211 lack of suppression by lactation, 188-189 lactational fertility suppression, 209-21 l, 212 menstrual cycle control, 209 suppression by estradiol, 211 suppression during pregnancy, 209 Food deprivation amenorrhoea and, 195, 215 delayed puberty and, 215 infertility and, 17, 215 litter size and, 218 neuropeptide Y upregulation, 222 Food hoarding, nucleus accumbens and, 273 Food intake see also appetite; feeding patterns; food deprivation; hyperphagia CCK and satiety, 216 estrous cycle and, 217, 220 fertility and, 215-216 hyperphagic effects of prolactin, 162 lactation and, 16, 217-219, 220, 221-222, 224 leptin and, 216, 220, 221-223 neuropeptide Y and, 222 orexigenic peptides and feeding, 217 pregnancy and, 217-219, 220-222, 224 prolactin and, 16 reproductive cycle and, 217-219 Fos expression maternal behavior and c-fos, 11-12 maternal behavior and Fos B, 10-11, 62-63 mating and, 174-177 parturition and c-fos, 21, 22-23 prolactin surges and, 174-177, 179 stress hyporesponsiveness and c-fos, 118-120, 121 y-aminobutyric acid (GABA) see also GABA synapses; GABAA receptors; GABAergic afferents burst firing of oxytocin neurons and, 20 depression and, 305 inhibition of oxytocin neurons, 54 inhibition of parturition, 22-23 kyphosis and, 272 memory and, 304 stress hyporesponsiveness and, 244 GABA synapses frequency in supraoptic nucleus (SON), 51 sprouting in adult neuronal systems, 52 GABAA receptors c~1 subunit mRNA expression in pregnancy, 42

349 c~4 subunit, seizures, and anxiety, 43 allopregnanolone action on, 23, 40-45 anxiety and, 43 benzodiazepine sensitivity of, 43 cerebral cortex expression, 44 channel open time, 40 oxytocin neuron inhibition, 54 parturition and, 23, 42 phosphorylation and allopregnanolone sensitivity of, 43 pregnancy and, 23, 42-44 regulation by progesterone, 42-43 GABAergic afferents hypothalamic magnocellular nuclei, 51 oxytocin neurons, 19, 42, 51, 54 Galactopoiesis arcuate nucleus and, 15 glucocorticoids and, 113 Gamma-aminobutyric acid see y-aminobutyric acid (GABA) Gene knockout mice inhibition of maternal behavior, 259-260 oxytocin knockout, 14, 17-18, 25, 61-63 Genetics, maternal behavior, 10-11 Genomic imprinting see also Peg3 gene brain development, 280-281 embryonic growth and, 283 mammalian development, 279-281 maternal behavior and, 10, 281-282 maternal brain and, 279-284 Mest gene, 281 parental conflict theory, 280, 282, 283-284 Peg3 gene, 14, 281-282 similarities to allelic exclusion, 283 theories of, 280, 282-284 Gestation see pregnancy Gestation length, maternal stress and, 134 Gestational antinociception see antinociception Glial elements, synaptic remodelling and, 19, 52-53 Glomerular filtration, increase in pregnancy, 229 Glucocorticoid receptor mRNA expression, non-genomic transmission, 292 Glucocorticoid receptors (GR) diurnal rhythm and, 117-118 lactation and, 117-118 Glucocorticoids see also corticosterone; cortisol fear responses and, 242 galactopoiesis and, 113 neonatal brain programming, 113 pregnancy levels, 242 regulation of hypothalamo-pituitary-adrenal (HPA) axis activity, 117-118 secretion during lactation, 101-102 Glucokinase, brain glucose sensor, 201 Glucose deficiency, suppression of luteinizing hormone (LH) secretion, 200-201 Glucose sensor, 200-201 Glutamatergic synapses frequency in supraoptic nucleus, 51-52 immunocytochemistry,51 oxytocin neurons, 52, 54 vasopressin neurons, 52 Gonadotropin-releasing hormone (GnRH) fertility suppression, 6, 16-17, 164, 209-212 follicle-stimulating hormone (FSH) secretion and, 209-211 induction by leptin, 217 inhibition by fasting, 197-198 inhibition by lactation, 10, 16-17 lactational amenorrhoea and, 24, 211 luteinizing hormone (LH) secretion and, 189, 191, 193, 209-211 menstrual cycle control, 209 ovulation blockage and, 10, 189 pulsatile secretion, 189 suppression by suckling, 16-17, 190, 211,212 suppression during pregnancy, 209 Gonadotropins see also follicle-stimulating hormone (FSH); luteinizing hormone (LH) blockade of ovulation, 188 embryonic diapause and, 188 inhibition of secretion by progesterone, 191 Heart, effects of relaxin, 231 Heart disease quality of parental care, 287-288 stress and, 289 Hippocampus allopregnanolone control of, 41, 43 anxiety and, 43 seizure threshold and, 43 Hippocrates, recognition of puerperal psychosis, 333-334 Hormone-simulated pregnancy (HSP) antinociception, 86, 89-90, 91-92 pain thresholds, 88-89, 91-92

350 'Hormones of motherhood' s e e a l s o oxytocin; prolactin; sex steroids post-partum adaptations and, 9-10 Human development, fetal habituation, 136-137 Human fetus, learning ability, 136-137 Humans amenorrhoea and food deprivation, 215 central nervous system (CNS) development, 131-132, 136-137 cognitive changes in pregnancy, 14-15 decidual prolactin-like protein, 155-156 delayed puberty and food deprivation, 215 fetal behavior, 136-137 hyperphagia in pregnancy and lactation, 218 hypothalamo-pituitary-adrenal (HPA) axis, 8-9 leptin levels during pregnancy and lactation, 219-220 neuron number in cerebral cortex, 131 placental lactogens, 155 stress and development, 137 stress and pre-term delivery, 9 3c~-hydroxy-5~-pregnan-20-one s e e allopregnanolone Hyperphagia s e e a l s o appetite; food intake birds, 162 lactation, 217-219 paraventricular nucleus (PVN) and, 162-163 pregnancy, 217-219 prolactin and, 158, 162-163 Hyperprolactinaemia s e e a l s o prolactin induction by suckling, 156-157, 165 lactation, 156-157, 165 mood and, 165-166 pregnancy, 157, 165 tyrosine hydroxylase induction, 177 Hypervigilance, stress and, 288 Hypogastric nerve gestational antinociception and, 91-92 nociception, 93 Hypothalamo-pituitary-adrenal (HPA) axis basal activity, 112-114, 117-118 corticosteroid production, 111-112 corticotropin-releasing hormone (CRH) and, 241-242 diurnal rhythm, 112-113, 117-118 effects of maternal psychosocial processes, 134135 effects of vasopressin, 244-245, 246 endogenous opioids and, 68-69 fetal programming and, 7-8 glucocorticoid regulation of, 117-118 human, 8-9 lactation and, 7-8, 10, 112-115, 117-118, 120122 mood and, 306 noradrenaline and, 121 noradrenergic modulation during lactation, 120122 opioid receptors in, 68-69 oxytocin and, 7-8, 122-123, 147, 149, 244-245, 246 parturition and, 67, 74, 143, 149-150 peripartum responses, 114-115, 122-123 placental corticotropin-releasing hormone (CRH) and, 135-136 post-natal depression and, 25-26 post-partum resetting, 26, 243 pregnancy and, 7-8, 26, 73-74, 112, 117-118, 242-243 prenatal programming, 8 stimulation by suckling, 113 stress and, 7-8, 10, 243, 245, 289-290, 297 stress hyporesponsiveness, 7, 112, 115, 116-119, 143-144, 147-148 stress hyporesponsiveness in lactation, 7, 100-108 timing of delivery, 9-10 Hypothalamo-pituitary-gonadal axis, endocrine aspects of suppression, 188-191 Hypothalamus maternal behavior and, 11 M e s t gene expression, 281 prolactin receptor expression, 159-161, 165 prolactin-releasing peptides (PrRP) in, 183 stimulation by suckling, 193 Illness, stress and, 288-289, 297 Immediate-early gene s e e Fos expression Immune system, stress and development, 8 Implantation s e e a l s o embryonic diapause chorionic gonadotrophin and, 3 delayed, 188,223 estrogen and, 188 lactational inhibition, 188, 223 progesterone and, 3, 188 prolactin and, 188 suppression by suckling, 188 Imprinted genes s e e genomic imprinting

351 Infant behavior, prenatal stress and, 137, 138 Infant morbidity, interbirth interval and, 208 Infertility see also amenorrhoea; fertility suppression; menstrual cycle food deprivation and, 17,215 gonadotrophin-releasing hormone (GnRH) and, 16-17 lactational, 16-17, 222-223 obesity and, 215, 217 prolactin and, 17 reversal by leptin, 217 Inhibin B, follicle-stimulating hormone (FSH) secretion and, 209, 211 Insulin, homology to relaxin, 5 Interbirth interval, infant morbidity and mortality, 208 Interleukin-2, monoallelic expression and regulation, 283 International Classification of Diseases (ICD), puerperal psychosis, 333-334 K-opioid receptors gestational antinociception and, 87, 89-92 oxytocin neurons, 68 Kumar, Ramesh ('Channi') 339-341 Kyphosis see also maternal behavior; nursing behavior dopamine and, 273 dorsolateral funiculus and, 272 GABAergic regulation, 271-272 gamma-aminobutyric acid (GABA) and, 272 nucleus accumbens and, 273-274 nursing behavior, 10, 11-12, 264, 269-272 periaqueductal gray and, 11-12, 269-272 spino-mesencephalic tract and, 272 Kyphotic nursing see kyphosis Labour see parturition Lactation see also breastfeeding; milk ejection; nursing behavior; suckling adipose tissue and, 16 adrenocorticotropic hormone (ACTH) secretion, 100, 104, 114 aggression and, 10, 12, 14, 145 anxiety and, 7-8, 14, 145 appetite and, 16 arcuate nucleus and, 15, 16, 216 bed nucleus of the stria terminalis (BNST) and, 104-108 body fluid regulation, 16 central nucleus of the amygdala (CeA) and, 104-108 corticosteroid feedback regulation, 117 corticosteroid-binding globulin (CBG) and, 117 corticosterone and, 7, 112-114 corticotropin-releasing hormone (CRH) and, 100-101,102-105, 107-108, 113-114 delayed implantation and, 223 drinking and, 16 energy requirements, 193 estrogen and, 15, 222, 307 fertility suppression, 16-17, 187-202, 207-213 food intake, 16, 217-219, 220, 221-222, 224 genomic imprinting and, 282 glucocorticoid receptors (GR) and, 117-118 glucocorticoids and, 100, 101 gonadotropin-releasing hormone (GnRH) inhibition, 10, 16-17 hyperphagia, 217-219 hyperprolactinaemia, 156-157, 165 hypothalamo-pituitary-adrenal (HPA) axis and, 7-8, 10, 100-108, 112-115, 117-118, 120-122 implantation inhibition, 188 inhibition of ovarian cyclicity, 188 initiation, 10, 15 lack of follicle-stimulating hormone (FSH) suppression, 188-189 leptin and, 219-224 limbic CRF neurons and, 104-108 luteinizing hormone (LH) suppression, 188-189, 191-193 magnocellular oxytocin system, 102 maintenance by suckling, 15-16 maternal aggression and, 145 metabolic resetting, 16 mineralocorticoid receptors (MR) and, 117-118 mood changes, 9-10, 14-15 neuroendocrine stress responses, 99-108 neuropeptide Y expression, 222 ovulation suppression, 188-189 oxytocin and, 8, 10, 16-20, 22, 25, 54, 104, 245 paraventricular nucleus (PVN) neurons and, 100-102 parvocellular PVN expression of vasopressin, 101 Peg3 gene and, 14, 25,282

352 pituitary sensitivity, 100-101,102-104 placental lactogen and, 15 post-partum depression and, 245-246 progesterone and, 15 prolactin and, 10, 15-16, 156-157, 179-183, 246 prolactin receptors and, 159-161,165 reduction of startle reflex, 100, 104 relaxin and, 16 stress and, 7-8, 243-244 stress hyporesponsiveness, 7, 99-108, 114-117, 118-123, 244 suppression of luteinizing hormone (LH) secretion, 191-193 synaptic plasticity and, 49-54 tuberoinfundibular dopaminergic (TIDA) neurons and, 15-16 tyrosine hydroxylase and, 156, 179 vasopressin and, 16, 100-104, 114 Lactational amenorrhoea s e e a l s o amenorrhoea; fertility suppression; lactational anestrus contraception and, 208 gonadotropin-releasing hormone (GnRH) and, 211 pregnancy and, 208 Lactational anestrus s e e a l s o amenorrhoea; fertility suppression; lactational amenorrhoea energy demand and, 193 luteinizing hormone (LH) and, 193-201 metabolic pathways, 190-191,201-202 non-metabolic pathways, 190-191, 201-202 rat model, 191 suckling and, 187-202 Lactational infertility, leptin and, 222-223 Lactogenic hormones s e e a l s o decidual prolactin-like protein; placental lactogens; prolactin maternal behavior and, 12-13, 255-259 Lateral habenula, maternal behavior and, 268 Lateral septum, maternal behavior and, 268 Learning s e e a l s o cognition; memory ability, human fetus, 136-137 explicit, pregnancy and, 308 incidental, pregnancy and, 308 Leptin access to brain, 216 appetite reduction, 6-7, 216 diurnal rhythm in pregnancy, 219, 220, 223-224 effect of suckling, 202 energy regulation and, 216 estrous cycle and, 219-220 fertility and, 216, 217, 222-223 follicle-stimulating hormone (FSH) induction, 217 food intake and, 216, 219, 220-222 gonadotropin-releasing hormone (GnRH) induction, 217 infertility and, 222-223 lactation and, 219-224 luteinizing hormone (LH) induction, 217 mechanism of action, 215-217 neuropeptide Y inhibition, 216, 222 orexigenic neuropeptide inhibition, 216-217, 222 parturition and, 219, 220 pregnancy and, 219-222 puberty induction, 217 resistance to in pregnancy, 221 reversal of infertility in obese mice, 217 satiety factor, 216-217, 221 sites of synthesis, 220-221 Leptin receptors distribution in brain, 216 mutations in obese mice, 216 Limbic CRF neurons, changes during lactation, 7, 104-108 Limbic motor system s e e periaqueductal gray Limbic system, maternal behavior and, 11 Litter size, food deprivation and, 218 Locus coeruleus, stress and, 289, 291 Lordosis s e e a l s o mating GABAergic regulation, 271-272 periaqueductal gray and, 269-272 Lumbar spinal cord increased dynorphin in pregnancy, 84-86 prohormone convertase 2 (PC2) levels in pregnancy, 85 Luteinizing hormone (LH) blockade of ovulation, 188 energetic regulation of release, 190-202 estradiol and, 209, 211 gonadotropin-releasing hormone (GnRH) secretion and, 209-211 induction by leptin, 217 inhibition by fasting, 195-200 lactational anestrus and, 193-201 lactational fertility suppression, 209-211, 212 lactational suppression, 191-193 menstrual cycle control, 209 ovulation blockage, 189 pulsatile secretion, 189, 191,193

353 suppression after birth, 209-211 suppression by suckling, 188-189, 190, 191-193, 201 suppression during pregnancy, 209 suppression of release by estrogen, 198-200, 201-202 Luteotrophic function, prolactin and, 154 tx-opioid receptors enhancement by progesterone, 75-76 mating-induced prolactin surges and, 175-176 oxytocin neurons, 68 Magnocellular neurons see also oxytocin neurons; vasopressin neurons burst firing of oxytocin neurons, 20 F3 glycoprotein, 53 GABA synapses, 51 GABAergic afferents, 51 glutamatergic afferents, 51-52 lactation and, 102 milk ejection and, 20, 24 parturition and, 22, 24 shared synapses, 50 synaptic plasticity, 50, 52-53 Mammalian development, genomic imprinting, 279-281 Mammary glands, leptin production, 220-221 Mania see bipolar disorder; puerperal psychosis Marsupials, lactational inhibition of implantation, 188 Maternal aggression estrogen and, 146 lactation and, 10, 12, 14, 145 mouse, 265 periaqueductal gray and, 269-272 progesterone and, 146 rat, 144-145, 264-265 suckling and, 265 testosterone and, 146 Maternal behavior see also maternal aggression; maternal care; mother-infant interactions; nursing behavior; parental care; pronurturance amygdala and, 295 anxiety and, 294 bed nucleus of the stria terminalis (BNST) and, 268, 295 behavioral changes, 14-15 cognitive changes, 14-15 corticotropin-releasing hormone (CRH) and, 295 cues from pups, 264, 295 dopamine and, 12, 273 environmental regulation, 295-298 estrogens and, 12-13, 252, 254-255, 259, 294 Fos B and, 10-11, 62-63 GABAergic regulation, 271-272 genetic basis, 10-11 genomic imprinting and, 10, 281-282 hypothalamus and, 11 imprinting of Mest gene, 10, 281 imprinting of Peg3 gene, 14, 25, 281-282 induction by offspring, 12 induction by pregnancy hormones, 254-255 inhibition, 255, 259-260 intergenerational transmission, 291-293 lactogenic hormones and, 12-13,255-259 lateral habenula and, 268 lateral septum and, 268 limbic system and, 11 maintenance, 260 medial preoptic area (MPOA) and, 11-13, 60-61, 258, 259, 268-269, 294-295 medio-basal hypothalamus and, 61 Mesh gene and, 62-63 modification by postnatal handling, 290-291,292 mouse, 14, 24-25, 61-63,259-260 neonatal care, 10-15 neural basis for differences, 294-295 neural circuitry, 11-12 non-genomic inheritance, 287-298 nucleus accumbens and, 12, 268 olfactory bulb and, 60, 61 olfactory cues, 12, 21-22 onset, 252, 254-260 oxytocin and, 12-14, 24-25, 59-64, 282, 294-295 paraventricular nucleus (PVN) and, 60-61,295 Peg3 gene and, 25 periaqueductal gray and, 11-12, 268, 271 placental lactogens and, 252, 254, 258-259, 294 prepubertal rats, 251 primary somatosensory cortex (SI) and, 268 progesterone and, 12-13, 252-255, 259, 294 prolactin and, 10, 13, 148-149, 157-158, 162, 252, 254-259, 294 prolactin receptors and, 62-63, 259-260 pup retrieval, 264, 268, 273 rat, 10-14, 60, 62, 251,252-259, 260

354 sensory cues from offspring, 12 sex steroids and, 10, 12-13 sheep, 10, 12-14, 24, 60-61, 62 tocinoic acid and, 60, 61 vagino-cervical stimulation and, 60, 61 vasopressin and, 60 ventral striatum and, 12 ventral tegmental area and, 60 ventromedial nucleus and, 258-259 virgin rats, 251,255, 256-257, 259 Maternal care see also maternal behavior; motherinfant interactions; nursing behavior; parental care; pronurturance environmental effects, 297 maternal responsivity and, 294 Maternal psychosocial processes birth outcomes and, 134, 138 effects on hypothalamo-pituitary-adrenal (HPA) axis, 134-135 neuroendocrine axis and, 134-135 prematurity and, 138 Maternal responsivity fearfulness and, 294 rat, 294 Maternal separation ascending serotonergic system development and, 290 corticotropin-releasing hormone (CRH) and, 290 stress responses, 289-290 Maternal social support, fetal growth and, 134 Maternal stress see also stress hyporesponsiveness fetal development and, 132-133 infant birth weight and, 134 length of gestation and, 134 Maternal-fetal communication, role of placenta, 133 Maternity blues see also depression; mental illness; post-natal depression characteristics, 334 estrogens and, 310 frequency, 321,322 post-partum corticotrophin-releasing hormone (CRH) suppression and, 243 progesterone and, 310 symptoms, 321,322 Mating ~-endorphin neurons and, 175-176 blockage of prolactin increase by naloxone, 175 dopamine neurons and, 176-177, 273 establishment of pregnancy in rat, 3 immediate-early gene expression and, 174-177 induction of prolactin surges, 6 Medial preoptic area (MPOA) maternal behavior and, 11-13, 60-61,258-259, 268-269, 294-295 mating-induced prolactin surges and, 174, 177 Mest gene expression, 281 prolactin receptors, 159, 160 pronurturance and, 267, 268-269 pup retrieval and, 267, 268 relaxin action and, 232, 235 suckling-induced quiescence, 272 Medio-basal hypothalamus, maternal behavior and, 61 Medulla oblongata, prolactin-releasing peptides (PrRP) in, 183 Melanin-concentrating hormone, 202 Memory see also amnesia; cognition; learning gamma-aminobutyric acid (GABA) and, 304 peri-partum deficit, 309 pregnancy and, 25, 308-309 semantic, 309, 311 stress and, 288 verbal episodic, long-term effects of pregnancy, 311-312 Menstrual cycle lactational inhibition, 188 normal control, 209 proportions of sex steroids, 5 resumption after birth, 208, 209 resumption after suckling, 209 seizure threshold, 43 Menstruation breastfeeding and, 188 lactational inhibition, 188 Mental illness see also bipolar disorder; post-natal depression; puerperal psychosis pregnancy-related, 10, 25-26 quality of parental care and, 287 Mesh gene, maternal behavior and, 62-63 Mest gene genomic imprinting, 281 maternal behavior and, 10, 281 regulator of embryonic growth, 10, 281 Metabolic resetting, lactation, 16 Metabolism, effects of progesterone, 305 Microtus ochrogaster see prairie voles

355 Milk ejection see also breastfeeding; lactation; oxytocin BNST (bed nuclei of the stria terminalis) and, 20 burst firing of oxytocin neurons, 70 genomic imprinting and, 14, 282 inhibition by relaxin, 231,232, 234 inhibition by stress, 245 magnocellular neurons and, 20, 24 nursing posture and, 269 oxytocin and, 20, 54, 62, 272, 282 Peg3 gene and, 14, 25, 282 suckling and, 264, 272 Milk production see galactopoiesis Mineralocorticoid receptors (MR) diurnal rhythm and, 117-118 lactation and, 117-118 Monkeys lactational anestrus, 188 mother-infant interactions, 297 Mood see also bipolar disorder; depression; mood changes; post-natal depression androgens and, 310 bottle feeding and, 244, 246 breastfeeding and, 244, 246 cognition and, 309 corticotropin-releasing hormone (CRH) levels, 289 dehydroepiandrosterone (DHEA) and, 313 effects of sex steroids, 305-307, 310, 312-314 hypothalamo-pituitary-adrenal (HPA) axis and, 306 post partum, 39, 311 pregnancy and, 39, 303-308, 309-314 progesterone and, 39, 305, 310, 313-314 prolactin and, 165-166, 246 steroid hormones and, 15 Mood changes see also mood lactation, 9-10, 14-15 oxytocin and, 15 pregnancy, 9-10, 14-15 sex steroids and, 15 Morbidity, infant, interbirth interval and, 208 Morphine, obstetric analgesia, 71-72 Mortality, infant, interbirth interval and, 208 Mother-infant interactions see also maternal behavior; maternal care; nursing behavior; parental care; pronurturance breastfeeding and, 243-244 monkeys, 297 oxytocin release, 242 Motilin, 202 Mouse see also rat; rodents fearfulness in BALB/c, 293 infertility in obese, 217 lactational inhibition of implantation, 188 maternal aggression, 265 maternal behavior, 14, 24-25, 259-260 oxytocin knockout, 14, 17-18, 25, 61-63 oxytocin and maternal behavior, 14, 24-25, 61-63, 64 prolactin receptor gene knockout, 259-260 prolactin receptor isoforms, 158 Naloxone acceleration of parturition, 24, 71, 72, 74 inhibition of opioids, 24, 69, 70, 71 no effect on lactation, 75 oxytocin secretion and, 24, 71, 74 prolactin blockage, 175, 180 Naltrexone, abolition of gestational antinociception, 86 Neonatal brain programming, glucocorticoids and, 113 Neonatal care see maternal behavior Neonatal morbidity, prematurity and, 138 Neurohypophysial system, endogenous opioids, 68-69 Neurological disorders, prenatal origins, 138-139 Neuropathic pain, insensitivity to opioids, 93 Neuropeptide Y arcuate nucleus, 216 food intake and, 222 inhibition by leptin, 216, 222 lactation and, 222 obesity and, 216 suckling and, 102, 202 upregulation by food deprivation, 222 Neurosteroids see allopregnanolone; dehydroepiandrosterone; pregnenolone Neurotrophic effects, steroid hormones, 304-305 Nipples, relaxin and development of, 230 Nitric oxide, parturition and, 23-24 Nociception see also antinociception; opioids; pain thresholds hypogastric nerve and, 93 inhibition of oxytocin neurons, 69 Non-genomic inheritance

356 corticotropin-releasing hormone (CRH) levels, 292 fearfulness, 292, 293 glucocorticoid receptor mRNA expression, 292 interaction with genetic factors, 293 maternal behavior, 287-298 spontaneously hypertensive rat (SHR), 292-293 Noradrenaline a2-receptors, and gestational antinociception, 90-92 hypothalamo-pituitary-adrenal (HPA) axis and, 120-122 stimulation of oxytocin secretion, 54 stress and, 120-122, 289 Noradrenergic afferents oxytocin neurons, 19, 52, 54 paraventricular nucleus (PVN) and supraoptic nucleus (SON), 52 Noradrenergic neurons luteinizing hormone (LH) suppression in fasting animals, 197-200 prolactin-releasing peptides (PrRP) in, 183 Novelty, responses to and postnatal handling, 291 Nucleus accumbens food hoarding and, 273 kyphotic nursing and, 273-274 maternal behavior and, 11, 12, 268 pronurturance and, 273-274 pup retrieval and, 273 Nucleus of the solitary tract (NTS) luteinizing hormone (LH) suppression in fasting animals, 197-200 parturition and, 21, 22-23 prolactin-releasing peptides (PrRP) in, 181, 183 Nursing behavior s e e a l s o kyphosis; lactation; maternal behavior; pronurturance active role of young, 264 dopamine and, 272-274 forebrain regulation, 267-269 kyphosis, 10, 11-12, 264, 269-272 periaqueductal gray and, 11-12, 269, 271 posture, milk ejection and, 10, 269 quiescence during, 264, 267, 269, 272 rat, 10, 264-274 satiety or hunger of pups, 267 somatosensory determinants, 264-267 Obesity s e e a l s o body fat infertility and, 215 neuropeptide Y and, 216 Olfactory bulb activation in parturition, 21-22 maternal behavior and, 60, 61, 63-64 oxytocin receptors, 63-64 prolactin receptors, 165 Olfactory signalling maternal behavior and, 12, 21-22 parturition, 21-22 Olfactory system development, prolactin and, 165 Opiates s e e morphine; pethidine Opioid gene expression paraventricular nucleus (PVN), 76-77 parturition and pregnancy, 76-77 supraoptic nucleus (SON), 76-77 Opioid receptors s e e a l s o 3-opioid receptors; K-opioid receptors; ~-opioid receptors amygdala, 69 hypothalamo-pituitary-adrenal (HPA) axis, 68-69 neurohypophysial system, 68-69 Opioids s e e a l s o ~-endorphin; dynorphins; ~-endorphin; enkephalins antinociception in pregnancy, 9, 24, 86, 90 CRH neurons, actions on, 68 fertility suppression and, 24, 211-212 hypothalamo-pituitary-adrenal (HPA) axis, 6869, 73-74 increase of delivery time, 7 l, 72 naloxone inhibition, 69, 70, 71 neurohypophysial system, 68-69 oxytocin neurons, control of, 24, 69-73 oxytocin secretion and, 9, 24 pregnancy and, 70-71, 73-74, 76-77 pregnancy analgesia, 9 restraint of oxytocin neurons, 24 sex steroid interactions, 75-76 sites of synthesis, 68 suckling-induced prolactin secretion, 24, 180 Orexigenic neuropeptides feeding induction in rat, 217 inhibition by leptin, 216-217, 222 Organum vasculosum of the lamina terminalis (OVLT) relaxin action and, 235 relaxin binding sites, 232, 234 Osmotic stimuli, synaptic plasticity, 49 Ovulation s e e a l s o amenorrhoea; fertility; fertility suppression; infertility; menstrual cycle

357 inhibition by fasting, 195 lactational inhibition, 16-17, 188-189 suppression by suckling, 188 Oxytocin s e e a l s o oxytocin neurons anxiety and, 7-8, 146-147, 295 anxiolytic action, 146-147, 245 corticotropin (ACTH) secretion during lactation and, 104 dynorphin regulation, 68 effect of opioids on secretion, 9, 24 enkephalin regulation, 68 gene expression and progesterone, 18 hypothalamo-pituitary-adrenal (HPA) axis and, 7-8, 122-123, 147-149, 244-246 increase of secretion by naloxone, 71, 75 inhibition of secretion by enkephalins, 68 lactation and, 8, 10, 16-20, 22, 25, 54, 245 maternal behavior and, 12-14, 24-25, 59-64, 282, 294-295 milk ejection and, 20, 54, 62, 272, 282 mood changes and, 15 naloxone control of secretion, 24 parturition and, 9, 17-24, 62, 67, 71-73, 75 pituitary dynorphins and, 70-71 pregnancy and, 8-9, 16, 41-42, 70, 73-75 production in posterior pituitary, 18 prolactin and, 10, 19, 158, 164 reproduction and, 17-18 sexual receptivity and, 61, 63 sites of synthesis, 242 social behavior and, 61, 63 social memory in mice, 63 species differences in function, 63-64 stimulation of secretion by noradrenaline, 54 stress and, 9, 73, 74-75, 122, 245 stress hyporesponsiveness and, 122-123, 144, 147-148, 244 structure, 59 suckling and, 19, 20, 122, 272 synaptic remodelling and, 53 synthesis, 18 Oxytocin neurons s e e a l s o oxytocin; magnocellular neurons allopregnanolone control of, 23, 41-43 burst firing, 19-20, 24, 42, 54, 70, 75 control by endogenous opioids, 24, 68-73 excitation by relaxin, 72 GABA inhibition of, 54 GABAergic input, 20, 42, 51 glutamatergic synapses, 52 inhibitory post-synaptic currents (IPSCs), 42 ~:-opioid receptors, 68 Ix-opioid receptors, 68 nociceptin inhibition, 69 noradrenergic afferents, 19, 52, 54 parturition and, 41-42, 70, 72 P e g 3 gene and development, 14, 25,282 plasticity in pregnancy, 19 progesterone and, 41-42 synaptic plasticity, 19, 49-54 Oxytocin receptors distribution in central nervous system (CNS), 63-64, 242 expression patterns and flanking sequences, 64 Pain, control during parturition, 9 Pain thresholds s e e a l s o antinociception cervical mechanostimulation and, 93 hormone-simulated pregnancy (HSP), 88-89, 91-92 pregnancy, 86, 87, 90 pseudopregnancy, 88 Paraventricular nucleus (PVN) s e e a l s o corticotropinreleasing hormone corticotropin-releasing hormone (CRH) expression, 101-103, 105, 107-108, 241 endogenous opioid synthesis, 68 hyperphagia and, 162-163 lactation and, 7, 100-102 lactational hyporesponsiveness to stress, 114-115, 118-121 luteinizing hormone (LH) suppression in fasting animals, 198-200 maternal behavior and, 60-61,295 neuropeptide synthesis, 101 noradrenergic afferents, 52 opioid gene expression, 76-77 opioid receptors, 69 opioid synthesis, 68 parturition and, 22, 23 prolactin receptors, 164 shared synapses, 52-53 stress and, 289, 290 synaptic plasticity, 19, 50, 52-54 vasopressin secretion, 103 Parent-child relationships

358 socioeconomic factors, 297-298 stress and, 297 Parental bonding alcoholism and, 288 anxiety and, 287-288 depression and, 287-288 intergenerational transmission, 291 Parental care s e e a l s o maternal behavior; maternal care child development and, 287-288 chronic stress and, 297 diabetes and, 287-288 heart disease and, 287-288 quality and risk of mental illness, 287 Parental conflict theory, genomic imprinting, 280, 282, 283-284 Parthenogenetic embryos, developmental failure in mammals, 279 Parturition s e e a l s o pre-term delivery acceleration by naloxone, 71, 72 adrenocorticotropic hormone (ACTH) levels, 74, 149 antinociception, 9, 93 behavioral activation in rats, 149 burst firing of oxytocin neurons, 24, 70 c - f o s and, 21, 22-23 corticosterone levels, 74, 149 dynorphin levels in spinal cord, 84 effect of relaxin, 72 endogenous opioid control of oxytocin neurons, 71-73 Ferguson reflex, 20 GABAA receptors and, 23, 42 hypothalamo-pituitary-adrenal (HPA) axis and, 67, 74, 149-150 inhibition by GABA, 22-23 inhibition by opioids, 71,72 inhibition by progesterone, 22-23 inhibition by stress, 245 leptin levels and, 219, 220 magnocellular neurons and, 22, 24 nitric oxide and, 23-24 nucleus of the solitary tract (NTS) and, 21, 22-23 olfactory signalling, 21-22 opioid gene expression, 76-77 oxytocin and, 9, 17-24, 41-42, 62, 67, 71-73, 242 pain control, 9 paraventricular nucleus (PVN) and, 22, 23 risk factor for psychiatric disorder, 322 slowing by opioids, 71, 72 stress and oxytocin secretion, 75 supraoptic nucleus (SON) and, 21, 22-23 synaptic plasticity and, 49, 53-54 Peg1 gene s e e M e s t gene P e g 3 gene s e e a l s o genomic imprinting genomic imprinting, 14, 281-282 lactation and, 14, 25,282 maternal behavior and, 14, 25, 281-282 milk ejection and, 14, 25, 282 oxytocin neuron development, 14, 25, 282 tumour necrosis factor (TNF) signalling pathway, 282 Periaqueductal gray kyphotic nursing, 11-12, 269-272 lordosis and, 269-272 maternal aggression and, 269-272 maternal behavior and, 11-12, 268, 271 nursing behavior and, 11-12, 269, 271 proceptivity and, 271 pronurturance and, 271 suckling and, 268, 269, 271,272 Peripartum period anxiolytic action of brain oxytocin, 146-147 behavioral adaptations, 144-145 hypothalamo-pituitary-adrenal (HPA) axis responses, 114-115 Periventricular nucleus, prolactin receptors, 159 Pethidine, obstetric analgesia, 71-72 Photoperiod, effect on drinking, 233 Pigs s e e sows Pituitary, anterior s e e ACTH; gonadotropins; prolactin Pituitary, posterior s e e oxytocin; vasopressin Placenta leptin production, 220 maternal-fetal communication and, 133 overdevelopment in androgenetic embryos, 279 sex steroid production, 304 underdevelopment in parthenogenetic embryos, 279 Placental corticotropin-releasing hormone (CRH) environmental regulation, 133 fetal learning ability and, 137 human pregnancy, 133, 135-136 hypothalamo-pituitary-adrenal (HPA) axis and, 135-136

359 prematurity and, 136, 138 Placental lactogens s e e a l s o decidual prolactin-like protein; lactogenic hormones; prolactin absence in pigs, rabbits and dogs, 155 action on maternal brain, 5, 6 ~-endorphin neuron regulation, 177-179 dopamine neuron regulation, 177-179 human, 155 inhibition of pituitary prolactin secretion, 154 lactation and, 15 maternal behavior and, 252, 254, 258-259, 294 pregnancy and, 5, 154-155, 252, 254 prolactin secretion surges and, 177 prolactin suppression in late pregnancy, 6 rodents, 155 Plasma osmolality, decrease in pregnancy, 229, 230, 237 Plasma volume, expansion in pregnancy, 229 Polysialylated neural cell adhesion molecule (PS-NCAM), synaptic remodelling, 53 POMC, gene expression in pregnancy, 77 Post partum cognitive changes, 14-15, 311 depression, 309-310, 311-312 estrogen levels, 307 mood and, 311 progesterone levels, 307 semantic memory, 311 testosterone levels, 308 Post-natal depression s e e a l s o depression; maternity blues; mental illness breastfeeding and, 246 corticotropin-releasing hormone (CRH) and, 25 -26 estrogen treatment, 340 frequency, 25, 321,322, 334 hypothalamo-pituitary-adrenal (HPA) axis and, 25 -26 lactation and, 245-246 sex steroids and, 25, 310 sleep disruption and, 245 symptoms, 321,322 Post-partum blues s e e maternity blues Post-partum psychosis s e e puerperal psychosis Postnatal handling corticotropin-releasing hormone (CRH) and, 290, 291 endocrine responses, 289-290 modification of maternal behavior, 290-291,292 responses to novelty and, 291 stress responses, 289-290 Prairie voles oxytocin inhibition of hypothalamo-pituitaryadrenal (HPA) axis, 245 pair-bond establishment, 273 Pre-menstrnal stress estradiol and, 313-314 progesterone and, 313-314 Pre-synaptic elements, synaptic remodelling and, 52-53 Pre-term delivery, 9 s e e a l s o prematurity corticotropin-releasing hormone (CRH) and, 9 stress and, 8-9 timing and hypothalamo-pituitary-adrenal (HPA) axis, 9-10 5c~-pregnan-3c~-ol-20-ones e e allopregnanolone Pregnancy s e e a l s o hormone-simulated pregnancy (HSP) adipose tissue accumulation, 7 adrenocorticotropic hormone (ACTH) and, 8 aggression in rats, 144-145 allopregnanolone concentrations, 40 alterations in GABAA receptors, 42-45 amnesia and, 15 analgesia s e e antinociception antinociception, 9, 24, 83-94 anxiety and, 7, 144 appetite regulation, 6, 7, 217-222, 224 13-endorphin increase, 77 behavioral changes, 14-15 body fluid increase, 6, 16 body weight increase, 6 brain dysfunction and progesterone, 45 brain size and, 309 cardiovascular changes, 229 chorionic gonadotrophin increase, 3-5 cognitive changes, 14-15, 303-309, 311-314 corticosteroid-binding globulin (CBG) and, 117 corticosterone and, 7 dehydroepiandrosterone (DHEA) levels, 307-308 depression, 309-310 dynorphin levels, 84-86, 92 effects of relaxin, 72 endocrine state, 242-243, 252-254 endogenous opioids and, 69-71, 76-77 establishment, 3-4

360 estradiol:progesterone ratio, 254 estradiol levels, 253, 307 estrogen levels, 252-253 fertility suppression, 5-6 fluid balance, 6-7, 16 follicle-stimulating hormone (FSH) and, 209 GABAA receptor expression, 44 gonadotropin-releasing hormone (GnRH) and, 209 hyperprolactinaemia, 157, 165 hypothalamo-pituitary-adrenal (HPA) axis and, 7-8, 26, 73-74, 112, 117-118, 135-136, 242-243 leptin and, 219-220, 221-222, 223-224 luteinizing hormone (LH) and, 209 maternal pituitary-adrenal function, 135-136 memory and, 25, 308-309, 311-312 mental illness and, 10, 25-26 metabolic changes, 6-7 mood changes, 9-10, 14-15, 39, 303-308, 309-314 opioid gene expression, 76-77 oxytocin and, 8, 16, 19, 41-42, 69-71, 73, 74-75 pain thresholds, 86, 87, 90 placental corticotropin-releasing hormone (CRH) and, 135-136 placental lactogens and, 5, 154-155, 252, 254 POMC expression, 77 progesterone and, 3-5, 39, 146, 252-253, 307 prohormone convertase 2 (PC2) increase, 85, 86 prolactin and, 154-156, 165,252, 174-177, 254 prolactin receptors and, 159-161,165 reduced response to CRH, 73 relaxin and drinking, 229-237 relaxin increase in, 5 resistance of TIDA neurons to prolactin, 156 seizure threshold, 43 sex steroids and, 3-5, 307-308 sodium retention, 16 stress and, 7-8, 9, 73, 74-75, 100 stress hyporesponsiveness, 7, 115-116 synaptic plasticity and, 19 testosterone levels, 146, 307, 308 uteroplacental signalling, 3-5 Pregnenolone, sulphation and neuroactivity, 45 Prematurity see also pre-term delivery maternal psychosocial processes and, 138 neonatal morbidity and, 138 placental corticotropin-releasing hormone (CRH) and, 136, 138 Prenatal processes chronic degenerative diseases and, 139 neurological disorders and, 138-139 programming, hypothalamo-pituitary-adrenal (HPA) axis, 8 stress, infant behavior and, 137, 138 Primary somatosensory cortex (SI), maternal behavior and, 268 Proceptivity, periaqueductal gray and, 271 Progesterone see also allopregnanolone; sex steroids anti-seizure effects, 43 antinociception and, 83, 88-89, 92-93 anxiety and, 43, 146 cognitive effects, 306 effects on nervous system, 39 embryonic diapause in wallabies, 188 enhancement of ~t-opioid receptors, 75-76 establishment of implantation, 3 fertility suppression during pregnancy, 6 increase of 13-endorphin in hypothalamus, 75 inhibition of gonadotropin secretion, 19 ! lactation and, 15 maternal aggression and, 146 maternal behavior and, 12-13,252-255, 259, 294 maternity blues and, 310 metabolic effects, 305 mood and, 39, 305,310, 313-314 oxytocin gene expression and, 18 oxytocin neurons and, 41-42 oxytocin response to stress, 9 parturition inhibition, 22-23 post-natal depression and, 310 post-partum levels, 307 pre-menstrual stress and, 313-314 pregnancy and, 3-5, 39, 45, 146, 252-253, 307 regulation of GABAA receptors, 42-43 secretion by corpus luteum, 3, 6 stress hyporesponsiveness and, 116 Prohormone convertases (PC), 85, 86 Prolactin see also decidual prolactin-like protein; placental lactogens; prolactin receptors; prolactin secretion surges; prolactin-releasing peptides action of prolactin-releasing peptides (PrRP), 15, 180-183 access to brain, 157, 165 actions in brain, 157-165

361 anxiety-related behavior and, 148-149 appetite increase, 158, 162-163 body temperature and, 158 central nervous system (CNS) access, 157, 165 cerebrospinal fluid (CSF) levels, 157, 165 effect of endogenous opioids on secretion, 180 effects in fetal brain, 165 embryonic diapause in wallabies, 188 establishment of pregnancy in rats, 3 fertility suppression and, 158, 164, 212 food intake and, 16 induction by mating, 6 infertility and, 17 inhibition of release by bromocriptine, 256 inhibition of secretion by placental lactogen, 154 lactation and, 10, 15-16, 156-157, 179-183, 246 luteotrophic function and, 154 maintenance of lactational corpus luteum, 191 maternal behavior and, 10, 13, 157-158, 162, 252, 254-259, 294 medial amygdala and, 6 mood and, 246 naloxone blockage, 175, 180 olfactory system development, 165 oxytocin and, 19, 158, 164 peripartum behavioral adaptations, 148-149 peripartum neuroendocrine adaptations, 148-149 pre-partum surge, 154-155 pregnancy and, 154-156, 165, 252, 254 regulation of maternal behavior, 148-149 regulation of secretion by dopamine, 175-176 REM sleep stimulation, 158 reproductive behavior and, 158 stimulation of secretion by t3-endorphin, 174-176 stress hyporesponsiveness and, 149, 158, 164 suckling and tuberoinfundibular dopamine neurons, 179-180 suckling-induced secretion, 10, 156-157, 164, 179-180, 212 suppression by placental lactogen in late pregnancy, 6 suppression of luteinizing hormone (LH) release, 191 transport across blood-brain barrier, 257 transport into cerebrospinal fluid (CSF), 157 tuberoinfundibular dopamine (TIDA) neurons and, 6, 156, 158-159, 164-165, 175-176, 179-180 Prolactin receptor antagonists, inhibition of maternal behavior, 259 Prolactin receptors brain, 158-161,258 choroid plexus, 159, 165, 258 diestrous rats, 159 dopamine neurons, 177 expression in hypothalamus during pregnancy, 159-161, 165 fetal brain, 165 gene knockout mice, 259-260 hypothalamus, 159-161, 165 isoforms, 158 lactation and, 159-161, 165 maternal behavior and, 62-63,259-260 olfactory bulb, 165 regulation, 161 Prolactin surges dopamine and, 177 Fos expression and, 174-177, 179 induction by mating, 6 medial amygdala (MeA) and, 6 placental lactogens and, 177 pregnancy, 154, 174-177 tuberoinfundibular dopamine (TIDA) neurons and, 177 tyrosine hydroxylase and, 176, 177 vaginocervical stimulation and, 3, 174 Prolactin-releasing factors, 157 Prolactin-releasing peptides (PrRP) colocalization with tyrosine hydroxylase, 183 distribution in brain, 181,183 noradrenergic neurons, 183 nucleus of the solitary tract, 181,183 stimulation of prolactin release, 15, 180-183 Pronurturance see also maternal behavior; maternal care; nursing behavior dead infants, 267 dopamine and, 272-274 forebrain and, 267-269 medial preoptic area (MPOA) and, 267, 268-269 nucleus accumbens and, 273-274 periaqueductal gray and, 271 rat, 264, 265, 267 Pseudopregnancy pain thresholds, rat, 88 Puberty delayed by food deprivation, 215, 217

362 induction by leptin, 217 Puerperal psychosis, 310, 321-329 s e e a l s o mental illness; post-natal depression biological aetiological factors, 325-326 bipolar diathesis, 326, 329 bipolar disorder and, 245, 323-325, 326-327, 329 characteristics, 334-335 dopamine systems and, 326 epidemiological evidence, 335-336 estrogens and, 326, 327 familial factors, 325 frequency, 25, 321-322, 335 genetic factors, 324-325, 326, 327 history, 333-334 mania and, 323-324 and neuroendocrine-neurotransmitterdysfunctions, 336-337 outcome, 324 prospective studies, 336-337 psychosocial factors and, 325 puerperal trigger, 322, 325, 326, 327 recurrence, 324 serotonin systems and, 326 serotonin transporter gene (SERT/5-HTT) and, 327 symptoms, 322 Pup retrieval s e e matemal behavior; nursing behavior Rat s e e a l s o mouse, rodents anxiety-related behavior, 144-149 behavioral activation at parturition, 149 concaveation, 12, 294 delayed implantation, 188, 223 dipsogenic response to relaxin, 231-233 diurnal drinking patterns, 233 diurnal feeding patterns, 217 drinking in pregnancy, 229-237 food intake and puberty, 217 food intake and reproductive cycle, 217-219 hyperphagia, 218-219 hypothalamo-pituitary-adrenal (HPA) axis, 7, 73-75, 122-123 induction of prolactin surges by mating, 6 kyphotic nursing, 264, 269-272 lactation and, 188, 218-219 leptin and food intake in pregnancy, 221 leptin levels during pregnancy and lactation, 219 litter size and food deprivation, 218 maternal aggression, 144-145, 264-265 maternal behavior, 10-14, 251-259, 260 prepubertal, 251 virgin females, 251,255,256-257,259 mating and establishment of pregnancy, 3 model for lactational anestrus, 191 neonatal care, 10 nipple attachment and temperature, 264 nursing behavior, 10, 263-274 oxytocin and maternal behavior, 13, 24, 60, 62, 63 pituitary prolactin secretion, 154-155 placental lactogens, 154 pregnancy and, 3, 154, 174-177, 218-219, 221 prolactin receptor isoforms, 158 prolactin surges in pregnancy, 154, 174-177 pronurturance, 264, 265,267,273 pseudopregnancy, 88 pup retrieval, 10, 264, 268, 273 Rat, spontaneously hypertensive (SHR), epigenetic factors in expression, 292-293 Relaxin action on maternal brain, 5, 231 actions on non-reproductive tissues, 230-231 angiotensin II and, 232, 233, 235,236 binding sites in brain, 232 birth and, 229-230 circumventricular organs and, 232-233, 234-235 dipsogenic response, 231-233 drinking in pregnant rats, 229-237 effects on heart, 231 estrogen and, 235-236 increase of oxytocin secretion, 72 increase in pregnancy, 5 inhibition of milk ejection, 231,232, 234 insulin homology, 5 lactation and, 16 molecular structure, 230, 232 nipple development and, 230 opioid inhibition at parturition, 72 physiological actions, 6, 230, 234-235 pregnancy and, 72, 230 pregnancy-induced hypertension and, 237 regulation of fluid balance in pregnancy, 6, 16 relaxation of cervix and birth canal, 229-230 sites of synthesis, 230, 233,234 sodium retention in pregnancy, 16 species differences, 230 REM sleep, stimulation by prolactin, 158, 164 Reproduction, oxytocin and, 17-18

363 Rodents s e e a l s o mouse; rat; prairie vole cannibalization of young, 10 distribution of oxytocin receptors in brain, 63 food deprivation and infertility, 215 maternal separation and stress, 289-290 olfaction and parturition, 21-22 oxytocin and social behavior, 61 placental lactogens, 155 postnatal handling and stress, 289-290 prolactin secretion in pregnancy, 154 Seizures, 43 Serotonergic system development depression and, 290 maternal separation and, 290 Serotonin systems, puerperal psychosis and, 326, 327 Serotonin transporter gene (SERT/5-HTT), puerperal psychosis and, 327 Sex differences, antinociception, 94 Sex steroids s e e a l s o androgens; estradiol; estrogens; progesterone; testosterone amnesia and, 15 analgesia of pregnancy and, 9 antinociception and, 9, 83, 92-93 brain functioning and, 304-305 changes in maternal brain and, 4-5 depression and, 306 effects on cognition, 305-307, 312-313 effects on mood, 305-307, 310, 312, 313 endogenous opioid interactions, 75-76 maternal behavior and, 10, 12-13 mood changes and, 15 post-partum depression and, 25, 310 pregnancy and, 3-5, 15, 307-308 proportions during menstrual cycle, 5 Sexual behavior s e e mating Sexual receptivity, oxytocin and, 61, 63 Shared synapses hypothalamic magnocellular nuclei, 50 paraventricular nucleus (PVN), 52-53 supraoptic nucleus (SON), 52-53 Sheep bonding with offspring, 10 maternal behavior, 12, 13, 14 olfaction and parturition, 21 oxytocin and maternal behavior, 13, 24, 60-61, 62, 63 Sleep disruption, post-partum depression and, 245 Social behavior, oxytocin and, 61, 63 Social memory, oxytocin and, 61, 63 Social support, fetal growth and, 134 Socioeconomic factors, parent-child relationships, 297-298 Sodium, retention in pregnancy, 16 Sows, inhibition of return of estrus by suckling, 188 Spinal cord dynorphin levels in parturition, 84 dynorphin levels in pregnancy, 84-86 Spino-mesencephalic tract, kyphosis and, 272 Startle reflex, reduction in lactating females, 100, 104 Steroid hormones s e e allopregnanolone; androgens; cortisol; corticosterone; dehydroepiandrosterone; estradiol; progesterone; sex steroids; testosterone Stress s e e a l s o stress hyporesponsiveness adrenocorticotropic hormone (ACTH) and, 289, 291 amygdala and, 289, 291 anxiety and, 289 c - f o s mRNA induction, 118-120, 121 cognitive responses to, 288 corticotropin-releasing hormone (CRH) and, 289-290, 291 depression and, 289, 297 development and, 132 development of immune system, 8 diabetes and, 289 drug addiction and, 289 estrogens and, 9 gene expression in medial amygdala (MeA), 119 gene expression in ventrolateral septum (VLS), 119 heart disease and, 289 human parental care and, 297 hypervigilance and, 288 hypothalamo-pituitary-adrenal (HPA) axis and, 7-8, 10, 67-68, 73-75, 100-108, 243, 245, 289-290, 297 illness and, 288-289, 297 inhibition of labour, 245 inhibition of milk ejection, 245 lactation and, 7-8, 99-108, 243-244 locus coeruleus and, 289, 291 maternal stress, 132-134 memory and, 288 noradrenergic systems and, 120, 289 oxytocin and, 9, 73, 74-75, 122, 245

364 oxytocin secretion and parturition, 75 paraventricular nucleus (PVN) and, 289 parent-child relationships and, 297 physiological responses to, 288 pre-term delivery and, 8-9 pregnancy and, 7-8, 9, 73, 74-75, 100 progesterone and, 9 Stress hyporesponsiveness see also stress adrenocorticotropic hormone (ACTH) and, 115 c-fos mRNA and, 118-119 catecholamine release and, 244 corticosteroid receptors and, 117-118 corticotropin-releasing hormone (CRH) and, 115 estrogen:progesterone ratio, 116 gamma-aminobutyric acid (GABA) and, 244 hypothalamo-pituitary-adrenal (HPA) axis, 7, 115, 116-118, 143-144, 147-148 lactation and, 99-108, 114-117, 118-123, 244 noradrenaline and, 120-122 oxytocin and, 7, 122-123, 144, 147-148, 244 pregnancy and, 7, 115-116 prolactin and, 149, 158, 164 recovery from, 116-117 Subfornical organ estrogen receptors, 235-236 relaxin and, 232, 234-235 Suckling see also breastfeeding; lactation burst firing of oxytocin neurons, 19 effect on brain peptide levels, 202 fertility suppression, 16-17, 158, 164, 207-213 GABA release, 244 GnRH/LH suppression, 190, 201 gonadotropin-releasing hormone (GnRH) suppression, 16-17, 211,212 hyperprolactinaemia induction, 156-157, 165 hypothalamo-pituitary-adrenal (HPA) axis stimulation, 113 implantation suppression, 188 lactational anestrus and, 16-17, 187-202 lactational hyperphagia and, 218-219 loss of body fat, 218, 220 luteinizing hormone (LH) release suppression, 188-189, 191-193 maintenance of lactation, 15-16 maternal aggression and, 265 milk ejection and, 264, 272 neuroendocrine regulation, 100, 102 neuropeptide Y expression, 102 nipple attachment and temperature, 264 ovulation suppression, 188 oxytocin and, 8, 19, 20, 122, 242, 272 periaqueductal gray and, 268, 269, 271 prolactin and, 10, 24, 156-157, 164, 179-180, 212 quiescence during, 264, 267, 269, 272 reduced anxiety and, 7 stimulation of hypothalamus, 193 tuberoinfundibular dopamine (TIDA) neurons and, 179-180 Suprachiasmatic nucleus, prolactin receptors, 159 Supraoptic nucleus (SON) GABA synapse frequency, 51-52 GABAergic somata, 51 noradrenergic afferents, 52 opioid gene expression, 68, 76-77 parturition and, 21, 22-23 prolactin receptors, 159, 164 synaptic plasticity, 19, 50, 52-54 Synaptic plasticity see also synaptic remodelling hypothalamic magnocellular nuclei, 50, 52-53 lactation and, 49-54 neuropil synaptic density, 50 osmotic stimuli, 49 oxytocin neurons, 19, 49-54 paraventricular nucleus (PVN), 50, 53-54 parturition and, 49, 53-54 pregnancy and, 19 speed of synaptogenesis, 49, 50 supraoptic nucleus (SON), 19, 50, 52-54 Synaptic remodelling see also synaptic plasticity cellular factors, 52-53 F3 glycoprotein, 53 glial elements and, 52-53 inductive factors, 53-54 oxytocin and, 53 permissive factors, 53 polysialylated neural cell adhesion molecule (PS-NCAM), 53 Telencephalon, maternally expressed alleles in development, 280 Testosterone see also androgens; sex steroids anxiety-related behavior and, 146 cognitive effects, 306 depression in women, 306 levels during pregnancy, 307, 308 maternal aggression and, 146

365 mood and, 313-314 post-partum levels, 308 pregnancy levels, 146 Tocinoic acid s e e a l s o oxytocin maternal behavior and, 60, 61 Tuberoinfundibular dopamine (TIDA) neurons s e e a l s o dopamine; dopamine neurons lactation and, 15-16 prolactin and, 6, 10, 156, 158-159, 164-165, 175-176, 179 prolactin secretion surges and, 177 suckling-induced prolactin secretion, 10, 156, 164-165, 179-180 Tumour necrosis factor (TNF) signalling pathway, P e g 3 gene and, 282 Tyrosine hydroxylase colocalization with prolactin-releasing peptides (PrRP), 183 induction by hyperprolactinaemia, 177 lactation and, 156, 165, 179 prolactin secretion surges and, 176, 177 suckling-induced prolactin secretion, 180 Unipolar disorder s e e a l s o bipolar disorder; depression; maternity blues; post-natal depression, diagnostic criteria, 323 Uterine contractility, regulation by corticotropinreleasing hormone (CRH), 5 Uteroplacental signalling, pregnancy, 3-5 Vaginocervical stimulation s e e cervical mechanostimulation Vasopressin s e e a l s o vasopressin neurons; magnocellular neurons adrenocorticotropic hormone (ACTH) secretion and, 114 anxiogenic effects, 245 body fluid regulation during lactation, 16 effects on hypothalamo-pituitary-adrenal (HPA) axis, 243-245, 246 lactation and, 16, 114 maternal behavior and, 60 pituitary sensitivity in lactating females, 100, 102-104 production in parvocellular CRF neurons, 101102, 103 sites of synthesis, 242 Vasopressin neurons s e e a l s o vasopressin; magnocellular neurons glutamatergic synapses, 52 noradrenergic afferents, 52 Vasopressin receptors, distribution in central nervous system (CNS), 242 Ventral forebrain, M e s t gene expression, 281 Ventral striatum, maternal behavior and, 12 Ventral tegmental area, maternal behavior and, 60 Ventrolateral preoptic nucleus, prolactin receptors, 159, 160 Ventrolateral septum (VLS), stress and gene expression, 119 Ventromedial nucleus (VMH), maternal behavior and, 258-259 mating-induced prolactin surges and, 174 Ventromedial preoptic nucleus, prolactin receptors, 159, 160 Verbal ability, estradiol and, 306 Wallaby, lactational inhibition of implantation, 188 Weaning depression and, 246 stress hyporesponsiveness and, 116-117