Estrogen Actions in the Central Nervous System*

BRUCE S. MCEWEN AND STEPHEN E. ALVES

Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, Rockefeller University,
New York, New York 10021
I. Introduction
II. Mechanisms of Estrogen Action
A. Genomic and nongenomic mechanisms
B. Steroid hormone actions on gene expression
C. Subtypes of estrogen receptors
D. Steroid hormone actions on putative receptors on
membranes
E. Rapid actions of steroids on neuronal excitability
F. Steroid hormone actions via second messengers
G. Neuroprotective effects of estrogens
H. Summary
III. Areas of the Brain Affected Outside of the Hypothala-
mus
A. Studies of hypothalamic and extrahypothalamic ac-
tions of estrogens
B. Estrogens and the cholinergic system
C. Estrogens and the serotonergic system
D. Catecholaminergic neurons
E. Spinal cord
F. Hippocampus
G. Glial cells, endothelial cells, andthe blood-brain bar-
rier
H. Summary
IV. Effects of Estrogens on Learning and Memory
V. Estrogens, Neuroprotection, and Alzheimers Disease
VI. Conclusions
I. Introduction
G
ONADAL hormones affect the nervous systemin ways
that extend beyond their essential actions of regulat-
ing gonadotropin and PRL secretion and modulating sexual
behavior. For example, estrogens and androgens have been
reported to influence verbal fluency, performance on spatial
tasks, verbal memory tests, and fine motor skills (14); and
they affect the coordination of movement in animals (5) and
symptoms of Parkinsons disease and tardive dyskinesia in
human subjects (6). Estrogens are also linked to symptoms of
depression and treatment of depressive illness (710).
Many estrogen effects differ qualitatively or quantitatively
between the sexes, suggesting that they may be subject to
sexual differentiation during pre- or early postnatal devel-
opment. In addition, circulating hormone levels may con-
tribute differentially in adult males and females. Sex differ-
ences in brain function also include gender differences in the
incidence of psychopathologies such as depressive illness,
which is more common in women, substance abuse and
antisocial behavior, which are more common in men, as well
as pain sensitivity (see Ref. 11 for review).
The diversity of these effects implies that regions of the
brain are involved outside of the hypothalamus, which has
been the traditional site for the study of ovarian steroid
receptors and their role in the control of reproductive func-
tion. For example, the hormonal influences on memory pro-
cesses appear to involve actions on brain structures such as
hippocampus and basal forebrain, while the effects on nor-
mal and abnormal motor activity undoubtedly involve brain
structures such as the caudate-putamen, nucleus accumbens,
and substantia nigra and ventral tegmental, A9 and A10,
respectively, and dopaminergic nuclei of the midbrain; and
those on mood involve, at least in part, the serotonergic
system of the midbrain raphe nuclei. Indeed, mapping of
intracellular receptors, which modulate genomic actions, has
revealedthe presence of estrogen and/or progestin receptors
in regions such as the olfactory lobe, amygdala, hippocam-
pus, cortex, locus ceruleus, dorsal raphe, midbrain central
gray, and cerebellum. Although the density of such receptors
is sometimes lower and more diffuse in many of these brain
areas compared with hypothalamus and amygdala, the ex-
istence of prominent estrogen and progestin effects requires
a careful examination of the role of the cells that do express
intracellular receptors in these brain regions, as well as a
consideration of possible alternative mechanisms of steroid
action, involving membrane receptors. There are also indi-
cations that steroid receptor expression is developmentally
regulated and transient in some brain regions.
This article will review the neural actions of estrogens in
the central nervous system (CNS), focusing on brain struc-
tures outside of the hypothalamus and on neurally mediated
processes other than reproductive behavior and reproduc-
tive neuroendocrine events. We do this, however, in the
context of what is known about estrogen actions in the hy-
pothalamus. In this summary of the recent scientific litera-
ture, we place particular emphasis on estrogen and progestin
effects in the hippocampal formation, as well as basal fore-
brain of the rat, because these brain structures are prominent
in learning and memory and also are sites of neural degen-
eration in dementing illnesses such as Alzheimers disease.
We will also discuss ovarian steroid influences in the mid-
Address reprint requests to: Bruce S. McEwen, Ph.D., Laboratory of
Neuroendocrinology, Rockefeller University, 1230 York Avenue, New
York, New York 10021 USA. E-mail: mcewen@rockvax.rockefeller.edu
* Work in the authors laboratory on this topic is supported by NIH
Grants NS-07080 (to B.Mc.) and by NRSA Fellowship F32 NS-10047 (to
S.E.A.) as well as NIH Grant NS-30105 to Dr. Nancy Weiland, a labo-
ratory colleague who has made important contributions to this ongoing
story that are covered in this review.
0163-769X/99/$03.00/0
Endocrine Reviews 20(3): 279307
Copyright 1999 by The Endocrine Society
Printed in U.S.A.
279
brain and brainstem monoaminergic systems, and spinal
cord, in view of their widespread involvement with brain
functions that subserve affective state andmovement, as well
as analgesia and nociception. First, however, we will sum-
marize the cellular mechanisms of estrogen action in neural
tissue.
II. Mechanisms of Estrogen Action
A. Genomic and nongenomic mechanisms
It has been customary to distinguish between steroid hor-
mone actions that are delayed in onset and prolonged in
duration and are called genomic effects and other steroid
hormone actions that are rapid in onset and short in duration
and are called nongenomic (12). This is because the dis-
covery of intracellular steroid hormone receptors in the early
1960s created, for a number of decades, a single-minded
focus on the long lasting effects of steroids on cell function,
even though rapid actions of steroids were known since the
1930s from anesthetic effects of progesterone (13).
While rapid and delayed effects of steroids are clearly
distinguishable from each other at their extremes in terms of
mechanism, there is a gray area of uncertainty for actions that
have onset times of minutes, as to whether genomic or
nongenomic mechanisms apply. Genomic actions of glu-
cocorticoids on lymphocytes were reported with onset la-
tencies of 20 min (12). Thus, changes in neural activity re-
corded in vivo after systemic administration of steroids could
have onset latencies of minutes, leading to uncertainty as to
whether the lag was due to a delay in steroid reaching the
tissue or to an intrinsic delay in the mechanismof action (14).
Still further uncertainty about mechanism of action was
provided by demonstrations of rapid, but apparently
genomic, actions of steroids affecting neuronal excitability
and promoting or suppressing long-term potentiation (15
19). On the other hand, at least several steroid actions on
membranes involve either a demonstrated coupling to G
proteins or an effect that resulted in the generation of a
second messenger (20), raising the possibility that a mem-
brane steroid receptor may regulate gene expression indi-
rectly via a second messenger-regulated DNA-binding pro-
tein such as a member of the cAMP response element-
binding protein (CREB) family (21).
Even more in contradiction to the simple stereotype, it has
become apparent that some important steroidactions require
the coparticipation of certain neurotransmitters, involving
hormone actions on cells that do not appear to have the
genomic steroid receptors inside of them; rather, the effects
may be transmitted via other steroid-sensitive neurons. An
important example is the GnRHsystemof the hypothalamus.
The activity of GnRH neurons is regulated by the ovarian
steroids; yet, in vivo, these cells have not been found to
concentrate estrogen (22) or express the classical ER, ER,
or PR, in any species studied (2325). However, distinct
populations of adjacent cells, immunoreactive to neurotensin
(23), galanin (26), -aminobutyric acid (GABA), or glutamate
(24), have been shown to express ER and/or PR protein.
Furthermore, it is known that GnRH release is regulated by
hypothalamic amino acid transmitter systems (27, 28). Col-
lectively, these findings point to an indirect, transsynaptic
regulation of GnRH neurons by estrogen and progesterone
in vivo, although it should be pointed out that GT17 cells,
immortalized mouse GnRH neurons, do express seemingly
functional ER (29). A similar, possibly transsynaptic regu-
lation by estrogen of synapse formation occurs in hippocam-
pus, as will be discussed below in Section III.F. The finding
of estrogen induction of synapses in hippocampus and also
hypothalamus has challenged the notion of a morphologi-
cally stable adult brain by showing that steroids alter struc-
tures of the adult brain, including remodeling of synapses,
changes in dendritic structure, and neurogenesis, as will be
reviewed below in Section III.F.
B. Steroid hormone actions on gene expression
The identification and mapping of cells expressing the
genomic steroid receptors by binding, immunocytochemis-
try, and in situ hybridization have provided the target sites
for investigation of hormonal control of gene expression.
Nevertheless, it is only a starting point, because the quali-
tative nature of hormonal regulation of gene expression can-
not be predicted with any certainty from one brain region to
another. For example, vasopressin is an important neuropep-
tide system that is subject to gonadal hormone regulation,
and vasopressin mRNA levels are induced by androgens in
the bed nucleus of the stria terminalis (30) and are sup-
pressed by glucocorticoids in the paraventricular nuclei (31).
CRH gene expression is suppressed by glucocorticoids in
paraventricular nuclei, but induced in placenta (32, 33); in
addition, there are brain areas that contain both glucocorti-
coid receptors and CRH in which there is no apparent glu-
cocorticoid regulation of CRH gene expression (32).
Sexual differentiation involves more than sex differences
in structure and wiring of the brain. There are also sex dif-
ferences in gene expression, in which the male and female
brain respond differently to the same hormone (34). For
example, estradiol has a double roleas an ovarian steroid
in females and as the product of the aromatization of tes-
tosterone in males. Therefore, it is not surprising that estra-
diol can produce somewhat different effects on the male and
female brain, e.g., inducing prodynorphin mRNA in the an-
terioventral periventricular nucleus of female, but not male,
rats (35). Moreover, some of these sex differences are known
to be reversed by the hormonal conditions during early life
that reverse the sex differences in sexual behavior. For ex-
ample, in anterioventral periventricular nucleus, male rats
express more preproenkephalin mRNA than females,
whereas the reverse is true for prodynorphinmRNA; females
that are androgen sterilized at birth show male patterns of
neuropeptide gene expression (35). Alternatively, some
genes appear to be similarly regulated by estradiol in both
sexes, such as the hypothalamic oxytocin receptor (36), the
serotonin 2A receptor (37), and the -form of the estrogen
receptor (ER) in some brain regions (38, 39).
C. Subtypes of ERs
The discovery and cloning of the -isoformof the estrogen
receptor (ER) (4042) radically changed our view of estro-
280 MCEWEN AND ALVES Vol. 20, No. 3
gen action and provided, among other things, a basis for
understanding how the knockout of ER (ERKO) (43, 44)
could result in a viable organism and a continued respon-
siveness of at least some tissues to estrogens. Before the full
recognition of ER, a mapping study was carried out in the
ERKO brain using [
125
I]estrogen, and estrogen induction of
progestin receptor (PR) was also mapped by in situ hybrid-
ization of the mRNA (45). A low level of residual estrogen
binding was found in the medial preoptic nucleus, arcuate
nucleus, bed nucleus of the stria terminalis, and amygdala,
and a significant estrogen-induced up-regulation of PR
mRNA was found in the medial preoptic nucleus (45). Sub-
sequent attempts to map ER have confirmed that residual
estrogen binding and action in ERKO mice might be due to
ER, and these studies have also provided some novel sites
for ER as well as some overlap with ER (4648). As for PR
regulation and the functionality of residual ER (particularly
ER) in ERKOmice, a recent immunocytochemical study has
shown estrogen induction of PRimmunoreactivity in several
hypothalamic nuclei and in amygdala (49).
A recent study by Krege and colleagues (50) has reported
the generation of mice lacking functional ER. Interestingly,
ER knockout females and males appear to develop nor-
mally, exhibit normal sexual behavior, and are reproduc-
tively competent, although females do exhibit reduced fer-
tility (i.e., fewer and smaller litters), apparently due to
decreased ovarian efficiency. This is in sharp contrast to
ERKO mice, which are sterile and do not display normal
sexual behavior (5154). Thus, it appears that ER, more so
than ER, is necessary for the estrogen-mediated regulation
of reproductive physiology, including the behavioral com-
ponents.
Distributions of ER and ER in the body differ quite
markedly, with moderate to high expression of ER in pi-
tuitary, kidney, epididymis, and adrenal, moderate to high
expression of ER in prostate, lung, and bladder, and over-
lapping high expression in brain, ovary, testis, and uterus
(48, 55, 56). It is now known that at least several isoforms of
ER are expressed (5760). The best characterized of these
variants has been termed ER2, vs. the originally identified
ER1, and this isoform appears to have a lower affinity for
estrogens (61), presumably due to an 18-amino acid insertion
in the ligand-binding domain (58). The ER2 variant was
found at levels equal to ER1 in ovary, prostate, pituitary,
and muscle; in brain, expression was found in cortex, hy-
pothalamus, and hippocampus, although at lower levels
thanER1 (57). Despite diminishedligandbinding, ER2 can
bindat the ERE, andit apparently acts as a negative regulator
of estrogen action, as it was found to suppress ER and
1-mediated transcriptional activation in a dose-dependent
manner (58). Moreover, both ER1 and ER are reported to
interact with the estrogen-dependent coactivator, SRC-1,
whereas ER2 does not do so and requires 100- to 1000-fold
higher 17-estradiol concentrations to activate a promotor
containing the estrogen response element (ERE) (62). Human
ER isoforms 25, with alterations in the ligand-binding
domain, have also been identified, and they can form homo-
and heterodimers with ER1 and ER (42, 59). Another vari-
ant, termed ERcx, has a truncated C terminus, but has 26
additional amino acids due to alternative splicing (60). This
form appears to specifically inhibit ER-induced transcrip-
tion; however, ERcx has not yet been found in brain (60).
The genomic effects of estradiol via intracellular ER are de-
picted in the top panel of Fig. 1.
In brain, the distribution of ER is fairly well established,
but there is less certainty and more controversy surrounding
the localization of functional ER. The original autoradio-
graphic maps of [
3
H]estradiol uptake and retention in brain
(63, 64) reflect binding to all forms of the ER, particularly the
ER and the ER1 isoform, which have similar affinities for
17-estradiol (55). In situ hybridization data suggest wide-
spread distribution of ER mRNA throughout much of the
FIG. 1. Schematic diagram of intracellular estrogen action via ER
and ER, as well as possible cell surface effects of putative membrane
ERs that produce neuroprotection (top) or affect intracellular signal-
ing (bottom) via the cAMP and MAP kinase pathways. Top panel,
Estradiol exerts its effects intracellularly via two principal receptor
types, ER and ER, and these are characterized by a distinct spec-
ificity for 17-estradiol over 17-estradiol. Estrogens also exert neu-
roprotective effects in part via a mechanism in which 17-estradiol
has equal or greater potency compared with 17-estradiol. Bottom
panel, Estradiol acts either via cell surface receptors or an intracel-
lular ER to activate two different second messenger pathways, one
involving the MAP kinase cascade and the other involving cAMP.
Both pathways result in activation of gene transcription via at least
three possible response elements: CRE, SRE, and AP-1. Note that in
the case of intracellular second messengers there is some uncertainty
concerning the involvement of ER and ER in the signaling process
vs. the role of other, as yet uncharacterized, receptors (see text). AC,
Adenylate cyclase; CREB-P, phosphorylated form of CREB; ras, ras
oncogene; MAPK, mitogen-activated protein kinase; MAPKK, mito-
gen-activated protein kinase kinase; fos-jun, fos-jun heterodimer.
June, 1999 ESTROGEN ACTIONS IN THE CNS 281
brain (46, 47) while results from immunocytochemical stud-
ies (see below) tend to indicate a more restricted localization
of detectable protein, raising questions about the specificity
of the in situ hybridization procedure, on the one hand, and
the efficacy of the antibodies usedfor immunocytochemistry,
on the other.
The initial commercially available polyclonal antiserumto
ER (no. 310, Affinity BioReagents, Inc.), raised against a
short fragment of the C terminus of the receptor, has pro-
duced a consistent pattern of strong cell nuclear label in the
medial amygdala, paraventricular nucleus (PVN), and pre-
optic area, and striking cytoplasmic/fiber label in cells of the
lateral septum(6567). Detection of cell nuclear ER labeling
in the supraoptic nucleus (SON) may be dependent upon the
use of acrolein in the fixation procedure (65, 67). If the C
terminus antiserum is preabsorbed with the synthetic pep-
tide in a 1:1 (wt/vol) ratio, labeling is completely absent in
these brain regions (6567). However, in other brain regions
such as hippocampus and cortex, immunolabeling is not as
consistent, and such labeling is not always obliterated by
preabsorption of the antiserum (S. E. Alves and B. S.
McEwen, unpublished results). This is particularly true for
the N terminus antiserum (no. 311, Affinity BioReagents,
Inc., Golden, CO), which produces both cytoplasmic and
nuclear labeling. Factors such as fixative (acrolein vs. para-
formaldehyde), gonadal state of the animal (gonadectomized
vs. intact), or stage of the estrous cycle, appear to affect ER
protein detection in the brain. Thus, caution must be taken
in the interpretation of ER immunoreactivity in brain re-
gions other than the hypothalamus and amygdala using
these antibodies.
Moreover, reports of the neurochemical phenotypes of
ER-containing cells in some brain regions, particularly the
PVN and SON, have been somewhat conflicting. While all
studies thus far have identified subpopulations of oxytocin
neurons in the PVNand/or SONto contain ER (46, 6668),
differences have been reported in the specific cell popula-
tions, as well as for other peptide systems. For example, two
studies measuring either ER mRNA (8) or protein (7) have
reported colocalization with vasopressin, particularly in the
SON; another study has reported that colocalization between
ER mRNA and vasopressin was only seen in scattered cells
of the parvocellular PVN (46). This later study also reported
that more than half of the CRH neurons of the caudal par-
vicellular PVN contain ER mRNA. In contrast, Alves and
co-workers (67) observed only few scattered parvicellular
CRH-positive neurons to contain ER protein, using the C
terminus antiserum.
Several viable explanations for discrepancies between
message and protein data exist. Considering the existence of
receptor variants, one possible explanation is that a form of
ER, not recognized by the short C terminus antiserum, is
expressed in these cells, which would result in an underes-
timation of ER-expressing cells when using this antiserum.
Alternatively, perhaps not all ER mRNA is translated into
functional proteinor it is translatedina transient manner that
differs between cell types and developmental stages. On the
other hand, it is also possible that the discrepancies in the
results of different laboratories are due to false-positives with
the existing probes to ER.
However, if the reported CNS distribution of ER mRNA
is found to reflect the expression of some functional ER
protein in those brain regions, this would certainly help to
explainnumerous estrogenactions inbrainregions withlittle
or no ER. This includes areas such as the olfactory bulbs,
cerebellum, and cerebral cortex, in which ER mRNA has
been abundantly detected (48). It is hoped that future inves-
tigations into the seemingly complex detection and expres-
sion of ER will provide a clearer picture of the distribution
and phenotype of cells that contain functional ER.
As noted above, ER and ER1 are similar not only in
affinity for a number of estrogens and estrogen antagonists
(55), but also in their ability to regulate genes in which the
ERE is the primary site of interaction (69) (see top panel of Fig.
1). The major differences between ER and ER1 concern
their ability to regulate transcription via the AP-1 response
element. For interactions of ER with AP-1, 17-estradiol, as
well as a number of antiestrogens, activated transcription;
however, for ER1 interacting with AP-1, 17-estradiol
failed to activate transcription but antiestrogens activated
transcription (69). As mentioned above, ER and ER1 can
form heterodimers when expressed in the same cells, thus
giving rise to additional possible variants of gene regulation
(42). Thus far, the endogenous colocalization of ER and ER
has beenreportedrecently inthe hypothalamic preoptic area,
bed nucleus of the stria terminalis, and medial amygdaloid
nucleus (68).
The agonist effects of estrogen antagonists bring to mind
earlier studies in which estrogen antagonists produced es-
trogen-like effects on some neurochemical endpoints and
antagonistic effects on others. The antagonistic effects for
CI-628, a tamoxifen-like estrogen antagonist, were seen in
terms of PR induction and lordosis behavior (70, 71) (see Fig.
2), whereas the agonist-like effects of CI-628 were seen for
choline acetyltransferase regulation and monoamine oxidase
A regulation, but not for the regulation of glucose-6-phos-
phate dehydrogenase in pituitary and uterus (72) (see Fig. 3).
The molecular mechanisms underlying the differences in
these antiestrogen effects remain to be explored, and they
may reflect the operation in some of the cases of a response
element other than the ERE and perhaps even the operation
of heterodimers of ER and , but the diverse effects of
CI-628 indicate that the nonsteroidal antiestrogens do not
have uniform agonist-like or antagonist-like effects in the
brain. This is an important consideration for the therapeutic
use of estrogen antagonists of this type.
D. Steroid hormone actions on putative receptors
on membranes
Membrane ERs have been reported on pituitary, uterine,
ovarian granulosa cell, and liver cell membranes, but they
have been characterized only partially and have not yet been
shown to be linked to signal transduction mechanisms (73
80). For membrane fractions frompituitary andovariangran-
ulosa cells, the specificity of the binding sites shows equal
potency of 17- and 17-estradiol, estriol, and estrone; for
liver and uterine cells, there is a preference for 17- over
17-estradiol (73, 75, 76, 79, 80). However, for GH3/B6 pi-
tuitary cells, a 17-estradiol binding site was identified using
282 MCEWEN AND ALVES Vol. 20, No. 3
an estrogen-BSA conjugate; but, in contrast to the other re-
ports suggesting a novel membrane ER (cited above), mono-
clonal antibodies to the intracellular ER, H226, and H222,
as well as the polyclonal antiserum, ER21, each recognizing
a unique epitope onER, labeledsites onthese cells inor near
the cell surface (77, 81). A more recent report used transient
transfection of both ER and ER cDNA into Chinese ham-
ster ovarian cells and demonstrated both types of ER ex-
pressed in both cell membrane and nuclear fractions; the
binding affinities for estradiol were similar in both mem-
brane nuclear fractions, and, in membranes, estradiol acti-
vated Gq and Gs proteins in the membrane and rapidly
simulated, respectively, inositol phosphate production and
adenylate cyclase activity (82).
These limited findings can be viewed in relation to data
about other membrane steroid receptors. The best under-
stood membrane receptor for a steroid is the GABA-A re-
ceptor. Anesthetic effects of progesterone derivatives (13)
led, after many years, to the recognition of a unique mem-
brane recognition site on many subunit combinations of the
GABA-A-benzodiazepine receptor system (83, 84). A-ring-
reduced metabolites of progesterone and deoxycorticoste-
rone are among the most active steroids affecting the
GABA-A receptor system, and such metabolites are pro-
duced in the body, including the brain, from the parent
steroid. The effects of these steroid metabolites include not
only anesthetic effects but also antiepileptic, sedative-hyp-
notic, and anxiolytic actions (83). The efficacy of these me-
tabolites in normal physiology is suggested by experiments
on progesterone facilitation of lordosis in the hamster, in
which it was found that local application of GABA-A-active
derivatives of progesterone to the midbrain ventral tegmen-
tal area (VTA) of the estrogen-primed hamster was able to
facilitate lordosis (8587). Moreover, while GABA-A recep-
tor-active pregnane steroids, appliedto the ventral tegmental
area, facilitate lordosis behavior, inhibitors of 5-reductase,
the first step in A-ring reduction, prevented systemically
applied progesterone from facilitating lordosis (20).
Other membrane receptors for steroid hormones are not so
well characterized (20). One exception is the membrane-
binding site for 1,25-dihydroxyvitamin D
3
in basal-lateral
membranes of chick intestinal epithelium, which demon-
strates pharmacological specificity for a receptor modulating
nongenomic transport of calcium (88). Another example is a
membrane steroid receptor site for corticosterone in the
FIG. 2. Effect of the estrogen antagonist, CI-628 (nitromiphene ci-
trate; -[4-pyrrolidin-oethoxyl] phenyl-4-methoxy--nitrostilbene),
on the induction of cytosol PRs in the hypothalamic/preoptic region of
ovariectomized female rats by estradiol (E
2
) or estradiol benzoate
(EB). Treatment protocols are referred to as follows: 1 DAY: 5 g E
2
and 2 mg CI-628 at 0 h, killed at 24 h; 2 DAYS: 2 g EB at 0 h and
CI-628 at 0 h and 24 h, killed at 48 h; 3 DAYS: 15 g EB and 2 mg
CI-628 at 0, 24, 48 h, sacrificed at 72 h. Cytosolic receptors were
assayed using
3
H-labeled R 5020, a synthetic progestin. Black bar
indicates increase above control levels. It can be seen that estrogen
treatment increased PR binding and that CI-628 at least partially
antagonized the effects of estrogen treatment under all conditions. In
the lower right panel, a Scatchard analysis of PR binding of rats
treated with 2 g EB at 0 h vs. 2 g EB at 0 h plus two injections of
2 mg CI-628 at 0 h and 24 h, killed at 48 h (2 DAYS protocol).
[Reprinted with permission from E. Roy et al.: Endocrinology 104:
13331336, 1979 (70). The Endocrine Society.]
FIG. 3. Effect of the estrogen antagonist, CI-628, on estradiol ben-
zoate (EB)-dependent enzyme changes inuterus, pituitary, and brain.
Ovariectomized rats were injected for 5 days with sesame oil vehicle,
CI-628 (18 mg/kg), or EB (140 g/kg) either alone or in combination.
Enzyme activities are reported as mean SEM for choline acetyl-
transferase (CAT) in preoptic area, type Amonoamine oxidase (MAO)
in amygdala, glucose-6-phosphate dehydrogenase (G6PDH) in pitu-
itary and uterus. Details of the assay may be found in the original
publication (72). Differences among groups were tested by Newman-
Keuls procedures: *, Different from OVX, P 0.05; **, P 0.01;
different from EB CI, P 0.05. For uterus, all groups were sig-
nificantly different from one another. [Reproduced with permission
from V. Luine and B. S. McEwen: Endocrinology 100:903910, 1977
(72). The Endocrine Society.] It can be seen that CI-628 exerted
agonist-like effects on CATand MAOactivity, whereas it antagonized
estrogen induction of G6PDH activity in pituitary and uterus.
June, 1999 ESTROGEN ACTIONS IN THE CNS 283
newt, Taricha granulosa. Using both autoradiography and
binding assays on isolated membrane fractions, the cortico-
sterone site has been shown to be coupled to a G protein and
to have the characteristics expected of a site involved in the
rapid inhibition of sexual behavior (89). However, it has been
difficult to obtain satisfactory binding data for putative ste-
roid receptors on rat brain membranes. Approaches using
progesterone linked to
125
I-labeled BSA have met with some
success in labeling putative progestin sites on brain mem-
branes (90). However, the majority of the evidence to date is
based on functional studies using electrophysiology or other
endpoints, and some of this will be summarized below as it
applies to the coupling to second messenger systems.
E. Rapid actions of steroids on neuronal excitability
Estrogens and other steroids affect neuronal excitability,
and one of the challenges in studying estrogen actions on
neurons is to identify whether genomic or nongenomic
mechanisms are involved. For the induction of the so-called
MINKpotassiumchannel by estrogens (91), a genomic mech-
anism is clearly implicated. On the other hand, estradiol has
been reported to rapidly excite neurons in cerebellum, ce-
rebral cortex, and the CA1 pyramidal neurons of hippocam-
pus (see below) by a mechanism that seems not to involve
intracellular ERs, which do not appear to be found in the
responding neurons. However, as discussed above in Section
II.C, the presence of ER mRNA in these brain areas leaves
open the possibility of some functional ER receptor. Nev-
ertheless, the rapidity of many of these effects makes a
genomic mechanism unlikely.
For example, direct application of 17-estradiol rapidly
decreases the spontaneous firing of neurons in medial pre-
optic area (92) and rapidly increases the firing rate of pitu-
itary cells (93). 17-Estradiol also increases excitatory
postsynaptic potentials in the hippocampus, apparently by
increasing currents mediated by kainate receptors in hip-
pocampal neurons (9496), and 17-estradiol increases re-
sponses to applied glutamate in the cerebellum (97, 98). 17-
Estradiol also causes rapid hyperpolarization of neurons in
medial amygdala (99, 100), and it suppresses -opioid and
GABA-B receptor-based hyperpolarization of hypothalamic
arcuate neurons in the guinea pig (101). Furthermore, 17-
estradiol directly potentiates potassium-stimulated dopa-
mine release in the rat nucleus accumbens (102). These effects
are very rapid, occurring within seconds; moreover, in one
instance, the estrogen antagonist, tamoxifen, was shown not
to mimic or block estrogen actions on the kainate currents
(96). Furthermore, mice lacking ER showthe same estrogen
effect on the kainate current, providing further evidence that
a separate type of ER may be involved (103). However, there
is no extensive data with estrogen antagonists on many of
these rapid estrogen effects, and thus they remain relatively
uncharacterized pharmacologically.
Other steroid effects occur rapidly, albeit within minutes
and not seconds, and yet they are blocked by antagonists of
intracellular steroid receptors; yet, in most cases, specific
gene products are not yet identified. This is true for the
actions of adrenal steroids through type I receptors to dis-
inhibit CA1 pyramidal neurons from serotonin 1A (5-HT1A)
receptor-mediated inhibition and to suppress via type II re-
ceptors noradrenaline-mediated facilitation of CA1 excit-
ability (104) (see Ref. 105 for review). Asimilar story has been
described for adrenal steroid effects on the induction of long-
term potentiation (LTP) and its relative, primed burst po-
tentiation (PBP) (see Ref. 106 for review).
F. Steroid hormone actions via second messengers
Estrogens and other steroids affect the activity of second
messenger systems and may do so via genomic as well as
nongenomic mechanisms. Three categories of second mes-
sengers will be considered from the standpoint of evidence
for receptor mechanisms involved, both genomic and non-
genomic. Because these second messenger pathways interact
with each other, the phenomenology described below is not
mutually exclusive. Moreover, the phenomena described be-
low may be related to the membrane-binding sites described
above and/or to the estrogen effects on excitability described
in the previous section.
1. cAMP regulation. The finding that progesterone stimulates
accumulation of cAMP in frog oocytes and stimulates phos-
phoinositol turnover in sperm (107110) raised the possibil-
ity that membrane actions of certain steroids on certain target
cells might regulate second messenger formation. The iden-
tification of membrane receptors for corticosterone in Taricha
granulosa that are coupled to G-proteins further strengthens
this possibility (89).
Addition of estrogen to MCF-7 or uterine cells in culture
evoked an increase in cAMP levels; and, although 17-es-
tradiol and other nonestrogenic steroids were without effect,
a number of estrogen antagonists mimicked the estradiol
effect, and protein and RNA synthesis blockade failed to
prevent the effect (111). In some brain regions, estrogen treat-
ment increases phosphorylation of CREB via a cAMP-de-
pendent mechanism (112, 113). Estrogen-induced depolar-
ization of hypothalamic neurons involves cAMP and also
attenuates potassium conductance (99, 100); in pituitary, es-
tradiol was reported to inhibit GTPase activity, although this
effect was also produced by testosterone and progesterone
(114). The so-far limited evidence for estrogenic regulation of
cAMP levels, which in many cases lack definitive structure-
activity studies or the use of antagonists, is nevertheless
consistent with an alternative or indirect pathway for gene
regulation by which steroid-driven second-messenger re-
sponses, possibly via a novel receptor mechanism, regulate
gene expression via DNA-binding proteins such as the phos-
phorylate form of CREB (PCREB) (21). The effects of estro-
gens involving a second messenger systemare schematically
summarized in the bottom panel of Fig. 1, although it must be
pointed out that there are many unknowns about the nature
of the intracellular sites for these effects and the receptors
that may be involved.
2. Mitogen-activated protein (MAP) kinase regulation. In addi-
tion to protein kinase A and cAMP, the MAP kinase system
has been implicated in estrogen action. In human mammary
cancer MCF-7 cells, 17-estradiol was reported to induce
immediate and transient activation of the Src/p21ras/Erk
pathway via a mechanism that is blocked by the pure es-
284 MCEWEN AND ALVES Vol. 20, No. 3
trogen antagonist ICI 182780 and which, therefore, appears
to involve an intracellular ER(115117). In a follow-upstudy,
progesterone activation of this pathway was shown to occur
by an association of the PR with an N-terminal region of ER
and not with c-Src directly (116).
In neuroblastoma SK-N-SH cells and in cortical explants,
17-estradiol was reported to activate the MAP kinase and
phosphorylate and activate two of them, ERK-1 and ERK-2
(118). In contrast to MCF-7 cells, the response in SK-N-SH
neuroblastoma cells and cortical explants was not blocked by
ICI 182780 or by tamoxifen (118), indicating that a classical
ERmay not be involved. Further support for this in SK-N-SH
cells came from the finding that 17-estradiol conjugated to
BSA activates a reporter gene driven by the mouse c-fos
protooncogene, which responds to MAP kinase activation,
but does not activate transcription mediated by a promotor
containing the ER response element, ERE (118). A possible
sequence of signaling events is summarized in the bottom
panel of Fig. 1. Estrogen-dependent activation of MAP ki-
nases, ERK-1 and ERK-2, has also been studied in embryonic
cerebral cortical explants grown in culture (119, 120). The
activation of ERK was blocked by the MEK-1 inhibitor,
PD98059, but not by ER antagonist ICI 182780, again sug-
gesting that a conventional ER may not be involved (119).
3. Calcium homeostasis. Calcium ions also constitute an im-
portant player in second messenger pathways, and the effect
of estrogen on calcium channels and calcium release from
intracellular stores has also emerged as a possible cellular
mechanism of estrogen action that is relevant to excitability
and neuronal vulnerability to damage. There appear to be at
least three distinct pathways for estrogen action on calcium
homeostasis that have been documented in different cell
systems, each involving a different type of membrane ER,
and at least one pathway for affecting calcium homeostasis
via a genomic mechanism.
In the first of the nongenomic pathways, 17-estradiol
activates a G-protein-coupled receptor in rat neostriatal neu-
rons and, within seconds, suppresses currents mediated by
L-type calcium channels (121). 17-Estradiol is considerably
less potent than 17-estradiol, as are other steroids, and the
estrogen antagonist, tamoxifen, mimicked estrogen action
and did not block them (121), thus further supporting a
unique ER on the cell surface. Interestingly, these estrogen
effects were sex specific, occurring more robustly in neurons
from female rats (121). A similar effect of 17-estradiol to
inhibit currents mediated by L-type calcium channels was
reported in aortic smooth muscle, providing a basis for the
well known effects of estradiol to regulate vascular tone
(122).
In a second nongenomic pathway, as shown for liver and
uterine endometrial cell membranes, estradiol binds ste-
reospecifically (17 17), and these sites may be respon-
sible for estrogenic stimulation of calcium influx into these
cells (7476, 78, 79). In contrast, via a third pathway, in
chicken ovarian granulosa cells, 17- and 17-estradiol fa-
cilitated release of intracellular calcium stores equipotently
in a concentration range of 10
10
to 10
6
m (80). Estriol and
estrone were also effective in the same range, but progestins
and androgens were ineffective; moreover, estrogen actions
were not blocked by tamoxifen or by RNA and protein syn-
thesis inhibitors (80), thus defining an estrogen membrane
site of broader specificity than that seen in liver or endome-
trial cells or striatal neurons.
There are, however, reported estrogen effects on calcium
currents that are more consistent with an intracellular,
genomic action of estradiol. In a study on GH3 pituitary cells,
17-estradiol treatment increased low voltage-activated cal-
cium currents over 24 h by a mechanism requiring protein
synthesis (123). Moreover, in a hippocampal slice study, in
vivo treatment with estradiol increased in vitro both the sus-
tained and transient calcium currents, while in vivo proges-
terone acutely amplified the estrogen effects over 4 h; in
contrast, potassium currents were not altered by these same
treatments (124). These results appear to be more consistent
with the genomic actions of estradiol that are related to
synaptogenesis and will be discussed below.
G. Neuroprotective effects of estrogens
Estrogens exert protective effects on neuronal cells in cul-
ture that may be mediated, at least in part, by their ability to
alter free radical production and/or free radical action on
cells. However, as was also the case for the second messenger
systems, the evidence for involvement of intracellular ERs vs.
novel membrane receptors is controversial, although tending
to point to a nontraditional receptor mechanism. The dis-
tinction between neuroprotective effects of estrogens medi-
ated by intracellular receptors and those mediated by puta-
tive receptors located in other parts of the cell are
summarized in the top panel of Fig. 1 and is based on the
different estrogen structure-activity profile, as will be de-
scribed below.
The first neuroprotective actions of estradiol were de-
scribed in relation to the effects of serum deprivation on
neuronal survival in cell culture (125129). In one of these
studies (127), picomolar levels of 17-estradiol enhanced
fetal rat hypothalamic neuronal survival in a serum-free
medium in the presence or absence of glial cells by a mech-
anism that was blocked by tamoxifen and that, presumably,
involves intracellular ERs. A similar example will be given
at the end of this section regarding estrogenic neuroprotec-
tion from glutamate toxicity (130).
In serum-free medium, embryonic cortical neurons were
shown to survive better in the presence of nanomolar con-
centrations of 17-estradiol; in fact, 17-estradiol facilitated
neurite outgrowth by a process that was blocked by AP5, an
N-methyl-d-aspartate (NMDA) receptor antagonist, but not
by ICI 182,780, an ER antagonist, suggesting a possible non-
genomic mechanism (131).
In another series of studies (128, 129), neuroblastoma SK-
N-SHcells were protectedby concentrations of both 17- and
17-estradiol in serum-free media. In the first of these studies
(128), 17-estradiol concentrations of 2 m enhanced total
live cell number for up to 48 h without increasing thymidine
incorporation, indicating an effect on cell survival and not
cell division. In the second study in this series (129), 17- and
17-estradiol in the range of 0.22 nm protected SK-N-SH
cells in culture, and a 10-fold molar excess of tamoxifen
antagonized only one-third of the neuroprotective effect.
June, 1999 ESTROGEN ACTIONS IN THE CNS 285
Taken together, the absence of stereoselectivity of 17- vs.
17-estradiol and the weak antagonism by tamoxifen argue
against involvement of the classical intracellular ERs in neu-
roprotection of SK-N-SH cells.
The other situationinwhichneuroprotectionby estrogenic
steroids has been described is in relation to oxidative dam-
age, and, once again, the weight of evidence is against a role
for the classical intracellular ERs. Before discussing estrogen
effects on cell survival, we note a chemical study carried out
in the absence of living cells or cellular extracts, in which the
addition of 200 nm 17-, 17-estradiol, or estriol each re-
duced the generation of free radicals, whereas other steroids
were ineffective (132). This suggests that features of the es-
trogen A ring, involving the 3 hydroxyl group, have the
ability to interfere with free radical production in the absence
of proteins or other cellular materials.
There have been a number of studies investigating a neu-
roprotective role of estrogenfromfree radical damage incells
in culture. In the first of these, exposing cloned mouse HT22
hippocampal cells to amyloid-, hydrogen peroxide or glu-
tamate resulted in oxidative damage and cell loss that was
reduced by preincubating cells for 20 h with 10 m 17-
estradiol or 200 umvitamin E, but not by 10 mprogesterone,
aldosterone, corticosterone, or cholesterol; 17-estradiol at a
concentration of 0.1 m was not effective in these studies
(133). Afollow-upstructure-activity study by the same group
revealed a broad spectrum of estrogen specificity, with 17-
and 17-estradiol as well as estriol and estrone all being
effective and pointing to the C3 -hydroxyl group on the
steroid A ring as being important as well as implying that
intracellular ERs are not involved (134).
Dissociated embryonic hippocampal neurons were also
sensitive to estrogen-mediated protection against amyloid-
toxicity, glucose deprivation, glutamate treatment, or FeSO
4
toxicity; in this study, the effective steroid concentration
range was 100 nm to 10 m, and estriol and progesterone
were also effective, whereas corticosterone enhanced neu-
rotoxicity across this concentration range (135).
In contrast to these studies using high, supraphysiological
concentrations of estrogens, another recent investigation
showed that as little as 0.22 nm estradiol, either 17 or 17,
protected SK-N-SH cells against -amyloid toxicity (136).
This raises the question of how different investigators have
found such different concentrations of estrogens to be effec-
tive (136). These discrepancies in effective concentration
ranges of estrogens are difficult to understand. However, one
possible clue points to the concentration of natural reducing
agents inthe culture medium, i.e., a recent report showedthat
the addition of glutathione to HT22 cells in culture, which
lack functional ER, reduced the dose range of estrogen neu-
roprotection by 400-fold (137).
In contradistinction to all of the above mentioned neuro-
protection studies, a report on glutamate toxicity in primary
cortical neurons in culture suggests involvement of an in-
tracellular ER; 24-h pretreatment with 1550 nm 17-estra-
diol reduced glutamate-induced toxicity, measured by lac-
tate dehydrogenase release, an effect that was blocked by the
estrogen antagonist, tamoxifen. Thus, this finding suggests
that there are situations in which estrogen neuroprotection
may involve the activation of intracellular ER (130).
In a related study, the toxicity of gp120 for hippocampal
cells in culture is inhibited over 72 h by 17-estradiol at
concentrations of 1 nm or above (138). Since gp120 exerts its
effects via excitatory amino acids and calcium ions, culmi-
nating in free radical-induced damage (138), it is noteworthy
that estradiol reduces the free radical accumulation induced
by gp120 (R. Sapolsky, personal communication). However,
there are no experiments with this system to indicate
whether a conventional ER mechanism or a novel type of
receptor is involved.
Finally, there is another mechanism potentially involved
in neuroprotective estrogen effects, namely, the regulation of
the Bcl-2 family of genes (139). Some members of this family,
such as Bcl-2 and Bcl-X
L
, suppress programmed cell death,
whereas others such as Bax.Bad and Bid act as positive reg-
ulators of apoptosis (see Ref. 139). In arcuate nucleus neurons
of female rats, estrogen treatment up-regulated expression of
Bcl-2 immunoreactivity (139). Estrogen up-regulation of
Bcl-X
L
was also reported both in vivo and in vitro in hip-
pocampal and cortical cells (140). Thus, estradiol may also
increase gene expression that inhibits programmed cell
death, although the mechanism by which estrogen regulates
Bcl expression is not known at this time; intracellular ER
and/or ER may very well be involved in such genomic
regulation, as both receptors are foundin these brain regions.
H. Summary
Estrogen actions on brain cells occur through at least two
types of intracellular receptors as well as other mechanisms
for which receptor sites are not yet clearly identified. Indeed,
for a number of processes, there are conflicting reports, based
upon structure-activity studies with different estrogens and
the actions of estrogen antagonists, as to whether intracel-
lular receptors are involved. For estrogen actions on some
aspects of calcium homeostasis, activation of certain second
messenger systems, and some features of neuroprotection, a
novel receptor mechanism may exist in which stereospeci-
ficity for 17- over 17-estradiol is replaced by a broader
specificity for the 3-hydroxyl group on the A ring.
III. Areas of the Brain Affected Outside of
the Hypothalamus
A. Studies of hypothalamic and extrahypothalamic actions
of estrogens
Until recently, the hypothalamus has been the focus of
much of the attention regarding neural effects of gonadal
hormones. Ovarian hormone actions on neurons of the ven-
tromedial hypothalamus, which are important for the reg-
ulation of sexual behavior in female rats, include regulation
of neuropeptide gene expression(141) andsecondmessenger
systems (142) and induction of oxytocin receptors, PR, and
the regulation of cyclic synaptogenesis (36, 143145). There
are also developmentally programmed sex differences in-
volving both neuronal wiring as well as programming of
responses to hormonal activation of gene expression (144,
146). All of these actions occur in neurons that express high
levels of ER and PR, in contrast to the hippocampus, mid-
286 MCEWEN AND ALVES Vol. 20, No. 3
brain raphe, basal forebrain, brainstem, and spinal cord, in
which ER and PR, if detected at all, appear to be relatively
scarce and are found in many cases in interneurons or scat-
tered principal neurons. As noted in Section II.C, the extra-
hypothalamic distribution of ER protein is still unclear, but
the presence of ER mRNA in brain regions such as the
cerebellum, hippocampus, cerebral cortex, and olfactory
bulbs (47, 48) suggests that ER must be regarded as a po-
tential mediator of estrogen action in those brain areas.
This is a very important consideration, since gonadal hor-
mones, and, inparticular, estrogens, have many effects onthe
nervous system that extend beyond their very important
actions in hormonal regulation of reproductive function.
Moreover, many of these estrogen effects differ qualitatively
or quantitatively between the sexes, suggesting that they
may be influenced by the process of sexual differentiation
during early pre- or postnatal development and/or by dif-
ferent levels of circulating sex hormones. In considering
these nonreproductive actions of estrogens, we must also
consider other brain regions outside of the hypothalamus.
However, this does not mean that the hypothalamus is con-
cerned only with reproduction, nor does it mean that the
extrahypothalamic brain regions are not contributing to re-
productive functions. Indeed, the hypothalamus is con-
cerned with many aspects of autonomic and neuroendocrine
control, and extrahypothalamic systems such as serotonin
and the catecholamines play important supporting roles in
reproductive endocrinology. Nevertheless, in addition to re-
production there is much more to brain function that is
influencedby estrogens andby sex differences, andthis is the
primary focus of the following discussion.
Sex differences in brain function also include gender dif-
ferences in the incidence of psychopathologies such as de-
pressive illness, which is more common in women, and sub-
stance abuse and antisocial behaviors, which are more
common in men, as well as pain sensitivity (11). Sex differ-
ences andestrogen effects upon the serotonergic, cholinergic,
dopaminergic, andnoradrenergic systems all may contribute
to many aspects of brain function that are affected by ovarian
hormones, including affective state (7), movement disorders
(6), and cognitive function (2, 147). Furthermore, the discov-
ery of estrogen-induced synapse formation in hippocampus
and hypothalamus, which are described below, are relevant
to postmenopausal changes in brain function, including de-
cline of short-term verbal memory (147), as well as to the
occurrence of dementia, which becomes more prevalent in
women after the menopause as well as in men as they age
(148). Recent epidemiological studies have suggested a pos-
sible protective role for postmenopausal estrogen therapy
against Alzheimers disease (149, 150). Furthermore, estro-
gen treatment trials have shown some benefit to demented
woman as far as global cognitive function and mood (151,
152) as well as to normal women (3, 4, 153) as far as verbal
memory. A recent study also suggests a positive role for
estrogen therapy on cognitive function in multiple sclerosis
(154). Moreover, estrogen and progestin-induced regulation
of synapse formation and excitability may play a role in
catamenial epilepsy, which varies in frequency during the
menstrual cycle (155). The presence or absence of hormones
also contributes to aging of the brain, e.g., loss of hippocam-
pal neurons as a result of elevated glucocorticoid activity
(156, 157); and consequences of estrogen loss in females may
include loss of synaptic connections in hippocampus (158) or
decline in basal forebrain cholinergic function in the absence
of circulating estrogens (159). An additional aspect of estro-
gen action is the regulation of neurogenesis in the dentate
gyrus, which continues to produce newneurons in adult life.
A recent report indicates that female rats have a higher rate
of neurogenesis than males and that neurogenesis varies
during the estrous cycle with a peak on the day of proestrus
(160).
Sex differences in brain structures and mechanisms are
programmed early in life by gonadal hormones and are per-
manent for the life of the individual. Sex differences occur in
brain regions other than the hypothalamus, such as hip-
pocampus, and they appear to be involved in aspects of
cognitive function and other processes that go beyond the
reproductive process itself. In this review, we refer to sex
differences and the process of sexual differentiation but
not to sexual dimorphism, which is a term that refers to
nonoverlapping differences in phenotype between the sexes.
This is because true sexual dimorphisms are very rare, and
the more common pattern of sex differences involves over-
lapping, but significantly different, distributions of pheno-
typic traits.
Understanding the cellular and molecular basis of sex
differences and of sex differences in the actions of gonadal
hormones is vitally important for assessing how pharma-
ceutical agents differentially affect the brains of males and
females (161), as well as in understanding other male-female
differences relevant to health and disease, such as the higher
incidence of depression in women and of substance abuse in
males (7). There are also sex differences in the severity of
brain damage resulting fromtransient ischemia (162) and sex
differences in the response of the brain to lesions (163) and
to severe, chronic stress (164, 165).
The diversity of these effects implies that regions of the
brain are involved outside of the hypothalamus. Indeed, as
we have noted in Section II.C above, mapping of intracellular
receptors, which modulate genomic actions, has revealed the
presence of ER and/or PR expression in regions such as the
olfactory lobe, hippocampus, cortex, locus ceruleus, mid-
brain raphe nuclei, and midbrain central gray and cerebel-
lum. Although the density of ER is often lower and more
diffuse in many of these brain areas compared with hypo-
thalamus and amygdala, the existence of prominent estrogen
and progestin effects in many of these brain areas requires a
careful examination of the role of the cells that express in-
tracellular receptors in these brain regions. We have noted in
Section II above that the localization and expression of ER
is an important consideration, along with a consideration of
possible alternative mechanisms of steroid action. We now
consider a number of the extrahypothalamic brain regions
that are sensitive to estrogens and progestins.
B. Estrogens and the cholinergic system
The basal forebrain contains cholinergic neurons that
project to cerebral cortex and hippocampus, where they play
an important role in cognitive function. Studies of estrogen
June, 1999 ESTROGEN ACTIONS IN THE CNS 287
effects on the expression of cholinergic enzymes were among
the first that pointed to nonreproductive actions of gonadal
steroids (166). Experiments with ovariectomy and estrogen
replacement therapy revealed an induction of choline acetyl-
transferase (ChAT), the rate-limiting enzyme for acetylcho-
line formation, within624 hinbasal forebrainof female rats.
In addition, estrogen treatment increased ChAT activity in
projection areas of the basal forebrain 10 days after hormone
injection, suggesting that estrogen-induced ChAT was trans-
ported from cell bodies to nerve endings in the cerebral
cortex and hippocampus (166). 17-Estradiol treatment also
induced acetylcholinesterase, as well as ChAT activity, im-
plying that a general trophic effect onthe cholinergic neurons
might occur (166). The estrogen induction of ChAT was
mimicked by the estrogen antagonist, CI-628 (see Fig. 3),
suggesting that a different type of interaction with the ge-
nome is involved than the traditional one involving an ER
operating via the ERE (see Section II.C) (72).
Arecent investigation of long-term(528 wk) ovariectomy
and long-term estrogen replacement in rats revealed a de-
cline in high-affinity choline uptake and in ChAT activity in
frontal cortex and hippocampus that was at least partially
prevented by estrogen treatment (167). Estrogen treatment
also increased the acetylcholine released by potassium de-
polarization (168). Estrous cycle variations in ChAT mRNA
levels were also reported in the basal forebrain cholinergic
system (168). Along with these effects, long-term ovariec-
tomy caused a decline in learned performance of active
avoidance behavior that was prevented by estrogen replace-
ment therapy (167).
One possible candidate as a regulator of the cholinergic
system of the basal forebrain is nerve growth factor (NGF),
which is produced by the hippocampus and transported
retrogradely to basal forebrain neurons to produce trophic
effects. Although the effects of estrogen treatment on NGF
levels in hippocampus remain to be investigated, ER have
been reported to colocalize with low-affinity NGF receptors
in cholinergic neurons of the basal forebrain of the newborn
rat (169). Moreover, estrogen replacement in both young and
aged female rats increases both trkA (NGF receptor) mRNA
and ChAT mRNA expression in basal forebrain (170).
The basal forebrain of male rats failed to show the same
response to estrogen treatment as females, and postnatal
estrogen treatment of females or blockade of aromatization
in males failed to change this sex difference (166, 171), in-
dicating that the sexual differentiation of the cholinergic
system either occurs earlier in development or does not in-
volve the aromatization of testoserone to estradiol. Addi-
tional studies revealed that the basal forebrain cholinergic
system differs between male and female rats, with females
having smaller and more densely packed cholinergic neu-
rons compared with untreated males (172). Moreover, ap-
plication of T
3
to newborn male and female rats, creating
transient hyperthyroidism during the first week of postnatal
life, revealed further indications of sexual differentiation of
the basal forebrain cholinergic system in which male rats
responded to the treatment while females did not (172). For
example, treatment with T
3
increased cholinergic cell density
and induced increased ChAT activity and muscarinic recep-
tor binding in the septum/diagonal band region of males.
Females did not respond to T
3
in most respects, except in
medial septum where they showed the opposite effect to
males, namely, an increased cholinergic cell body area (172).
This finding suggests that there is an interaction between the
prenatal effects of testosterone in the development of the
cholinergic system of the basal forebrain (173) and the post-
natal effects of T
3
(172).
On the other hand, in another study of sex differences in
the cholinergic system, female rats showedlarger effects than
males to the cholinergic lesions producedin hippocampus by
the specific cholinergic neurotoxin, AF64A, andfemales were
particularly sensitive when the toxin was administered into
the lateral ventricles on the day of proestrus (174). A recent
clinical study of estrogen replacement in relation to Alzhei-
mers disease revealed that the beneficial effects of tacrine, a
cholinergic-enhancing drug, were evident in women on es-
trogen replacement therapy and not in women who did not
receive estrogen replacement therapy (175).
Taken together, these results point to a sexually differen-
tiated organization of the basal forebrain cholinergic system
in the rat, involving a prenatally programmed difference in
the neuroanatomical organization as well as sex differences
in response to estradiol as far as cholinergic enzyme induc-
tioninadult life andthe effects of T
3
treatment withinthe first
week of postnatal life. These differences may underlie, at
least in part, the sex differences in spatial learning that are
discussed below.
C. Estrogens and the serotonergic system
Serotonin neurons of the midbrain/brainstem raphe nu-
clei are among the earliest neuronal phenotype to become
differentiated during CNS development, and serotonin is
believed to act as a regulatory/developmental agent (176,
177). The more rostral nuclei (primarily the dorsal and me-
dial raphe) form ascending projections, densely innervating
such forebrain regions as the hypothalamus, hippocampus,
and cortex. Thus, the serotonergic system is involved in the
regulation of such diverse functions as reproduction, mood,
sleep, and cognition. While serotonergic activity is regulated
by the ovarian steroids, the mechanisms by which such reg-
ulation occurs are not fully understood. Here, we will briefly
review findings that suggest involvement of both presyn-
aptic and postsynaptic actions.
A sex difference in the serotonin system of the rat brain is
established by the end of the second postnatal week (178).
Female rats demonstrate higher serotonin levels and/or syn-
thesis measured in whole brain (179), forebrain (180), raphe
(181), frontal cortex (182), hypothalamus (181183), and hip-
pocampus (182, 184) compared with the male rat brain. A
similar sex difference in rat brain serotonin turnover, an
indication of serotonergic activity, has also been reported
(180, 185). Furthermore, brain serotonin levels and activity
are altered during periods of physiological ovarian hormone
fluctuation, including the estrous cycle, pregnancy, or the
postpartum period (186190) in the rodent. In addition, es-
trogen and/or progesterone treatment of ovariectomized
rats has been shown to positively affect the serotonergic
system of the female rat brain (51, 191202).
In addition to reporting significant increases in hippocam-
288 MCEWEN AND ALVES Vol. 20, No. 3
pal serotonin levels and synthesis rate in females, Haleem
and colleagues (184) found that female rats are much more
responsive to the 5-HT1A receptor-mediated inhibition of
serotonin synthesis. That is, after the administration of the
5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-
tetralin, female rats exhibited a decrease in hippocampal
serotonin synthesis that was twice that seen in males. This
may be partly explained by the finding that estrogen treat-
ment increases the efficiency of the 5-HT1A receptor to in-
hibit cAMP formation in isolated membrane fractions in the
hippocampus (203).
With regard to the estrogen sensitivity of serotonergic
neurons, the direct or indirect nature of hormone action is
only nowbeginning to emerge at the level of the raphe nuclei.
Estrogen-concentrating cells, determined by autoradiogra-
phy, have been previously reported in the raphe nucleus in
the male and female lizard, Anolis carolinensis, but it was not
determined whether the cells were serotonergic (204). More
recently, in rhesus macaques, Bethea (205) demonstrated the
presence of estrogen-inducible PR in a majority of serotonin
neurons, as well as in nonserotonin cells, in the dorsal and
ventral (medial) raphe of intact and spayed estrogen- and
progesterone-treated macaques. Because progesterone treat-
ment of estrogen-primed macaques increases PRL release via
a serotonergic mechanism (206, 207), the finding of PR in
serotonin neurons provides a direct means by which the
ovarian steroids can regulate serotonergic function. In ad-
dition, Betheas group has demonstrated that ovarian hor-
mones increase the expression of tryptophan hydroxylase
(TPH), the key enzyme in serotonin biosynthesis, and sup-
press expression of the serotonin transporter (SERT) in the
macaque raphe nuclei (208, 209). For a recent review, see
(210). While TPHmRNAlevels do not appear to be regulated
by estrogen or progesterone in the rat dorsal raphe (S. Alves
and B. McEwen, unpublished results), SERT mRNAhas been
reported to be increased by estrogen in the dorsal raphe of
this rodent species (211). It is not presently known whether
this difference in SERT regulation between the macaque and
the rat is due to difference(s) in species and/or length of
hormone treatment.
Curiously, the rat does not show localization of ER or PR
in serotonergic neurons (38) in spite of the ample evidence
for ovarian hormone regulation of serotonergic function in
the rat brain. However, a number of ER and/or PR immu-
noreactive neurons are found within the female and male rat
dorsal raphe, adjacent to the serotonin cells (Fig. 4), suggest-
ing transsynaptic regulation; females were found to have
significantly more PR-containing cells, but no sex difference
in the number of ER-labeled cells was observed (38). Recent
data indicate that a subpopulation of these steroidtarget cells
demonstrate immunoreactivity to the excitatory amino acids,
glutamate andaspartate (212). ERmRNAhas been reported
within the dorsal raphe of the rat (47), although ER protein
has not yet been detected (38) (see Section II.C for discussion
of possible explanations for this type of discrepancy). Yet
there are estrogen effects in the rat serotonin systemthat may
eventually be explained by the presence of functional ER.
Estrogen and progesterone treatment alters the expression
of several genes within the rat dorsal raphe nucleus that are
involved in serotonergic transmission: the postsynaptic
5-HT2A receptor (37, 213) and the presynaptic SERT (211)
and vesicular monoamine transporter (VMAT2) (214). These
data suggest that ovarian steroids are likely to modulate
serotonergic transmission at the dorsal raphe by regulating
both nonserotonergic and serotonergic cells. Interestingly,
recent findings indicate that the former two genes appear to
be similarly regulated by estrogen in females and males (37,
215), which is in agreement with the lack of a gender dif-
ference in the number of ER-containing cells within this
nucleus. Ovarian steroid regulation of VMAT2 has been re-
ported only in females, and in this study it was demonstrated
that progesterone, either alone or in combination with es-
trogen treatment, decreased VMAT2 mRNA to a similar ex-
tent. It would be interesting to investigate whether such
regulation occurs in males, considering the gender difference
in PR immunoreactive cells within the dorsal raphe (38).
Thus far, the only conclusion to be drawn is that ovarian
hormones may work indirectly in the rat brain through
adjacent neurons that express ER and/or PR, and per-
haps both directly and indirectly in the macaque raphe
nuclei, to influence serotonergic function at the midbrain
level. However, as noted, the demonstration of functional
ER in the dorsal raphe could change this interpretation,
particularly for the rat. Moreover, the rat may be unusual,
in that preliminary evidence from the mouse suggests that
ER or PR immunoreactivity occurs in some TPH-labeled
neurons, and abundantly in non-TPH cells in the dorsal
raphe, suggesting direct steroid regulation of at least a
subpopulation of serotonin cells in this rodent species
(212). It should be mentioned that while estrogen-induced
PR have been identified in serotonin neurons in the ma-
caque (as described above), ERs have not been detected
in the macaque raphe (C. L. Bethea, personal communi-
cation), once again raising the issue of a problemin antigen
detection and/or rather that ER may be the functional ER
in this species. Recent evidence from the ERKO mouse
indicates abundant estrogen binding in the dorsal raphe
(216), strengthening the idea that ER may play an im-
portant role in this brain region.
Thus, by a multiplicity of pre- and postsynaptic mecha-
nisms, ovarian steroids affect serotonergic function in a sex-
ually dimorphic fashion, and these actions are relevant to the
actions of estrogens on mood and cognition. High doses of
estrogens were reported to have antidepressant effects in
human subjects (217), and estrogen treatment influences the
response to antidepressant drugs in animal models (8) and
in clinical studies (10). Moreover, estrogen treatment of
ovariectomized rats led to less struggling or immobility, and
more time swimming, in the forced swim test, a measure of
anxiety; and estrogen treatment reduced the number of cells
expressing the immediate early gene, c-fos, during the forced
swim test (218). Both of these results are consistent with an
antianxiety effect of estrogens in the rat and human. Indeed,
results from a recent clinical trial of fluoxetine (Prozac, Eli
Lilly, Indianapolis, IN) indicated that women receiving es-
trogens as well as Prozac were the most responsive (10).
However, in view of the small sample size in that study, this
is a finding that needs to be replicated in a larger study.
June, 1999 ESTROGEN ACTIONS IN THE CNS 289
D. Catecholaminergic neurons
1. Noradrenergic system. In addition to the cholinergic and
serotonergic systems, catecholaminergic systems respond to
estrogens, i.e., brainstemcatecholaminergic neurons (A6 and
to a lesser extent A5 and A7) contain small numbers of ER
(219), and estrogen treatment after gonadectomy exerts com-
plex, time-dependent effects on the level of tyrosine hydrox-
ylase mRNA(220). Moreover, recent studies of rats (221) and
sheep (222) indicate that A1 and A2 noradrenergic neurons
specifically express the ER and show cyclical and estrogen-
dependent patterns of immediate early gene expression (223,
224). In the rat locus ceruleus, galanin is coexpressed in many
noradrenergic neurons, andestrogen treatment increasedthe
expression of galanin mRNA, leading to the speculation that
estrogen treatment might reduce noradrenergic tone in the
absence of separate effects on tyrosine hydroxylase expres-
sion by enhancing the cosecretion of galanin, which reduces
noradrenaline release (225).
2. Dopaminergic systems. Incertohypothalamic dopamine neu-
rons are distributed in the rostral, periventricular, caudal,
FIG. 4. Map of ER- and PR-containing neurons in the rat midbrain dorsal raphe region and surrounding periaqueductal gray (PAG). Each dot
represents approximately two immunoreactive cells. A, Nuclear ERs (ER) in gonadectomized (GDX) rats. B, Nuclear PRs in GDXrats treated
with estradiol benzoate for several days. The brain levels depicted are measured in distances from Bregma (B). Note the higher density of cells
containing ER immunoreactivity at the more rostral levels of the dorsal raphe nucleus (DRN) but the higher concentration of PR-immuno-
reactive cells specifically within the lateral wings (LW) of the DRN, depicted at level B 8.30. In contrast to most other regions of the DRN
examined, this population of neurons maintains rather abundant PR immunoreactivity without E priming. AQ, Cerebral aqueduct; ELi, caudal
linear raphe nucleus; V4, fourthventricle. [Reproduced withpermissionfromS. Alves et al.: JComp Neurol 391:322334, 1998 (38). Wiley-Liss,
Inc., a subsidiary of John Wiley & Sons, Inc.]
290 MCEWEN AND ALVES Vol. 20, No. 3
and dorsomedial regions of the hypothalamus and represent
an internal source of dopamine innervation for the hypo-
thalamus and preoptic region (226). The incertohypotha-
lamic dopamine neurons express sex differences in neuron
number and function (227). Estrogen and PRL have hetero-
geneous effects on dopamine turnover, increasing it in dor-
somedial nucleus and decreasing it in rostral periventricular,
medial preoptic, and preoptico-suprachiasmatic nuclei (228).
In the midbrain dopaminergic projections to the corpus
striatum and nucleus accumbens, there are sexually dimor-
phic actions of estrogens and progestins, involving both pro-
and antidopaminergic effects that depend on the dose and
time course of estrogen administration and are manifested in
both the nigrostriatal and mesolimbic dopaminergic systems
(229, 230). Estrogen facilitates amphetamine- or apomor-
phine-stimulateddopamine release andlocomotor activity in
rats unilaterally lesioned by 6-hydroxydopamine (231233),
and this activity is responsive to natural fluctuations in es-
tradiol and generally increased during late proestrus and
early estrus (232234). Spontaneous sensorimotor activity is
also influenced by estrogens, e.g., in a tight-rope walking
task (235). Coordination of locomotor activity may also in-
volve estrogen actions in other brain regions, such as cere-
bellum, where membrane actions of the steroidare suspected
on the basis of rapidity of effects and absence of known
intracellular receptors (1, 98). In spite of their rapidity, these
effects must still be considered in terms of the presence of
ER mRNA in cerebellum and cerebral cortex (48) even
though functional ER protein has not yet been demon-
strated (see Section II.C).
In striatum, ovariectomy decreases, and administration of
estradiol potentiates, the depolarization-induced release of
dopamine as well as rotational behavior in a sexually di-
morphic pattern(236240). Male rats showsmaller responses
to estrogen than females, and castration of males does not
affect the amphetamine stimulation of rotational behavior or
striatal dopamine release (234, 241, 242).
In females, no classical ERhave been identified in striatum
(63, 64, 243); nevertheless, intrastriatal application of estra-
diol rapidly causes rotational behavior (244) and enhances
sensorimotor performance (235). Estrogen directly potenti-
ates potassium-stimulated dopamine release from rat nu-
cleus accumbens (102), and estrogen pretreatment increases
the firing rate of neostriatal neurons in response to dopamine
(245), possibly via changes in D1 or D2 receptor coupling
(246). A variety of estrogen effects on dopamine receptor
binding have been reported (see Refs. 229 and 230 for re-
views).
Estrogen actions in the striatum that do not involve the
classical ER have been proposed on the basis of four types of
evidence: 1) the lack of intracellular ER in striatum; 2) the
rapidity of estrogen effects; 3) the pharmacological profile of
estrogen action, particularly the ineffectiveness of diethyl-
stilbestrol; and 4) the ability of estradiol conjugated to BSA
to mimic effects of free estradiol (247). One possible expla-
nation, already described above, are the actions of estradiol
to reduce L-type calcium channel activity in striatal neurons
via a G-protein-coupled receptor (121).
The dopamine system shows declining function in the
aging brain (248), and clinical observations indicate antido-
paminergic effects of moderate to high doses of estrogens.
Relatively high levels of estrogens, including oral contracep-
tives and estrogen replacement therapy, exacerbate symp-
toms of Parkinsons disease (6, 249, 250), pointing to anti-
dopaminergic actions that are opposite to the actions of
physiological levels of estradiol. Asimilar antagonistic effect
of chronic or high-dose estrogen was found in male and
female rats for drug-induced motor activity (251, 252).
E. Spinal cord
The spinal cord contains limited numbers of cells dem-
onstrating intracellular ER, and there is also evidence for
antinociceptive and analgesic actions of estrogens, with a
large sex difference that may be mediated at the spinal level
or at other levels of the neuraxis (for discussion see Refs. 253
and 254). However, the information is rather limited regard-
ing possible genomic or nongenomic mechanisms, and func-
tional studies do not coincide with the information about ER
localization.
ERs have been colocalized by immunocytochemistry
with enkephalin in many neurons in the medullary and
spinal dorsal horn, particularly in the superficial laminae
where they could be involved in modulating sensory and
nociceptive processing (253, 254). A moderate concentration
of labeled cells expressing ER mRNA has been reported in
lamina II of the spinal cord, whereas scattered cells express-
ing ER mRNA were found in laminae I and II, the medial
portion of laminae VI and VII, and in lamina X near the
central canal (47).
Pain sensitivity differs strikingly between men and
women and in women in different reproductive hormone
states (see below and Refs. 253 and 254). Sex differences in
analgesia have been reported in mice along with sex-specific
effects of estrogens. In particular, nonopioid analgesia pro-
ducedby swimstress was different between male andfemale
Swiss-Webster mice and became equalized by ovariectomy;
estrogen replacement of ovariectomized females reversed
the effect, but estrogen treatment of intact or castrated males
had no effect, indicating an insensitivity of this system to
estrogens in the male mouse (254). In a follow-up study,
quantitative trait locus (QTL) mapping was carried out and
led to the identification of a female-specific QTL on chro-
mosome 8 (255). This female-specific mechanism, which is
sensitive to estrogen modulation, is consistent with a gene
that is turned off by testosterone exposure during sexual
differentiation (256). Because it involves nonopioid analge-
sia, this form of estrogen-sensitive analgesia is unlikely to be
related to the enkephalin/estrogen colocalization described
above or to NMDA-receptor mediated analgesia to which
mice are also insensitive; rather, a novel form of nonopioid,
non-NMDAanalgesia is indicated (254, 255). The role of ER
mRNAexpression in spinal cord and its relationship to func-
tional ER receptors in this structure remain to be estab-
lished.
F. Hippocampus
1. Cyclic synaptogenesis on hippocampal neurons. While syn-
apses are formed and eliminated during development, syn-
June, 1999 ESTROGEN ACTIONS IN THE CNS 291
aptogenesis was, until recently, believed to be more limited
in the adult nervous system. Estrogens regulate synapse
density in the adult rat hypothalamic ventromedial nucleus
that differs between males and females (145, 257, 258). This
discovery led to the finding that the ovarian cycle regulates
cyclic synaptogenesis on excitatory spines in hippocampal
CA1 pyramidal neurons in female but not in male rats (259,
260). Synaptogenesis is cyclic, and fluctuations in synapse
density occur throughout the estrous cycle of the female rat
(158). The increase in synapses on dendritic spines after
estrogen treatment is shown in Fig. 5, along with the decrease
in spine synapse density that occurs between the days of
proestrus and estrus in cycling female rats. Male rats show
much less estrogen-induced synapse formation unless they
are treated at birth with an aromatase inhibitor (260). This
suggests that the developmentally regulated expression of
ERs and aromatase activity in hippocampus (261, 262) is
involved in programming the response of the adult hip-
pocampus.
Cyclic synaptic turnover in the hippocampus and hypo-
thalamus during the estrous cycle of a female rat shows a
high degree of specificity. For example, in hypothalamus,
neurons of the ventromedial nucleus show cyclic synapto-
genesis directed by ovarian steroids. In CA1 pyramidal neu-
rons of the hippocampus, estrogen-induced synaptogenesis
occurs on dendritic spines and not on shafts, and there are
no estrogen effects on dendritic length or branching; more-
over, as far as one can tell, such synaptic plasticity is ex-
tremely specific and does not occur on CA3 pyramidal neu-
rons or dentate gyrus granule neurons (263). The discreteness
and specificity of this synapse formation imply that molec-
ular markers may be very specific or subtle and that the
mechanism may involve changes in a limited number of
cellular events, including transcription of discrete structural
genes and posttranscriptional events such as translation of
mRNAs for structural proteins. Moreover, local regulation,
as via afferent input or interneurons, may be very important.
One of the surprises of the synaptogenesis story is that
estrogen induction of synapses is blocked by NMDA recep-
tor antagonist treatment, indicating that excitatory amino
acids and NMDA receptors are involved in synapse forma-
tion (264, 265). Progesterone secreted at the time of ovulation
appears to be responsible for down-regulation of estrogen-
induced synapses in the CA1 region (266), and the cellular
location of PRs, as well as of the ERs, is a prime question.
2. Localization of intracellular ERs. The presence of the classical
ER and the recently discovered ER complicates the story
of estrogen action. ERs have been identified by immuno-
cytochemistry in scattered GABA-ergic interneurons in the
rat hippocampus (39), and this distribution of ER is in agree-
ment with autoradiography of [
3
H]estradiol uptake (267), so
that one does not need to postulate the existence of another
high-affinity intracellular ER. The localization of ER is sum-
marized for the hippocampus and adjacent cerebral cortex in
Fig. 6. ER expression has been claimed in pyramidal neu-
rons by immunostaining and also mRNA expression (46, 47,
65), although, as mentioned previously, our laboratory has
not seen consistent ER immunostaining in hippocampus
(N. Weiland, S. E. Alves, V. Lopez, and K. Bulloch, unpub-
lished). Clearly, more studies are needed on this issue. There
are a number of plant estrogens, genestein and daidzein,
with approximately 20-fold higher affinities for ER than
ER, which makes them useful to discriminate between the
two receptor types (55, 268), and these may be useful in
further studies on the role of ER in the hippocampus.
3. Role of the NMDA receptors. Antagonists of NMDA recep-
tors blocked estrogen-induced synaptogenesis on dendritic
spines in ovariectomized female rats (264, 265). Because es-
trogen treatment increases the density of NMDAreceptors in
the CA1 region of hippocampus (265, 269, 270), it is possible
that activation of NMDA receptors by glutamate is the pri-
mary actionthat induces newexcitatory synapses to develop.
Spines are occupied by asymmetric, excitatory synapses, and
they are sites of Ca

ion accumulation and thus ideal sites

for NMDA receptors (271). NMDA receptors are expressed
in large amounts in CA1 pyramidal neurons and can be
imaged by conventional immunocytochemistry as well as by
confocal imaging (270), in which individual dendrites and
spines can be studied for colocalization with other markers
(272, 273). NMDA receptor mRNA can also be measured by
in situ hybridization, and four different forms showdifferent
regional patterns and developmental regulation (274).
FIG. 5. Depiction of ovarian steroid
regulation of the density of excitatory
spine synapses in the CA1 region of the
female rat hippocampus. Estimated
density of synapses on dendritic spines
in the stratum radiatum of the CA1 re-
gion of the hippocampus, in panel A, of
an ovariectomized (OVX) rat treated
with oil (O) or estradiol (E) or, in panel
B, of intact rats in the proestrus or es-
trus phase of the estrous cycle. Values
represent mean SEM obtained using
the Disector method. Note that in each
case, higher estradiol levels are corre-
lated with a greater density of syn-
apses. Data were analyzed with un-
paired, two-tailed t tests, n 4 in each
case. *, P 0.025. [Reproduced with
permission from C. Woolley and B. S.
McEwen: J Neurosci 12:25492554,
1992 (158).]
292 MCEWEN AND ALVES Vol. 20, No. 3
NMDA receptors are implicated in other morphogenetic
processes in the adult brain such as suppressing neurogen-
esis in the dentate gyrus (275), and they are also involved in
the developing nervous system as facilitators of neuronal
migration (276, 277). However, there is a noteworthy para-
dox, in that NMDA receptors are implicated during visual
system development in the reduction of synaptic contact in
the developing retinal axon arbors (278), and NMDA recep-
tor blockade results in rapid acquisition of dendritic spines by
visual thalamic neurons (279). It appears likely that hip-
pocampus and visual system neurons respond in opposite
ways to NMDAreceptors, since a recent report on embryonic
hippocampal neurons in culture (see Section III.F.5 below)
indicates that NMDA receptor blockade prevents estrogen-
induced synaptogenesis (280).
4. Genomic vs. nongenomic actions of estrogens on synapse for-
mation. The paradoxical estrogen effects on hippocampal py-
ramidal neurons that do not appear to have intracellular ER
or show uptake and cell nuclear retention of [
3
H]estradiol
might be explainable if there were cell surface ERs. Rapid
estrogen effects on CA1 pyramidal neurons of the hippocam-
pus have beendescribedbyin vitro electrophysiological stud-
ies on slices fromthis brain region, and these appear to involve
non-NMDA excitatory amino acid receptors (94, 95) that are
very likely to be AMPA (-amino-3-hydroxy-5-methyl-4-isox-
azole propionic acid) receptors (96). One approach to rule in or
out nongenomic actions of estrogen would be to study ERKO
mice lacking intracellular ER (43), and a recent study with
mice lacking ER has shown that estrogen actions on kainate-
stimulated ionic currents are still present (103). An ER double
knockout would be even better, provided there are only two
intracellular ER genes.
Another approach to discriminate between classical intra-
cellular ER and membrane ER is to use antiestrogens that
bind to the intracellular ER but which mimic, rather than
block, the rapid membrane effects, such as was the case for
estrogen effects on calcium currents in neurons from the
corpus striatum (121). Antiestrogens also have another use,
namely, to discriminate between the response elements that
the ER uses to activate transcription. As noted above, the
major differences between ER and ER1 concern their abil-
ity to regulate transcription via the AP-1 response element.
For interactions of ER with AP-1, 17-estradiol as well as a
number of antiestrogens activated transcription; however,
for ER1 interacting with AP-1, 17-estradiol failed to acti-
vate transcription but antiestrogens activated transcription
(69).
Estrogen antagonists have been very useful in testing al-
ternatives to conventional genomic actions of estrogen on
hippocampal synapse formation by providing pharmacolog-
ical evidence in favor of a particular pathway of hormone
action and against other possible mechanisms (281). The
antiestrogen, CI-628, has previously been shown to enter the
brain and block estrogen induction PR (see Fig. 3). The same
dose of CI-628 that blocked PR induction was also able to
block spine synapse induction by estrogen in the hippocam-
pus, and CI-628 did not have any agonist-like activity of its
own (281) (see Fig. 7). An agonist-like action of CI-628 would
have been expected had it exerted its action nongenomically
FIG. 6. Map of ER immunoreactivity in the hippocampus and cortex
of the rat brain. [Drawings are modified from L. W. Swanson, Brain
Maps: Computer Graphic Files (version 1.0), and represent coronal
sections from three different levels of brain measured from bregma.]
Each dot represents one ER-immunoreactive cell (total number is
mean from male and female rats, which do not differ significantly
from each other). Note the ER immunoreactivity cells are interneu-
rons, not pyramidal neurons, in agreement with previous autoradio-
graphic studies (see text). DGlb, Lateral blade of the dentate gyrus;
ENT, entorhinal cortex; fc, fasciola cinerea; fi, fimbria; hf, hilar fis-
sure; mo, molecular layer of the dentate gyrus; PAR, parietal cortex;
po, polymorph layer (hilus) of the dentate; RSP, retrosplenial cortex;
sg, stratum granulosum; so, stratum oriens; sp, stratum pyramidale;
sr, stratumradiatum; SUB, subiculum; v3, third ventricle; vip, velum
interpositum; vl, lateral ventricle. [Reproduced with permission from
N. G. Weiland et al.: J Comp Neurol 388:603612, 1997 (39). Wiley-
Liss, Inc., a subsidiary of John Wiley & Sons, Inc.]
June, 1999 ESTROGEN ACTIONS IN THE CNS 293
via calcium channels, as has been shown for striatal neurons
(121). An agonist-like action might also have occurred via
ER or -, or a heterodimer, acting via another response
element than the ERE (see Section II.C). The fact that CI-628
blocked, rather than mimicked, estrogen action is inconsis-
tent with any known nongenomic effect and is similar to the
estrogen induction of PRs that is believed to involve an ERE
(282). Moreover, it is consistent with an action of estrogen via
the intracellular ERs that are known to exist in hippocampal
interneurons, although, again, it should be pointed out that
ER could also mediate actions via an ERE and that the
presence of some functional ER in hippocampus is still a
distinct possibility given the presence of ER mRNA in this
brain region (see Section II.C).
5. Synapse formation in cultured hippocampal neurons. Recent
studies on hippocampal neurons in culture have revealed
that estrogen induces spines on dendrites of dissociated hip-
pocampal neurons in culture by a process that is blocked by
an NMDA receptor blocker and not by an AMPA/kainate
receptor blocker (283). In a subsequent study, estrogen treat-
ment was found to increase expression of PCREB and CREB,
and a specific antisense to CREB prevented both the forma-
tion of dendritic spines and the elevation in PCREB (284).
ERs have been located on glutamic acid carboxylase
(GAD)-immunoreactive cells in vitro that constitute approx-
imately 20% of neurons in the culture (39), and this is con-
sistent with in vivo data summarized above. 17-Estradiol
treatment of cultured cells caused GAD content and the
number of neurons expressing GAD to decrease, and mim-
icking this decrease with an inhibitor of GABA synthesis,
mercaptopropionic acid, caused an up-regulation of den-
dritic spine density, simulating the effects of 17-estradiol
(285). Figure 8 summarizes the hypothesized interaction be-
tween these GABA interneurons and the pyramidal neurons
upon which the synapses are induced by estrogen treatment.
Both cell culture data and in vivo studies summarized above
are consistent with this model.
An additional factor in the formation of dendritic spines
in the in vitro cell culture model is the neurotrophin, brain-
derived neurotropic factor (BDNF), which is expressed in
GABA interneurons in hippocampal cell cultures (286). In
addition to down-regulating GABA in these interneurons,
estrogen treatment also reduced BDNF by 60% within 24 h
(286). Since exogenous BDNF blocked estrogen induction of
dendritic spines and BDNF depletion with an antisense or
blockade with BDNF antibodies both mimicked estrogen in
inducing spine density, the authors suggest that BDNF is an
important player in the regulation of GABA inhibition,
which in turn blocks activity-dependent regulation of den-
dritic spines in hippocampal neurons (286). It is interesting
to note that neurotrophins such as BDNF and NT-3 also
increase the function of inhibitory and excitatory synapses in
hippocampal cell cultures, and BDNF causes an increase in
axonal branching and length of GABA-ergic interneurons
(287).
6. Developmentally regulated sex differences in the hippocampus.
The hippocampus is one of a number of extrahypothalamic
brain structures that shows subtle sex differences. For ex-
ample, there are sex differences in the density of apical den-
dritic excrescences and branching of dendrites of CA3 py-
ramidal neurons. Treatment with T
3
during the first week of
postnatal life enhanced these differences (288). Excrescences
on the proximal region of apical dendrites receive input from
mossy fiber synapses from granule neurons of the dentate
gyrus. Therefore, the greater density of excrescences in males
is consistent with a report that male rats have a greater
number of mossy fiber synapses than females (289). Other
studies have pointed to sex differences in hippocampal mor-
phology that are dependent on the rearing environment
(290).
The dentate gyrus of mice and rats also shows sex differ-
ences. In mice, there are strain-dependent sex differences: in
strains with large numbers of granule neurons, males have
more neurons than females, while in strains with fewer gran-
ule neurons, the sexes do not differ fromeach other in neuron
FIG. 7. Effect of the estrogen antagonist, CI-628, on estrogen induc-
tion of increased dendritic spine density on CA1 pyramidal neurons.
CI-628 (10 mg/kg) was given at 0, 24, and 48 h, and estradiol benzoate
(EB, 10 g/kg) was given at 24 and 48 h to ovariectomized female rats,
whichwere thenkilled at 72 h. The single-sectionGolgi procedure was
carried out to stain dendrites of CA1 pyramidal neurons for visual-
ization of dendritic spines. Data are expressed as the number of
spines/10 m length on secondary dendrites that were greater than
10 min length and located 150200 maway fromthe cell body. Six
CA1 neurons fulfilling the criteria described in the original publica-
tion (281) were analyzed for each rat brain. The number of rats per
group was six for the CI628 EB group and five for each of the other
treatments. The error bars show the SEM; this is based on the mean
and variance calculated across animals, with data for the six neurons
of each animal in a treatment compiled into a single average. Sta-
tistical analysis of data revealed that there was an overall treatment
effect, P 0.0001, by one-way ANOVA. A Tukey post hoc comparison
revealed a clear E induction of spines, in which ovx E was different
from each of the other groups (P 0.001). Moreover, CI-628 partially
blocked the E effect, in that ovx E CI-628 was significantly
elevated compared withOVX(P0.01) and significantly less that ovx
E(P0.001). There was no agonist effect of CI-628 by itself onspine
density (P 0.92). [Reproduced with permission from B. S. McEwen
et al.: Endocrinology 140:10441047, 1999 (281). The Endocrine
Society.]
294 MCEWEN AND ALVES Vol. 20, No. 3
number (291). Male rats have a larger and more asymmetric
dentate gyrus than females, and neonatal testosterone treat-
ment caused the genetically female dentate gyrus to appear
male like (292). Neonatal testosterone treatment in female
rats also improved spatial learning ability in a Morris water
maze (292).
How do these sex differences come about during devel-
opment? Like the cerebral cortex, the rat hippocampus ex-
presses ER transiently during perinatal development (261,
293). The presence of these receptors in hippocampus coin-
cides with the transient expression of the aromatizing en-
zyme system that converts testosterone to estradiol (294); as
a result, ER in male rats would be exposed to locally gen-
erated estradiol, and this could lead to sexual differentiation
of hippocampal structure and function. Consistent with this
scenario are data showing that, while neonatal castration of
male rats produced female-like learning curves in a Morris
water maze, the administration of estradiol to newborn fe-
male rats produced a male-like learning curve (295).
It should be noted that the cell culture model described
above for studying estrogen-induced synaptogenesis in vitro
lies right on the interface between developmental actions of
estrogens and the activational effects in mature neurons.
That is, the cell cultures are generated from late fetal brain
tissue before the stage of sexual differentiation has been
completed; however, the fact that the hippocampal cell cul-
tures are allowed to mature in vitro, differentiate into exci-
tatory and inhibitory neurons, and form synaptic connec-
tions makes them more like the mature nervous system. It is
interesting to consider whether application of gonadal ste-
roids during the differentiation and formation of synaptic
connection might mimic aspects of the sexual differentiation
of hippocampal circuits and functions described above and
might lead, for example, to a permanent male-like inability
of the cultures to show synapse induction in response to
estradiol.
7. Comparison with other forms of structural plasticity. The hip-
pocampus also undergoes two other forms of plasticity, in
which circulating hormones and excitatory amino acids act-
ing via NMDA receptors are involved. One of these is the
ongoing neurogenesis in the adult rat dentate gyrus, which
continues for at least 1 yr after birth and can be increased
either by adrenalectomy or by treatment with an NMDA
receptor antagonist (275). Although the male dentate gyrus
is larger than that of the female (296), there are data for the
prairie vole (297) and rat (298) indicating that estrogens in-
crease neurogenesis of granule neurons in the female. Thus,
it remains to be established for males and females what the
balance is between neurogenesis and programmed cell death
to account for sex differences in overall neuron number be-
tween the sexes.
Dentate gyrus granule neurons innervate the CA3 region
of Ammons horn, and stress causes apical dendrites of CA3
pyramidal neurons to undergo atrophy by a process that is
dependent in part on circulating adrenal steroids and in part
on excitatory amino acids acting via NMDA receptors (299).
Stress-induced dendritic atrophy is also reversible (A. M.
Magarinos and B. S. McEwen, unpublished), but severe and
prolonged social stress (in vervet monkeys) and cold-swim
stress (in rats) causes CA3 pyramidal neuron loss in males
that is not evident in females (164, 165). Thus, there is the
possibility that intrinsic sex differences in hippocampal mor-
phology or in response to hormones or excitatory amino
acids may have a protective role in the female.
A recent study indicates that female rats are also resistant
to the stress-induced atrophy of CA3 pyramidal neurons in
hippocampus (300). In addition to the larger dentate gyrus
of the male (296), male CA3 neurons have more excrescences
for mossy fiber contacts, while female CA3 apical dendrites
are more extensively branched (288). However, it is not clear
howthese differences might contribute to the sex differences
in the effects of stress.
FIG. 8. This model of synaptogenesis in
the hippocampus emphasizes the role of
NMDA receptors and the key role of in-
hibitory GABA interneurons. ER is
present in interneurons, and its pres-
ence coincides with the distribution of
ER-binding sites fromin vivo [
3
H]estra-
diol autoradiography. According to the
best evidence to date, based upon im-
munocytochemistry of hippocampus
and cell culture studies, estrogens sup-
press GABA function transiently and
lead to disinhibition of a large number
of innervated CA1 neurons resulting in
up-regulation of NMDA receptors and
synapse formation. Blocking NMDA re-
ceptors prevents estrogen-induced syn-
apse formation.
June, 1999 ESTROGEN ACTIONS IN THE CNS 295
8. The functional significance of synaptogenesis in the hippocam-
pus. The functional significance of synaptogenesis in the
hippocampal CA1 region has been shown in electro-
physiological studies indicating that estrogen treatment of
ovariectomized rats produces a delayed facilitation of syn-
aptic transmission in CA1 neurons that is NMDA mediated
(95) and leads to an enhancement of voltage-gated Ca

currents (94, 95). This approach has nowbeen taken to a new

level by Woolley, who has used biocytin injection and im-
munostaining after recording from CA1 pyramidal neurons
to visualize estrogen induction of spines; she found that
spine density correlates negatively with input resistance and
that input/output curves show an increased slope under
conditions in which NMDA receptor-mediated currents pre-
dominate, whereas there is no increased slope where AMPA
receptor currents predominate (265). Moreover, in intact fe-
male rats, there is a peak of LTP sensitivity on the afternoon
of proestrus in female rats at exactly the time when excitatory
synapse density has reached its peak (301).
Proestrus is also the time of the estrous cycle when seizure
thresholds in dorsal hippocampus are the lowest (302). Be-
cause activation of NMDA receptors in hippocampus is en-
hanced via AMPA receptors in some cases but not in others
(303), it remains to be seen how plastic the AMPA receptor
system is to ovarian steroid manipulations or whether the
estrogen-induced synapses are so-called silent synapses or
ones in which AMPA receptors are induced by LTP. Block-
ade of AMPA receptors with 6-nitro-7-sulfamobenzo(f) qui-
noxaline-2,3-dione during estrogen treatment failed to block
synaptogenesis (264), which suggests that AMPA receptors
do not play a major role in the operation of the estrogen-
induced synapses.
G. Glial cells, endothelial cells, and the blood-brain barrier
A separate category of estrogen actions concern the glial
cells of the brain and the endothelial cells, thus blood-brain
barrier, since both cell types affect the entry of vital sub-
stances such as glucose into the brain. Glial cells are affected
by estrogens in vivo and in vitro (304, 305). In addition, es-
trogens regulate specific genes such as Apolipoprotein E
within astrocytes and microglia (306, 307). Apolipoprotein E
is a lipophilic protein involved in cholesterol transport, and
its absence has recently been linked to a deficit in synaptic
sprouting in the hippocampus (307). Estrogen treatment also
regulates morphology of astrocytes in hypothalamus (308)
and hippocampus (309312), and these changes may reflect
a role of glial cells in normal synaptic plasticity as well as
lesion-induced plasticity.
Gonadal steroids, including estrogen, regulate the expres-
sion of glial fibrillary acidic protein (GFAP). In the arcuate
nucleus, estrogenelevationat proestrus transiently increased
expression GFAP mRNA in female mice (313). However,
removal of gonadal steroids increases GFAP expression in
hippocampus (314), andthis mirrors the cyclicity of astroglial
morphology in the CA1 region of hippocampus, with in-
creases of astrocytic volume on diestrus when estrogen levels
are low and decreases of astrocytic volume on proestrus
when estrogen levels are elevated (312). It is noteworthy that
levels of GFAP mRNA and protein increase throughout the
brain as it ages, regardless of gender or species (313, 315), and
thus the effect of gonadal steroids in both sexes opposes the
effect of aging. Schipper (316) has recently reviewed the
relationship between astrocytes and brain aging.
Astroglia play a role in synaptic retraction during the
ovulatory cycle in the adult hypothalamic arcuate nucleus.
During the preovulatory and ovulatory phases of the female
rat estrous cycle, there is a transient disconnection of inhib-
itory synaptic inputs to arcuate nucleus neurons (317). This
remodeling is mimicked by estrogen, blocked by progester-
one, and begins with the onset of puberty in female rats;
moreover, neonatal testosterone during the sensitive period
for sexual differentiation of the brain alters the pattern of
synaptic contacts in the arcuate nucleus (317). Astroglia reg-
ulate this process of synaptic remodeling by controlling the
amount of neuronal membrane available for synaptic con-
tacts and by releasing soluble factors, such as insulin-like
growth factor I (IGF-I) (317, 318). The decline in synaptic
inputs to arcuate neurons between the morning of proestrus
and the morning of estrus was blocked by an IGF-I receptor
antagonist (318). Moreover, there seems to be a reciprocal
interaction between IGF-I and estrogen, and one speculation
is that estrogens may act in arcuate nucleus neurons to reg-
ulate the production of a factor, possibly GABA, that, in turn,
regulates the expression of IGF-I by astroglia (tanycytes) in
the arcuate nucleus region (Refs. 317 and 318; M. Garcia-
Segura, personal communication).
The mechanisms of estrogen action on glial cell function
remain unclear. Central glial cells have been reported to
express ER (304, 305), although receptor protein is not usu-
ally detectable in vivo within glia at the light microscopy level
(B. S. McEwen, N. Weiland, S. E. Alves, and K. Bulloch,
unpublished results). The widespread distribution of ER
mRNA within the CNS may eventually be shown to include
expression within astrocytes, oligodendrocytes, and/or mi-
croglia.
Glial cells and vascular epithelium also play another role
in the adult brain, namely, in relation to glucose uptake and
energy metabolism. Ovarian steroids also play a role in the
ability of the female brain to utilize glucose as its primary
energy source. While ovariectomized rats show a signifi-
cantly decreased capacity for glucose utilization, estrogen
treatment increases this capacity by 2039% (319, 320). In
studies on postmenopausal women with or without estrogen
replacement therapy, there were significant enhancing ef-
fects of estrogen on verbal and figural memory tests as well
as enhancements of cerebral blood flow during the memory
tasks (321). One potential mechanismfor these effects may be
the reported estrogen induction of increased glucose trans-
porter-1 in the endothelial cells of the blood-brain barrier
(322). Moreover, one study has reported immunoreactivity
for ER in cerebrovascular endothelia (304). It has been sug-
gested that decreased capacity to remove glucose from the
bloodmay be a factor that contributes to the cascade of events
in Alzheimers disease (323325), and, indeed, glucose sup-
plementation is beneficial to cognitive function in the aging
brain (326, 327).
296 MCEWEN AND ALVES Vol. 20, No. 3
H. Summary
Estrogens have effects on many brain regions involved in
a host of nonreproductive brain functions. Whereas actions
of estradiol on hippocampal synaptogenesis appear to be
attributable to intracellular ER or -, actions of estrogens in
the striatum and accumbens on dopaminergic activity ap-
pear to be mediated by membrane actions that are not char-
acterized as yet in terms of receptors. Estrogen actions on
noradrenergic, serotonergic, and hypothalamic dopaminer-
gic systems, on the other hand, are likely to be mediated by
known intracellular ER either within these cells and/or in
adjacent neurons. The spinal cord also has intracellular ER,
but the reported effects on nociception and analgesia do not
directly relate to those receptor sites in enkephalin-express-
ing spinal neurons. Moreover, endothelial cells and at least
some glial cells express ERand must be considered as targets
for estrogen action that affect glucose uptake and mecha-
nisms that support the replenishment of cell membranes and
possibly also synaptogenesis and other forms of structural
plasticity. Finally, as will be seen in the next section, estrogen
effects on memory have been reported in animal models and
in studies on humans. The memories affected are ones in
which the hippocampus plays a role along with the basal
forebrain cholinergic system.
IV. Effects of Estrogens on Learning and Memory
In addition to reducing seizure thresholds and enhancing
LTP in hippocampus, estrogen treatment is reported to exert
effects on memory, particularly those types of memory in
which the hippocampus plays a role. Four types of effects
have been reported. First, estrogen treatment of ovariecto-
mized rats has been reported to improve acquisition, as well
as choice accuracy, on a radial maze task as well as in a
reinforced T-maze alternation task (328330). Second, sus-
tained estrogen treatment is reported to improve perfor-
mance in a working memory task (331) as well as in the radial
arm maze (329, 332). Third, estrogen treatment is reported to
promote a shift in the strategy that female rats use to solve
an appetitive two-choice discrimination, with estrogen in-
creasing the probability of using a response as opposed to a
spatial strategy (333). Fourth, aging female rats that have low
estrogen plasma levels in the estropause are reported to
perform significantly more poorly in a Morris water maze
than female rats with high estrogen levels (334). The effects
of sustained estrogen replacement in rats are reminiscent of
the effects of estrogen treatment in women whose ovarian
function has been eliminated by surgical menopause or by a
GnRH antagonist used to shrink the size of fibroids before
surgery (3, 4, 153).
In contrast to the effects of sustainedestrogen treatment on
memory processes, it should also be noted that, in the natural
estrous cycle of the female rat, it has been difficult to detect
cyclicity of performance in spatial tasks, with either no effect
reported (335), or differences reported in motivational or
attentional parameters (336), or an impairment reported in
performance on proestrus (337). In female mice, the back-
ground strain showed impairment of a spatial memory task
by estrogen treatment, whereas the ERKO genotype was
unresponsive to estrogen (338). These authors suggest that
ER activation is responsible for inhibition of spatial dis-
crimination in female mice (338).
In human subjects, there is also some evidence that estro-
gen treatment has a negative effect on performance of spatial
tasks in women while enhancing verbal performance (2, 3,
339). The inhibitory effects of estrogen on spatial memory,
while estrogen also appears to have positive effects on de-
clarative memory, may indicate that spatial memory is af-
fected differently from declarative memory. Yet there are
additional dimensions to this growing story.
There is evidence for acute actions of estrogen on hip-
pocampal-dependent memory, particularly in relation to the
cholinergic system. Posttraining, intrahippocampal injec-
tions of estrogen, immediately after training but not 2 h later,
enhanced memory in a Morris water maze measured 24 h
later, and these effects could be blocked by peripheral ad-
ministration of the cholinergic antagonist, scopolamine (340,
341). A similar, rapid effect on this memory task was found
using systemic estrogen treatment (342). In addition, estro-
gen treatment of ovariectomized rats also improved perfor-
mance of a reinforced T-maze alternation task and counter-
acted the amnestic effects of scopolamine administration
systemically or into the hippocampus (343). Moreover, es-
trogen replacement of ovariectomized rats attenuated the
effects of scopolamine and lorezepam to cause deficits in
acquisition of a passive avoidance memory task (344), an-
other memory task in which there is a hippocampal involve-
ment (345). Although it is tempting to attribute the cholin-
ergic involvement in these results as effects on memory,
another point of view is that the basal forebrain cholinergic
system is concerned primarily with selective attention (346).
As far as potential relevance of the cholinergic system to
attentional and memory processes in humans, it should be
notedagainthat the beneficial effects of tacrine, a cholinergic-
enhancing drug, were evident in women on estrogen re-
placement therapy but not in women who did not receive
hormone replacement (175).
Taken together, estrogens exert complex and time-depen-
dent effects on spatial and declarative memory in animals
and humans. The basal forebrain cholinergic system and
hippocampus are two brain systems that appear to be in-
volved in both attentional and memory effects of estrogens.
At the same time, the inhibition of spatial memory task
performance under some experimental conditions in animal
models and in human subjects raises questions about the
relationship of spatial memory to other forms of memory.
The complexity of the estrogen effects on memory suggest
that other estrogen-sensitive brain systems, in addition to the
cholinergic system and the hippocampus, are also involved.
V. Estrogens, Neuroprotection, and
Alzheimers Disease
We have seen that estrogens affect multiple brain regions
and do so via multiple mechanisms that have different con-
sequences for cell function and survival. The decline of ovar-
ian activity after surgical and natural menopause appears to
affect a number of brain functions, including mood and cer-
June, 1999 ESTROGEN ACTIONS IN THE CNS 297
tain types of memory, as discussed above. However, these
effects appear to be largely reversible and distinguishable
from the long-term degenerative changes associated with
Alzheimers disease, and, for that reason, we consider this
topic separately.
It stands to reason that the loss of ovarian hormones may
increase the vulnerability of brain cells to damage and de-
generation. Indeed, there are recent and somewhat contro-
versial findings that estrogen treatment of postmenopausal
women may have a protective effect on the brain toward
Alzheimers disease. These findings and the concerns raised
by them will be reviewed below followed by a discussion of
possible mechanisms.
Regarding the evidence for a link between estrogens and
Alzheimers disease, some, but not all, recent retrospective
and prospective epidemiological studies have suggested a
possible protective role for postmenopausal estrogen ther-
apy toward Alzheimers disease (148150, 347350). There
have also been several prospective studies showing that hor-
mone replacement therapy benefits normal cognitive func-
tion in postmenopausal women (351, 352). However, not all
studies of this kind have revealed significant effects (353),
and justified caution has recently been expressed against any
firm conclusions until large, placebo-controlled studies are
carried out (350).
Anumber of estrogen treatment trials have indicatedsome
benefit to demented women as far as improving measures of
global cognitive function and mood (151, 152, 354, 355) and
also improvements of verbal memory performance in non-
demented women (3, 4, 153). However, all of the published
trials to date have been quite small in size, short in duration,
unrandomized, and uncontrolled, which has prompted cau-
tion against overenthusiasm until more data are collected
(350).
In spite of the reservations, it is appropriate to ask the
following question: By what mechanisms might estrogen
exert neuroprotective and cognitive or mood-enhancing ef-
fects? Based upon the information covered in the preceding
sections of this reviewarticle, one can envision two principal
pathways: 1) maintaining neural functions and 2) protection
against damage.
First, as far as maintaining neural functions, estrogens, as
we have seen, regulate neural functions in a wide range of
brain structures, including cholinergic and monoaminergic
systems that ramify and affect many brain regions, including
the hippocampus. As estrogen levels decline over the meno-
pause, these systems and the cognitive and other behavioral
processes that depend upon them also decline in their func-
tional capacities; but they are, at least in principle, subject to
reversal by estrogen replacement therapy unless irreversible
degenerative changes take place.
Second, regarding degeneration and neuroprotection, the
absence of estrogens may increase vulnerability of brain cells
to insults and to the effects of other age-related changes in
neural function. We have seen above that the A ring of the
estrogen molecule appears to have special properties with
respect to the formationof free radicals andspecial protective
effects on cells in culture that are deprived of serum or
exposed to free radical generators (see above). In vivo studies
of estrogen-mediated neuroprotection have reported suc-
cessful reduction of lesion size by Silastic implants of 17-
estradiol in male rats subjected to middle cerebral artery
occlusion (356). In another study, a single injection of 17-
estradiol reduced damage to hilar neurons in the hippocam-
pal dentate gyrus of female rats caused by kainic acid treat-
ment (357).
Inadditionto these interactions of estrogens withneuronal
survival in cell culture and in vivo, there is another mecha-
nism that points to a unique action of estrogens in relation
to Alzheimers disease, namely, the regulation of the secreted
formof the amyloidprecursor protein andsuppression of the
toxic -amyloid protein (358, 359). This effect has been dem-
onstrated in both fibroblasts and neural cell lines but is thus
far without a mechanistic explanation as far as involvement
of intracellular ER or some of the nongenomic estrogen ac-
tions described above. It should be noted that the -amyloid
protein produces its toxic effects via the generation of free
radicals (360), whereas the secreted form of the -amyloid
precursor protein is actually neuroprotective against the
toxic -amyloid protein (361). Recent data from in vivo stud-
ies of estrogen action in relation to neuroprotection from
ischemia caused by middle cerebral artery occlusion indicate
that the increased expression of -amyloid precursor protein
caused by the ischemia is reduced by a single dose of 17-
estradiol 2 h before the ischemic episode (362).
Another aspect of brain aging is how the presence or
absence of hormones also contributes to aging of the brain,
e.g., loss of hippocampal neurons as a result of elevated
glucocorticoid activity (156, 157) and consequences of estro-
gen loss in females, which may include loss of synaptic
connections in hippocampus (158) or decline in basal fore-
brain cholinergic function in the absence of circulating es-
trogens (159). In addition to the loss of circulating hormones
and degeneration of the neural systems that are maintained
by them, there are also sex differences in the severity of brain
damage resulting from transient ischemia (162) and sex dif-
ferences in the response of the brain to lesions (163) and to
severe, chronic stress (164, 165).
VI. Conclusions
Estrogens have numerous effects on the brain throughout
the lifespan, beginning during gestation and continuing on
into senescence, and they do so via multiple mechanisms in
which both genomic and cell surface receptors appear to be
involved. For genomic effects, both the ER and ER genes
are expressed in brain tissue, and mapping studies continue
to reveal new ER-containing cells in regions of the nervous
systemnot previously thought to be estrogen targets. For cell
surface actions, receptors are not well characterized, but
many actions are reported, ranging from second messenger
systems to effects on neuronal excitability and ion channels,
as well as calciumion homeostasis. In addition, estrogens are
reported to have neuroprotective effects against free radical-
induced damage and to do so, at least in part, via a non-
genomic mechanism.
In addition to affecting the hypothalamus and other brain
areas related to reproduction, ovarian steroids have wide-
spread effects throughout the brain: on catecholaminergic
298 MCEWEN AND ALVES Vol. 20, No. 3
neurons and serotonergic pathways and the basal forebrain
cholinergic system, as well as the hippocampus, spinal cord,
nigrostriatal and mesolimbic systems, in addition to glial
cells and the blood-brain barrier. Regulation of the seroto-
nergic systemappears to be linkedto the presence of estrogen
and progestin-sensitive neurons in the midbrain raphe,
whereas the ovarian steroid effects on cholinergic function
involve induction of ChAT and acetylcholinesterase accord-
ing to a pattern that differs between the sexes. Because of the
widespread influences of these various neuronal systems,
ovarian steroids have measurable effects on mood and affect
as well as on cognition, with implications for dementia. One
of the most surprising estrogen effects is the regulation of
synapse turnover in the CA1 region of the hippocampus
during the 4- to 5-day estrous cycle of the female rat. For-
mation of new excitatory synapses is induced by estrogen
and involves NMDA receptors, whereas down-regulation of
these synapses involves intracellular PRs. There are devel-
opmentally programmed sex differences in hippocampal
structure that may help to explain differences in the strate-
gies which male and female rats use to solve spatial navi-
gation problems. Collectively, the multiple sites and mech-
anisms of estrogen action in brain underlie a variety of
important effects on cognitive and other brain functions as
well as possible protection in Alzheimers disease.
Therefore, estrogens are now increasingly recognized as
multipurpose messengers to many regions of the brain, and
they influence many processes and many brain regions
throughout the entire lifespan. Estrogen actions on the brain,
originally believed to act via a single type of intracellular
receptor, are now known to take place through the products
of at least two distinct genes for intracellular ER, as well as
via a host of actions on the cell surface that are mediated by
as yet uncharacterized receptor sites. Because of these wide-
spread and diverse actions throughout the nervous system,
it is not so surprising that estrogen actions now include
effects on cognitive function, coordination of movement,
pain, and affective state, among other processes, and that
estrogen withdrawal after natural or surgical menopause can
lead to a host of changes in brain function and behavior. The
putative neuroprotective effects of estrogens are among the
most puzzling and novel, and rapid progress in this area
offers some hope for preventing or retarding Alzheimers
disease, although this aspect must be treated somewhat ten-
tatively until more data are in hand.
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