Dopamine Research Advances

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DOPAMINE RESEARCH ADVANCES

DOPAMINE RESEARCH ADVANCES

AKIYAMA WATANABE EDITOR

Nova Biomedical Books New York

Copyright © 2008 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA 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

CONTENTS Preface

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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

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vi Chapter VII

Chapter VIII

Chapter IX

Index

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 Bermúdez-Echeverry, Ana Carolina Issy, and Elaine A. Del-Bel Dopamine Receptors Regulation by Non-Dopaminergic Mechanisms Jaromír Mysliveček and Anna Hrabovská

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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 Parkinson’s 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 Parkinson’s disease and, possibly, other nocturnal movement disorders. Finally, the critical

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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

Preface

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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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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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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 Parkinson’s 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 δ2 glutamate receptors and thus changes in other receptor systems are discussed. Transgenic mice reveal cerebellar degeneration and learning impairment. The

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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 (α1-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.

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 Patrick M. Fuller∗ and Jun Lu¥ 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 Parkinson’s 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 Parkinson’s 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: [email protected]. 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: [email protected].

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Patrick M. Fuller and Jun Lu 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, Tourette’s 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 Parkinson’s 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

Dopamine Control of Sleep and Arousal

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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 Tourette’s 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 Parkinson’s 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 Parkinson’s 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: PARKINSON’S DISEASE Patients with Parkinson’s 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 Parkinson’s disease or dementia with Lewy bodies (Olson et al., 2000) and the majority of Parkinson’s 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 Rye’s 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 Parkinson’s 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 Freud’s 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 Alzheimer’s 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 Rexed’s 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 DA’s 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 DA’s 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 α2 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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Fleetwood-Walker SM, Hope PJ, Mitchell R. Antinociceptive actions of descending dopaminergic tracts on cat and rat dorsal horn somatosensory neurones. J Physiol. 399: 335–348 (1988). Fuller PM, Sherman D, Chen X, Saper CB, Lu J. Neuronal Substrates of the Ascending Arousal System In submission (2007). Gallopin T, Luppi PH, Rambert FA, Frydman A, Fort P. Effect of the wake-promoting agent modafinil on sleep-promoting neurons from the ventrolateral preoptic nucleus: an in vitro pharmacologic study. Sleep 27:19-25 (2004). Gerashchenko D, Chou TC, Blanco-Centurion CA, Saper CB, Shiromani PJ. Effects of lesions of the histaminergic tuberomammillary nucleus on spontaneous sleep in rats. Sleep 27:1275-1281 (2004). Gladwell SJ, Coote JH. Inhibitory and indirect excitatory effects of dopamine on sympathetic preganglionic neurones in the neonatal rat spinal cord in vitro. Brain Res 818: 397–407 (1999). Hara J et al., Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity. Neuron 30:345-354 (2001). Hasue RH and Shammah-Lagnado SJ. Oriigin of the dopaminergic innervation of the central extended amygdala and accumbens shell: combined retrograde tracing and immunohistochemical study in the rat. J Comp Neurol 454:15-33 (2002). Hobson DE, Lang AE, Martin WRW, et al. Excessive daytime sleepiness and sudden-onset sleep in Parkinson disease. JAMA. 287: 455–463 (2002). Hokflet T, Martensson R, Bjorklund A, Kleinau S, Goldstein M. Distribution of tyrosinehydroxylase-immunoreactive neurons in the rat brain. In: Handbook of chemical neuroanatomy (Bjorklund A, Hokfelt T, eds), pp 277-379. Amsterdam: Elsevier Science (1984). Jaber M, Robinson SW, Missale C, and Caron MG. Dopamine receptors and brain function. Neuropharmacology 35:1503-1519 (1996). Jellinger KA. Postmortem studies in Parkinson’s disease: is it possible to detect brain areas for specific symptoms? J Neural Transm Suppl 56:1-29 Jones BE, Harper ST, Halaris AE. Effects of locus coeruleus lesions upon cerebral monoamine content, sleep-wakefulness states and the response to amphetamine in the cat. Brain Res 124:473-496 (1977). Kume K, Kume S, Park SK, Hirsh J, Jackson FR. Dopamine is a regulator of arousal in the fruit fly. J Neurosci 25:7377-7384 (2005). Lai YY, Shalita T, Hajnik T, W JP, Juo JS, Chia LG, Siegel JM. Neurotoxic N-methyl-Daspartate lesion of the ventral midbrain and mesopontine junction alters sleep-wake organization. Neuroscience 90:469-483 (1999). Larson JP and Tandberg E. Sleep disorders in patients with Parkinson’s disease: epidemiology and management. CNS Drugs 15:267-275 (2001). Lee RS, Steffensen SC and Henriksen SJ. Discharge profiles of ventral tegmental area GABA neurons during movement, anesthesia, and the sleep-wake cycle. J Neurosci 21(5):17571766 (2001). Lu J, Chou TC and Saper CB. Identification of Wake-Active Dopaminergic Neurons in the Ventral Periaqueductal Gray Matter. J Neurosci 26(1):193-202 (2006).

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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: [email protected]; 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.

Theory of DA Modulation of PFC Activity

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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 D’Esposito 2003; D’Esposito 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


21

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 D’Esposito 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 D’Esposito 2005). Kimberg and D’Esposito (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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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

τp

+ 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 τ n ( z) where x p and xn are the population activities of the pyramidal neurons and the interneurons, respectively,

τ p and τ 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


23

⎧ f tanh( x) x ≥ 0 f ( x) = ⎨ max 0 x 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.


27

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 author’s 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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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

-0.02

2 0.0

-0.01 70 60

0.0 1

0

50 40

0

0

30 20

0

10

0

PFC activity [a.u.]

90

0

-0.01

1

-0.02

2

4

6

8

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

A

0.005 0 -0.005 -0.01

0

20

40

60

80

100

p

dx /dt

0.02

B

0.01 0 -0.01 -0.02

0

20

40

60

80

100

p

dx /dt

0.05

C 0

-0.05

0

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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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


31

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


33

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,

34

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


35

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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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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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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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”, Università 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”, Università degli Studi di Palermo, Corso Tuköry 129, 90134 Palermo, Italy. Tel.: +39 091 6555821; Fax: +39 091 6555823; [email protected].

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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: Parkinson’s Neuroprotection.

Disease,

Neurogenesis,

Neuroinflammation,

Apoptosis,

Parkinson’s 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 γaminobutyric 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 µm 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

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expressed [38,39]. According to cytoarchitectural, topographical, and chemical criteria, González-Hernández and Rodríguez [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

DAergic Neuron Life Cycle

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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-β (TGFβ). 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 ½ (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

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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 α-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


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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-α (TNF-α), 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

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DA can potentiate α-synuclein’s 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 α-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,


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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 Alzheimer’s disease (AD), Huntington’s 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, IL1β, IL-6, TNF-α and interferon γ (IFN-γ), 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].

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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-1β, IL-6 and TNF-α 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-α, IL-1α, IL-1β 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-κB (NF-κB), 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 α 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


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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-κB 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-κB from any endogenous inhibitor, permitting the translocation of free NF-κB from the cytoplasm to the nucleus. Consequently, NF-κB 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-κB 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-κB 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-γ (PPARγ) seems relevant to neuroprotection [138]. This receptor PPARγ is a ligand-activated inhibitory transcription factor that antagonizes the activity of NF-κB, 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 PPARγ in microglia, reducing the Aβ-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

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(BACE1) mRNA and protein levels [143]. In addition, PPARγ depletion potentiates betasecretase mRNA levels by increasing BACE1 gene promoter activity. Conversely, overexpression of PPARγ, as well as NSAIDs and PPARγ activators, reduced BACE1 gene promoter activity. These recent results suggest that PPARγ 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-κB, 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 don’t 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


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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, aspirin’s 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

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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]. Sánchez-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. Celocoxib’s 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


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of NSAIDs, since PD is much more common in people over 75 years old, an age group not included in the Chen team’s 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.

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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-γ, and NF-κB), 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 Βcl-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


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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]

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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 α (TGFα), 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.


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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

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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, Granny’s 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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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 Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, “G. Pagano”, Università degli Studi di Palermo, 90134 Palermo, Italy.

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: [email protected].

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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

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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 Parkinson’s 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 Parkinson’s 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 raphé (MR) [22-28] (Figure 1). Serotonin-containing cell bodies of the raphé 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

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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 raphé 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


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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 non–DA (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

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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


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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].

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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


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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.

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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


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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].

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Parkinson’s 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 Parkinson’s 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 Parkinson’s 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,


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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

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neuropsychiatric illness such as schizophrenia, Parkinson’s 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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[156] McMahon, L.R. & Cunningham, K.A. (2001) Antagonism of 5-Hydroxytryptamine2A receptors attenuates the behavioral effects of cocaine in rats. J Pharmacol Exp Ther, 297, 357-363. [157] Fletcher, P.J., Phil, D., Grottick, A.J., Higgins, G.A. (2002) Differential effects of the 5-HT2A receptor antagonist M100,907 and the 5-HT2C receptor antagonist SB242,084 on cocaine-induced locomotor activity, cocaine self-administration and cocaineinduced reinstatement of responding. Neuropsychopharmacology, 27, 576-586. [158] McCreary, A.C. & Cunningham, K.A. (1999) Effects of the 5-HT2C/2B antagonist SB 206553 on hyperactivity induced by cocaine. Neuropsychopharmacology, 20, 556-564. [159] Rocha, B,A., Goulding, E.H., O’Dell, L.E., Mead, A.N., Coufal, N.G., Parsons, L.H., Tecott, L.H. (2002) Enhanced locomotor, reinforcing, and neurochemical effects of cocaine in serotonin 5-hydroxytryptamine 2C receptor mutant mice. J Neurosci, 22, 10039-10045. [160] Grottick, A.J., Fletcher, P.J., Higgins, G.A. (2000) Studies to investigate the role of 5HT2C receptors on cocaine- and food-maintained behavior. J Pharmacol Exp Ther, 295, 1183-1191. [161] Tomkins, D.M., Joharchi, N., Tampakeras, M., Martin, J.R., Wichmann, J., Higgins, G.A. (2002) An investigation of the role of 5-HT2C receptors in modifying ethanol selfadministration behaviour. Pharmacol, Biochem Behav, 71, 735-744. [162] Grottick, A.J., Corrigall, W.A., Higgins, G.A. (2001) Activation of 5-HT2C receptors reduces the locomotor and rewarding effects of nicotine. Psychopharmacology, 157, 292–298.



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: [email protected]; [email protected]

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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; Jĕzová 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…

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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.

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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 Millonig’s phosphate buffer at pH 7.4 at 4°C, rinsed in buffer, and postfixed in 1% OsO4 (2h, 4°C), 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/μm2, the mean number of NE and E granules/μm2; the NE/E granule numeric ratio; the mean number of intermediate granules/μm2, 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:


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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 µl for aldosterone and 30 to 40 µl 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 µl of pure methanol. Methanol extracts were dried at 37° under vacuum and redissolved in 1,400 µl 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 µl serum. For catecholamine extraction, 50 µl 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 Duncan’s test for multigroup comparison and Student’s 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.


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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 μm; c: 1,1 μm; d: 2 μm; e: 1.2 μm; f: 2 μm.

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Anna Capaldo, Flaminia Gay, Salvatore Valiante et al. 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/ μm2

Intermediate granules/ μm2

NE granules/ μm2

E granules/ μm2

NE/ E ratio

Lip/ cyt.ratio

8.27 ± 2.38 8.39 ± 2.49 8.19 ± 2.24 3.15 ± 1.02●a 4.50 ± 1.33●b

0.07 ± 0.003

4.11 ± 1.26 4.14 ± 1.34 4.06 ± 1.19 0.16 ± 0.01◊a 0.17 ± 0.02◊b

1.01/1

0.33

1.00/1

0.34

1.01/1

0.33

2.43 ± 0.99◊a 0.08 ± 0.00

4.09 ± 1.57 4.15 ± 1.60 4.08 ± 1.33 0.56 ± 0.06◊a 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 ± 1.06◊a 4.47 ± 1.19◊b

7.25 ± 2.31 7.40 ± 2.20 7.29 ± 2.25 3.22 ± 0.96●a 2.19 ± 0.94◊b

1.31 ± 0.26 1.24 ± 0.2 1.27 ± 0.84 0.60 ± 0.04●a 0.54± 0.08●b

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 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.

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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 NO–DA 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

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Cristiane Salum, Marcela Bermúdez-Echeverry, Ana Carolina Issy et al.

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-), NO’s 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 Célia 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, Márcio Lazzarini, Prof. Francisco S. Guimarães and Prof. Marcus L. Brandão. The authors were recipients of CNPq, FAPESP and CAPES/COFECUB fellowships.

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changes in prepulse inhibition of startle and its disruption by apomorphine. Psychopharmacology (Berl), 122, 35-43. [210] Lipska, B.K. & Weinberger, D.R. (1994). Subchronic treatment with haloperidol and clozapine in rats with neonatal excitotoxic hippocampal damage. Neuropsychopharmacology, 10, 199-205. [211] Weiner, I. (2003). The "two-headed" latent inhibition model of schizophrenia: modeling positive and negative symptoms and their treatment. Psychopharmacology (Berl), 169, 257-297. [212] Black, M.D., Simmonds, J., Senyah, Y., & Wettstein, J.G. (2002). Neonatal nitric oxide synthase inhibition: social interaction deficits in adulthood and reversal by antipsychotic drugs. Neuropharmacology, 42, 414-420. [213] Hauber, W. (1998). Involvement of basal ganglia transmitter systems in movement initiation. Prog Neurobiol, 56, 507-540. [214] Sanberg, P.R., Bunsey, M.D., Giordano, M., & Norman, A.B. (1988). The catalepsy test: its ups and downs. Behav Neurosci, 102, 748-759. [215] Del Bel, E.A. & Guimaraes, F.S. (2000). Sub-chronic inhibition of nitric-oxide synthesis modifies haloperidol-induced catalepsy and the number of NADPHdiaphorase neurons in mice. Psychopharmacology (Berl), 147, 356-361. [216] Moore, N.A., Blackman, A., Awere, S., & Leander, J.D. (1993). NMDA receptor antagonists inhibit catalepsy induced by either dopamine D1 or D2 receptor antagonists. Eur J Pharmacol, 237, 1-7. [217] Cavas, M. & Navarro, J.F. (2002). Coadministration of L-NOARG and tiapride: effects on catalepsy in male mice. Prog Neuropsychopharmacol Biol Psychiatry, 26, 69-73. [218] Araki, T., Mizutani, H., Matsubara, M., Imai, Y., Mizugaki, M., & Itoyama, Y. (2001). Nitric oxide synthase inhibitors cause motor deficits in mice. Eur Neuropsychopharmacol, 11, 125-133. [219] Marras, R.A., Martins, A.P., Del Bel, E.A., & Guimaraes, F.S. (1995). L-NOARG, an inhibitor of nitric oxide synthase, induces catalepsy in mice. Neuroreport, 7, 158-160. [220] Salter, M., Duffy, C., & Hazelwood, R. (1995). Determination of brain nitric oxide synthase inhibition in vivo: Ex vivo assays of nitric oxide synthase can give incorrect results. Neuropharmacology, 34, 327-334. [221] Del Bel, E.A., da Silva, C.A., Guimaraes, F.S., & Bermudez-Echeverry, M. (2004). Catalepsy induced by intra-striatal administration of nitric oxide synthase inhibitors in rats. Eur J Pharmacol, 485, 175-181. [222] Kriegsfeld, L.J., Eliasson, M.J., Demas, G.E., Blackshaw, S., Dawson, T.M., Nelson, R.J., & Snyder, S.H. (1999). Nocturnal motor coordination deficits in neuronal nitric oxide synthase knock-out mice. Neuroscience, 89, 311-315. [223] Starr, M.S. & Starr, B.S. (1995). Do NMDA receptor-mediated changes in motor behaviour involve nitric oxide? Eur J Pharmacol, 272, 211-217. [224] Dzoljic, E., De Vries, R., & Dzoljic, M.R. (1997). New and potent inhibitors of nitric oxide synthase reduce motor activity in mice. Behav Brain Res, 87, 209-212. [225] Del Bel, E.A., Souza, A.S., Guimaraes, F.S., da Silva, C.A., & Nucci-da-Silva, L.P. (2002). Motor effects of acute and chronic inhibition of nitric oxide synthesis in mice. Psychopharmacology (Berl), 161, 32-37.


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[226] Deutsch, S.I., Rosse, R.B., Paul, S.M., Tomasino, V., Koetzner, L., Morn, C.B., & Mastropaolo, J. (1996). 7-Nitroindazole and methylene blue, inhibitors of neuronal nitric oxide synthase and NO-stimulated guanylate cyclase, block MK-801-elicited behaviors in mice. Neuropsychopharmacology, 15, 37-43. [227] De Oliveira, C.L., Del Bel, E.A., & Guimaraes, F.S. (1997). Effects of L-NOARG on plus-maze performance in rats. Pharmacol Biochem Behav, 56, 55-59. [228] Dere, E., Souza Silva, M.A., Topic, B., Fiorillo, C., Li, J.S., Sadile, A.G., Frisch, C., & Huston, J.P. (2002). Aged endothelial nitric oxide synthase knockout mice exhibit higher mortality concomitant with impaired open-field habituation and alterations in forebrain neurotransmitter levels. Genes Brain Behav, 1, 204-213. [229] Kulig BM, Vanwersch RA, & Wolthuis OL. (1985). The automated analysis of coordinated hindlimb movement in rats during acute and prolonged exposure to toxic agents. Toxicol Appl Pharmacol, 80, 1-10. [230] Ebadi, M., Srinivasan, S.K., & Baxi, M.D. (1996). Oxidative stress and antioxidant therapy in Parkinson's disease. Prog Neurobiol, 48, 1-19. [231] Rice, M.E. (2000). Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci, 23, 209-216. [232] Lazzarini M, Salum C, & Del Bel EA (2005). Combined treatment of ascorbic acid or alpha-tocopherol with dopamine receptor antagonist or nitric oxide synthase inhibitor potentiates cataleptic effect in mice. Psychopharmacology, 181, 71-79.



Chapter IX

DOPAMINE RECEPTORS REGULATION BY NON-DOPAMINERGIC MECHANISMS Jaromír Mysliveček1,∗ and Anna Hrabovská2 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 δ2 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 Jaromír Mysliveček, 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: [email protected].

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Jaromír Mysliveček and Anna Hrabovská 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 (α1adrenoceptors, 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 (Mysliveček, 1997), memory and emotions. It participates in various neurological and psychiatric disorders such as Parkinson´s disease, schizophrenia, amphetamine and cocaine addiction (Hantraye, 1998), as well as depression and GillTourette´s 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; O’Dowd, 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

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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

Jaromír Mysliveček and Anna Hrabovská

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(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 γ7 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


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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 δ2 glutamate receptor (GluRδ2) that is predominantly expressed in cerebellar Purkinje cells and plays a crucial role in cerebellar functions. Despite their importance, the mechanism by which GluRδ2 participates in cerebellar functions is still unexplained. GluRδ2 is predominantly expressed at distal dendrites of Purkinje cells where parallel fibers form synapses (Yuzaki, 2004). It is important to note that GluRδ2 does not form functional glutamate-gated ion channels when expressed, either alone or with other glutamate receptors. Despite that functional exceptionality, GluRδ2 is crucial in cerebellar function: mice that lack the gene encoding GluRδ2 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 (Vožeh et al., 1999, Křížková and Vožeh, 2004) and lower resistance to the neurotoxin 3acetylpyridine (Caddy and Vožeh, 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 δ2 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 (Mysliveček 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-

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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.


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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 α (α2-α10) and β (β2-β4) subunits. Combinantion of these subunits is tissue specific. Neuronal form consists of α and β subunits or is represented by α homomers), ganglionic type is mainly α homomer and muscular type

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combines α, β, δ and γ 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. Parkinson’s 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


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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 (Pacák 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 (Nováková et al., 2006) in the peripheral tissues (heart and lungs) and in the central nervous system (cerebral cortex, cerebellum; Beneš et al., 2006). Muscarinic receptors (i.e. those mainly expressed in the central nervous system – odd numbered receptors) and α1-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 β-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 α1-adrenoceptor and muscarinic receptor binding sites (to 184 %, and 234 %, respectively). The level of other receptors, i.e. β-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 α1-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

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nervous system (an increase in α-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 (Beneš 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.


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Following conclusions could be made for cFos mutated mice: a) c-fos gene disruption affects the level of receptors which activate phospholipase Cβ/proteinkinase C pathway exclusively. The G protein-coupled receptors which activate adenylyl cyclase (i.e. D1-like dopamine receptors and β-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 Alzheimer’s 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 Huntington’s 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 Parkinson’s 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-α, 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

225

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