134-Dopamine Research Advances-Akiyama Watanabe Arcangelo Benigno-1600218202-Nova Biomedical Book

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DOPAMINE RESEARCH ADVANCES
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DOPAMINE RESEARCH ADVANCES
AKIYAMA WATANABE
EDITOR
Nova Biomedical Books

New York
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Copyright 2008 by Nova Science Publishers, Inc.

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Dopamine research advances / Akiyama Watanabe (editor).
p. ; cm.
Includes bibliographical references and index.
ISBN-13: 978-1-60692-764-9
1. Dopamine. 2. Dopamine--Physiological effect. I. Watanabe, Akiyama.
[DNLM: 1. Dopamine--pharmacokinetics. 2. Dopamine--physiology. 3. Blood-Brain
Barrier--physiology. 4. Dopamine Agents--metabolism. 5. Dopamine Agents--pharmacokinetics.
WK 725 D6924 2007]
QP563.D66D664 2007
612.8'042--dc22
2007024086
Published by Nova Science Publishers, Inc.
New York
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CONTENTS
Preface
vii
Chapter I
Dopamine Control of Sleep and Arousal

Patrick M. Fuller and Jun Lu
Chapter II
A Circuit Dynamics Theory of Complex Dopaminergic

Modulation of Prefrontal Cortical Activity and
its Relevance to Schizophrenia
Shoji Tanaka
Chapter III
Chapter IV
Chapter V
Chapter VI
The Life Cycle of the Dopaminergic Neurons

in the Substantia Nigra
Vincenzo Di Matteo, Massimo Pierucci,
Arcangelo Benigno, Ennio Esposito,
and Giuseppe Di Giovanni
Electrophysiological and Neurochemical in vivo Studies
on Serotonin 5-HT2C Control of Central
Dopaminergic Function
Vincenzo Di Matteo, Giuseppe Di Giovanni,
Massimo Pierucci, and Ennio Esposito
Dopamine Effects on the Adrenal Gland of the Newt
Triturus Carnifex (Amphibia, Urodela)
Anna Capaldo, Flaminia Gay, Salvatore Valiante,
Vincenza Laforgia, Lorenzo Varano and Maria De Falco
Serotonin 5-HT2C Receptor and Dopamine
Function in Depression
Giuseppe Di Giovanni, Vincenzo Di Matteo,
Massimo Pierucci, and Ennio Esposito
17
51
87
113
131
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vi
Chapter VII
Chapter VIII
Chapter IX
Contents
A Possible Role for Intracellular Pathways Activation
in the Modulation of Learning and Memory Processes
by the Dopaminergic and Opioid Systems Interaction
M. Costanzi, V. Cestari and C. Castellano
Dopamine System and its Modulation by Nitric Oxide:
Approaches in Experimental Parkinson and Schizophrenia
Cristiane Salum, Marcela Bermdez-Echeverry,
Ana Carolina Issy, and Elaine A. Del-Bel
Dopamine Receptors Regulation
by Non-Dopaminergic Mechanisms
Jaromr Mysliveek and Anna Hrabovsk
Index
145
153
193
209
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PREFACE
Dopamine is a phenethylamine naturally produced by the human body. In the brain,
dopamine functions as a neurotransmitter, activating the five types of dopamine receptor D1, D2, D3, D4 and D5, and their variants. Dopamine is produced in several areas of the
brain, including the substantia nigra. Dopamine is also a neurohormone released by the
hypothalamus. Its main function as a hormone is to inhibit the release of prolactin from the
anterior lobe of the pituitary. Dopamine can be supplied as a medication that acts on the
sympathetic nervous system, producing effects such as increased heart rate and blood
pressure. However, since dopamine cannot cross the blood-brain barrier, dopamine given as a
drug does not directly affect the central nervous system. To increase the amount of dopamine
in the brains of patients with diseases such as Parkinson's disease and Dopa-Responsive
Dystonia, L-DOPA (levodopa), which is the precursor of dopamine, can be given because it
can cross the blood-brain barrier.
This book presents new research in the field.
Chapter I - The traditional account of the central dopaminergic system includes the
important role for dopamine (abbr. DA), a catecholamine neurotrasmitter, in the regulation of
a myriad of neurobiologic, physiologic and pathophysiologic processes, including: cognition,
motivation, memory, salience detection, motor disturbances of Parkinsons disease,
depression, schizophrenia, and hypophyseal function. More recently, however, an important
role for DA in the regulation of sleep-wakefulness and cortical arousal has been established,
challenging the traditional view that DA is the only central aminergic group not involved in
regulating sleep. To this end, wake-active DA neurons of the ventral periaqueductal area
(vPAG) appear to exert a potent arousal influence through a mutually inhibitory interaction
with the ventrolateral preoptic nucleus (VLPO) as well as through less-well defined
interactions with components of the ascending arousal system, e.g., locus coeruleus and
lateral hypothalamus. In addition to the vPAG DA neurons, recent electrophysiogical work
has revealed increased activity of ventral tegmental area (A10) DA neurons during rapid-eye
movement sleep (also called paradoxical sleep), providing further evidence linking changes
in the activity of DA neurons with changes in behavioral state. Recent data has also
suggested, but not demonstrated empirically, that alterations in DA neurotransmission may
form the etiological bases of REM behavior disorder, the excessive sleepiness of evolving
Parkinsons disease and, possibly, other nocturnal movement disorders. Finally, the critical
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Akiyama Watanabe
role for DA in mediating the wake-promoting effects of psychostimulants (e.g.,

methamphetamines and modafinil) has begun to emerge and is discussed herein. Taken
together, these observations reinforce the notion that the functional role of the DA system and
attendant implications for sleep-related disorders reach beyond the traditional view of the role
of the central dopaminergic system in neurobiology.
Chapter II - Working memory and other cognitive functions depend on dopaminergic
transmission. A number of functional imaging studies have suggested that the prefrontal
cortex (PFC) is the center for working memory. Working memory processing would be
mediated centrally by the circuit in the PFC. Then the research on the dynamics of the PFC
circuit under dopaminergic modulation would be crucial for the understanding of functioning
and dysfunctioning of the cognitive system. In this chapter, the authors develop a circuit
dynamics theory of the dopaminergic modulation of PFC activity. Persistent activity with
target selectivity over seconds is the essential dynamics of the maintenance of working
memory, and is known to have an inverted-U shaped profile of dopaminergic modulation.
However, the dynamics is not always stable along the inverted-U shaped curve. Under
hypodopaminergic conditions, the prefronto-mesoprefrontal system with cortical
dopaminergic modulation switches over from a negative to a positive control system, making
the PFC circuit unstable. This would be relevant to schizophrenia, in which cognitive
dysfunction is associated with the hypodopaminergic transmission in the PFC. Because of
this instability of the PFC circuit, the activity of the PFC tends to be largely fluctuated, as
often observed in human functional imaging studies. Beyond the inverted-U shape region of
dopaminergic modulation, in contrast, the PFC circuit has bistability, and a hyperactive mode
of PFC activity would emerge, depending on the strength of the input to the PFC. The
emergence of the hyperactivity of the PFC is due to disinhibition in the circuit and would be
relevant to psychotic states in schizophrenia and other psychiatric diseases. This is consistent
with the finding that GABAergic transmission through parvalbumin-positive GABA neurons
in the PFC is downregulated in schizophrenia. The theory predicts that the PFC has such a
complex profile of dopaminergic modulation and argues that it is relevant to complex
symptomatology of schizophrenia.
Chapter III - Since the 1950s, when dopamine (DA) was discovered in the mammalian
central nervous system (CNS), an enormous amount of experimental evidence has revealed
the pivotal role of this biogenic amine in a number of cognitive and behavioural functions
including voluntary movement and a broad array of behavioural processes such as mood,
reward, addiction, and stress. Moreover, dopaminergic neurons, although their numbers are
few, are of clinical importance because it is implicated in several psychiatric disorders, such
as schizophrenia, depression, and anxiety. The lost of dopaminergic neurons of the substantia
nigra compacta (SNc) is associated with one of the most prominent human neurological
disorders, Parkinson's disease (PD). Moreover, the mechanisms whereby nigral dopaminergic
neurons may degenerate still remain controversial. Hitherto, several data have shown that the
earlier cellular disturbances occurring in dopaminergic neurons include oxidative stress,
excitotoxicity, inflammation, mitochondrial dysfunction, and altered proteolysis. These
alterations, rather than killing neurons, trigger subsequent death-related molecular pathways,
including elements of apoptosis. In rare incidences, PD may be inheritated; this evidence has
opened a new and exciting area of research, trying to shed light on the nature of the more
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Preface
ix
commune idiopathic PD form. In this review, the characteristics of the SNc dopaminergic
neurons and their life cycle from birth to death are reviewed. In addition, of the mechanisms
by which the aforementioned alterations cause neuronal dopaminergic death, particular
emphasis will be given to the role played by inflammation, and the relevance of the possible
use of anti-inflammatory drugs in the treatment of PD. Finally, the new evidence of a
possible de novo neurogenesis in the SNc of adult animals and in PD patients will also be
examined.
Chapter IV - Central serotonergic, and dopaminergic systems play a critical role in the
regulation of normal and abnormal behaviours. Recent evidence suggests that dysfunction of
dopamine (DA) and serotonin (5-HT) neurotransmitter systems contribute to various mental
disorders including depression and schizophrenia. This chapter was undertaken to summarize
the authors and other works that have extensively explored the role of 5-HT2C receptors in the
control of DA systems both in basal and drug-induced conditions, using in vivo
electrophysiological and microdialytic techniques. The physiology, pharmacology and
anatomical distribution of the 5-HT2C receptors in the CNS will be firstly reviewed.
Moreover, experimental data regarding the effect of 5-HT2C selective agents on the neuronal
activity of DA neurons of the ventral tegmental area (VTA) and substantia nigra pars
compacta (SNc) as well as the changes of basal DA release in the striatum and nucleus
accumbens are discussed. Finally, the potential use of 5-HT2C agents in the treatment of
depression, schizophrenia, Parkinson's disease and drug abuse will be also discussed.
Chapter V - The existence of intra-adrenal paracrine interactions of functional relevance
between chromaffin and steroidogenic tissues has been shown in mammals as well as in
lower vertebrates. In Triturus carnifex, an urodele amphibian, recent studies showed that two
tissues may influence each other as well; moreover, both epinephrine and norepinephrine
exert a stimulatory effect on epinephrine and norepinephrine release, whereas the effects of
two amines on steroidogenic tissue are different from one another: epinephrine inhibits and
norepinephrine stimulates aldosterone release. To date, data are lacking about dopamine role
in this species; therefore, the aims of the present study were 1) to evaluate the influence of
dopamine on the adrenal gland of the newt 2) to compare the effect of dopamine with those
of the other two amines, in order to study in depth intraadrenal paracrine interactions in
urodele amphibians.
In April and June, adult male newts were given intra-peritoneal (ip) injections of
dopamine (1.25 mg/100 g body wt/day for 4 consecutive days); the effects, after two and
twenty-four hours, were evaluated by examination of the ultrastructural morphological and
morphometrical features of the tissues as well as the serum levels of aldosterone,
corticosterone, epinephrine and norepinephrine. In both periods, dopamine exerted an
inhibitory effect on steroidogenic tissue, always significantly decreasing serum corticosterone
levels, and in April serum aldosterone levels too. Only twenty-four hours later, steroidogenic
cells showed signs of renewal of biosynthetic activity. Dopamine administration increased
serum levels of catecholamines (epinephrine in April, norepinephrine in June). Chromaffin
cells, in both periods, showed clear signs of increased biosynthetic activity, like a high
development of R.E.R. and a significant increase in the number of intermediate granules (i.e.,
granules in different stages of biosynthetic pathway leading to catecholamines). The results
of this study indicate that 1) dopamine may influence both tissues of newt adrenal gland 2)
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Akiyama Watanabe
dopamine plays an inhibitory role on steroidogenic activity, like epinephrine, and a

stimulatory role on the chromaffin tissue, like both catecholamines 3) the chromaffin tissue
may modulate the activity of the steroidogenic one.
Chapter VI - Several hypotheses regarding the physiopathology of major depression
exist. Attention has been focused on cerebral monoaminergic systems, the dysfunction of
which is thought to underlie various aspects of depressive symptomatology. There is an
extensive literature describing the involvement of serotonergic and dopaminergic systems in
the mechanism of action of antidepressant drugs. However, a unitary analysis of the data in
terms of interaction between different monoaminergic systems is still lacking. Among the
multiple classes of 5-HT receptors described in the central nervous system, much attention
has been devoted to the role of 5-HT2 receptor family in the control of central dopaminergic
activity, because of the moderate to dense localization of both transcript and protein for 5HT2 receptors in the substantia nigra (SN) and ventral tegmental area (VTA), as well as their
terminal regions. Recent studies have focused on the functional interaction between the
serotonergic and dopaminergic systems to explain the mechanism of the antidepressant action
of SSRIs and 5-HT2 antagonists. In this article, the most relevant data regarding the role of
these receptors in the control of brain DA function are reviewed, and the importance of this
subject in the search of new antidepressant drugs is discussed.
Chapter VII - Dopaminergic and opioid mechanisms have been extensively studied for
their role in modulating learning and memory processes. The dopaminergic system plays an
important role in the emotional response to rewarding stimuli as well as in learning and
memory processes following psychostimulant administration. As concerns the intracellular
pathway activated by psichostimulant drugs, it has been shown that the administration of
dopaminergic agonists (i.e. amphetamine and cocaine) activated ERK proteins in the
striatum. A number of studies have shown that the opioid system modulates the memory
consolidation processes and that this activity is related to the dopaminergic function.
Recently, it has been observed that opioid receptor stimulation induces ERKs phosphorilation
through G protein-coupled activation in the striatal neurons. Taken together these findings
suggest a pivotal role for ERKs in the intracellular mechanisms involved in the long lasting
behavioural modification induced by drugs of abuse that contribute to the development of
addiction. Thus, the ERK proteins might represent a possible candidate for intracellular
modulation of the interaction between opioid and dopaminergic systems in learning and
memory processes linked to the addicted behaviour.
Some preliminary results obtained in the authors laboratory showed that ERK1 null
mutant mice submitted to the active avoidance task are not affected by the posttraining
administration of D1 dopamine receptor antagonist (SCH 23390), as well as by mu opioid
receptor agonist (morphine), while both treatments improve the performance of wild type
mice. Thus, the possible pivotal role of ERK1 on the behavioural effect exerted by both
dopaminergic and opioid system can not be ruled out.
Overall, the understanding of the intracellular mechanisms involved in the possible
interaction between these neuromodulatory systems might be crucial for both studying and
developing new strategies to better clarify the learning-reward processes linked to the
addicted behaviour.
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Preface
xi
Chapter VIII - The influence of dopamine (DA) in mammalian and invertebrate neural
processes has been extensively documented. The mesencephalic dopaminergic neurons have
key roles in sensorimotor integration, motor behavior and in the modulation of behavioral
responses to positive and negative reinforcement. It is now known that nitric oxide (NO), an
atypical neurotransmitter, mediates a number of neuronal processes including the regulation
of dopaminergic neurotransmission. NO has been implicated in several behavioral
pathologies concerned with dopaminergic imbalance, such as Parkinsons disease (PD) and
schizophrenia. Although the nature of the NO-mediated modulatory influence on DA
neurotransmission have some conflicting neurochemical observations, a growing body of
literature indicates that NO, by its signaling mechanisms and effector pathways, exerts a
primary facilitatory influence over tonic and phasic dopaminergic neurotransmission under
physiological conditions. There is considerable evidence indicating that NO also inhibits DA
uptake, thus modulating DA-controlled behaviors. Additionally, NO may interact with DA
modifying not only its regulatory actions but also producing oxidants and free radicals that
are likely to trigger toxic pathways in the nervous system. Thus, the chemical interaction
between DA and its metabolites with NO components constitutes a source of neurotoxic
molecules, which may contribute to the cellular process of neurodegeneration. Consequently,
the interaction between these systems has become a potential target for exploring the
neurochemical basis of some neuropsychiatric diseases. In particular, there is a great interest
in investigating PD and schizophrenia by the underlying processes which control motor
behavior, attentional and information processing deficits. Increased mesolimbic DA
following administration of amphetamine-like drugs to rodents is coupled with
hyperlocomotion, deficit in sensorimotor filter, stereotyped behaviors and also provokes
attentional dysfunction. Inhibition of nitric oxide synthase (NOS) has been shown to prevent
many of these effects. Moreover, the cataleptic effect of DA antagonists, like haloperidol, can
be mimicked by NOS inhibitors. This chapter first summarizes neurochemical aspects of DA
and NO neurotransmissions and reviews a broad spectrum of mechanisms by which nitrergic
system may influence the dopaminergic neurotransmission. Supporting evidence is presented
for the involvement of NO in behavioral conditions controlled by DA. Finally, the
modulation of dopaminergic functions by NO in behavioral models of neuropsychiatric
diseases is demonstrated focusing on motor and attentional dysfunctions which can occur in
PD and schizophrenia, respectively.
Chapter IX - Dopamine receptors are widely distributed in the central nervous system
and are responsible for many physiological, pharmacological and pathological functions such
as movement coordination, cognition or drug abuse. Dopamine receptors belong to the G
protein - coupled metabotropic receptor family. Five different dopamine receptors have been
characterized so far. These can be classified as either D1-like or D2-like, based on their
structure, signal transduction pathway and pharmacological characteristic. The activity and
the level of dopamine receptors depend on the presence of its ligand dopamine. However,
other mechanisms can be involved in the dopamine receptors regulation.
This article focuses on the non-dopaminergic regulation of dopamine receptors. It
summarizes and concludes results obtained in studies with genetically modified animals. (1)
First, the mutation in 2 glutamate receptors and thus changes in other receptor systems are
discussed. Transgenic mice reveal cerebellar degeneration and learning impairment. The
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Akiyama Watanabe
authors have found that dopaminergic system is affected in these mutants. (2) Second, the
dopaminergic consequences of acetylcholinesterase deletion are followed. It was shown in
past that the level of muscarinic receptors is significantly changed in animals with null
acetylcholinesterase activity. Receptors are down-regulated due to over-stimulation by excess
of acetylcholine. Muscarinic receptor subtypes are co-expressed with dopamine receptors on
striatal projection neurons. The authors results uncovered a dramatic decrease in striatal
dopamine receptors levels. (3) At last, the lack of gene for transcription factor c-fos is
examined. Its deletion did not cause changes in D1-like and D2-like receptors in cerebral
cortex and cerebellum, although other receptor subtypes (1-adrenoceptors, muscarinic
receptors) were affected. These data show that dopamine receptors are regulated by nondopaminergic mechanisms and serve to cope with changes in the central nervous system.
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In: Dopamine Research Advances

Editor: Akiyama Watanabe, pp. 1-15
ISBN: 978-1-60021-820-0
2008 Nova Science Publishers, Inc.
Chapter I
DOPAMINE CONTROL OF SLEEP AND AROUSAL

Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Institutes of
Medicine, Room 814, 77 Louis Pasteur Avenue, Boston, MA 02115, USA.
ABSTRACT
The traditional account of the central dopaminergic system includes the important
role for dopamine (abbr. DA), a catecholamine neurotrasmitter, in the regulation of a
myriad of neurobiologic, physiologic and pathophysiologic processes, including:
cognition, motivation, memory, salience detection, motor disturbances of Parkinsons
disease, depression, schizophrenia, and hypophyseal function. More recently, however,
an important role for DA in the regulation of sleep-wakefulness and cortical arousal has
been established, challenging the traditional view that DA is the only central aminergic
group not involved in regulating sleep. To this end, wake-active DA neurons of the
ventral periaqueductal area (vPAG) appear to exert a potent arousal influence through a
mutually inhibitory interaction with the ventrolateral preoptic nucleus (VLPO) as well as
through less-well defined interactions with components of the ascending arousal system,
e.g., locus coeruleus and lateral hypothalamus. In addition to the vPAG DA neurons,
recent electrophysiogical work has revealed increased activity of ventral tegmental area
(A10) DA neurons during rapid-eye movement sleep (also called paradoxical sleep),
providing further evidence linking changes in the activity of DA neurons with changes in
behavioral state. Recent data has also suggested, but not demonstrated empirically, that
alterations in DA neurotransmission may form the etiological bases of REM behavior
disorder, the excessive sleepiness of evolving Parkinsons disease and, possibly, other
nocturnal movement disorders. Finally, the critical role for DA in mediating the wake
Correspondence concerning this article should be addressed to: Patrick M. Fuller, PhD, Harvard Medical School,
Department of Neurology, Beth Israel Deaconess Medical Center, 77 Ave. Louis Pasteur, HIM 819 Boston, MA
02115, U.S.A. Voice: +1 (617) 667 0823; Fax: +1 (617) 667 0810; email: pfuller@bidmc.harvard.edu.
Correspondence concerning this article should be addressed to: Jun Lu, PhD, MD, Harvard Medical School,
Department of Neurology, Beth Israel Deaconess Medical Center, 77 Ave. Louis Pasteur, HIM 819 Boston, MA
02115, U.S.A. Voice: +1 (617) 667 0489; Fax: +1 (617) 667 0810; email: jlu@bidmc.harvard.edu.
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promoting effects of psychostimulants (e.g., methamphetamines and modafinil) has
begun to emerge and is discussed herein. Taken together, these observations reinforce the
notion that the functional role of the DA system and attendant implications for sleeprelated disorders reach beyond the traditional view of the role of the central
dopaminergic system in neurobiology.
ANATOMY OF THE CENTRAL DOPAMINERGIC SYSTEM

The central dopaminergic system comprises three major and well-described tracts, all of
which originate in ventral mesencephalic neurons (designated A8-A10; For Review, Saper,
2000). Collectively, these three midbrain DA nuclei, which include the ventral tegmental area
(VTA; A10), the substantia nigra (SN; A9, including the pars reticulata and pars compacta)
and the retrorubral area (A8), contain ~85% of the CNS DA neuron population and, through
the medial forebrain bundle, give rise to ascending projections to the striatum, forebrain and
cerebral cortex (Figure 1). In addition to being distributed across several midbrain nuclei, DA
neurons differ with respect to the inputs they receive, their morphology, receptors they
express, firing characteristics (i.e., spontaneous firing rate, burst firing), neuropeptides they
colocalize and in their projection fields (Lu et al., 2006; Jaber et al, 1996). Thus, for example,
two of these projection systems, the mesolimbic and mesocortical pathways, originate in DA
cell bodies in the VTA and SN and project to structures in the ventral striatum,
hypothalamus, nucleus accumbens and other limbic structures and the prefrontal association
cortex. Mesolimbocortical neurotransmission is implicated in mediating motivated behaviors,
addiction, salience detection and in the pathogenesis of several neurological conditions,
including schizophrenia, Tourettes syndrome and depression. By contrast, the nigro-striatal
pathway connects DA cell bodies in the substantia nigra with the dorsolateral striatum
(caudate and putamen), which are important input stuctures of the basal ganglia. This
extrapyramidal motor system modulates movement and degeneration of this pathway, as
occurs in the pathogenesis of Parkinsons disease, results in motor abnormalities, including
ridigity, resting tremor and akinesia.
In addition to the major central dopaminergic tracts described, several other
dopaminergic systems exist, including: 1) the diencephalic A11-15 cell groups, which are
located in the dorsal-posterior hypothalamus (A11; sole source of spinal DA), arcuate (A12;
tuberoinfundibular control of prolactin secretion), incertohypothalamic region (A13) and
periventricular region (A14); 2) the retinal interplexiform cells (A16); 3) the periglomerular
cells of the olfactory bulb (A17); and 4) the wake-active DA neurons located in the ventral
periaqueductal grey (vPAG).
PHARMACOLOGY OF CENTRAL DOPAMINE

As described, dopamine (3,4-dihydroxyphenylethylamine) is a catecholamine
neurotransmitter and thus shares a biosynthetic pathway with norepinephrine and epinephrine
(For extensive review, Cooper et al., 2002). Accordingly, the rate limiting enzyme for DA
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synthesis is tyrosine hydroxlyase (TH). TH immunoreactivity is widely considered a useful

marker for DA neurons and their projections, but only in areas lacking adrenergic inputs
(Hokfelt et al., 1984). DA-ergic axons are also characterized by the presence of varicosities,
with many synaptic junctions occuring in an en passant configuration. DA receptors are
located on the perikarya, dendrites and axon terminals of DA neurons (autoreceptors) as well
as postsynaptically on a variety of different neuronal populations including, but not limited
to, GABA-ergic, glutamatergic, cholinergic, serotinergic and peptidergic, i.e., neurotensin
neurons.
DA is stored in synaptic vesicles and is released in a Ca+2-dependent manner. Five DA
receptors (designated D1-D5) mediate the actions of DA neurotransmission in the brain (For
review, Jabr et al., 1996). All five DA receptors are G-protein coupled receptors (GPCR),
which can be further grouped into two classes based upon their effect on adenylate cyclase
(AC) activity. In general, activation of D1-type receptors (D1 and D5) increases AC activity
via Gs-type G proteins (increasing cAMP) whereas activation of D2-type receptors
(D2,D3,D4) decreases AC activity via Gi-type G proteins (decreasing cAMP). DA receptors
also couple with other second messenger systems to regulate intracellular Ca+2 levels and K+
currents. Although all 5 subtypes of DA receptors can can localize to the postsynaptic
membrane, only D2 and D3 receptors function as autoreceptors, i.e., are found
presynaptically. Accordingly, autoreceptor activation generally inhibits DA
neurotransmission by decreasing DA release.
In addition to classic synaptic neurotransmission, several findings suggest that volume
transmission may be a primary mechanism of DA neurotransmission (Pickel, 1996). In
general, the following facts support of this concept: 1) DA is released from both synaptic and
extrasynaptic sites; 2) the vast majority of DA receptors are not located in postsynaptic
densities; and 3) DA transporters (DAT) are not concentrated exclusively around synapses.
DA reuptake by the DAT is a rapid, selective and sodium-dependent process, which can be
reversed in the presence of some drugs such as amphetamines. The DAT is found in highest
density at DA terminal regions of the striatum, hypothalamus and basal forebrain and
mesopontine DA neuron groups, including the ventral periaqueductal (vPAG). As discussed
below, DAT may play an important regulatory role in sleep homeostasis and in the wakepromoting action of stimulants.
Disrupted DA neurotransmission is the presumed etiologic basis for several neurological
disorders, including schizophrenia, major depression and Tourettes syndrome. Based upon
this hypothesized role for DA in these pathophysiologic states, drugs targeting the central DA
system, i.e., neuroleptics/anti-psychotics, have been used clinically with varying success. For
example, the canonical typical antipsychotic drug, Haloperidol, antagonizes D2 DA receptor
activity, resulting in reduced mesocorticolimbic DA neurotransmission (NB: almost all
clincially effective antipsychotic drugs have moderate to high affinity for D2 receptors).
Unfortunately, Haloperidol (and other typical antipsychotics) also block neurotransmission in
the nigrostriatal pathway, producing highly undesireable neurological side-effects involving
the extrapyramidal motor system, including Parkinsonism, akathisia, acute dystonia, and the
later-appearing syndrome, tardive dyskinesia. By contrast, newer atypical antipsychotics are
more selective for mesolimbic D2 receptors (and also exhibit a moderate affinity for other
classes of receptors, e.g., adrenergic, serotinergic, histaminergic) and exhibit a lower
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incidence of extrapyramidal side effects. Patients taking these atypical antipsychotic must
however be closely monitored for agranulocytosis (an acute and possibly fatal leukopenia).
Finally, several drugs of abuse which can induce psychotic episodes, including cocaine and
amphetamines (see below), promote the release of and/or block DA reuptake, implicating the
central DA system in the mood-altering, psychomotor and addictive properties of these drugs.
Figure 1. A) illustrates the afferent and efferent projections of the midbrain dopaminergic (A8-A10)
system, including the vPAG dopaminergic neurons. The A8-A10 neurons form the origins of the
mesolimbic, mesocortical and nigro-striatal projections. The wake-active vPAG DA neurons are
reciprocally connected with several components of the sleep regulatory and arousal systems, including
the ventrolateral preoptic nucleus, the locus coeruleus, the pontine laterodorsal tegmental nucleus, the
lateral hypothalamic orexin neurons, the midline and intralaminar thalamus, the basal forebrain
cholinergic cells and the prefrontal cortex. B) illustrates the projections of the diencephalic
dopaminergic system, including the descending A11 projection to the dorsal horn at all levels of the
spinal cord.
DOPAMINE AND SLEEP REGULATION

In contrast to other monoaminergic and cholinergic systems, the central DA system has
historically been ascribed only a limited role in the regulation of sleep-wakefulness and
cortical EEG arousal (Miller et al., 1983; Lee et al., 2001). Here we review recent work,
including our own, that has challenged this popular conception. Our work has firmly
established an important role for DA in the regulation of both sleep-wake behavior and
electrocortical arousal as well as identified, for the first time, the neuroanatomical locus of
wake-active DA neurons.
It has been long recognized that A10 (and to some extent A9) neurons respond to alerting
stimuli, leading many investigators to speculate that mesocorticolimbic DA transmission
originating in these cell groups might be critically involved in both behavioral and EEG
arousal. Yet, surprisingly, neurotoxic lesions of the A10 cell group do not decrease
behavioral wakefulness (Lai et al., 1999). Moreover, recording studies have revealed that the
firing patterns of mesencephalic DA neurons do not correlate with overall levels of
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behavioral wakefulness (Miller et al., 1983). By contrast, all other monoaminergic cell
groups exhibit robust state-dependent activation. Although collectively these observations
seem to contradict a role for midbrain DA neurons and mesocorticolimbic DA transmission in
sleep regulation, an important role for DA in the regulation of sleep-wakefulness is
nevertheless indicated by several findings. First, it has been demonstrated that DAT knockout
mice have ~20% more wakefulness than control mice and are refractory to psychostimulantinduced (presumably DA-mediated; see below) arousal (Wisor et al., 2001). A similar
phenotype (i.e., increased wake) resulted from mutation of the Drosophila DAT gene (Kume,
2005). Second, administration of exogenous dopaminomimetics affects the sleep-wake state
in a complex dose- and receptor-dependent manner (Larson and Tandberg, 2001). For
example, in general, lower dopaminomimetic doses have a soporific effect, presumably
mediated by presynaptic D2-like inhibitory autoreceptors, whereas higher doses (which
typically promote an attendant increase in locomotor activity and suppress REM sleep)
enhance arousal, likely via postsynaptic D1-like receptors. Third, patients with Parkinsons
disease exhibiting extensive loss of DA neurons in the substantia nigra and less extensive loss
of DA neurons in the VTA, often demonstrate excessive daytime sleepiness, which is made
worse by D2 receptor agonists (which activate inhibitory presynaptic autoreceptors on DA
neurons, and therefore inhibit the firing of DA neurons). Finally, arousal and waking
behaviors are associated with increased forebrain DA secretion.
Figure 2. A) is a photomicrograph showing the distribution of wake-active (Fos see arrows) DA

neurons (TH brown stain) in the vPAG. B) is a photomicrograph showing midbrain DA neurons of
the VTA (A10) and SN (A9), which do not demonstrate state-dependent activation, i.e., wake- or sleepactive DA neurons.
Recent work by our laboratory has uncovered a previously unrecognized group of wakeactive (i.e., Fos positive) DA neurons in the ventral periaquetuctal gray (vPAG) that may
provide the long-sought ascending dopaminergic waking influence (Lu et al., 2006; see
Figure 2). In our study, 6-hydroxydopamine induced lesions of vPAG DA neurons (sparing
intermingled dorsal raphe serotonergic neurons, the VTA and SNc) resulted in an increase in
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total daily sleep (NREM and REM) of ~ 20%. Importantly, the magnitude of sleep increase
seen following vPAG DA lesions was significantly larger than that produced by lesions of
other monoaminergic and cholinergic cell groups thought to be important in arousal,
including: the locus coreleus (LC), lateral dorsal tegmentum (LDT), dorsal raphe (DRN),
lateral hypothalamic (LH) orexin neurons, basal forebrain cholinergic neurons and the
histaminergic tuberomammillary nucleus (TMN) (Jones et al., 1977; Webster and Jones,
1988; Mouret and Coindet, 1980; Hara et al., 2001; Wenk et al., 1994; Gerashcenko et al.,
2004).
The presence of DA neurons in the vPAG has been identified previously in humans and
rats (Saper and Petito, 1982; Hokfelt et al., 1984). Yet because the vPAG DA neurons share
many efferent projections with A10 DA neurons, such as the ventral striatum and prefrontal
cortex, these vPAG DA neurons have long been considered a rostral extension of the A10
DA group. Anatomical and physiological characteristics of the vPAG DA neurons suggest,
however, that these cells constitute a functionally distinct neuronal population. For example,
unlike VTA DA neurons, vPAG DA neurons project heavily to the central and extended
nucleus of the amygdala as well as to the ventrolateral preoptic nucleus (VLPO), a critical
sleep-promoting center (Chou et al., 2002; Hasue and Shammah-Lagnado, 2002). These
vPAG DA neurons also project heavily to (and receive reciprocal innervations from) most of
the major components of the sleep-wake and arousal systems, including the VLPO, BF, LH
orexin cells, LC, the LDT cholinergic cells and the midline and intralaminar thalamus. Also,
as mentioned, unlike the vPAG DA neurons, VTA DA neurons do not demonstrate statedependent changes in firing. Taken together, these findings indicate that vPAG contain a
functionally distinct group of DA neurons which likely form the origin of a potent
dopaminergic arousal system. Thus, for example, loss/dysfunction of vPAG DA neurons may
underlie excessive daytime sleepiness in Parkinsons disease (see below). Although it
remains unclear how DA influences sleep regulation and arousal, it is likely through both
inhibition of sleep active neurons in the VLPO, i.e., as a component of the flip-flop switch for
sleep-wake control, and activation of the basal forebrain and monoaminergic systems, i.e., as
part of the extrathalamic cortical arousal system (Lu et al., 2006). Finally, recent
electrophysiogical work has revealed increased bursting of ventral tegmental area (A10) DA
neurons during REM sleep (also called paradoxical sleep), providing further evidence
linking changes in the activity of DA neurons with changes in behavioral state (Dahan,
2006).
DOPAMINERGIC DISORDERS AND SLEEP:

PARKINSONS DISEASE
Patients with Parkinsons disease (PD) suffer a progressive loss of DA neurons, largely
in the SNc and VTA, leading to a marked reduction in DA content in the basal ganglia and
ultimately manifesting in motor abnormalities that include akinesia, rigidity, resting tremor
and postural instability (Jellinger KA, 1999). In addition to motor disturbances, PD patients
often complain of sleep disturbances ranging from sleep fragmentation to abnormal motor
activity to excessive daytime sleepiness (Matheson and Saper, 2003). These problems tend to
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worsen with disease progression. During the earlier stages of PD, dopaminomimetic drugs,
e.g. L-3,4-dihydroxyphenyalamine (L-DOPA) can treat these sleep disturbances;
unfortunately, patients become refractory to L-DOPA treatment during the more advanced
stages of PD.
Ascertaining the contribution of central DA dysfunction to the sleep disturbances of PD
is complicated by several factors. First, patients with PD exhibit neurochemical changes in
cholinergic and monoaminergic systems, both of which are implicated in sleep regulation.
Second, medications used to treat PD may themselves alter sleep. Finally, nocturnal tremors
and inappropriate phasic motor bursting (which are common in PD and discussed in detail
below) may produce fragmented sleep. Nevertheless, several compelling lines of evidence
provide support for the concept that DA dysfunction significantly contributes to the
pathological sleep disturbances of PD, the most common of which are excessive daytime
sleepiness and nocturnal sleep disruption in the form of involuntary motor disturbance, e.g.,
REM behavior disorder and Restless Leg Syndrome.
Excessive Daytime Sleepiness

Significant hypersomnia in PD, manifesting as excessive daytime sleepiness (EDS), is
common and yet under-recognized with respect to diagnosis and treatment (Rye, 2004).
Clinic-based objective measurements of sleepiness employing the standardized multiple sleep
latency test (MSLT) has revealed a high rate of EDS (ca. 20-50%) in PD patients (Hobson et
al., 2002). As indicated above, the determinant(s) of EDS in PD are difficult to resolve as
EDS is likely secondary to severe sleep fragmentation in PD, which itself may be attributable
to other motor (e.g., abnormal nocturnal movements; see below) or respiratory (e.g.,
obstructive sleep apnea) disturbances that accompany the pathology of PD. It has recently
been hypothesized that degeneration of mesothalamic projections (collaterals of A8-A10
neurons that innervate the striatum) may lead to a decrease in thalamocortical activity,
resulting in decreased arousal and altered sleep-wake behavior (Rye et al., 2003). Although
an attractive hypothesis, the observation of normal sleep-wake and cortical arousal in
thalactomized rats and cats contradicts an important role for these mesothalamic projections
in normal or pathologic sleep, i.e., the sleep disturbances of PD (Fuller et al., 2007).
REM Behavior Disorder

REM sleep behavior disorder (RBD) is a parasomnia that typically manifests as dream
enactment behavior, i.e., involuntary nocturnal movements that include kicking, punching,
shouting and screaming during REM sleep (For review, Boeve and Saper, 2006). RBD may
represent an early pathophysiologic manifestation of evolving PD and other Lewy body
diseases (LBD), e.g., dementia with Lewy bodies and pure autonomic failure. Indeed, RBD
typically manifests a decade prior to the motor and cognitive sequela of PD and thus the
diagnosis of RBD may provide an early therapeutic window for delaying or preventing the
full development of PD.
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Of note, because Lewy bodies and Lewy neurites are composed of alphasynucleinopathies, these disorders (and others, e.g., multiple system atrophy) are considered
synucleinopathies (Dickson, 1999). Although the brainstem is clearly implicated in RBD
pathogenesis, the identity of the neural networks that become dysfunctional to manifest RBD
is currently unknown. Because about 50% of people with RBD develop Parkinsons disease
or dementia with Lewy bodies (Olson et al., 2000) and the majority of Parkinsons patients
have RBD, it has been suggested that the intrinsic pathology of PD, i.e., severe nigrostriatal
DA neuron loss, may contribute to the development of excessive nocturnal movement, in
particular RBD. Dr. David Rye and colleagues have advanced the hypothesis that DA may
produce RBD by modulating brainstem circuits affecting REM sleep atonia. They propose a
multi-synaptic route linking the basal ganglia output pathways, i.e., Globus Pallidus internal
(GPi) and SNr collaterals of the pallidothalamic pathway, with pontomedullary reticulospinal
pathways via the PPN and midbrain extrapyramidal area (MEA), areas that contain putative
REM-on and REM-off neurons. In turn, these REM-regulating neurons project to
ventromedullary reticulospinal neurons (the so-called bulbospinal inhibitory zone), which
then project to glycinergic interneurons of the spinal cord to produce REM atonia. Despite
the intuitive appeal of Ryes hypothesis, preliminary data from our laboratory (Fuller and Lu,
unpublished observations) suggest instead that neither the PPN nor the caudal ventromedial
medulla play a critical role in the development of REM without atonia in rats or mice (RBD
equivalent in animal models). Our recent studies have, however, suggested a critical role for
the subcoerulus region (SC; equivalent to the sublateraldorsal nucleus (SLD) in rats) and,
possibly, the intermediate region of the ventromedial medulla, (see below) in generating
atonia during REM sleep. These regions also project to spinal glycinergic interneurons that
project to motor neurons. SC/SLD dysfunction, as the neuropathologic substrate for RBD, is
seemingly consistent with the temporal pattern of neuronal degeneration in PD and other
LBD, which starts in the brainstem and includes the coeruleus-subcoerules complex in earlier
stage I-II of Parkinsons disease, and progresses inexorably rostrally towards the forebrain
(Braak et al., 2001, 2006). The temporal pattern of lesions, i.e., caudal to rostral progression,
is also consistent with RBD (secondary to SC/SLD degeneration) as an early manifestation of
these neurodegenerative conditions. Nevertheless, although SC/SLD dysfunction has been
demonstrated in PD it remains unclear if RBD in evolving PD is caused by SC/SLD
degeneration or loss of critical inputs such as DA nigrostriatal projections.
Remarkably, the RBD has also been identified as one of the main independent risk
factors for the presence of psychotic disorders in PD, e.g., hallucinations and delusions. At
present, however, the neurobiological determinants of these psychotic states in PD, including
the role of DA, remain unresolved. We hypothesize that the association between RBD and
psychotic disturbances, in particular hallucinations, in PD may reflect pathologic changes in
REM-off circuitry located in the pontine tegmentum. In this context, failure of REM-off
inputs may disinhibit patterns of neuronal firing that normally occur only during REM sleep,
thus producing dream-like states (i.e., hallucinations) at inappropriate times (i.e., against a
waking backdrop). In some respects, our hypothesis, although untested, is reminiscent of
Freuds prediction made more than one century ago: the intrusion of the sleeping mind on the
conscious mind forms the basis of psychosis.
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Restless Leg Syndrome

In addition to RBD, DA dysfunction has also been implicated in the pathogenesis of
disordered sleep in the form of other types of excessive nocturnal movement in PD (Rye and
Bliwise, 1997). An important example of a non-RBD nocturnal movement disorder in PD is
Restless Leg Syndrome (RLS), which often takes the form of involuntary, periodic limb
movements during sleep (PLMS) and resting wakefulness (PLMW). Similar to RBD, RLS
occurs more commonly in PD than in conditions not involving the nigrostriatal system,
including Alzheimers disease and aging. According to the International Classification of
Sleep Disorders, RLS is a sensorimotor disorder characterized by a complaint of a strong,
nearly irresistible, urge to move the legs. The desire to move is often accompanied by other
uncomfortable paresthesias felt deep in the legs. Leg movement typically brings immediate
relief from the paresthesias. Like RBD, it is tempting to speculate that the pathogenesis of
RLS is related to altered DA modulation of brainstem circuits controlling REMS atonia,
although at present there is little data to support this proposal.
Alternatively, it has been proposed that dysfunction of the diencephalo-spinal DA system
may form the pathological basis of RLS (Fleetwood et al., 1998; Rye, 2003; Gladwell and
Coote, 1999). Specifically, several groups of investigators have proposed a role for the A11
DA cell group, located in the dorsal-posterior hypothalamus in the pathophysiology of RLS.
The A11 DA group projects to the spinal cord with collaterals extending to all of Rexeds
laminae with heaviest innervation at the level of the sympathetic preganglionics in the
intermediolateral column (IML) and the sensory-related dorsal horn (Skagerber and Lindvall,
1985). This supraspinal DA input is hypothesized to reduce spinal nociceptive processing and
sympathetic outflow and enhance motor output, likely via D2-like receptor mechanisms.
Despite its intuitive appeal, this hypothesis is contradicted by two observations: 1) the A11
DA group is rarely pathologically involved in PD and 2) video-EEG analysis of rats with
bilateral 6-OHDA lesions of the A11 DA group has revealed no evidence of PLMS during
slow-wave sleep (Fuller and Lu, unpublished observations). On the other hand, lesions of the
intermediate ventromedial medulla produced periodic leg and tail movements during REM
sleep (Fuller and Lu, unpublished data; see Figure 3).
DOPAMINE AND STIMULANT-INDUCED AROUSAL

Drugs such as amphetamines and amphetamine-like stimulants (e.g., cocaine,
methylphenidate, methamphetamines) have potent wake-promoting effects (For review see,
Seiden et al., 1993). These drugs are thought to produce their arousal effects by blocking DA
reuptake/transport, i.e., acting on the cell membrane DAT and/or stimulating DA release,
resulting in increased synaptic DA concentrations. Determining DAs contribution to
behavioral arousal in this context has nevertheless been complicated by the fact that
psychostimulant administration also promotes the synaptic accumulation of other
monoamines, in particular NE. This is particularly true at higher doses, where amphetamines
interact with vascular monoamine transporter-2 to increase the cytoplasmic pool of
monoamines. In fact, for many years, the wake-promoting effect of amphetamine-like
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stimulants was attributed almost exclusively to NE mechanisms, as adrenergic signaling is

known to modulate arousal state. Further reinforcing this notion is the fact that locus
coeruleus (LC) neurons (the major source of brain NE) display robust state-dependent
activity and discharge most rapidly during enhanced arousal (Aston-Jones and Bloom, 1981).
Although taken together, these observations suggest NE signaling might form the molecular
basis for the wake-promoting actions of psychostimulants, several recent studies have
suggested a more dominant role for DA than NE in this regard. Centrally acting DA
antagonists, for example, cause drowsiness and drugs that selectively block the DAT are
more effective in promoting wakefulness than drugs that selectively block the NE transporter
(NET). Indeed, both desipramine and nisoxetine, two potent and selective NET blockers,
have minimal effects on EEG arousal in narcoleptic canines (even at high doses which
suppress REM sleep) (Nishino and Mignot, 1997). The wake-promoting effect of
amphetamine-like stimulants is also abolished in mice with deletion of the DAT gene (Wisor
et al., 2001). Conversely, the wake-promoting effect of amphetamines is preserved following
lesions of the locus coerulus (and hence a dramatic reduction in central NE) in cats and
following chemical ablation of NET-bearing NE forebrain projections from the LC in mice
(Wisor and Erikkson, 2005). The potency of most wake-promoting psychostimulants is also
best predicted by binding affinity to DAT. Finally, elevated arousal levels correlate with
increases in synaptic DA but not NE. Taken together, these findings support the concept that
presynaptic modulation of DA transmission is the key pharmacological mechanism by which
amphetamines and their derivatives mediate cortical and behavioral arousal.
The anatomical substrates for DAs effects on arousal also remain unresolved, although
several lines of experimental evidence suggest this may occur through inhibition of sleeppromoting systems. For instance, as previously indicated, a mutually inhibitory circuit
between wake-active vPAG DA neurons and sleep-active neurons of the VLPO has recently
been elucidated (Lu et al., 2006). Moreover, in vitro administration of DA inhibits neurons of
the VLPO. Curiously, however, DA mediated inhibition of the VLPO is blocked by 2
receptor antagonists but not by D2 receptor antagonists. Although these findings appear
difficult to reconcile, they likely indicate, at minimum, cross-talk between these CNS
catecholaminergic systems. Thus, for example, the wake-promoting drug modafinil (which,
as identified above, shares the wake-promoting properties of traditional psychostimulants)
hyperpolarizes VLPO neurons in vitro, presumably by blocking the NET. Yet DAT-knockout
mice are unresponsive to modafinil treatment, suggesting instead that DA neurotransmission,
i.e., blockade of the DAT, underlies the wake-promoting effect of modafinil. Combined,
these findings suggest a possible dual mode of action for modafinil, i.e., interference with
both NE and DA uptake, and this may explain why modafinil can exert its wake-promoting
effects without inducing dopaminergic side-effects such as addiction. Indeed, recent data has
suggested that modafinil may produce its arousal effects through dopaminergic-dependent
adrenergic signaling (Wisor and Erikkson, 2005). For example, DA may function as a
physiological ligand at adrenergic receptors. In support of this concept, DA stimulation of NE
receptors has been documented previously in the pontine brainstem. For now, however, the
mechanism of action by which DA produces its effects on cortical and behavioral arousal
remains unresolved.
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In addition to inhibiting VLPO neurons, amphetamine-like stimulants may also promote

wakefulness by activating, for example, the wake-promoting basal forebrain cholinergic
neurons and lateral hypothalamic orexin neurons. To this end, DA has been reported to
stimulate cholinergic cells in the basal forebrain by D1-like receptors, i.e., D1 and D5 in
vitro. Recent work has also uncovered an extended network of basal forebrain and preoptic
sites that mediate amphetamine-induced increases in behavioral and electrocortical arousal
(Berridge et al., 1999). Remarkably, however, although the anatomical resolution of this
mapping study was limited, close inspection of the data reveals that infusion of amphetamine
into the region best approximating the location of the VLPO produced one of the largest
increases in arousal, providing further support for the concept that the VLPO may be a
critical structure for mediating the arousal-promoting effects of amphetamines and
amphetamine-like stimulants.
Figure 3. Lesions of the ventromedial medulla produce phasic leg and tail movements during REM
sleep in rats (Fuller and Lu, unpublished observations). A) is a photomicrograph showing the extent of
the lesions (see black outline in intermediate ventromedial medulla). B) is the EEG and FFT power
spectrum from the same lesioned animal during a REM sleep bout (low voltage/high frequency EEG,
high theta (see FFT) and atonia (see EMG)). The EMG trace evidences abnormal phasic activity in the
EMG during REM sleep, which appear periodic in nature. C) shows time-locked video capture of this
same animal, which demonstrated tail flicking corresponding to the spike in the EMG (see white
arrows).
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SUMMARY
Work over the past decade has firmly delineated an important role for DA in sleep-wake
regulation (both normal and pathologic) and behavioral and electrocortical arousal. Wakeactive DA neurons of the vPAG likely exert their potent arousal influence through a mutually
inhibitory interaction with the VLPO as well as less-well defined interaction with
components of the ascending arousal system, e.g., basal forebrain, locus coereleus and lateral
hypothalamus. Based upon more recent findings, it is tempting to speculate that alterations in
DA neurotransmission may underlie the excessive sleepiness of evolving PD as well as the
manifestation of abnormal nocturnal movements in the form of RBD and RLS. Finally, a
critical role for DA in mediating the wake-promoting effects of psychostimulants has begun
to emerge, although the indirect or direct mechanism by which this occurs remains to be
clarified.
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Wisor JP et al. Dopaminergic role in stimulant-induced wakefulness. J Neurosci 21:17871794 (2001).
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ISBN: 978-1-60021-820-0
Chapter II
A CIRCUIT DYNAMICS THEORY OF COMPLEX

DOPAMINERGIC MODULATION OF PREFRONTAL
CORTICAL ACTIVITY AND ITS RELEVANCE TO
SCHIZOPHRENIA
Shoji Tanaka
Department of Information and Communication Sciences,
Sophia University, 7-1 Kioicho,
Chiyoda-ku, Tokyo 102-8554, Japan.
ABSTRACT
Working memory and other cognitive functions depend on dopaminergic
transmission. A number of functional imaging studies have suggested that the prefrontal
cortex (PFC) is the center for working memory. Working memory processing would be
mediated centrally by the circuit in the PFC. Then the research on the dynamics of the
PFC circuit under dopaminergic modulation would be crucial for the understanding of
functioning and dysfunctioning of the cognitive system. In this chapter, we develop a
circuit dynamics theory of the dopaminergic modulation of PFC activity. Persistent
activity with target selectivity over seconds is the essential dynamics of the maintenance
of working memory, and is known to have an inverted-U shaped profile of dopaminergic
modulation. However, the dynamics is not always stable along the inverted-U shaped
curve. Under hypodopaminergic conditions, the prefronto-mesoprefrontal system with
cortical dopaminergic modulation switches over from a negative to a positive control
system, making the PFC circuit unstable. This would be relevant to schizophrenia, in
which cognitive dysfunction is associated with the hypodopaminergic transmission in the
PFC. Because of this instability of the PFC circuit, the activity of the PFC tends to be
Correspondence concerning this article should be addressed to: Shoji Tanaka, Department of Information and
Communication Sciences, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan. email: tanakas@sophia.ac.jp; fax: +81-3-3238-3321; phone: +81-3-3238-3331.
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Shoji Tanaka
largely fluctuated, as often observed in human functional imaging studies. Beyond the
inverted-U shape region of dopaminergic modulation, in contrast, the PFC circuit has
bistability, and a hyperactive mode of PFC activity would emerge, depending on the
strength of the input to the PFC. The emergence of the hyperactivity of the PFC is due to
disinhibition in the circuit and would be relevant to psychotic states in schizophrenia and
other psychiatric diseases. This is consistent with the finding that GABAergic
transmission through parvalbumin-positive GABA neurons in the PFC is downregulated
in schizophrenia. The theory predicts that the PFC has such a complex profile of
dopaminergic modulation and argues that it is relevant to complex symptomatology of
schizophrenia.
INTRODUCTION
The pioneering work by Brozoski et al. (1979) and the succeeding studies have suggested
the involvement of dopamine (DA) in cognitive functions. Dopaminergic modulation of
signal transmission and neuronal excitability alters circuit dynamics of the brain, thereby
influencing cognitive functions, such as working memory. Neurophysiological studies of
nonhuman primates with DA agonists and antagonists found that DA modulated the
persistent neuronal activity for working memory in the dorsolateral prefrontal cortex
(DLPFC) with an inverted-U shaped profile (Goldman-Rakic et al. 2000; Williams and
Castner 2006). Both DA agonists and antagonists could impair working memory by
insufficient neuronal activity in the DLPFC (Arnsten 1997, 1998; Arnsten et al. 1994, 1995;
Cai and Arnsten 1997; Sawaguchi 2001; Sawaguchi and Goldman-Rakic 1991, 1994;
Sawaguchi et al. 1990a, b, 2001), suggesting a critical range of dopaminergic transmission in
which cognitive functions work properly.
Early functional imaging studies suggested reduced responses of the DLPFC or
hypofrontality in patients with schizophrenia (Andreasen et al. 1992; Carter et al. 1998;
Paulman et al. 1990). Recently, however, many studies have reported overactivation of the
DLPFC during the performance of working memory tasks (Callicott et al. 2000, 2003;
Manoach 2003; Manoach et al. 1999, 2000; Weinberger et al. 2001). Patients with
schizophrenia, whose brains commonly exhibit altered dopaminergic transmission, have
cognitive symptoms (e.g., Goldberg and Green 2002). The revised DA hypothesis of
schizophrenia postulates hypodopaminergic transmission in the cortex and
hyperdopaminergic transmission in the subcortical structures (Kahn and Davis 2000). It
would therefore be the hypodopaminergic transmission in the cortex that impairs working
memory and other cognitive functions, which is supposed to be generated largely from
neuronal interactions in the cortical network. According to this hypothesis, schizophrenic
brains with hypodopaminergic transmission would have a leftward shift of the operating
point of the cortical circuit along the inverted-U shaped curve. A recent computational study
suggests that the PFC circuit tends to be unstable when the PFC is hypodopaminergic
(Tanaka 2006, 2007), which has led the author to the proposal of the instability theory of
schizophrenia (Tanaka 2006, 2007). This theory would account for the seemingly
inconsistent overactivation and underactivation of the DLPFC in patients with schizophrenia
that have been observed in human imaging studies.
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Theory of DA Modulation of PFC Activity
19
In contrast to the low baseline DA tone in the PFC, transient DA levels could be high in
patients with schizophrenia as well as drug addiction. Because acute administration of
psychostimulants, such as amphetamine and cocaine, increases the DA release in the PFC, the
psychotic state would be associated with hyperdopaminergic transmission in the PFC.
Psychotic brains, either in schizophrenia or drug addiction, show selective hyperactivation of
the PFC (Mattay et al. 1996; Uftring et al. 2001). The inverted-U shape characteristic,
however, does not account for this activation pattern because it predicts that
hyperdopaminergic transmission yields hypoactivation of the PFC. This indicates that the
activation profile of the PFC under dopaminergic modulation might not be just inverted-U
shaped. In fact, a recent computational study suggested the possibility of the emergence of a
hyperactive mode of PFC activity with hyperdopaminergic transmission (Tanaka et al. 2006).
This chapter develops a circuit dynamics theory of such complex activation of the PFC under
dopaminergic modulation. This theory associates this complex modulation of PFC activity by
DA with symptomatology of schizophrenia.
CIRCUIT DYNAMICS FOR COGNITION

Cortical Activation for Cognitive Processing
In humans, functional imaging studies have shown that the DLPFC is consistently
activated during performing working memory and other cognitive tasks, suggesting that the
DLPFC works as the central commander for cognitive processing (Curtis and DEsposito
2003; DEsposito et al. 1999, 2000; Leung et al. 2002; Owen 1997; Rowe et al. 2000; Smith
and Jonides 1999). The notion that cognitive functions critically depend on dopaminergic
activity is supported by many studies since Brozoski et al. (1979). Because cognitive
functions would be largely owing to the control of circuit dynamics of the PFC that is under
dopaminergic modulation, it is necessary to know how DA modulates the circuit dynamics
for cognition.
Dopaminergic Modulation of Neurotransmission

DA reduces glutamate release from pyramidal neurons in the PFC (Gao et al. 2001;
Seamans et al. 2001). In the cortex, D1 receptor stimulation decreases excitatory
neurotransmission through non-NMDA receptors (Gao et al. 2001; Seamans et al. 2001;
Seamans and Yang 2004). Urban et al. (2002) further suggested that this reduction of
excitatory synaptic transmission is circuit or target specific; excitatory inputs to PFC
pyramidal neurons were reduced by the bath application of DA, but the inputs from nearby
pyramidal neurons were unaffected by DA. DA had no effect on excitatory transmission to
fast-spiking (FS) interneurons in the PFC (Gao and Goldman-Rakic 2003). Similar target
specificity of dopaminergic modulation of excitatory neurotransmission was found also in
inhibitory neurotransmission. DA depressed inhibitory neurotransmission between FS
interneurons and pyramidal neurons but enhanced inhibition between non-FS interneurons
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Shoji Tanaka
and pyramidal neurons PFC (Gao et al. 2003). In contrast to non-NMDA receptors, D1
receptor stimulation increases excitatory neurotransmission through NMDA receptors in the
cortex as well as in the striatum and hippocampus (e.g., Seamans et al. 2001; Seamans and
Yang 2004). This dopaminergic effect is mediated via D1-Gs-cAMP-PKA phosphorylation of
DARPP32 (Greengard 2001) and is characterized by its delayed onset and prolonged duration
(Seamans and Yang 2004). However, how the selective modulation of excitatory
transmission in the PFC circuit is related to the dopaminergic control of cognitive functions is
unclear. Moreover, DA directly enhanced excitability of the FS interneurons, indicating
important contribution of GABAergic inhibition in the dopaminergic control of cognitive
functions (Gao and Goldman-Rakic 2003; Gorelova et al. 2002). Therefore, there remain
several issues to be clarified, such as: the circuit mechanism by which dopaminergic
transmission changes the activity of the PFC; which receptor subtypes contribute to which
aspects of cognitive processing; and how the abnormality in dopaminergic transmission is
related to cognitive impairments in schizophrenia and other psychiatric diseases.
Neuropharmacological Studies
The effects of DA on working memory activity were studied in monkeys using D1
receptor agonists and antagonists (Sawaguchi 2001; Sawaguchi et al. 1988, 1990a, b).
Iontophoretic application of DA into the DLPFC enhanced delay-period activity during a
visuospatial working memory task, whereas D1 receptor antagonist, SCH 23390, suppressed
in most of the neurons tested (Sawaguchi et al. 1988, 1990a, b). Williams and GoldmanRakic (1995) reported enhanced spatially tuned delay-period activity of pyramidal neurons of
monkey PFC by iontophoretic application of D1 antagonists, such as SCH 39166, at low
doses. In contrast, iontophoretic application of D1 agonists, such as SKF 38393, suppressed
the delay-period activity (Williams and Goldman-Rakic 1995). As D1 receptor activation
level increases, the increment of the excitability of GABAergic interneurons would surpass
the increase in the excitatory signal transmission through NMDA receptors. This has led
Muly and Goldman-Rakic (1998) to propose a neurophysiological model for the inverted-U
shaped characteristic of dopaminergic modulation of working memory activity of PFC
neurons (Goldman-Rakic et al. 2000; Williams and Castner 2006). Unlike D1 receptors, D2
receptors have little influences on delay-period activity but seem to regulate response-period
activity (Wang et al. 2004).
Functional Modulation by DA
The relationship between the neuronal activity of the PFC and cognitive functions is
uncertain. Besides the neuronal activity, the performance of working memory tasks also
depends on the DA tone with an intermediate level for best performance. This has been
demonstrated by psychopharmacological studies with monkeys: D1 antagonists impair
working memory dose dependently (Sawaguchi and Goldman-Rakic 1991, 1994). The deficit
was sensitive to the delay period of the working memory task, suggesting a selective role of
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D1 receptors in working memory maintenance. Increased DA turnover also impairs cognitive

functions (Murphy et al. 1996a). Acute stress reversibly impairs working memory by
increasing the DA release in the PFC (Arnsten and Goldman-Rakic 1998; Murphy et al.
1996b). Chronic stress, in contrast, reduces the DA release in the PFC, which impairs
working memory in rats via D1 receptor hypostimulation (Mizoguchi et al. 2000). These
results suggest that moderate stimulation of D1 receptors is required for optimum working
memory processing (Murphy et al. 1996b).
Human Studies
In contrast to monkey studies, the distinctive roles of D1 and D2 receptors in working
memory processing are less clear in human studies. Pergolide, a mixed D1/D2 receptor
agonist, demonstrated an enhancement of working memory performance (Muller et al. 1998),
a beneficial effect on performance for subjects with greater working memory capacities
(Kimberg and DEsposito 2003), or no effect on performance (Bartholomeusz et al. 2003;
Roesch-Ely et al. 2005). Bromocriptine, a D2 agonist, facilitated visuospatial working
memory performance (Luciana et al. 1992; Mehta et al. 2001). This effect was for spatial but
not object working memory (Luciana and Collins 1997). Kimberg et al. (1997), however,
observed no effect of bromocriptine on spatial working memory but improvement of other
executive functions in a subgroup of subjects with low verbal working memory capacity in a
reading span task. Subjects with high verbal working memory capacity performed more
poorly on the drug, suggesting critical dependence of the drug effect on working memory
capacity. Kimberg et al. (2001) reported that bromocriptine resulted in task-specific
modulations of task-related activity while overall effect of bromocriptine across tasks was to
reduce task-related activity. A succeeding study in the same laboratory suggested further that
bromocriptine decreased activity in the task network at coding and increased activity at
response (Gibbs and DEsposito 2005). Kimberg and DEsposito (2003) further reported that
pergolide had effects on only delayed response tasks among a variety of cognitive tasks.
Muller et al. (1998) reported that only pergolide, but not bromocriptine, facilitated
visuospatial working memory performance. This result is in accordance with monkey studies,
confirming a selective role of D1 receptors in the PFC for working memory modulation.
However, the study by Ellis et al. (2005), reporting that acute tyrosine depletion in healthy
men did not impair working memory performance on any of the tasks they tested and that
stimulation of D1/D2 receptors under the depleted conditions caused a subtle impairment in
spatial working memory, would indicate a complex relationship between the cognitive
functions and dopaminergic transmission.
Computational Studies
Robustness of working memory representation depends on dopaminergic transmission.
Computational studies have suggested that D1 receptor stimulation increases the robustness
of working memory representation (Durstewitz and Seamans 2002; Durstewitz et al. 1999,
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Shoji Tanaka
22
2000). Computer simulations of dopaminergic modulation of the PFC circuit for working
memory by Tanaka (2002a, b) have shown that the PFC circuit treats the same set of cue
inputs for working memory differently when the activation level of D1 receptors is different:
Low D1 receptor activation makes working memory that is easily replaced by a newly loaded
item. With slightly higher activation of D1 receptors, both old and new working memory
items are represented in the same circuit. When D1 receptor activation is increased further,
represented working memory rejects new working memory items to be loaded. From these
results, Tanaka (2002a) has proposed the hypothesis that DA can control fundamental
cognitive operations by changing D1 receptor activation in the PFC (the operational control
hypothesis of dopamine). It is noteworthy that the robustness of working memory
representation and the switching of operations of working memory critically depends on the
glutamatergic transmission efficacy through the NMDA receptors on the neurons in the
circuit. Computational studies have suggested that NMDA hypofunction results in the failure
of maintenance of working memory (Brunel and Wang 2001; Durstewitz and Seamans 2002;
Durstewitz et al. 1999, 2000; Tanaka 2002a, b).
PFC CIRCUITRY
Prefrontal Cortical Circuit
To analyze the dynamics of the PFC circuit below, we introduce a simplified model of
the PFC circuit. The architecture of the model, we employ here, is depicted in Figure 1. The
model PFC has two neuron populations, the pyramidal neurons and the GABAergic
interneurons, which are connected reciprocally. Each population has self-innervations. Both
populations of neurons are under dopaminergic modulation. The pyramidal neurons receive a
transient external input, which triggers the dynamics of the circuit. The state equations of the
population activities (Tanaka 2006; Yamashita and Tanaka 2005) are given by
dx p
dt
xp
+ W pp ( z ) f ( x p ) Wnp f ( x n ) + I cue
(1)
dx n
x
= n + W pn ( z ) f ( x p ) Wnn f ( x n )
dt
n ( z)
where x p and xn are the population activities of the pyramidal neurons and the
interneurons, respectively,
p and n are time constants of these neurons, Wij (i, j = p, n) is
the synaptic efficacy from population i to j, and I cue is the transient external input that
mimics the cue input. The parameters that depend on z are subject to dopaminergic
modulation, where z is the D1 receptor activation. The activation function is given by
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f tanh( x) x 0
f ( x) = max
0
x<0
(2)
where f max is the maximum firing rate. This activation function is assumed to be common to
both populations of neurons.
Working memory
loading
Phasic input
Pyramidal
neurons
Working memory
representation
DLPFC
Interneurons
Dopamine
Dopaminergic modulation
Figure 1. A schematic diagram of the model. The DLPFC contains pyramidal neurons and GABAergic
interneurons, which are connected reciprocally and have also self-innervations. Both populations of
neurons are under dopaminergic modulation via D1 receptors. The phasic input to the pyramidal
neurons triggers the dynamics of the circuit.
Dopaminergic Modulation via D1 Receptors

The activation of D1 receptors affect channel conductance, such as g AMPA , g NMDA and
g K (for reviews: Seamans and Yang 2004; Yang et al. 1999). These change the efficacy of
glutamatergic signal transmission ( W pp and W pn ) and the excitability of the interneurons
( n ) (Tanaka 2006; Yamashita and Tanaka 2005). According to Gorelova and Yang (2000),
the excitability of the PFC inhibitory interneurons increases with D1 receptor activation. This
leads to a model in which the time constant of the interneurons, n , decreases with D1
receptor activation (Tanaka 2006; Yamashita and Tanaka 2005). Taken together, our model
describe these effects simply by
W pp ( z ) = W pp (0)(1 + az )
W pn ( z ) = W pn (0)(1 + bz )
(3)
n ( z ) = n (0)(1 + cz )
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Shoji Tanaka
24
where a, b and c are constants. The values of the parameters in the above equations (Eqs. (1)(3)) used in the simulation below are: f max = 100 sp/s, W pp (0) = 0.00055, W pn (0) =
0.000375, Wnp = 0.0005, Wnn = 0.00025,
p = 20.0, n (0) = 5.0, a = 0.2, b = 0.5, c = 0.3.
The Equilibrium State of the Prefrontal Cortex

By putting dxp/dt = dxn/dt = 0 in Eqs. (1), we have the equilibrium state of this model
circuit as
x p = p W pp ( z ) f ( x p ) Wnp f ( xn )
xn = n ( z ) W pn ( z ) f ( x p ) Wnn f ( xn )
(4)
From the above equations, we have
x p = p W pp ( z ) f ( x p ) W np
W
Wnn
f n ( z )W pn ( z ) nn W pp ( z ) f ( x p ) +
x p
pW np
W np
(5)
W pp ( z )
x n + nn W pp ( z ) W np f ( x n ) Wnn f ( x n )
x n = n ( z ) W pn ( z ) f p
W
n ( z )W pn
pn
These equations give the expressions of the equilibrium state for the pyramidal neurons and
the interneurons, respectively.
THE INVERTED-U REGION

The Inverted-U Shape Modulation
The model introduced in the last section shows a rather complex profile of dopaminergic
modulation, as we will see later in this chapter. In this section, however, before going into it,
we restrict ourselves to the region with moderate D1 receptor activation, which is the
inverted-U region. As mentioned earlier, working memory is formed in the PFC circuit when
the D1 receptors are activated within the optimum region; too low or too high activation of
the D1 receptors results in working memory deficits. The so-called inverted-U shape
characteristic of dopaminergic modulation has been used to interpret functional imaging data
from patients with schizophrenia. Because the PFC is in a hypodopaminergic state, according
to the revised DA hypothesis (Kahn and Davis 2000), the operating point of the PFC circuit
would be considered to be leftward shifted from the central or the optimum point (Tanaka
2006). Glutamatergic transmission and GABAergic transmission are also increasing with D1
receptor activation (Williams and Castner 2006; Yamashita and Tanaka 2002, 2003, 2005).
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Interestingly, therefore, the leftward shift is also compatible with the hypoglutamatergic
hypothesis and the hypoGABAergic hypothesis of schizophrenia. This simple interpretation
thus became a starting point for the mechanistic explanation of the PFC activity in
schizophrenia.
An interesting and important feature of the hypodopaminergic state is the existence of
instability. This is illustrated theoretically by using a closed-loop model of the prefrontomesoprefrontal system, which is composed of the PFC and the midbrain DA nuclei that are
reciprocally connected (Tanaka 2006, 2007). The closed-loop circuitry of the prefrontomesoprefrontal system (Frankle et al. 2006; Sesack and Carr 2002) can work as a "regulator"
of the PFC activity for working memory if the system is stable (Tanaka, 2006). The stability
of the PFC circuit depends on the DA tone in the PFC, and a conventional stability analysis
(e.g., Khalil 2000) shows that the closed-loop circuit is indeed stable when the DA level is
around the optimum point or higher (Tanaka, 2006). Under hypodopaminergic conditions, in
contrast, the system becomes unstable and no longer works as a regulator of the PFC activity.
This is because the closed-loop gain of the system varies with the DA level in the PFC, and
the switchover from a negative feedback system to a positive feedback system occurs at a
certain point as the cortical DA level decreases.
A Control System Example

The switching between negative feedback and positive feedback is illustrated as follows:
Let us consider a simple closed-loop or feedback system shown in Figure 2. The transfer
function of the plant is simply given by G(s) = 1/(s + 1), where s is the Laplace variable (e.g.,
Phillips and Harbor 1996). This transfer function represents the PFC and the feedback loop
represents the cortical dopaminergic system. The feedback gain represents the action of DA
in the PFC. We here use a bit of the basic control theory (e.g., Phillips and Harbor 1996). The
transfer function of the whole system is
T (s) =
Y ( s)
G ( s)
1
=
=
R( s ) 1 KG ( s ) s + 1 K
(6)
The characteristic equation of this system is
s +1 K = 0
(7)
and the root of the characteristic equation is
s = K 1
(8)
The condition of this system being stable is s < 0, which gives K < 1. In other words, as long
as K is less than 1, the system is stable. When K exceeds 1, in contrast, the system becomes
unstable.
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Shoji Tanaka
26
This system works as a negative feedback control system when the feedback gain is
negative (K < 0). An increase in the PFC activity (it is actually the response of pyramidal
neurons to the transient input) increases the activity of the DA unit, thereby increasing the
DA release in the PFC. Under hyperdopaminergic conditions, this increase in the DA tone
decreases the neuronal activity of the PFC. In other words, the PFC activity is controlled by a
negative feedback scheme. The feedback gain, K, corresponds to the slope of the inverted-U
shaped curve. The slope is negative under the hyperdopaminergic conditions (i.e., in the right
half of the inverted-U shaped curve) (Figure 3). The value of K increases as the operating
point on the inverted-U shaped curve approaches to the optimum point (i.e., the maximum
point of the curve). It becomes positive in the hypodopaminergic region (i.e., the left half of
the inverted-U shaped curve). Then the system turns to be a positive feedback system.
Interestingly, however, the positive feedback system is stable when 0 < K < 1. This explains
the reason why there remains a stable region in the left half of the inverted-U shaped curve.
In other words, the critical point (K = 1) is to the left of the point with maximum PFC activity
(K = 0). Once K exceeds unity (K > 1), this system becomes unstable, defining the unstable
region in hypodopaminergic conditions.
PFC circuit
Input to the PFC
R(s)
PFC activity
G(s)
Y(s)
+
K
The DA action through the
prefronto-mesoprefrontal loop
Figure 2. A block diagram of a simple closed-loop or feedback control system, which illustrates
conceptually the dopaminergic modulation of PFC activity. The transfer function G(s) exemplifies the
PFC circuit. The feedback loop represents the DA action through the prefronto-mesoprefrontal system.
When the feedback gain is negative (K < 0), this system works as a negative feedback control system.
The system becomes unstable when K > 1.
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Figure 3. A control system explanation of the stability of the prefronto-mesoprefrontal system. The
feedback gain of the system (illustrated in Figure 2), K, determines its stability. The system becomes
unstable when K > 1. The critical point (K = 1) is to the left of the point with maximum PFC activity (K
= 0).
Implications of Hypodopaminergic Instability

As illustrated above, this instability is due to the effectively positive feedback control of
the prefronto-mesoprefrontal closed-loop system under the hypodopaminergic condition.
Thus caused instability of the prefrontal cortical circuit makes the activity of the prefrontal
cortex largely fluctuated. Because of this, even a slight increase in the dopamine releasability
causes a catastrophic jump of the activity of the PFC from a very low level to a high level
(Tanaka 2006, 2007). Under the hyperdopaminergic condition, in contrast, the prefrontal
cortical circuit is stable, exerting negative feedback to control the dopamine release in the
prefrontal cortex. Even in this case, however, a decrease in GABAergic inhibition can make
the circuit unstable, causing hyperactivity of the PFC, as we will see below.
PFC ACTIVITY WITH WIDE VARIATION OF

D1 RECEPTOR ACTIVATION
Mode Diagram
To the authors knowledge, the circuit dynamics of the PFC beyond the inverted-U
region has never been argued. This section is devoted to see the profile of the dopaminergic
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Shoji Tanaka
28
modulation of the PFC activity over wider variation of D1 receptor activation. The simulation
with the model shows that two distinct stable modes exist over a wide range of the D1
receptor activation. One emerges when the D1 receptors are moderately activated, which is
considered to correspond to the conventional mode with an inverted-U shape profile (we call
this mode the inverted-U mode). The other mode emerges when the D1 receptors are
activated highly (to be more than 100% higher than the optimum level). This mode is
characterized by high neuronal activity with high D1 receptor activation, and termed the H
mode (Tanaka et al. 2006, 2007). The mode diagram of the dopaminergic modulation of
PFC activity is shown in Figure 4. The region in which the D1 receptors are activated at the
level of between 0.95 and 4.25 shows the inverted-U mode. Beyond 4.25, the mode
disappears and then emerges again when the D1 receptor activation level exceeds 6.0. In this
hyperdopaminergic region, the diagram shows two branches. The upper branch is stable
while the lower branch is unstable. Across this lower, unstable branch, the circuit is stable
when either the PFC activity is on the upper branch or the PFC is inactive (bistability).
80
-0.02
01
-0.
0.
0
-0.02
2
0.0
-0.01
70
60
0.0
1
50
40
30
20
10
PFC activity [a.u.]
90
-0.01
-0.02
10
D1 receptor activation [a.u.]

Figure 4. The mode diagram of the PFC with respect to dopaminergic modulation via D1 receptors. The
vertical axis is the population activity of the pyramidal neurons in the PFC in arbitrary unit, and the
horizontal axis is the D1 receptor activation level in arbitrary unit. Only the lines that are numbered 0
show the equilibrium state of the PFC circuit. See text for the method of drawing of this diagram.
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dx p /dt
0.01
0.005
0
-0.005
-0.01
20
40
60
80
100
dx /dt
0.02
0.01
0
-0.01
-0.02
20
40
60
80
100
dx /dt
0.05
C
0
-0.05
20
40
60
80
100
PFC activity [a.u.]

Figure 5. Operating points of the system. The curves of dxp/dt vs PFC activity or f(xp) are depicted for
different levels of z (A: 3.0, B: 5.0, C: 7.0). The intersections of these curves and the line of dxp/dt = 0
are fixed points, of which those marked with circles are stable fixed points and those marked with
crosses are unstable fixed points. The stable fixed points are the operating points of the system in the
equilibrium states. In other words, these points give the PFC activity in equlibrium.
Operating Points
In drawing the mode diagram (Figure 4), dxp/dt is obtained as a function of f(xp) and z.
Actually, Figure 4 is a contour plot of dxp/dt. The contours with dxp/dt = 0 give the mode of
PFC activity in equilibrium (the curves numbered by 0 in the figure). To see how the PFC
activity is determined, dxp/dt vs f(xp) or PFC activity is plotted in Figure 5 with three
different values of z (3.0, 5.0, 7.0). When z = 3.0 (Figure 5A), dxp/dt > 0 for f(xp) < 39. The
condition of f(xp) < 39 corresponds to that the state is under the inverted-U shaped curve.
That dxp/dt > 0 when the state is under the inverted-U shaped curve means that the activity is
increasing as long as the state is under the curve. When the state is above the inverted-U
shaped curve, which corresponds to the condition of f(xp) > 39, the activity is decreasing
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Shoji Tanaka
30
because dxp/dt < 0 in this case. In either side of the curve, the activity goes to f(xp) = 39, and
the activity level is fixed to be f(xp) = 39 because dxp/dt = 0 at this point. This means that the
point of f(xp) = 39 is a stable fixed point. When z = 5.0 (Figure 5B), on the contrary, dxp/dt <
0 for any activity levels, f(xp). Then, the activity decreases and goes down to zero eventually.
When z = 7.0 (Figure 5C), there appear two intersections between the dxp/dt curve and the
line of dxp/dt = 0. For 48 < f(xp) < 87, the activity is increasing (dxp/dt > 0). When either f(xp)
< 48 or f(xp) > 87, the activity decreases (dxp/dt < 0). Therefore, the point of f(xp) = 87 is a
stable fixed point, whereas the point of f(xp) = 48 is an unstable fixed point because the state
goes away from this point unless the activity level is exactly 48. These stable and unstable
fixed points give the upper and lower branch of the equilibrium contour (dxp/dt = 0), as
shown in Figure 4. When the activity is less than the lower branch, the PFC becomes
inactive. Therefore, the PFC has two stable fixed points in this case, one is the upper branch
of the equilibrium contour and the other is the inactive state (f(xp) = 0). In such situation, only
strong inputs can drive the PFC to cross the unstable fixed point, bringing the state to the
upper, stable fixed point. The emergence of the H mode thus depends on the strength of the
input.
THE H MODE
Circuit Dynamics of the H Mode
The circuit dynamics of the PFC under hyperdopaminergic conditions has several notable
characteristics. First, it is characterized by its bistability, and, because of this, the PFC has
two states; i.e., the hyperactive state and the inactive state. Both states are stable, and the
circuit can take either one of them. The hyperactivity of the H mode is primarily due to
hyperglutamatergic transmission. In our model, the glutamatergic transmission increases by
50% in the pyramidal to pyramidal connection (from W pp (3) = 1.6 W pp (0) to
W pp (7) = 2.4 W pp (0) ) and by 80% in the pyramidal to interneuron connection (from
W pn (3) = 2.5W pn (0) to W pn (7) = 4.5 W pn (0) ). This means that the input to the inhibitory
interneurons is strong in the H mode. Due to the nonlinearity of the activation function of
neurons, however, the activity of the interneurons tends to be saturating earlier than the
pyramidal neurons. This changes the dynamics of the circuit from balanced excitation and
inhibition to relatively weaker inhibition in such hyperdopaminergic situations. Therefore,
once the circuit receives a strong excitatory input, the state crossed the unstable branch of the
H mode, and then reaches the upper branch of the H mode. The second notable characteristic
is that, because of the bistability, the H mode has critical input dependency. That is, the
bifurcation to either the H mode or the inactive mode depends on the input to the PFC circuit.
Figure 6 shows the time courses of PFC activity profiles over D1 receptor activation with low
and high inputs. Only the high input induces the H mode. The time course of the inverted-U
mode depends on the strength of the input. However, both cases have the same profile of the
inverted-U mode in equilirium: The activity profiles at t = 1000 ms are not identical to those
shown in Figure 4 because the circuit has not reached the equilibrium states, but they
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eventually become identical. Figure 6 also shows the difference in the time courses of the
inverted-U mode and the H mode: In contrast to the slow time course of the inverted-U mode,
the transition to the H mode is quick. The input dependency of the H mode has important
implications; it indicates that the H mode can be prevented if the input is regulated to be
lower than the threshold. We will return to this issue later.
Figure 6. Time courses of high input vs low input activation of the PFC in the three dimensional
graphics of the activity profiles over D1 receptor activation. The strengths of the input, Icue in Eqs. (1),
are 0.025 (A) and 0.15 (B) in arbitrary unit. Only the stronger input induces the H mode. Note that, in
contrast to quick transition to the H mode (B) as well as to the inactive mode (A), the transition to the
inverted-U mode is slow. The circuit has not reached the equilibrium state at t = 1000 ms. The profiles
of the inverted-U mode in equilibrium for the two different strengths of the input eventually become the
same. The overall profiles in the equilibrium states are identical to those shown in Figure 4.
Evidence for H-Mode Activity
The H mode of PFC activity, which has not been explicitly described yet, could actually
occur because of the following possibilities: (i) Psychostimulants increase the extracellular
DA level not only in the subcortical areas but in the PFC. Psychostimulants also increase the
neuronal activity of the PFC. (ii) If the D1 receptors in the PFC are upregulated or
supersensitive, the z value increases (or the operating point shifts rightward). (iii) Psychosis,
which is characterized enhanced DA release and selective hyperactivation of the PFC. (iv)
Excessive or unfiltered thalamocortical input to the PFC may cause hyperactivation of the
PFC. (v) Upregulation of D2 receptors would also contribute to the transition to the H mode.
(vi) Stress enhances DA release in the PF. (vii) People with epilepsy are susceptible to
schizophrenia-like psychosis.
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Shoji Tanaka
i) Enhancemenet of DA Release by Psychostimulants

Acute administration of psychostimulants, such as amphetamines and cocaine, increases
the extracellular DA level significantly not only in the subcortical areas but in the PFC
(Shoblock et al. 2004; Stephans and Yamamoto 1995). A microdialysis study reported that
intraperitoneal administration of 2 mg/kg of amphetamine induced six-fold increase in the
baseline DA concentration (Shoblock et al. 2004), which would be expected to highly
activate D1 receptors in the PFC. Psychostimulants also increase task-dependent cortical
activation (Daniel et al. 1991; Mattay et al. 1996; Uftring et al. 2001). High doses of
psychostimulants could, therefore, induce the H mode of PFC activity.
ii) Upregulation/Supersensitivity of D1 Receptors
In contrast to acute administration, chronic administration of psychostimulants lowers the
extracellular level of DA in the PFC (Castner et al. 2005; Pierce and Kalivas 1997). This
induces sensitization. In patients with schizophrenia, using [11C]NNC 112 as a radiotracer,
Abi-Dargham et al. (2002) reported an increase in the binding potential of D1 receptors.
However, the study by Okubo et al. (1997), which used [11C]SCH 23390, showed reduced
binding of D1 receptors in the PFC. Later, Abi-Dargham and coworkers examined the effect
of acute and subchronic DA depletion on the in vivo binding of [11C]NNC 112 and [3H]SCH
23390 in rats (Guo et al. 2003). Acute DA depletion did not affect [11C]NNC 112 in vivo
binding, but paradoxically decreased [3H]SCH 23390 in vivo binding. Subchronic DA
depletion increased [11C]NNC 112 in vivo binding and decreased [3H]SCH 23390 in vivo
binding. Therefore, the increase in the binding potential of D1 receptors in patients with
schizophrenia, reported by Abi-Dargham et al. (2002), would reflect a chronically reduced
DA concentration and an increase in the density of D1 receptors or supersensitivity of DA
receptors by increasing the proportion of high affinity states of the receptors (Rubinstein et al.
1990; Seeman et al. 2005, 2006). Upregulation or sensitization of D1 receptors would be
involved in schizophrenia, and an increase in the DA releasability or the responsivity of DA
neurons has been suggested (Laruelle 2000; Lieberman et al. 1997). In this case, again, the z
value in the model increases accordingly, thereby increasing susceptibility to the H mode.
iii) Psychosis
In either schizophrenia or drug addiction, psychosis is associated with selective or focal
activation of the cortex (Breier et al. 1997; Daniel et al. 1991; Mattay et al. 1996; Uftring et
al. 2001). Functional magnetic resonance imaging (fMRI) studies of patients with
schizophrenia using a verbal fluency task showed that increasing task demand produced
greater activation of the PFC with higher error rates in psychotic states compared with
remission (Fu et al. 2005). It is postulated that before experiencing psychosis, patients
develop an exaggerated release of dopamine, independent of and out of synchrony with the
context (Kapur 2003). Psychostimulants increase the DA release in the PFC. Therefore,
psychosis would be associated with selective hyperactivation with hyperdopaminergic
transmission in the PFC.
iv) Enhanced Thalamocortical Inputs
Because of the bistable nature of the H mode, the occurrence of the H mode critically
depends on the strength of the input. It is mediated by corticocortical and thalamocortical
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afferents to the PFC, and would be modulated by several ways, including dopaminergic
modulation. DA has also been suggested to have sensorimotor gating function in PFC and
subcortical circuits (Braver et al. 1999; Montague et al. 2004). Actually, there are many
studies reporting deficits in the sensorimotor gating function in patients with schizophrenia
(for reviews: e.g., Braff and Freedman 2002; Geyer et al. 2001; Swerdlow et al. 2001) and,
interestingly, in amphetamine-sensitized animals (Tenn et al. 2003). When the input is
dysregulated or unfiltered input is given to the PFC, the PFC could respond to it with
hyperactivity. Recent neurophysiological study in monkey reported an enhancement of the
response-period activity of DLPFC neurons, but no effect on delay-period activity, by the
stimulation of the D2 receptors in the DLPFC (Wang et al. 2004). This may suggest that D2
receptors gate afferent input to the DLPFC circuit for working memory and other cognitive
functions.
v) Upregulation/Supersensitivity of D2 Receptor
In connection with the above issue, hyperactivation of D2 receptors could contribute to
the enhancement of the input to the PFC. If D2 receptors are upregulated or supersensitive
(Seeman et al. 2006), the H mode would more readily emerge. Being consistent with the
neurophysiological study in monkeys (Wang et al. 2004), suggesting a gating function, the
D2 receptors in the DLPFC, especially in its upregulated or supersensitive forms, may have a
critical role in the transition to the H mode.
vi) Stress
Acute stress increases DA turnover in the PFC, thereby impairing cognitive functions
(Arnsten and Goldman-Rakic 1998; Hutson et al. 2004). It seems that metabolic activity of
DA neurons innervating the PFC is increased selectively in the PFC (Deutch et al. 1991). The
administration of the stressor FG 7142 also increases DA turnover in the PFC (Murphy et al.
1996a, b). Chronic stress induces hypodopaminergic states, and, again, impairs cognitive
functions (Mizoguchi et al. 2000). In this case, Bmax or the density of D1 receptors in rat PFC
was significantly increased (from 14.5 with 2.9 SD to 22.3 with 3.5 SD). Interestingly, either
the hyperdopaminergic state or the hypodopaminergic state with D1 upregulation could lead
to the H mode, according to the above arguments.
vii) Epilepsy
Epilepsy is accompanied by excessive excitation of neuronal circuits in the brain (Avoli
et al. 2005; Fisher et al. 2005). People with epilepsy are susceptible to schizophrenia-like
psychosis (Qin et al. 2005; Sachdev 1998; Toone 2000). The association between epilepsy
and schizophrenia-like psychosis has long attracted much attention (Adachi et al. 2002;
Kanemoto et al. 2001), and would be interesting to know the commonalities between epilepsy
and schizophrenia and the mechanisms underlying both diseases. Many studies have
suggested selective alterations in GABAA receptor subtypes in patients with epilepsy (Bower
et al. 2002; Loup et al. 2000). DeFelipe (1999) proposed the hypothesis that the chandelier
cell is a key component of cortical circuits in the establishment of epilepsy. Links to
dopaminergic mechanisms have also been suggested (Ando et al. 2004; Starr 1996). Using
whole-cell recording and voltage-sensitive dye imaging techniques in the rat PFC,
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Shoji Tanaka
Bandyopadhyay et al. (2005) demonstrated that bath application of SKF 81297, a selective
D1 receptor agonist, enhanced spatiotemporal spread of activity in response to weak
stimulation and previously subthreshold stimulation resulted in epileptiform activity that
spread across the whole cortex. This result indicates that DA, via a D1 receptor-mediated
mechanism, enhances spatiotemporal spread of neuronal activity and lowers the threshold for
epileptiform activity in local circuits within the PFC. A rat study suggested that the
supersensitivity of the DA systems, developed in the chronic phase of the kainate-induced
temporal lobe epilepsy, is responsible for the genesis of epileptic psychosis (Ando et al.
2004). The H mode hypothesis is consistent with all of these results.
SCHIZOPHRENIA
Baseline Dopaminergic Transmission
The revised DA hypothesis of schizophrenia states that the baseline dopaminergic

transmission in the PFC is reduced. A consequential association between the
hypodopaminergic transmission and cognitive impairment leads the notion that
hypodopaminergic transmission is responsible for cognitive deficits in schizophrenia. The
circuit dynamics theory developed in this chapter is consistent with this notion, proposing
further that the circuit instability would cause a large fluctuation of the PFC activity.
Dysregulation of PFC activity thus occurs under hypodopaminergic conditions. The stability
of the PFC circuit depends on the DA tone in the PFC, and the dysregulation of PFC activity
due to circuit instability may have more direct links to schizophrenia than dopaminergic
transmission itself (the circuit instability theory of schizophrenia).
COMT
The hypodopaminergic state of the PFC in patients with schizophrenia would have
several distinct origins. One of the candidates is catechol-O-methyltransferase (COMT)
polymorphism (e.g., Diaz-Asper et al. 2006; Harrison and Weinberger 2005; Mannisto and
Kaakkola 1999; Matsumoto et al. 2003). COMT is a methylation enzyme that converts DA to
inactive 3-methoxytramine. It distributes in an extrasynaptic space, regulating the DA
concentration. The COMT gene has three polymorphic variations: Val/Val, Val/Met and
Met/Met. The Val allele of COMT is associated with reduced tonic DA levels in the PFC
because of its high activity (Bilder et al. 2002, 2004). Many studies have suggested that
COMT polymorphism is differentially related to cognitive performance, being consistent
with the inverted-U shaped characteristics of dopaminergic modulation (e.g., MeyerLindenberg and Weinberger 2006; Meyer-Lindenberg et al. 2006). The Met allele is
associated with better performance on the Wisconsin Card Sorting Test (WCST) (Egan et al.
2001; Malhotra et al. 2002; Weinberger et al. 2001). In accordance with the tonic DA levels,
cognitive performance using WCST is highest in the Met/Met group examined and is lowest
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in the Val/Val group (Egan et al. 2001). Because of these features, COMT has been
suggested to have links to schizophrenia (Harrison and Weinberger 2005). In patients with
schizophrenia, the percent correct on working memory tasks is generally lower than healthy
controls and is lowest for the patients in the Val/Vel group (Diaz-Asper et al. 2006;
Weinberger et al. 2001). Therefore, the Val allele, which is associated with reduced DA
release in the PFC and declined cognitive functions, increases susceptibility to schizophrenia
(Bilder et al. 2002; Egan et al. 2001; Weinberger et al. 2001). Another study, however,
suggested that this is the case for siblings of patients with schizophrenia but not for the
patients (Rosa et al. 2004). A COMT inhibitor, tolcapone, improved set-shifting performance
in rats by increasing stimulated DA release in the PFC (Tunbridge et al. 2004). COMT
inhibition increases the tonic DA level, which is then considered to be effective for cognitive
and negative symptoms of schizophrenia. Therefore, COMT could be a therapeutic target for
ameliorating cognitive symptoms in schizophrenia (Tunbridge et al. 2004, 2006).
Glutamate
Glutamatergic transmission itself is also suggested to be altered in schizophrenia, which

has been theorized as the glutamate hypothesis of schizophrenia (e.g., Coyle et al. 2003; Goff
and Coyle 2001; Jentsch and Roth 1999; Meador-Woodruff and Kleinman 2002; Tsai and
Coyle 2002). This hypothesis is supported by the facts that NMDA antagonists, such as
phencyclidine and ketamine, worsen positive, negative and cognitive symptoms in patients
with schizophrenia (e.g., Jentsch and Roth 1999; Lahti et al. 2001; Malhotra et al. 1997) and
that subanesthetic administration of ketamine to healthy subjects produces various cognitive
and behavioral deficits that are similar to schizophrenia and dissociative states (e.g., Adler et
al. 1999; Krystal et al. 1994; Radant et al. 1998; Umbricht et al. 2000). Acute administration
of NMDA antagonists markedly increases the release of DA and glutamate in the PFC, which
would be responsible for observed impairment of cognitive functions (Moghaddam et al.
1997; Verma and Moghaddam 1996). Ketamine-induced psychosis in healthy volunteers is
accompanied by focal activation of the PFC (Breier et al. 1997). In addition to the complex
interaction between the glutamatergic system and the dopaminergic system, which was
illustrated recently by a computational study (Tanaka 2005), the interaction between the
glutamatergic system and the GABAergic system is another important issue. The
glutamatergic hyperfunction may be caused by GABAergic hypofunction (Krystal et al.
2003). This could be the case given that GABAergic interneurons seem to be more sensitive
to NMDA blocking than pyramidal neurons (Grunze et al. 1996; Krystal et al. 2003;
Maccaferri and Dingledine 2002).
Functional Roles of GABA
Collaborative interactions among the dopaminergic, glutamatergic and GABAergic

systems determine the dynamics of the PFC circuit. Because the dopaminergic system
innervates both pyramidal neurons and GABAergic interneurons, DA actions in the circuit
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Shoji Tanaka
are both excitatory and inhibitory with nonlinear network effects, as argued above in this
chapter. GABAergic interneurons have many subtypes, such as basket cells, chandelier cells,
neurogliaform cells, Martinotti cells, and double bouquet cells (Kawaguchi and Kubota
1997). Each subtype has a specific form of dendritic and axonal arborization, suggesting
functional specificity, though the function of each subtypes of GABAergic interneurons is not
fully understood. Studies of monkeys performing visuospatial working memory tasks
suggested two distinct types of inhibition in the DLPFC: iso- and cross-directional inhibition
(Rao et al. 1999, 2000). The roles of these types of inhibition was investigated
computationally, which suggested that the isodirectional inhibition has a primary role for
stabilization of sustained activity by preventing excessive firing and the cross-directional
inhibition primarily contributes to the directional selectivity by sharpening the tuning curve
of working memory activity (Tanaka 2000a, b). Both stability and selectivity are obviously
critical features for proper working memory processing, and the above arguments indicate
that DA could regulate these features not only directly but indirectly via the control of the
intracortical inhibition.
GABAergic Inhibition in Schizophrenia
Many studies so far have suggested alteration of GABAergic transmission in

schizophrenia (Benes 1995; Benes and Berretta 2001; Benes et al. 1992). Benes and
colleagues found decreased densities of interneurons in layer II of the PFC and layers II-IV of
the cingulate cortex of patients with schizophrenia (Benes et al. 1991). The GABA synthesis
is also suggested to be reduced in schizophrenia. These indicate the hypofunction of the
GABAergic inhibition. Benes and colleagues also suggested upregulation of GABAA
receptors in the PFC (Benes et al. 1992, 1996). Given that the intracortical GABAergic
inhibition has the above mentioned functions (i.e., the regulation of stability and selectivity),
its alteration would cause dysregulation of the circuit dynamics, resulting in the impairment
of working memory. The alteration of GABAergic transmission in the cortex seems to be
selective for subtypes of the interneurons (Beasley et al. 2002; Guidotti et al. 2005; Reynolds
and Beasley 2001; Reynolds et al. 2001). Recent postmortem studies have consistently
suggested reduced levels of mRNA for GAD67, the 67-kilodalton isoform of glutamate acid
decarboxylase, in the DLPFC of patients with schizophrenia (Lewis et al. 2005; Volk and
Lewis 2002). The GABA neurons showing such reduction of GAD67 express parvalbumin
(PV) (Hashimoto et al. 2003), which constitute about 25% of GABA neurons in the primate
DLPFC. The PV-positive GABA neurons contain chandelier cells, which is characterized by
their synapsing exclusively on the axonal initial segment of pyramidal neurons. The
chandelier cells are, therefore, considered to suppress action potentials at axon hillocks. The
neurophysiological study of rats by Zhu et al. (2004) suggested that chandelier cells, whose
spontaneous activity is fairly low, are reserved to prevent excessive activation of neurons in
the circuit. The preferential loss of this type of interneurons might, therefore, be a key
component in cortical circuits in the establishment of epilepsy (DeFelipe 1999). Finally, it is
noteworthy that GABA-modulating drugs differentially affect working memory performance
in patients with schizophrenia: Lorazepam, a benzodiazepine type drug, impaired
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performance and flumazenil, a benzodiazepine antagonist, enhanced it (Menzies et al. 2007).

Imidazenil, a positive allosteric modulator at GABAA receptors, could increase cortical
GABAergic transmission, thereby ameliorating symptoms associated with specific cortical
GABAergic downregulation (Guidotti et al. 2005).
Gamma-Band Oscillations
The association of the GABA alterations in schizophrenia with gamma-band oscillations

(30-80 Hz) is interesting because gamma-band oscillations have been suggested to play roles
in cognitive as well as sensory processing in the brain (e.g., Herrmann et al. 2004). Patients
with schizophrenia show gamma-band oscillations with several altered parameters, including
reduced amplitudes, while performing a working memory task (Cho et al. 2006; Haig et al.
2000). Similar alterations have been observed also in sensory and perceptual processing in
patients with schizophrenia (Kwon et al. 1999; Spencer et al. 2004). Traub and colleagues
have suggested critical involvement of GABAergic interneurons in the generation of gammaband oscillations (Lee et al 2003; Traub et al. 1996). Therefore, dysfunction of gamma-band
synchronization may be one of the reasons that the GABA alterations in schizophrenia cause
cognitive impairment. All of the issues argued in this chapter, including the abnormalities of
dopaminergic, glutamatergic and GABAergic transmission in schizophrenia, may be
interrelated in the alterations of gamma-band oscillations in schizophrenia (Lee et al. 2003),
but this chapter does not argue this issue any further.
The Circuit Dynamics Theory of the PFC
The circuit dynamics theory of the PFC as described in this chapter, link PFC activity
with dopaminergic transmission. The fundamental perspectives underlying this theory are
that mental states critically depend on the dynamics of the circuits in the PFC and that the
circuit dynamics is different with different DA tones. These lead the hypothesis that the
circuits of the brains of the patients with schizophrenia have dynamics that is different from
that of healthy controls. According to the instability theory of schizophrenia, described in this
chapter, the hypodopaminergic state of the PFC is not necessarily associated with
hypoactivity. On the contrary, the theory proposes that the PFC could be activated at a high
level due to a slight increase in DA releasability. Both activity states could occur under
hypodopaminergic conditions. This would account for seemingly paradoxical activation of
the DLPFC in schizophrenia. However, many issues are yet to be addressed. For example, the
DLPFC of patients with schizophrenia seems to have complex patterns rather than just hypo
or hyper (Callicott et al. 2003) and the complex patterns are not restricted to the DLPFC
(Glahn et al. 2005). Furthermore, hyperdopaminergic transmission would induce a bistable
hyperactive mode or the H mode of PFC activity, according to the circuit dynamics theory of
the PFC. We have argued that this activity mode might be associated with psychotic states.
The circuit dynamics theory thus describes different dynamical aspects that are considered to
be relevant to schizophrenia.
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Shoji Tanaka
CONCLUSION
This chapter has proposed a circuit dynamics theory of complex dopaminergic
modulation of the PFC activity in relation to cognitive functions. With this theory, we have
argued how the circuit dynamics of the PFC alters with the DA tone in the PFC. This theory
contains two important points. First, the PFC circuit with hypodopaminergic transmission
tends to be unstable due to an effective positive feedback control scheme. This would be
consistent with the DA hypothesis of schizophrenia and functional imaging studies of
patients with schizophrenia showing both hypo- and hyperactivation of the PFC. Second, the
PFC circuit with hyperdopaminergic transmission is bistable. Strong inputs to the PFC could
induce the H mode or hyperactivity of the PFC. We argued that this might be associated with
psychotic states (the H mode hypothesis of psychosis).
The modulation profile that this theory predicts is complex, rather than just an invertedU. Given the pathological complexity of schizophrenia, this seems reasonable. The invertedU shaped profile might be too simple to account for such complex dynamics underlying
schizophrenic as well as normal brain circuits. Therefore, this theory would have potency to
provide deeper insights into the DA hypothesis of schizophrenia, although further study is
obviously necessary.
The H mode has critical input dependency. Provided that D2 receptors in the PFC gate
the input to the PFC, this is consistent with the notion that D2 receptors are involved in the
process of psychosis. In other words, it is in accordance with that antagonists of D2 receptors,
as most antipsychotic drugs actually are, are effective in ameliorating positive symptoms of
schizophrenia. Interestingly, however, the D2 receptors in the above argument are ones in the
PFC rather than in the striatum. This study hence proposes a new perspective that the
antipsychotic effect on positive symptoms might be mediated, if not solely, by blocking
cortical D2 receptors in the PFC rather than in the striatum.
ACKNOWLEDGEMENTS
This work was supported partly by the Human Information Science Research Project,
Sophia University and the Ministry of Education, Science and Technology.
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ISBN: 978-1-60021-820-0
Chapter III
THE LIFE CYCLE OF THE DOPAMINERGIC

NEURONS IN THE SUBSTANTIA NIGRA
Vincenzo Di Matteo1, Massimo Pierucci1, Arcangelo Benigno2, Ennio
Esposito1, and Giuseppe Di Giovanni1,2,
1
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud,

Santa Maria Imbaro (Chieti), Italy;
2
Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, G. Pagano,
Universit degli Studi di Palermo, 90134 Palermo, Italy.
ABSTRACT
Since the 1950s, when dopamine (DA) was discovered in the mammalian central
nervous system (CNS), an enormous amount of experimental evidence has revealed the
pivotal role of this biogenic amine in a number of cognitive and behavioural functions
including voluntary movement and a broad array of behavioural processes such as mood,
reward, addiction, and stress. Moreover, dopaminergic neurons, although their numbers
are few, are of clinical importance because it is implicated in several psychiatric
disorders, such as schizophrenia, depression, and anxiety. The lost of dopaminergic
neurons of the substantia nigra compacta (SNc) is associated with one of the most
prominent human neurological disorders, Parkinson's disease (PD). Moreover, the
mechanisms whereby nigral dopaminergic neurons may degenerate still remain
controversial. Hitherto, several data have shown that the earlier cellular disturbances
occurring in dopaminergic neurons include oxidative stress, excitotoxicity, inflammation,
mitochondrial dysfunction, and altered proteolysis. These alterations, rather than killing
neurons, trigger subsequent death-related molecular pathways, including elements of
apoptosis. In rare incidences, PD may be inheritated; this evidence has opened a new and
exciting area of research, trying to shed light on the nature of the more commune
Correspondence concerning this article should be addressed to Dr. Giuseppe Di Giovanni, Dipartimento di
Medicina Sperimentale, Sezione di Fisiologia Umana, G. Pagano, Universit degli Studi di Palermo, Corso
Tukry 129, 90134 Palermo, Italy. Tel.: +39 091 6555821; Fax: +39 091 6555823; g.digiovanni@unipa.it.
52
Vincenzo Di Matteo, Massimo Pierucci, Arcangelo Benigno et al.

idiopathic PD form. In this review, the characteristics of the SNc dopaminergic neurons
and their life cycle from birth to death are reviewed. In addition, of the mechanisms by
which the aforementioned alterations cause neuronal dopaminergic death, particular
emphasis will be given to the role played by inflammation, and the relevance of the
possible use of anti-inflammatory drugs in the treatment of PD. Finally, the new evidence
of a possible de novo neurogenesis in the SNc of adult animals and in PD patients will
also be examined.
Keywords: Parkinsons
Neuroprotection.
Disease,
Neurogenesis,
Neuroinflammation,
Apoptosis,
Parkinsons disease (PD) is the second most common neurodegenerative disorder in the
elderly population for which, unfortunately, there is no cure as yet. Idiopathic PD is a
progressive disorder the impact of which reaches far beyond the clinical signs and symptoms
exhibited by those afflicted. This neurodegenerative disorder not only places a severe burden
on the patients but also on their family, friends and society.
It is estimated that close to 4 million people worldwide suffer from PD. The disease
afflicts both sexes equally, and the initial symptoms typically appear when people are in their
late 50s or early 60s. Indeed, nearly 1% of the population over the age of 65 is estimated to
suffer from the disease. Moreover, the number of Parkinson sufferers is expected to grow as
the general population in the Western world ages. Accordingly, the costs of treatment (health
and social care), estimated at between 560,000 and 1.6 million per 100,000 population, is
expected to rise [1-3].
Clinical features at presentation include the asymmetric onset of cardinal motor
symptoms such as tremor at rest, bradykinesia, muscular rigidity, stooped posture and
instability [4]. These are the result of the loss of dopaminergic (DAergic) neurons in the
substantia nigra pars compacta (SNc), which causes a consequent reduction of dopamine
(DA) levels in the striatum [5-7]. Regrettably, the symptoms of PD do not appear until up to
80% of the DAergic nerve cells have been lost [8,9] In the early stages of the disease, DA
replacement therapy, using the dopamine precursor levodopa, is effective but the dose
response decreases with disease progression and motor complications (dyskinesias) and other
side effects (e.g. mood disorders, sleep disturbances) arise after chronic treatment. These
complications may be due either to the advanced stage of the disease when degenerating
DAergic neurons can not buffer the fluctuating plasma levels of levodopa, resulting in
pulsatile stimulation of the dopamine receptors, or to the further degeneration in non-DAergic
regions [10,11]. Since the underlying mechanisms of neuronal loss in patients are not known,
current therapies are mainly symptomatic and do not halt the progression of the disease [12].
It appears clear that understanding the etiopathogenesis of PD; the modalities whereby
the neurodegenerative process begins and progresses, is fundamental for the development of
drugs to slow or prevent the progression of PD. There have certainly been major advances in
these areas over the past few years, but, the modalities whereby the neurodegenerative
process begins and progresses remain unclear. The situation is complicated further by the
large number of factors that seem to be involved in the onset of this disease, such as aging,
genetic vulnerability, exogenous or endogenous toxins, hydroxyl radicals (OH) production,
DAergic Neuron Life Cycle

neuronal metabolic disturbances and inflammation [4,13-16]. Thus, the cumulative
insults attributable to these metabolic stress factors may promote premature SNc
degeneration through the activation of apoptotic programs [17-19]. However, the
and sequential neuroapoptotic events associated with premature, progressive SNc
atrophy remain undefined.
53
neuronal
DAergic
specifics
neuronal
THE SNC DAERGIC NEURONS

Dopamine is one of the most intensively studied neurotransmitters in the brain due to its
involvement in several mental and neurological disorders, such as schizophrenia, depression
and PD. The most prominent DAergic cell group resides in the ventral part of
mesencephalon, which contains approximately 90% of the total number of brain DAergic
cells. Essentially, they are restricted to two nuclei, the ventral tegmental area of Tsai (VTA;
A10) and the lateral SNc (A9). Nevertheless, cells expressing tyrosine hydroxylase (TH), the
rate-limiting enzyme in the biosynthesis of catecholamines, have also been described in the
striatum of rodents, monkeys and even humans [20-23]. The DAergic neurons localised in the
SNc preferably project to the caudate nucleus and the putamen, i.e. the dorsal part of the
striatum, and therefore this pathway is often called nigrostriatal DAergic system. More
medial to this pathway are the mesolimbic and mesocortical DAergic systems, which arise
from DAergic cells present in the VTA [24]. The substantia nigra (SN) has been
cytoarchitecturally divided into three different parts: the SNc, a horizontal sheet of densely
packed medium and large cells that occupies its dorsal one-third; the SN pars reticulata
(SNr), a more diffuse and cell-poor division, containing small and medium neurons lying
between the SNC and the cerebral peduncles, and the SN pars lateralis (SNL), a small cluster
of medium cells that extends rostrocaudally along the lateral border of SNC and SNR [2528]. According to their neurotransmitter, nigral neurons were classified into DAergic and aminobutyric acid (GABA)-ergic neurons [29-31]. Most DAergic neurons are localized in the
SNC, some of them in the SNr, and to a lesser extent, in the SNL [32-35]. The majority
(>90%) of cells in the SNc are medium sized aspiny DAergic neurons with sparsely
branching dendritic trees. There appear to be two distinct dendritic arborizations. The largest
stays mostly within pars compacta, and consists of medium length dendrites from 300 to
about 500 m in length. Most pars compacta DAergic neurons also send one or two very long
dendrites ventrally into pars reticulata that may be over 1 mm in length. The axon is thin and
unmyelinated, and does not give off local collaterals. In addition, there is a much smaller
number of small to medium sized non-DAergic interneurons whose connectivity and function
are not well understood. Electrophysiologically, DAergic neurons in the SNc display the
typical firing properties of DAergic neurons i.e. broad action potential (mean biphasic = 2.17
ms), slow firing rate (mean = 4.15 Hz) and regular firing pattern [36]. They are composed of
subsets of neurochemically different neurons, and these chemical differences may be
involved in their physiological properties and vulnerability to aggression. Cholecystokinin
(CCK), is expressed in DA-cells in the rostral half of the SNc, but not in those of its caudal
half and SNr [37]. Calbindin-D28k (CB), calretinin (CR), and parvalbumin (PV), three
calcium-binding proteins which act as buffers or transporters of intracellular Ca++, are also
54
expressed [38,39]. According to cytoarchitectural, topographical, and chemical criteria,

Gonzlez-Hernndez and Rodrguez [40] identified five different cell groups: a cell group in
the dorsomedial portion of the SNc which contains CCK, CR, and CB (dmSNC); DA-cells in
the SN pars lateralis (SNL) which also contain CCK, CR and CB; DA-cells in the rostral half
of the SNC containing CCK and CR (rSNC); DA-cells in the SNr and the caudal half of the
SNc which only express CR (cSNC-SNR), and a DA-cell group in the lateral part of the SNc
that contains none of the markers studied (lSNC) [40]. Considering exclusively the
distribution of imureactivity for CB, the SNc is compartmentally organized along the lines of
a nigrosome-matrix [41-43]. According to Damier and colleagues [42,43], 60% of DA
neurons in the human SNc are sparsely distribuited whithin the large region of intense CB
staining, which they named nigral matrix; the other 40% of DA-containing neurons are
included within 5 different nigrosomes, numered from 1 to 5. In conclusion, DA-nigral
cells are far from being a homogeneous group, on the contrary they form a mosaic of
neurochemically different subnuclei which are likely to differ in their physiological and
pharmacological properties and vulnerability to aggression.
THE BIRTH OF SNC DAERGIC NEURONS

The number of nigral neurons declines during normal aging, and it is possible that many
normal elderly subjects, as well as would-be PD cases, do not develop signs of the condition
because DAergic cell loss has not reached the threshold level. Hence, it is theoretically
feasible that individuals born with smaller numbers of nigral neurons might be more
susceptible to reaching the critical level of neuronal loss, for example by exposure to
environmental toxins or even through aging. The idea that early DAergic neurogenesis might
have long-term effects on the onset of PD was strengthened by the discovery of the role of
specific transcription factors that control DAergic neurogenesis during brain development. It
is widely accepted that immature neurons die by programmed cell death as a result of trophicfactor deprivation, owing to their inability to form proper synapses with their targets, whereas
mature neurons die because of toxin insult. The quality of the contact and/or the degree of
trophic support in early life might be important in determining the number, health and length
of survival of cells such as DAergic neurons. Moreover, the transcription factors involved are
expressed throughout life in the basal ganglia, suggesting that they have a role in maintaining
the health of specific neurons. Recent advances in molecular biology and mouse genetics
have helped to unravel the mechanisms involved in the development of mesodiencephalic
DAergic (mdDA) neurons, including their specification, migration and differentiation, as well
as the processes that govern axonal pathfinding and their specific patterns of connectivity and
maintenance [44]. Such insights into the molecular biology of mdDA neurons have facilitated
the development of embryonic stem (ES) cell-replacement strategies, whereby ES cells are
induced to differentiate into a specific neuronal phenotype and transplanted into the brain for
the treatment of PD and potentially other disorders affecting the mdDA neurons. Successful
cell-replacement strategies begin with knowledge of how to make the appropriate mdDA
neuron. The precise time point of origin of the first postmitotic mdDA neurons is still a
matter of debate. The problem has been complicated by the fact that the mdDA system is not
55
a homogenous group of neurons. The development of mdDA neurons follows a number of

stages marked by distinct events. The first mDA neurons are born around embryonic day
(E)12 in Sprague-Dawley rats [44-46], and develop from a single embryological cell group
that originates at the mesencephalic-diencephalic junction, known as the isthmus [47,48].
Developmental studies of the pathways involved, have led to the identification of several
factors that influence the final formation of midbrain DA-neurons in the adult animal. The
specification of the permissive region for DA neuron generation occurs through the secretion
by the isthmus of two secreted signalling proteins; the sonic hedgehog (SHH) and the
fibroblast growth factor 8 (Fgf8). This permissive region is also defined by a specific pattern
of gene expression in the mesodiencephalon ventricular zone; i.e. orthodenticle homologue 2
(OTX2), gastrulation brain homeobox 2 (GBX2) and transforming growth factor- (TGF).
While transcription factors that are specifically expressed in the proliferating DAergic
progenitor cells have yet to be identified, others important for post-mitotic DAergic cell
development have already been characterized. These developmental factors induce mitotic
cells in this region to become postmitotic young neurons that are destined to become fully
differentiated mdDA neurons. These include LIM homeobox transcription factor (LMX)
1A/B, OTX1/2, NK6 transcription factor related, locus 1 (NKX6.1), SHH, nuclear receptorrelated 1 (NURR1), paired-like homeodomain transcription factor 3 (PITX3), and engrailed
(EN1/EN2). NURR1 appears to be strictly coupled to neurotransmitter synthesis which
regulates several proteins that are required for DA synthesis and regulation, such as tyrosine
hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), dopamine transporter
(DAT) and RET receptor tyrosine kinase (cRET) [49], where as LMX1B is necessary for the
expression of PITX3 [50]. PITX3 is expressed in all mesencephalic DAergic neurons in the
CNS and it is involved in the terminal differentiation and/or early maintenance of SNc
neurons [51,52]. Moreover, PITX3 cooperate with NURR1 inducing the late maturation of
mdDA neurons [51]. This cooperativity offers a potential mechanism for the relatively celltype-specific expression of late markers of midbrain DA neurons maturation. Studies
analyzing the functions of these transcription factors have not only increased the
understanding of how DAergic neurons are generated in vivo, but also allowed for the
development of new strategies in stem cells for engineering DAergic neurons in vitro. These
results may be significant in terms of the development of future therapies for PD patients [5153].
THE DEATH OF THE SNC DAERGIC NEURONS

Considerable differences exist in the numbers of midbrain DAergic cell bodies in various
mammals ranging from about 45,000 in the rat, 165,000 in the macaca monkey, to 590,000 in
human beings [34]. This latter number applies to humans in their fourth decade of life but
drops to an average of about 350,000 during the sixth decade of life [54]. Such an agedependent decrease in the numbers of SNc DA-cells has also been reported for nonhuman
primates. It is intriguing to note that parkinsonian neurodegeneretion it is not simply an
accelerated form of cell loss seen during the normal aging, even though they share some
56
pathological characteristics. For example, the pattern of loss is opposite to ageing, with
greatest in the ventral part of SNc [55].
DAergic neurons are peculiarly prone to oxidative stress due to their high rate of oxygen
metabolism, low levels of antioxidants, and high contents of iron and neuromelanin pigment
(NM). Moreover, DA is thought to be capable of generating toxic reactive oxygen species
(ROS) via both its enzymatic and non-enzymatic catabolism [56,57]. Specifically, DA
oxidation can occur either spontaneously in the presence of transition metal ions or via an
enzyme-catalyzed reaction involving monoamine oxidase (MAO). Oxidation of DA via MAO
generates a spectrum of toxic species including H2O2, oxygen radicals, semiquinones, and
quinones [56,57]. Conditions that increase brain concentration and/or turnover of DA could
potentially increase the formation of reactive metabolites especially under conditions in
which the ratio of available DA to antioxidant capacity is high [58]. Furthermore, the
midbrain region that encompasses the SN is particularly rich in microglia [59,60], therefore,
activation of nigral microglia and the release of these pro-inflammatory neurotoxic factors
may be a crucial component of the degenerative process of DAergic neurons in PD.
We are far from seeing the whole picture; the mechanisms responsible for nigral DA cell
death are only beginning to be understood. Nevertheless, the future is promising, and it is
likely that the most crucial evidence will come from those investigations designed to
understand the propensity of different DA neurons to undergo cell death according to the
different mesencephalic structures in PD. In fact, the VTA and the central grey substance
(CGS) neurons are prone to death, albeit to a lesser degree compared to the SNc nerve cells.
In PD the SNc are the most affected (80-90% cell loss), in the CGS most DA neurons are
spared (2-3% lost) and in the VTA, the cell loss is intermediate (40-50%) [13]. In addition,
differences among DA neurons exist within the same SNc. It is well known that DAergic
neurons degenerate in PD in a disease-duration pattern [42,61,62]. Human DA nigral neurons
in the calbindin-D28k-poor nigrosomes in contrast to those in the calbindin-D28k-rich matrix
are more likely to degenerate in PD. Within the nigrosomes, cell loss follows a strict order,
depletion being maximal, with a maximum cell loss of 98%, in nigrosome 1 located in the
caudal and mediolateral part of the SNc and then to other nigrosomes and finally to the
matrix. In addition, the degree of loss of DAergic neurons in the SNc is related to the
duration of the disease, with a pattern of neuronal loss consistent from one parkisonian SN to
another [43,61,62]. A similar pattern of cell death has been recently confirmed in a partially
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmoset model of
PD where the majority of surviving DA neurons was confined to nigral calbindin-D28k-rich
regions and resistant DA terminals were contained within calbindin-D28k-rich striatal matrix
[63]. The reason for such differential vulnerability of DAergic neurons is not known. Yet, the
different nigral compartments may differ in terms of their content of growth factor, receptors,
compounds related to exocitoxicity, agents involved in oxidative metabolism, and activity of
potentially predisposing genes such as those for -synuclein and parkin. Moreover, a
difference in gene expression patterns has been shown between the SNc and the VTA in
C57Bl/6 mice [64] and in Lewis rats [65]. In the study by Chung et al. [64], 103 genes with a
higher than twofold difference were identified and six genes of interest were retained for
further functional analysis. Greene et al. [65] identified 161 transcripts expressed differently
by VTA and SNc DAergic neurons. A subsequent pathway analysis revealed that genes
57
involved in energy metabolism were expressed more highly in the SN than in the VTA, in
accordance with previous knowledge regarding a central role for mitochondrial dysfunction
in PD pathogenesis. Recently, further evidence has been given by Lu and colleagues in a
post-mortem study [62]. They have investigated the expression of 8 genes implicated in cell
survival in DA neurons from the SNc and the CGS of subjects without history of neurological
or psychiatric disease. The amyloid precursor-like protein 2 (APLP2) was the only gene
expressed preferentially in SNc DAergic neurons, suggesting a probable deleterious influence
of this gene on cell survival [62]. No clear evidence, hitherto, exists for a genetic difference
among DAergic mesencephalic neurons. Recent data indicate that groups of midbrain
neurons vary dramatically in their vulnerability to injury and these differences are
attributable, at least in part, to a varying susceptibility of DAergic cell populations to
oxidative stress [66]. Consistently, much attention has been given to the role of NM, which
has long been recognized as a marker of increased oxidative stress [67]. A dual role of NM
has been recently proposed in the pathogenesis of PD [68]. In the early stages, NM synthesis
and iron-chelating properties may act as a powerful protective mechanism, delaying symptom
appearance and/or slowing disease progression. Once these systems have been exhausted, the
pathogenic mechanisms affecting cytoplasmic organelles other than NM destroy NMharboring neurons, with consequent pouring out of NM granules. These in turn activate
microglia, causing release of nitric oxide, interleukin (IL)-6 and tumor necrosis factor-
(TNF-), thus becoming an important determinant of disease aggravation. SNc DAergic
neurons are large, melanized differently from the CGS DA neurons that do not carry NM and
are generally significantly smaller [62]. Interestingly, the distribution pattern of NM within
the SNc is inverse to the density of calbindin-D28k-immunostaining intensity and clearly
overlaps the degeneration disease-duration pattern showed by Damier et al. [42]. In so far as
ventral SNc DAergic neurons of nigrosome 1, the first to degenerate in PD, contain a high
degree of pigmentation [13,69]. NM pigment could be toxic to aminergic neurons as it
physically interferes with intracellular communication [70,71], causing a macromolecular
crowding effect [72], thereby interfering with the synthesis and degradation of cellular
proteins. NM pigment formation is related to the level of the vesicular monoamine
transporter-2 (VMAT2) in the DAergic neurons [69]. In vitro data indicate that NM pigment
is formed from the excess cytosolic catecholamine that is not accumulated into synaptic
vesicles by VMAT2. In midbrain DAergic neurons, there is an inverse relationship between
NM pigment content and VMAT2 immunoreactivity. Neurons with high levels of VMAT2
immunoreactivity are located in the VTA in the region of the exit of the third nerve, in the
dorsal portion of the SN, and in the retrorubral field. On the other hand, neurons in the
ventral subdivision of the SN have lower levels of VMAT2 immunoreactivity than the VTA
neurons and higher levels of NM pigment. The lowest levels of TH and VMAT2 are found in
the somata of nigrosome 1 neurons. In vitro studies have found that, when synaptic vesicles
contain more VMAT2 molecules, they can store and release more catecholamine [73].
Although the SN neurons possess relatively low levels of TH, they must also possess levels
of VMAT2 that allow a pool of DA to exist that is not stored in synaptic vesicles, which
ultimately, can be oxidized to form NM pigment. Being as NM pigment can take up over
50% of the cytoplasmic volume of many DAergic neurons by the sixth decade of life, this
pigment may play an important role in macromolecular crowding such that the cytoplasmic
58
DA can potentiate -synucleins tendency to form toxic protofibrillar and fibrillar species
leading to cell death [74,75]. It can be hypothesized that, when a sizeable nonvesicular pool
of amine exists in the cytoplasm for a prolonged period, reactive oxygen species (ROS) are
formed along with toxic DA adducts. The ROS and adducts are stored in lysosomal
structures, which constitute the NM pigment granule [76]. It may be that, after the neuron has
accumulated an excessive amount of NM pigment, additional ROS and toxic DA adducts can
no longer be stored in the form of NM pigment, and, once the toxins are in the cytoplasmic
compartment, they inhibit proteasome function, and the neuron becomes poisoned.
Consistently with this hypothesis, old monkeys treated with the DA neurotoxin MPTP exhibit
a preferential loss of NM-containing DAergic neurons vs. DAergic neurons without NM [77].
Another important factor that can contribute to aminergic cell death in PD relates to an
impairment of mitochondrial function. Because ATP is required for VMAT2 to pump amine
into synaptic vesicles, individuals with a complex I (NADH-ubiquinone reductase complex,
one of the five enzyme complexes of the inner mitochondrial membrane involved in
oxidative phosphorylation) defect may have higher than normal levels of cytoplasmic DA,
which hastens NM pigment formation in vitro [76] and macromolecular crowding, along with
-synuclein-containing Lewy body formation [78], and leads to premature cell death.
Mitochondrial dysfunction in PD is also supported by the findings on complex I deficiency in
the nigro-striatum of the postmortem brain from PD patients and of complex I inhibition in
the SN of animal PD models produced by treatment with neurotoxins such as MPTP [79] or
the insecticide rotenone [80]. The discovery of MPTP causing PD in humans suggests the
neurotoxin hypothesis, in which MPTP-like exogenous or endogenous neurotoxins acting
together with presumed PD-susceptibility genes are assumed to be the cause of PD. On the
other hand, recent molecular genetic studies on mutations of the causative genes of autosomal
dominant or recessive familial PD, especially mutations in 5 genes (a-synuclein, parkin, DJ-1,
PINK1, and LRRK2), have led to a new hypothesis, that familial PD is caused by the
accumulation and/or aggregation of misfolded proteins due to dysfunction of the ubiquitin
proteasome system [81-83]. The discovery of the causative genes of familial PD may also
give important clues to elucidating the signaling pathway of cell death in PD [81-85].
NEUROINFLAMMATION
Decades of research on the aetiology of PD have resulted in much information, but little
has been gained in establishing the events causing the initiation and progression of the
disease. Recently, the involvement of inflammation and microglial activation in the
pathogenesis of PD has been emphasized [86,87]. The brain had been considered an immune
privileged site, i.e., one free from immune reactions, since it is protected by the blood-brainbarrier. However, accumulating findings have revealed that immune responses may occur in
the brain, especially due to activation of the microglia, cells which are known to produce proinflammatory cytokines. This inflammatory process is now thought to be fundamental to, if
not at first the initiator of, the progression of PD pathogenesis. Not only DA neurons but also
other non-DA neurons may be affected by this process, dysfunction of which may negatively
impact on DA and non-DA pathways in PD patients. Results of neurotoxin models of PD,
59
corroborating findings obtained in transgenic animal models and epidemiological studies,

strongly support the hypothesis that this neurodegenerative disease is not purely neuronal, as
it has been previously considered [88,89]. Thus, DAergic neuronal degeneration is the likely
result of multiple pathogenic factors occurring both within and outside the cell. The crosstalk between neurons and glia is becoming more and more important for the understanding of
brain pathophysiology. This new finding, unfortunately, does not allow us to diagnose the
disease any earlier because the neuroinflammatory process is silent and unnoticed due to the
absence of pain fibres in the brain, but it at least gives a glint of hope for new potential
therapeutic targets for the slowing of neuronal degeneration.
Neuroinflammation is not a distinctive characteristic of PD but it has been clearly
revealed in a broad spectrum of neurodegenerative diseases that share with it a common
pathological process, such as Alzheimers disease (AD), Huntingtons disease (HD),
amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) [87,90]. The scenario is still
obscure, but inflammation in PD is not any longer considered a non-specific consequence of
neuronal degeneration as it was originally thought to be. Indeed, neuroinflammation may
aggravate the course of the disease and, as it has recently been suggested, may be a primary
factor in some cases of PD [16,87,88,91]. Indeed, postmortem examinations have shown that
neuronal degeneration in PD is associated with massive gliosis due to a subset of activated
glial cells, the microglia [56,92-94], evidence that has been confirmed in MPTP-induced
parkisonism in monkeys and humans [79]. Interestingly, the SN, usually prone to the
deleterious effects of oxidant stress, containing DA neurons high in iron and low in
glutathione [16], is also one of the brain regions more sensitive to inflammation. Indeed,
healthy SN exhibits the highest concentration of microglia in the brain especially in the
ventral tier of the pars compacta [59,60]. Normally, very few microglial cells are detected in
the vicinity of DAergic neurons, and when present, they appear to be resting with fine, long
processes. Neuronal damage, aggregated proteins with abnormal conformations present in
Lewy bodies and other unknown factors increase the number and change the shape of glial
cells, to such an extent that they can be found in proximity to DAergic cells with short
cellular processes. Activated microglia are recruited to the SNc from various structures and
finally stuck to DA neurons. It has been shown that glial cells once activated become
phagocytes that ingest degenerating DA neurons piece-by-piece. This occurs early in
neuronal degeneration, starting at the extending fibres, such as the neurite which extend into
the SN reticulata [95]. Hence, activated glial cells release detrimental compounds such as, IL1, IL-6, TNF- and interferon (IFN-), which may act by stimulating inducible nitric oxide
synthase (iNOS), or which may exert a more direct deleterious effect on DAergic neurons by
activating receptors that contain intracytoplasmic death domains involved in apoptosis
[56,96-103]. Microglia can also induce neuritic beading [104] or synaptic stripping along
dendrites [105] leading to synaptic disconnection and loss of trophic support and cell death
[106]. Animal studies using MPTP have shown that the immune reaction might evolve,
ultimately leading to the infiltration of lymphocytic CD4+ and CD8+ T cells into the injured
SN and striatum, given that glial cells are potent activators in lymphocyte invasion.
Moreover, activated lymphocytes present in the SN could start an immune-mediated
inflammation [103,107].
60
Nevertheless, such activation of microglia is not only disadvantageous to neurons.

Indeed, some investigations indicate that activated microglial cells and macrophages tend to
synthesise and produce neurotrophic factors (brain-derived neurotrophic factor, BDNF and
glia-derived neurotrophic factor, GDNF) through certain compensatory mechanisms
following neuronal injury and induce sprouting surrounding the wound in the striatal DA
terminals [90,108]. Moreover, activated glia play a role in gradually removing the dead DA
neurons as a defence mechanism, although some healthy DA neurons might be also
phagocytosed during the process [17,109]. Therefore, inflammation has been rightly defined
as a double-edged sword. It normally starts as a defence reaction but, for the failure of its
control mechanism, can lead to an uncontrolled and continuous extremely damaging immune
response. A brief pathogenic insult, furthermore, can induce an ongoing inflammatory
response and the toxic substances released by the glial cells may be involved in the
propagation and perpetuation of neuronal degeneration. This theory is plausible, corroborated
by the evidence that several years after exposure to MPTP, increased levels of factors such
as, IL-1, IL-6 and TNF- have been found in the basal ganglia and cerebral spinal fluid
(CSF) of patients with toxin-induced PD [86].
So far, among the plethora of toxic factors released by the reactive glia it is not clear
which one of them is responsible for the DAergic neuronal death. Reactive oxygen species
(ROS), .OH, NO and its peroxinitrite (ONOO-), are the likely candidates. From this evidence
it appears clear that inflammatory process and oxidative stress derived from DA metabolism,
constitute a vicious cycle that lead to the final demise of nigral DA cells [88]. Furthermore,
experimental evidence has also shown that inflammatory loss of DA nigro-striatal neurons
might be mediated by apoptosis [109-113]. Indeed, inflammation induced by intranigral
injection of LPS could be mediated, at least in part, by the mitogen-activated protein kinase
p38 (MAPK p38) signal pathway leading to activation of iNOS and cysteine protease
caspase-11 [110]. Consistent with this evidence, it has recently been shown that LPS-induced
inflammation causes apoptosis in the SNc due to increased pro-inflammatory cytokine levels
of mRNA for TNF-, IL-1, IL-1 and IL-6, and the apoptosis-related genes Fas and Bax and
caspase-3 immunoreactivity [109]. These data have been confirmed also in a MPTP mouse
model, neurotoxic effect seems to be mediated via activation of the caspase-11 cascade and
inflammatory cascade, as well as the mitochondrial apoptotic cascade [111].
The link between inflammation and apoptotic signalling cascade could follow other
pathways. In a chronic MPTP model of PD, activation of the nuclear transcription factor
nuclear factor-B (NF-B), that is well-known for its role in preventing apoptotic cell death,
has been revealed [114], this in turn, promotes the synthesis of cyclooxygenase types 2
(COX-2) [115]. COX-2 induction increases inflammatory response with ROS formation by
the arachidonic acid (AA) cascade, thus triggering a vicious circle. The release of AA also
inhibits glutamate (GLU) uptake contributing to the neurodegenerative processes seen in PD
[116]. In addition, COX-2 could also be induced by pro-inflammatory cytokines such as TNF
via the c-Jun N-terminal kinase (JNK) pathway [56,117,118].
The above discussion makes it plausible that drugs with the capacity to rescue DA
neurons from microglia toxicity and inflammatory processes may result in an amelioration of
parkisonian symptoms by delaying the onset and slowing the progression of the disease
[91,119,120]. Several agents have been shown to inhibit microglial or monocytic cell
61
neurotoxicity [119,121]. Among them much attention has been devoted to nonsteroidal antiinflammatory drugs (NSAIDs) since it has been shown by experimental and clinical
observation that they may represent a possible new therapeutic approach for treating PD.
Non steroidal anti-inflammatory drugs (NSAIDs) are a heterogeneous group of
compounds which share many pharmacological properties (and side effects) and are the main
drugs used as analgesics and antipyretics to reduce the untoward consequences of
inflammation. NSAIDs are capable of halting eicosanoids synthesis and suspending
inflammatory process progression. NSAIDs inhibit COX activity inducing a diminution of
PGs levels, accompanied by a compensatory increase in levels of leucotriens (LTs). Although
many of the NSAIDs pharmacological actions are related to the ability to inhibit
prostaglandin (PG) biosynthesis, some of their beneficial therapeutic effects are not
completely understood. NSAIDs are able to inactivate the transcription factors NF-B and
factor activator protein 1 (AP-1) which is critical for the induction of neoplastic
transformation and the induction of multiple genes involved in inflammation and infection
[122-128]. Diverse noxious cellular stimuli free NF-B from any endogenous inhibitor,
permitting the translocation of free NF-B from the cytoplasm to the nucleus. Consequently,
NF-B binds to DNA and activates a number of genes involved in the inflammatory and
immune responses. Some of these gene products, such as TNF could exert cytotoxic effects
by switching on apoptotic self-destruct programs [129,130]. Furthermore, aspirin and
salicylate at therapeutic concentrations inhibit COX-2 protein expression pointing towards a
possible (cell-specific) target of NSAIDs upstream to COX-2 enzyme activity through
interference with the binding of CCAAT/enhancer binding protein beta (C/EBPbeta) to its
cognate site on COX-2 promoter/enhancer. Expression of other genes, such as iNOS and IL4, may be inhibited by NSAIDs through a C/EBP-dependent mechanism or inhibiting NF-B
activation [131, 132].
In addition, it has been shown that NSAIDs in neuronal cells, might directly and dosedependently scavenge ROS and reactive nitrogen species (RNS) blocking their detrimental
effects [123,133]. Moreover, NSAIDs can reduce NO brain levels inhibiting its production
through multiple mechanisms (i.e., inhibition of iNOS activity and/or expression, inhibition
of NF-B production) [122,133-135]. Interestingly, we have recently shown that pretreatment with aspirin as well as 7-nitroindazole, a NOS inhibitor, blocks the toxic effect of
MPP+ almost completely in a rat model of PD [136,137], confirming a possible direct effect
of NSAIDs on NOS enzymes. Furthermore, the agonistic activity shown at high
concentration by some NSAIDs such as ibuprofen and indomethacin toward the peroxisome
proliferator-activated receptor- (PPAR) seems relevant to neuroprotection [138]. This
receptor PPAR is a ligand-activated inhibitory transcription factor that antagonizes the
activity of NF-B, AP-1, signal transducer and activator of transcription 1 (STAT-1) and
nuclear factor of activated T cells (NFAT) [139,140]. Its cellular activation is associated with
a reduction in the expression of several inflammatory genes [141] and the production of
inflammatory cytokines (i.e., IL-1, IL-6, TNF) [140]. In vitro studies have shown that the
selective agonists pioglitazone, indomethacin and ibuprofen can activate PPAR in microglia,
reducing the A-mediated secretion of inflammatory cytokines and neurotoxicity, decreasing
the number of activated microglia and reactive astrocytes [142,143]. NSAIDs treatment
reduces the expression of the proinflammatory enzymes COX-2, iNOS and beta-secretase-1
62
(BACE1) mRNA and protein levels [143]. In addition, PPAR depletion potentiates betasecretase mRNA levels by increasing BACE1 gene promoter activity. Conversely,
overexpression of PPAR, as well as NSAIDs and PPAR activators, reduced BACE1 gene
promoter activity. These recent results suggest that PPAR could be a repressor of BACE1
binding to a PPRE located in the BACE1 gene promoter [144]. These effects may explain the
overexpression of BACE1 in the brain under inflammatory conditions and emphasize the
hypothesis that neuroinflammatory mechanisms significantly contribute to the pathogenesis
of PD. This could be a potential mechanism by which NSAIDs have a protective effect
against the development of PD.
Several studies have been carried out in which the effects of NSAIDs have been tested on
animal (mouse and rat) models of PD and cell cultures and almost all of them have shown a
neuroprotective effect for this pharmacological class of drug. Differently, conflicting results
have been obtained about the mechanism through which they act. Further experiments will
clarify the role of the classical versus non classical effects that still remains controversial.
Their broad sites of action and pharmacological effects (from anticancer to antipyretic) might
be the basis on which their efficacy in neurodegnerative disease is founded.
The first to propose a non classical mechanism for aspirin and its metabolite salicylic
acid in their protective effect against GLU-neurotoxicity were Grilli and co-workers [123].
The common molecular target for aspirin and salicylic was identified as COX-independent
and involved specific inhibition of GLU-mediated induction of NF-B, suggesting, for the
first time, a link between neuroprotection and the nuclear event [123]. Moreover, aspirin, its
soluble lysine salt and salicylic acid have been shown to have neuroprotective effects in the
MPTP mouse model of PD probably not due to COX inhibition, conversely to ROS
scavenging activity [145-147]. Salicylic acid demonstrates a clear antioxidant action blocking
toxin-induced glutathione (GSH) and DA depletion acting as an .OH scavenger in the brain.
The neuroprotective effects of salicylic acid dont seem to be linked to the possible blockade
on the production of MPP+ from MPTP, being as the MAO-B enzyme is not inhibited by this
anti-inflammatory drug [147]. The anti MAO-B effect in the action of salicylic acid and
aspirin might be ruled out completely, if it is taken into consideration that they posses a
protective activity even in the model of PD induced infusing MPP+ directly into the striata of
rats [136,148,149]. In this PD model, pretretment with salicylic acid [148] and aspirin [136]
protect animals against MPP+-induced DA depletion with a significant attenuation of severe
DA depletion (>65%). The failure of celecoxib, diclofenac and meloxicam, selective COX-2
inhibitors, to protect animals against MPP+-induced DA depletion, indicate the absence of the
involvement of PGs in MPP+ action and give further proof of a non classical mechanism for
aspirin and salycilic acid that is mostly dependent on their antioxidant activity [136,148]. We
have confirmed these findings also in vitro, in a human neuroblastoma cell culture line. In
fact, aspirin, but not meloxicam, inhibited cell death induced by treatment with MPP+, in a
dose-dependent manner (unpublished observation). We showed that pretreatment of rats with
aspirin, also protected DA neurons against 6-hydroxydopamine (6-OHDA) toxin as indicated
by electrochemical and TH immunostaining evidence, whereas meloxicam was still devoid of
any activity. The mechanism of action of aspirin seemed to be different in each toxin-model
since it was associated with ROS scavenging activity in the 6-OHDA one, but not in the
MPP+-model that surprisingly did not induce any .OH formation at the concentration used in
63
our study. Results obtained in cultures of embryonic rat mesencephalic neurons treated with
6-OHDA and MPP+ showed that these two neurotoxins act differently in the killing of DA
neurons. Neuronal COX-2 activity and PG production is involved only in the 6-OHDAneurotoxic effect whereas MPP+ toxicity does not require COX involvement [150]. This
evidence comes from experiments carried out with ibuprofen, a non selective COX inhibitor,
SC-560 a COX-1 selective inhibitor and two selective COX-2 inhibitors, NS-398 and
Cayman 10404, showing that COX-2, but not COX-1, is involved in 6-OHDA toxicity. Since
ibuprofen attenuated both 6-OHDA and MPP+-neurotoxicity, the authors proposed that this
drug has additional COX-independent effects as yet not well identified [150]. Therefore, it is
likely that the protective effect exerted by aspirin, in vivo, may be due to inhibition of MPP+
toxicity at the cell level, possibly by blocking NF-kB or caspase activation, thus providing
further evidence that the neuroprotective effect of NSAIDs might be independent from COX2 inhibition. However, other mechanisms, such as .OH scavenging activity, as in the model of
6-OHDA-induced damage, cannot be ruled out [136]. In addition, aspirin and paracetamol
might act on a different molecular target: the mitochondrion. In fact, these NSAIDs prevented
MPP+-induced inhibition of the mitochondrial electron transport chain and complex I activity
and significantly attenuated MPP+-induced superoxide anion generation [151]. The non
classical mechanism seems important also in the effect of indomethacin. This drug protected
SNc DAergic neurons against the MPTP effect in the mouse model of PD and it was
associated with diminished microglial activation and lymphocytic infiltration in the damaged
areas, behaving as a scavenger of ROS. Reduced inflammation by indomethacin might result
in less damage of DAergic neurons. However, microglial and lymphocytes accumulation was
decreased only in association with less neuronal impairment, when indomethacin was given
before MPTP. Indomethacin in a higher dose or given 24 h after intoxication did not decrease
inflammatory reaction. However, indomethacin appeared to be toxic in high doses indicating
that doses of NSAIDs should be considered carefully in clinical trials [107].
Notably, aspirin appears to offer an adjuvant as well as a prophylactic therapy for PD.
Indeed, aspirin given after MPP+ administration, completely blockaded MPP+-induced striatal
DA depletion. Similar treatment with paracetamol resulted instead only in a partial
protection. Aspirin and paracetamol acted mainly as antioxidants, they were also capable of
blocking .OH production and lipid peroxidation in vitro, but in this action aspirin was the
weaker when compared to paracetamol. Thus, aspirins adjuvant as well as prophylactic
effect is only partially mediated by ROS scavenging properties [149].
The experimental evidence reviewed so far suggest that NSAIDs act as neuroprotectants
essentially through a nonclassical mechanism. Against this trend, the role of COX-1 and
COX-2 enzymes was reassessed by Teismann and colleagues [56,152], who proposed the use
of COX-2 inhibitors as a new non-DAergic therapy for PD. Their assumption was based on
the effects of some NSAIDs, in an in vivo MPTP mouse model of PD, an in vitro study from
MPTP-treated mice and post-mortem PD samples. These drugs, at higher dosages, showed an
almost complete protection against MPTP toxicity. COX-2 isoenzyme is up-regulated in the
SNc DAergic neurons in both animal and human samples, COX-2-mediated
neurodegeneration might be correlated to its catalytic activity through the production of PGs
and maybe also to the oxidation of catechols such as DA [57]. Aspirin, salicylic acid, and
meloxicam valdecoxib pretreatment attenuate the reduction of TH immunereactivity of the
64
SNc and the MPTP-induced decrease in locomotor activity [147,152,153]. Treatment with
rofecoxib, before and after MPTP-injection, blocked the increase of PGE2 in the midbrain,
doubled the number of the surviving TH-positive neurons, and prevented the rise in protein
cysteinyldopamine, an index of DA quinones production [56]. Rofecoxib either alone or in
combination with creatine, that facilitates metabolic channelling and shows antiapoptotic
properties [154] protected against striatal DA depletions and loss of SN TH-immunoreactive
neurons. Administration of rofecoxib with creatine produced significant additive
neuroprotective effects against DA depletions in MPTP model of PD in mice. These results
suggest that a combination of a COX-2 inhibitor with creatine might be a useful
neuroprotective strategy for PD [155]. It is also worth noting that in the MPTP model of PD
in mice, rofecoxib has no neuroprotective effect when it is given after MPTP intoxication,
even for a long period, revealing that the time of COX-2 inhibition is critical to achieve a
protective effect. Consequently, COX-2 activity, PGs production and oxygen species
formation might not play a detrimental role in neuronal cells death, at least when the injury
process has started already. Nonetheless, the inhibition of COX-2 activity could be harmful to
neurons injured by MPTP. Indeed, the authors showed that, in later stages of injury, COX-2,
through the formation of cyclopentenone PGs derived from PG D2 (PGD2), may participate
in the resolution of inflammation and even in the regeneration process [94]. Accordingly,
neither pharmacological nor genetic abrogation of COX-2 activity mitigates inflammatory
processes [56].
Snchez-Pernaute and colleagues [156], in a 6-OHDA rat model of PD, showed that
selective inhibition of COX-2 by treatment (pre and post lesion) with celocoxib is protective
against the neurotoxin effect. The authors evaluated celocoxib effects using micro PET and
immunohistochemical techniques, and observed a decrease in microglial activation in the
striatum and ventral midbrain associated with a prevention of the progressive degeneration
seen in the intrastriatal 6-OHDA retrograde lesioned rats treated with the vehicle. The benefit
of COX-2 activity inhibition might be attributed to a selective decrease of the harmful glial
cells and to the no effect on the protective astroglia. Celocoxibs rescue of DA toxin-insulted
neurons from death could be mediated by both neuronal and glial COX-2, but in any case the
effect obtained by this drug is to create favourable conditions for the prevention of
progressive neurodegenerative cascades during and after neuronal injury similar to that seen
in PD [156].
Despite the evidence of inflammation in the brains of patients with PD, and in animal
models of PD, NSAIDs have not yet been formally tested in PD. Hitherto, only few
epidemiological studies have been carried out analyzing the association between regular use
of NSAIDs and the risk of PD with conflicting results.
The first piece of evidence was provided by Chen et al. [157] who investigated
prospectively the potential benefit in humans of the use of NSAIDs in reducing the risk of
PD. These researchers found that regular users of these drugs had a lower risk of PD than
non-users. The risk of developing PD was 45% lower among regular users of non-aspirin
NSAIDs compared to non-users. A similar decrease in risk was also found among
participants who took two or more tablets of aspirin per day compared to non-users.
Additionally, increasing benefits were observed with longer duration of use of non-aspirin
NSAIDs [157]. It is worth noting, that the Chen study may underestimate the protective effect
65
of NSAIDs, since PD is much more common in people over 75 years old, an age group not
included in the Chen teams data. Therefore, benefits of even greater magnitude might be
demonstrable if this intervention were applied to the same population as it aged beyond 75
years. Chen and coinvestigators continued examining the relationship between NSAIDs use
and risk of PD this time, utilizing another large cohort. Ibuprofen was associated with 35%
lower risk of PD. In contrast to the previous study, no significant associations were found for
aspirin, other NSAIDs or paracetamol [158]. These discrepancies might be simply explained
by the fact that considerably more people in the cohort used ibuprofen than other
medications. However, the authors also did not exclude that there may be an ibuprofenspecific effect against PD, related to its unique molecule. Recently, a case-control study on
subjects with no history of PD or parkinsonism-related drug use at baseline reported a
surprising finding: non-aspirin NSAIDs use reduces PD risk in men but not in women. Use of
non-aspirin NSAIDs was associated with a 20% reduction in the incidence of PD among
men, and a 20% increase in the incidence of PD among women [159]. Less promising
insights have been provided by a population-based, case-control study. Consistent with the
previous epidemiological studies [157-159], Bower and colleagues found that cases of PD
used NSAIDs (excluding aspirin) less frequently than controls, however, the difference did
not reach significance. This trend was similar for both NSAIDs and steroidal agents
considered separately. The use of aspirin was not significantly associated with PD as shown
previously [158,159]. These investigators also showed a significant association between preexisting immune-mediated diseases and the later development of PD. The association was
stronger for women and for earlier onset of PD cases, but neither of these differences reached
significance. These results support the hypothesis that there is an inflammatory component in
the pathogenesis of PD and provide a rationale for the use of NSAIDs as neuroprotectants
capable of delaying onset or slowing progression of the disease [160]. Since patients with
diseases of immediate-type hypersensitivity are genetically predisposed to initiate a humoral
response to low levels of antigens, they might also be predisposed to initiate
neuroinflammatory responses as well and play a role in the aetiology of PD [161].
The latest available data on the subject is a population-based case-control study in which
the investigators did not observe a significant association between PD and NSAIDs in
reducing the risk of PD. These results provide only limited support for the hypothesis that use
of aspirin may reduce the risk of this disease, but this association was statistically imprecise
and no clear trend according to the number of aspirin prescriptions was observed. In addition,
no indication of any protection from other NSAIDs such as ibuprofen was revealed [162].
These findings offer, at most, a limited support for the hypothesis of neuroprotection
from aspirin, and no indication of protection from other NSAIDs. Larger studies that include
medication records and over-the-counter medication use will clarify these associations.
Nevertheless, these unclear indications must be clarified and corroborated by clinical trial
before any firm conclusions can be drawn. Furthermore, the role of selective COX-2
inhibitors might be investigated since only the effect of traditional NSAIDs has been
analysed by epidemiological studies. Indeed, selective COX-2 inhibitors have not been in use
long enough for epidemiological data to be collected.
66
HOW THE DAERGIC NEURONS DIE

Two major mechanisms of neuronal demise have been discussed in neurodegeneration:
apoptosis and (oncotic) necrosis. These cell death types are different, frequently divergent,
but sometimes overlapping cascades of cellular breakdown. The modulation of these cascades
by cellular available energy, may cause the cells to use diverging execution pathways of
demise [163]. Apoptosis, a specific form of gene-directed programmed cell death (pcd)
brings about the removal of unnecessary, aged or damaged cells and is distinguished by
distinct morphological and biochemical features. It is performed by an intrinsic suicidal
machinery of the cell and can be set off by environmental stimuli including irradiation
leading to DNA damage, oxidative stress, toxins, viruses, withdrawal of neurotrophic
support, etc. [164]. Although pcd has often been likened to apoptosis, it is becoming evident
that nonapoptotic forms of pcd also exist: for example, the developmental cell deaths,
"autophagic" cell death and "cytoplasmic" cell death, bear no resemblance to apoptosis [165].
Neuronal cell death (neuroapoptosis) has been widely studied in the developing brain under
natural neurogenesis as well as in the adult brain under pathological conditions [166,167]. It
is well known that adult mature neurons die at a low rate during the normal aging process but
at an accelerated pace in cases of neurodegenerative disorders, like PD. Some groups have
reported that dying neurons displaying the morphological features of apoptosis are present in
the post mortem human brain of patients with neurodegenerative disorders. These features
include cell shrinkage, chromatic condensation, DNA fragmentation, and increased
expression of both proapototic (c-Jun, c-Fax, Bax, p53, APO-1/Fas-CD95, Fas, Fas-L,
caspase 8 and 9, activated caspase 3, IF-, and NF-B), and antiapoptotic proteins (Bcl-2,
Bcl-x), or DNA repair enzymes, such as Ref-1 and the co-expressed GADD45 [168-171].
However, other groups have observed little or no evidence of apoptotic neuronal cell death
associated with neurodegenerative diseases [172-175].
The current view about the apoptotic mechanism underlying nigrostriatal DA neuron
degeneration in PD is quite mixed. Recent evidence in experimental models of PD, point to
the fact that neuroapoptosis might quite possibly be an early pathological event, and may or
may not be present at the end of disease stage, when postmortem samples are collected and
analyzed [17]. Experimental models of PD have provided strong evidence of a role for
apoptosis in SNc cell death, since systemic administration of the neurotoxin MPTP produces
DNA fragmentation with induction of caspase-3 activity [176,177], while inhibitors of the
downstream cellular substrate of caspase-3, protect against MPTP-mediated neurotoxicity
[177]. MPTP administration in mice increases nigrostriatal activity of both c-10 Jun and cJun NH2-terminal kinase (JNK), members of the stress-induced protein kinase (SAPK)
pathway, which is attenuated by a JNKspecific inhibitor also reducing DAergic cell loss in
the SN [178]. In another animal model for PD, intracerebral injection of 6-OHDA causes
both apoptotic and necrotic cell death of DAergic SN neurons [179,180]. In DAergic cell
cultures, however, 6-OHDA mediates apoptosis via activation of caspases but, in contrast,
not all DAergic neurotoxins (e.g. MPP+) appear always to induce apoptosis [181].
Expression of Bcl-2 and Bax can prevent the toxin induced apoptosis, suggesting that cl-2
related proteins may show a specific interaction with a distinct partner protein or cell-death
pathway determining its role as a positive or negative modulator of cell death [182]. In the
67
last 5 years or so, the understanding of the pathophysiology of animal models of

neurodegenerative diseases has progressed admirably, much more than that of the diseases
themselves. However, the lack of clinical efficacy of two apoptosis inhibitors targeting
different elements of the apoptotic pathway function that is thought to be involved in the
death of DA neurons in PD, raises serious concerns as to the suitability of currently available
models, e.g., the classical 6-OHDA and MPTP models of PD as a basis for the preclinical
evaluation and prioritization of apoptosis inhibitors designed to slow or halt progression of
PD based on novel cellular mechanisms and in vitro cellular activity [183].
THE SNC DAERGIC NEURONS RESURRECTION

Recently, another dogma of science has been disproved; the adult mammalian brain does
have the potential to generate new neurons and to integrate them into existing circuits.
Neurogenesis has been shown at least in two rather discrete areas of the brain, the dentate
gyrus of the hippocampal formation and the subventricular zone (SVZ) and its projection
through the rostral migratory stream to the olfactory bulb, where they become interneurons
[184]. Neuroblasts born in the adult subgranular zone (SGZ) of the dentate gyrus migrate into
the adjacent granular layer, where they become granular neurons. The constitutive
neurogenesis that occurs in the SVZ and SGZ is thought to be of functional importance in
olfaction, mood regulation and memory processes [185-188]. Low levels of neurogenesis also
occur in various other regions of the adult CNS including the SNc [189,190], a finding,
which may have profound implications for the treatment of PD. Whether DAergic
neurogenesis occurs in the adult substantia nigra in normal brain or in PD animal models is
still a matter of debate. The existence of endogenous neurogenesis in the striatum and the
subventricular zone in PD would open possibilities for a new cell-based approach to the
treatment of neurodegeneration in PD patients, bypassing the need for transplantation.
Unfortunatrly, initial enthusiasm for the usefulness of persuing cell-based approaches was
dampened by the failure of the transplantation of human fetal mesencephalic tissue from
aborted fetuses, rich in primary DAergic neurons, in the putamen or caudate nucleus of PD
patients [191] and turned out to be less effective than deep brain stimulation [192,193].
However, there is some evidence supporting neurogenesis or a more general degree of
plasticity in the brains of PD patients and of PD animal models [194]. For example, a huge
increase in astrocytes and glial cells was associated with neuronal death in neurodegenerative
diseases as well as in cases of induced brain insult [13,195]. Additionally, a restoration of DA
to nearly normal levels in the striatum was observed a month post lesion in an MPTP-model
of PD [196], this was due essentially to a collateral sprouting from uninjured DAergic
neurons. Moreover, a marked increase in the number of TH-positive cells in the striatum of
PD patients, has been reported [197]. This evidence has been corroborated by some results in
rats and mice and also in monkeys, where nigrostriatal MPTP and 6-OHDA toxins induced
degeneration caused a clear augmentation of TH-immunoreactive cells in the dorsal striatum
[198-200]. It is plausable that both TH-positive and astroglial cells are either newly generated
from precursor cells in the striatum or migrate from the SVZ. Indeed, a recent study in
macaques revealed a topographically organized projection from the SNc to the SVZ [201]
68
and a significant decrease in the number of proliferating cell nuclear antigen (PCNA)+ cells
and polysialylated (PSA) form of neural cell adhesion molecule (PSA-NCAM)+ neuroblasts
in the SVZ after MPTP lesioning. Importantly, close contact between DAergic fibers and
epidermal growth factor receptor (EGFR)+ cells is conserved in the SVZ in humans [202].
On the one hand, it has been postulated that DAergic differentiation occurs at a very low
level in the SN of healthy mice and that this process increases after MPTP lesioning [190].
Basal levels of neurogenesis, increased proliferation and DAergic differentiation after MPTP
administration were also demonstrated in nestin-LacZ transgenic mice [203], although the
levels of DAergic differentiation were very low. On the other hand, recently Frielingsdorf
and colleagues did not find any evidence of new dopaminergic neurons in the substantia
nigra, either in normal or 6-OHDA-lesioned hemi-parkinsonian rodents, even after growth
factor treatment. Also, they found no evidence of neural stem cells emanating from the
cerebroventricular system and migrating to the substantia nigra [204]. Moreover, results from
several laboratories demonstrated that 6-OHDA lesioning in rats or MPTP lesioning in mice
resulted in cell proliferation in the SN without DAergic differentiation [189,205]. So, even if
DAergic neurogenesis takes place in the substantia nigra, it will only become therapeutically
relevant if the levels can be boosted considerably. To this end it might be possible to generate
DAergic neurons in the striatum either by recruitment of endogenous progenitors from the
SVZ or stimulation of resident cells in the striatum. After which, since the SN harbors
proliferating cells, it may be feasible to stimulate differentiation into DAergic neurons, but,
restoration of nigrostriatal projections may be a major challenge here. To date the optimal
approach for endogenous stem cell therapy remains unknown. Perhaps, stimulation of cell
proliferation or induction of DAergic differentiation may be mediated by viral vectormediated local overexpression of either growth or transcription factors or by pharmacological
intervention. Alternatively, alteration of the local microenvironment by overexpression of
growth factors may increase cell survival and help in DAergic differentiation. Since it is
difficult to predict if intrinsic and extrinsic signals or a combination of both will be
necessary, careful investigations in animal models of PD are required to shed light on the
possibility of combining cell therapy with gene or pharmacotherapy to induce DAergic
differentiation. In animals, it has been shown that SVZ progenitors can be recruited to the
striatum after the administration of several growth factors (e.g. Transforming Growth Factor
(TGF), Brain Derived Neurotrophic factor (BDNF)) [206,207]. Although this study
suggests that recovery could be stimulated by DAergic differentiation from endogenous
precursor cells, it should be noted that no clear correlation between newly-born DAergic
neurons and functional recovery was demonstrated. Also, platelet derived growth factor
(PDGF) and BDNF were also able to recruit new cells to the striatum and the SNc in 6OHDA lesioned rats [208]. These initial studies have thus demonstrated that cell recruitment
to the striatum is possible in animal models of PD. However, the next challenge lies in
stimulating the proliferated cells to differentiate into dopamine-secreting cells and
demonstrating a clear correlation between DAergic differentiation and functional recovery. It
will be of great interest to study the role of these differentiation factors with or without
additional growth factors after DAergic denervation in animal models of PD.
69
EXPERT COMMENTARY AND FIVE-YEARS VIEW

From the large amount of literature here reviewed it appears evident that the progress in
understanding the neuropathological process that induce DAergic cell demise in PD has been
impressive. However, despite these advances, the processes that initiate cell death remain
unclear. Whether they involve energy metabolism deficiencies, inadequate control of the
redox state, low amounts of neurotrophic support and/or the action of environmental and
endogenous toxins, remains to be elucidated. Clearly, a better understanding of the DAergic
cell biology, the mode of cell death in PD, and the molecular mechanisms that controls it, is
required. Indeed, DAergic neurons are sui generis cells, involved in a large number of
physiological and pathological conditions and are also very delicate. SNc DAergic cells for
some unknown reason are prone to degenerate and they are very sensitive to oxidative stress
and inflammation. A better comprehension of the difference in resistance among DAergic
cells of the mesencenphalic region will bring further insight into their peculiar characteristics.
Recently, neuroinflammation, a processes orchestrated and sustained by activated
resident microglia cells, has been suggested as a possible cause of the demise of nigral DA
cells, perpetuating the neurodegenerative phenomenon. A large body of information on the
molecular and cellular mechanisms whereby inflammation might induce neuronal death has
been generated in the past few years by researchers in the neuroscience community.
Nevertheless, further clarification of the role of inflammation in the pathophysiology of basal
ganglia disorders is required, since the overall picture is still confusing. The situation is
complicated by the fact that inflammation is a double-edged sword and probably starts as a
beneficial defence mechanism that at some point evolves into a destructive and
uncontrollable chronic reaction. Thus, the ideal approach would be to inhibit the deleterious
effects associated with neuroinflammation while preserving the inflammatory pathways that
lead to neuroprotection. From the above discussion it seems clear that drugs inhibiting
inflammation and microglial activation might be an important feature of the treatment of PD
and also the dementia, often associated with the disease [89,91,119,120,209]. Consequently,
a rational use of NSAIDs could be useful as therapeutic intervention in PD and in other major
neurological diseases with a similar etiopathology, such as AD, ASL and MS. Nonetheless,
despite the fact that experimental and epidemiological evidence has been provided for future
use of anti-inflammation agents, they have not been rigorously corroborated in trial studies
for the treatment of motor disorders as yet. Furthermore, most of the data have yielded
contradictory results. Indeed, it is quite possible that NSAIDs are ineffective once the
pathological process has started, the pharmacological intervention should start very early in
the pre-symptomatic period, according to some experimental a epidemiological evidence
[97,157-159]. Due to the complexity of the disease, it is possible that combination therapy
with concomitant use of agents with nonoverlapping or even synergistic mechanisms of
action, may represent the best means available to enhance treatment effectiveness. Some
results could be achieved, therefore, by combining NSAIDs with other rescue agents, such as
MAO inhibitors (rasagiline, safinamide); mitochondrial function enhancers (coenzyme Q10,
creatine); antiapoptotic agents; protein aggregation inhibitors and neurotrophic factors [210].
Although this hypothesis is worthy of consideration, it remains largely undocumented and
certainly deserves further discussion. Furthermore, NSAIDs might be a beneficial adjuvant to
70
L-DOPA therapy counteracting the toxicity induced by its long-term use, through antiinflammatory action and the reduction of DA quinones generated by L-DOPA therapy itself
[57].
There are also many avenues that remain unexplored, so there are undoubtedly further
advances to be made. In the next few years, we believe that novel approaches [211,212] will
support the current dopamine-replacement therapy for PD. Furthermore, early diagnosis,
early symptomatic treatment and particularly the introduction of neuroprotective therapies
will improve PD pharmacological management. Indeed, disease modification remains the
most important goal in PD. Consequently, compounds inhibiting neuroinflammation and
apoptosis represent an important starting point that could lead us to the identification for the
first time of disease-modifying agents for this devastating disease. These will be supported by
cell-replacing therapy i.e. cell transplantation and endogenous neurogenesis. The latter stem
cell therapy for PD offers several potential advantages. In fact, immunological reactions are
circumvented and ethical issues surrounding the use of embryonic stem cells are avoided.
However, many challenges still need to be overcome before this strategy can be brought into
the clinic.
Hitherto, Grannys advice to us, to modify our life style, by increasing our level of
physical exercise, changing to a varied fresh food low calorie diet and augmenting our dietary
intake of natural antioxidants still remains the best advice to reduce the risk of developing
PD.
ACKNOWLEDGEMENTS
This work was supported in part by the Ateneo di Palermo research founding, project
ORPA068JJ5, coordinator: Di Giovanni G. We want to thank Ms. Samantha Austen for the
English revision of the manuscript.
DISCLOSURE SECTION
The authors declare they have no competing interests.
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ISBN: 978-1-60021-820-0
Chapter IV
ELECTROPHYSIOLOGICAL AND
NEUROCHEMICAL IN VIVO STUDIES ON
SEROTONIN 5-HT2C CONTROL OF CENTRAL
DOPAMINERGIC FUNCTION
Vincenzo Di Matteo1,, Giuseppe Di Giovanni1,2, Massimo Pierucci1,
and Ennio Esposito1
1
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030
Santa Maria Imbaro (CH), Italy;
2
ABSTRACT
Central serotonergic, and dopaminergic systems play a critical role in the regulation
of normal and abnormal behaviours. Recent evidence suggests that dysfunction of
dopamine (DA) and serotonin (5-HT) neurotransmitter systems contribute to various
mental disorders including depression and schizophrenia. This chapter was undertaken to
summarize our and other works that have extensively explored the role of 5-HT2C
receptors in the control of DA systems both in basal and drug-induced conditions, using
in vivo electrophysiological and microdialytic techniques. The physiology,
pharmacology and anatomical distribution of the 5-HT2C receptors in the CNS will be
firstly reviewed. Moreover, experimental data regarding the effect of 5-HT2C selective
agents on the neuronal activity of DA neurons of the ventral tegmental area (VTA) and
substantia nigra pars compacta (SNc) as well as the changes of basal DA release in the
striatum and nucleus accumbens are discussed. Finally, the potential use of 5-HT2C
Correspondence concerning this article should be addressed to Dr. Vincenzo Di Matteo, Istituto di Ricerche
Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy,
Telephone: (+39) 0872-5701; telefax: (+39) 0872-570416; e-mail: vdimatteo@negrisud.it.
88
Vincenzo Di Matteo, Giuseppe Di Giovanni, Massimo Pierucci et al.

agents in the treatment of depression, schizophrenia, Parkinson's disease and drug abuse
will be also discussed.
Keywords: 5-HT2C receptors, Dopaminergic function, Mesocorticolimbic system,

Nigrostriatal system, Antidepressants, Antipsychotics, Drug addiction.
INTRODUCTION
There is now an extensive scientific literature regarding the functional interaction
between serotonin (5-HT) and dopamine (DA)-containing neurons in the brain. In recent
years, research on this matter has been spurred by new acquisition of important insights on
the molecular biology of 5-HT receptor subtypes and by the availability of 5-HT receptor
knockout mice [1,2].
Central serotonergic and dopaminergic systems play an important role in regulating
normal and abnormal behaviors [3-5]. Moreover, dysfunctions of 5-HT and DA
neurotransmission are involved in the pathophysiology of various neuropsychiatric disorders
including schizophrenia, depression and drug abuse [3-6]. Thus, the development of a
number of relatively selective pharmacological agents with agonist or antagonist activity at 5HT2C receptor subtype, has allowed investigators to better understand the functional role of
this receptor in the control of central DA-ergic function, as it widely contributes to the
serotonergic regulation of a number of behavioral and physiological processes involving both
limbic and striatal DA pathways [7-10]. Therefore, the physiology, pharmacology and
anatomical distribution of the 5-HT2C receptors in the CNS, as well as experimental data
regarding the effect of 5-HT2C selective agents on the neuronal activity of DA neurons of the
ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) and the changes of
basal DA release in the striatum and nucleus accumbens will be reviewed in this chapter,
which will be introduced by a brief description of the functional neuroanatomy of
dopaminergic and serotonergic systems. Finally, the potential use of 5-HT2C agents in the
treatment of depression, schizophrenia, Parkinson's disease, and drug abuse will be also
examined. Inasmuch as it is the most prominent receptor by which the serotonergic system
affects both mesolimbic and nigrostriatal DA function, and it is consequently involved in the
regulation of a number of behavioral and physiological processes.
DOPAMINE SYSTEMS
Dopamine-containing neurons of the ventral mesencephalon have been designated as A8,
A9 and A10 cell groups: these neurons can be collectively designated as the
mesotelencephalic DA system [11]. Historically, the mesolimbic DA system was defined as
originating in the A10 cells of the ventral tegmental area (VTA) and projecting to structures
closely associated with the limbic system. This system was considered to be separated from
the nigrostriatal DA system, wich originates from the more lateral substantia nigra (A9 cell
group) [11-15].
5HT2C Control of DA Function
89
The mesolimbic and mesocortical DA system appear critically involved in modulation of

the functions subserved by cortical and limbic regions such as motivation, emotional control
as well as cognition [16]. Substantial evidence indicates that the mesolimbic pathway,
particularly the DA cells innervating accumbal areas, is implicated in the reward value of
both natural and drug reinforcers, such as sexual behavior or psychostimulants, respectively
[5,17]. Furthermore, animal studies have shown that lesion of DA terminals in the nucleus
accumbens induces hypo-exploration, enhanced latency in the initiation of motor responses,
disturbances in organizing complex behaviors and inability to switch from one to another
behavioral activity [16]. Hence the mesolimbic DA system seems important for acquisition
and regulation of goal-directed behaviors, established and maintained by natural or drug
reinforcers [16,18].
The medial prefrontal cortex is generally associated with cognitive functions including
working memory, planning and esecution of behavior, inhibitory response control and
maintenance of focused attention [16]. In addition, the mesolimbic DA pathway is sensitive
to a variety of physical and psychological stressors [19]. Indeed, recent studies have indicated
that stress-induced activation of the mesocortical DA neurons may be obligatory for the
behavioral expression of such stimuli [20].
The nigrostriatal DA system, wich originates from the substantia nigra (A9 cell group), is
one of the best studied because of its involvement in the pathogenesis of Parkinsons disease
[21]. In mammals, the substantia nigra (SN) is a heterogeneous structure that includes two
distinct compartments: the substantia nigra pars compacta (SNc) and the substantia nigra pars
reticulata (SNr). The SNc represent the major source of striatal DA and, as already
mentioned, its degeneration causes Parkinsons disease. On the contrary, the SNr mainly
contains GABA-ergic neurons which constitute one of the major efferences of the basal
ganglia [21].
SEROTONIN SYSTEMS
Virtually all parts of the central nervous system receive innervation from serotonergic
fibers arising from cell bodies of the two main subdivisions of the midbrain serotonergic
nuclei, the dorsal (DR) and the median raph (MR) [22-28] (Figure 1). Serotonin-containing
cell bodies of the raph nuclei send projections to dopaminergic cells both in the VTA and
the SN, and to their terminal fields in the nucleus accumbens, prefrontal cortex and striatum
[22-27]. Electron microscopy demonstrates the presence of synaptic contacts of [3H]5-HT
labeled terminals with both dopaminergic and non-dopaminergic dendrites in all subnuclei of
the VTA, and the SN pars compacta and reticulata [14,22,25].
The precise nature of the interaction between 5-HT and DA has been difficult to
elucidate, in that both inhibitory and excitatory roles for 5-HT have been suggested.
However, these discrepancies may be attributable to the differential distribution and to the
diverse functional roles of 5-HT receptors subtypes within the dopaminergic systems [29,30].
Thus, much attention has been devoted to the role of 5-HT2 receptor family in the control of
central DA activity, because of the moderate to dense localization of both transcript and
protein for the 5-HT2A and 5-HT2C receptors in the SN and VTA as well as in DA terminal
90
regions of the rat forebrain [31-34]. It is therefore of interest to briefly review the principal
characteristics of the 5-HT2 receptor family.
Figure 1. Schematic representation of serotonin-dopamine interaction in the mesocorticolimbic and

nigrostriatal DA-ergic system. Serotonin-containing cell bodies of the raph nuclei send projections to
dopaminergic cells both in the ventral tegmental area (VTA, A10) and the substantia nigra (SN, A9),
and to their terminal fields in the nucleus accumbens, prefrontal cortex and striatum.
The 5-HT2 Receptor Family
5-HT2 receptors form a closely related subgroup of G-protein-coupled receptors,

functionally linked to the phosphatidylinositol hydrolysis pathway and currently classified as
5-HT2A, 5-HT2B and 5-HT2C subtypes [29,30,35], based on their close structural homology
and pharmacology [29,30,35]. There is an high sequence homology (> 80% in the
transmembrane regions) between the mouse, rat and human 5-HT2C receptors [29], and it is
not surprising that many compounds bind with high affinity all these three receptor subtypes.
5-HT2C receptors are widely distribuited throughout the brain and have been proposed as the
main mediators of the different actions of 5-HT in the central nervous system [29,30,35].
High levels of 5-HT2C mRNA or protein expression have been found in the choroid plexus,
the frontal cortex, in limbic structures such as hippocampus, septum and hypotalamus, and
also in the striatum, nucleus accumbens, rhombencephalon and spinal cord. The presence of
these receptors has also been demonstrated on DA and non-DA cells in the VTA, SNc and
the SNr [21,36-39]. The regional and cellular distribution of 5-HT2C receptors was also
investigated in the human brain. The main sites of mRNA 5-HT2C receptors or protein
expression were the choroid plexus, cerebral cortex, hippocampus, amygdala, some
91
components of the basal ganglia and other limbic structures [32,40], suggesting that this
receptor might be involved in the regulation of different human brain function, and might
play a role in the pathophysiology of several mental disorders [7-10,41,42].
There is now evidence that the 5-HT2C receptor is mainly located postsynaptically within
dopaminergic, GABA-ergic, cholinergic, substance P, dynorphin and other systems
[29,43,44]. Interestingly, the studies by Eberle-Wang et al. [43] showed the presence of 5HT2C mRNA within inhibitory GABA-ergic interneurons making direct synaptic contact with
SNc and VTA dopaminergic cell bodies. Other immunohistochemical and
electrophysiological studies demonstrated an important role of 5-HT2C receptors, localized on
non-DA neurons, presumably GABA-ergic, in the regulation of DA cells in the VTA [45,46],
as well as in the SNc [47,48] (Figure 2).
Figure 2. Distribution of 5-HT2A and 5-HT2C receptors on GABA- and DA-containing neurons in the
midbrain. 5-HT2A receptors are expressed on a subpopulation of DA-containing neurons, and on nonDA neurons whose neurochemical identity is as yet unknown (indicated by the question mark). 5-HT2C
receptors are expressed on GABA-containing neurons in both the substantia nigra pars reticulata (SNr)
and the ventral tegmental area (VTA). (The scheme is based on data from references 33,34,43).
Recent studies found a somatodentritic localization of 5-HT2A receptors on DA neurons

in both the parabrachial and paranigral subdivisions of the VTA [33,34], which project
mainly to the prefrontal cortex and nucleus accumbens, respectively. In addition, 5-HT2A
immunoreactivity was also expressed on non-DA cells in the VTA, providing a potential
anatomical basis for the modulation of DA neurons in the VTA either directly, by 5-HT2A
receptors localized on DA cell or indirectly, through receptors present on nonDA
(presumably GABA-ergic) neurons [33,34]. These receptors were also found at high
concentrations in various cortical regions [33,39]. It is likely that 5-HT2A receptors could
affect DA function by acting at the level of dopaminergic nerve terminals, although no direct
evidence for the presence of 5-HT2A receptors on such terminals has been provided so far.
Using sensitive techniques, several groups have also shown the presence of both 5-HT2B
receptor mRNA [49] and protein [50] in the rat brain, including midbrain regions. Although
there are regional differences in the distribution of these receptors, they are all expressed in
92
the brain with extensive pharmacological and functional similarities, so that it is often
difficult to ascribe particular functions to a receptor subtype.
5-HT2C RECEPTORS AND DOPAMINE FUNCTION

Several studies have focused on the role of 5-HT2 receptors in the regulation of forebrain
DA function and highlighted their potential as a target for improved treatments of
neuropsychiatric disorders related to central DA neuron dysfunction [7-10,51-70]. The
involvement of 5-HT2C receptor subtypes in the control of mesocorticolimbic and
nigrostriatal DA neuron activity is now well established [52-70], and evidence has been
provided that they exert both tonic and phasic modulation of central dopaminergic function
[52-70].
Initially, in our laboratory, it was found that the firing rate of DA neurons in the VTA
was reduced by mCPP and trifluoromethylphenylpiperazine (TFMPP), two mixed 5HT1B/2A/2B/2C receptor agonists [30], whereas these neurons were stimulated by mesulergine
[52]. Based on those findings, it was suggested that 5-HT could exert an inhibitory action on
DA neurons in the VTA by acting through 5-HT2 receptors [52]. However, these data did not
allow to distinguish the relative contribution of each 5-HT2 receptor subtype in the control of
central DA function. Subsequently, our and other studies clearly indicated a selective
involvement of 5-HT2C receptors for the suppressive influence of 5-HT on the activity of
mesocorticolimbic and nigrostriatal dopaminergic pathways. In fact, a series of in vivo
electrophysiological and neurochemical studies showed that 5-methyl-1-(3pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f ]indole) (SB 206553), a selective 5HT2C/2B receptor inverse agonist [68,71], and 6-chloro-5-methyl-l-[2-(2-methylpyridiyl-3oxy)-pyrid-5-yl carbomoyl] indoline (SB 242084), the most potent and selective 5-HT2C
receptor antagonist available [72], increased the basal firing rate and the bursting activity of
VTA DA neurons, and enhanced DA release in both rat nucleus accumbens and prefrontal
cortex [55-59]. Conversely, systemic administration of (S)-2-(chloro-5-flouro-indo-l-yl)-lmethylethylamine 1:1 C4 H4 O4 (RO 60-0175), a selective 5-HT2C receptor agonist [73] had
opposite effects [54,56,57,59,61]. SB 206553 and SB 242084 were also found to potentiate
pharmacological-induced accumbal DA release [63,65,69], and stress-stimulated DA outflow
in the rat prefrontal cortex [64], while stimulation of 5-HT2C receptors by RO 60-0175 in the
VTA suppressed it [64], suggesting a role of these receptors on evoked accumbal DA release
also. On the other hand, 5-HT2C receptor agonists such as mCPP, MK 212 [6-chloro-2-(1piperazinyl)piperazine], and RO 60-0175 did not significantly affect the activity of SNc DA
neurons and the in vivo DA release in the striatum [56,60]. Moreover, the mixed 5HT2B/2C
antagonist SB 206553 caused only a slight increase in the basal activity of DA neurons in the
nigrostriatal pathway [57], suggesting that the serotonergic system controls both basal and
stimulated impulse flow dependent release of DA preferentially in the mesocorticolimbic
system by acting through 5-HT2C receptors.
Consistently, a study carried out in our laboratory has shown that mCPP excites non-DA
(presumably GABA-containing) neurons both in the SNr and the VTA by activating 5-HT2C
receptors [47]. One interesting finding of that study was the differential effect exerted by
93
mCPP on subpopulations of SNr neurons. Thus, mCPP caused a marked excitation of

presumed GABA-ergic SNr projection neurons, whereas it did not affect SNr GABAcontaining interneurons that exert a direct inhibitory influence on DA neurons in the
substantia nigra [47]. On the other hand, all non-DA neurons in the VTA were equally
excited by mCPP. It is tempting to speculate that this differential response to mCPP might be
the basis of the preferential inhibitory effect of 5-HT2C agonists on the mesocorticolimbic
versus the nigrostriatal DA function. Other in vivo electrophysiological and neurochemical
studies have confirmed and extended the above mentioned data, that 5-HT exerts a direct
excitatory effect on GABA-ergic neurons in the substantia nigra pars reticulata and VTA by
acting on 5-HT2C receptors [74,75]. In fact, about 50% of SNr neurons are excited by the
selective 5-HT2C receptors agonist RO 60-0175 and this effect is counteracted by the new and
selective 5-HT2C inverse agonist SB 243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) [76,77], in addition,
microiontophoretic application of RO 60-0175 clearly showed a direct effect of the 5-HT2C
receptors on the SNr neurons, antagonized by SB 243213. Infusion of RO 60-0175 and
mCPP by reverse-dialysis significantly increased extracellular levels of GABA in the SNr
[74]. Nevertheless, intra-VTA infusion of SB 206553 has been shown to attenuate MDMAinduced increase GABA levels in the VTA and to potentiate the concurrent increase in
accumbal DA release [75].
Although recent studies showed that systemic administration of 5-HT2C receptors
agonists, including RO-600175, do not significantly decrease the activity of nigrostriatal DAergic neurons [56,60], such treatment decreases DA efflux in the striatum [59,67,68], while,
systemic administration of SB 206553 and SB 242084 enhance it [55,57,65,69]. A recent
study has shown that the 5-HT2C receptor inverse agonist-induced increase in accumbal and
striatal DA release is insensitive to the depletion of extracellular 5-HT, suggesting that
constitutive activity of the 5-HT2C receptors participates in the tonic inhibitory control that
they exert upon DA release in both the nucleus accumbens and striatum [68]. Furthermore,
biochemical evidence indicates that both VTA and accumbal 5-HT2C receptors participate in
the phasic inhibitory control exerted by central 5-HT2C receptors on mesoaccumbens DA
neurons, and that the nucleus accumbens shell region constitutes the major site for the
expression of the tonic inhibitory control involving the constitutive activity of 5-HT2C
receptors [70]. There is also evidence that 5-HT2C receptors can modulate the phasic activity
of the DA-ergic nigrostriatal system. Indeed, SB 206553 has been shown to potentiate
cocaine- and morphine-induced increases DA outflow in the rat striatum [65,69] and systemic
administration of RO 60-0175 was found to attenuate haloperidol-induced DA release in the
same area [69], as well as nicotine-induced increase in DA activity in the nigrostriatal system
[78,79].
94
5-HT2C RECEPTORS AND PSYCHIATRIC DISORDERS

Depression
Although dopamine has received little attention in biological research on depression, as

compared to other monoamines such as serotonin and noradrenaline, current research on the
dopaminergic system is about to change this situation. It is now well established that
disturbances of mesolimbic and nigrostriatal DA function are involved in the
pathophysiology of depression [3,6]. Moreover, stress promotes profound and complex
alterations involving DA release, metabolism and receptor densities in the mesolimbic system
[80,81]. It seems that exposure to unavoidable/uncontrollable aversive experiences leads to
inhibition of DA release in the mesoaccumbens DA system as well as impaired responding to
rewarding and aversive stimuli. These alterations could elicit stress-induced expression and
exacerbation of some depressive symptoms in humans [81]. Thus, in view of the hypothesis
that disinhibition of the mesocorticolimbic DA system underlies the mechanism of action of
several antidepressant drugs [82-87] the disinhibitory effect of SB 206553 and SB 242084 on
the mesolimbic DA system might open new possibilities for the employment of 5-HT2C
receptor antagonists as antidepressants [8,53,56,85,87]. This hypothesis is consistent with the
suggestion that 5-HT2C receptor blockers might exert antidepressant activity [7,8,10,42,87].
In this respect, it is interesting to note that several antidepressant drugs have been shown to
bind with submicromolar affinity to 5-HT2C receptors in the pig brain and to antagonize
mCPP-induced penile erections in rats, an effect mediated through the stimulation of central
5-HT2C receptors [7,88,89]. Based on those findings, Di Matteo et al. [85] have carried out
experiments showing that acute administration of amitriptyline and mianserin, two
antidepressants with high affinity for 5-HT2C receptors, enhances DA release in the rat
nucleus accumbens by blocking these receptor subtypes, in addition to their other
pharmacological properties. Interestingly, amitriptyline and mianserin have been tested in the
chronic mild stress-induced anhedonia model of depression and were found to be effective in
reversing the stress effects [90,91]. The antianhedonic effects of tricyclic antidepressants,
mianserin, and fluoxetine were abolished by pretreatment with D2 /D3 receptor antagonists,
thus indicating an involvement of DA in the antidepressant effect of various drugs in this
model [90,92]. The ability of antidepressants, such as tricyclics, SSRIs and mianserin, to
affect DA systems, via indirect mechanisms, was also reported by studies of Tanda et al.
[93,94] suggesting that potentiation of DA release in the rat cortex may play a role in the
therapeutic action of antidepressants. The chronic mild stress procedure, which induce a
depression-like state in animals, was shown to enhance 5-HT2C receptor mediated function, as
measured in vivo by mCPP induced penile erections. In contrast, two different antidepressant
treatments (72-h REM sleep deprivation and 10-day administration of moclobemide, a
reversible inhibitor of monoamine oxidase type A) resulted in a reduction of this 5-HT2C
receptor-mediated function [95]. This was interpreted as an indication that the 5-HT2C
receptor may be altered, and preasumably may exist in a dysregulated (hypersensitive) state
in depressive illness. Thus, adaptive processes resulting from chronic antidepressant
treatment (i.e. desensitization and/or downregulation of 5-HT2C receptors) may play an
95
important role in reversing the 5-HT2C receptor system supersensitivity resulting from a
depressive state [7,96].
In contrast to most other receptors, 5-HT2C is not classically regulated. Indeed, 5-HT2C
receptors appear not only to decrease their responsiveness upon chronic agonist stimulation,
but also and paradoxically after chronic treatment with antagonists [97,98]. This mechanism
appears to be related to an internalisation process that removes activated cell surface
receptors from the plasma membrane involving a phosphorylation step and possible
degradation in lysosomes [97]. As a large number of psychotropic drugs, including atypical
antipsychotics, antidepressants, and anxiolitics, can all induce down-regulation of 5-HT2C
receptors, it has been suggested that this receptor adaptation plays a role in the therapeutic
action of these drugs [97,98].
In this respect, it is interesting to note that chronic treatment with 5-HT2 agonists or
antagonists resulted in a paradoxical down-regulation at the 5-HT2A and 5-HT2C receptors
[96-101] and it seems that the down-regulation state occurring after chronic exposure to
mianserin in isolated systems as well as in cell cultures, is a direct receptor-mediated
mechanism of this drug at these receptors [101]. Therefore, the down-regulating capacity of
5-HT2C agonists and antagonists may play a particularly important role in treating the
supersensitivity of 5-HT2C receptors resulting from a depressive state [7,96,98].
The possible involvement of 5-HT2C receptors in the pathogenesis of depressive
disorders and in the mode of action of antidepressants is further substantiated by several other
observations. For example, acute administration of fluoxetine caused a dose-dependent
inhibition of the firing rate of VTA DA neurons [102], and decreased DA release in both the
nucleus accumbens and the striatum [103], but it did not affect the activity of DA cells in the
SNc [102]. A similar effect, though less pronounced, has been observed with citalopram
[102]. Furthermore, mesulergine, an unselective 5-HT2C receptor antagonist [35], as well as
the lesion of 5-HT neurons by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), prevented
fluoxetine-induced inhibition of VTA DA cells [102]. These results indicate that fluoxetine
inhibits the mesolimbic DA pathway by enhancing the extracellular level of 5-HT, which
would act through 5-HT2C receptors [102]. This study also demonstrated that fluoxetineinduced inhibition of DA neurons in the VTA was no longer observed after chronic treatment
(21 days) with this drug. Interestingly, mCPP inhibited the firing activity of VTA DA
neurons in control animals but not in those chronically treated with fluoxetine [102]. The
authors suggested that 5-HT2C receptors might be down-regulated after repeated fluoxetine
administration. Consistent with this hypothesis is the evidence that chronic treatment with
sertraline and citalopram, two selective serotonin reuptake inhibitors (SSRIs), induce
tolerance to the hypolocomotor effect of mCPP [104]. This hyposensitivity of 5-HT2C
receptors might be a key step for the achievement of an antidepressant effect. Indeed, it is
possible to argue that the acute inhibitory effect of fluoxetine on mesolimbic DA system
would mask its clinical efficacy in the early stage of treatment. This masking effect would
disappear when the hyposensitivity of 5-HT2C receptors occurs. A series of studies carried out
in our laboratory have shown that acute administration of SSRIs such as paroxetine,
sertraline, and fluvoxamine causes a slight but significant decrease in the basal firing rate of
VTA DA neurons [105]. Therefore, it is conceivable that, similar to fluoxetine, these SSRIs
could reduce mesocorticolimbic DA transmission by activating 5-HT2C receptors.
96
Furthermore, employing complementary electrophysiological and neurochemical approach,

and both acute and chronic administration route, it was found that mirtazapine, nefazodone
and agomelatine, three effective and innovative antidepressants, elicit a robust and
pronounced enhancement in the activity of mesocorticolimbic DA pathways. These actions
were ascribed to their antagonistic properties at inhibitory, tonically active 5-HT2C receptors,
that desensitize after repeated drug administration [106-108].
Interestingly, agomelatine, has shown antidepressant efficacy in clinical trials [109-111],
and, indeed, it was found to be effective in treating severe depression associated with anxiety
symptoms, with a better tolerability and lower adverse effects than other antidepressants such
as paroxetine [109].
Schizophrenia
Both hypo- and hyperfunction of dopaminergic systems may occur in schizophrenic

patients, perhaps even simultaneusly, albeit in a region specific manner [112-114]. Thus,
whereas a dopaminergic hyperfunction of the mesolimbic system may underlie the
development of positive symptoms, a dopaminergic hypofunction of the cortical projections
may well be related to the negative symptomatology in schizophrenia. Given the critical role
of cortical DA in cognitive functioning [115,116], the hypothesized cortical DA
hypofunction may therefore also be implicated in the cognitive disturbances frequently
experienced by schizophrenic patients. Hence, it appears likely that both the negative
symptoms and cognitive disturbances of schizophrenia may be associated with a
hypofunction of the mesocortical DA system.
Currently used antipsychotic drugs are usually divided into two main classes, on the
basis of their liability to induce neurological side effects after long-term treatment. Drugs
defined as typical antipsychotics (e.g. chlorpromazine, haloperidol, trifluopromazine) are
known to induce, following repeated administration, various extrapyramidal side effects
(EPS) including Parkinson-like syndrome and tardive dyskinesia [117]. On the other hand,
chronic treatment with atypical antipsychotic drugs (e.g. clozapine, risperidone, sertindole,
zotepine) is associated with a low incidence of neurological side effects [117]. Moreover,
atypical antipsychotic drugs do not increase plasma prolactin levels in humans [117]. The
hypothesis that typical antipsychotics produce their clinical effects, as well as EPS, by
blocking DA D2 receptors in the mesolimbic and nigrostriatal systems, respectively [117], is
now generally accepted. In contrast, the mechanisms responsible for the clinical effects of
atypical antipsychotic drugs are still not clear. The most relevant hypothesis on the mode of
action of the atypical antipsychotics is that their action depends on their interaction with
central 5-HT2A or 5-HT2C receptor subtypes, more than with D2 receptors [4,117,118].
Numerous studies show that several antipsychotic drugs exhibit appreciable affinity for
central 5-HT2 receptors [117,119] and induce significant blockade of these receptors in
human brain [120]. Early clinical studies indicated that the selective 5-HT2A/2C receptor
antagonist ritanserin [121,122] could ameliorate negative symptoms as well as attenuate
exciting EPS in schizophrenics treated with classical antipsychotic drugs [123,124]. The
importance of 5-HT2 receptor antagonism in the pharmacology of schizophrenia is further
97
underlined by the fact that clozapine is indeed a potent 5-HT2A receptor antagonist and
exhibit a high ratio of 5-HT2A to D2 receptor affinities [125,126]. In fact, by examining in
vitro receptor binding data, Meltzer et al. [118] found that typical and atypical antipsychotics
could be distinguished on the basis of their 5-HT2A to D2 receptor binding ratios.
Accordingly, they suggested that the mechanism of action of atypical antipsychotic drugs is
based on their ability to achieve a balanced 5-HT2A to D2 receptor antagonistic action and not
on their absolute affinity for these receptors per se. Such hypotheses have pressed to develop
novel antipsychotic drugs with combined antiserotonergic and antidopaminergic properties.
Indeed, agents acting at multi-receptor sites appear to be more promising as antipsychotic
drugs, and recent data show that blockade of DA receptors and combined antagonism at 5HT2A as well as 5-HT2C receptors may be involved in the therapeutic effects of novel
antipsychotics [127-129]. In this respect, it is noteworthy to mention recent data showing that
atypical antipsychotic drugs (clozapine, sertindole, olanzapine, ziprasidone, risperidone,
zotepine, tiospirone, fluperlapine, tenilapine), which produce little or no EPS while
improving negative symptoms of schizophrenia, exert substantial inverse agonist activity at
5HT2C receptors [130,131]. Thus, 5-HT2C receptor inverse agonism might underlie the unique
clinical properties of atypical antipsychotic drugs [130].
Antagonism at 5-HT2C receptors by several antipsychotics was also observed in vivo.
Indeed, clozapine produces an increase in extracellular levels of DA in the nucleus
accumbens [132,133], reverses the inhibition of accumbal DA release induced by the 5-HT2C
agonist RO 60-0175 [132] and blocks the hypolocomotion induced by the 5-HT2C agonist
mCPP [134]. It is worth noting that clozapine, like several atypical APDs, behaves as a
5HT2C inverse agonist in heterologus expression systems in vitro [130,131,135] and in vivo
[135]. Thus, the 5-HT2C receptor inverse agonism might underlie the unique clinical
properties of atypical APDs [130,135]. The modification of 5-HT2C receptors constitutive
activity may also participate in the effects of the typical APD haloperidol. Indeed, it has been
reported that the increase in striatal DA release induced by haloperidol is dramatically
potentiated by the 5-HT2C inverse agonist SB 206553 [135]. Therefore, bearing in mind that
haloperidol does not bind to 5-HT2C receptors, it was suggested that it could act at the level of
a common effector pathway [135].
A preferential increase of DA release in medial prefrontal cortex seems to be a common
mechanism of action of atypical antipsychotic drugs, an effect which might be relevant for
their therapeutic action on negative symptoms of schizophrenia [136]. In this respect, it is
important to note that the selective 5-HT2C receptor antagonist SB 242084 [72] markedly
increases DA release in the frontal cortex of awake rats [54,59]. Thus, it is possible to argue
that blockade of 5-HT2C receptors might contribute to the preferential effect of atypical
antipsychotics on DA release in the prefrontal cortex. Interestingly, there is preclinical
evidence indicating that 5-HT2C receptor blockade is responsible for reducing EPS: 5-HT2C
but not 5-HT2A receptor antagonists were capable of inhibiting haloperidol-induced catalepsy
in rats [137].
98
Parkinsons Disease
Another interesting application of the data regarding the functional role of 5-HT2C
receptors in the basal ganglia is the possible use of 5-HT2C receptor antagonists in the
treatment of Parkinsons disease, and 5-HT2C agonists to reduce the problems of levodopainduced dyskinesia [138,139]. The neural mechanisms underlying the generation of
parkinsonian symptoms are thought to involve reduced activation of primary motor and
premotor cortex and supplementary motor areas, secondary to overactivation of the output
regions of the basal ganglia, i.e. SNr and globus pallidus internus (GPi) [140], largely
because of excessive excitatory drive from the subthalamic nucleus (STN). Therapy of
Parkinsons disease consists mainly of amelioration of the symptoms with classical
dopaminomimetics [141]. This treatment, however, is characterized by declining efficacy and
occurrence of disabling side-effects [142]. Functional inhibition of GPi or STN, has provided
an alternative to lesioning, by deep brain stimulation associated with modest side-effects
[143]. As already mentioned, 5-HT2C receptors are located in the SNr and medial segment of
the pallidal complex in the rat and human brain [28,40], and enhanced 5-HT2C receptormediated transmission within the output regions of the basal ganglia in parkinsonism appears
to contribute to their overactivity [138]. In addition, 5-HT2C-like receptor binding is increased
in a rat model of parkinsonism [144] and in human parkinsonian patients [145]. Interestingly,
systemic administration of SB 206553 enhanced the anti-parkinsonian action of the DA D1
and D2 agonists in the 6-hydroxydopamine-lesioned rats [146,147], suggesting that the use of
a 5-HT2C receptor antagonist in combination with a DA receptor agonist may reduce the
reliance upon dopamine replacement therapies and may thus reduce the problems associated
with long term use of currently available antiparkinsonian agents [138].
Drugs of Abuse
Substantial evidence indicates that the mesolimbic pathway, particularly the

dopaminergic system innervating accumbal areas, is implicated in the reward value of both
natural and drug reinforcers, such as sexual behavior or psychostimulants, respectively
[5,17,148]. The fact that drugs of abuse act through different cellular mechanisms leads to the
possibility that their effects on DA release could be modulated differentially by each of the 5HT2 receptor subtypes. As an example, it has been reported that the increased locomotor
activity, as well as the accumbal DA release, elicited by phencyclidine is further enhanced by
the blockade of 5-HT2C receptors [63], while antagonism at 5-HT2A receptors had opposite
effects [149]. A similar picture emerges when considering at the influence of these receptors
on 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-induced effects on DA neuron
activity. Thus, the selective 5-HT2A antagonist MDL 100,907 significantly reduced the
hyperlocomotion and stimulated DA release produced by MDMA while the selective 5-HT2C
antagonists SB 242084 and SB 206553 potentiated it [150-153].
It was recently found that SB 206553 administration potentiates both the enhancement of
DA release in the nucleus accumbens and striatum, and the increased DA neuron firing rate
induced by morphine both in theVTA and the SNc [65]. Consistent with these findings,
99
stimulation of central 5-HT2C receptors has been shown to inhibit morphine-induced increase
in DA release in the nucleus accumbens of freely moving rats [154]. A series of studies
showed that blockade of 5-HT2A or 5-HT2C receptors had opposite effects on cocaine-induced
locomotor activity. Thus, 5-HT2A receptor blockade with M100,907 attenuated cocaineinduced locomotion, whereas 5-HT2C blockade with SB 242084 or SB 206553 enhanced
cocaine-induced activity [155-158]. Consistent with these data obtained in rats, 5-HT2C
receptor null mutant mice showed enhanced cocaine-induced elevations of DA levels in the
nucleus accumbens, and marked increase in locomotor response to cocaine as compared to
wild-type mice, suggesting that selective 5-HT2C receptor agonist treatments may represent a
promising novel approach for treating cocaine abuse and dependence [159]. In line with this
hypothesis, it was previously found that RO 60-0175 reduced cocaine-reinforced behavior by
stimulating 5-HT2C receptors [160]. Moreover, these authors also showed that RO 60-0175
reduced ethanol- and nicotine-induced self-administration and hyperactivity [161,162].
Consistent with these evidences, we showed that the selective activation of 5-HT2C receptors
by RO 60-0175 blocks the stimulatory action of nicotine on SNc DA neuronal activity and
DA release in the corpus striatum [78,79]. The mesolimbic DA system appeared to be less
sensitive to the inhibitory effect of 5-HT2C receptors activation on nicotine-induced
stimulation, indeed a higher dose of RO 60-0175 was necessary to prevent the enhancement
of VTA DA neuronal firing elicited by acute nicotine. Furthermore, pretreatment with the 5HT2C agonist did not affect nicotine-induced DA release in the nucleus accumbens [78,79].
Interestingly, in animals treated repeatedly with nicotine, pretreatment with RO 60-0175
reproduced the same pattern of effects on the enhancement in DA neuronal firing caused by
challenge with nicotine, resulting effective only at a higher dose in preventing nicotine
excitation in the VTA compared to the SNc. Furthermore, the 5-HT2C receptors agonist
counteracted nicotine-induced DA release both in the striatum and in the nucleus accumbens
in rats chronically treated with this alkaloid, even if this effect was observed only with the
highest dose of RO 60-0175 [78,79]. Therefore, we hypothesized that after repeated nicotine
exposure an up-regulation of 5-HT2C receptors occurs only in the DA mesolimbic system and
the blocking of its hyperfunction by 5-HT2C receptor activation might be a useful approach in
reducing nicotine reward, and eventually helping in smoking cessation.
CONCLUSION
Serotonergic and dopaminergic systems are closely related in the central nervous system,
and the involvement of 5-HT2 receptor family in the control of central DA activity is now
well established. Twenty five years of 5-HT2C receptors research have generated detailed
information on the molecular biology, regional and cellular localization of these receptors. A
series of studies have shown that the serotonergic system exerts phasic and tonic control on
DA function in the mesocorticolimbic system by acting through 5-HT2C receptors. Based on
these findings, it has been suggested that 5-HT2C receptor antagonists might be useful in the
treatment of depression. This hypothesis has been confirmed by preliminary clinical trials
showing antidepressant activity of drugs acting as 5-HT2C receptor antagonists. Several other
studies indicate that selective 5-HT2C ligands may serve for the treatment of other
100
neuropsychiatric illness such as schizophrenia, Parkinsons disease, and drug abuse. In

addition, many atypical antipsychotic drugs display antagonism at both 5-HT2C and 5-HT2A
receptors, which might be the basis of their capability to ameliorate negative symptoms, as
well as to attenuate EPS in schizophrenic patients treated with classical antipsychotic drugs.
It has also been proposed a combination of 5-HT2C antagonists and dopamine agonists to
reduce the problems associated with the long term use of currently available antiparkinsonian
agents. However, the possible use of 5-HT2C agonists for the treatment of drug addiction, is
still under investigation.
ACKNOWLEDGMENTS
We wish to dedicate this chapter to the Laboratorio di Neurofisiologia del Consorzio
Mario Negri Sud.
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ISBN: 978-1-60021-820-0
Chapter V
DOPAMINE EFFECTS ON THE ADRENAL GLAND

OF THE NEWT TRITURUS CARNIFEX
(AMPHIBIA, URODELA)
Anna Capaldo*, Flaminia Gay, Salvatore Valiante,
Vincenza Laforgia, Lorenzo Varano and Maria De Falco
Department of Biological Sciences- Section of Evolutive and Comparative Biology.
Faculty of Sciences. University Federico II. Via Mezzocannone 8, 80134 Naples, Italy
ABSTRACT
The existence of intra-adrenal paracrine interactions of functional relevance between
chromaffin and steroidogenic tissues has been shown in mammals as well as in lower
vertebrates. In Triturus carnifex, an urodele amphibian, recent studies showed that two
tissues may influence each other as well; moreover, both epinephrine and norepinephrine
exert a stimulatory effect on epinephrine and norepinephrine release, whereas the effects
of two amines on steroidogenic tissue are different from one another: epinephrine inhibits
and norepinephrine stimulates aldosterone release. To date, data are lacking about
dopamine role in this species; therefore, the aims of the present study were 1) to evaluate
the influence of dopamine on the adrenal gland of the newt 2) to compare the effect of
dopamine with those of the other two amines, in order to study in depth intraadrenal
paracrine interactions in urodele amphibians.
In April and June, adult male newts were given intra-peritoneal (ip) injections of
dopamine (1.25 mg/100 g body wt/day for 4 consecutive days); the effects, after two and
twenty-four hours, were evaluated by examination of the ultrastructural morphological
and morphometrical features of the tissues as well as the serum levels of aldosterone,
corticosterone, epinephrine and norepinephrine. In both periods, dopamine exerted an
inhibitory effect on steroidogenic tissue, always significantly decreasing serum
* Corresponding author: A. Capaldo; Department of Biological Sciences, Section of Evolutive and Comparative
Biology, Via Mezzocannone 8, 80134 Naples, Italy; Tel.: + 39-081-2535173; Fax: + 39-081-2535035; E-mail:
nevotta16@libero.it; anna.capaldo@unina.it
114
Anna Capaldo, Flaminia Gay, Salvatore Valiante et al.

corticosterone levels, and in April serum aldosterone levels too. Only twenty-four hours
later, steroidogenic cells showed signs of renewal of biosynthetic activity. Dopamine
administration increased serum levels of catecholamines (epinephrine in April,
norepinephrine in June). Chromaffin cells, in both periods, showed clear signs of
increased biosynthetic activity, like a high development of R.E.R. and a significant
increase in the number of intermediate granules (i.e., granules in different stages of
biosynthetic pathway leading to catecholamines). The results of this study indicate that 1)
dopamine may influence both tissues of newt adrenal gland 2) dopamine plays an
inhibitory role on steroidogenic activity, like epinephrine, and a stimulatory role on the
chromaffin tissue, like both catecholamines 3) the chromaffin tissue may modulate the
activity of the steroidogenic one.
Keywords: Adrenal gland; Electron microscopy; Dopamine; HPLC; Lipid/cytoplasm ratio;

NE/E numeric ratio; Newt; RIA.
INTRODUCTION
The existence of intra-adrenal paracrine interactions between chromaffin and
steroidogenic tissues has been shown in mammals [Bornstein et al., 1997; Ehrart-Bornstein et
al., 2000; Hodel, 2001; Nussdorfer, 1996; Sicard et al., 2006; Wurtman, 2002] as well as in
lower vertebrates [Gfell et al., 1997; Leboulenger et al., 1993; Mazzocchi et al., 1998;
Montpetit and Perry, 1999; Reid et al., 1998; Sheperd and Holzwarth, 1998]. In Triturus
carnifex, an urodele amphibian, recent studies showed that both steroidogenic tissue may
influence the chromaffin one [Capaldo et al., 2004a, 2006] and chromaffin tissue may affect
the activity of the steroidogenic one. As a matter of fact, both epinephrine (E) and
norepinephrine (NE) may exert a stimulatory effect on E and NE release, whereas they have
an opposite influence on steroidogenic activity: NE increases [Capaldo et al., 2004b] and E
inhibits [Capaldo et al., 2004c] aldosterone release.
Evidence shows that dopamine may be involved in modulation of mammalian adrenal
gland activity. Exogenous dopamine [King, 1969] and dopaminergic agonists [Borowsky and
Kuhn, 1992; Jzov et al., 1985] increase ACTH and corticosterone levels in rat; moreover
dopamine causes a dose-dependent increase in cortisol secretion from cultured bovine zfr
cells, through nonspecific stimulation of adrenergic beta-receptors [Bird et al., 1998].
Conversely, dopamine was shown to exert a prevalently inhibitory effect on the zona
glomerulosa and aldosterone secretion in many mammalian species, including humans and
rats [Nussdorfer, 1996]. In normal human adrenal gland both D1-like and D2-like receptors
are expressed; in vitro, only a minor inhibition of the secretion of adrenal hormones was
found [Pivonello et al., 2004]. Wu et al. [2001] demonstrated in normal human adrenal gland
the existence of both D2 and D4 receptors; moreover, they showed that dopamine exerts in
cultured NCI-H295 cells dual effects on aldosterone secretion, D4 receptors increasing and D2
inhibiting aldosterone release.
Dopamine is involved in the modulation of the physiological secretory process in the
chromaffin cells of rat adrenal gland [Artalejo et al., 1985]. Dopamine and epinephrine, but
not norepinephrine, may be catecholaminotropic in the rat [Epple et al., 1988]. Conversely,
Dopamine Effects on the Adrenal Gland of the Newt Triturus Carnifex
115
dopamine receptors played a role as inhibitory modulators of adrenal catecholamine release

from bovine chromaffin cell cultures [Bigornia et al., 1988]; the inhibition was found to be
mediated by D4 and D5 dopamine receptors on the chromaffin cells [Dahmer and Senogles,
1996].
In lower vertebrates, the effect of dopamine on adrenal gland activity has been studied in
reptiles, amphibians and fish. Dopamine increased plasma ACTH and corticosterone levels in
the lizard Podarcis sicula; moreover, in the chromaffin tissue, a strong increase in the number
of epinephrine cells, and a decrease in the number of norepinephrine cells were observed,
suggesting a stimulatory effect on the activity of PNMT (phenylethanolamine-N-methyltransferase) enzyme, converting norepinephrine into epinephrine [Capaldo et al., 2004d].
Morra et al. [1990, 1992] showed that dopamine causes a clear-cut inhibition of the basal
release of both corticosterone and aldosterone by perifused frog adrenals, acting via the DA1
and DA2 receptor subtypes, positively and negatively coupled to both phospholipase A2 and
phospholipase C [Morra et al., 1989, 1991]. In Anguilla rostrata, dopamine was found to be
in vivo catecholaminotropic, enhancing epinephrine and norepinephrine release [Epple and
Nibbio, 1985; Reid et al., 1998].
To date, data are lacking about the role of dopamine in the adrenal gland of the newt T.
carnifex. Therefore, the aims of the present study were 1) to evaluate the influence of
dopamine on the adrenal gland and 2) to compare the effect of dopamine with those of the
other two amines, norepinephrine and epinephrine, in order to study in depth intraadrenal
paracrine interactions in urodele amphibians. Adult newts were given dopamine in vivo; the
effects were evaluated by means of the ultrastructural morphological features of
steroidogenic and chromaffin tissues, as well as the serum levels of aldosterone,
corticosterone, epinephrine and norepinephrine.
MATERIALS AND METHODS

Animals and Experimental Design
Adult male specimens of Triturus carnifex (mean weight 8.0 g), captured in the field
around Naples, Italy, were kept in aquaria at seasonal temperature and photoperiod, fed
minced cow liver and used after an acclimation period of 2 weeks. The experiments were
performed in April and June, corresponding to different stages of the chromaffin cell
functional cycle [Laforgia and Capaldo, 1991]. The specimens were given intra-peritoneal
(ip) injections of dopamine. Based on the results of preliminary dose-response and timecourse tests, the minimum effective dose and time of treatment were chosen for the following
experiments. Fifty animals were collected two weeks prior to the April experiment and
another 50 collected prior to the June experiment. In both periods, the newts were treated as
follows:
Twenty animals were injected with dopamine (dopamine hydrochloride; SigmaAldrich, St. Louis, MO, U.S.A.) (1.25 mg./100 g body wt/day for 4 consecutive
days), dissolved in 0.64% NaCl, with an injection volume of 0.1 ml.
116
Twenty animals received ip injections of carrier solution (0.64% NaCl).

Ten animals were untreated.
The newts were anaesthetized by hypothermia, chilling them in chipped ice, within 5 min
after capture. Blood was immediately collected over 3 min by heart puncture, from 1)
untreated specimens and from 2) 10 treated and 10 carrier specimens, 2 h after the last
injection, and from 3) 10 treated and 10 carrier specimens, 24 h after the last injection,
between 11 AM and 2 PM. Blood was centrifuged for 15 min at 2,000g and serum was
collected and stored at 22 C until assayed. Institutional committees (Department of Health)
approved the experiments, which were organized to minimize the stress and the number of
animals used.
Transmission Electron Microscopy
The animals were killed by decapitation immediately after collection of blood samples.
The adrenals and adjacent nephric tissue were excised and fixed in 2.5% glutaraldehyde in
Millonigs phosphate buffer at pH 7.4 at 4C, rinsed in buffer, and postfixed in 1% OsO4 (2h,
4C), dehydrated in ethanol, cleared in propylene oxide, embedded in epoxy resin, and
polymerized. Ultrathin sections (30 nm) were cut with glass knives on a Reichert-Jung
ultracut ultramicrotome (SUPER NOVA), collected on formvar-coated copper grids, stained
with solutions of uranyl acetate and lead citrate, and observed with a Philips EM 301
transmission electron microscope at the Interdepartmental Center of Services for Electron
Microscopy (Naples).
For each specimen from each group, ten low-power micrographs of the chromaffin tissue
and ten of the steroidogenic tissue, each containing at least four cells, were processed for
morphometric investigation by a computerized image analysis system (KS 300 for Windows
98, Zeiss). In the chromaffin cells we evaluated: the mean total number of chromaffin
granules/m2, the mean number of NE and E granules/m2; the NE/E granule numeric ratio;
the mean number of intermediate granules/m2, i.e. the secretory granules that cannot be
considered as NE or E granules, but represent granules in different stages of the byosinthetic
pathway leading to the end-product, NE or E. The sampling criteria to selectively
discriminate between NE, E and intermediate granules were the following: granules
recognized as NE granules were of variable shape, with a very electron-dense and compact
core filling the granule; the core was separated from the limiting membrane. Granules
identified as E granules were roundish, homogeneous, with a finely granular core of medium
electron density, separated from the limiting membrane by a narrow electron-lucent space.
The granules not showing these distinctive features were considered intermediate granules
[Laforgia and Capaldo 1991].
In the steroidogenic cells, the area occupied by the lipid droplets was evaluated,
according to the formula:
117
Hormone Assay
Serum levels of aldosterone and corticosterone were determined by radioimmunoassay

(RIA) as previously described [Andreoletti et al., 1988; Capaldo et al., 2006]. Briefly,
nonhemolyzed serum samples (80 l for aldosterone and 30 to 40 l for corticosterone) were
incubated for 30 min at 37 C with known amounts of radioactive steroids (3H-aldosterone,
and 3H-corticosterone from Bio-Rad, Hercules, CA) in 0.06 M Na-phosphate buffer
containing 0.01 EDTA disodium salt and 0.1% BSA pH 7.4. Samples were applied to an
extraction column (Sep-Pak C18, Waters, Milford, MA) and washed with 500 l of pure
methanol. Methanol extracts were dried at 37 under vacuum and redissolved in 1,400 l of
PBS. An aliquot was taken to determine the labeled hormone recovery and on two other
aliquots aldosterone and corticosterone were assayed by RIA. After incubation with rabbit
antiserum (Biogenesis, Poole, UK) for 30 min at 37 C and for another 2 h in an ice bath,
dextran-coated charcoal was used to separate free from bound steroids. After immersion for
10 min in an ice-bath and centrifugation (2,000 rpm), a supernatant aliquot was counted with
a liquid scintillation spectrometer (Tri-Carb Packard, GMI, Albertville, MN, USA).
Extraction yields ranged from 80%-90% for both hormones. Data were obtained through a
standard calibration curve linearized with a log-logit method and corrected for individual
extraction yield. Sensitivity was 5 pg/tube for aldosterone and corticosterone. Intraassay
coefficient of variation was 10%, and interassay coefficient of variation was 12% for both
steroids.
Norepinephrine and epinephrine levels were determined in 150 l serum. For
catecholamine extraction, 50 l of dihydroxybenzylamine were added as an internal standard.
Ten milligrams activated aluminium oxide (Sigma, St. Louise, MO) was used as adsorbent
for catecholamines and the internal standard. After 15 min shaking and centrifugation, the
supernatant was removed and the aluminium oxide containing the adsorbed catecholamines
and the internal standard was washed three times with 1 ml distilled water by shaking,
centrifuging, and discarding the supernatant- extracted samples using high performance
liquid chromatography (HPLC), with electrochemical detection, according to the method
previously used in Triturus carnifex [Kloas and Hanke, 1992; Capaldo et al., 2006].
Electrochemical HPLC detection was carried out using an acid eluant; NE and E levels were
calculated in comparison to the internal standard (dihydroxybenzylamine). The detection
limit for NE and E was around 20 pg.
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Statistical Analysis
All data were expressed as means standard error of mean (S.E.M.). The control and
experimental data of all the groups were tested together for significance using one-way
analysis of variance (ANOVA), followed by Duncans test for multigroup comparison and
Students t test for between group comparison. Differences were considered significant when
P < 0.05.
RESULTS
Transmission Electron Microscopy
The adrenal gland of urodeles includes numerous discrete bodies scattered on the
ventral surface of the functional opistonephric kidney, close to its medial margin. The bodies
contain tightly intermingled steroidogenic and chromaffin cells. The steroidogenic cells
contain numerous mitochondria, a smooth endoplasmic reticulum arranged in tubules and
vesicles, a well-developed Golgi apparatus and a large amount of lipid vacuoles [Hanke,
1978]. In both periods control steroidogenic cells showed a cytoplasm rich in lipid droplets
and numerous mitochondria with tubular cristae (Fig. 1a, b); the smooth endoplasmic
reticulum appeared more developed in April (Fig. 1a) than in June (Fig. 1b). Values of
lipid/cytoplasm ratio were 0.33 in April and 0.45 in June (Table 1). Two hours after last
dopamine injection, steroidogenic cells showed in April (Fig. 1c) a decrease in lipid droplet
content, whereas in June (Fig.1d) lipid content was almost unchanged, as shown by the
evaluation of lipid/cytoplasm ratio (Table 1). Twenty-four hours after the last dopamine
injection (Fig. 1e,f), steroidogenic cells, in both periods, showed a lipid droplet content and a
value of lipid/cytoplasm ratio like that of carrier-injected ones (Table 1). Moreover, cells
showed signs of weak activity, such as an enlarged smooth endoplasmic reticulum (S.E.R.),
mitochondria increased in size and with cristae less closely packed than in the carrier-injected
specimens.
119
Figure 1. Electron micrographs of steroidogenic cells of Triturus carnifex adrenal gland. (a) April and
(b) June control specimens. The cells showed numerous mitochondria (M) and lipid droplets (L) filling
the cytoplasm. Smooth endoplasmic reticulum (S.E.R.) appears more developed in April specimens. (c)
April and (d) June treated-2 h specimens: cells show in April a decrease in lipid (L) content, whereas in
June lipid (L) content is similar to control ones. (e) April and (f) June treated-24 h specimens: lipid
content is like that of control specimens. Moreover, mitochondria (M) with cristae less closely packed
than in control specimens and a marked development of smooth endoplasmic reticulum (S.E.R.) are
present. Scale bar = a, b: 1.5 m; c: 1,1 m; d: 2 m; e: 1.2 m; f: 2 m.
120

Table 1. Mean SE of the different parameters evaluated
in control and treated specimens
April
Control
Carrier 2 h
Carrier-24 h
Treated 2 h
Treated 24 h
June
Control
Carrier-2 h
Carrier-24 h
Treated 2 h
Treated 24 h
Chromaffin
granules/
m2
Intermediate
granules/
m2
NE
granules/
m2
E
granules/
m2
NE/
E ratio
Lip/
cyt.ratio
8.27
2.38
8.39
2.49
8.19
2.24
3.15
1.02a
4.50
1.33b
0.07 0.003
4.11
1.26
4.14
1.34
4.06
1.19
0.16
0.01a
0.17
0.02b
1.01/1
0.33
1.00/1
0.34
1.01/1
0.33
2.43
0.99a
0.08
0.00
4.09
1.57
4.15
1.60
4.08
1.33
0.56
0.06a
4.25
1.00
3.50/1
0.25
25.00/1
0.30
0.003
0.0009
0.001
0.0005
0.008
0.0001
3.08
1.06a
4.47
1.19b
7.25
2.31
7.40
2.20
7.29
2.25
3.22
0.96a
2.19
0.94b
1.31
0.26
1.24
0.2
1.27
0.84
0.60
0.04a
0.54
0.08b
5.50/1
0.45
5.96/1
0.47
5.74/1
0.46
5.36/1
0.42
4.05/1
0.43
8.56
2.45
8.64
2.51
8.57
2.34
6.90
1.93
7.20
2.02
0.09 0.005
0.05 0.006
a Significantly (P < 0.05) different from carrier-2 h values

a Significantly (P < 0.001) different from carrier-2 h values
b Significantly (P < 0.05) different from carrier-24 h values
b Significantly (P <0.001) different from carrier-24 h values
The adrenal gland of Triturus carnifex has a single type of chromaffin cell, having an
annual cycle with seasonal variations in the norepinephrine/epinephrine granule ratio within
the cells. In the periods of the year characterised by extreme temperatures (winter or
summer), there is a strong prevalence of NE granules; in the milder periods (autumn or
spring), E and NE granules are present in the cells in almost equal quantities. Norepinephrine
granules are of variable shape and have a very electron-dense core, sometimes not separated
from the limiting membrane. Epinephrine granules are rounded and have a fine granular core
of medium electron density, with a clear halo between the core and the limiting membrane.
The NE/E granule ratios within the cells vary during the year [Laforgia and Capaldo, 1991].
In April (Fig. 2a), NE (4.09 1.57 granules/m2) and E (4.11 1.26 granules/m2) granules
were both present in the chromaffin cells of control specimens in almost equal quantities,
whereas in June (Fig. 2b) chromaffin cells of control specimens contained almost exclusively
NE vesicles (NE: 7.25 2.31 granules/m2; E: 1.31 0.26 granules/m2). In both periods the
number of intermediate granules, i.e. granules in different stages of the byosinthetic pathway
leading to the end-product, NE or E, was scant (Table 1). Two hours after the fourth
dopamine injection, in April (Fig. 2c) a significant decrease in the mean total number of
121
chromaffin granules/m2, in the mean number of NE granules/m2 and in the mean number
of E granules/m2, leading to an increase in NE/E granule ratio, was observed; moreover, a
significant increase in the number of intermediate granules/m2 was found (Table 1).
Twenty-four hours after dopamine treatment (Fig. 2d), the number of intermediate
granules/m2 and the mean number of NE granules/m2 appeared like the carrier ones; the
mean total number of chromaffin and epinephrine granules/m2 appeared still significantly
lower than carrier one (Table1). Moreover, a considerable development of rough endoplasmic
reticulum (R.E.R.) was always evident in the chromaffin cells (Fig. 2c,d).
Figure 2. Electron micrographs of the chromaffin cells of Triturus carnifex adrenal gland. (a) April and
(b) June control specimens. In April the cytoplasm presents norepinephrine (NE), very electron-dense,
and epinephrine (E) granules, of medium electron-density, in almost equal quantities. June specimens
show the cytoplasm crowded with norepinephrine (NE), very electron-dense, vesicles. In both periods
intermediate (i) granules are few (c) April treated-2 h specimens, showing a decrease in the total content
of chromaffin vesicles and in the presence of NE and E granules, and a strong increase in the number of
intermediate (i) granules. Rough endoplasmic reticulum (R.E.R.) appears considerably enlarged. (d)
April treated-24 h specimens. In the cytoplasm the presence of NE granules is like the control
specimens, whereas E and intermediate (i) granules are few; rough endoplasmic reticulum (R.E.R.) is
well developed. (e) June treated-2 h specimens. In the chromaffin cells, the number of NE and E
granules appears decreased, whereas the presence of intermediate (i) granules is high. (f) June treated24 h specimens. In the cytoplasm an enlarged S.E.R. and a great quantity of intermediate (i) granules
are evident. Scale bar = a: 1.3 m; b: 1.2 m; c: 0.8 m; d: 1 0 m; e, f: 0.6 m.
122
In June, dopamine treatment slightly affected the mean total number of chromaffin
granules/m2 and the NE/E ratio (Table 1); two hours after the fourth dopamine injection
(Fig. 2 e), a significant decrease in the mean number of NE and E/granules/m2 was found,
whereas the number of intermediate granules/m2 appeared significantly increased (Table 1).
Twenty-four hours after dopamine (Fig. 2f), the mean number of NE and E/granules/m2
appeared still lower than carrier values, whereas the number of intermediate granules/m2
appeared still higher than carrier one (Table 1). As in April, chromaffin cells showed a highly
developed R.E.R.(Fig. 2e,f).
Hormone Assay
In April dopamine significantly decreased, two hours after the last injection, serum
aldosterone (P < 0.05) levels, that were still lower than carrier ones twenty-four hours after
dopamine treatment; in June aldosterone serum levels did not undergo any consistent change
instead (Fig. 3).
Figure 3. Serum aldosterone levels in April and June untreated, carrier-injected and treated specimens.
Values are means SE of the mean. a Significantly (P < 0.05) different from carrier-2 h values. b
Significantly (P < 0.05) different from carrier-24 h values.
123
Corticosterone serum levels appeared significantly decreased 2 h after the last dopamine
injection in April (P < 0.001) and in June (P < 0.05), whereas twenty-four hours after the last
administration, serum levels were like the carrier ones in both periods (Fig. 4).
Figure 4. Serum corticosterone levels in April and June untreated, carrier-injected and treated
specimens. Values are means SE of the mean. a Significantly (P < 0.05) different from carrier-2 h
values. a Significantly (P < 0.001) different from carrier-2 h values.
Catecholamine serum levels were affected by dopamine, but in a different way: in April
serum E levels increased 2 h (P < 0.05) and 24 h (P < 0.001) after the last injection, whereas
NE serum levels were unchanged (Fig. 5); in June, 2 h after dopamine, an increase (P <
0.001) in NE serum levels, and a decrease in E serum levels were found; both values became
normal again 24 h later (Fig. 6).
Figure 5. Serum catecholamine levels in April untreated, carrier-injected and treated specimens. a
Significantly (P < 0.05) different from carrier-2 h values. b Significantly (P < 0.001) different from
carrier-24 h values.
124
Figure 6. Serum catecholamine levels in June untreated, carrier-injected and treated specimens. a
Significantly (P < 0.05) different from carrier-2 h values. a Significantly (P < 0.001) different from
carrier-2 h values.
CONCLUSION
The present results show that dopamine was able to influence both tissues of Triturus
carnifex adrenal gland.
Dopamine seemed to exert an inhibitory influence on steroidogenic tissue in both
periods. Indeed, in April, 2 h after dopamine administration, aldosterone and corticosterone
serum levels, and the value of lipid/cytoplasm ratio, appeared significantly lower than carrier
ones. Twenty-four hours after dopamine, only aldosterone serum levels were still low,
whereas corticosterone serum levels and the value of lipid/cytoplasm ratio appeared like the
control ones. Moreover, the morphological features of steroidogenic cells, such as the
enlargement of S.E.R., the increase in size of mitochondria and the arrangement of their
cristae, suggested an increase of their biosynthetic activity. In June, 2 h after dopamine
administration, only a significant decrease in corticosterone serum levels was found, whereas
the other values were almost unchanged. Twenty-four hours after dopamine, aldosterone and
corticosterone serum levels, and the value of lipid/cytoplasm ratio, appeared like the control
ones; also in this case, steroidogenic cells showed signs of increased activity.
125
Dopamine affected mainly corticosterone serum levels, that significantly decreased both
in April and June, 2 h after dopamine administration, and became normal again 24 h later; on
the contrary, aldosterone serum levels were affected only in April, when they remained still
low 24 h after dopamine, whereas in June only a slight decrease was observed. In amphibians
corticosterone is the predominant circulating corticosteroid [Norris, 1997]; moreover, in T.
carnifex, Zerani and Gobbetti [1993] found that the seasonal pattern of plasma corticosterone
shows two peaks, one in January and another in July, and a progressive increase in plasma
values from April to June. Our data, showing in April lower serum corticosterone levels than
in June, agree with the seasonal pattern previously found [Zerani and Gobbetti, 1993].
In comparison with the decrease in serum corticosterone levels observed in June (P <
0.05), the major decrease (P < 0.001) found in April, when usually lower serum
corticosterone levels are present, may suggest that the biosynthetic pathway leading to
corticosterone was probably more sensitive to dopamine inhibition than in June. Bearing in
mind that corticosterone is an aldosterone precursor, the decrease in corticosterone
concentration affected aldosterone levels too. Indeed, 24 h after dopamine, aldosterone serum
levels were still low, whereas corticosterone serum levels became normal again. In June,
when higher serum concentrations of corticosterone are normally present, the effect of
dopamine on the biosynthetic pathway was probably lower than in April, and only a slight,
non significant decrease in aldosterone levels was found.
Dopamine may have different effects on steroidogenic tissue: it increases ACTH and
corticosterone levels in rats [Borowsky and Kuhn, 1992; Jzov et al., 1985; King, 1969] and
cortisol secretion from cultured bovine zfr cells [Bird et al., 1998]. Conversely, dopamine
was found to exert a prevalently inhibitory effect on the zona glomerulosa and aldosterone
secretion in many mammalian species, including humans [Nussdorfer, 1996; Pivonello et al,
2004]. Moreover, dopamine was shown to exert in humans dual effects on aldosterone
secretion, depending on which receptor, D2 or D4, mediates its action [Wu et al., 2001]. As far
as dopamine role in lower vertebrates is concerned, this amine has been shown to increase
plasma ACTH and corticosterone levels in lizards [Capaldo et al., 2004d], whereas a direct
inhibitory action of dopamine on the spontaneous secretion of corticosterone and aldosterone
from frog adrenocortical cells, likely through activation of a D2 receptor subtype, was found
[Morra et al., 1990; 1992]. Our present results, showing a decrease in corticosteroid serum
levels, are in agreement with the inhibitory role before evidenced in amphibians. Moreover,
the inhibitory effect of dopamine on corticosteroid serum level appears like that of
epinephrine, that gave rise to a decrease in aldosterone serum levels in T. carnifex ; in this
species, steroidogenic cells showed signs of lowered activity after dopamine administration
[Capaldo et al., 2004c]. On the contrary, the role of dopamine appears different from that of
norepinephrine, that instead was found to increase serum aldosterone levels and to stimulate
the activity of steroidogenic cells [Capaldo et al., 2004b].
The anatomical arrangement of amphibian adrenal gland, such as T. carnifex, where the
steroidogenic and chromaffin cells are tightly intermingled, represents the condition
necessary to the existence of paracrine relationships between steroidogenic and chromaffin
tissues, already evidenced in many Vertebrates [Bornstein et al., 1997; Ehrart-Bornstein et
al., 2000; Gfell et al., 1997; Hodel, 2001; Leboulenger et al., 1993; Mazzocchi et al., 1998;
Montpetit and Perry, 1999; Nussdorfer, 1996; Reid et al., 1998; Sheperd and Holzwarth,
126
1998; Sicard et al., 2006; Wurtman, 2002;]. Our present results suggest that dopamine,
synthesized and released by chromaffin cells, may be involved in modulation of
corticosteroid secretion. Taken together with previous results of epinephrine and
norepinephrine administration [Capaldo et al., 2004b,c], our present results confirm that in
the newt T. carnifex chromaffin tissue may influence the activity of steroidogenic one.
The chromaffin tissue was also affected by dopamine administration in both periods; in
April dopamine increased epinephrine serum levels, that were still higher than normal ones
24 h after treatment. Consistently, in the chromaffin cells the content of secretory vesicles
and that of E and NE granules decreased, leading to an increase in the NE/E ratio. Bearing in
mind that norepinephrine is the precursor of epinephrine, and that serum norepinephrine
levels were in April almost unchanged after dopamine treatment, the strong decrease in NE
granules in the cells probably indicates a rise in the conversion of norepinephrine into
epinephrine, necessary to support the increase in E serum levels, induced by dopamine.
Moreover, the intense development of R.E.R. and the increase in the number of intermediate
granules suggest a rise in biosynthetic activity of chromaffin cells. Twenty-four hours after
dopamine administration, the presence of secretory vesicles and E granules in the cytoplasm
was still low, whereas the number of intermediate and NE granules appeared normal, and the
value of NE/E ratio appeared further increased.
In June dopamine affected serum levels of both catecholamines, increasing
norepinephrine serum levels and decreasing epinephrine serum levels; they both became
normal again 24 h later. In the chromaffin cells, the content of secretory vesicles slightly
decreased, whereas the presence of E and NE granules diminished and remained still low 24
h later, without changing the NE/E numeric ratio. The strong decrease in the number of NE
granules in the cells reflects the increase in NE serum levels; consistently, a minor amount of
NE was probably turned into E, and indeed the presence of E granules in the cells, and E
serum levels, appeared both decreased. In this period too, R.E.R. development and the high
number of intermediate granules indicates a high biosynthetic activity of the chromaffin cells.
The different effect of dopamine administration (rise in epinephrine or norepinephrine
serum levels) in April and June, respectively, may depend on the relationships between the
annual reproductive cycle and the chromaffin cell functional cycle of Triturus carnifex. The
presence in the chromaffin cells of large amounts of epinephrine in April is probably related
to an increase in metabolism necessary for the onset of spermatogenesis, a main event of the
annual reproductive cycle of the newt starting in February-April [Laforgia and Capaldo,
1992].Therefore, the stimulating action of dopamine in April is likely to affect epinephrine,
because in this period this amine is necessary to the increased metabolism. In June, when the
chromaffin cells produce almost exclusively norepinephrine, dopamine is likely to influence
primarily this amine.
Dopamine was found to be catecholaminotropic in the rat [Epple et al., 1988] and in fish
[Epple and Nibbio, 1985; Reid et al., 1998]; in reptiles the amine increased the conversion of
norepinephrine into epinephrine in the adrenochromaffin tissue, suggesting a stimulatory
effect on PNMT enzyme [Capaldo et al., 2004d]. Conversely, in bovine chromaffin cell
cultures dopamine receptors behaved like inhibitory modulators of adrenal catecholamine
release [Bigornia et al., 1988; Dahmer and Senogles, 1996]. Our results, showing an increase
in serum catecholamine levels in both periods, are in agreement with the catecholaminotropic
127
effect observed in rat and fish [Epple and Nibbio, 1985; Epple et al., 1988; Reid et al., 1998].
Moreover, the effect of dopamine on chromaffin tissue in Triturus carnifex appears like that
exhibited from the other two catecholamines; indeed, both norepinephrine and epinephrine
were shown to play in the newt a catecholaminotropic role, increasing NE or E serum levels,
according to the period of chromaffin cell functional cycle [Capaldo et al., 2004b,c].
In conclusion, our results indicate that dopamine may.influence the adrenal gland of the
newt. Dopamine inhibitory effect on steroidogenic tissue appears like that of epinephrine; the
stimulatory influence of the amine on the chromaffin tissue appears like that of both
norepinephrine and epinephrine. These findings confirm the involvement of chromaffin tissue
in modulation of the activity of the steroidogenic one in the adrenal gland of the newt
Triturus carnifex.
ACKNOWLEDGEMENTS
The authors thank Dr. T. Criscuolo and Dr. M. Faggiano for assistance in the
determination of serum hormone levels.
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ISBN: 978-1-60021-820-0
Chapter VI
SEROTONIN 5-HT2C RECEPTOR AND

DOPAMINE FUNCTION IN DEPRESSION
Giuseppe Di Giovanni1,2, Vincenzo Di Matteo1, Massimo Pierucci1,
and Ennio Esposito1,
1
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030
Santa Maria Imbaro (Chieti), Italy;
2
ABSTRACT
Several hypotheses regarding the physiopathology of major depression exist.
Attention has been focused on cerebral monoaminergic systems, the dysfunction of
which is thought to underlie various aspects of depressive symptomatology. There is an
extensive literature describing the involvement of serotonergic and dopaminergic systems
in the mechanism of action of antidepressant drugs. However, a unitary analysis of the
data in terms of interaction between different monoaminergic systems is still lacking.
Among the multiple classes of 5-HT receptors described in the central nervous system,
much attention has been devoted to the role of 5-HT2 receptor family in the control of
central dopaminergic activity, because of the moderate to dense localization of both
transcript and protein for 5-HT2 receptors in the substantia nigra (SN) and ventral
tegmental area (VTA), as well as their terminal regions. Recent studies have focused on
the functional interaction between the serotonergic and dopaminergic systems to explain
the mechanism of the antidepressant action of SSRIs and 5-HT2 antagonists. In this
article, the most relevant data regarding the role of these receptors in the control of brain
DA function are reviewed, and the importance of this subject in the search of new
antidepressant drugs is discussed.
Correspondence concerning this article should be addressed to Dr. Ennio Esposito, Istituto di Ricerche
Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy,
Telephone: (+39) 0872-570274; telefax: (+39) 0872-570416; e-mail: esposto@negrisud.it.
132
Giuseppe Di Giovanni, Vincenzo Di Matteo, Massimo Pierucci et al.
Keywords: 5-HT2C receptors, Serotonergic function,

Mesocorticolimbic system, Antidepressants, SSRIs.
Dopaminergic
function,
INTRODUCTION
Several hypotheses about the pathophysiology of major depression exist. Attention has
been focused on cerebral monoaminergic systems, whose dysfunction is thought to underlie
various aspects of depressive symptomatology. There is an extensive scientific literature
describing the involvement of serotonergic and dopaminergic systems in the mechanism of
action of antidepressant drugs. However, a unitary analysis of the data in terms of interaction
between different monoaminergic systems is still lacking. Therefore, in this paper a
description of the functional interaction between serotonin (5-HT) and dopamine (DA)
systems in the brain will precede the review of the studies reporting the biochemical,
behavioral and clinical effects of tricyclic antidepressants (TCA), monoamine oxidase
inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), selective blockers of
presynaptic dopamine (DA) receptors, and antagonists of 5-HT2 receptors. Moreover, a brief
review of the most relevant data regarding the involvement of 5-HT or DA in the mechanism
of action of antidepressant drugs will be carried out before going into a detailed analysis of 5HT/DA interaction, with a particular emphasis on its relevance for the mechanism of action
of novel antidepressant drugs.
SEROTONIN/DOPAMINE INTERACTION
There is an extensive scientific literature regarding the functional interaction between 5HT- and DA-containing neurons in the brain. Although this subject has been investigated for
a period of almost three decades, the exact mechanisms by which 5-HT control dopaminergic
functions are still unclear. Research on this matter has been spurred by recent acquisition of
important insights on the molecular biology of 5-HT receptor subtypes and by the availability
of 5-HT receptor knock out mice [1,2]. Increasing the knowledge on 5-HT/DA interaction
might be of great interest in that there is evidence that central serotonergic and dopaminergic
systems play an important role in regulating normal and abnormal behaviors [3-5]. Moreover,
a number of studies suggest that dysfunctions of 5-HT and DA neurotransmission are
involved in the pathophysiology of various neuropsychiatric disorders including depression,
schizophrenia and drug abuse [3-8]. The present knowledge about the functional interaction
between the serotonergic and dopaminergic system is derived from a number of anatomical,
biochemical, behavioural and electrophysiological studies. The most relevant data regarding
this very interesting area of research will be reviewed in this paper, which will be introduced
by a brief description of the functional neuroanatomy of serotonergic and dopaminergic
systems.
5HT2C-DA in Depression
133
5-HT2 RECEPTOR FAMILY, DEPRESSION AND

ANTIDEPRESSANT DRUGS
Dopamine and Depression
Because mesocorticolimbic DA pathways are involved in physiological functions

regarding motivation and reward, this makes them a possible candidate as a neurobiological
substrate of the depressive syndrome [3]. Indeed, a DA impairment in corticolimbic areas
leads to anhedonia as well as loss of motivation (lack of interest): two symptoms usually
present in depression [9]. Thus, a decreased DA turnover has been found in patients with
depressive disorder [10,11]. Particularly evident is the reduction of homovanillic acid (HVA)
concentrations in the CSF of a subgroup of patients with psychomotor retardation [10,12,13].
Also, the high incidence of depression in Parkinsons disease is a further indication of a
relationship between DA dysfunction and the depressive syndrome [14]. Moreover, there is
striking similarity between the mood effects of neuroleptics reported in healthy volunteers
(dysphoria, paralysis of volition and fatigue) and some of the depressive symptoms [15].
Taken together, these data suggest that a DA dysfunction may be involved, at least in part, in
the depressive syndrome.
Dopamine and Antidepressant Drugs
As already mentioned, mesolimbic DA neurons arising from the ventral tegmental area
(VTA) have long been implicated in reward and incentive motivation [3]. For instance, it is
now known that intracranial self-stimulation (ICSS) obtained with electrodes implanted in the
VTA is mediated by an increased activity of DA neurons arising from this structure.
Interestingly, Fibiger and Philipps [16] demonstrated that chronic desipramine treatment
potentiated the ICSS. Moreover, chronic treatment with other antidepressants was capable of
enhancing amphetamine-induced locomotor activity, a behaviour mediated by mesolimbic
DA pathway [17-19]. Furthermore, TCA facilitated the behaviour elicited by local injection
of amphetamine into the nucleus accumbens [20]. Of particular interest is the fact that
desipramine selectively facilitated the amphetamine-induced DA release in the nucleus
accumbens only after 21 days of treatment [18]. These data argue in favour of sensitization of
DA response after antidepressant treatment. Many studies have focused on the potential
adaptative changes involved in the enhancement of DA responsivity after antidepressant
treatment. Since animals treated with antidepressant were hypersensitive to direct DA
agonists (apomorphine and quinpirole), it has been suggested that postsynaptic DA receptors
are up regulated and/or hypersensitized [18,21]. However, binding studies have provided
controversial results, in that some authors have found either an increased number or affinity
of mesolimbic DA D2 receptors after chronic treatment with various antidepressants [21,22],
whereas others have not found any significant alteration of the affinity or the number of these
receptors in the nucleus accumbens [19,23]. Nevertheless, the possibility cannot be ruled out
that the mechanisms underlying the hypersensitivity of these receptors lie downstream with
respect to the receptor, e.g. receptor/G-protein or G-protein/effector coupling efficacy.
134
Although the hyposensitivity of DA autoreceptors has long been proposed to explain the
enhancement of sensitivity observed in the mesolimbic DA pathway [24,25], studies are
largely discrepant [3]. In particular, studies which used microdialysis to directly assess DA
release in the terminal field failed to show any modification of DA response to small doses of
apomorphine (thought to selectively stimulate presynaptic receptors) after desipramine
treatment [3,26]. An additional mechanism causing DA hypofunctioning in major depression
is a reduced DA release by dopaminergic nerve terminals, which is evident especially in the
mesolimbic system. This reduction in DA release is accompanied by a decrease in the number
of presynaptic DA transporters (DAT) [27-29]. Based on the evidence that antidepressants
elevate mood only in depressed patients, and not in healthy volunteers, some authors
proposed that studying antidepressant effect in animal models of depression would be much
more informative [9]. Among the various animal models of depression so far developed,
chronic mild stress (CMS) is likely the best validated [9]. However, a possible limitation of
this test is that it expresses mostly anhedonia which nevertheless is a cardinal symptom of
depression. A down-regulation of D2/D3 receptors was reported in the ventral striatum (e.g.
nucleus accumbens) after CMS [9,30]. This neurochemical adaptative change is correlated to
a subsensitivity of these stressed animals to locomotor stimulant- as well as to the rewarding
effect of direct or indirect DA agonists [9,31]. This set of data strengthens the hypothesis of a
dysfunction of the mesolimbic DA pathway in anhedonia. More interestingly, these authors
recently demonstrated that chronic antidepressant treatment reverses the CMS-induced
D2/D3 receptor down-regulation. Furthermore, it appears that a wide range of antidepressant
treatments (TCA, MAOI, ECT and SSRIs), but not unrelated drugs, reverses CMS-induced
anhedonia [9,26,32-34]. Using the sucrose intake paradigm, Muscat et al. [26] provided
evidence of a direct link between sensitization of D2/D3 receptors and anti-anhedonia effect
of antidepressants (fluoxetine and maprotiline). Since most of the drugs used in the
aforementioned studies have little, or no, direct effect on DA activity, one may suggest that
their reversal of DA receptor subsensitivity may be achieved by acting on other
neurotransmitter pathways (e.g. 5-HT; see table 1).
Serotonin/Dopamine Interaction and Antidepressant Drugs
As mentioned above, a great deal of data has demonstrated the existence of a functional
interaction between central 5-HT and DA systems. Moreover, the importance of the
mesolimbic dopaminergic system as a neurobiological substrate involved in the
pathophysiology of depressive illness has been already stressed. Thus, in view of the
hypothesis that disinhibition of the mesolimbic DA system underlies the mechanism of action
of several antidepressant drugs [35-37] the disinhibitory effect of SB 206553 and SB 242084
on the mesolimbic DA system might open new possibilities for the employment of 5-HT2C
receptor antagonists as antidepressants [38-42]. This hypothesis is consistent with the
suggestion that 5-HT2C receptor blockers might exert antidepressant activity [43]. In this
respect, it is interesting to note that several antidepressant drugs have been shown to bind
with submicromolar affinity to 5-HT2C receptors in the pig brain and to antagonize mCPPinduced penile erections in rats, an effect mediated through the stimulation of central 5-HT2C
135
receptors [44-46]. Based on those findings, Di Matteo et al. [40] have carried out experiments
showing that acute administration of amitriptyline and mianserin, two antidepressants with
high affinity for 5-HT2C receptors, enhances DA release in the rat nucleus accumbens by
blocking these receptor subtypes, in addition to their other pharmacological properties.
Interestingly, amitriptyline and mianserin have been tested in the chronic mild stress-induced
anhedonia model of depression and were found to be effective in reversing the stress effects
[33,47]. The antianhedonic effects of tricyclic antidepressants, mianserin and fluoxetine were
abolished by pretreatment with D2/D3 receptor antagonists, thus indicating an involvement
of DA in the antidepressant effect of various drugs in this model [9,47]. The chronic mild
stress procedure, which induces a depressionlike state in animals, was shown to enhance 5HT2C receptormediated function, as measured in vivo by mCPP induced penile erections. In
contrast, two different antidepressant treatments (72-h REM sleep deprivation and 10-day
administration of moclobemide, a reversible inhibitor of monoamine oxidase type A) resulted
in a reduction of this 5-HT2C receptor-mediated function [48], supporting the hypothesis that
the 5-HT2C receptor may be altered, and presumably may exist in a dysregulated
(hypersensitive) state in depressive illness [49]. In this respect, it is interesting to note that
chronic treatment with 5-HT2 agonists or antagonists resulted in a paradoxical downregulation of 5-HT2A or 5-HT2C receptors [49-52] and it seems that the down-regulation state
obtained after chronic exposure to mianserin in isolated systems as well as in cell cultures, is
a direct receptor-mediated mechanism of this drug at these receptors [52]. Therefore, the
down-regulating capacity of 5-HT2C agonists and antagonists may play a particularly
important role in reversing the 5-HT2C receptor system supersensitivity which presumably
results from a depressive state [44,49]. The possible involvement of 5-HT2C receptors in the
pathogenesis of depressive disorders and in the mode of action of antidepressants is further
substantiated by several other observations. For example, acute administration of fluoxetine
caused a dose-dependent inhibition of the firing rate of VTA DA neurons, but it did not affect
the activity of DA cells in the SNc [53]. A similar effect, though less pronounced, has been
observed with citalopram [53]. Furthermore, mesulergine, an unselective 5-HT2C receptor
antagonist [54], as well as the lesion of 5-HT neurons by the neurotoxin 5, 7-DHT, prevented
the fluoxetine-induced inhibition of VTA DA cells [53]. These results indicate that fluoxetine
inhibits the mesolimbic DA pathway by enhancing the extracellular level of 5-HT, which
would act through 5-HT2C receptors [53]. This study also demonstrated that fluoxetineinduced inhibition of DA neurons in the VTA was no longer observed after chronic treatment
(21 days) with this drug. Interestingly, mCPP inhibited the firing activity of VTA DA
neurons in control animals but not in those chronically treated with fluoxetine [47]. The
authors suggested that 5-HT2C receptors might be down regulated after repeated fluoxetine
administration. Consistent with this hypothesis is the evidence that chronic treatment with
fluoxetine, sertraline, citalopram, paroxetine and fluvoxamine, five selective serotonin
reuptake inhibitors (SSRIs), induces tolerance to the hypolocomotor effect of mCPP [55-59].
This hyposensitivity of 5-HT2C receptors might be a key step for the achievement of an
antidepressant effect. Indeed, it is possible to argue that the acute inhibitory effect of
fluoxetine on mesolimbic DA system would mask its clinical efficacy in the early stage of
treatment. This masking effect would disappear when the hyposensitivity of 5-HT2C receptors
occurs. In this regard, it is interesting to note that a series of studies carried out in our
136
laboratory have shown that acute administration of SSRIs such as paroxetine, sertraline, and
fluvoxamine causes a slight but significant decrease in the basal firing rate of VTA DA
neurons [60]. Therefore, it is conceivable that, similarly to fluoxetine, these three SSRIs
could reduce mesocorticolimbic DA transmission by activating 5-HT2C receptors. Behavioral
effects of antidepressant drugs also support the role of this receptor in the pathogenesis of
depression: in the modified rat forced swimming test, three selective 5-HT2C receptor
agonists, WAY 161503, RO 60- 0175 and RO 60-0332, were demonstrated to have
antidepressant activity, similar to that of fluoxetine [61]. In addition, interactions between
several doses of RO 60-0175 and antidepressant drugs, such as tricyclics and SSRIs were
found effective in the mouse forced swimming test [62] and antagonism at 5-HT2C receptors
significantly enhanced the anti-immobility effects of imipramine in the same mouse test [63].
It was also demonstrated that a 5-HT2C receptor mutation may contribute to the pathogenesis
of major depression and bipolar disorder [64]. Interestingly, in a recent clinical study [65,66]
agomelatine, an agonist of the human cloned melatonergic (MT) receptors as well as
antagonist of the human cloned 5-HT2C receptors was found effective in treating severe
depression associated with anxiety symptoms, with a better tolerability and lower adverse
effects than other antidepressants such as paroxetine. It is now well established that the
antidepressant effect of agomelatine, which has been clearly shown in several pre-clinical
behavioural tests in laboratory animals, depends on its combined activation of MT receptors
and blockade of 5-HT2C receptors [67-69], giving rise to the MASSA concept (Melatonin
Agonist and Selective Serotonin Antagonist). Interestingly, agomelatine significantly
increases extracellular DA and noradrenaline (NA) levels, as measured by intracerebral
microdialysis, in the frontal cortex of freely moving rats [70]. The increases of DA and NA
extracellular levelswere unaffected by the selective MT receptor antagonist N-[2-(5-ethylbenzo[b]thien-3-yl)ethyl] acetamide (S22153) and likely reflect disinibition of dopaminergic
and noradrenergic pathways innervating the frontal cortex, as a consequence of 5-HT2C
receptor blockade by agomelatine [70]. The antidepressant efficacy of agomelatine has been
demonstrated in comparison with placebo at various levels of severity of depression. Indeed,
it appears that the treatment effect of agomelatine tends to increase with the severity of the
depression [71]. The results also indicate an early improvement of depressive symptoms and
good response rates. Agomelatine has also been shown to have a comparable efficacy profile
to the SSRI paroxetine and the SNRI venlafaxine. The novel mode of action of agomelatine,
involving melatonergic agonism and 5-HT2C antagonism, has interesting consequences in
terms of clinical benefits. The low rate of adverse events observed with agomelatine contrasts
with the SSRIs and SNRIs, for which the release of serotonin and noradrenaline can cause
gastrointestinal, central nervous system, and cardiovascular side effects. Furthermore,
agomelatine avoids treatment-related sexual dysfunction and has positive effects on the relief
of sleep disturbances in depression. Indeed, agomelatine improves sleep quality without
sedation, and even improves daytime condition [72]. Together with the absence of a
discontinuation syndrome upon abrupt cessation of treatment, this favourable profile should
have positive consequences on the adherence to treatment of depressed patients. In
conclusion, agomelatine appears as a clear option in the treatment of MDD patients for whom
antidepressant efficacy is required without the tolerability problems of the currently available
antidepressants. Thus, agomelatine might represent the prototypical compound of a new class
137
of antidepressant drugs with a mechanism of action completely different from that of the
antidepressants available so far [71,73]. Another emerging characteristic of 5-HT2C receptor
antagonists is their newly discovered capability to potentiate the effects of SSRIs [130]. Thus,
agomelatine was observed to produce a robust augmentation of citalopram-, fluoxetine-, and
sertraline-induced elevations of hippocampal extracellular 5-HT levels [130]. The
potentiation of SSRI-induced increases in hippocampal 5-HT levels was reproduced by the 5HT2C receptor-selective antagonists SB 242084 and RS 102221, but not by the 5-HT2A
receptor-selective antagonist MDL 100,907. Although 5-HT2C receptor antagonists
potentiated the actions of SSRIs, they had no effect on extracellular 5-HT levels or tail
suspension responses when administered alone. These results were in good accord with
independent findings using a line of 5-HT2C receptor null mutant mice [74]. Although this
mutation did not affect baseline extracellular 5-HT levels or tail suspension test (TST)
behaviour, it enhanced fluoxetine-induced effects on 5-HT levels and immobility in the TST.
Since 5-HT2C receptors have recently been found to be expressed on GABAergic neurons
(but not on 5-HT-containg cells) of the anterior raphe nuclei [75], it is conceivable that
blockade of these receptors could disinhibit serotonergic neurons. This hypothesis is
strengthened by the evidence that stimulation of 5-HT2 receptors by serotonin activates local
GABA inhibitory inputs to 5-HTcontaing neurons in the dorsal raphe nucleus, an effect
which is partly mediated by 5-HT2C receptor subtypes [76]. Whether 5-HT/DA interaction is
involved in the mechanism of augmentation of SSRI antidepressant effect exerted by 5-HT2C
receptor antagonists is presently unclear, and it remains to be determined. Nevertheless, it is
tempting to speculate that blockade of 5-HT2C receptors would increase the serotonergic tone
to VTA DA neurons, which would be excited by the excess of 5-HT acting on free 5-HT2A
receptors. Thus, the pharmacological blockade of 5-HT2C receptors would favor an
unbalanced excitatory effect of 5-HT acting through 5-HT2A receptors whose stimulation is
known to cause a direct depolarization of DA-containing neurons in the VTA [77]. However,
this hypothesis needs experimental demonstration to be confirmed. Another important
contribution to the research regarding 5-HT/DA interaction and the effects of antidepressant
drugs comes from the data obtained on a strain of laboratory rats called Flinders Sensitive
Line (FSL) which represents a suitable animal model of depression [78]. The characteristic
marker for depressive-like behavior in FSL rats is increased immobility time during the
forced swimming test, which is normalized to the level of control Sprague-Dawley rats by
daily treatment with nefazodone, desipramine or paroxetine for 7 or 14 days [78-80].
Moreover, FLS rats have low basal extracellular levels of DA, which can be restored to the
level of Sprague-Dawley rats by 14-day desipramine treatment [80]. However, 7-day
treatment with nefazodone (a serotonin/noradrenaline reuptake inhibitor and 5-HT2C
antagonist) as well as 7-day and 14-day treatments with the tricyclic antidepressant
desipramine increased extracellular DA levels in the nucleus accumbens of FLS rats [80]. On
the basis of these data, Dremencov et al. [80] have suggested that the absence of 5-HTinduced DA release that was observed in the nucleus accumbens of FLS rats might be
explained by an increased inhibitory-like effect of the 5-HT2C receptor on DA release.
Repeated antidepressant treatment may restore the impaired 5-HT-induced DA release of FLS
rats to the level of Sprague-Dawley rats by attenuating the increased inhibitory-like activity
of 5-HT2C receptors [8].
138

Table 1. Second Generation Antidepressants
Selective serotonin reuptake inhibitors
- Fluoxetine
- Fluvoxamine
- Paroxetine
- Sertraline
- Citalopram
Selective noradrenaline reuptake inhibitors
- Reboxetine
- Nisoxetine
- Atomoxetine
Serotonin and noradrenaline reuptake inhibitors
- Venlafaxine
- Duloxetine
- Milnacipram
Serotonin/noradrenaline reuptake and 5-HT2 receptor inhibitors
- Trazodone
- Nefazodone
Noradrenergic and specific serotonergic antidepressant
- Mirtazapine
5-HT1A receptor partial agonists
- Buspirone
- Ipsapirone
- Gepirone
Presynaptic DA receptor blockers
- Amisulpride
- Supiride
Melatonin receptor agonist/5-HT2C antagonist
- Agomelatine
CONCLUSIONS
This review has focused on the involvement of 5-HT and DA in the mechanism of action
of antidepressant drugs, in particular of novel antidepressant compounds. It is now well
established that antidepressant drugs exert their therapeutic action only after chronic
139
treatment. This peculiar effect is ascribed to their ability, following repeated administration,
to induce adaptative changes of central monoaminergic transmission and, in particular, of the
5-HT and DA systems. It is suggested that drugs acting primarily on the serotonergic system
such as the SSRIs and 5-HT2C receptor antagonists would exert their antidepressant action by
enhancing dopaminergic transmission in the mesolimbic system. It is concluded that the use
of compounds which disinhibit mesolimbic DA transmission might be useful in the treatment
of depression.
ACKNOWLEDGEMENTS
This work was supported by the Italian MIUR (Ministero Istruzione Universit Ricerca)
L488/92 project n. s209-p/f.
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ISBN: 978-1-60021-820-0
Chapter VII
A POSSIBLE ROLE FOR INTRACELLULAR

PATHWAYS ACTIVATION IN THE MODULATION
OF LEARNING AND MEMORY PROCESSES BY THE
DOPAMINERGIC AND OPIOID SYSTEMS
INTERACTION
M. Costanzi1,, V. Cestari1,2 and C. Castellano1
1
Institute of Neuroscience CNR, via del Fosso di Fiorano, 64 00143 Roma, Italy;
Facolt di Scienze della Formazione, LUMSA University, Piazza delle Vaschette, 101
00193 Roma, Italy.
ABSTRACT
Dopaminergic and opioid mechanisms have been extensively studied for their role in
modulating learning and memory processes. The dopaminergic system plays an
important role in the emotional response to rewarding stimuli as well as in learning and
memory processes following psychostimulant administration. As concerns the
intracellular pathway activated by psichostimulant drugs, it has been shown that the
administration of dopaminergic agonists (i.e. amphetamine and cocaine) activated ERK
proteins in the striatum. A number of studies have shown that the opioid system
modulates the memory consolidation processes and that this activity is related to the
dopaminergic function. Recently, it has been observed that opioid receptor stimulation
induces ERKs phosphorilation through G protein-coupled activation in the striatal
neurons. Taken together these findings suggest a pivotal role for ERKs in the
intracellular mechanisms involved in the long lasting behavioural modification induced
by drugs of abuse that contribute to the development of addiction. Thus, the ERK
proteins might represent a possible candidate for intracellular modulation of the
Correspondence concerning this article should be addressed to: M. Costanzi, Institute of Neuroscience CNR, via
del Fosso di Fiorano, 64 00143 Roma, Italy. E-mail: m.costanzi@ipsifar.rm.cnr.it.
146

interaction between opioid and dopaminergic systems in learning and memory processes
linked to the addicted behaviour.
Some preliminary results obtained in our laboratory showed that ERK1 null mutant
mice submitted to the active avoidance task are not affected by the posttraining
administration of D1 dopamine receptor antagonist (SCH 23390), as well as by mu
opioid receptor agonist (morphine), while both treatments improve the performance of
wild type mice. Thus, the possible pivotal role of ERK1 on the behavioural effect exerted
by both dopaminergic and opioid system can not be ruled out.
Overall, the understanding of the intracellular mechanisms involved in the possible
interaction between these neuromodulatory systems might be crucial for both studying
and developing new strategies to better clarify the learning-reward processes linked to
the addicted behaviour.
The corticostriatal dopaminergic system is involved in long-term plasticity and rewardrelated learning (Berke and Hyman, 2000; Hyman and Malenka, 2001; Hyman et al., 2006).
The importance of studying the role of dopaminergic system in memory modulation comes
also from researches showing that the process of addiction shares similarities with neural
plasticity associated to learning and memory (see Kelly, 2004 for a review). In particular,
many studies indicate that chronic (or even acute) exposure to drugs of abuse modifies, in a
long-term manner, some intracellular signalling proteins in brain structures that are relevant
for learning and memory (Yao et al., 2004; Mato et al., 2004; Saal et al., 2003; Ghasemzadeh
et al., 2003; Melis et al., 2002; Ungless et al., 2001).
Considering memory formation, several studies have shown that post-training
administration of psychostimulants increases the consolidation process (Simon and Setlow,
2006; Brown et al., 2002; Puglisi-Allegra et al., 1994; see also Di Chiara et al., 2004 for a
review). Since these drugs are active on the dopaminergic system a role of dopamine in
memory consolidation has been envisaged (Di Chiara et al., 2004; Castellano et al., 1994;
Castellano et al., 1991; see also Castellano et al., 1996 for a review). For instance, there are
two classes of dopamine receptors in the central nervous system of vertebrates: D1-types
(that include D1 and D5 dopamine receptors) and D2-types (that include D2, D3 and D4
dopamine receptors). The striatum has a very high density both in D1 and D2 dopamine
receptors (Neve and Neve, 1997).
Post-training administration of both D1 (SKF 38393) and D2 (LY 171555) dopaminergic
receptor agonists dose-dependently impaired memory formation in DBA mice submitted to
an inhibitory avoidance task while the D1 (SCH 23390) and D2 ((-)-sulpiride) dopaminergic
receptor antagonists improved memory formation for this task (Castellano et al., 1991;
Castellano et al., 1994). The opposite effect was observed in C57 mice submitted to the same
experimental procedure (Ciamei et al., 2000; Castellano et al., 1999), suggesting that
different strain-dependent distribution of D1 and D2 receptors in the brain might explain the
opposite effect observed in C57 and DBA mice. Alternatively, an opposite strain-dependent
effect of dopamine receptor activation on second messengers cannot be ruled out (PuglisiAllegra et al., 1994; Castellano et al., 1991).
As concerns the intracellular mechanisms modulated by stimulation of dopamine
receptors, it has been observed that D1 and D2 receptors play opposite roles on the activation
of downstream pathways. D1 receptors are coupled to G-proteins that stimulate the adenylate
147
cyclase followed by an increase in cAMP second messenger whereas D2 receptors are

coupled to G-proteins that inhibit the adenylate cyclase decreasing the downstream proteins
phosphorilation and early genes induction. (see Berke and Hyman, 2000 for a review).
Recently, it has been extensively demonstrated that the behavioural effects exerted by
drugs of abuse, such as cocaine, are mediated by activation of ERK proteins into
mesocorticolimbic projection areas (i.e. nucleus accumbens, prefrontal cortex, amygdala and
bed nucleus of stria terminalis) that originate by the ventral tegmental area (Janab et al.,
2005; Radwanska et al., 2005; see also Lu et al., 2006 for a review).
Valijent and collaborators found that acute administration of cocaine activates ERK
proteins in the striatum and that this activation was blocked by a pre-treatment with a D1
receptors antagonist (SCH 23390) but not by a D2 receptors antagonist S(-)-raclopride.
Moreover, the MEK/ERK inhibitor (SL327) administration decreased both the
hyperlocomotion and the rewarding effect exerted by cocaine, suggesting a role for the ERK
pathway in the intracellular events underlying behavioural responses induced by cocaine
administration (Valijent et al., 2000).
More recently it has been observed that ERKs activation in the striatum is a common
effect of the action of different drugs of abuse and that their activation requires coincident
stimulation of both D1 dopamine- and NMDA glutamate receptors, providing a basis for
integration of the signals generated by mesostriatal and corticostriatal pathways (Valjient et
al., 2005; 2004; 2000). For instance, it has been shown that the stimulation of mu opioid
receptors modulates the phosphorilation of the ERK/MAPK cascade through G proteincoupled receptor kinase in striatal neurons (Macey et al., 2006; Haberstock-Debic et al.,
2005). Interestingly, some studies have shown that the dopaminergic system have a pivotal
role in mediating the effect exerted by systemic administration of cannabinoid and opioid
receptors agonists on memory formation in mice (Castellano et al., 1994; Costanzi et al.,
2004; see also Castellano et al., 1991 for a review).
Taken together, the above reported results suggest that the ERK/MAPK cascade could
play a key role in the intracellular integration of signals coming from different receptors
known to mediate the effect of drugs of abuse like morphine and cocaine.
In our laboratory, it has been observed that mice lacking ERK1 protein showed higher
levels of both memory consolidation (Mazzucchelli et al., 2002) and reconsolidation (Cestari
et al., 2006) in comparison to wild type mice in tasks characterized by a strong emotional
component. Moreover, these mice showed an enhanced place preference conditioning for
morphine and a significant increase of long-term potentiation recorded in striatal neurons that
correlate with a stimulus-dependent increase of ERK2 signalling at the cellular level
(Mazzucchelli et al., 2002). This observation suggests the existence of a regulatory role for
ERK1 in the long-term changes underlying striatum-dependent behavioural plasticity and
drug addiction. Moreover, it has recently been observed that ERK1 deletion facilitates the
development of both cocaine-induced psychomotor sensitization and cocaine place
preference in ERK1 knockout mice (Ferguson et al., 2006).
These results show the importance of combining pharmacological studies with the gene
targeting approach in order to clarify the role of the intracellular mechanisms necessary for
the establishment of neuronal changes produced by drugs of abuse.
148
In particular, the understanding of the intracellular mechanisms involved in the

interaction between dopaminergic and opioid systems might be crucial to better elucidate the
learning processes linked to the addiction behaviour.
In this context, we carried out a preliminary study in order to better clarify the possible
role in procedural learning of the two ERK proteins in the intracellular transduction of signals
coming from both the dopaminergic and the opioid systems. For this purpose, we have
intraperitoneally injected ERK1 mutant mice with either the D1 dopaminergic antagonist
(SCH23390) or the mu opioid agonist (morphine). Both ERK1 knockout and control mice
injected with vehicle, morphine (20mg/kg, i.p.) or SCH23290 (0.1 mg/kg, i.p.) have been
submitted to a two-way avoidance test.
The two-way avoidance is an operant conditioning task particularly sensitive to the longterm synaptic modification occurring into the nucleus accumbens (Schutz and Izquierdo,
1979; Taghzouti et al., 1985; Salamone, 1994). This behavioural paradigm is a measure of
associative emotional learning and reinforcer-driven control of motor activity (Clincke and
Webrouck, 1993; Mazzucchelli et al., 2002). In our procedure, all mice submitted to the
training procedure (5 days, 100 trials per day) were injected with drugs immediately after
every daily session.
The results (Figure 1) show that both the mu opioid agonist and the D1 dopaminergic
antagonist administration enhances the procedural memory only in wild type mice, while no
significant differences are found between vehicle- and drugs-injected ERK1 knockout mice.
These results suggest that ERK1 has a pivotal role for the intracellular transduction of the
signals coming from both opioid and dopamine receptors during learning and memory
processes.
Since ERK1 null mutation increases under stimulation the intracellular ERK2 activation
and both morphine and SCH23290 administration enhance memory consolidation only in
wild type mice submitted to a two-way avoidance task, some hypotheses can be made.
First, considering the blockade of D1 receptor by the antagonist administration, it seems
reasonable to hypothesize that the behavioural effects related to dopamine transmission on
procedural memory formation are D2 mediated. Thus it is possible that G-proteins coupled to
the D2 receptors inhibit ERK1 phosphorilation followed by an increase in ERK2 activity.
This mechanism could explain the memory enhancement in wild type mice, but not in the
ERK1 null mutant mice.
Second, since it has been observed that the administration of drugs of abuse, such as
heroin, morphine, cocaine and amphetamine increases the extracellular levels of dopamine in
the shell of the nucleus accumbens (Cadoni and Di Chiara, 1999; Pontieri et al., 1996; 1995)
it can be hypothesized that the dopamine increase in the nucleus accumbens due to morphine
administration might inhibit ERK1 phosphorilation. Also in this case, the effects observed in
this study on memory consolidation could be ascribed to an increase of ERK2 activity in wild
type mice but not in ERK1 null mutant mice.

90
AVOIDANCE RESPONSES (% +/- SEM)
80
+/+, sal
+/+, sch 0.1 mg/Kg
-/-, sal
70
149
-/-, sch 0.1 mg/Kg
60
50
40
30
20
10
0
day1
day2
day3
day4
day5
90
+/+, mo 20 mg/Kg
80
+/+, sal
-/-, mo 20 mg/Kg
AVOIDANCE RESPONSES (% +/- SEM)
70
-/-, sal
60
50
40
30
20
10
0
day1
day2
day3
day4
day5
Figure 1. Effects of posttraining SCH 23390 (0.1 mg/Kg, i.p.; Sch 0.1) and morphine (20 mg/Kg, i.p.,
mo 20) administrations in ERK1 knockout (-/-) and wild type (+/+) mice submitted to a two-way
avoidance task. A) Posttraining administration of SCH23390 enhanced the performance in +/+ mice.
Statistical analysis (ANOVA three-way) showed a significant interaction between genotype and
pharmacological treatment (F(1,64) = 7.725 p<.05), a significant effect of the training (days)
(F(4,256)=84.54 p<.001) and a significant interaction between the three factors (F(4,256)= 2.13 p<.05).
Post hoc analysis (Duncans test) showed that SCH administration enhanced the performance in +/+
mice (open circles) in comparisons to saline-injected +/+ mice (filled circles). No significant differences
have been found between saline- (filled squares) and SCH-injected (open squares) -/- mice. B)
Posttraining administration of morphine enhanced the performance in +/+ mice. Statistical analysis
(ANOVA three-way) showed a significant interaction between genotype and pharmacological treatment
(F(1,62) = 3.69 p<.05), a significant effect of the training (days) (F(4,248)=130 p<.001) and a
significant interaction between the three factors (F(4,248) = 2.54 p<.05). Post hoc analysis (Duncans
test) showed that mo20 administration enhanced the performance in +/+ mice (open circles) in
comparisons to saline-injected +/+ mice (filled circles). No significant differences have been found
between saline- (filled squares) and mo20-injected (open squares) -/- mice * p<.05.
150
However, further studies have to be carried out in order to better clarify the actual role of
ERK1 in the effects exerted by dopaminergic and opioid systems and their interaction on
memory consolidation. The experimental approach used in this study can contribute to
elucidate these mechanisms and the learning-reward processes linked to the addicted
behaviour.
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Costanzi M. et al., (2004) Neurobiol. Learn. and Mem., 81: 144-9
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Ferguson S.M. et al., (2006) Neuropsychoprmacol. 31(12): 2660-8
Ghasemzadeh M.B. et al., (2003) Eur. J. Neurosci., 18: 1645-51
Haberstock-Debic H. et al., (2005) J. Neurosci., 25:7847-57.
Hyman S.E. and Malenka R.C. (2001) Nat. Rev. Neurosci., 2: 695-703
Hyman S.E. et al., (2006) Annu. Rev. Neurosci., 29: 565-98
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Lu L. et al., (2006) Trends in Neurosci. 29(12): 697
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Melis M. et al., (2002) J. Neurosci, 22: 2074-2082
Neve K.A. and Neve R.L. (1997) In The Dopamine Receptors (Human Press) p27-76
Pontieri F.E. et al., (1995) Proc. Natl. Acad. Sci. USA, 92: 12304-8
Pontieri F.E. et al., (1996) Nature, 382: 255-257
Puglisi-Allegra S. et al.(1994) Psychopharmacol 115: 157-62.
Radwanska K. et al., (2005) Eur. J. Neurosci., 22: 939-948
Saal D. et al., (2003) Neuron, 37: 577-82
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Schutz R.A. and Izquierdo I. (1979) Physiol. Behav. 23: 97105.
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Valjent E. et al., (2000) J. Neurosci., 20: 8701-9
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151

ISBN: 978-1-60021-820-0
Chapter VIII
DOPAMINE SYSTEM AND ITS MODULATION BY

NITRIC OXIDE: APPROACHES IN
EXPERIMENTAL PARKINSON AND
SCHIZOPHRENIA
Cristiane Salum, Marcela Bermdez-Echeverry, Ana Carolina Issy,
and Elaine A. Del-Bel
Department MEF- Physiology, FORP, University of So Paulo, Ribeiro Preto, SP,
Brazil.
ABSTRACT
The influence of dopamine (DA) in mammalian and invertebrate neural processes
has been extensively documented. The mesencephalic dopaminergic neurons have key
roles in sensorimotor integration, motor behavior and in the modulation of behavioral
responses to positive and negative reinforcement. It is now known that nitric oxide (NO),
an atypical neurotransmitter, mediates a number of neuronal processes including the
regulation of dopaminergic neurotransmission. NO has been implicated in several
behavioral pathologies concerned with dopaminergic imbalance, such as Parkinsons
disease (PD) and schizophrenia. Although the nature of the NO-mediated modulatory
influence on DA neurotransmission includes some conflicting neurochemical
observations, a growing body of literature indicates that NO, by its signaling mechanisms
and effector pathways, exerts a primary facilitatory influence over tonic and phasic
dopaminergic neurotransmission under physiological conditions. There is considerable
evidence indicating that NO also inhibits DA uptake, thus modulating DA-controlled
behaviors. Additionally, NO may interact with DA modifying not only its regulatory
actions but also producing oxidants and free radicals that are likely to trigger toxic
Correspondence concerning this article should be addressed to: Cristiane Salum, Department MEF- Physiology,
FORP, Campus USP, Av. Caf S/N, 14040-904, Ribeiro Preto, SP, Brazil. Phone: +55-16-36024050; Fax: 163633 2301; Email: crisalum@uol.com.br.
154
Cristiane Salum, Marcela Bermdez-Echeverry, Ana Carolina Issy et al.

pathways in the nervous system. Thus, the chemical interaction between DA and its
metabolites with NO components constitutes a source of neurotoxic molecules, which
may contribute to the cellular process of neurodegeneration. Consequently, the
interaction between these systems has become a potential target for exploring the
neurochemical basis of some neuropsychiatric diseases. In particular, there is a great
interest in investigating PD and schizophrenia via the underlying processes which control
motor behavior, attentional and information processing deficits. Increased mesolimbic
DA following administration of amphetamine-like drugs to rodents is coupled with
hyperlocomotion, deficit in sensorimotor filter, stereotyped behaviors and also provokes
attentional dysfunction. Inhibition of nitric oxide synthase (NOS) has been shown to
prevent many of these effects. Moreover, the cataleptic effect of DA antagonists, like
haloperidol, can be mimicked by NOS inhibitors. This chapter first summarizes
neurochemical aspects of DA and NO neurotransmission and reviews a broad spectrum
of mechanisms by which nitrergic system may influence the dopaminergic
neurotransmission. Supporting evidence is presented for the involvement of NO in
behavioral conditions controlled by DA. Finally, the modulation of dopaminergic
functions by NO in behavioral models of neuropsychiatric diseases is demonstrated
focusing on motor and attentional dysfunctions which can occur in PD and
schizophrenia, respectively.
INTRODUCTION
Dopamine (DA), initially considered to be only an intermediate in the biosynthesis of
norepinephrine and epinephrine, is the most recently discovered catecholamine transmitter in
the mammalian brain. Early studies demonstrated that in Parkinsons disease (PD) the amount
of DA depletion in the brain, most of which is confined to the basal ganglia, is directly
correlated to motor deficits observed [1]. This led to the hypothesis that DA might have a key
involvement in motor control. Those findings were the starting point for a massive series of
investigations over the past four decades, which are still in progress, guiding the development
of L-dihydroxyphenylamine (L-DOPA) therapy and other types of medications to improve
PD patients symptoms.
In the meantime, the discovery of neuroleptics as effective drugs to reduce schizophrenia
symptoms and as potent DA receptor blockers provided support for a dopaminergic
overactivity hypothesis of schizophrenia [2]. Neuroleptics were soon found to induce severe
extrapyramidal side effects, later named parkinsonism-like syndrome. Further evidence for
the DA hypothesis came from the observations that DA agonists, like amphetamines, can
induce paranoid psychosis and exacerbate schizophrenia [3].
DA has also been linked to behavior concerning reinforcement learning, motivation and
drug abuse. In this respect, DA neurons have been shown to fire in response to reward and
reward-predicting stimuli [4,5]. Additionally, several studies have suggested that selfadministration of psychostimulants in animals such as amphetamine (Amph) and cocaine
depends on activation of the dopaminergic system in the nucleus accumbens [6]. Therefore,
the dopaminergic system has been crucially involved in the use of reward information for
learning and maintaining approach and consummatory behavior. Besides, there is evidence
showing that DA can be released in the nucleus accumbens during stress, by aversive stimuli
Dopamine System and its Modulation by Nitric Oxide
155
and stimuli conditioned to them [7]. In fact, DA neuron activation appears to correlate with
the detection of salient stimuli, and DA release is claimed not to be determined by the
direction or subjective quality of the stimulus, but rather by its behavioral relevance [8].
Neurochemical Aspects of Dopamine
DA synthesis originates from the amino acid tyrosine, which is converted to L-DOPA by
the enzyme tyrosine hydroxylase (TH) using molecular oxygen and tetrahydrobiopterin (BH4)
[9]. Subsequently, L-DOPA is decarboxylated, by the enzyme L-aromatic amino acid
decarboxylase, to form DA. Thus, the rate of DA synthesis is modulated by TH activity (a
rate-limiting enzyme). In turn, TH activity can be attenuated by (1) activation of synthesismodulating autoreceptors, (2) end-product inhibition by intraneuronal DA, and controlled by
BH4 availability. DA can be released in a phasic way, in response to behaviorally relevant
stimuli that activate DA neurons causing a large influx of Ca2+. Grace [10] proposed that
phasic DA activates postsynaptic DA receptors but is rapidly removed from the synaptic
space by fast, low-affinity/ high-capacity reuptake systems. There is also a tonic DA release
mediated by glutamate (GLU) stimulation of N-methyl-D-aspartate (NMDA) receptors
located on DA neuron terminals. This tonic mechanism of DA release is proposed to exhibit a
prolonged time-course and underlie the background, steady-state level of extracellular DA in
subcortical structures. The tonic DA would contribute to regulatation of the intensity of
phasic DA release by activating presynaptic DA autoreceptors implicated in inhibiting DA
synthesis and release. This hypothesis has been extended by West and coworkers [11] by
introducing a nitric oxide (NO) regulation mechanism of DA neurotransmission, further
discussed in this chapter. Alternatively, another mechanism of DA release has been proposed
to involve the reverse-transport of the cytosolic amine by the carrier or DA transporter
(DAT), ordinarily responsible for uptake, which can operate in a reverse direction, thus
releasing DA from the nerve terminal [12]. In fact, DAT has an important role in the
inactivation and recycling of DA release into the synaptic cleft.
Conceptually, DAT is an integral membrane protein in DA nerve endings where it
provides the critical function of terminating the synaptic activity of DA through transport into
the presynaptic site [13]. DAT is a member of the SLC6 solute carrier gene family and its
structure includes a total of 13 Cys residues, arrayed throughout its membrane domains,
which are potential targets for peroxynitrite (ONOO-) [14], DA and Amph reactions [15]. It
has been found that Amph decreases DAT cell surface expression, which leads to a decrease
in DA uptake and possibly contributes to its ability to increase extracellular DA in vivo [15].
Since DA uptake capacity is dependent on (1) the DAT turnover rate, (2) the affinity of the
transporter for DA, and (3) the number of functional transporters at the cell surface,
regulation of DAT cell surface expression is important for tuning DA neurotransmission [16].
Advances in molecular biology revealed a higher degree of complexity within DA
receptors than previously thought by the application of gene-cloning procedures to receptors.
There are currently five subtypes of DA receptors identified in the human nervous system,
which have been classified in two receptor families according to their biochemical
characteristics [17]. The D1-receptor family comprises D1 and D5 subtypes which both
156
stimulate adenylyl cyclase via the Gs protein to increase cAMP formation and the activity
cAMP-dependent protein kinase. The D2-receptor family comprises D2, D3 and D4 subtypes
which act by activating Gi inhibitory G-proteins, thereby inhibiting the formation of cAMP.
Specific subtypes of DA receptors have a distinct distribution in the brain [18]. Due to the
lack of ligands specific for each receptor subtype, in situ hybridization has been extensively
used to study the distribution of DA receptor mRNAs in the brain. In the DA cell body
regions, including substantia nigra (SN) and ventral tegmental area (VTA), a high density of
D2, but not D1, mRNA is detected. The absence of D1 receptors mRNA in these areas argues
against these receptors playing a role as autoreceptors. D2 receptors are also abundantly
expressed in the caudate putamen, nucleus accumbens and olfactory tubercle as postsynaptic
receptors. D1 receptors are also richly situated in caudate putamen, nucleus accumbens,
olfactory tubercle and additionally in amygdala. D3 and D4 receptors appear to be
concentrated within parts of the limbic system, specially nucleus accumbens and frontal
cortex, respectively. Finally, D5 receptors have a limited and unusual distribution mainly
detected in the hippocampus, thalamus and hypothalamus.
Dopaminergic neurons in the SN pars compacta, the VTA, and the hypothalamus give
rise to the three major dopaminergic pathways: nigrostriatal, mesolimbic and mesocortical.
The nigroestriatal tract, primarily involved in motor control, projects from the SN (A9) in the
midbrain to the (dorsal or neo) striatum, in particular the caudate nucleus and putamen.
Manifestations of Parkinsons disease (PD) are attributed to reduced dopaminergic input into
the striatum, due to neuronal degeneration in the pars compacta of the SN. Etiological factors
are complex. Environmental toxins and oxidative stress and mitochondrial dysfunction may
exert a contribution to nigral degeneration of dopaminergic neurons. In a comparable way,
parkinsonian side-effects of classical antipsychotics arise from the blockage of DA receptors
in the termination of the nigrostriatal tract. The mesolimbic tract has its origin in cell bodies
of the VTA (A8 and A10) adjacent to the SN. These cells project to several parts of the
limbic system, including the nucleus accumbens, amygdala, hippocampus, cingulated cortex
and entorhinal cortex. As mentioned above, the nucleus accumbens has an important role in
the reinforcing effects of certain sorts of stimuli and goal-directed behaviors, so it has been
involved in addictive behavior [19]. Additionally, the hyperactivity of this tract has been
proposed to be the cause of the positive symptoms of schizophrenia [20]. The neuronal cell
bodies of the mesocortical system are also located in the VTA. Their axons send excitatory
projections to the prefrontal cortex affecting functions such as formation of short-termmemories, motivation, attention, planning, and strategy preparation for problem solving
[21,22]. Several findings led Daniel Weinberger and coworkers [23] to postulate that the
latter two systems are differently affected in schizophrenia. First, there is an increased
activity of the mesolimbic pathway (probably mediated by the receptors D2, D3, and
particularly by D4 receptors), which drives the manifestation of positive symptoms. Second, a
reduced activity of the mesocortical connections with the prefrontal cortex would lead to the
manifestation of negative symptoms. According to this model, an imbalance between
dopaminergic neurotransmissions in cortical and subcortical regions would be the base for
the development of schizophrenia.
It was first proposed that DA plays a role in the limbic/motor interaction through
projections from the nucleus accumbens to the SN [24]. Studies using retrograde and
157
anterograde tracers revealed that rather than a direct limbic-motor connection, different
striatal subdivisions are linked by overlapping feedback to DA neurons forming an ascending
spiral between regions [25]. As suggested by these authors, such an anatomical arrangement
could account for the parallel psychomotor, affective, and cognitive disturbance seen in
several psychiatric disorders.
The striatum provides a powerful feedback regulation of DA neuron firing. In turn, the
striatal response to DA is heterogeneous and determined by multiple variables including
complex interactions between several receptors [26]. One system in particular that seems to
affect striatal activation, leading to an alteration in DA neuron activity, is the NO system.
Consequently, NO is one of the main candidates found to be linked to schizophrenia,
Parkinsons disease and other behavioral pathologies involved with dopaminergic imbalance.
Figure 1. Structural domains of NOS enzymes (modified from [31]). The three NOS isoforms have an
oxygenase and a reductase domain and consensus sites for the binding of different biochemical
cofactors including flavin mononucleotide (FM), flavin adenine dinucleotide (FAD), nicotinamide
adenine dinucleotide phosphate (NADPH) and calmodulin (CA). Four different nNOS peptides (, , ,
) are also shown.
Neurochemical Aspects of Nitric Oxide
NO is a reactive and highly diffusible free radical gas that is now recognized as a major
messenger molecule. NO regulates numerous physiological processes, including neuronal
158
communication, blood vessel modulation and immune response. In mammals, NO was first
identified as the endothelial-derived relaxing factor by Furchgott and Zawadzki [27]. NO
readily permeates cell membranes and it is rapidly removed by its diffusion through tissues
into red blood cells, where it is converted to nitrate by reaction with oxyhemoglobin. Unlike
most others endogenous chemical mediators, NO is not stored in vesicles and its signaling
specificity must be controlled at the level of synthesis. Indeed, the members of the nitric
oxide synthase (NOS) family are amongst the highly regulated known enzymes [28]. NOS
generates NO via the catalytic combination of aminoacid L-arginine and molecular oxygen,
with stoichiometric formation of citrulline. There are two constitutive isoforms of NOS:
neuronal NOS (nNOS or NOS1 the first cloned and purified) regulated by Ca2+ and
calmodulin and mainly localized in neuronal tissue, and endothelial NOS (eNOS or NOS3),
that is also a Ca2+/calmodulin-requiring enzyme isolated from endothelium; the inducible
isoform, Ca2+-independent NOS (iNOS or NOS2), is found in a variety of cells following
induction with inflammatory mediators and bacterial products [29]. There are different nNOS
mRNA splice isoforms that lead to the generation of four different peptides (nNOS , , , )
[30]. In the brain, the 160kDa nNOS is the predominant splice variant, and contains an Nterminal PSD/Discs-large/ZO-1 homologous (PDZ)-binding domain, which anchors this
complex to the postsynaptic density in the vicinity of the NMDA glutamate receptor (Figure
1) [31]. The isoforms of NOS are highly complex structures and all three NOS use
nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. In
formaldehyde-fixed tissue NADPH-diaphorase (NADPH-d) histochemistry provides the
localization of NOS activity [32,33]. The molecular structure, enzymology and pharmacology
of these enzymes have been well defined. Studies of NOS enzymes using knockout and
transgenic mouse models have provided an extensive contribution to define the biological
function of NO in mammals. Additionally, agents that modulate the activity of NO NOS
inhibitors or donors, may be of considerable therapeutic value.
Since NO has a relatively short half-life, quantitative assessment of its production has
generally relied on the indirect measurement of its oxidized products, nitrite and nitrate,
suitable markers of NO generation [34]. In the brain, NO acts as a neuromodulator to control
behavioral activity [35], influence memory formation, and intensify responses to painful
stimuli [36]. The discovery that NO is produced by neurons and regulates synaptic activity
has challenged the definition of a neurotransmitter. NO is synthesized postsynaptically and
does not act at conventional receptors on the surface of adjacent neurons. As mentioned, NO
released in the brain is typically linked to the activation of NMDA GLU receptors (Figure 2),
which is found in close physical proximity to the nNOS, and trigger long-term potentiation
(LTP) that may participate in learning and memory functions by the hippocampus and other
types of synaptic plasticity [37]. NO from nNOS can act as a retrograde messenger diffusing
to the presynaptic terminal and eliciting changes in the neurotransmitter release machinery, as
well as postsynaptic effects. The heme group of the soluble guanylyl cyclase (sGC) is the
major intracellular target of NO. It activates this enzyme and promotes the synthesis of cyclic
guanosine monophosphate (cGMP) (Figure 2). Subsequent binding of cGMP to protein
kinase, ion channels, phosphodiesterases and other proteins translates NO signals into long
and short-term functional alterations, including downstream mechanisms that lead to changes
in synaptic strength [38]. However, NO can exert its biological effects through other
159
mechanisms, such as modulating monoamine transporter function and S-nitrosylation of

receptors [39]. The S-nitrosylation occurs when NO reacts with the sulphur from a cysteine
thiol surrounded by specific amino acids, which favor nitrosylation [40]. It has been
demonstrated that NO can S-nitrosylate the NMDA receptor leading to its down-regulation
[41]. While NO normally functions as a physiological neuronal mediator, excessive
production of NO mediates brain injury. Overactivation of GLU receptors associated with
cerebral ischemia and other excitotoxic processes results in a massive release of NO. As a
free radical, NO mediates cellular toxicity by damaging critical metabolic enzymes and by
reacting with superoxide to form an even more potent oxidant ONOO- [42]. There are
scavenging enzymes called superoxide dismutases that may remove superoxide. However, in
normal conditions NO and superoxide will usually collide and form ONOO- in a reaction that
does not require enzymes [43]. Pathological conditions can greatly increase the production of
ONOO- resulting in substantial oxidation and potential destruction of host cellular
constituents. Therefore, the production of ONOO- can be an important feature in the
development of many pathological processes in vivo [43]. In the last two decades
uncountable researches tried to understand the nature of physiological and pathological NO
function.
Figure 2. Scheme of NO synthesis and its possible effects. Glutamate activates the NMDA receptors.
This causes Ca2+ influx via the NMDA receptor channel and the increase in intracellular Ca2+ activates
NO synthase. NO can diffuse from where it is synthesized into surrounding cells where it will activate
soluble guanylate cyclase (sGC) in the target tissue to produce cGMP. In turn, cGMP activates cGMP
dependent kinases, ion channels and phosphodiesterases to regulate diverse activities. NO can generate
peroxynitrite which may cause considerable oxidation, peroxidation and DNA damage.
Regulation of Dopaminergic Neurotransmission by NO
NO may influence dopaminergic neurotransmission or metabolism by: i) entering the cell

and stimulating the production of cGMP by the activation of sGC; ii) the nitrosylation of
thiol groups; iii) NO autoxidation; iv) NO-mediated oxidation of DA through the formation
of ONOO-; or v) interfering with monoamine transporters (Figure 3) [44-46].
It has been shown by multiples studies that the addition of NOS substrate(s) to biological
systems stimulates the production of endogenous NO beyond basal levels [47]. Zhu and Luo
[48] first demonstrated that NOS substrate infusions caused an increase in endogenous NO,
160
leading to DA overflow from striatal slice preparations. These findings have been replicated
and extended by many other studies using in vivo [49,50] and in vitro [44,51,52]
preparations. The NO-mediated increase in extracellular DA levels has been found to depend
on neuronal NOS activity, since it can be blocked by NOS inhibitors as 7-nitroindazole (7NI) or oxyhemoglobin [49,53]. Additional support for NO-mediated facilitation of DA
release stems from observations that the NOS inhibitors NG-nitro-L-arginine (L-NOARG) and
NG-nitro-L-arginine methyl ester (L-NAME) inhibited the methamphetamine-induced DA
release [54]. In contrast, there is evidence sustaining an inhibitory role of NO on DA release
[55]. NO donors may decrease DA and dihydroxyphenylacetic acid (DOPAC) during
methamphetamine exposure [54] and 7-NI may enhance Amph-evoked release of DA in rat
striatum [56]. Despite these results, the most consistent effect of NO on DA release is of
facilitation (see West et al., 2002 for a review). It is argued that the effect of NO and NO
donors on DA release may depend on a balance between the amount of NO diffusing across
the cell membrane and the amount of NO which either undergoes extracellular autoxidation
or induces extracellular DA nitration (Figure 3) [44,57,58]. DA-mediated release by
exogenous NO, both in vivo [59] and in vitro [50], appears to be dependent on external Ca2+.
In addition, it is a mechanism dependent on the activation of the NO/ sGC/ cGMP pathway,
given that the increase in DA concentrations elicited by NO-donors such as the S-nitroso-Nacetylpenicillamine (SNAP) may be inhibited by the sGC inhibitor 1H[1,2,4]
oxadiazolo[4,3]quinoxalin-1-one (ODQ) [44,60]. In fact, a recent in vitro study with
microdialysis from PC12 cell suspensions [44] showed that the infusion of the NO-donor
NOR-3 was only able to induce an increase in DA concentrations in the presence of ascorbic
acid. Since this antioxidant may inhibit either NO autoxidation, DA autoxidation, or NOmediated DA oxidation, the authors suggested that ascorbic acid has a key role in modulating
NO profiles. Therefore, the composition of the endogenous environment in which NO is
generated seems to regulate its biological actions [61]. Another investigation using in vivo
microdialysis revealed that NO can modulate extracellular concentrations of striatal
transmitters both by stimulating formation of cGMP and through conversion to ONOO- via
superoxide [60]. Overall, the predominant effects of NO (via sGC or ONOO-) has been used
to explain why NO and NO donors may increase DA concentrations through activation of
sGC [44,48-53] and decrease it through ONOO- formation [55,56,62].
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Figure 3. Scheme of possible ways by which NO may influence DA neurotransmission or metabolism,

depending on the redox state of where NO is generated. Release of GLU stimulates NMDA receptors
and the concomitant influx of Ca2+ (not shown) activates nNOS. NO synthesized by the enzyme spreads
over and may i) enter the cell and stimulate the production of cGMP; ii) undergo autoxidation; iii)
inhibit the function of DAT. Inside the DA neuron, NO activates the sGC / cGMP pathway, which may
result in extracellular Ca2+ entry and consequent activation of DA release. Secreted DA may undergo
NO-mediated oxidation, by a reaction with ONOO- (not shown), or nitration, under acid conditions. By
inhibiting DAT, NO may reduce DA uptake, thus increasing extracellular DA.
Endogenous NO is also able to inhibit the function of monoamine transporters [45]. The
effect of NO on monoamine transporters is believed to represent a new form of interneuronal
communication, that is, a nonsynaptic interaction which does not involve classical receptors
[63]. This nonsynaptic interaction may occur by influencing the function of a wide variety of
proteins. Pogun and colleagues [64] found that the NO-donor, sodium nitroprusside (SNP),
decreased [3H]DA uptake into rat striatal synaptosomal preparations and that this decrease
was due to a decrease in the velocity (Vmax) of DA transport. This was also determined to be
dose-, time- and temperature-dependent and could be prevented by hemoglobin, a NO
scavenger. A similar study [65] gave support to these findings demonstrating that the NOdonor S-nitroso-L-cysteine (NO-CYS) reduced DA uptake by diminishing the capacity of the
DA transporter without having a significant effect on the affinity, thus suggesting that the site
of action of the NO-donor is not identical to the DA recognition site of the transporter. In this
study, it was also suggested that striatal DA release-stimulation by NO-CYS was mediated by
reverse transport. Corroborating this concept, Kiss and Vizi [45] have shown that SNP
inhibited DA uptake, while L-NAME increased it in striatal tissue. These authors have
previously suggested a role for NO in the regulation of DA transporter function based on the
finding that L-NAME decreased the basal release of DA in the striatum of anesthetized rat,
and this effect was completely diminished in the presence of the DA uptake inhibitor
nomifensine [66]. In contrast, it has been suggested that the NO produced via NOS from the
infusion of L-arginine may interact with DAT leading to an increase in the velocity (Vmax) of
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DA transport [67]. This contradiction has been frequently attributed to differences in the
methodology used in each study, as well as the experimental conditions [68]. However,
recent studies have indicated that these divergences may be related to the redox state of the
brain tissue or preparation, which could affect the reaction of NO generated, the neurotoxic
or neuroprotective nature of NO and so the observed effects. In fact, NO generated by NO
donors may engender different effects and operate through different mechanisms of action
than NO produced from NOS. It has been suggested that DAT activity may be modulated by
interaction of NO with DAT Cys residues [64]. A possible explanation for NO inhibition of
monoamine transporters is by nitrosylation of cysteine (Cys) groups in DAT [69]. Moreover,
NO may interact with DA and other catecholamines modifying not only its regulatory actions
but also producing oxidants and free radicals that are likely to trigger toxic pathways in the
nervous system [70]. Thus, the chemical interaction between DA and its metabolites with NO
components constitutes a source of neurotoxic molecules, which may contribute to the
cellular process of neurodegeneration. Specifically, neurotoxicity as a result of oxidative
stress of basal ganglia structures has gained prominance in the etiology of Parkinsons
disease.
Basal Ganglia: DA and NO in the Modulation of the Striatal Complex
The basal ganglia have been the subject of major reviews for almost four decades,
considering their biochemical and functional organization implicated in motor control and
movement disorders. They comprise a group of gray matter structures deep in the brain
adjacent to the thalamus and hypothalamus. It is understood that they are responsible for
modulating and facilitating motor and cognitive programs [71].
The main structures controlling movement include the caudate nucleus and putamen (the
caudate-putamen are fairly undifferentiated in the rat but are separated by the internal capsule
in primates), the globus pallidus internal (GPi) and external (GPe) segments, the subthalamic
nucleus, and the SN (pars compacta-SNpc and pars reticulate-SNpr). The latter two structures
are generally considered to be part of the basal ganglia because they are closely related to the
striatopallidal neuronal circuitry [72]. There is evidence of three distinct pathways from
striatum to thalamus named the direct, the indirect and the hyperdirect pathways. The direct
pathway is inhibitory and passes monosynaptically from the striatum to GPi. The indirect
pathway reaches the same destination but synapses first in the GPe and then in the
subthalamic nucleus. Recently there has been growing evidence of a direct cortico
subthalamic nucleuspallidal pathway described as the hyperdirect pathway [73]. The basal
ganglia receive inputs from the neocortex and project massively to thalamic nuclei, which in
turn project to the frontal cortex (corticocortical loop). Striatal information is transferred also
to the SNpc which projects to the medial part of the thalamus complex (the parafascicular
nucleus), going back to the caudate nucleus (Nauta-Mehler's loop). GPi neurons provide
inhibitory inputs to the thalamus, the pedunculopontine nuclei and the superior colliculus.
The subthalamic nucleus (corpus Luysi), a relatively small nucleus located ventrally to the
zona incerta and dorsally to the cerebral peduncle, is a relay nucleus controlling pallidal
function [74,75].
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Considerable effort has been devoted to experimental analysis of the various striatal
neuroanatomical and neurochemical compartments with the expectation that the complex role
of the striatum in the organization of behavior and in particular for motor function may
possibly be illuminated. Striatum (caudate-putamen) is formed by two major divisions (i.e.,
dorsal and ventral striatum) that differ in the information processed and the particular
topography of their efferents. The ventral striatum (ventromedial or limbic striatum)
involves the nucleus accumbens and portions of the olfactory tubercle receiving allocorticalmesocortical input. The dorsal striatum (dorsolateral) comprises caudate nucleus or dorsal
striatum, which receives afferents from the sensorimotor cortex and DA-containing inputs
from the SNpc (neocortical input) [76,77]. Also, a comprehensive study has been carried out
to understand the striosomal (limbic circuit) and matrix (sensorimotor/associative circuit)
functional organization of the dorsal striatum [78].
In recent years, neurophysiological studies provided a great deal of information of striatal
activity concerning not only the physiological action of DA in the striatum, but also the
effects of nigral DA denervation. An imbalance in DA, the principal neurotransmitter
involved in the motor control, can cause extrapyramidal symptoms. A single dopaminergic
input to the striatum terminates at several locations on the dendritic spines of medium spiny
neurons [79,80]. Thus, cortical afferents that terminate on the heads of the dendritic spines
can be influenced by a single SNpc neuron [81] (Figure 4).
The action of DA on striatal neurons depends on the type of DA receptor involved. Both
D1 and D2 receptors are located on medium spiny cells. D2 autoreceptors are also on
corticostriatal terminals and on other dopaminergic terminals [82]. DA can modulate the flow
of information arising from the cerebral cortex, acting via both pre and postsynaptic
receptors, by mechanisms of long-term depression (LTD) and LTP [83]. There is evidence
suggesting that D1 receptors are preferentially located on cells that project to GPi or SNpr
(direct pathway) and that D2 receptors are located on cells that project to GPe (indirect
pathway) [84].
Figure 4. Afferent inputs to medium spiny striatal neurons. A: Representation of dendritic spines with
cortical afferents on the spine heads, and dopaminergic input on their shafts (modified from [81]. B:
Photomicrography of rat brain positive neurons labeled for NADPH-d located in the striatum. * Internal
Capsulae fibers.
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Figure 5. Photomicrographs of rat embryonic mesencephalic cell cultures (DIV12) labeled for tyrosine
hydroxylase (TH, green) and neuronal nitric oxide synthase (nNOS, red). The arrows indicate
colocalizations of TH and nNOS positive neurons and apposition among their fibers. (A) nNOS positive
neurons; (B) TH positive neurons; (C) double labeling for TH and nNOS; (D) colocalization of TH and
nNOS positive neurons.
Vincent and Kimura [85], using NADPH-d histochemical technique, demonstrated that
NOS is present not only in DA terminal regions like striatum and nucleus accumbens, but
also at sites of origin of DA cells, like SNpc (A9) and VTA (A10) (Figure 5), as well as in
other regions involved in motor control like cortex, pedunculopontine and tegmental nucleus.
NOS-positive interneurons represent 12% of striatal neurons and are spiny cells, 1225 m
in diameter, with fusiform or polygonal somata. The medium spiny neurons are projection
neurons, representing 95% of the total neuronal population in the striatum. Their function is
profoundly influenced by the level of dopaminergic activity [32,78,86]. The role of NOSpositive interneurons in the striatum is still not clear. Among others their postulated functions
are (i) to control local blood flow in the striatum by releasing NO acting directly on sGC in
the vascular smooth-muscle and causing vasodilatation; (ii) to produce NO that acts as a
neurotransmitter and affects striatal activity, either through direct interactions with ligandgated channels or by influencing surrounding striatal projecting neurons by the stimulation of
second messenger systems [63,87].
Additionally, studies showed that striatal NOS/NADPH-d positive neurons contain
several transmitters and co-transmitters (i.e. somatostatin, neuropeptide Y, serotonine)
[33,85]. It was demonstrated that the NO donor SNP activates sGC in GABAergic
striatonigral terminals leading to the enhancement of cGMP-dependent protein kinases and
phosphorylation of the DA and cAMP-regulated phosphoprotein (DARPP-32) [88]. This
interaction suggested that NO may modulate motor behavior, probably by interfering with
dopaminergic, serotonergic, and cholinergic neurotransmission in the striatum. However, the
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precise role of NO in dopaminergic transmission and, consequently, in motor control remains

uncertain.
West and coworkers [11] proposed a model including NO effector pathways in the
striatal dopaminergic regulation of striatal output. Their model considers that glutamatergic
afferents maintain tonic extracellular DA levels directly via activation of ionotropic GLU
receptors located on DA terminals, and indirectly, via pathways involving striatal NOS
interneurons. It accounts for a regulatory mechanism and control over the modulation of DA
release. Under resting conditions without motor activation, NO would be produced by
glutamatergic activation of NOS interneurons and would increase DA release either by
intensifying GLU release or by influencing the activity of DAT, decreasing DA uptake and
possibly causing reverse release of DA. As described before, this increase in tonic DA would
regulate phasic release by activating presynaptic DA autoreceptors. In contrast, during
behavioral arousal, a strong production of NO, caused by intense glutamatergic corticostriatal
transmission, would result in the inhibition of NMDA receptor function and consequent
decrease in tonic extracellular DA levels which, in turn, would produce less inhibition of
phasic DA release via disinhibition of the autoreceptors. Therefore, NO could regulate tonic
and phasic extracellular DA levels differentially, in a manner dependent on the arousal state
of the animal. In line with this model, it has been recently shown that the tonic NO-cGMP
signaling pathway may have a facilitatory influence on striatal medium spiny neuron activity
[89] and that DA D1 and D2 receptor activation may regulate striatal NOS activity in
opposing manners. Phasic DA transmission may produce nNOS activation via a mechanism
which is dependent on D1/5 receptors [90]. Additionally, nNOS activation is down-regulated
via a D2 receptor-dependent mechanism [91]. These findings provide support for a role of NO
signaling in maintaining the homeostatic balance between tonic DA levels, controlled by low
levels of glutamatergic and nitrergic activity, and the stimulus-driven, spike-dependent phasic
DA neurotransmission [11]. It is suggested that a dysfunction within corticostriatal NOdependent signaling pathways could disrupt the operation of those feedback circuits involved
in regulating DA transmission. It could account for altered sensorimotor information
processing in medium spiny projection neurons and result in inappropriate selections or
inactivations of striatal output circuits involved in the control of motor behavior.
Accordingly, experimental evidence supporting a key role for abnormal NO signaling (i.e. by
an imbalanced neurotransmission and/or by its neurotoxic etiology) in basal ganglia
pathophysiology [93].
Although questions about the exact way that these regions function remain unanswered,
diseases of the basal ganglia result in a variety of abnormal movements ranging from
hypokinesia to hyperkinesia. One of the most studied movement disorders in human is
Parkinsons disease which can be modeled in animals using standardized procedures that
recreate specific pathogenic conditions.
NO, DA and Parkinson Disease
Various experimental models have been proposed to explore the basic molecular
mechanisms of neurodegeneration in Parkinsons disease (PD). However, a perfect animal
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model, able to investigate behavioral, basic cellular, biochemical and anatomical interactions
underlying the symptoms of PD, remains unavailable.
PD is a motor neurodegenerative disorder characterized by progressive death of
nigrostriatal dopaminergic neurons that mainly results in bradykinesia (slowness of
movement), rigidity, and tremor as major motor abnormalities. The motor symptoms of this
disorder may be significantly reduced with therapy of dopaminergic replacement, at least in
the first and middle phases of the disease, although this treatment does not delay/arrest the
progress of neuronal injury. The symptomatology appears when striatal DA content is
reduced by about 80% [93].
The pathophysiological mechanisms of neuronal cell death are still unclear. The most
investigated hypotheses for the PD etiology is that oxidative stress is responsible for
neurodegeneration in the SNpc. According to this hypothesis, reactive oxygen species (ROS),
including the superoxide radical (O2), NO radical (NO) and, most significantly, the
hydroxyl radical (OH), damage essential components of the dopaminergic neuron, including
DNA, mitochondria, protein structures and the cell membrane, resulting in functional
disruption and ultimately cell death [94]. These features are correlated with the accumulation
of aberrant proteins, and ubiquitin-proteasome system dysfunction (inclusions termed Lewy
bodies and dystrophic neuritis-Lewy neurites) that commonly underlie the pathogenesis of
sporadic and familial forms of PD [95,96].
In addition, increased NO release by glia, microglia or macrophages could elevate local
ROS [97]. It can also react with the superoxide radical to form the ONOO, a potently
oxidative radical, which in turn decomposes into OH and NO2, potent initiators of lipid
peroxidation. As a contrast, L-DOPA, which is the typical PD treatment, has also been
reported to induce ROS, in vitro and in rodents with nigrostriatal tract lesions [98,99]. NO
also inhibits directly mitochondrial respiration (mainly at the level of complex IV, but also at
complex I), and liberates ferritin-bound iron, thereby promoting lipid peroxidation [94]. In
PD, a decline of about 30% in complex I activity is reported in the SNpc and striatum, though
not necessarily with mitochondrial DNA changes. Furthermore, iNOS activity is increased in
the SNpc in PD, possibly secondarily to reactive gliosis. However, the increased iNOS
activity is considered an indicative of increased NO-induced damage [100].
Evidence using a rat model of PD may provide further support to the view that NOS
stimulation and enhanced NO formation take place after partial injury of the nigrostriatal DAergic system. NO may be a reactive radical involved in cell death by producing highly toxic
hydroxyl radicals reducing the glutathione levels [101]. Li and coworkers [102] suggested
that sGC/cGMP pathway activation is required for the nerve cell death caused by glutathione
depletion. Chalimoniuk and coworkers [103] provided evidence for the involvement of the
NO/cGMP pathway in the neurodegeneration of SNpc DA-ergic neurons. Toxicity by MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrehydropyridine) and its MAO-B metabolite 1-methyl-4phenyl-pyridine (MPP+) produces PD symptoms with severe motor impairments, striatal DA
depletion, and loss of TH immunoreactivity in humans, monkeys, and various other species
[104-106]. NO increases the toxicity of MPTP by rendering the irreversible inhibition of
mitochondrial complex I activity by MPP+ [107,108] (Figure 6). MPTP-induced changes
include increases in both nNOS and iNOS activities suggesting a role for these two enzymes
in PD-related neurodegeneration. This may explain why the specific inhibitor of nNOS, 7-NI,
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is protective in the MPTP-induced neurodegeneration in mice [109]. The hypothesis that

relates decreased activation of the NO/cGMP pathway to reduced neurodegeneration of DAergic neurons finds additional support in results showing that MPTP neurotoxicity is
diminished in knock-out mice lacking the nNOS gene [110]. Mutant mice lacking neuronal
NOS are more resistant to MPTP-induced neurotoxicity than their wild-type littermates. It
has been shown that 7-NI prevents the conversion of MPTP to MPP+ [111,112].
Figure 6. MPTP-induced dopaminergic cell death. MPP+, an active metabolite of MPTP, enters into DA
neurons by high affinity binding with the DA transporter (DAT). In mitochondria, MPP+ inhibits
complex I, leading to superoxide anion (O2-) formation. O2- reacts with NO (produced by nNOS and
iNOS) to form peroxynitrite (ONOO-), which damages intracellular proteins and DNA causing cell
death. Poly (ADP-ribose) polymerase (PARP) is activated by damaged DNA. It depletes energy stored
through decrements in NAD and ATP, leading to cell death (modified from [108]).
Microinjection of the neurotoxin 6-hydroxydopamine (6-OHDA) is one of the most

widely used experimental animal models of PD, first described by Ungerstedt [113]. This
catecholaminergic neurotoxin is transported into the cell bodies and fibers of both
dopaminergic and noradrenergic neurons. It causes gradual degeneration of nerve terminals
and can affect cell body regions. Administration of 6-OHDA into the medial forebrain bundle
(MFB), SNpc, or striatum can produce variable results regarding the extent of the lesion and
the injured pathways [114]. It results in nearly total depletion of DA and denervation super
sensitivity of the postsynaptic DA receptors in the ipsilateral CPu, and in a characteristic
functional asymmetry with quantifiable turning behavior, contralateral to injection side in
response to the direct DA agonist apomorphine [113]. The lesion in MFB, SNpc, or striatum
produces unilateral hypokinesia and sensorimotor disintegration, mimicking the symptoms of
human PD [115].
In addition, recent work from our laboratory showed the relation between NO and 6OHDA [116] by describing that 6-OHDA lesion induced a significant decrease in the number
of NADPH-d/NOS positive cells in the ipsilateral SNpc related to the lesion side, in contrast
with cell number increase in the ipsilateral dorsal striatum. By contrast, 6-OHDA-treated
animals showed a decrease in the number of NOS immunoreactive cells in the contralateral
nucleus accumbens. It was concluded that populations of NO-synthesizing neurons are
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differentially regulated in PD induced by different experimental procedures. Nevertheless, De

Vente and coworkers [117] reported that lesion of the dopaminergic innervation also using 6OHDA resulted in a 50% decrease in NOS activity of the injured frontal cortex and caudateputamen. Barthwal and coworkers [118] reported that pretreatment with the NOS inhibitor,
L-NAME significantly blocked Amph-induced rotations and restored striatal DA in the 6OHDA lesioned rats. A protective effect of 7-NI against methamphetamine-induced
dopaminergic neurotoxicity in mice has also been reported [119]. Nonetheless, controversial
results regarding the role of NO in PD also exist. For instance, L-NAME did not show
protection for MPTP toxicity in some studies [110,120]. In addition, Ponzoni and coworkers
[121] found that NO synthesis inhibition potentiated the lesion with manganese chloride in
the SNpc and reverted the NADPH-d cell number increase induced by lesion.
Other PD animal model is the chronic systemic exposure to rotenone (an inhibitor of
mitochondrial NADH dehydrogenase, a naturally occurring toxin and a commonly used
pesticide) through jugular vein cannulation which reproduces many features of PD in rats,
including nigrostriatal dopaminergic degeneration and formation of alpha-synuclein-positive
cytoplasmic inclusions in nigral neurons [122]. Bashkatova and coworkers [123] have shown
that only chronic administration (not acute) of rotenone over a long period is capable of
increasing NO in the cortex and striatum and mimics PD-like behavioral symptoms, such as
akinesia and rigidity in rats. In agreement with these results, there are changes like depleted
DA and enhanced catalepsy time described by Alam and Schmidt [124]. It might be
suggested that oxidative stress associated with partial inhibition of complex I is a crucial
factor for neurodegeneration occurring under rotenone administration. These results may
contribute to understanding the mechanism of pesticide action in the pathogenesis of PD and,
in particular, the role of oxidative damage in the pathophysiology of this disease.
Therefore, NO radical released by glial elements might be increased following exposure
to a primary toxin, or secondarily caused by an infection or other inflammatory incident. In
addition, the role of NO in motor transmission, and the search for the initial cause of this
disease is also a challenge that has occupied the attention of neuroscientists. A fact that
aggravates this picture is that not all neuronal losses in PD are dopaminergic neurons. Also,
massive losses of cholinergic cells in the nucleus basalis Meynert have been associated with
PD and dementia [125]. Recent evidence suggests that NO is involved in the regulation of
acetylcholine (ACH) release in the caudate putamen [117]. In addition, studies of Bashkatova
and coworkers [126] concluded that Amph enhances ACH release through increased NO
synthesis in the nucleus accumbens and that 7-NI can abolish this effect.
In recent years, GLU has received emphasis in PD pathogenesis. Results showed effects
of the NO donor SNP and the NOS inhibitor L-NAME, increasing and decreasing,
respectively, the firing activity of GLU and GABA neurons in the striatum [127]. LTP and
LTD in the striatum have been described as a result of stimulation of the massive
glutamatergic innervation arising from most cortical areas, conveying sensorimotor, limbic
and cognitive information [128,129]. A synaptic depotentiation, a NMDA-dependent
synaptic phenomenon, is required to erase redundant information. Interestingly, in rats treated
with 6-OHDA, the striatal synaptic plasticity has been shown to be impaired, though chronic
treatment with L-DOPA is able to restore this process. However, recordings from the
dyskinetic group of rats (induced by chronic administration of L-DOPA) demonstrated a
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selective impairment of the synaptic depotentiation phenomenon [130]. In spite of this

evidence, the relationships between DA, GLU and NO after unilateral lesion of the
nigrostriatal and in L-DOPA-induced dyskinesia have not been elucidated yet.
In conclusion, there is ample experimental evidence supporting the fact that NO is
implicated in the pathogenesis of PD, with increased ONOO- formation and/or an imbalance
in motor neurotransmission, and altered synaptic plasticity. In line with this, drugs that
prevent these phenomena, such as NOS inhibitors or ONOO- scavengers, are under intense
research in order to provide more effective pharmacological interventions.
Besides the role of NO in PD, this molecule may also be involved in the etiology of other
chronic neuropsychiatric/neurodegenerative diseases, such as schizophrenia. A large series of
studies have pointed to the ventral striatum (including the nucleus accumbens), the ventral
pallidum and the corresponding medial parts of the DA-containing cell groups of the VTA in
this pathogenesis. The close connection between frontal cortex and the basal ganglia has
provided support for a fundamental role of basal ganglia in schizophrenia. Interestingly,
Graybiel [86] has proposed a concept that the basal ganglia act as cognitive pattern
generators, in that they form a set of common neural circuits that regulate both motor and
mental action. Based on this concept, Graybiel and colleagues have proposed that similar
circuitry to that used to coordination of motion sequences may be used to coordinate
thinking, planning, and other cognitive acts. Therefore, the involvement of NO in the basal
ganglia described above may also influence schizophrenia.
Evidence of NO Involvement in Schizophrenia
Schizophrenia is a severe mental disorder, which is characterized by thought disturbance,

abnormal perception, impaired cognition and bizarre behavior. Patients usually experience
different symptoms, often divided into positive (hallucinations, delusions, thought
disorganizations), negative (loss of motivation, social withdrawal, anhedonia, flattening of
affect) and cognitive (deficits in attention, memory and executive functions) [131,132].
In addition to the antipsychotic D2 antagonism and psychotogenic effects of DA agonists,
recently several brain imaging studies have illustrated increased occupancy of D2 receptors in
untreated patients [133] giving support for the idea of dopaminergic hyperactivity in
schizophrenia. On the other hand, noncompetitive NMDA antagonists, such as phencyclidine
(PCP) and ketamine, exacerbate some psychotic symptoms in schizophrenic patients and
have psychotomimetic effects in normal humans [134,135]. These facts suggest that some
aspects of schizophrenia may relate to abnormal glutamatergic function.
NO has been functionally linked to both dopaminergic and glutamatergic
neurotransmission in the brain [11]. Both biochemical and anatomical evidence indicate a
role for NO in schizophrenia; however these findings remain still unclear. Abnormalities in
populations of cells containing NADPH-d have been detected in schizophrenics. Postmortem
analyses demonstrate a low density of NADPH-d neurons in the frontal and temporal grey
cortical areas and an increased density of these cells in the frontal or subcortical white matter
[136]. Both decreases and increases in NOS activity, NOS protein and mRNA content were
found in postmortem brains of schizophrenic patients. Some studies revealed decreased
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activity of nNOS in the prefrontal cortex of these patients [137]. A similar reduction in NOS
immunoreactive neurons was detected in the hypothalamic paraventricular and
suprachiasmatic nucleus [46]. In contrast, Baba and coworkers [138] reported increased
nNOS mRNA levels in schizophrenic brains. In the same direction, NO was found
significantly increased in the plasma of schizophrenic patients [139,140]. In addition,
diminished levels of nitrite and nitrate were detected in cerebrospinal fluid of schizophrenic
patients [34,139].
Given that excessive NO concentrations are associated with neurotoxicity [141,142],
inhibition of NOS activity in schizophrenia could be neuroprotective against certain
mechanisms of neuronal damage. There is substantial evidence suggesting that oxidative
stress may play a role in neuropsychiatric disorders, including schizophrenia [43,143]. ROS,
including NO, can cause cellular injury when excessively generated, as they are hazardous to
lipids, proteins, carbohydrates and nucleic acids. Both increased plasma xanthine oxidase
activity, which generates superoxide anions, and decreased superoxide dismutase activity
have been found in plasma of neuroleptic-treated schizophrenic patients [144]. These results
suggest that increased oxidative stress and diminished enzymatic antioxidants may be related
to the pathophysiology of schizophrenia. However, this hypothesis should be taken carefully
considering that it is not clear whether oxidative stress is part of this pathophysiology or if it
depends on the chronic administration of antipsychotic drugs.
In spite of the contradictory findings about NO metabolism in schizophrenia, an
expressive effort has been made using experimental models in order to comprehend the
possible participation of NO in schizophrenia.
Despite the difficulties and limitations of developing animal models which are suitable
for investigating psychiatric disorders, a number of them have been extremely important to
progress in understanding the physiological and pharmacological mechanisms underlying
these disorders.
Traditionally, most animal models of schizophrenia have focused primarily on
phenomena linked to DA, because of the implication of the dopaminergic system in this
disorder, as described above. However, a number of structural lesions, environmental, genetic
and pharmacological models have been developed and are used in the screening of potential
antipsychotic drugs [145]. These models include acute challenge and chronic treatments with
DA agonists or other hallucinogens, such as NMDA receptor antagonists, or developmental
interventions which mimic certain aspects of the illness. Some DA-based models involve
behavioral paradigms that were inspired by antipsychotics, such as catalepsy, and others
reproduce phenomena isomorphic with selected characteristics of schizophrenia such as
motor behaviors, attentional and information processing deficits [132].
These DA-linked behaviors, although not specific for or uniquely prominent in
schizophrenia, can be detected and precisely quantified in non-human species and have been
useful in screening drugs with a predicted mechanism of action (e.g., DA blockade).
Animal models of psychiatric disorders can be classified as having predictive, face or
construct validity, depending on a variety of criteria [146]. Particularly, a number of
experimental cognitive tasks of information processing and selective attention which are
evaluated in animals have been demonstrated to be deficient in schizophrenics. These include
sensorimotor gating tasks such as prepulse inhibition (PPI) [147,148] and tasks involving
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selective attention, such as Kamin blocking (KB) [149-151], latent inhibition (LI) [152,153]
and habituation [154,155]. It has been suggested that LI, as well as PPI, fulfil many of the
criteria for construct validity (i.e., similar underlying neurophysiological concept), face
validity (i.e., similarity of measured characteristics in clinical and experimental models) and
predictive validity (i.e., similar pharmacological profile in clinical and experimental models)
[131,156-158].
DA and NO in Attentional and Information Processing Tasks
The importance of dopaminergic mechanisms in LI has been widely demonstrated by the

effect of several DA agonists on this test. The most robust pharmacological finding refers to
the disruption of LI by the indirect DA agonist Amph, both in animals and humans
[8,156,159,160]. LI consists of the retardation of an associative learning about a stimulus
which was previously presented without any consequence [161]. Numerous protocols exist
for demonstrating LI but in all cases there is a pre-exposure phase, during which a stimulus is
presented without consequence, followed by a conditioning phase, where the same stimulus
is paired with the unconditioned stimulus [156]. It describes a phenomenon in which the
difficulty in forming a subsequent association with a stimulus is directly proportional to the
amount of previous experience. Amph-induced disruption of LI can be reversed by typical
and atypical antipsychotic drugs [160,162,163]. A number of studies have investigated more
precisely the behavioral and neurochemical mechanism of Amph-induced disruption of LI
and the influence of the dopaminergic system on it. 6-OHDA lesions of the DA terminals
confined to the nucleus accumbens potentiate LI [164]. Bilateral local injections of the DA
antagonist haloperidol into the nucleus accumbens before conditioning also potentiate LI, and
reverse the disruptive effect of systemic Amph on LI. However, intra-accumbens injection of
Amph before conditioning disrupted LI only if it was preceded by a prior systemic Amph
injection the day before. It is suggested that the attenuation of LI by increased DA function in
the nucleus accumbens is brought about by impulse-dependent release of the neurotransmitter
occurring at the time of conditioning. Similar effects were found using nicotine in place of
Amph [8]. These results combined with previous literature confirm that the nucleus
accumbens is the critical site for dopaminergic modulation of LI. Considering the evidence
for a close relation between dopaminergic and glutamatergic systems, the latter is also
thought to play a major role in schizophrenia [165]. Initially, it was reported that NMDA
antagonists do not disrupt LI [166]. Recently, there is divergent evidence implicating GLU
receptors in LI. For instance, the NMDA channel blocker, MK-801, has shown to induce
deficits in LI, which can be reversed by a selective Type 4 cyclic adenosine monophosphate
(cAMP) phosphodiesterase inhibitor, rolipram, implicating cAMP signalling in this task
[167]. In the same way, the non-competitive NMDA antagonist, ketamine, when injected at
the pre-exposure stage, was found to abolish LI at a dose that was able to induce
hyperlocomotion, stereotypies and ataxia in a conditioned-fear paradigm [168]. In contrast,
another non-competitive NMDA receptor antagonist, PCP, was found to potentiate LI when
administered acutely prior to the conditioning stage using a taste aversion conditioning
procedure [169]. Interestingly, this effect could be blocked by pretreatment with L-NAME
172
[170]. L-NAME was found to abolish LI when administered alone. It was suggested that the
effect of PCP, to some extent, may depend on dopaminergic mechanisms. However, the same
study showed no interaction between L-NAME and Amph in LI.
Schizophrenic deficits in attention are often described as an inability to attend selectively
to relevant, as opposed to irrelevant, aspects of the environment [171,172]. Considering that
LI measures the ability to learn to ignore an irrelevant stimulus, it has been suggested that the
KB effect is a closer analogue of the schizophrenic attention disorder. In KB the subject must
select between relevant and irrelevant stimulus. This model refers to the learning
phenomenon whereby prior learning to one stimulus, CSA, inhibits learning to another
stimulus, CSB, when both stimuli are later associated to a reinforcer as a compound stimuli,
CSAB [173]. Schizophrenics show impaired performance in the related task KB [149151,174]. Systemic administration of Amph has also been shown to disrupt KB in animals
[174-177] and this disruption can be reversed by the typical antipsychotic, haloperidol [176].
This has led investigators to suggest that KB may represent an animal model with construct
validity for the symptoms of schizophrenia [178]. However, so far there is no evidence of NO
influence on this model.
Another consequence of the attentional deficit in schizophrenia may be an impaired
ability to screen out irrelevant stimuli [179], leading to a deficit in habituation response.
Habituation refers to the decrease in response to repeated presentation of identical stimuli,
which is considered to be the simplest form of learning. It has been demonstrated that
schizophrenic patients exhibit deficits in this non-associative form of learning [154,155].
Psychotomimetic drugs, such as PCP, MK-801 and D-AMP, impaired the habituation of
acoustic startle in mice [180]. The typical antipsychotic, haloperidol, reversed the effects of
PCP and D-AMP, but not that of MK-801. The NOS inhibitor, L-NAME, alone did not affect
habituation or startle amplitude, suggesting that NO is not a critical component of the
intrinsic habituation circuitry. Notably, the disruptive effects on habituation of acoustic
startle, induced by PCP, MK-801 and D-AMP were shown to be all reversed by L-NAME,
indicating that both glutamatergic and dopaminergic mechanisms modulating habituation
convene on NO in mice.
PPI is a cross-species phenomenon that has been used as an operational measure of
sensorimotor gating. Its dysfunction has been related to attentional deficits occurring in
schizophrenia [181,182]. PPI is the inhibition of the acoustic startle reflex, a contraction of
the skeletal and facial muscles in response to a sudden and intense auditory stimulus, when
the startling stimulus is preceded (30300 ms) by a non-startling stimulus or prepulse
[183]. PPI is suggested to measure pre-attentional filtering mechanisms that filter or gate
internal and external stimuli during critical periods of information processing [182]. PPI
occurs naturally in man and most experimental animals, but is diminished in some psychiatric
disorders such as schizophrenia [147]. A similar deficit in PPI is seen in rodents by the
administration of DA direct agonists or DA releasers [184,185]. Additionally, mice with
excess synaptic DA levels via genetic deletion of the DA transporter exhibit robust PPI
deficits [186]. Dopaminergic models of disrupted PPI are frequently used, reflecting the early
emphasis on the postulated dopaminergic hyperactivity in schizophrenia. The predictive
validity of the DA agonist model of PPI disruption has been considered given that the
administration of DA releasers, such as Amph or the D2 direct agonist bromocriptine, disrupts
173
PPI in humans as they do in rodents [187-190] and DA antagonists pretreatment prevents this
disruption [147,191]. Because PPI requires very similar stimuli parameters for both humans
and rodents, it has been employed with the aim of understanding the underlying
pathophysiological mechanisms involved in the development of psychiatric diseases and also
of predicting antipsychotic effectiveness [192]. PPI is a relevant model to investigate the
neural control exerted by cortical and limbic structures on gating processes and the possible
deregulation of these processes in neuropsychiatric disorders [181]. Whereas the startle reflex
is controlled by brain structures at the level of the brainstem, the mechanism of its inhibition
by the prepulse requires forebrain structures, such as the nucleus accumbens, hippocampus,
amygdala and prefrontal cortex [181,193-195]. Dopaminergic modulation of PPI is complex
and variable across species [196,197]. The disrupting effect of DA agonists on PPI involves
the stimulation of more than one subtype of DA receptors for its full manifestation. D2subtype receptors appear to be the most important dopaminergic receptors for PPI expression
in rats [198]. However, a possible synergism between D1 and D2 receptors has been suggested
to regulate this task. Furthermore, PPI depends on the animal strain and on experimental
parameters [197,199]. In fact, in mice the role of D2 receptors in the regulation of PPI is
uncertain. The D2 agonists quinpirole and quinelorane failed to disrupt PPI in various strains
of inbred and outbred mice [200].
Recent studies developed in our laboratory have shown that the NOS inhibitor, LNOARG, can prevent the disruption of PPI produced by Amph in Wistar rats [190]. We
suggest that NO may interact with DA on the modulation of sensorimotor gating, probably by
a presynaptic mechanism, since this NOS inhibitor did not affect the PPI of rats treated with
the direct DA receptor agonists, apomorphine, bromocriptine and SKF38393. Moreover,
NOS inhibitors do not seem to affect PPI when administered alone.
Administration of the NMDA antagonist PCP also disrupts PPI in experimental animals.
In rats, this PCP-induced PPI deficit can be attenuated by pretreatment with NOS inhibitors
such as L-NOARG and 7-NI [201]. In mice, the PCP-induced decrease in PPI is blocked by
pretreatment with the non selective NOS inhibitor, L-NAME [202-204], and the more
selective nNOS inhibitor, NG- propyl L-arginine (NPLA) [205]. Conversely, in nNOS
knockout mice PCP caused a dose-related increase in PPI and did not alter acoustic startle. It
is possible that nNOS deficient mice develop compensatory mechanisms to their nNOS
deficiency during neurodevelopment and this process could change PPI in the adult mouse
[170]. It was also found that methylene blue, an inhibitor of sGC prevents the decrease in PPI
caused by PCP in a dose-related manner [206]. These findings indicate that some of the
behavioral effects of PCP are mediated by NO/sGC pathway. In view of these facts, the
interaction between NO, DA agonists and NMDA antagonists in PPI model supports the
hypotheses that NO may play an important role in schizophrenia via a connection between
nitrergic, dopaminergic and glutamatergic systems.
Another approach for the detection of antipsychotic drug efficacy, in line with the
neurodevelopmental hypothesis of schizophrenia, is the lesion model developed by Lipska et
al. [207], which consists of lesioning the ventral hippocampus of neonatal rats. Bilateral
excitotoxic lesions of the ventral hippocampus performed on 7-day-old rat pups result in
animals that demonstrate at puberty or adulthood hyper-responsiveness to stress, diminished
PPI, and increased sensitivity to DA agonists and NMDA antagonists [207-209]. This
174
behavioral profile is presumed to reflect overactivity of the mesolimbic dopaminergic system

and is ameliorated by antipsychotic drugs [210]. However, the precise neuroanatomical and
neurochemical substrates that mediate the emergence of these behaviors have yet to be
established.
In addition, non invasive manipulations, such as environmental animal models including
early handling, maternal separation and social isolation have also been shown to affect PPI
and latent inhibition [192,211].
Interestingly, neonatal treatment with a NOS inhibitor affects an animal's sensitivity to
Amph and PCP. Furthermore, these treated rats at adulthood (PD56 and older) show a deficit
in social interaction which can be reversed by atypical antipsychotics, clozapine and
olanzapine, but not by haloperidol [212].
These models may be suitable for a variety of investigations at the anatomical,
electrophysiological or neurochemical levels of schizophrenia pathophysiology including the
involvement of DA, NO and GLU functions in this disease.
NO in Catalepsy
Catalepsy test is widely used to evaluate motor effects of drugs that act on the
extrapyramidal system, such as the typical antipsychotics [213]. It is defined as a failure to
correct an externally imposed posture. Drugs that decrease dopaminergic neurotransmission
in the striatum, such as neuroleptics, induce catalepsy in rodents and Parkinsonian symptoms
in humans [214]. Cataleptic effects can be detected both in the hanging-bar and in the wirering tests.
Results with the catalepsy test showed that NOS inhibitors as well as the DA D2 receptor
antagonist haloperidol can induce motor deficits in rodents [215], reinforcing the hypothesis
that nNOS plays an important role in the control of motor behavior. Systemic injections of LNOARG and 7-NI, non-specific and specific inhibitors of neuronal NOS, respectively [216],
induced catalepsy in mice (Figure 7), and had an additive effect with haloperidol [215,217219]. These effects were obtained with doses similar to those that significantly inhibited
neuronal NOS (> 10 mg kg-1) [220]. Similar to the effects obtained after systemic
administration, catalepsy was also induced after intracerebroventricular (i.c.v.) [221] or intrastriatal injection of NOS inhibitors such as NG-monomethyl-L-arginine (L-NMMA), 7-NI, LNOARG, and L-NAME in rats. The effect of L-NOARG i.c.v. injection was completely
prevented by pretreatment with L-arginine but not by D-arginine. Both i.c.v. and intra-striatal
injection of L-NOARG or L-NAME produced bell-shaped dose-response curves. These
results confirm that interference with NO within the striatal formation induces significant
motor effects in rats.
175
Figure 7. Photograph of Swiss mouse in cataleptic position. Catalepsy induced by L-NOARG i.c.v.
microinjected in mice, one hour before the test. The cataleptic effect with 30 nmol was observed one
and two hours following application.
Additionally, mutant mice for the neuronal NOS isoform have altered locomotor abilities
[222], whereas rats and mice treated with various NOS inhibitors show problems with fine
motor control [218,223-225]. Studies showed that basal locomotor activity induced by D1 and
D2 agonists and NMDA receptor antagonists was decreased after blockade of NO signaling
[223,226]. Non-specific NOS inhibition reduced the open arm exploration of an elevated plus
maze accompanied by a decrease in the number of enclosed arm entries, a measure related to
general exploratory activity in this test [227]. Similarly, in the open field test L-NOARG and
7-NI decreased exploratory behavior [225]. Consistently, eNOS knockout mice were
hypoactive during the exposure to the open field test and showed improved motorcoordination. These observations suggest that eNOS-derived NO might be involved in the
control of general activity or in the motivation to explore novel environments [228]. Analyses
of locomotion pattern show that drugs that produce ataxia in humans, such as ethanol or
diazepam, induced deficits in coordinated hind limb movements [229]. In contrast, rats tested
while walking under L-NOARG treatment do not show any modification in their locomotion
pattern [225]. Considering the use of antioxidants in clinical trials, alpha-tocopherol (vitamin
E) therapy is proposed to retard the progression of the degenerative process in patients with
PD [230], and ascorbic acid (vitamin C) as well as vitamin E have been proposed as possible
neuroprotective agents [231]. Lazzarini and coworkers [232] showed that vitamins C and E
potentate the catalepsy produced by NOS inhibitors and haloperidol. These results support an
involvement of dopaminergic and nitrergic systems in motor behavior control and provide
compelling evidence that combined administration of the antioxidants vitamins C and E with
either haloperidol or NOS inhibitors exacerbates extrapyramidal effects.
CONCLUSION
Taken together, the neurochemical and behavioral evidence described above demonstrate
that NO plays an important role in modulating DA neurotransmission and, consequently,
influencing DA-controlled behaviors. Given the role of DA circuitry in many
neuropsychiatric and neurodegenerative disorders, such as PD and schizophrenia, it is
claimed that NODA interactions are critical for the pathophysiology of these diseases. It is
likely that NO exerts its effects via multiple circuits. NO signaling appears to have an
176
important function in maintaining the homeostatic balance between tonic and phasic DA. As
mentioned above, studies using in vitro and in vivo preparations converge to the most agreed
conclusion that NO facilitates DA release and inhibits its uptake. These may happen through
different mechanisms including a direct action of NO on the DA transporter, by activating
sGC/cGMP pathway, or also via oxidative mechanisms. Since the NO radical can be oxidized
into the neuroprotective nitrosonium cation (NO+) or reduced to NO (which forms ONOO-),
NOs biological actions will be determined by the redox state of the endogenous environment
where it is generated. Accordingly, one of the likely roles of NO in the etiology of PD and
schizophrenia is related to neurodegenerative processes. It is also suggested that a
dysfunction within corticostriatal NO-dependent pathways could account for sensorimotor
deficits and inappropriate control of motor behavior by disrupting the operation of feedback
circuits involved in regulating DA transmission. Thus, the interaction between dopaminergic
and nitrergic systems has become a potential target for exploring the neurochemical basis of
neuropsychiatric diseases. In particular, there is a great interest in investigating PD and
schizophrenia by the underlying processes which control motor behavior, attentional and
information processing deficits. There is considerable evidence for a role for NOS inhibitors
not only in preventing DA-agonists- or NMDA-antagonists-disruptive effects in selective
attentional or sensorimotor tasks, but also in potentiating or mimicking DA-antagonistinduced motor deficits. Finally, understanding of the DA and NO interaction in the striatum,
and in other signaling pathways involved in the integration of sensorimotor information
makes this an attractive goal for a future pharmacotherapy of these pathologies.
ACKNOWLEDGEMENTS
We would like to deeply thank Prof. Alasdair Gibb for the manuscript revision. We
acknowledge the expert technical assistance provided by Clia A. da Silva and Miso
Mitkovski. We thank our coworkers who participated in many of the experiments described
in this manuscript, particularly Fernando E. Padovan, Margarete Zanardo Gomes, Mrcio
Lazzarini, Prof. Francisco S. Guimares and Prof. Marcus L. Brando. The authors were
recipients of CNPq, FAPESP and CAPES/COFECUB fellowships.
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ISBN: 978-1-60021-820-0
Chapter IX
DOPAMINE RECEPTORS REGULATION

BY NON-DOPAMINERGIC MECHANISMS
Jaromr Mysliveek1, and Anna Hrabovsk2
1
Institute of Physiology, 1st Faculty of Medicine, Charles University, Prague,

Czech Republic;
2
Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy,
Comenius University, Bratislava, Slovakia.
ABSTRACT
Dopamine receptors are widely distributed in the central nervous system and are
responsible for many physiological, pharmacological and pathological functions such as
movement coordination, cognition or drug abuse. Dopamine receptors belong to the G
protein - coupled metabotropic receptor family. Five different dopamine receptors have
been characterized so far. These can be classified as either D1-like or D2-like, based on
their structure, signal transduction pathway and pharmacological characteristic. The
activity and the level of dopamine receptors depend on the presence of its ligand
dopamine. However, other mechanisms can be involved in the dopamine receptors
regulation.
This article focuses on the non-dopaminergic regulation of dopamine receptors. It
summarizes and concludes results obtained in studies with genetically modified animals.
(1) First, the mutation in 2 glutamate receptors and thus changes in other receptor
systems are discussed. Transgenic mice reveal cerebellar degeneration and learning
impairment. We have found that dopaminergic system is affected in these mutants. (2)
Second, the dopaminergic consequences of acetylcholinesterase deletion are followed. It
was shown in past that the level of muscarinic receptors is significantly changed in
animals with null acetylcholinesterase activity. Receptors are down-regulated due to
Correspondence concerning this article should be addressed to Jaromr Mysliveek, Institute of Physiology, 1st
Faculty of Medicine, Charles University Albertov 5, 12800 Prague, Czech Republic. Phone: 420-2-24968485;
Fax: +420-2-24918816; e-mail: jmys@lf1.cuni.cz.
194

over-stimulation by excess of acetylcholine. Muscarinic receptor subtypes are coexpressed with dopamine receptors on striatal projection neurons. Our results uncovered
a dramatic decrease in striatal dopamine receptors levels. (3) At last, the lack of gene for
transcription factor c-fos is examined. Its deletion did not cause changes in D1-like and
D2-like receptors in cerebral cortex and cerebellum, although other receptor subtypes (1adrenoceptors, muscarinic receptors) were affected. These data show that dopamine
receptors are regulated by non-dopaminergic mechanisms and serve to cope with changes
in the central nervous system.
1. INTRODUCTION
Dopaminergic system is one of the most important transmitter systems in the central
nervous system. It is involved in many physiological functions, as cognition, movement
coordination, learning (Mysliveek, 1997), memory and emotions. It participates in various
neurological and psychiatric disorders such as Parkinsons disease, schizophrenia,
amphetamine and cocaine addiction (Hantraye, 1998), as well as depression and GillTourettes syndrome (for review see Dziedzicka-Wasylewska, 2004). Furthermore, dopamine
plays an important role in long-term potentiation (LTP) of hippocampal prefrontal synapses
and their plasticity. It participates in a long-lasting inhibition of LTP in respond to stress on
cognitive functions (Jay et al., 2004). In brain, dopamine acts not only as a neurotransmitter
but also as a neuromodulator (on presynaptic level). Beside its function in the central nervous
system, dopamine receptors were shown to have a role in multiple periphery organs, such as
kidney (Yamaguchi et al., 1993), retina, parathyroid, and peripheral vascular beds of the
kidney and heart (Niznik et al., 1989; Felder et al., 1989; Sandrini et al., 1986).
2. DOPAMINE RECEPTORS SUBTYPES AND SIGNALIZATION

CASCADES
According to their structural similarities, dopamine receptors are divided into the two
groups (for review see Emilien et al., 1999): D1-like (D1 and D5 subtypes) and D2-like (D2, D3
and D4 subtypes) see Table 1. The families of dopamine receptors differ in the coupling to
G proteins and subsequent steps of intracellular signalization. While D1-like receptors
activate adenylyl cyclase via Gs protein, D2-like family (mainly pre-synaptic D2 receptors)
inhibits adenylyl cyclase via Gi protein activation. Moreover, coupling with Gq protein allow
D2 receptor subtype to activate phospholipase C (see note about receptor variants above). The
genes for D1-like and D2-like families differ in the presence of introns in their coding
sequence. While D1-like family does not contain introns (Civelli et al., 1993; ODowd, 1993),
D2-like family does (Giros et al., 1989; Grandy et al., 1989; Sokoloff et al., 1990; Van Tol et
al., 1991). This fact allows generation of receptor variants, long and short D2 receptor
isoforms. These two isoforms exhibit largely similar pharmacological characteristics, but
their differences G protein coupling (Liu et al., 1996), suggest different functions (Picetti et
al., 1997).
Dopamine Receptors Regulation by Non-Dopaminergic Mechanisms
195
Table 1. The properties of dopamine receptor subtypes

subtype
Length of
aminoacid
chain in
human
Chromosomal
Location
G protein
coupling
Effect of G
protein
activation
Main
localization in
CNS
D1
446
D2
short: 414
long: 443
D3
400
D4
386
D5
477
5q35.1
11q23
3q13.3
unknown
4p15.2
Gs
Gi
Gq/11
cAMP
(modulation)
IP3/DAG
striatum,
hypothalamus,
hippocampus,
Gi
Gi
Gs
cAMP
(modulation)
cAMP
(modulation)
cAMP
striatum,
substantia nigra
frontal cortex,
hippocampus,
parafascicular
thalamic nucleus,
lateral
mammillary
nucleus
Other
localization in
CNS
neocortex,
hippocampus,
amygdala
frontal, temporal,
cingulate and
entorhinal
cortices, lateral
septal nucleus,
medial preoptic
nucleus,
hippocampus,
cerebellar granule
cells, pituitary,
retina
Pathology/mai
n function
Incentive learning
schizophrenia,
attention deficit
hyperactivity
disorder,
delusional
disorder
Blood pressure
regulation (D5
KO mice are
hypertensive)
cAMP
striatum,
olfactory tubercle,
islands of Calleja
entopeduncular
nucleus,
olfactory
tubercle,
septum,
cerebral
cortex,
substantia
nigra,ventral
tegmental area,
pituitary
Parkinson
disease
Islands of Calleja,
shell of nucleus
accumbens,
archicerebellum,
subependymal
ventricular zone,
ventral tegmental
area
yawning,
hypothermia,
drug addiction
Description of dopamine receptor subtypes - the chromosomal location, aminoacid chain length,
signalization and localization in the CNS. Note, that peripheral tissue localization is not described.
More information, and for references see comprehensive review by Emilien et al. (1999).
2.1. D1-Like Family
D1-like family is the main element of the dopamine post-synaptic action (despite its
presynaptic localization). Its members, D1 and D5 dopamine receptors, are pharmacologicaly
indistinguishable. However, the affinity of D5 dopamine receptors to the agonists is up to 10
times higher than that of D1 ones (Weinshank et al., 1991). This fact could be of importance
when one transmitter is supposed to have two effects one through the high affinity sites and
the second one through the low affinity sites in tissue expressing both subtypes. This could
explain the different functions of striatal D1 and D5 dopamine receptors in synaptic plasticity
196
(Centonze et al., 2003). From another differences between these two subtypes, it is interesting
to mention, that D5 dopamine receptor, unlike D1 subtype, is constitutively (agonistindependently) active (Tiberi and Caron, 1994). Moreover, D1 dopamine receptors couple
preferentially to G protein heterotrimers that contain 7 subunits (Wang et al., 2001). D1
dopamine receptor can also couple with another G protein, Golf (which also stimulates
adenylyl cyclase) that is highly expressed in some brain areas, such as caudate nucleus,
nucleus accumbens, and olfactory tubercle. Coupling of D1 dopamine receptors with Golf was
even suggested to be preferential (Herve et al., 1993). Generation of D5 dopamine receptor
knockout mouse uncovered possible involvement of this subtype in the pathology of
hypertension, as the mutant mice were hypertensive (Hollon et al., 2002).
2.2. D2-Like Family
D2 dopamine receptors are as D1 dopamine receptors (Weiner et al., 1991) localized both
pre- and postsynaptically. D2 dopamine receptor has relatively low (nanomolar) affinity for
dopamine, which supports its importance as modulatory (pre-synaptic) receptor. D2 receptor
isoforms (long and short) are differently distributed and thus may possess distinct functions.
Short isoform seems to serve as an autoreceptor, whereas the long isoform is primarily a
postsynaptic receptor (Khan et al., 1998). Using genetically targeted deletion of D2 dopamine
receptor gene in mouse revealed that other members of the receptor family were not affected
(Saiardi et al., 1998) and these mutants had reduced locomotion and less coordinated
movement (Saiardi et al., 1998).
D3 subtype of dopamine receptors appears to have similar distribution as D2 dopamine
receptor (Emilien et al., 1999). Similarly to D2 dopamine receptors, alternative splicing
variants of D3 dopamine receptors were observed. These variants were hypothesized to play a
role in availability of active D3 dopamine receptors in some psychiatric conditions (Schwartz
et al., 1993). Hypothesis suggests that inactive D3 dopamine receptors affect ligand binding
to the active D3 dopamine receptors and thus influence their function.
The D4 dopamine receptor has high densities in cerebral cortex, amygdala hypothalamus
and pituitary (Dziedicka-Wasylewska, 2004). In striata, the occurrence of D4 dopamine
receptor is much lower than the D1 and D2 subtypes (Patel et al., 1996).
In summary, dopaminergic system is one of the most important neurotransmitter systems
in the central nervous system. It acts mainly by activation of D1-like receptor family at the
target cell. In addition, fine tuning of the signal is achieved via pre-synaptic modulation by
D2-like receptor family.
3. G PROTEIN COUPLED RECEPTOR REGULATION

In addition to signal modulation via pre-synaptic regulation, the cell signalization though
the G protein-coupled receptors can be modified by post-synaptic mechanisms, such as
homologous and heterologous regulation. Typical case of homologous regulation is receptor
desensitization, internalization and down-regulation due to its hyperstimulation (Morris and
197
Malbon 1999, Fukunaga et al., 2006). The heterologous regulation is comprehended as a

process in which the normaly activated receptors are regulated through secondary
mechanisms, (Dhami and Ferguson 2006). This means that receptors can be regulated via
other receptors, i.e. dopamine receptors can be regulated via activation of another receptors
such as ghrelin receptors (Jiang et al., 2006). Vice versa, NMDA currents can be amplified
via dopamine receptor activation (Chen et al., 2004).
The process of regulation consists of multiple steps such as receptor desensitization,
receptor internalization - receptor endocytosis, and finaly receptor down-regulation (Grady et
al. 1997, Liang & Fishman 2004, Fukunaga et al. 2006, Bernard et al. 2006). Although the
terms of these events are not consistently defined, it is possible to simplify that
desensitization is process in which the receptor capabilities to activate G proteins/enzymes
producing second messengers is diminished. Internalization is process in which the receptors
are sequestered from the membrane but not yet degraded as in the process called receptor
down-regulation.
4. REGULATION OF DOPAMINE RECEPTOR LEVELS VIA OTHER

RECEPTORS
4.1. Glutamate Receptors (Lurcher Mutants)
Lurcher mutants are mice with functional mutation in the 2 glutamate receptor (GluR2)
that is predominantly expressed in cerebellar Purkinje cells and plays a crucial role in
cerebellar functions. Despite their importance, the mechanism by which GluR2 participates
in cerebellar functions is still unexplained. GluR2 is predominantly expressed at distal
dendrites of Purkinje cells where parallel fibers form synapses (Yuzaki, 2004). It is important
to note that GluR2 does not form functional glutamate-gated ion channels when expressed,
either alone or with other glutamate receptors. Despite that functional exceptionality, GluR2
is crucial in cerebellar function: mice that lack the gene encoding GluR2 display ataxia and
impaired motor-related tasks such as eye-blink conditional learning and adaptation of the
vestibulo-ocular reflex. Moreover, these mutants display impaired cognitive functions (Voeh
et al., 1999, Kkov and Voeh, 2004) and lower resistance to the neurotoxin 3acetylpyridine (Caddy and Voeh, 1997).
Lurcher mutant mice are heterozygote animals (+/Lc) which suffer from progressive and
complete loss of cerebellar Purkinje and granule cells, and inferior olive neurons. By
postnatal day 26 there is only 10 % of Purkinje cells remaining, by postnatal day 90, there are
no Purkinje cells. On that day, granule cells represent 10 % and inferior olive neurons are as
little as 25 % of the wild type value (Caddy and Biscoe, 1979). Purkinje cells die in
consequence of a functional mutation in the 2 glutamate receptor gene and this is a type of
excitotoxic apoptosis (Zuo et al., 1997). Affected homozygotes (Lc/Lc) are unable to survive.
We have investigated the role of dopamine transmitter receptor system in these mice
with olivocerebellar degeneration (Mysliveek et al., 2007). First, the behavioral effects of D1
dopamine receptors activation and inhibition on spatial learning in Lurcher mutant and wild
type mice derived from C57Bl/7 strain were followed. Second, the density of D1-like and D2-
198
like dopamine receptor in three brain structures (hippocampus, striatum, cerebellum) was
investigated. Third, strain differences between C57Bl/7 and C3H mice were studied in both
genotypes.
Lurcher mutants revealed worse results in the Morris water maze than control wild type
mice. D1 receptor agonist SKF 38393 caused no changes in latencies of reaching criterion in
comparison to animals treated with saline, both in wild types and Lurcher mice. On the other
hand, the animals of both genotypes that were given the D1 receptor antagonist SCH 23390
showed significantly worse results in reaching criterion in comparison to control mice treated
with physiological saline.
When looking at dopamine receptor properties, we have found substantial increase in
both D1-like and D2-like dopamine receptors in hippocampus in C57Bl strain. In cerebellum,
D1-like dopamine receptors were decreased and D2-like dopamine receptors were not
affected. In striatum, receptor densities were similar to the wild type counterparts. In C3H
strain, the only change comparing to wild type was an increase in D1-like dopamine receptors
in hippocaompus.
It is important to note that application of D1-like dopamine receptor antagonist SCH
23390 had similar effect in wild type and Lurcher mice. This finding, together with observed
changes in the number of D1-like and D2-like dopamine receptors, suggest that dopamine
receptors are the tool to cope with olivocerebellar degeneration. In case of defect in dopamine
receptors (i.e. D1-like post-synaptic and D2-like pres-synaptic dopamine receptors), one
would expect that the spatial learning should be affected differently. However, although the
Lurchers are worse in spatial learning, the function of dopamine receptor system is preserved
as the changes of reaching criteria were similar in Lurchers and wild types. Our results are in
good agreement with findings that dopamine transporter (uptake sites) were similar to
controls, except for a decrease in the subthalamic nucleus (Strazielle et al., 1998). These data
strengthen the hypothesis about the main role of dopamine receptors in coping with changed
condition in olivocerebellar degeneration. Similarly to that finding, no decrease was found
for aspartate, gamma-aminobutyric acid (GABA), glycine, as well as dopamine and its
metabolites (Reader et al., 1998).
Similar to our results with spatial learning in Lurchers, there was an improvement in the
distance traveled on the suspended horizontal string after dextromethorphan (an NMDA
antagonist) and L-dopa/carbidopa, but not after D1 dopamine receptor partial agonist SKF
77434 (Thullier et al., 1999).
Taken together, it is possible to conclude that dopaminergic system plays an important
role in the mice with olivocerebellar degeneration and that dopamine receptors are, in
contrast to dopamine transporters, subject of changes in these mutants. Moreover, data
described above reveal that dopamine receptor system is preserved in Lurchers as supported
by similar changes in case of both genotype in spatial learning after D1 dopamine antagonist
SCH23390.
199
4.2. Acetylcholine Receptors (AChE KO)
Acetylcholinesterase (AChE) is one of the key elements of the cholinergic system. Its
physiological function is the hydrolysis of neurotransmitter acetylcholine (ACh) and thus
termination of the cholinergic transmission at the synapses in the central nervous system and
neuromuscular junction (NMJ). AChE was long time considered to be an essential enzyme
for the living organisms based on the fact that its inhibition leads to severe pathological
changes or even death. However, generation of AChE knock-out (AChE-/-) mouse
contradicted this dogma. AChE -/- mouse is born alive and survives till adulthood (Xie et al.,
2000). It develops severely changed phenotype comparing to wild-type siblings (Duysen et
al., 2002, Duysen et al., 2006). Mutant animals are smaller than their littermates, with lower
body weight and impaired thermoregulation. They show many symptoms of hypecholinergic
activity, e.g. miosis. They lack the grip strength resulting probably from the general muscle
weakness. This may be as well the cause of their abnormal gait, hunched body posture and
spread legs. The AChE-/- pups can be easily recognized at birth by the whole body tremor.
Average life span of mutant mice is shorter than the life span of their wild-type siblings.
Mutant animals die prematurely during the tonic phase of grand mal seizure, with the peak of
death around the age 30 days (Hrabovska et al., 2005).
Even if phenotype of these animals has been well described, it is still unclear how the life
without AChE is possible. To answer this question, one of the first hypothesis proposed that
the lack of AChE activity is buffered by butyrylcholinesterase (BChE). BChE is a sister
enzyme which physiological function in the body is not fully understood yet. However, its
ability to hydrolyze ACh suggests the possibly to substitute the cholinergic action of missing
AChE. Indeed, AChE -/- mice are more sensitive to BChE inhibitors than the wild-type
siblings (Xie et al., 2000). However, the level of expressed protein is not changed in
nullizygous animals. The second hypothesis to be tested was modification of ACh synthesis
and release. To address this question, choline acetyltransferase (ChAT) acitivity, vesicular
ACh transporter (vAChT) and high-affinity choline transporter (CHT) levels were
determined. There was no difference in the activity of ChAT or the level of expressed vAChT
which reports no changes in presynaptic de novo ACh synthesis or its synaptic release
(Volpicelli-Daley et al, 2003). The level of CHT protein was elevated in AChE-/- mice
(Volpicelli-Daley et al, 2003), which suggest decreased re-uptake of choline, probably due to
its low synaptic level. This fact together with unchanged BChE expression level suggest an
insufficient participation of BChE in ACh hydrolysis resulting in elevated level of ACh at the
synapse and NMJ.
Indeed, in vivo microdialysis confirmed dramatically increased level of extracellular
ACh in the AChE -/- brain (Hartmann et al., 2007). Such an elevation of the neurotransmitter
level can not be without consequences on the receptor systems. Cholinergic neuronal
transmission is mediated through the muscarinic (MR) and nicotinic receptors (NR). Five MR
subtypes (M1 M5) have been described so far. They are G-protein coupled receptors. While
M1, M3 and M5 MR stimulate phospholipase C; M2 and M4 MR inhibit adenylyl cyclase.
Nicotinic receptors are ligand-gated channels formed by (2-10) and (2-4) subunits.
Combinantion of these subunits is tissue specific. Neuronal form consists of and subunits
or is represented by homomers), ganglionic type is mainly homomer and muscular type
200
combines , , and subunits of nicotinic receptor. Multiple biochemical and microscopic

studies showed drastic changes of all types of cholinergic receptors in the CNS and at the
NMJ of AChE -/- mice (Bernard et al., 2003; Li et al., 2003; Volpicelli-Daley et al., 2003).
Described changes in cholinergic system can partially explain the viability and phenotype of
AChE nullizygote mice. However, only little attention has been paid to noncholinergic
adaptation changes in the lack of AChE which could help to make a full picture of survival
mechanisms in AChE-/- mice. Experimental and clinical data suggest the strong interaction
between dopaminergic system and cholinergic muscarinic system in CNS. Its pathological
imbalance is linked to many diseases, e.g. Parkinsons disease, Alzheimer disease,
schizophrenia, cocaine dependence. (Tanda et al., 2007). Especially in the striatum, dopamine
and ACh strongly interact at several presynaptic and postsynaptic sites, while their action
seems to be in opposite manner (Di Chiara et al., 1994). ACh is released in the striatum from
the large aspiny neurons - interneurons, which represent 1-2 % of the striatal population
(Calabresi et al., 2000, Kawaguchi et al., 1995). Striatal interneurons express both D1-like
and D2-like dopamine receptors. D2 mRNA is expressed by all of them, 90% express D5
receptor subtype mRNA (rather than D1 subtype) while D3 and D4 mRNA has been reported
to be undetectable (Pisani et al., 2000). Dopamine has a prominent inhibitory effect on the
ACh release, even if it modulates striatal cholinergic tone by both excitation (through D1-like
DR) and inhibition (through D2-like DR). On the other hand, M1 MR agonists and antagonists
change the level of extracellular dopamine in striatum and cortex. (Tanda et al., 2007)
Activation of M1 muscarinic receptors excites dopaminergic neurons at the somatic level that
probably results in increased striatal dopamine release (Calabresi et al; 2006). Based on such
a tight connection between cholinergic and dopaminergic systems in striatum we
hypothesized changes in dopaminergic system of AChE -/- mice comparing to the wild-types.
To test this hypothesis, the D1-like and D2-like dopamine receptors in wild-type and
AChE-/- striatum from adult mice (60 days old) were quantified. Specific radiolabeled
ligands binding study confirmed our hypothesis. While binding properties of the receptors
were not changed, total number of receptors was drastically decreased, D1-like receptors in
AChE-/- striatum were reduced to 5% of normal, while D2-like receptors in the AChE-/striatum were almost undetectable under our conditions. Surprisingly, no changes were
observed by immunobloting of DR in striatal homogenates. However, the results from in situ
immunohistochemistry of striatal DRs confirmed the decreased level of dopamine receptors
in mutants. This severe alteration of the dopamine pathway was accompanied by a 40 to 64%
reduction in muscarinic receptors in striatum (Volpicelli-Daley et al., 2003; Bernard et al.,
2003). Such a drastic changes in both systems led to the hypothesis about neurodegeneration
of striata. However, DNA-specific staining in the striata did not show any differences in size,
density and distribution between two genotypes. Moreover, no differences in the AChE -/brain structure were observed by the light microscopy (Xie et al., 2000; Mesulam et al. 2002).
This ruled out possible depletion of striatal neurons in the AChE-/- brain. We can conclude
that the lowered binding of the specific ligands in both systems must be caused by the
decreased level of the receptors on the membrane, due to their internalization or even downregulation.
Similar changes in dopamine and muscarinic receptor systems were described in
pathology of some diseases. For example, decreased expression of D1 dopamine receptors, D3
201
dopamine receptors and D4 dopamine receptors is observed in cortex of Alzheimer disease

patients (Kumar et al., 2007). There is a parallel reduction in muscarinic receptors and D2
dopamine receptor binding in these patients (Piggott et al. 1998; Piggott et al. 1999). Loss of
D2 dopamine receptor in the striatum has been observed in the normal human brain with
aging (Kumar et al., 2007). Reduced D2 dopamine receptor binding has also been reported in
Parkinson disease (Volkow et al. 1998).
5. REGULATION OF DOPAMINE RECEPTORS BY MOLECULES

AFFECTING INTRACELLULAR SIGNALIZATION (C-FOS KO)
c-Fos is a third messenger activating the target genes (Kovacs, 1998). Secondly, it can be
comprehended as an inducible transcription factor (Hedegren and Leah, 1998). Third, it is a
product of an immediate early gene (Huges and Dragunov, 1995). Fourth, it is an ubiquitous
molecule that can give us a picture of cell activation (Pack and Palkovits, 2001). In respect
to G-protein coupled receptors, c-Fos is activated by multiple pathways, i.e. it can be
activated by both proteinkinase C activation (by diacylglycerol induced by phospholipase C)
and by Ca2+ increase (by inositoltrisphosphate induced by phospholipase C) (Dampney and
Horiuchi, 2003). Expression of c-Fos can be enhanced by many different factors, among the
others by D1-like and D2-like dopamine receptors (Pennypacker et al., 1995)
On the other hand, our knowledge about the reverse pathways (i.e. about the effects of cFos protein on the receptor G protein - coupled receptors) is limited. Therefore, we have
investigated the effects of c-Fos on the distribution of some G-protein coupled receptors in
mouse with targeted disruption of c-fos gene (Wagner, 2002).
In addition to dopamine receptors, we have studied the effects of c-fos gene disruption on
muscarinic receptors and adrenoceptors (Novkov et al., 2006) in the peripheral tissues
(heart and lungs) and in the central nervous system (cerebral cortex, cerebellum; Bene et al.,
2006). Muscarinic receptors (i.e. those mainly expressed in the central nervous system odd
numbered receptors) and 1-adrenergic receptors activate phospholipase C via Gq protein and
then proteinkinase C that can affect the c-Fos protein expression in the cell. D1-like dopamine
receptors and -adrenoceptors activate adenylyl cyclase via Gs protein. As have been
mentioned above, D2-like dopamine receptors inhibit adenylyl cyclase via Gi protein.
Therefore, with respect to the limits of the chosen set of receptors, we could deduce the
role of c-Fos in receptor regulation.
In the cerebral cortex, there was an increase in the number of 1-adrenoceptor and
muscarinic receptor binding sites (to 184 %, and 234 %, respectively). The level of other
receptors, i.e. -adrenoceptors, D1-like dopamine receptors and D2-like dopamine receptors
did not change in comparison to wild type animals.
In cerebellum, as in the structure with important connection to motion regulation and
equilibrium maintenance, we have been able to find changes only in 1-adrenoceptor number.
No other studied receptors were affected.
Our results have shown that disruption of c-fos gene can dramatically change the number
of binding sites (the functional receptor expression, in fact) both in the peripheral tissues (an
increase in muscarinic receptors and adrenoceptors in lungs and heart) and also in the central
202
nervous system (an increase in -adrenoceptors in cortex and cerbellum and an increase in
muscarinic receptors in cortex).
The data about the cerebral cortex are more interesting. The results imply the role of cFos in regulation of receptors that activate phospholipase C and consequently proteinkinase
C. This pathway can be comprehended as the major process in c-fos activation. Therefore
these receptors are affected by c-fos gene disruption but the other ones (activating nonphospholipase C signalling pathways) are not. Moreover, it is also interesting that despite the
increase in receptor number, the phospholipase C activity was decreased while
inositoltrisphosphate receptor (IP3 receptor) mRNA was increased (Bene et al., 2007). It is
also important to note, that in the cerebellum, the main subtype of muscarinic receptors
belong to M2 muscarinic receptors. This fact makes the hypothesis about c-fos gene
disruption effects on phospholipase C linked receptors more probable. The effects in
periphery can be explained by cross-regulation of the receptors between each other (i.e. by
heterologous regulation) while in the central nervous system the c-fos gene disruption could
be explained by effects of c-fos on the receptors.
In contrast to the changes in dopamine receptor level in Lurcher and AChE -/- mice,
dopamine receptor system remains preserved in case of c-fos gene disruption. It means that
dopamine receptor system is specificaly changed in different conditions and this way
participates in the homeostasis maintenance.
6. CONCLUSION
In detail, the experiments with Lurcher mice could be concluded as follows:
a) significantly higher density of D1 and D2 DA receptors in Lurcher mutants
hippocampus compared to wild type mice could be related to the differences in the
spatial learning,
b) No modification in D1 and D2 DA receptors density in striata of Lurcher mutants
questions the hypothesis about their impaired motor functions due to the changes in
dopaminergic transmission.
c) The same effect of D1 dopamine receptor antagonist on spatial learning in both
genotypes
Knocking out the gene for AChE-/- in mouse uncovered following facts:
a) BChE may partially substitute the function of missing AChE in mouse
b) ACh hydrolysis and ACh release is not changed in AChE -/- mice. However,
upregulated high-affinity choline transporter suggests decreased re-uptake of
choline, probably due to its low synaptic level.
c) Muscarinic receptors are downregulated in AChE -/- mice
d) D1-like and D2-like DR in AChE -/- striatum are dramatically reduced, probably due
to internalization or even down-regulation.
e) Striatal neurons are not depleted in AChE-/- brain.
203
Following conclusions could be made for cFos mutated mice:

a) c-fos gene disruption affects the level of receptors which activate phospholipase
C/proteinkinase C pathway exclusively. The G protein-coupled receptors which
activate adenylyl cyclase (i.e. D1-like dopamine receptors and -adrenoceptors) are
not affected,
b) the consequent steps in the intracellular signalization are affected differently: while
the phospholipase C is decreased, the IP3 receptor mRNA is increased.
Based on our results from the experiments with genetically modified mice, it is possible
to conclude that dopamine transmitter receptor system plays an important role in the
maintanance of homeostasis.
ACKNOWLEDGEMENTS
Work in our laboratories is suported by Grant of Ministry of Education of Slovak
Republic VEGA 1/2271/05 (to A.H.), by Grant GAUK 36/04 from Grant Agency of Charles
University (to J.M.) and by Czech-Slovak Grants CZ-112/ SK-87 and SK-CZ-08706 (to A.H.
and J.M.). We would like to thank dr. Zsolt Csaba for hepful comments to our manuscript.
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INDEX
6
6-OHDA, 9, 62, 64, 66, 67, 80, 82, 84, 167, 168,
171, 183, 191
A
AC, 3, 44
acetaminophen, 80
acetylcholine, xii, 168, 191, 194, 199, 203, 205, 206
acetylcholinesterase, xii, 193, 203, 204, 205, 207
achievement, 95, 135
acid, 36, 45, 53, 60, 62, 63, 72, 78, 80, 117, 129,
133, 140, 144, 155, 160, 161, 191, 198, 207
ACTH, 114, 115, 125, 128
action potential, 36, 53
activated receptors, 79, 197
activation, vi, x, 3, 5, 6, 19, 20, 22, 23, 24, 27, 28,
30, 31, 32, 35, 36, 37, 40, 41, 42, 45, 49, 50, 53,
56, 58, 60, 61, 63, 64, 66, 69, 73, 78, 79, 80, 81,
82, 89, 98, 99, 103, 104, 105, 106, 125, 136, 142,
145, 146, 147, 148, 154, 155, 157, 158, 159, 160,
161, 165, 166, 191, 194, 195, 196, 197, 201, 202,
207
activity level, 30
adaptation, 95, 197, 200
addiction, viii, x, 2, 10, 19, 32, 51, 88, 100, 146, 147,
148, 194, 195
adenine, 157, 158, 183, 185, 191
adenoma, 129
adenosine, 171, 191
adhesion, 68
administration, ix, x, xi, 5, 9, 10, 19, 32, 33, 35, 63,
66, 68, 76, 77, 92, 93, 94, 95, 96, 98, 104, 105,
106, 107, 109, 111, 114, 123, 124, 125, 126, 127,
128, 135, 139, 140, 141, 142, 143, 145, 146, 147,
148, 149, 154, 168, 170, 172, 174, 175, 184, 187,
188, 190, 191
ADP, 167, 191
adrenal gland, ix, 113, 114, 115, 118, 119, 120, 121,
124, 125, 127, 128, 129
adrenaline, 128
adrenoceptors, xii, 194, 201, 203
adrenocorticotropic hormone, 128
adulthood, 173, 174, 190, 191, 199
adverse event, 136
aetiology, 58, 65
affective disorder, 100, 142, 143
age, 39, 52, 55, 65, 199, 207
agent, 13
aggregation, 58, 69
aggression, 53
ag(e)ing, 9, 52, 54, 55, 56. 66, 75, 83, 201
aging process, 66
agonist, x, 21, 34, 42, 43, 44, 45, 80, 88, 92, 93, 95,
97, 98, 99, 104, 105, 107, 109, 110, 136, 138,
140, 141, 143, 146, 148, 167, 171, 172, 188, 189,
191, 196, 198, 204, 206, 207
agranulocytosis, 4
akathisia, 3, 108
akinesia, 2, 6, 168, 191
aldosterone, ix, 113, 114, 115, 117, 122, 124, 125,
127, 128, 129
allele, 34
alpha-tocopherol, 175, 191
ALS, 59
alternative, 98, 196
alters, 13, 18, 38
aluminium, 117
Alzheimers disease, 9, 59
210
Index
Alzheimer's disease, 76, 205

amines, ix, 113, 115
amino acid(s), 155, 159, 180, 191, 207
amphetamines, 3, 4, 9, 11, 32, 154, 177, 191
amphibia(ns), ix, 113, 114, 125
amplitude, 172, 191
amygdala, 6, 13, 90, 147, 156, 173, 178, 188, 191,
195, 196
amyotrophic lateral sclerosis, 59
analgesics, 61
anatomy, 72, 109, 139
animal models, 8, 59, 64, 67, 134, 167, 170, 174,
186, 187, 188, 191
animals, ix, xi, 33, 49, 52, 62, 68, 94, 95, 99, 115,
116, 133, 135, 154, 165, 167, 170, 171, 172, 173,
191, 193, 197, 198, 199, 201, 204
anion, 63, 80, 167, 191
ANOVA, 118, 149
antagonism, 46, 96, 98, 99, 108, 110, 136, 169, 191
Antiapoptotic, 78
antibody, 102
antidepressant(s), x, 83, 94, 95, 96, 99, 106, 107,
131, 132, 133, 134, 138, 140, 141, 142, 143, 144
antigen, 68
anti-inflammatory, ix, 52, 61, 62, 70, 74, 76, 77, 78,
79, 80, 81
anti-inflammatory agents, 78
anti-inflammatory drugs, ix, 52, 61, 74, 77, 78, 79,
80, 81
antioxidant, 56, 62, 63, 70, 160, 170, 175, 185, 191
antipsychotic (drugs), 3, 38, 96, 97, 99, 108, 109,
139, 169, 170, 171, 172, 173, 174, 186, 190, 191,
207
antipsychotic effect, 38, 173, 191
antipyretic, 62
anxiety, viii, 51, 96, 105, 136
anxiolytic, 101, 105
AP, 61, 78
apoptosis, viii, 51, 59, 60, 66, 70, 71, 78, 79, 81, 82,
197
apoptotic pathway, 67
arachidonic acid, 60, 78, 129
arginine, 158, 160, 161, 173, 174, 180, 185, 189, 191
argument, 38
arousal, vii, 1, 4, 6, 7, 9, 10, 11, 12, 13, 165, 191
arrest, 166, 191
ascorbic acid, 160, 175, 191
aspartate, 13, 46, 155, 191, 198
aspirin, 61, 62, 63, 64, 65, 78, 79, 80
assessment, 46, 158, 186, 191
astrocytes, 61, 67
asymmetry, 167, 191
ataxia, 171, 175, 191, 197
ATP, 58, 167, 183, 191
atrophy, 8, 53
attacks, 14
attention, x, 33, 57, 61, 89, 94, 131, 156, 168, 169,
171, 172, 186, 187, 191, 195, 200
atypical antipsychotic agents, 139
autophagy, 81
autosomal dominant, 58
availability, 88, 132, 155, 196
avoidance, x, 146, 148, 149
axon terminals, 3, 101
axons, 3, 156, 181, 191
B
basal forebrain, 3, 4, 6, 11, 12, 14
basal ganglia, 2, 6, 8, 45, 54, 60, 69, 71, 72, 84, 89,
91, 98, 102, 109, 139, 154, 162, 165, 169, 181,
190, 191, 205, 207
Basal ganglia, 178, 191
basket cells, 36
behavior, vii, xi, 1, 4, 7, 12, 14, 71, 84, 89, 98, 99,
111, 129, 137, 144, 154, 156, 163, 167, 169, 175,
176, 178, 191
behavioral effects, 83, 110, 143, 173, 191, 197
behavioral models, xi, 154, 191
beneficial effect, 21
benefits, 64, 136
benzodiazepine, 36, 40
binding, 10, 32, 39, 43, 47, 53, 61, 62, 73, 97, 98,
108, 110, 133, 140, 141, 157, 158, 167, 191, 196,
200, 201, 206
biochemistry, 45
biological systems, 159, 191
biosynthesis, 53, 61, 75, 154, 191
bipolar disorder, 40, 136
birth, ix, 52, 54, 199
blocks, 61, 97, 99, 105, 189, 191
blood, vii, 41, 58, 79, 116, 158, 164, 191
blood flow, 41, 164, 191
blood pressure, vii
blood-brain barrier, vii
body weight, 199
bradykinesia, 52, 166, 191
brain, vii, x, 3, 10, 12, 13, 14, 18, 33, 37, 38, 39, 44,
45, 46, 49, 53, 54, 56, 58, 59, 60, 61, 62, 66, 67,
71, 72, 73, 74, 76, 77, 81, 82, 83, 84, 88, 90, 91,
Index
94, 96, 98, 100, 102, 103, 105, 110, 131, 132,
134, 144, 146, 154, 156, 158, 162, 163, 169, 173,
178, 179, 180, 181, 182, 183, 184, 185, 190, 191,
194, 196, 198, 199, 200, 201, 202, 204, 205, 207,
208
brain activity, 49
brain development, 54, 81
brain stem, 8, 9, 10, 12, 72, 100 173, 191
brain structure, 146, 173, 191, 198, 200
branching, 53
Brazil, 153, 191
breakdown, 66
buffer, 52, 116, 117
Bupropion, 140
C
Ca2+, 155, 158, 159, 160, 161, 177, 179, 191, 201
calcium, 39, 47, 53, 73, 127, 180, 185, 191
calibration, 117
calorie, 70
candidates, 34, 60, 157, 191
capsule, 162, 191
carbohydrates, 170, 191
carrier, 116, 118, 120, 121, 122, 123, 124, 155, 191
caspases, 66, 82
catabolism, 56
catalytic activity, 63
catecholamines, ix, x, 53, 75, 114, 117, 126, 127,
128, 162, 180, 191
cation, 176, 191
cats, 7, 10
CD8+, 59
CD95, 66
CE, 41, 42
cell, 2, 4, 6, 9, 14, 33, 43, 53, 54, 55, 56, 59, 60, 61,
62, 66, 67, 69, 70, 71, 73, 74, 77, 81, 82, 83, 84,
88, 89, 90, 91, 95, 100, 102, 104, 107, 108, 115,
120, 126, 129, 135, 142, 155, 156, 158, 159, 160,
161, 164, 166, 167, 169, 182, 191, 196, 201, 203
cell adhesion, 68
cell body, 156, 167, 191
cell culture, 62, 66, 95, 115, 126, 135, 164, 191
cell death, 54, 56, 59, 60, 62, 66, 69, 71, 77, 81, 82,
166, 167, 182, 191
cell line, 77, 84, 107, 142
cell membranes, 158, 191
cell surface, 95, 155, 191, 203
cell transplantation, 70
211
central nervous system, vii, viii, x, xi, xii, 51, 89, 90,
99, 100, 102, 103, 131, 136, 146, 182, 191, 193,
194, 196, 199, 201, 202
cerebellar granule cells, 195
cerebellum, xii, 194, 198, 201, 202
cerebral blood flow, 41
cerebral cortex, xii, 2, 78, 90, 163, 191, 194, 195,
196, 201, 202
cerebral ischemia, 159, 191
cerebrospinal fluid, 140, 170, 178, 191
Chalmers, 81
channel blocker, 171, 191
channels, 164, 179, 191, 197, 199
chemical interaction, xi, 154, 162, 191
chloride, 168, 180, 184, 191
cholecystokinin, 73
cholinergic neurons, 6, 11, 183, 191
choroid, 90, 107, 142
chromaffin, ix, x, 113, 114, 115, 116, 118, 120, 121,
122, 125, 126, 127, 128, 129
chromatography, 117
chromosome, 204
citalopram, 95, 107, 135, 140, 142
classes, x, 3, 96, 131, 146
classification, 43, 102, 140
clinical trials, 63, 96, 99, 175, 191
cloning, 155, 191, 207, 208
clozapine, 96, 97, 108, 109, 110, 174, 186, 190, 191,
207
CNS, viii, ix, 2, 10, 13, 51, 55, 67, 87, 88, 101, 102,
106, 107, 141, 195, 200
cocaine, x, 4, 9, 19, 32, 93, 99, 104, 110, 111, 145,
147, 148, 154, 191, 194, 200
cocaine abuse, 99
coding, 21, 194
coefficient of variation, 117
coenzyme, 69
cognition, vii, xi, 1, 19, 41, 45, 48, 49, 89, 169, 187,
191, 193, 194
cognitive deficit, 34, 49
cognitive deficits, 34, 49
cognitive dysfunction, viii, 17, 49, 203
cognitive function, viii, 17, 18, 19, 20, 21, 33, 35,
38, 39, 89, 96, 194, 197
cognitive impairment, 20, 34, 37, 44, 46
cognitive performance, 34, 39, 108
cognitive process, 19, 20
cognitive processing, 19, 20
cognitive system, viii, 17
cognitive tasks, 19, 21, 170, 191
212
Index
cohort, 46, 65
collateral, 67, 101
combination therapy, 69
communication, 57, 158, 161, 179, 191
community, 69, 70
complex behaviors, 89
complex interactions, 157, 191
complexity, 38, 69, 155, 191
complications, 52
components, vii, xi, 1, 4, 6, 12, 91, 154, 162, 166,
181, 191
composition, 160, 191
compounds, 56, 59, 61, 70, 90, 109, 138
comprehension, 69
computed tomography, 39
Computer simulation, 22
COMT inhibitor, 35, 45
concentration, 32, 34, 56, 59, 61, 62, 75, 125, 178,
191
conception, 4
condensation, 66
conditioning, 147, 148, 171, 177, 186, 187, 191
configuration, 3
connectivity, 53, 54, 101
consciousness, 14
consensus, 157, 179, 191
consolidation, x, 145, 146, 147, 148, 150
construct validity, 170, 172, 187, 191
control, viii, ix, x, xi, 2, 5, 6, 17, 19, 20, 22, 25, 26,
27, 36, 38, 40, 41, 46, 47, 49, 50, 54, 60, 65, 69,
74, 81, 87, 88, 89, 92, 93, 95, 99, 100, 103, 118,
119, 120, 121, 124, 129, 131, 132, 135, 141, 148,
154, 158, 164, 165, 173, 174, 175, 176, 191, 198,
205
convergence, 43
conversion, 126, 160, 167, 191
copper, 116
correlation, 68
correlations, 71, 176, 191
cortex, viii, xii, 2, 4, 6, 17, 18, 19, 27, 32, 34, 36, 39,
40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 78, 89, 90,
91, 92, 94, 97, 98, 101, 103, 104, 106, 108, 109,
127, 128, 136, 147, 156, 163, 164, 168, 191, 194,
195, 196, 200, 201, 202, 204
cortical neurons, 46, 47, 48
corticosterone, ix, 76, 113, 114, 115, 117, 123, 124,
125, 127
corticotropin, 206
cortisol, 114, 125, 127, 185, 191
costs, 52, 70
coupling, 133, 194, 195, 205

COX-2 enzyme, 61, 63
COX-2 inhibitors, 62, 63, 65
creatine, 64, 69, 80
creatinine, 80
critical period, 172, 191
CSF, 60, 133, 139
culture, 62
cyclooxygenase, 60, 79, 80
cyclooxygenase-2, 79
cytokines, 58, 60, 61, 79
cytoplasm, 58, 61, 114, 118, 119, 121, 124, 126
Czech Republic, 193
D
death, viii, 51, 54, 55, 56, 59, 60, 62, 64, 66, 67, 69,
71, 77, 81, 82, 166, 167, 191, 199
deaths, 66
deep brain stimulation, 67, 98
deficiency, 14, 58, 173, 191
deficit, xi, 20, 40, 49, 154, 172, 173, 174, 187, 191,
195
definition, 158, 191
degenerate, viii, 51, 56, 69, 74
degradation, 57, 81, 95, 183, 191, 206
delivery, 203
delusions, 8, 169, 191
demand, 32, 42
dementia, 7, 8, 69, 84, 168, 191, 206
dendrites, 3, 53, 59, 89, 197
dendritic arborization, 53
dendritic spines, 163, 191
density, 3, 32, 33, 57, 107, 116, 120, 121, 142, 146,
156, 158, 169, 191, 197, 200, 202
dentate gyrus, 67, 83
depolarization, 137
depression vii, viii, ix, x, 1, 2, 3, 51, 53, 87, 88, 94,
96, 99, 100, 105, 106, 107, 131, 132, 133, 134,
135, 139, 140, 141, 142, 143, 144, 163, 184, 185,
191, 194
depressive symptomatology, x, 131, 132
depressive symptoms, 94, 133, 136
deprivation, 54, 94, 135
deregulation, 173, 191
derivatives, 10, 181, 191
desensitization, 94, 196, 197, 204
desire, 9
destruction, 110, 159, 191
detection, vii, 1, 2, 81, 117, 155, 173, 191
Index
developing brain, 66
developmental delay, 205
developmental factors, 55
DG, 41, 43
diacylglycerol, 201
dialysis, 93, 104
diet, 70, 204
dietary intake, 70
differentiation, 54, 68, 74, 84
diffusion, 158, 191
direct action, 176, 191
discrimination, 83
disease progression, 7, 52, 57
disinhibition, viii, 18, 46, 94, 134, 165, 191
disorder, vii, 1, 7, 9, 12, 14, 39, 40, 52, 71, 107, 133,
136, 143, 166, 170, 172, 186, 187, 191, 195
distilled water, 117
distribution, ix, 5, 46, 54, 57, 71, 73, 74, 87, 88, 89,
90, 91, 146, 156, 185, 191, 196, 200, 201, 206
diversity, 100, 139, 178, 191, 204
division, 53
DNA, 61, 66, 79, 159, 166, 167, 182, 191, 200
DNA damage, 66, 159, 182, 191
DNA repair, 66
donors, 158, 160, 162, 191
dopamine agonist, 100, 141, 189, 191
dopamine antagonists, 47, 106, 108, 142
dopamine precursor, 52
dopaminergic neurons, viii, xi, 4, 51, 68, 71, 72, 73,
74, 77, 79, 82, 83, 84, 101, 102, 103, 107, 143,
153, 156, 166, 168, 178, 184, 191, 200
Dopa-Responsive Dystonia, vii
dorsal horn, 4, 9, 13
dorsolateral prefrontal cortex, 18, 40, 45
down-regulation, 95, 107, 134, 135, 142, 159, 191,
196, 197, 200, 202
dream, 7, 8
Drosophila, 5
drowsiness, 10
drug abuse, ix, xi, 88, 99, 132, 154, 191, 193
drug action, 109
drug addict(ion), 19, 32, 100, 147, 195
drug discovery, 82
drug therapy, 76, 109
drug treatment, 71, 140
drug use, 65, 81
drugs, ix, x, xi, 3, 7, 9, 36, 38, 45, 52, 60, 61, 63, 64,
69, 74, 77, 78, 80, 81, 94, 95, 96, 97, 98, 99, 106,
108, 109, 131, 132, 134, 138, 142, 145, 146, 147,
213
148, 154, 169, 170, 172, 174, 175, 176, 187, 188,
191
DSM, 43
duration, 20, 56, 64
dyskinesia, 3, 96, 98, 169, 191
dysphoria, 133
dystonia, 3
E
ecstasy, 98
Education, 38, 203
EEG, 4, 9, 10, 11
eicosanoids, 61
elderly, 52, 54, 182, 191
elderly population, 52
electrochemical detection, 117
electrodes, 133
electron, 63, 116, 120, 121, 158, 191
electron density, 116, 120
electrophysiological properties, 182, 191
electrophysiological study, 103
email, 1, 17
embryonic stem cells, 70, 74
EMG, 11
emission, 39, 45, 108
emotion, 177, 191
emotions, 194
employment, 94, 134
encoding, 197
endocrinology, 128, 129
endocytosis, 197, 204
endogenous progenitors, 68
endothelial progenitor cells, 79
endothelium, 158, 191
energy, 57, 66, 69, 167, 191
enlargement, 124
enthusiasm, 67
entorhinal cortex, 156, 191
environment, 83, 84, 160, 172, 176, 179, 191
environmental stimuli, 66
enzyme, 2, 34, 53, 56, 58, 61, 62, 115, 126, 155,
158, 161, 177, 191, 199
enzymes, 61, 63, 66, 157, 158, 159, 166, 191, 197
epidemiology, 13
epidermal growth factor, 68, 83
epilepsy, 31, 33, 36, 38, 39, 40, 41, 42, 43, 44, 46,
47, 48, 49
epinephrine, ix, 2, 113, 114, 115, 117, 120, 121, 125,
126, 127, 129, 154, 191
Index
214
epithelial cells, 107, 142

epoxy, 116
equilibrium, 24, 28, 29, 30, 31, 201
ERK, x, 145, 147, 148
ERK proteins, x, 146
ERK1, x, 146, 147, 148, 149, 150
ester, 160, 191
ethanol, 99, 111, 116, 117, 175, 191
ethical issues, 70
etiology, 71, 162, 165, 166, 169, 176, 177, 191
evidence, vii, viii, ix, xi, 1, 6, 7, 9, 10, 14, 41, 43, 47,
48, 51, 56, 59, 60, 62, 63, 64, 66, 67, 69, 72, 84,
87, 89, 91, 92, 93, 95, 97, 98, 104, 109, 128, 129,
132, 134, 135, 140, 153, 154, 160, 162, 163, 165,
166, 168, 169, 170, 171, 172, 175, 178, 191
examinations, 59
excitability, 18, 20, 23, 177, 191
excitation, 30, 33, 42, 50, 78, 93, 99, 200
excitotoxicity, viii, 51, 81
execution, 66
executive function, 21, 169, 178, 191
executive functions, 21, 169, 178, 191
executive processes, 48
exercise, 70
experimental condition, 162, 191
exposure, 54, 60, 75, 94, 95, 99, 135, 146, 160, 168,
171, 175, 184, 191
extraction, 117
extrapyramidal effects, 175, 191
eye movement, vii, 1, 14
F
face validity, 171, 191
facial muscles, 172, 191
FAD, 157, 191
failure, 7, 8, 22, 60, 62, 67, 174, 191
family, x, xi, 52, 89, 90, 99, 102, 131, 141, 155, 158,
191, 193, 194, 195, 196
fatigue, 133
fear, 171, 191
feedback, 25, 26, 27, 38, 157, 165, 176, 191
ferritin, 166, 191
FFT, 11
fibers, 68, 89, 163, 164, 167, 191, 197
fibroblast growth factor, 55
fibroblasts, 79
fine tuning, 196
fish, 115, 126, 128, 129
fluctuations, 12
fluid, 60, 140

fluoxetine, 94, 95, 107, 134, 135, 140, 142
fluvoxamine, 95, 135, 142
fMRI, 32, 41, 42, 45, 49
focusing, xi, 154, 191
food, 70, 111
forebrain, 2, 3, 4, 5, 6, 8, 10, 11, 12, 14, 73, 76, 84,
90, 92, 141, 167, 173, 183, 191
formaldehyde, 158, 191
fragmentation, 6, 7, 66, 79
free radicals, xi, 153, 162, 191
frontal cortex, 43, 47, 90, 97, 103, 104, 136, 156,
162, 168, 169, 191, 195
frontal lobe, 41, 48, 185, 191
functional analysis, 56
functional imaging, viii, 17, 18, 19, 24, 38
functional MRI, 42
G
G protein, x, 194, 196, 201
GABA, viii, 3, 13, 18, 35, 36, 37, 43, 46, 53, 72, 89,
91, 92, 103, 105, 137, 144, 168, 184, 191, 198
GABAergic, viii, 18, 20, 22, 23, 25, 27, 35, 36, 37,
39, 40, 43, 47, 72, 73, 103, 137, 144, 164, 191
gait, 83, 199
Ganglia, 162, 181, 191
gastrulation, 55
gene, xii, 5, 10, 34, 43, 45, 47, 55, 56, 61, 62, 66, 68,
79, 80, 148, 155, 167, 191, 194, 196, 197, 201,
202, 203, 204, 205, 206, 207, 208
gene expression, 43, 55, 56, 204, 205
gene promoter, 62, 80
gene targeting, 148
generation, 37, 49, 55, 63, 80, 98, 101, 141, 158,
180, 185, 191, 194, 199
genes, 43, 56, 60, 61, 76, 147, 194, 201, 205
genetics, 40, 50, 54
genotype, 41, 149, 198
gland, ix, 113, 114, 115, 118, 119, 120, 121, 124,
125, 127, 128, 129, 206
glass, 116
glia, 59, 60, 72, 166, 191
Glial, 71, 82
glial cells, 59, 60, 64, 67
globus, 98, 162, 181, 191
glucocorticoids, 128
glutamate, xi, 19, 35, 36, 42, 48, 60, 147, 155, 158,
179, 180, 185, 186, 187, 191, 193, 197, 204, 208
glutathione, 59, 62, 166, 191
Index
glycine, 198
goal-directed behavior, 89, 156, 191
G-protein, 3, 90, 129, 133, 147, 148, 156, 191, 199,
201, 203
granule cells, 195, 197
granules, ix, 57, 114, 116, 120, 121, 122, 126
gray matter, 162, 191
grids, 116
groups, 2, 3, 4, 6, 9, 14, 54, 57, 66, 74, 88, 91, 118,
159, 162, 169, 185, 191, 194
growth, 55, 56, 68, 77, 83, 84, 129, 205
growth factor, 55, 56, 68, 77, 83, 84, 129
growth factors, 68, 129
growth hormone, 205
H
habituation, 171, 172, 186, 187, 191
half-life, 158, 191
hallucinations, 8, 169, 191
Harvard, 1
HD, 59
HE, 40
health, 52, 54, 70, 71, 179, 191
heart rate, vii
heme, 158, 191
hemoglobin, 161, 191
heroin, 148
heterozygote, 197
hippocampus, 20, 83, 90, 156, 158, 173, 189, 191,
195, 198, 202
histochemistry, 158, 191
HLA, 76
homeostasis, 3, 202, 203
homovanillic acid, 133
hormone, vii, 117, 127, 128, 129, 205, 206
host, 159, 191
housing, 142
HPLC, 114, 117, 179, 191
human brain, 14, 39, 45, 66, 71, 73, 90, 96, 98, 102,
201
human subjects, 43
Huntingtons disease, 59
Huntington's disease, 77
hydrolysis, 90, 199, 202
hydroxyl, 52, 166, 191
hyperactivity, viii, 18, 27, 30, 33, 38, 99, 110, 111,
156, 169, 172, 189, 191, 195
hyperkinesia, 165, 191
hypersensitivity, 65, 133
215
hypersomnia, 7
hypertension, 196
hypodopaminergic, viii, 17, 18, 24, 25, 26, 27, 33,
34, 37, 38
hypokinesia, 165, 167, 191
hypothalamus, vii, 1, 2, 3, 9, 12, 127, 156, 162, 191,
195, 196
hypothermia, 116, 195
hypothesis, 7, 8, 9, 18, 22, 25, 33, 34, 35, 37, 38, 40,
43, 58, 59, 62, 65, 69, 71, 94, 95, 96, 99, 110,
134, 154, 155, 166, 167, 170, 173, 174, 176, 177,
178, 186, 191, 198, 199, 200, 202
I
ibuprofen, 61, 63, 65, 80
identification, 55, 70, 180, 191, 206
identity, 8, 91
idiopathic, ix, 14, 52
IFN, 59
IL-6, 59, 60, 61
image analysis, 116
imaging, viii, 18, 19, 24, 32, 33, 38, 43, 44, 74, 169,
184, 191
imaging techniques, 33
immersion, 117
immune reaction, 58, 59
immune response, 58, 60, 61, 158, 191
immune system, 81
immunocytochemistry, 72
immunohistochemistry, 73, 200
immunoreactivity, 3, 47, 57, 60, 91, 166, 191
impairments, 20, 80, 166, 191
impregnation, 72
in situ, 156, 191, 200
in situ hybridization, 156, 191
in vitro, 10, 11, 13, 55, 58, 62, 63, 67, 97, 108, 114,
129, 144, 160, 166, 176, 179, 191
in vivo, ix, 32, 43, 55, 63, 73, 77, 79, 82, 87, 92, 93,
94, 97, 103, 104, 105, 108, 109, 115, 127, 135,
140, 155, 159, 160, 176, 179, 180, 182, 190, 191,
199, 203
incidence, 4, 65, 81, 96, 133
inclusion, 12
inclusion bodies, 12
indication, 65, 94, 133
indices, 185, 191
indirect measure, 158, 191
indomethacin, 61, 63, 79
216
Index
induction, 60, 61, 62, 66, 68, 76, 78, 79, 84, 147,
158, 191
infection, 61, 168, 191
inflammation, viii, 51, 53, 58, 59, 60, 61, 63, 64, 69,
71, 76, 77, 80, 83
inflammatory mediators, 158, 191
inflammatory response, 60
information processing, xi, 154, 165, 170, 172, 176,
181, 191
ingest, 59
inhibition, 6, 10, 19, 27, 30, 35, 36, 40, 42, 43, 44,
48, 49, 58, 61, 62, 64, 71, 77, 81, 94, 95, 97, 98,
107, 114, 115, 125, 128, 135, 144, 155, 162, 165,
166, 168, 170, 172, 175, 177, 181, 183, 185, 186,
188, 189, 190, 191, 194, 197, 199, 200
inhibitor, 35, 61, 63, 64, 66, 80, 94, 135, 140, 141,
147, 160, 161, 166, 168, 171, 172, 173, 174, 180,
187, 189, 190, 191
inhibitory effect, ix, 93, 95, 99, 113, 114, 125, 127,
135, 140, 200
initiation, 58, 89, 190, 191
injections, ix, 14, 47, 108, 113, 115, 116, 171, 174,
191
injury, 57, 60, 64, 75, 83, 159, 166, 170, 182, 191
inositol, 129
insecticide, 58
insight, 69
instability, viii, 6, 17, 18, 25, 27, 34, 37, 52
insulin, 128
integration, xi, 147, 153, 176, 191
intensity, 57, 155, 191
interaction, vi, vii, x, xi, 1, 12, 35, 66, 88, 89, 90, 96,
103, 127, 131, 132, 134, 144, 145, 146, 148, 149,
150, 154, 156, 161, 164, 172, 173, 174, 176, 184,
188, 190, 191, 200
Interaction, 78, 132, 134
interactions, vii, ix, 1, 14, 18, 35, 48, 101, 105, 113,
114, 115, 128, 136, 140, 164, 166, 175, 181, 189,
191, 204
interface, 178, 191
interference, 10, 61, 174, 191
interferon, 59, 81
Interleukin-1, 77
internalization, 196, 197, 200, 202, 204
interneuron, 30
interneurons, 8, 19, 20, 22, 23, 24, 30, 35, 36, 37, 39,
40, 42, 44, 46, 53, 67, 91, 93, 164, 165, 191, 200,
204
interpretation, 25
intervention, 65, 68, 69, 204
intoxication, 63, 64, 77

introns, 194
ion channels, 158, 159, 191, 197
ions, 56
ipsilateral, 167, 191
iron, 56, 57, 59, 166, 179, 191
irradiation, 66
Israel, 1
Italy, 51, 87, 113, 115, 131, 145
J
Japan, 17
K
K+, 3
kidney, 118, 194
kidneys, 208
killing, viii, 51, 63
kinase, 55, 60, 66, 78, 82, 147, 156, 158, 177, 191
kinetics, 40
L
labeling, 101, 164, 191
latency, 7, 89
latent inhibition, 171, 174, 177, 186, 187, 188, 190,
191
lateral sclerosis, 59
lead, 7, 33, 37, 60, 69, 70, 116, 156, 158
learning, vi, x, xi, 45, 145, 146, 148, 150, 154, 158,
171, 172, 177, 191, 193, 194, 195, 197, 198, 202,
205, 206, 207
learning process, 148
lectin, 72
lesions, 4, 5, 8, 9, 10, 11, 13, 14, 72, 74, 77, 84, 166,
170, 171, 173, 183, 187, 191
leukopenia, 4
levodopa, vii, 52, 71, 98, 109
Lewy bodies, 7, 8, 59, 166, 191, 206
life cycle, ix, 52
life span, 199
life style, 70
ligand, xi, 10, 61, 164, 191, 193, 196, 199, 204, 206
ligands, 79, 99, 156, 191, 200
limbic system, 88, 156, 181, 191
limitation, 134
links, 34, 35
Index
lipid peroxidation, 63, 80, 166, 180, 184, 191
lipids, 170, 191
liquid chromatography, 117
literature, x, xi, 69, 88, 131, 132, 153, 171, 191
liver, 115
localization, x, 14, 46, 89, 91, 99, 102, 129, 131,
158, 182, 191, 195, 205
location, 11, 195
locomotor activity, 5, 64, 98, 99, 110, 111, 133, 175,
188, 189, 191
locus, vii, 1, 4, 6, 10, 12, 13, 55
London, 39, 128, 129, 185, 191, 203
LPS, 60
LTD, 163, 168, 191
LTP, 158, 163, 168, 191, 194
lying, 53
lymphocytes, 59, 63
lysine, 62
M
machinery, 66, 158, 191
macrophages, 60, 77, 166, 191
magnetic resonance, 32
magnetic resonance imaging, 32
major depression, x, 3, 131, 132, 134, 136, 144
major depressive disorder, 107, 143
mammalian brain, 67, 84, 154, 191
management, 13, 70, 71
manganese, 168, 184, 191
mania, 100, 106, 139
manipulation, 41
manners, 165, 191
mapping, 11, 72, 181, 191
masking, 95, 135
matrix, 54, 56, 73, 163, 181, 191
maturation, 55, 73
measures, 172, 191
median, 89, 102
medication, vii, 44, 65
medicine, 178, 191
medulla, 8, 9, 11, 127
MEK, 147
melanin, 14
melatonin, 107, 143
memory, vi, vii, viii, x, 1, 17, 18, 19, 20, 21, 24, 25,
33, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 67, 83, 89, 108, 145, 146, 147, 148,
150, 158, 169, 191, 194, 206
memory capacity, 21, 43
217
memory formation, 146, 147, 148, 158, 191

memory performance, 21, 36, 42, 45, 46
memory processes, vi, x, 67, 145, 148
men, 21, 65
mental disorder, ix, 87, 91, 169, 191
mental state, 37
mental states, 37
mesencephalon, 53, 74, 88
messenger RNA, 102
messengers, 146, 197
meta-analysis, 42
metabolic disturbances, 53
metabolism, 43, 56, 60, 69, 80, 94, 126, 140, 159,
161, 170, 191
metabolites, xi, 56, 139, 154, 162, 182, 191, 198
metabotropic receptor, xi, 193
metal ions, 56
methamphetamines, viii, 2, 9
methanol, 117
methylation, 34
methylene, 173, 189, 191
methylphenidate, 9
mice, x, xi, 5, 8, 10, 13, 47, 56, 63, 66, 67, 72, 73,
76, 77, 80, 83, 84, 88, 99, 111, 132, 137, 143,
146, 147, 148, 149, 167, 168, 172, 173, 174, 175,
182, 183, 188, 189, 190, 191, 193, 195, 196, 197,
198, 199, 200, 202, 203, 204, 205, 206, 207, 208
microdialysis, 32, 73, 79, 103, 104, 134, 136, 160,
177, 179, 180, 191, 199
microenvironment, 68
microglia, 56, 57, 58, 59, 60, 61, 69, 74, 76, 77, 166,
191
microglial cells, 59, 60
microscope, 116
microscopy, 89, 114, 200
midbrain, 2, 4, 5, 8, 13, 25, 55, 56, 57, 64, 73, 74,
75, 89, 91, 101, 102, 104, 105, 142, 156, 177,
182, 191
migration, 54, 84
milligrams, 117
Ministry of Education, 38, 203
miosis, 199
mitochondria, 78, 118, 119, 124, 166, 167, 191
mitochondrial damage, 80
mitochondrial DNA, 166, 191
mitochondrial dysfunction, viii, 51, 57, 156
mitochondrial membrane, 58
mitogen, 60, 78
mitotic, 55
mobility, 83
218
Index
moclobemide, 94, 135, 141

modafinil, viii, 2, 10, 13, 14
modeling, 190, 191
models, xi, 8, 42, 49, 58, 62, 64, 66, 67, 76, 78, 84,
100, 106, 134, 139, 165, 170, 172, 174, 183, 185,
187, 191
molecular biology, 45, 54, 79, 88, 99, 132, 155, 191
molecular mechanisms, 39, 69, 165, 191
molecular oxygen, 155, 158, 191
molecular structure, 158, 191
molecular weight, 206
molecules, xi, 57, 78, 154, 162, 191, 201
monkeys, 20, 33, 36, 39, 40, 46, 47, 53, 58, 59, 67,
108, 166, 191
monoamine oxidase, 56, 94, 132, 135, 184, 191
monoamine oxidase inhibitors, 132
mononuclear cells, 79
mood, viii, 4, 51, 52, 67, 106, 133, 134
mood disorder, 52, 106
morphine, x, 93, 98, 104, 110, 146, 147, 148, 149
morphology, 2, 72, 74
morphometric, 74, 101, 116
mortality, 191
mosaic, 54
motion, 169, 191, 201
motivation, vii, 1, 89, 133, 154, 156, 169, 175, 191
motor activity, 6, 140, 148, 190, 191, 203
motor behavior, xi, 84, 153, 164, 165, 170, 174, 175,
176, 187, 191
motor control, 154, 156, 162, 163, 164, 165, 175,
191
motor function, 163, 191, 202
motor neurons, 8
motor skills, 205
motor system, 2, 3
mouse model, 60, 62, 63, 76, 77, 80, 81, 82, 158,
183, 191
movement, vii, viii, xi, 1, 2, 8, 9, 13, 14, 51, 162,
165, 166, 190, 191, 193, 194, 196
movement disorders, vii, 1, 14, 162, 165, 191
MRI, 42
mRNA, 36, 60, 62, 72, 76, 82, 90, 91, 103, 107, 129,
142, 156, 158, 169, 178, 191, 200, 202, 203, 208
multiple sclerosis, 59
multiples, 159, 191
muscarinic receptor, xii, 193, 200, 201, 202, 203,
207
muscle cells, 79
muscle weakness, 199
mutant, x, 99, 111, 137, 146, 148, 175, 191, 196,

197, 199, 203, 204, 207
mutation, xi, 5, 40, 136, 148, 193, 197, 208
mutations, 58
N
NaCl, 115, 116
NAD, 167, 191
NADH, 58, 168, 191
narcolepsy, 13, 14
necrosis, 57, 66, 78
negative reinforcement, xi, 153, 191
neocortex, 162, 191, 195
nerve, 52, 56, 73, 77, 91, 134, 155, 166, 167, 191
nerve cells, 52, 56
nerve growth factor, 77
nervous system, vii, viii, x, xi, xii, 51, 89, 90, 99,
100, 101, 102, 103, 131, 136, 146, 154, 155, 162,
179, 188, 191, 193, 194, 196, 199, 201, 202, 205
network, 11, 18, 21, 36, 40, 41, 43, 45, 101
neural mechanisms, 98
neural network, 8, 45, 181, 191
neural networks, 8
neuritis, 166, 191
neurobiology, viii, 2, 42, 100, 106, 139, 182, 184,
191
neuroblastoma, 62, 107, 142
neuroblasts, 68
neurodegeneration, xi, 63, 66, 67, 71, 74, 76, 77, 80,
81, 154, 162, 165, 166, 168, 185, 191, 200
neurodegenerative diseases, 59, 66, 67, 76, 82, 169,
182, 191
neurodegenerative disorders, 66, 76, 78, 81, 83, 175,
191
neurodegenerative processes, 60, 176
neuroendocrine, 44, 206
neurogenesis, ix, 52, 54, 66, 67, 70, 83, 84
neurohormone, vii
neuroimaging, 42
neuroinflammation, 59, 69, 70
neuroleptics, 3, 108, 133, 154, 174, 191, 207
neurological condition, 2
neurological disease, 69
neurological disorder, viii, 3, 51, 53
neuromodulator, 158, 191, 194
neuronal apoptosis, 71
neuronal cells, 61, 64
neuronal circuits, 33
neuronal death, 60, 67, 69, 77
Index
neuronal degeneration, 8, 59, 60, 156, 191
neuronal excitability, 18
neuronal systems, 72
neuropeptide, 164, 191
neuropeptides, 2, 103, 128
neuropharmacology, 12
neuroprotection, 61, 62, 65, 69, 80, 110
neuroprotective, 62, 64, 70, 80, 162, 170, 175, 176,
191
neuroprotective agents, 175, 191
neuropsychological assessment, 46
neuropsychology, 184, 191
neuropsychopharmacology, 43
neuroscience, 44, 69
neurotensin, 3
neurotoxic effect, 60, 63
neurotoxicity, 61, 62, 66, 74, 80, 82, 162, 167, 168,
170, 177, 183, 184, 185, 191
neurotoxins, 58, 63, 66
neurotransmission, vii, xi, 1, 2, 3, 10, 12, 19, 46, 80,
88, 108, 132, 153, 155, 159, 161, 164, 165, 169,
174, 175, 177, 180, 191
neurotransmitter, vii, ix, xi, 2, 53, 55, 87, 134, 153,
158, 163, 164, 171, 177, 180, 191, 194, 196, 199,
205
neurotransmitters, 53, 204
neurotrophic factors, 60, 69
Newton, 78, 107, 142, 178, 191
nicotinamide, 157, 158, 183, 185, 191
nicotine, 93, 99, 105, 111, 171, 191
nigrostriatal, 3, 8, 9, 53, 66, 67, 71, 74, 82, 84, 88,
89, 90, 92, 93, 94, 96, 104, 110, 156, 166, 168,
169, 183, 184, 191
nitrate, 158, 170, 178, 191
nitric oxide, vi, xi, 57, 59, 76, 77, 79, 153, 155, 158,
164, 177, 178, 179, 180, 181, 182, 183, 184, 185,
187, 188, 189, 190, 191
, 191
nitric oxide synthase, xi, 59, 77, 79, 154, 158, 164,
178, 179, 180, 181, 182, 183, 184, 185, 187, 189,
190, 191
nitrogen, 61, 180, 191
nitrogen oxides, 180, 191
NMDA receptors, 19, 20, 22, 159, 161, 179, 191
nociceptive, 9
non-steroidal anti-inflammatory drugs, 79
noradrenaline, 71, 94, 103, 104, 128, 136, 138, 142
norepinephrine, ix, 2, 12, 113, 114, 115, 120, 121,
125, 126, 127, 154, 180, 191
normal aging, 54, 55, 66
219
NSAIDs, 61, 62, 63, 64, 65, 69, 78

nuclei, 2, 12, 14, 25, 53, 72, 89, 90, 102, 137, 144,
162, 191
nucleic acid, 170, 191
nucleus, vii, ix, 1, 2, 4, 6, 8, 12, 13, 53, 61, 67, 77,
87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 101,
103, 104, 106, 109, 110, 133, 135, 140, 141, 144,
147, 148, 154, 156, 162, 163, 164, 167, 168, 169,
171, 173, 177, 181, 184, 186, 187, 191, 195, 196,
198
nurses, 70
O
obesity, 13
observations, viii, xi, 2, 5, 8, 9, 10, 11, 95, 135, 153,
154, 160, 175, 191
obstructive sleep apnea, 7
oculomotor, 46, 47, 108
olanzapine, 97, 174, 191
olfaction, 67
open field test, 175, 191
operant conditioning, 148
opioid, vi, x, 145, 146, 147, 148, 150
organelles, 57
organization, 13, 46, 73, 127, 162, 163, 181, 191
oxidants, xi, 153, 162, 191
oxidation, 56, 63, 159, 160, 161, 191
oxidative damage, 168, 191
oxidative stress, viii, 51, 56, 57, 60, 66, 69, 74, 82,
156, 162, 166, 168, 170, 191
oxygen, 56, 58, 60, 64, 182, 191
oxyhemoglobin, 158, 160, 191
P
p53, 66, 71, 81
pain, 59
paradigm shift, 44, 82
paradoxical sleep, vii, 1, 6
paralysis, 133
parathyroid, 194, 206
paresthesias, 9
Parkinson disease, 13, 81, 82, 84, 195, 201
Parkinsons disease, vii, viii, ix, xi, 1, 2, 5, 6, 8, 12,
13, 14, 51, 52, 70, 71, 73, 74, 75, 76, 77, 78, 80,
81, 82, 83, 84, 85, 88, 89, 97, 98, 99, 109, 110,
133, 140, 153, 154, 156, 157, 162, 165, 182, 183,
184, 191, 200, 203, 206
220
Index
Parkinsonian symptoms, 174

Parkinsonism, 3, 65, 74, 75, 76, 82, 98, 108, 154,
182, 183, 184, 191
paroxetine, 95, 96, 135
parvalbumin, viii, 18, 36, 53, 73
pathogenesis, 2, 8, 9, 57, 58, 62, 65, 71, 75, 76, 89,
95, 135, 166, 168, 169, 185, 191
pathology, 7, 8, 12, 71, 82, 103, 139, 196, 200
pathophysiological mechanisms, 166, 173, 191
pathophysiology, 9, 41, 42, 44, 50, 59, 67, 69, 84,
88, 91, 94, 132, 134, 139, 165, 168, 170, 174,
175, 179, 180, 182, 186, 191
pathways, vi, viii, xi, 2, 8, 48, 51, 55, 58, 60, 66, 69,
75, 82, 88, 92, 96, 100, 104, 107, 133, 134, 136,
139, 143, 145, 147, 153, 156, 162, 165, 167, 176,
177, 178, 179, 183, 191, 201, 202, 204, 205, 206
PCP, 169, 171, 172, 173, 174, 191
PD, viii, xi, 6, 7, 8, 9, 12, 51, 52, 53, 54, 56, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 153, 154,
156, 165, 166, 167, 168, 169, 175
peptides, 157, 158, 191
perception, 48, 169, 191
perceptual processing, 37
performance, x, 18, 20, 21, 34, 37, 39, 42, 45, 46, 49,
108, 117, 146, 149, 172, 186, 191
peripheral blood, 79
peroxidation, 63, 80, 159, 166, 191
peroxynitrite, 155, 159, 167, 179, 180, 183, 191
pertussis, 129
pesticide, 75, 168, 184, 191
PET, 43, 46, 64
pH, 116, 117
pharmacological treatment, 149
pharmacology, ix, 43, 45, 87, 88, 90, 96, 100, 101,
102, 108, 139, 158, 186, 191
pharmacotherapy, 68, 176, 191
phencyclidine, 35, 43, 98, 104, 108, 110, 169, 187,
189, 191
phenethylamine, vii
phenomenology, 43, 184, 191
phenotype, 5, 54, 73, 75, 189, 191, 199, 200, 204
phenotypes, 40, 45
pheochromocytoma, 129
phosphate, 116, 117, 129, 157, 158, 181, 183, 185,
191
phosphorylation, 20, 58, 95, 164, 191
physical exercise, 70
physiology, ix, 79, 87, 88, 103, 139, 180, 191
physiopathology, x, 131
pilot study, 178, 191
pioglitazone, 61, 78, 80

placebo, 107, 108, 136, 143
planning, 46, 89, 156, 169, 191
plasma, 52, 95, 96, 115, 125, 128, 170, 185, 191
plasma levels, 52
plasma membrane, 95
plasticity, 67, 84, 146, 147, 194, 195, 203, 208
Platelet, 84
plexus, 90, 107, 142
PM, 13, 116, 129
point of origin, 54
polymerase, 167, 191
polymorphism, 34, 40, 45, 143
polymorphisms, 41
poor, 53, 56
population, 2, 6, 22, 28, 46, 52, 65, 164, 191, 200
positive feedback, 25, 26, 27, 38
positron, 45
postural instability, 6
posture, 52, 174, 191, 199
potassium, 180, 191, 206
power, 11, 116
precursor cells, 67
prediction, 8, 177, 191
predictive validity, 171, 172, 191
preference, 141, 147
prefrontal cortex, viii, 4, 6, 17, 18, 27, 39, 40, 41, 42,
43, 45, 46, 47, 48, 49, 50, 89, 90, 91, 92, 97, 101,
104, 106, 108, 109, 147, 156, 170, 173, 185, 187,
188, 191, 204
prefronto-mesoprefrontal, viii, 17, 25, 26, 27
pressure, vii, 195
prevention, 64
primate, 36, 41, 47, 49, 72, 83, 84
Primates, 42
problem solving, 156, 191
procedural memory, 148
processing deficits, xi
production, 52, 61, 62, 63, 79, 129, 158, 159, 161,
165, 191
progenitor cells, 55, 79, 83, 84
programming, 74
pro-inflammatory, 56, 58, 60
prolactin, vii, 2, 96
proliferation, 68, 84, 129
promote, 4, 5, 11, 53
promoter, 61, 62, 80
propagation, 60
prophylactic, 63
propylene, 116
Index
prostaglandin, 61, 79
prostaglandins, 129
protein, x, xi, 3, 41, 47, 57, 60, 61, 62, 64, 66, 69,
75, 76, 78, 81, 82, 89, 90, 91, 102, 103, 129, 131,
133, 145, 147, 155, 156, 158, 164, 166, 169, 183,
191, 193, 194, 195, 196, 199, 201, 203, 204, 206,
207
protein aggregation, 69
protein kinase C, 82
protein kinases, 78, 164, 191
protein structure, 166, 191
proteins, x, 3, 39, 53, 55, 57, 59, 66, 73, 82, 145,
146, 147, 148, 158, 161, 166, 167, 170, 191, 194,
197, 205
proteolysis, viii, 51
protocols, 171, 191
PSD, 158, 191
pseudogene, 208
psychiatric disorders, viii, 45, 51, 157, 170, 172,
184, 185, 191, 194
psychological stress, 89
psychological stressors, 89
psychopharmacology, 185, 191
psychoses, 38, 43, 49
psychosis, 8, 31, 32, 33, 35, 38, 39, 40, 43, 46, 47,
48, 49, 154, 191
psychostimulants, viii, 2, 10, 12, 19, 32, 46, 48, 89,
98, 146, 154, 191
psychotic states, viii, 8, 18, 32, 37, 38
psychotic symptoms, 44, 169, 191
psychotropic drug, 95
psychotropic drugs, 95
puberty, 173, 191
Purkinje cells, 197, 208
pyramidal cells, 46
Q
quality of life, 71
question mark, 91
quinone, 74
quinones, 56, 64, 70, 75
R
rain, 58
range, 18, 28, 44, 49, 107, 134, 143
reactive gliosis, 166, 191
reactive oxygen, 56, 58, 166, 191
221
reading, 21
receptor agonist, x, 5, 20, 21, 34, 40, 42, 43, 44, 92,
98, 99, 104, 106, 107, 110, 136, 138, 142, 146,
173, 188, 191, 198
receptor sites, 97
recognition, 161, 191
reconcile, 10
reconstruction, 72
recovery, 68, 117
recycling, 155, 191
red blood cell, 158, 191
red blood cells, 158, 191
reduction, 6, 10, 19, 36, 49, 52, 61, 63, 65, 70, 94,
133, 134, 135, 170, 191, 200, 201
refractory, 5, 7
regeneration, 64
regional, 40, 41, 74, 90, 91, 99
regulation, vi, vii, ix, xi, 1, 4, 5, 6, 7, 12, 36, 39, 42,
48, 55, 67, 78, 87, 88, 89, 91, 92, 95, 99, 100,
101, 105, 106, 107, 110, 128, 134, 135, 142, 153,
155, 157, 161, 165, 168, 173, 178, 179, 180, 187,
188, 189, 191, 193, 195, 196, 197, 200, 201, 202,
204, 206
regulators, 77
reinforcement, xi, 110, 154, 191
reinforcement learning, 154, 191
reinforcers, 89, 98
relationship, 20, 21, 57, 65, 75, 133, 186, 191
relationships, 125, 126, 169
relevance, ix, 17, 49, 52, 82, 102, 113, 132, 155,
178, 191
REM, vii, 1, 5, 6, 7, 8, 9, 10, 11, 12, 14, 94, 135
remission, 32
repair, 66, 83
repressor, 62
residues, 155, 162, 191
resistance, 69, 74, 197
resolution, 11, 64
respiration, 77, 166, 191
respiratory, 7
responsiveness, 95, 107, 142, 173, 191
retardation, 133, 171, 191
reticulum, 118, 119, 121
retina, 194, 195
reversal learning, 45
ribose, 167, 191
rigidity, 6, 52, 166, 168, 191
risk, 8, 41, 64, 65, 70, 81
risk factors, 8
risperidone, 96, 108, 109, 186, 191
Index
222
RNA, 102
robustness, 21
rodents, xi, 53, 68, 143, 154, 166, 172, 174, 191
rotations, 168, 191
Royal Society, 178, 191
S
SA, 41, 50
salt, 62, 117
sampling, 116
schizophrenia, vii, viii, ix, xi, 1, 2, 3, 17, 18, 19, 20,
24, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 87, 88, 96,
97, 99, 107, 108, 132, 153, 154, 156, 157, 169,
170, 171, 172, 173, 174, 175, 176, 177, 178, 179,
184, 185, 186, 187, 188, 190, 191, 194, 195, 200
Schizophrenia, vi, 34, 38, 43, 44, 45, 47, 96, 153,
169, 178, 184, 185, 189, 191
schizophrenic patients, 96, 100, 108, 169, 170, 172,
178, 185, 186, 191
science, 67, 74
sclerosis, 59
search, x, 131, 168, 191
seasonal variations, 120
secretion, 2, 5, 55, 61, 114, 125, 126, 127, 128, 129,
179, 191
seizure, 199
seizures, 42
selective attention, 170, 176, 187, 191
selective serotonin reuptake inhibitor, 95, 132, 135,
143
selectivity, viii, 17, 36, 74
senile dementia, 187, 191
sensation, 14
sensitivity, 40, 134, 167, 173, 174, 191, 206
sensitization, 32, 41, 44, 46, 133, 147
separation, 174, 191
septum, 90, 195
series, 92, 95, 98, 99, 135, 154, 169, 191
Serotonin, ix, 72, 84, 87, 88, 89, 90, 94, 95, 100,
101, 102, 103, 104, 105, 107, 108, 109, 110, 111,
131, 132, 134, 136, 138, 139, 140, 141, 143, 144
sertraline, 95, 107, 135, 142
serum, ix, 76, 113, 115, 116, 117, 122, 123, 124,
125, 126, 127, 186, 191
severity, 136
sexual behavior, 89, 98, 129
shape, viii, 18, 19, 24, 28, 59, 116, 120
shares, 2, 10, 146
short-term memory, 47
sibling, 47
siblings, 35, 199
side effects, 4, 52, 61, 96, 136, 154, 191
signal transduction, xi, 193, 206
signaling, xi, 10, 58, 79, 83, 153, 158, 165, 175, 177,
178, 182, 191, 204, 205, 206
signaling pathway, 58, 79, 165, 176, 182, 191
signaling pathways, 165, 176, 191
signalling, 55, 60, 146, 147, 171, 179, 191, 202
signals, 45, 68, 81, 147, 148, 158, 179, 191
signs, ix, 52, 54, 71, 114, 118, 124, 125
similarity, 133, 171, 191
simulation, 24, 28, 50
sites, 3, 11, 62, 90, 97, 101, 157, 164, 181, 191, 195,
198, 200, 201, 204
skills, 205
sleep apnea, 7
sleep disturbance, 6, 7, 52, 136
Slovakia, 193
smoking, 99
smoking cessation, 99
smooth muscle, 79
smooth muscle cells, 79
SN, x, 2, 5, 42, 53, 56, 59, 64, 66, 68, 89, 90, 131,
156, 162
SNAP, 160, 191
SNc, viii, ix, 5, 6, 51, 52, 53, 54, 55, 56, 59, 60, 63,
66, 67, 69, 87, 88, 89, 90, 91, 92, 95, 98, 135
SNP, 161, 164, 168, 184, 191
social care, 52
social isolation, 174, 191
social withdrawal, 169, 191
society, 52
sodium, 3, 78, 79, 80, 161, 191
somata, 57, 164, 191
somatosensory, 13
somatostatin, 164, 191
spatial learning, 197, 198, 202
spatial memory, 45, 83
species, ix, 56, 58, 60, 61, 64, 113, 114, 125, 166,
170, 172, 182, 189, 191
specificity, 19, 36, 158, 191, 206
spectrum, xi, 11, 56, 59, 154, 191
spermatogenesis, 126
spinal cord, 4, 8, 9, 13, 14, 90, 102
spine, 163, 191
Sprague-Dawley rats, 55, 137, 188, 191
sprouting, 60, 67, 77, 83
St. Louis, 115, 117
Index
stability, 25, 27, 34, 36
stabilization, 36, 41, 75
stages, ix, 7, 14, 52, 55, 57, 64, 114, 115, 116, 120
standard error, 118
stem cell therapy, 68, 70
stem cells, 55, 68, 70, 74
steroid hormone, 129
steroidogenic, ix, 113, 114, 115, 116, 118, 119, 124,
125, 127
steroids, 117
stimulant, 15, 134, 141
stimulus, 147, 155, 165, 171, 172, 191
storage, 44, 129
strain, 137, 146, 173, 189, 191, 197, 198
strategies, x, 43, 54, 84, 139, 146
strength, viii, 18, 30, 32, 199
stress, viii, 21, 33, 39, 45, 46, 51, 53, 56, 57, 59, 60,
66, 69, 74, 76, 82, 89, 92, 94, 104, 105, 106, 116,
129, 134, 135, 140, 141, 142, 143, 154, 170, 173,
191, 194, 205, 206
stress factors, 53
stressors, 89
striatal dopamine receptors, xii, 194
striatum, ix, x, 2, 3, 6, 7, 20, 38, 52, 53, 58, 59, 64,
67, 71, 72, 73, 77, 82, 83, 84, 87, 88, 89, 90, 92,
93, 95, 98, 103, 104, 134, 140, 145, 146, 147,
156, 157, 160, 161, 162, 163, 164, 166, 167, 168,
169, 174, 176, 178, 179, 180, 181, 182, 183, 184,
191, 195, 198, 200, 201, 202, 204
strong interaction, 200
subcortical structures, 18, 155, 191
substantia nigra, vii, viii, ix, x, 2, 5, 51, 52, 53, 67,
71, 72, 73, 74, 75, 76, 77, 82, 83, 84, 87, 88, 89,
90, 91, 93, 101, 103, 105, 110, 131, 156, 195
substantia nigra compacta, viii, 51
substantia nigra pars compacta, ix, 52, 71, 87, 88, 89
substrates, 10, 174, 191
sucrose, 134, 141
sulphur, 159, 191
summer, 120
Sun, 43
superoxide, 63, 80, 159, 160, 166, 167, 170, 191
suprachiasmatic nucleus, 170, 191
surprise, 187, 191
survival, 54, 57, 68, 200
susceptibility, 32, 35, 57, 74
susceptibility genes, 58
suspensions, 160
switching, 22, 25, 61
sympathetic nervous system, vii
223
symptom, 57, 134, 186, 191

symptomatic treatment, 70
symptoms, 13, 18, 35, 37, 38, 44, 52, 60, 94, 96, 97,
98, 100, 133, 136, 154, 156, 163, 166, 167, 168,
169, 172, 184, 190, 191, 199
synapse, 199
synaptic plasticity, 158, 168, 169, 184, 191, 195,
203, 208
synaptic strength, 158, 191
synaptic transmission, 19, 42, 182, 191
synaptic vesicles, 3, 57, 75
synchronization, 37
syndrome, 2, 3, 96, 133, 136, 154, 191, 194
synthesis, 3, 36, 55, 57, 60, 61, 75, 129, 140, 155,
158, 159, 168, 179, 183, 190, 191, 199
systems, vi, ix, x, xi, 2, 3, 4, 6, 7, 10, 14, 34, 35, 43,
46, 53, 57, 72, 87, 88, 89, 91, 94, 95, 96, 97, 99,
100, 101, 127, 131, 132, 134, 139, 145, 146, 148,
150, 154, 155, 156, 164, 171, 173, 175, 176, 190,
191, 193, 194, 196, 199, 200
T
T cell, 59, 61
tardive dyskinesia, 3, 96
targets, 54, 59, 85, 101, 155, 181, 191
taste aversion, 171, 187, 191
taxonomy, 81
technical assistance, 176, 191
temperature, 115, 161, 191
temporal lobe, 34, 39, 44
temporal lobe epilepsy, 34, 39, 44
terminals, 3, 56, 60, 74, 89, 91, 101, 134, 155, 163,
164, 165, 167, 171, 181, 191
TGF, 55
thalamus, 4, 6, 156, 162, 191
theory, viii, 17, 18, 19, 25, 34, 37, 38, 41, 60, 82,
186, 191
therapeutic approaches, 74
therapeutic targets, 59
therapeutics, 41, 108, 109
therapy, 52, 63, 68, 70, 71, 76, 78, 83, 109, 154, 166,
175, 191
thinking, 169, 191
three-dimensional reconstruction, 72, 181, 191
threshold, 31, 34, 54
threshold level, 54
time, 4, 11, 22, 23, 30, 54, 62, 64, 65, 70, 76, 105,
115, 137, 155, 161, 168, 171, 186, 191, 199
timing, 44
224
Index
tissue, ix, 67, 77, 113, 114, 115, 116, 124, 125, 126,
127, 158, 159, 161, 191, 195, 199
TNF, 57, 59, 60, 61, 79
TNF-, 57, 59, 60
Tokyo, 17, 129
tonic, xi, 34, 40, 92, 93, 99, 153, 155, 165, 176, 177,
191, 199
toxic effect, 61
toxic substances, 60
toxicity, 60, 63, 70, 109, 159, 166, 168, 183, 191
toxin, 54, 60, 62, 64, 66, 129, 168, 191
training, 146, 148, 149
transcription, xii, 54, 60, 61, 68, 73, 74, 78, 79, 194,
201, 205
transcription factors, 54, 61, 68, 205
transcripts, 56
transducer, 61
transduction, xi, 148, 193, 206
transformation, 61, 78
transforming growth factor, 55
transgenic, 59, 68, 80, 158, 191
transition, 31, 33, 56
transition metal, 56
transition metal ions, 56
translocation, 61, 179, 191
transmembrane region, 90
transmission, viii, 3, 4, 10, 14, 17, 18, 19, 20, 21, 23,
25, 30, 32, 34, 35, 36, 37, 38, 42, 48, 95, 98, 103,
107, 116, 136, 139, 141, 148, 165, 168, 176, 180,
182, 186, 191, 199, 202
Transmission Electron Microscopy, 116, 118
transplantation, 67, 70
transport, 9, 63, 72, 77, 155, 161, 179, 180, 191
trees, 53
tremor, 2, 6, 52, 166, 191, 199
trend, 63, 65
trial, 65, 69, 70, 143
tricyclic antidepressant, 94, 132, 135, 141
tricyclic antidepressants, 94, 132, 135
triggers, 22, 23
Triturus carnifex, ix, 113, 114, 115, 117, 119, 120,
121, 124, 126, 127, 128, 129
trophic support, 54, 59
tumor, 57
tumor necrosis factor, 57
turnover, 21, 33, 41, 46, 56, 133, 155, 191
tyrosine, 3, 13, 21, 53, 55, 72, 73, 155, 164, 177,
181, 191
Tyrosine, 72
tyrosine hydroxylase, 53, 55, 72, 73, 155, 164, 177,

181, 191
U
UK, 70, 117
unconditioned, 171, 177, 191
underlying mechanisms, 52
United States, 178, 191
users, 64
V
vacuum, 117
validity, 106, 139, 171, 191
values, 24, 29, 108, 120, 122, 123, 124, 125
variable, 25, 116, 120, 167, 173, 191
variables, 157, 191
variance, 118
variation, 27, 28, 45, 117
vector, 68
vein, 168, 191
velocity, 161, 191
venlafaxine, 136
ventral tegmental area, vii, ix, x, 1, 2, 6, 13, 53, 87,
88, 90, 91, 101, 102, 103, 107, 131, 133, 142,
143, 144, 147, 156, 182, 195
ventricular zone, 55, 195
verbal fluency, 32, 42
vertebrates, ix, 113, 114, 115, 125, 129, 146
vesicle, 75, 183, 191
virus, 205
viruses, 66
vitamin C, 175, 191
vitamin E, 175, 191
vitamins, 175, 191
VTA, ix, x, 2, 5, 6, 53, 56, 87, 88, 89, 90, 91, 92, 93,
95, 99, 131, 133, 135, 156, 164, 169
vulnerability, 52, 53, 56, 74, 75, 143, 182, 191
W
waking, 5, 8, 12, 14
walking, 175, 191
WCST, 34
weakness, 199
white matter, 169, 191
wild type, x, 146, 147, 148, 149, 197, 198, 201, 202,
203
Index
winter, 120
Wisconsin, 34
withdrawal, 66, 105
women, 65
workers, 62
working memory, viii, 17, 18, 19, 20, 21, 24, 25, 33,
35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 89, 108, 178, 191
X
xenon, 39
Y
yield, 117
yuan, 49
Z
ziprasidone, 97
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DOPAMINE RESEARCH ADVANCES

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DOPAMINE RESEARCH ADVANCES

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Dopamine Control of Sleep and Arousal

A Circuit Dynamics Theory of Complex Dopaminergic

The Life Cycle of the Dopaminergic Neurons

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role for DA in mediating the wake-promoting effects of psychostimulants (e.g.,

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dopamine plays an inhibitory role on steroidogenic activity, like epinephrine, and a

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In: Dopamine Research Advances

DOPAMINE CONTROL OF SLEEP AND AROUSAL

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Patrick M. Fuller and Jun Lu

ANATOMY OF THE CENTRAL DOPAMINERGIC SYSTEM

PHARMACOLOGY OF CENTRAL DOPAMINE

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Dopamine Control of Sleep and Arousal

synthesis is tyrosine hydroxlyase (TH). TH immunoreactivity is widely considered a useful

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DOPAMINE AND SLEEP REGULATION

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Dopamine Control of Sleep and Arousal

Figure 2. A) is a photomicrograph showing the distribution of wake-active (Fos see arrows) DA

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DOPAMINERGIC DISORDERS AND SLEEP:

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Dopamine Control of Sleep and Arousal

Excessive Daytime Sleepiness

REM Behavior Disorder

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Restless Leg Syndrome

DOPAMINE AND STIMULANT-INDUCED AROUSAL

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stimulants was attributed almost exclusively to NE mechanisms, as adrenergic signaling is

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In addition to inhibiting VLPO neurons, amphetamine-like stimulants may also promote

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Fleetwood-Walker SM, Hope PJ, Mitchell R. Antinociceptive actions of descending

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