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8oO-S2l-û6ûû

Studies on the expression of synaptic terminal proteins and neuroûansrnitter receptors in the rat brain FoIlowing amphetamine-induced behavioral sensitization

Sabarinath Subramaniam

Department of Psychiatrj McGilI University

05i2000

A thesis submitted to the Faculty of Graduate Studies and Research in partial fuIfiIlment of the requirernents of the de- of Master of Science

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I

ABSTRACT

Long-term abusers of the psychostimulant amphetamine, display symptoms of paranoid

psychosis that closely resemble schizophrenia (Connel, 1958; Ellinwood, 1967; Angrist and

Gershon, 1970; Grifith et al., 1972). Amphetamine addicts who develop psychosis also

display a long-lasting hyperresponsivity to the psychotomimetic properties of the d n ~ g

(Magos, 1969; Utena, 1974; Sato et al., 1983; Robinson and Becker, 1986; Sato et a1.,1992).

Repeated administration ofamphetamine has been shown to induce asimilar hypersensitivity

to the behavioral effects of amphetamine in animals (Segal and Schuckit, 1983; Robinson

and Becker, 1986; Strakowski et al., 1996). Hence, chronic amphetamine administration has

been extensively employed as an animal model for amphetamine-induced psychosis as well

as for certain aspects ofschizophrenia (Segal et al., 1981, Robinson and Becker, 1986). The

mesocorticolimbic system has been postulated to play a major role in mediating

amphetamine-induced behavioral sensitization (Clarke et al., 1988; Vezina and Stewart,

1990; Kalivas and Stewart, 1991; Antoniou et al., 1998).

This study employed a paradigm involving the administration of repeated intermittent

injections of low doses of amphetamine to adult male Sprague-Dawley rats to induce

behavioral sensitization. The persistent nature of behavioral sensitization suggests that

repeated exposure to amphetamine results in long-lasting neurochemical changes that would

mediate the behavioral hypersensitivity.

This study investigated alteration in the expression of syntaxinl, synaptophysin and

synapsinl, synaptic proteins involved in neurotransmitter release/synaptic plasticity. The

results of this study indicated significant reduction in the expression of syntaxin I and

synaptophysin in the core subregion of the nucleus accumbens along with a significant

ii

enhancement in syntaxin I expression in the shell subregion. Thus repeated administration

ofamphetamine induced differential changes in synaptic protein expression between thecore

and shell subregions of the nucleus accumbens. This result is consistent with several studies

reporting differential neurochemical changes occurring in the core and shell in response to

repeated psychostimulant administration (Cadoni and Di Chiara, 1999; 2000; Cadoni et al.,

2000).

This study also investigated possible alteration in glutamatergic (NMDA and kainate) and

dopaminergic (D3) receptor levels in response to amphetamine-induced behavioral

sensitization. The results of this study indicated a lack of significant difference in the

expression of these receptors following amphetamine sensitization.

iii

ROSUME

Les personnes qui abusent i long terme de I'amphCtamine, stimulant psychotique, ont des

symptdmes de type paranoi'de qui resemble i ceux rencontris chez les personnes atteintes

de la schizophrinie (Connel, 1958; Ellinwood, 1967; Angrist and Gershon, 1970; Griffith

et al., 1972). Les consommateurs d'amphitamine qui diveloppent une psychose dimontrent

aussi une hyper-r6ponse de longue dur6e aux propriitis psychomimitic de I'amph6tamine

(Magos, 1969; Utena, 1974; Sato eta]., 1983; Robinson and Becker, 1986; Sato et a1.,1992).

Des injections r6pit6es d'amphitamine 1 des animaux ont dimontri une hyper-sensitivite

persistante similaire aux effets de I'amph6taminesur le comportement (Segal and Schuckit, 1983;

Robinson and Becker, 1986; Shakowski et al., 1996). C'est pourquoi l'injection chronique

d'amphitamine a 6tC utilisee comme modi-le animal pour 6tudier la psychose induite par

I'amphitamineet igalement pourcertainaspectde laschizophrinie(Segal el al., 1981, Robinson

and Becker, 1986).

I1 a id proposi que le systtme m~socorticolimbique joue un r6le majeur dans le comportement

induit par I'amphitamine (Clarke et al., 1988; Vezina and Stewart, 1990; Kalivas and Stewart,

1991; Antoniou et al., 1998). Cene etude utilise un test comportemental bas6 sur l'injection

n5pCtee de doses intermittentes d'amphitamine sur des rats adultes de souche Sprague-Dawley

pour induire une sensitiviti de longue duke. L'effet persistent de ce comportement suggire que

I'exposition ripit6e d'amph6tamine ammtne deschangements neurochimiques de lonyesdurks

provoquant le comportement d'hyper-sensibilit6.

Les modifications des expressions des protiines synaptiques (Syntaxin I, Synapsin I et

Synaptophysin) impliquh dans la liberation de neumtransmeneurs et de la plasticit6 synaptique

ont it6 6tudites. Les risultats de cene itude dimontrent une dduction significative de

iv

I'expression de Syntaxin I et Synaptophysin dans la partie centnle du noyau accumbens, tandis

que pour la partie pkriphdrique du noyau accumbens, une augmentation significative de

I'expression de Syntaxin I et une tendance A I'augmentation de I'expression de Synaptophysin

ont CtC obtenues. Donc, une injection rdpCtCe d'amphdtamine induit un changement dans

I'expression des pmtdines synaptiques dans les diffh-entes sous-rkgions du noyau accumbens

(centrale et pCriphCrique). Ces rksultats sont en accord avec plusieurs Ctudes ddmontnnt diffdrents

changementsneurochimiquesayant lieux d m lapartiecentnledu noyau accumbens et lessous-

rCgions de la partie pCriphCrique du noyau accumbens en riponse au psycho-stimulant induisant

un comportement de sensibilisation (Cadoni and Di Chiara, 1999; 2000; Cadoni et a]., 2000).

Cene Ctude a aussi vCrifiCe les modifications possible de la densitC des ricepteurs

glutamatergiques (NMDA et Kainate) et dopaminergiques (D3) causdes par le comportement de

sensibilisation par I'amphdtamine. Les rdsultats de cene Ctude indiquent aucun changements

significatifs dans I'expression de ces recepteurs suite A une sensitisation de I'amph6tiunine.

ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr Lalit.K.Srivasfava for giving me the opportunity and

financial support to be part of this project as well as for his guidance and advice through these

years.

I would also like to thank all the members of Dr. Srivasfava's labontory who have always been

a great help for me during the project. I would like to personally thank Dr Eric Marcone for all

the help during the thesis preparation and image analysis. Special thanks to Dr Gonzalo Flores

for his expertise in receptor autondiognphy. Special thanks also to Dr Stbphane Bastimeno, Dr

Sanjeev Bhardwaj and Steve Landry for their help with my thesis.

Finally, words cannot express my p t i t ude to my wife, Subha who was always there for me and

continuously motivated me to strive harder.

TABLE OF CONTENTS ABSTRACT .....................................................................................................................................

ACKNOWLEDGEMENTS .......................................................................................................... v

... LIST OF ABBREVIATIONS ................................................................................................... VIII

.................................................... I. INTRODUCTION .1 ............................ 1.1. History of Amphetamine Use and Abuse .1

..................... 1.2 Chronic Amphetamine-induced Paranoid Psychosis .2 1.2.1 Clinical Features of amphetamine psychosis ................... .2

. . . . . . . . . . . . . . . . . . . 1.2.2 Amphetamine Psychosis and Schizophrenia . 3 1.3 Behavioral sensitization model of amphetamine psychosis . . . . . . . . . . . . . . . . .&

1.3.1 Behavioral features of acute amphetamine administration ......... .I 1.3.2 Characteristic features of the behavioral sensitization model ...... .6

................. 1.4 Neuroanatomical/Neurochemical basis of sensitization .u ...................... 1.4.1 Initiation and expression of sensitization 10

........................ 1.4.2 Neuroanatomical loci of sensitization fi 1.5 Neurochemical correlates of behavioral sensitization .................... &

1.5.1 Dopaminergic neurotransmission ........................... 14 ........................ 1 S.2 Dooamine rece~tors in sensitization .fi

1.5.3 The dopamine transporter and amphetamine sensitization ........ .u ................... 1.5.4 Glutamatergic involvement in sensitization .18 - ................................. 1.6 Synaptic proteins and sensitization .22

1.6.1 Synaptic vesicle-mediated neurotransmitter release .............. 22 1.6.2 Calcium transduction and amphetamine sensitization ........... .a

................. 1.6.3 Alterations in intracellular signalling cascade .%

2.1 Synaptic proteins mediating dopamine release in amphetamine sensitization . .a 2.2 GlutamatelDopamine receptors in amphetamine sensitization ............ .a

........................................................ 3.METHODS 2 .................................................... 3.1Animals E ................................................... 3.2 Materials .2

3.3 Establishment of the behavioral sensitization paradigm ................. - 30 .......................... 3.4 Western Blot analysis of Synaptic Proteins .a

.................. 3.4.1 Protein sample preparation and quantification 21 ............................... 3.4.2 Westem Blot hybridization .z

....................................... 3.5 Receptor Autoradiography .B

vii

3.5.1 Bnin Sample Processing ................................ .g 3.5.2 Autoradiography Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X

4. RESULTS.. ........................................................ .22 4.1 Establishment of behavioral sensitization paradigm ................... .= 4.2 Behavioral sensitization of 2 separate cohorts ...................... .22 4.3 Synaptic protein expression in amphetamine sensitization ................ 41

4.3.1 Syntaxin 1 expression in the nucleus accumbens core, shell and VTA ........................................

4.3.2 Synaptophysin expression in the nucleus accumbens core, shell and VTA .............................................. .a

4.3.3 Synapsin 1 expression in the nucleus accumbens core, shell and VTA LL ...................................................

4.4 Expression of glutamate NMDA, kainate receptors and dopamine D3 receptor ......................................................... @

4.4.1 NMDA receptor autoradiography .......................... .a 4.4.2 Kainate receptor autoradiognphy .......................... .a 4.4.3 Dopamine D3 receptor autondiognphy ..................... .a

5. DISCUSSION ...................................................... 5.1. Establishment of the behavioral sensitization paradigm ................ .z 5.2 Synaptic protein expression and amphetamine sensitization ............. .z

5.2.1 Differential properties of the core and shell subregions of Nucleus accumbens .......................................... Z

5.2.2 Sensitized dopamine release and chronic amphetamine administration ...................................................

5.2.3 Synapsin I expression and sensitized dopamine release ........ .z 5.2.4 Syntaxin 1 I Synaptophysin expression and sensitized dopamine release

................................................... 5.2.5 Synaptic proteins, disease and plasticity ..................... .a

5.3 Glutamatergic I dopaminergic receptors and amphetamine sensitization .... .a 5.3.1 NMDAKainate receptors and sensitization ................... .a 5.3.2 Dopamine D3 receptors and sensitization ..................... 89

6. REFERENCES ...................................................... .a

viii

AMPA ANOVA bFGF CFTR CREB DAT EAA FRA GABA GAP43 GluRl NMDA NSF PET PFC PKA PKC PPD SNAP SNAP-25 SNARE VAMP VTA

LIST OF ABBREVIATIONS

a-amino-3-hydroxy-5-methyl-4-isoxazole Analysis of Variance basic fibroblast growth factor Cystic fibrosis tmnsmembnne conductance regulator c-AMP response element binding protein Dopamine transporter Excitatory amino acids fos-related antigen Gamma-amino-butyric acid Growth associated protein43 Glutamate receptor 1 N-methyl-D-Aspartate N-ethylamaleimide sensitive factor Positron Emission Tomography Pre Frontal Cortex Protein kinase A Protein Kinase C Preprodynorphin Soluble NSF attachment protein Synaptosome-associated protein-25 Soluble NSF attachment protein receptor Vesicle associated membrane protein Ventral Tegmental Area

LIST OF FIGURES

Figure 1.1 Docking and Fusion steps of vesicular dopamine release.

Figure 4.1 Trial behavioral sensitization paradigm

Figure 4.2 Locomotor sensitization paradigm repeated for cohort of animals (N=30)

Figure 4.3 Locomotor sensitization paradigm repeated for second cohort of animals (N=24)

Figure 4.4 Autoradiographic representation of hybridization signals from syntaxin I labeling in the nucleus accumbens core.

Figure 4.5 Comparison of syntaxin I to a-tubulin expression in the nucleus accumbens core between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.6 Autoradiographic representation of hybridization signals from syntaxin 1 labeling in the nucleus accumbens shell

Figure 4.7 Comparison of syntaxin 1 to a-tubulin expression in the nucleus accumbens shell between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.8 Automdiographic representation of hybridization signals from syntaxin 1 labeling in the ventnl tegmental area

Figure 4.9 Comparison of syntaxin 1 to a-tubulin expression in theventnl tegmental area between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.10 Autoradiographic representation of hybridization signals from synaptophysin labeling in the nucleus accumbens core

Figure 4.1 1 Comparison ofsynaptophysin to a-tubulin expression in the nucleus accumbens core between d-amphetamine-pretrmted animals, saline-treated animals and untreated control animals

Figure 4.12 Autoradiographic representation of hybridization signals from synaptophysin labeling in the nucleus accumbens shell

Figure 4.13 Comparison of synaptophysin to a-tubulin expression in the nucleus

accumbens shell between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.14 Autondiographic representation of hybridization signals from synaptophysin labeling in the ventral tegmental area

Figure 4.15 Comparison of synaptophysin to a-tubulin expression in the ventral tegmental area behveen d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.16 Autondiognphic representation of hybridization signals from synapsin I labeling in the nucleus accumbens core

Figure 4.17 Comparison of synapsin to a-tubulin expression in the nucleus accumbens core between d-amphetamine-pretreated animals, saline-treated animals and untreated control $imals

Figure 4.18 Autoradiographic representation of hybridization signals from synapsin 1 labeling in the nucleus accumbens shell

Figure 4.19 Comparison of synapsinl to a-tubulin expression in the nucleus accumbens shell between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.20 Autoradiographic representation of hybridization signals from synapsin I labeling in the ventral tegmental area

Figure 4.21 Comparison of synapsin 1 to a-tubulin expression in the ventral tegmental area between d-amphetamine-pretreated animals, saline-treated animals and untreated control animals

Figure 4.22 Qmntitative analysis data of NMDA receptor levels in prekontal cortical regions following receptor autoradiography with [I1251 MK-801

Figure 4.23 Quantitative analysis data of NMDA receptor levels in nucleus accumbens ishiatal regions following receptor autoradiography with [I1251 MK-801

Figure 4.24 Quantitative analysis data of NMDA receptor levels in nigral and hippocampus regions following receptor autoradiography with [I1251 MK- 80 1

Figure 4.25 Quantitative analysis data of kainate receptor levels in prefrontal cortical regions following receptor autoradiography with [3H] kainic acid

Figure 4.26 Quantitative analysis data of kainate receptor levels in striatal/accumbal regions following receptor automdiognphy with [3H] kainic acid

Fiyre 4.27 Quantitative analysis data o f kainatc receptor levels in nignyhippocampus regions following receptor autondiognphy with [3H] kainic acid

Fiyrc 4.28 Quantitative analysis data of dopamine D3 levels in stiiataVaccumba1 regions following receptor autondiopphy with FOH-DPAT.

I. INTRODUCTION

1.1. History of Amphetamine Use and Abuse

Amphetamine (phenylisopropylamine) exists in three forms consisting of the isoforms, dextro-

or d-amphetamine and levo- or I-amphetamine and the N-methyl derivative, methamphetamine

(Biel and Bopp, 1978). Characterized in 1930, amphetamine has been found to possess a wide

range of sympathomimetic properties. Peripheral effects of amphetamine administration include

increased blood pressure and heart rate, relaxation of gastrointestinal tract and bronchiolar

muscles, increased metabolic rate and increased oxygen consumption (Morgan, 1978; Seiden et

al., 1993). More importantly, amphetamine induces potent stimulation of the central nervous

system resulting in a state of arousal characterized by feelings of euphoria, wakefulness,

alertness, increased interest and curiosity, mood elevation, inhibition of appetite, increased

initiative and elation, increased self confidence, decreased social inhibition, increased sexual

stimulation, self-esteem and energy as well as enhanced performance (Piness et al., 1930;

Leake, 1958; Groves and Rebec, 1976; Kuczenski, 1983; Seiden et al., 1993). These

stimulatory properties of amphetamine lead to its extensive usage for therapeutic purposes, first

in the 1930's as a nasal inhaler and later in the treatment of a variety of conditions including

narcolepsy, obesity (as an anorectic agent), asthma, motion sickness, enuresis and ADI-ID

(Prinzmetal and Bloomberg, 1935; Ersner, 1940; Angrist and Sudilovsky, 1978). With

prolonged usage however, amphetamine users develop dependence to the euphoric and

stimulatory properties of the drug and begin to administer progressively increasing doses,

eventually resulting in the abuse of the drug (Kramer et al., 1967). Long term abuse of

amphetamine induces several ill effects such as insomnia, nervousness, impulsiveness,

increased initability and anxiety, agitation, aggression as well as confused and violent behavior

(Knmer et al., 1967; Gawin and Ellinwood, 1988).

1.2 Chronic Amphetamine-induced Paranoid Psychosis

1.2.1 Clinical Features of atnpl~e~a~~~it~epsycl~osis

The most severe consequence of amphetamine abuse is amphetamine psychosis, a psychotic

condition bearing close resemblance to clinical characteristics of paranoid schizophrenia. A

hallmark feature of amphetamine-induced psychosis is paranoid ideation coexisting with well-

formed delusions (Connel, 1958; Ellinwood, 1967; Griffith et al., 1972). Delusions begin with

feelings of mild fear (an awareness or the suspicion of being watched) which develop into well-

formed thoughts and fear of persecution as the individual becomes increasingly convinced of

being plotted against (Ellinwood, 1967; Angrist and Gershon, 1970; Ellinwood et al., 1973).

Paranoid delusions are also accompanied by progressively increasing ideas of reference with

subjects reporting being talked about or referred to in unrelated conversations (Ellinwood, 1967;

Angrist and Gershon, 1970; Ellinwood et al., 1973).

Majority of individuals with amphetamine psychosis display hallucinatory behavior consisting

primarily of schizophrenia-like visual (peripheral as well as fully formed and stable) and

auditory (nonexistent noises, voices and conversations with voices) hallucinations (Ellinwood

et al., 1973; Bell, 1973; Janowsky and Risch, 1979). Other types of hallucinations such as

somatic or tactile (feelings of small animalslinsects beneath skin surface) as well as olfactory

hallucinations have also been reported in amphetamine psychotics (Angrist and Gershon,

1970; Kalant, 1973; Bell, 1973). Amphetamine psychotics also display persevemtive or

repetitive behavior characterized by compulsive examination, dismantling, sorting and

3

reassembling of objects as well as being excessively inquisitive about their immediate

environment (Bell, 1965; Ellinwood. 1967). Individuals become unable to perform complex

sequential tasks and examples of continuous repetitive behavior include reports of patients

disassembling objects and then attempting to 'repair' or put them back together (eg. repon of an

individual dismantling a dozen radios and then assembling the parts to make a single one

(Ellinwood, 1967). The stereotyped behavior is also chmcterized by aimless walkinddrifiing

for prolonged periods of time as well as continuous pacing back and forth (Gunne, 1977).

1.2.2 Arnphetan~ine Psychosis and Schkopkrenia

The clinical characteristics evident in amphetamine-induced psychosis closely mimic paranoid

schizophrenia which lead to several cases of misdiagnosis of amphetamine psychotics as being

schizophrenic. Visual and auditory hallucinations have been associated with both disorders and

similar to schizophrenia, amphetamine psychosis can also be effectively treated with

neuroleptics. Further, exposure to amphetamine has been clearly shown to exacerbate or

reinstate psychotic symptoms in individuals with prior history of mental illness (Liebermann et

al., 1987). The lint cases of amphetamine psychosis were described in 1938 by Young and

Scoville, who observed cases of paranoid psychosis among their subjects receiving

amphetamine as treatment for narcolepsy (Young and Scoville, 1938). However, the close

resemblance in symptomatology between paranoid schizophrenia and amphetamine psychosis

lead them to suggest that amphetamine only unmasks latent psychotic traits in these individuals

rather than induce psychosis. Several groups since have reported controlled studies describing

induction of psychotic symptoms in individuals with no previous history of mental illness

following exposure to repeated amphetamine(Ellinwood, 1967; Angrist and Gershon, 1970;

Bell, 1973). These studies provide evidence for the ability of amphetamine to induce paranoid

psychosis independent of schizophrenia. The similarities between amphetamine psychosis and

schizophrenia point to the involvement of similar neurochemical mechanisms in the induction

of both disorders.

Despite their similarities in symptomatology, certain clinical characteristics allow

differentiation between amphetamine psychosis and schizophrenia. Though both these disorders

are characterized by hallucinations, schizophrenic hallucinations are primarily auditory in nature

while those evident in amphetamine psychosis are both visual as well as auditory (Snyder,

1973, Bell; 1965). In addition, tactile and olfactory hallucinations have been reported to occur

frequently in amphetamine psychosis but rarely in schizophrenia (Snyder; 1973, Bell; 1965).

Further, schizophrenia has been associated with a formal thought disorder, a feature not evident

in the majority of amphetamine psychotics (Snyder; 1973, Bell; 1965).

13 Behavioral sensitization model of amphetamine psychosis

The parallelism in the symptomatology between amphetamine psychosis and schizophrenia

prompted interest in the development of animal models of amphetamine psychosis, which could

also, model aspects of schizophrenia. Several paradigms have been used to develop drug-

induced animal models for amphetamine psychosis (For Reviews refer Segal et al., 1981;

Robinson and Becker, 1986). Administration of these paradigms to animals has resulted in

different models that vary in their behavioral as well as neurochemical characteristics.

Examples of extensively applied drugemployed paradigms include a) the repeated interminent

5

administration of low doses of amphetamine (referred to as the behavioral sensitization

paradigm, b) the escalating dose or "binge" model of amphetamine administration and c) the

continuous administration of amphetamine, a paradigm that induces neurotoxicity in animals

(Segal et al., 1981; Robinson and Becker, 1986).

The paradigm that involves the administration of a constant low dose of amphetamine

intermittently has been widely accepted as the one most closely mimicking the behavioral and

neurochemical changes associated with amphetamine psychosis (Segal et al., 1981; Robinson

and Becker, 1986).

1.3.1 Behavioralfeaturer ofacule an~phetamine adminisrration

Administration of an acute low dose of amphetamine (0.5mgkg) in animals induces a dose-

dependent behavioral response characterized by general hyperactivity with increased incidence

of forward locomotion and rearing (Segal et al., 1981). As the dose is increased, the

hyperlocomotion is soon replaced with a period of focused stereotyped behavior characterized

by continous repeated head movements, sniffing, licking or biting (Randrup and Munkvad,

1967; Schiomng, 1971; Segal et al., 1981). Acute administration of high doses (3.5-7.5 mgkg)

of amphetamine results in a triphasic behavioral response pattern characterized by an initial

period of hyperlocomotion followed by a second stage of stereotyped behavior and finally a

repeat period ofhyperlocomotion (Segal et al., 1981).

1.3.2 Cl~aracieristicJeatures oJt11e behavioral sensitization aodel

I . Progressive enhancement in behavioral response

Chronic administration of amphetamine to animals induces alterations in the behavioral

response of the animal to a subsequent acute dose of amphetamine. Though repeated

administration of amphetamine results in the development of tolerance to certain aspects of the

behavioral response (eg. euphoria), the locomotor as well as stereotyped behavior are

progressively enhanced in a dose-dependent manner following the repeated intermittent

administration of the drug (Segal and Mandell, 1974; Segal, 1975; Segal et al., 1980). The

intensity and duration ofthe initial period ofhyperlocomotion and rearing induced by an acute

low dose of amphetamine is enhanced following the repeated adminishation of constant low

doses (Hitzemann et al., 1977; Hirabayashi and Alam, 1981; Leith and Kuczenski, 1982). With

repeated administration of higher doses of amphetamine, the stereotyped behavior becomes

favored with a progressive enhancement in both intensity (more focussed stereotypy) and

duration of stereotypy as well as a decrease in the time of onset of stereotypy (Ellinwood, 1974;

Sams-Dodd, 1998). Thus during chronic amphetamine administration, the animal becomes

increasingly hyperresponsive to the effects of amphetamine i.e. with each repeated injection, a

lower dose is sufficient to induce a behavioral response of similar magnitude. The progressive

behavioral hypersensitivity associated with the repeated intermittent administration of

amphetamine, referred to as behavioral sensitization is also chmcteristic of amphetamine

psychosis (Segal and Schuckit, 1983; Strakowski et al., 1996). With each repeated exposure to

the drug, amphetamine addicts develop a progressively increasing vulnerability to the

psychotomimetic properties of amphetamine (Segal and Schuckit, 1983; Robinson and Becker,

1986; Strakowski et al., 1996).

7

2. Persistent nature of behavioral alterations

The alterations in amphetamine-induced behavioral response resulting from the repeated

exposure to the drug are very long-lasting. The behavioral hypersensitivity associated with

repeated interminent administration of amphetamine to animals is enduring in nature and the

animal remains hyperresponsive to a challenge dose of the drug for up to several months

following cessation of treatment (Magos, 1969; Utena, 1974; Robinson and Becker, 1986).

This long-lasting nature of the behavioral hyperresponsivity in the animal model is analogous to

the persistent hypervulnerability to tho psychotogenie effects of amphetamine evident in addicts

in which addicts remain increasingly vulnerable to drug-induced psychosis even after several

years of abstinence (Sato et at., 1983; 1992).

3. Cross-sensitization to stress and cocaine

Similar to the behavioral sensitization resulting from chronic amphetamine administration,

animals exposed repeatedly to stressful stimuli develop sensitization to the behavionl effects of

stress (Antelman et al., 1980; Robinson et at., 1985). This behavioral sensitization to chronic

stress has been shown to be interchangeable with sensitization to repeated amphetamine i.e.,

chronic exposure to either amphetamine or stressful stimuli such as footshock and tail pinch

induces a similar long-lasting behavioral hypersensitivity to subsequent exposure to either a

stressor or amphetamine (Herman et al., 1984; Robinson et al., 1985; Hahn et at., 1986; Snyder-

Keller, 1990; Badiani et al., 1992; Henry et al., 1995). In addition to the cross sensitization of

behavioral characteristics, chronic exposure to d-amphetamine or stressful stimuli also results in

similar neurochemical alterations suggesting the involvement of common neuronal pathways in

stress- and amphetamine-induced sensitization (Eichler and Antelman, 1979; Robinson et al.,

1987; Lillrank et al., 1991; Steketee and Kalivas, 1991; Cador et al., 1992; Hamamura and

Fibiger, 1993; Hamamura et al., 1997). This cross sensitization behveen the behavioral effects

of repeated amphetamine and chronic stress evident in animals is analogous to similar changes

seen in humans. Former amphetamine addicts who are hyperresponsive to the psychotogenic

properties of amphetamine also remain vulnerable to relapse into psychosis following exposure

to stresshl stimuli even after several years of abstinence from the drug (Eichler and Antelman,

1979; Antelman, 1988). This cross sensitization behveen amphetamine and stress suggests a

role foramphetamine as a stressor in mediating its long-lasting behavioral effects. In addition to

cross-sensitization to stress, repeated amphetamine administration also sensitizes the animal to

the behavioral effects of subsequent exposure to other stimulants such as cocaine (Kalivas and

Weber, 1988; Zahniser et al., 1988; Peltier et al., 1996).

4. Context dependency of behavioral sensitization

Behavioral sensitization to amphetamine like psychostimulants has been shown to be

influenced by environmental stimuli associated with the circumstances of drug administration

(Tilson and Rech, 1973; Badiani et al., 1995; Anagnostans and Robinson, 1996). It has been

reported that rats treated in a novel test environment display a more potent sensitization to

amphetamine induced behavioral effects compared to rats treated in the same environment in

which they live (Badiani et al., 1997; Robinson et al., 1998). Thus experimental novelty can

enhance the magnitude of sensitization to the locomotor as well as stereotyped behavior

associated with amphetamine administration (Badiani et al., 1995; Fraioli et al., 1999). This

modulation of the susceptibility to behavioral sensitization by environmental stimuli has been

shown to occur in a dose-dependent manner (Browman et al., 1998). In addition to behavioral

augmentation, neurochemical changes associated with sensitization are also enhanced in a

context-dependent manner (Badiani et al, 1998; Ostrander et al, 1998).

5. Single dose is sufficient to induce behavioral sensitization

Though repeated amphetamine doses induce behavioral sensitization, even a single exposure to

amphetamine has often been reported to produce a longlasting hypersensitivity to subsequent

amphetamine challenge (Vanderschuren et al, 1999). A single injection of amphetamine has

been reported to enhance the locomotor activity, stereotypy, drinking and rotational behavior

produced by a subsequent injection of amphetamine given weeks later (Vanderschuren et al,

1999).

6. Relevance of the withdrawal period / treatment schedule

An important determinant of the magnitude of behavioral sensitization resulting from a chronic

amphetamine administration paradigm is the length of the withdrawal period. The withdrawal

period signifies the time interval between the last treatment dose and the challenge injection.

Several studies have been conducted in which behavioral sensitization was induced using

paradigms with different withdrawal periods ranging from 24-72 hours following last treatment

upto several weeks of abstinence. These studies showed that the magnitude of behavioral

augmentation increased with increase in the duration of the withdrawal period i.e., the longer

the withdrawal period, the more potent the sensitized response (Kolta, 1985; Paulson, 1991).

Animals withdrawn from amphetamine for 7 days and over showed much more robust

sensitization compared to animals administered the challenge dose 24-72 hours after last

heahnent injection (Kolta, 1985; Paulson, 1991). Animals withdnwn for 24-72 hours displayed

behavior alterations characteristic of the withdrawal syndrome (Hitzemann et al., 1977; Kolta,

1985). These behavioral changes however are hansient compared to the longlasting behavioral

effects associated with sensitization. Thus an appropriate paradigm necessary to induce potent

behavioral sensitization would utilize a comparatively longer withdrawal period (7-30 days). In

addition to an extended withdrawal period, the magnitude of sensitization is also affected by the

treatment schedule. Chronic d-amphetamine treatment administered intermittently has been

shown to induce more potent behavior sensitization when compared to daily injections (Post,

1980).

1.4 Neuroanatomical/Neurochemical basis of sensitization

1.4.1 Initiation and expression of sensitization

The sequence of events leading to persistent behavioral hyperactivity following chronic

exposure to amphetamine have been classified into a) the initiation and b) the expression of

behavioral sensitization. The initiation process of sensitization can be defined as the set of

events directly resulting from chronic amphetamine administration, that induce long-lasting

alterations in the neural circuitry leading to behavioral sensitization (Pierce and Kalivas, 1997).

Expression on the other hand refers to the set of molecular neurochemical adaptations resulting

from the initiation process that eventuate into enduring behavioral augmentation (Pierce and

Kalivas, 1997).

1.4.2 Nerrroa~~a~ornical loci of setnifizafion

1. The dopaminergic system

The dopaminergic system had been identified as the common locus for the reinforcing as well

as behavioral stimulant properties of amphetamine and other psychostimulants (Liebeman et

a]., 1990). Evidence for the role of dopamine in mediating the acute effects ofamphetamine laid

the focus for identifying the neuroanatomical loci involved in long term behavioral sensitization

primarily on the mesocorticolimbic dopaminergic system (Snyder, 1973; Lieberman et al.,

1990). Lesion studies have shown that ibotenic acid-induced and 6-hydroxydopamine-induced

lesioning of the mesolimbic dopaminergic neurons abolishes amphetamine-induced locomotor

activity (Creese and Iversen, 1974; Kelly and Iversen, 1976; Clarke et al., 1988; Antoniou et al.,

1998).

The dopamine neurons of primary importance in sensitization are the A10 (ventral tegmental

area; VTA) group of neurons of the ventral mesencephalon, though the A9 (substantia n i p )

group of neurons may also participate in some of the behavioral effects (Fallon and Moore,

1978). The A10 cell group, contributing to the mesocorticolimbic projections projects to the

nucleus accumbens as well as to the amygdala and the prefrontal cortex (PFC) while the A9

neurons project to the neo-striaturn (caudate and putamen) (Oades and Halliday, 1987; Fallon

and Moore, 1978; Swanson, 1982).

The distinct neuroanatomical loci involved in mediating the initiation and expression processes

of sensitization have been identified through direct intracerebral injection studies in which

amphetamine was directly microinjected into target areas and the effectiveness ofeach region in

12

inducing behavioral sensitization was assessed. These studies provided evidence for the specific

involvement of the AIOIA9 dopamine cell bodies (VTA and substantia n i p ) in the initiation

and the dopamine terminal fields (nucleus accumbens) in the expression of behavioral

sensitization (Kalivas and Weber, 1988; Vezina and Stewart, 1990; Kalivas and Stewart, 1991).

Repeated microinjection of amphetamine into A10 dopamine cell body regions resulted in the

sensitized response to a subsequent peripheral challenge dose of amphetamine (Vezina and

Stewart, 1990). This was in spite of the lack of behavioral activation following an acute

microinjection of amphetamine into the AlO cell bodies. The sensitized response was also

evident when the challenge dose of amphetamine was directly microinjected into the dopamine

terminal fields at the nucleus accumbens following repeated intra-VTA amphetamine

administration (Kalivas and Weber, 1988; Vezina and Stewart, 1990; Pemgini and Vezina,

1994).

Despite behavioral augmentation following an acute microinjection of amphetamine directly

into the dopamine terminal fields at the level of the nucleus accumbens, repeated microinjection

of amphetamine into the dopamine terminal fields did not cause sensitization to the behavioral

effects of amphetamine following a challenge dose (Hitzemann et al., 1980; Dougherty and

Ellinwood, 1981). This lack of sensitized response was evident following amphetamine

challenge administered both peripherally as well as directly into the terminal fields (Kalivas and

Weber, 1988; Vezina and Stewart, 1990). Thus amphetamine-induced alterations at the level of

the VTA during the initiation or development stage of sensitization induce long lasting

neurochemical changes in the nucleus accumbens which in turn mediate the persistent

behavioral modifications associated with expression of amphetamine sensitization. In addition

13

to evidence h m microinjection studies employing amphetamine, alterations to the

morphological characteristics of nucleus accumbens neurons following repeated amphetamine

(Robinson and Kolb, 1999) implies a major role for the nucleus accumbens in behavioral

sensitization to amphetamine.

2. Role of the prefrontal cortex (PFC) in sensitization

Though the focus in efforts to understand the neurobiological mechanism of amphetamine

sensitization has primarily been the dopaminergic system, evidence from studies using

glutamate receptor antagonists as well as PFC lesion studies point to a major involvement of

excitatory amino acid (EAA) transmission primarily from the PFC to the dopaminergic neurons

in chronic amphetamine-induced behavioral sensitization. Ibotenic acid-induced lesions of the

PFC has been shown to prevent the induction ofbehavioral sensitization to repeated intra-VTA

orperiphenl d-amphetamine administration (Wolf et al., 1995; Li and Wolf, 1997; Cador et al.,

1999; Tuchentke and Schmidt, 1999; Wolf and Xue, 1999). In addition to PFC lesions, studies

employing NMDA receptor antagonists have shown disruption of the sensitizing effects of

chronic d-amphetamine (Wolf et al., 1995; Tzschentke and Schmidt, 1999; Wolf and Xue,

1999). So also repeated administration of d-amphetamine has been shown to induce several

changes in the PFC including altered AMPA receptor mRNA levels (Lu et al., 1997; Lu and

Wolf, 1999), enhanced Calmodulin expression (Gnegy et al., 1991; Shimizu et al., 1997;

Michelhaugh et al., 1998), enhanced sbess-induced dopamine release (Hamamura and Fibiger,

1993) as well as long-lasting shuctural modifications to PFC neurons (Robinson and Kolb,

1997).

1.5 Neurochemlcal correlntes of behaviornl sensitization

1.5.1 Dopaminergic natrorransnlission

Dopaminergic neurotransmission in the nucleus accumbens plays an important role in

mediating the behavionl hyperactivity induced by amphetamine administration. In vivo

microdialysis studies in freely moving rats have shown that acute exposure to amphetamine and

other psychostimulants induces increased extracellular dopamine at the level of the nucleus

accumbens (Sharp et al., 1987; Di C h i m and Impento, 1988; Carboni et al., 1989; Kuczenski

and Segal, 1989; Maisonneuve et al, 1990). In addition to enhanced dopamine release in

response to an acute amphetamine injection, a persistent enhancement in amphetamine-induced

dopamine release has been shown to accompany behavioral sensitization resulting from

repeated exposure to amphetamine. This sensitized augmentation in dopamine release induced

by prior exposure to amphetamine has been reported both by in vitro studies examining

dopamine release from accumbal and striatal tissue slices (Robinson and Becker, 1982;

Nishakawa et al., 1983) as well as by in vivo microdialysis studies performed on h e l y moving

rats (Wilcox et al., 1986; Robinson et al., 1988; Kazahaya et al., 1989; Akimoto et al., 1990;

Pierce and Kalivas, 1995; Paulson and Robinson, 1995). Further, persistent enhancement in

dopamine neurotransmission has been proposed as the common neurophmacological basis for

the behavioral sensitizing effects of both amphetamine and cocaine (Akimoto et al., 1990).

Though a majority of studies report persistent increases in dopamine release in response to

repeated amphetamine and thus a role for enhanced dopamine in amphetamine sensitization,

some studies have also shown evidence for behavioral sensitization to repeated amphetamine

occurring in the absence of increased dopamine release at the nucleus accumbens or the caudate

putamen (Kuczenski et al., 1997a; 1997b).

15

1.5.2 Dopanline receptors in sensitizarion

Though the involvement of the dopaminergic system in amphetamine sensitization has been

well established, the role played by dopaminergic receptors in mediating the induction andlor

expression of sensitization is much less understood. The co-administration of dopamine

receptor antagonists along with the treatment injections of amphetamine has been employed in

several studies to determine the significance of dopaminergic receptors in the induction of

sensitization. Sensitization-induced augmentation in the behavioral response to systemic and

intra-VTA amphetamine has been shown to be blocked both by the systemic administration as

well as the direct intra-VTA microinjection of the Dl receptor antagonist, SCH-23390 prior to

each treatment dose (Stewart and Vezina, 1989; Vezina and Stewart, 1989; Bjijou et al., 1996;

Vezina, 1996; Pierre and Vezina, 1998). This is one of the most consistent results reported in

dopamine receptor studies associated with amphetamine sensitization and indicates a direct

involvement of the Dl receptor in the initiation of sensitization. So also studies employing

knockout mice deficient in the dopamine Dl receptor indicate a significant role in

psychostimulant induced sensitization (Moratalla et al., 1996; Cmwford et al., 1997).

On the other hand, studies involving the co-administration of D2 receptor antagonists along

with amphetamine have not consistently demonstrated a significant involvement of the D2

receptor in the initiation of sensitization (Vezina and Stewart, 1989; Drew and Glick, 1990;

Meng et al., 1998). A possible role for dopamine D4 receptors in behavioral sensitization has

been postulated by Feldpausch et al. who reported blockade of sensitizing properties of

amphetamine following co-administration of PNU-101387G, a D4 receptor antagonist

(Feldpausch et al., 1998).

16

Though a few studies have reported alterations in the characteristics of dopamine Dl, D2, and

D3 receptors in association with behavioral sensitization to amphetamine, no consistently

significant changes in dopamine receptor densities have been reported following repeated

amphetamine administration. Though receptor autondiography studies have reported an

increase in the density of dopamine Dl receptors in the substantia n i p following repeated

amphetamine administration, this effect has been shown to be hansient and does not persist

after longer withdrawal periods (Bonhomme et al., 1995; Brauer et al., 1997). Similarly, though

a reduction in dopamine D2 receptor density and function has been reported in the ventral

shiatum in amphetamine sensitization (Henry et al., 1995; Chen et al., 1999) the majority of

studies focusing on dopamine receptor characteristics report no persistent changes in Dl and

D2 receptor densities or mRNA levels following amphetamine sensitization (Richtand et al.,

1997). Though D3 receptor expression has been shown to be induced in behavioral sensitization

to chronic levodopa, an indirect dopamine receptor agonist (Bordet et al., 1997), D3 receptor

mRNA levels are reported to be unaltered following repeated amphetamine administration

(Hondo et al., 1999).

Since reduced sensitivity of pre-synaptic dopamine autoreceptors would disinhibit dopamine

neuron firing thereby resulting in increased dopamine rclease, dopamine autoreceptor

subsensitivity has been postulated as a mechanism involved in sensitization. Though

autoreceptor desensitization has been reported following repeated amphetamine administration,

these changes have been shown to be hansient in nature i.e. dopamine autoreceptor

subsensitivity becomes evident within 1-7 days after last treatment dose but disappears

following longer periods of withdrawal (White and Wang, 1984; Robinson and Becker, 1986;

17

Yamada et al., 1991; Seutin et al., 1991; Wolf et at., 1993). Hence its unlikely that transient

alterations in the dopamine autoreceptor sensitivity is sufficient to mediate the long lasting

effects associated with amphetamine sensitization.

1.5.3 The dopanline transporler and atnpl~e~an~ine sensilizalion

The primary hnction of the Na'ICI-dependent dopamine transporter is in the re-uptake of

released dopamine from the synaptic cleft back into the presynaptic terminal (Giros et at., 1992;

Giros and Caron, 1993; Jaber et al., 1997; Reith et a]., 1997). The involvement of the dopamine

transporter in mediating acute amphetamine-induced dopamine neurotransmission has been

well characterized (Pifl et a]., 1995; Giros et al., 1996; Jaber et al, 1997). Two models have

been proposed to explain the mode of amphetamine-induced dopamine release. The weak base

model postulates that amphetamine crosses the plasma membrane by lipophilic diffusion and

displaces stored dopamine from the secretory vesicles into the cytoplasm by disrupting the

vesicular proton gradient (Sulzer et al., 1993;1995). On the other hand, the exchange diffusion

model proposes that amphetamine acts as a substrate for the dopamine transporter and that

binding of amphetamine to the transporter increases the number of inward-facing transporter

biding sites (Fischer and Cho, 1979). Studies with dopamine transporter-knockout mice have

revealed that both the vesicle-depleting and reverse transport-mediating properties of

amphetamine are necessary to induce increased dopamine release. Thus the action of

amphetamine on the dopamine transporter results in the reverse transport of dopamine into the

synaptic cleft (Giros et at., 1996; Jaber et al., 1997; Jones et al., 1998).

Several studies have investigated possible alterations in dopamine transporter characteristics in

response to repeated or long-term exposure to amphetamine. Reduced levels of the dopamine

18

transporter in the nucleus accumbens, PFC and dorsal shiatum have been reported following

withdrawal from repeated cocaine administntion (Byrnes et al., 1993; Koff et al., 1994; Aloyo

et al., 1995; Claye et al., 1995; Boulay et al., 1996; Letchworth et al., 1997). Though PET

studies have revealed reductions in dopamine transporter density in the caudate and putamen

regions of abstinent methamphetamine users (McCann et al., 1998), results from animal studies

have been inconsistent with reports of upregulation, reduction and no change in dopamine

transporter mRNA in the substantia nign and VTA following amphetamine sensitization (Kula

and Baldessarini, 1991; Persico et al., 1993; Lu and Wolf, 1997; Shilling et al., 1997; Metzger

et al., 1998).

1.5.4 GIu~arnalergic involvetnent in sensilization

I. Glutamate neurotransmission

Enhanced glutamate neurohansmission has been postulated to play a major role in the initiation

of sensitization. Adminishation of psychostimulants as well as dopamine receptor agonists has

been reported to induce increased glutamate efflux at the level of the VTA. Enhanced

somatodendritic dopamine release in response to amphetamine has been proposed to induce the

stimulation of EAA release by Dl receptors located at the terminals of EAA neurons projecting

from the PFC to the VTA (White et a)., 1995; Kalivas and Duffy, 1995; 1997; Zhang et al.,

1997; Reid et al, 1997; Wolf and Xue, 1998; Dalia et al., 1998). The increased EAA release at

the VTA would then activate NMDA receptors located on the VTA dopamine neurons,

resulting in enhanced dopamine release at the level of the nucleus accumbens (Kalivas et al.,

1989; Kalivas and Alesdatter, 1993; Kalivas, 1995; Kalivas and Durn, 1995). Studies

19

mentioned earlier involving the prevention of sensitization to amphetamine following repeated

intra-VTA administration of Dl and NMDA receptor antagonists support this idea of an

interaction between glutamate release and Dl receptors at the level of the VTA and its

involvement in the initiation of sensitiz-ition (Carter et al., 1988; Stewart and Vezina, 1989;

Kalivas et al., 1989; Karler et al., 1990; 1994; Bjijou et al., 1996; Vezina, 1996).

2. Glutamate receptor antagonist studies

Several studies have shown that co-administration of N-methyl-D-aspartate (NMDA) receptor

antagonists prior to each treatment dose of amphetamine or cocaine prevents initiation of

behavioral sensitization to these psychostimulants (Karler et al., 1989; 1990; 1991; Wolf and

Khansa, 1991; Stewart and Dmhan, 1993; Wolf et al., 1995). In addition to blocking chronic

amphetamine-induced behavioral enhancement, NMDA antagonists have been reported to

prevent the neurochemical alterations associated with sensitization (Wolf et al., 1994a; Gnegy

et al., 1996). Administration of NMDA antagonists prior to challenge dose following

withdrawal h m repeated amphetamine has not been consistently reported to prevent the

expression of sensitization (Karler et al., 1991; Wolf, 1998; Rockhold, 1998). Thus most

findings are consistent with a preferential involvement for glutamate NMDA receptors in the

initiation of amphetamine-induced sensitization, suggesting similarities between behavioral

sensitization and other NMDAdependent forms of synaptic plasticity, such as long term

potentiation (Karler et al., 1989; Wolf and Khansa, 1991).

In contrast to NMDA, the involvement of other glutamate receptors such as u-amino-3-

hydroxy-5-methyl-4-isoxazole (AMPA) in sensitization is less understood, since amphetamine

sensitization studies involving the administration of AMPA rcceptor antagonists have provided

equivocal results. Co-adminishation of AMPA antagonists with treatment injections of

amphetamine has been shown to prevent sensitization in mice, suggesting the involvement of

AMPA receptors in the initiation of sensitization (Karler et al., 1991; Vanover, 1998). However,

rats administered repeated amphetamine along with AMPA antagonists did not show any

change in the sensitized response following challenge (Li et al., 1997). Similarly, though

AMPA antagonist administration prior to challenge amphetamine blocked the expression of

sensitization in mice, a similar paradigm in rats did not induce any alteration in the behavioral

response (Karler et al., 1991; Li et al., 1997; Mead and Stephens, 1998).

3. Alterations in glutamate receptors in sensitization

As mentioned earlier increased glutamate efflux at the VTA could play a role in sensitization by

inducing dopamine release via activation of VTA dopamine neurons. In support of this, several

studies have reported that repeated exposure to amphetamine like psychostimulants results in

increased basal firing rates of VTA dopamine neurons as well as hyperresponsiveness of VTA

dopamine neurons to glutamate induced excitatory effects following short periods of

withdrawal (Kalivas and D u e , 1995; Zhang et al., 1997; White et al., 1995). Since VTA

dopamine cells showed increased responsiveness to AMPA but not to NMDA or metabompic

glutamate receptor agonists, the enhancement in the excitatory properties of glutamate on

dopamine neurons following repeated amphetamine was proposed to be mediated by possible

alterations in AMPA receptors at the VTA (Zhang et al., 1997). However, studies focusing on

changes in AMPA receptor subunits in the VTA following repeated amphetamine have been

inconclusive, with most studies reporting no change or non-significant alterations in VTA

AMPA receptors in amphetamine sensitization (White et al., 1995; Fitzgerald et al., 1996,

Zhang et al., 1997).

In contrast to the hyperresponsiveness of VTA neurons to glutamate, repeated psychostimulant

administration has been shown to induce a transient subsensitive response to the excitatory

properties of glutamate in nucleus accumbens neurons (White et al., 1995; Kim and Vezina,

1997; 1998; Meeker et al., 1998). In support of this subsensitivity in the nucleus accumbens,

decreased levels of AMPA and NMDA receptor subunits in the nucleus accumbens has been

reported following repeated amphetamine and cocaine (White et al., 1995; Fitzgerald et al.,

1996; Lu et al., 1996; 1997; Lu and Wolf, 1997). However, other studies describe increased

levels of these receptoa at the level of nucleus accumbens following repeated cocaine

(Churchill et al., 1997; Ghasemzadeh et al., 1997).

As mentioned earlier, the PFC plays an important role in the initiation of repeated amphetamine

induced-sensitization. Chronic amphetamine treatment has been shown to induce increased

levels ofglutamateat the level of the ffontal cortex. Several studies have reported alterations in

the levels of expression of glutamate AMPA, NMDA and hinate receptors in the PFC

following administration ofchronic amphetamine (Kashiwabara et al., 1984; Eisch et al., 1996;

Lu et al., 1997). These receptor changes have been postulated to influence the activity of PFC

22

neurons that project to the VTA and nucleus accumbens thus resulting in the alteration of

dopamine neuronal activity.

1.6 Synaptic proteins and sensitization

The lack of consistent results reported by studies investigating the role of dopamine receptor or

transporter characteristics suggests that possible alteration in synaptic proteins directly involved

in neurotransmitter release could be responsible for the long term behavioral and neurochemical

changes associated with amphetamine sensitization.

1.6.1 Synaplic vesicle-mediated neurotransttlit!er release

1. Calcium influx-induced migration of synaptic vesicles

In the resting state, neurotransmitter-containing synaptic vesicles are distributed into two

distinct populations, one group not available for immediate release, immobilized at a distance

from the nerve terminal (known as the reserve pool) and a second group at closer proximity to

the terminal and readily available for neurohansminer release (the releasable pool) (Pieribone et

al., 1995). Synaptic vesicles in the reserve pool remain bound to the actin subunits of the

cytoskeleton by the unphosphorylated form of the phosphoprotein, synapsin I (Figure 1.1)

(Greengard et al., 1993; Ceccaldi et al., 1995). Calcium influx into the terminal following an

action potential, activates Ca2'/calmodulin-dependent kinase I1 leading to the phosphorylation

of synapsin I (Nielander et al., 1997; Llinas et al., 1991). Phosphorylation of synapsin I disrupts

the vesicleactin interaction and allows the translocation of the synaptic vesicles from the

reserve pool to the releasable pool (Bahler et al 1990; Greengard et al., 1993).

23

2. Docking, Priming and Fusion of synaptic vesicles

The second stage in vesicle-induced neurotransmitter release involves the docking of the

synaptic vesicles to the active zone on the plasma membrane, the region of active

neurotransmitter release (For reviews refer: Sollner and Rothman, 1994; Sodhof, 1995; Bums et

al., 1995, Catsicas et al, 1994). Following docking, the synaptic vesicles undergo a priming

reaction whereby they are made competent for calcium-induced fusion. The docking and

priming processes involves the formation of complex between specific synaptic vesicle

proteins, plasma membrane proteins as well as three cytosolic factors, N-ethylmaleimide

sensitive factor (NSF), soluble NSF attachment protein @-SNAP) and n-Secl (Muncl8). The

synaptic vesicle proteins involved are synaptophysin, synaptotagmin and synaptobrevin (vesicle

associated membrane protein, VAMP) while the plasma membrane proteins include syntaxin

and synaptosome-associated protein-25 (SNAP-25). These molecules act as receptors for

aSNAP and hence are referred to as SNARES (SNAP receptors).

synaptotagamin

synaptobrevin

s yntadn

synaptophysin a SNAP

!I

. - . syntaxin .:.. :., -. - . . neurotmmmitter release

Figure 1.1 Docking and fusion steps of neurotransmitter release. 1) n-secl detaches from syntaxin and synaptophysin detaches from synaptobrevin enabling the formation of the SNARE complex. 2) a-SNAP competes with synaptotagmin and binds to syntaxin. 3) The NSF protein also joins the complex and 4) following ATP hydrolysis, the complex starts to dissociate. 5) Calcium influx into the terminal stimulates the fusion of the vesicle with the plasma membrane resulting in release of the neurotransmitter.

25

Synaptophysin and synaptobrevin are bound together on the vesicle while on the plasma

membrane, n-secl is tightly attached to syntaxin. Prior to the priming reaction, n-secl detaches

from syntaxin and synaptophysin and synaptobrevin dissociate. This allows the formation ofthe

SNARE complex between syntaxin and SNAP-25 on the plasma membrane and synaptobrevin

and synaptotagmin on the vesicle. Following formation ofthe core complex, aSNAP competes

with and replaces synaptotagmin from binding sites on syntaxin. Binding of aSNAP to

syntaxin allows the NSF protein to join the complex. ATP hydrolysis then results in the

disruption of the complex and the vesicle is primed for calcium influx-stimulated fusion and

neurotransmitter release.

1.6.2 Calcium transduction and an~pltetarnine sensitization

Increased calcium influx and activation of calcium binding proteins mediate vesicular release of

neurotransmitters. Evidence from several studies demonstrate an important role for calcium

conductance in amphetamine sensitization. Adminishation of L-type calcium antagonists has

been shown to abolish the behavioral sensitized response to chronic amphetamine (Karler et al.,

1991). Also, microdialysis studies have shown that disruption of calcium hansmission prevents

the enhanced dopamine release at the nucleus accumbens following repeated amphetamine

(Westerink et al., 1989; Hurd and Ungerstedt, 1989). Chronic amphetamine treatment has also

been reported to alter the expression of calcium-activated phosphoproteins including

enhancement of Ca2'-calmodulin dependent protein kinase I1 mediated phosphorylation of

synapsin 1 (Iwata et al., 1997a; 1997b). Repeated amphetamine also induces alterations in

26

calmodulin protein content and calmodulin mRNA expression in the striaturn (Roberts-Lewis et

al., 1986; Gnegy et al., 1991; Ostrander et al., 1998; Michelhaugh et al., 1998; Kantor et al.,

1999). So also administration of calcium-calmodulin kinase I1 inhibitors has been shown to

shown to abolish sensitization to psychostimulants (Pierce and Kalivas, 1997).

1.6.3 Alferarions in intrucellular signalling cascude

In addition to focusing on alterations in neurotmnsmission or in receptors, several studies have

examined changes in second messenger systems involved in intracellular signal transduction. G

protein signal transduction has been postulated to contribute to the development of

sensitization. Repeated administration of cocaine has been shown to reduce G protein

expression in the VTA and nucleus accumbens (Nestler et al, 1990). So also, repeated

administration of G protein inhibitors has been shown to result in sensitization to subsequent

amphetamine administration (Steketee and Kalivas, 1991; Cunningham and Kelley, 1993). A

single administration of amphetamine has been shown to induce immediate but transient

induction of G protein signaling (Burchen et al., 1998). Stimulation of dopamine receptors has

been shown to result in the activation of adenylyl cyclase and protein kinase A (PKA).

Microinjection of selective PKA and adenylate cyclase activators directly into the VTA has

been shown to enhance amphetamine-induced behavioral sensitization while administration of

PKA and adenylate cyclase inhibitors prevented the induction of sensitization (Byrnes et al.,

1997; Tolliver et al., 1999). So also inhibitors of Protein Kinase C (PKC) were shown to block

amphetamine-induced dopamine release, in vitro @tor and Gnegy, 1998).

27

Exposure to psychostimulants has been shown to alter the expression of immediate early genes

such as c-jbs. Several studies have shown that acute adminishation of amphetamine induces

robust c-jos and c+n expression in the striatum and nucleus accumbens (Graybiel et al., 1990;

Snyder-Keller, 1991; Johansson et al., 1994). In contrast to c-fos and cc-jln, sensitization to

repeated amphetamine has been reported to induce enhanced phosphorylation of the CAMP

responsive element binding protein (CREB) and enhanced expression of fos-related antigen

(FRA) in the striatum and nucleus accumbens (Koluadi et al. 1994; Simpson et al,. 1995;

Turgeon et al., 1997). Thus changes in expression of transcription factors such as Fos and

CREB in response to amphetamine sensitization could be responsible for mediating the

longlasting changes associated with sensitization by altering the expression of relevant genes.

Amphetamine administration has been shown to induce differential changes to the mRNA

levels of zit7268, a member of the zinc finger family of immediate early genes and

preprodynorphin (PPD) (Wang and McGinty, 1995). Augmented PPD and reduced zit7268

mRNA have been found associated with the induction of behavioral sensitization. Further

evidence for the role of transcription factors in sensitization is evident from the blockade of

amphetamine- and cocaine- induced sensitization by inhibitors of protein synthesis (Karler et

al., 1993).

2.OBJECTIVES

2.1 Synaptic proteins mediating dopamine release in amphetamine sensitization

Given the critical role for synaptic proteins in neurotnnsmitter release and the evidence that

chronic amphetamine may alter the expressionlmodification of some of these molecules, the

present thesis explored further the specific roles of the synaptic proteins, synaptophysin,

syntaxinl and synapsinl in amphetamine sensitization. Western blotting technique was used to

ascertain expression levels of these synaptic proteins in key areas involved in amphetamine

sensitization, the nucleus accumbens and the VTA. The working hypothesis was that alteration

in the expression levels of these candidate proteins following chronic exposure to amphetamine

could indicate changes in the synaptic mechanisms directly involved in dopaminergic

neurotransmission. These long-term synaptic changes could thus mediate the long-lasting

augmentation in dopamine release evident in amphetamine sensitization.

2.2 Glutamate/Dopamine receptors in amphetamine sensitization

Behavioral sensitization resulting from repeated exposure to d-amphetamine has been shown to

be mediated primarily by the dopaminergic and glutamatergic systems. Though several studies

have reported changes in a number of neurochemical correlates associated with these systems,

these reported changes do not seem to occur consistently. So also, the behavioral paradigms

employed by some of these studies do not seem to accurately mimic all the behavioral aspects

of amphetamine sensitization. This study employed a repeated intermittent amphetamine

administration paradigm in order to induce behavioral sensitization. This paradigm, utilized

extensively by several groups, has been shown to closely mimic the behavioral characteristics

associated with chronic d-amphetamine-induced sensitization. The appropriateness of our

behavioral paradigm along with the lack of consistently reported neurochemical changes in

association with amphetamine sensitization lead us to investigate possible alterations in

dopaminergiclglutamatergic receptor densities in a repeated intermittent amphetamine-induced

behavioral sensitization model.

3. METHODS

3.1 Animals

Adult male Spraye-Dawley rats were obtained from Charles River Canada and housed in a

temperature- and humidity- controlled environment on a 12 hour lightldark cycle with free

access to standard laboratory food and water. Upon arrival the animals were left in the animal

housing for a period of at least 48 hours before use.

3.2 Materials

The primary antibodies used for the western blot analysis were

( I ) Monoclonal anti-Synaptophysin, (Clone SVP-38, Sigma)

(2) Polyclonal anti-Syntaxin 1 (Alomone labs)

(3) Polyclonal anti-Synapsin 1 (Calbiochem) and

(4) Monoclonal anti-a-Tubulin (Biodesign).

The secondary antibodies used were

(I) Anti-rabbit Ig: peroxidase-linked whole antibody (Amersham Life Sciences) and

(2) Anti-mouse IgG: peroxidase-linked whole antibody (Amersham Life Sciences).

The radioactive ligands used for the receptor autoradiography experiments were MK-801,

fH] Kainic acid and ['HI 7-OH DPAT all obtained ffom NEN Life Science Products, Boston,

MA.

All other chemicals used were of the highest analytical quality available.

30

3.3 Establishment of the behavioral sensitbation paradigm

Locomotor activity measurements were conducted in 12 activity chambers housed in a dimmed

room. Each chamber (30x40~40 cm) was equipped with two photoelectric switches; light beam

interruptions from each chamber were monitored and stored in a microcomputer. The animals

(N=30) were divided over 3 sessions (8-1 1 AM, l l AM-2 PM, and 2-5 PM) and their

spontaneous locomotor activity was measured over 3 hours for three consecutive days.

The animals were then assigned to one of t h e groups (Amphetamine, Saline or Novelty) such

that each group showed comparable mean total spontaneous locomotor activity. After 3 days of

locomotor activity measurements, the animals from each session received at the beginning of

each of 5 altemate days oftesting, one of the 3 conditions, d-amphetamine sulphate (1.5 mglkg,

I.P), an equal volume of saline vehicle (0.9%, LP) or no injection (control group), following

which the locomotor activity was monitored for 3 hours. Each animal was placed in the same

chamber throughout the duntion of locomotor activity testing.

Following 5 altemate days of testing, the animals were withdmwn from the treatment for 14

days during which time they were left undisturbed in the animal housing. After 2 weeks of

withdmwal, every animal received a challenge dose of d-amphetamine sulphate (0.5 mglkg l.P)

following which the locomotor activity was monitored for 3 hours.

Statistical comparisons between the three groups (amphetamine, saline and control) were based

on total locomotor activity scores, defined as the sum of photobeam intemptions recorded over

a 3 hour session. A one-way analysis of variance (ANOVA) was used to compare the total

locomotor activity counts of the 3 hour session following the challenge injection between the

31

three groups, Post-hoc tests were also performed using Newman-Keuls test to compare

significance between each of the three groups.

Following the establishment of a chronic amphetamine administration paradigm appropriate to

induce behavioral sensitization, the same paradigm was repeated with two separate cohorts of

rats (N=30 andN=24) with one exception. These 2 cohorts did not receive a challenge injection

14 days after the last treatment injection. Instead, at the end of the withdrawal period, the

animals were sacrificed by rapid decapitation and their brains surgically removed.

3.4 Western Blot analysis of Synaptic Proteins

3.4.1 Protein sample preparation and quan/ifcation

Animals from the first cohort (N=30) were sacrificed by rapid decapitation and their brains

surgically removed. These brains were sliced into Imm thick slices using surgical blades and a

rat brain mold. Relevant brain regions (the core and shell regions of the nucleus accumbens and

the ventral tegrnental area) were micropunched out (Figure 2.1) and the tissue homogenized in

Tris-EDTA homogenization buffer (50mM Tris, ImM EDTA, 0.2mM PMSF, IpM leupeptin

and lpM pepstatin, pH 7.4). For each group (n=IO), the tissue micropunched out from the

brains of 2 rats was combined as one sample i.e., combined to give a final sample number of 5

per group m=15). Protein concenwtion of each sample was determined using the Bradford

method, 200p1 of Bio-Rad protein assay reagent was mixed with lop1 of the sample and the

absorbance measured at 595nm using a ELx800 plate reader (Bio-Tek Instruments Inc). Each

homogenate sample was then diluted in homogenization buffer so as to give a fmal

32

concentration of 5mg in 15p1(0.33 mglpl). The samples were mixed 1:l with sample buffer

(0.25M Tris-HCI pH 6.8,20 % Glycerol, 4 % SDS, 10% P-Mercaptoethanol) and stored at - 2oUc.

3.4.2 Western Blot hybridiation

The samples were removed from -20°C and placed in a boiling water bath for 5 minutes, cooled

to room temperature and then centrifuged for 10 minutes at 12,000 rpm. Each sample (15~1 of

sample per well containing 5mg protein) was then individually loaded on a 15 well 4-20% Tris-

Glycine gel (Novex) assembled in an X-Cell I1 mini-cell gel electrophoresis assembly (Novex)

and then electrophoresed at 125V for 90 minutes in Tris-Glycine running buffer (16.5mM Tris,

0.135M Glycine, 0.1% SDS; pH 8.3). Following the electrophoresis step, the gel cassettes were

removed and thegels let? soaking in transfer buffer (25mM Tris, 0.2M Glycine, 20% methanol;

pH 8.3) for 10 minutes. The protein samples were then blot transferred onto a nitrocellulose

membrane (Hybond ECL, Amersham Pharmacia Biotech) using the X-cell I1 mini-cell blot

transfer assembly at 25V for2 hours. After the blot transfer, the membranes were placed in 5%

skim milk solution in TBS buffer (13.3mM Tris, 0.8%w/v NaCI; pH 7.6) containing 0.1%

Tween-20 (Sigma) and left shaking at room temperature for 1 hour. The membranes were then

hybridized with appropriate dilutions of the primary antibody (1:5000 for synaptophysin;

1:2000 for syntaxin 1 and 1:1000 for synapsin 1) in lOml of TBS-Tween-20 for 1 hour at mom

temperature. The membranes were then washed twice (15 minutes each) in TBS-Tween 20 at

room temperature and then hybridized with the secondary antibody (1:3000 dilution of anti-

mouse IgG: pemxidase-linked whole antibody for synaptophysin labeling and 1:2000 dilution

33

of anti-rabbit Ig: peroxidase-linked whole antibody for syntaxin and synapsin labeling) for I

hour at room temperature. The membranes were again washed in TBS-Tween-20 buffer (3 x 15

minutes). The antibody binding on the membrane was then detected using western blot

chemiluminescence detection reagents (Renaissance, NEN) and the membranes apposed to X-

ray imaging film (Kodak Scientific Imaging). The duration of exposure to X-ray film varied for

each antibody (15 seconds each for synaptophysin-labeled membranes, 30 seconds each for

syntaxin 1-labeled membranes and 45 seconds each for synapsin I-labeled membranes). The

films were then developed and the antibody-labeling was analyzed using a computerized image

analysis system (MCID-4, Imaging Research).

Following each hybridization reaction, the nitrocellulose membrane was placed in a stripping

solution (62.5mM Tris, 2% SDS and IOOmM P-Mercaptoethanol, pH 6.7) for 30 minutes at

50°C with occasional shaking. The stripped membrane was then washed with TBS-T buffer (3

x 10 minutes) and labeled with anti-tubulin antibody as the primary antibody (1:1000 dilution

in TBS-T buffer for Ihr at room temperature). AAer washing with TBS-T buffer, (2 x 10

minutes), the membrane was hybridized with anti-mouse IgG: pemxidase-linked whole

antibody (1 hour at room temperature). The membranes were again washed in TBS-T buffer (3

x 15 minutes). The antibody binding on the membrane was then detected using western blot

chemiluminescence detection reagents (Renaissance, NEN) and the membranes apposed to X-

ray imaging film (Kodak Scientific Imaging) for 30 seconds each. The films were then

34

developed and tubulin-labeling was analyzed using a computerized image analysis system

(MCID-4, Imaging Research).

For this study, a-fubulin was chosen as control for protein content since it is one of the most

ubiquitously expressed proteins and the majority of studies employing westem blot techniques

have reported using a-tubulin as control. Further, there is no direct evidence to suggest

alteration in a-tubulin expression following repeated amphetamine administration, although

treatment with antitumor drugs, hormones and psychotropic drugs have been reported to alter

microtubule dynamics and assembly (Poffenbarger and Fuller, 1977, Goh et al., 1998, Gastpar

et al, 1998).

3.5 Receptor Autoradiography

3.5.1 Brain Sample Processing

The animals fiom the second cohort (N=24) were sacrificed by rapid decapitation and the brains

were surgically removed and ffozen in 2-methyl butane maintained at -20UC and then stored at

-80UC until use. Frozen rat brains were sectioned at 20prn thickness using a Microtom cryostat.

Sections were collected on pre-cleaned, gelatin-coated microscopic slides, thaw-mounted,

desiccated under vacuum at 4'C overnight and then stored at -80UC until the day of experiment.

3.5.2 Autoradiography hperiments

Brain sections taken at the level ofprefrontal cortex, nucleus accumbens and VTA were used in

the following protocols. Brain regions were determined using a Paxinos and Watson rat brain

35

atlas (1986). For NMDA receptor binding, slides were incubated for 60 min at room

temperature with 200pM (+)-3-['2JI]Iodo-~~-801, 50mM sodium phosphatc/0.9% saline

solution (PBS) pH 7.4. Non specific binding was defined in adjacent sections by the addition of

5pM MK-801 to the incubation buffer. Following incubation, sections were rinsed in ice-cold

PBS and then washed twice for 15 minutes in ice-cold PBS. After a final brief dipping in ice-

cold distilled water, slides were dried at mom temperature and apposed to ['251]-Hype~lm

(Amersham, Toronto, Ontario) along with calibrated iodine standards (Amersham) for 8 hours.

For Kainate receptor binding, brain sections were first pre-incubated for 30 minutes at 4°C in

50mM Tris-citrate (pH 7.0) and then again for 10 minutes at 30°C in 50mM of Tris-citiate.

Sections were then incubated for 30 min at 4'C in the same buffer containing 20nM ['HIKainic

acid. Nonspecific binding was determined on adjacent brain sections by adding 50pM Kainic

acid to the binding buffer. The incubation was terminated by dipping the slides in ice-cold

buffer followed by four consecutive 5 second-washes in the same buffer. AAer a final dipping

in ice-cold distilled water, slides were dried at mom temperature and apposed to ['HI-Hyperfilm

(Amersham, Toronto, Ontario) for 3 weeks in the presence of ['HI-Micmscales calibrated

tritium standards (Amersham, Toronto, Ontario).

For Dopamine D3 receptor autoradiography the slides were first pre-incubated at room

temperature for 30 minutes in buffer containing 50mM Tris and 120 mM NaCl (pH 7.4).

Sections were then incubated for 120 min at room temperature in the same buffer containing

36

2nM ['HI 7-OH DPAT, 300pM GTP and 5 pM DTG (to block sigma sites). Non specific

binding was determined on adjacent brain sections by adding 1pM Dopamine (Sigma) to the

above buffer. The incubation was terminated by dipping the slides in ice-cold washing buffer

containing 50mM Tris twice for 10 minutes each. After a brief dipping in ice-cold distilled

water brain sections were rapidly dried and apposed to ['HI-Hyperfilm for 4 weeks along with

['HI-Microscales calibrated tritium standards.

A11 autoradiographic films were analyzed with a computerized image analysis system (MCID-

4, Imaging Research, Ste-Catherines, Ontario).

37 4. RESULTS

4.1 Establishment of behavioral sensitization paradigm

The locomotor activity counts of each of 3 groups (d-amphetamine-treated n=10, saline-

treated n=9 and untreated controls n=9) was measured over 180 minutes on 5 alternate

treatment days as well as following d-amphetamine challenge, 14 days after the last

treatment day. The total locomotor activity counts for each group (measured over 180

minutes and averaged over the number of animals in each group) for each of the 5 days of

treatment is illustrated in Figure 4.1. The mean locomotor activity of the animals treated

with 1.5mg/kg d-amphetamine on each treatment day was significantly greater than

repeated-saline-treated animals as well as the untreated controls

Following administration of a challenge injection of O.Smg/kg d-amphetamine after 14

days of withdrawal, animals pre-treated with d-amphetamine displayed significantly higher

locomotor activity compared to both saline-pre-treated @<0.01) as well as untreated

control animals @<0.001). So also, the locomotor activity of the d-amphetamine-pretreated

rats following challenge was greater than that during each of the 5 treatment days, though

the challenge dose of d-amphetamine administered (0.5mg/kg) was much lower than the

treatment dose (1.5mg/kg).

4.2 Behavioral sensitization of 2 separate cohorts

Following the establishment of a paradigm appropriate to induce long-lasting behavioral

sensitization to d-amphetamine, the same treatment paradigm (I .5mg/kg d-amphetamine or

- Control animals (n*)

Y Saline-trealad animals (n*)

~ ~ 2 3 Alrphetanine-treated anim$(n=lO)

CSA CSA CSA CSA CSA CSA day 1 day 2 day 3 day 4 day 5 challenge

Figure 4.1 Locomotor actidty counts (total number of beam interruptions over 18Omin * SEM) measured for 3 groups (d-amphetamine pretreated animals (A), saline-treated animals (S) and untreated control animals (C)) over 5 alternate days of treatment as well as following d-amphetamine challenge after 14 days of withdrawal. 'Ihe total locomotor actidty count of each treatment group has been averaged over the number of animals in each group. The locomotor actidty counts of 2 animals, one each from the saline-pretreated and control groups were excluded due to problems associated with the actidty chambers. Statistical significance was tested using one-way analysis of variance (ANOVA) followed by Newman Keuls test. During each of the five treatment days, d-amphetamine-treated animals displayed significantly higher levels of locomotor actinty compared to both the saline-treated as well as the untreated control animals. Following the challenge injection of d-amphetamine, the mean locomotor actidty counts of d-amphetamine-pretreated rats was significantly higher compared to both saline-pretreated animals as well as untreated control animals. ' indicates p<0.001 comparing d-amphetamine-pretreated animals to saline-treated animals on each treatment day: + indicating pc0.001 comparing d-amphetamine-pretreated group to untreated control animals; # indicating pc0.01 comparing d-amphetamine-pretreated to saline treated animals following d-amphetamine challenge and A indicating pc0.001 comparing d-amphetamine-pretreated animals with untreated control animals following d-amphetamine challenge.

2000 0 Control animls (n=9)

Saline-treated animls (n*) c 'B d-anphelaninelrealed anhls ( ~ 1 0 ) 0 z - g 1000 3

0

0 CSA CSA CSA CSA CSA:-: my1 m y 2 m y 3 m y 4 mys : ' 4 W :

Flgure 4.2 Locomotor activity counts p@$%t%ber of beam intemptions over 18Omin k SEMl measured for 3 orouos over 5 alternate davs of treatment. The total locomotor activiiy counts of each treatment group have been averaged over the number of animals in each group. Though 30 rats were tested, locomotor data from two rats, one each from the saline-trealed and control groups was excluded due to problems associated with the activity chambers. Statistical significance was tested using one-way analysis of variance (ANOVA) followed by Newman Keuls test. During each of the five treatment days, the damphelamine treated animals diplayed significantly increased locomotor activity compared to both saline-treated as well as untreated control animals. Saline-treated animals did not display significantly different lewis of locomotor actiuily compared to untreated control animals on any of the 5 days of treatment. Following 14 days of withdrawal after the treatment period, all animals were sacrificed, and the brains from this cohort of rats were used for the analysis of synaptic protein expression using Western blotting. indicates pe0.001 comparing d-amphetamine-treated animals with saline-treated animals: + indicates p<0.001 comparing d-amphetaminetreated animals to untreated control animals.

CSA my 1

CSA b y 3

CSA b y 5

CSA Day 7 -I4 14day 7

by ': withdrawal

r=l Control animls (nd) - Salinetreated anirrels (nd)

mzm d-anphelanine-lreated anirrels (n4)

Figure 4.3 Locomotor activity counts (total number of beam interruptions over 180min * SEM) measured for 3 groups over 5 alternate days of treatment. The total locomotor activity counts of each treatment group have been averaged over the number of animals in each group. Statistical significance was tested using one-way analysis of wiance (ANOVA) followed by Newman Keuls test. During each of the five treatment days, the d-amphetamine treated animals diplayed significantly increased locomotor activity compared to both saline-treated as well as untreated control animals. No significant difference was evident between the locomotor activity counts for saline-treated and untreated control animals. Following 14 days of withdrawal after the treatment period, all animals were sacrificed, and the brains from this cohort of rats were used for the analysis of neurotransmitter receptor densities using receptor autoradiography. * indicates pC0.001 comparing d-amphetamine-treated animals with saline-treated animals; + indicates pc0.001 comparing d-amphetamine-treated animals to untreated control animals.

4 1 saline for 5 alternate days along with untreated control animals) was repeated for 2 cohorts

of rats (N=30 and N=24). For both these cohorts, the animals were sacrificed after 14 days

of withdrawal following the treatment period without the administration of a challenge

dose of d-amphetamine. The mean values of locomotor activity counts (over 180 minutes)

of each group from the two cohorts (N=30, N=24) are illustrated in Figures 4.2 and 4.3

respectively. For each cohort, the locomotor activity profile during the 5 treatment days

resembled the profile from the previous (Figure 4. I) trial sensitization paradigm. For both

cohorts, d-amphetamine-treated animals showed significantly higher levels of locomotor

activity compared to saline-treated as well as control animals on each of the 5 days of

treatment.

The animals from the first cohort (N=30) were used for the analysis of synaptic protein

expression by western blotting while the second cohort of animals (N=24) were used for

receptor autoradiography.

4.3 Synaptic protein expression in amphetamine sensitization

4.3.1 Syntaxin 1 expression in the nucleus acawbens core, shell and VTA

Syntaxin 1 expression in the nucleus accumbens core and shell as well as the ventral

tegmental area was assessed by western blot hybridization using a polyclonal antibody

specific to syntaxin 1. Autoradiographic signals of syntaxin 1 and a-tubulin labeling in

representative samples from each of the three groups (d-amphetamine-treated,saline-treated

and untreated animals) measured for the three regions (nucleus accumbens core, shell and

CONTROL SALINE d-AMPH

SYNTAXIN 1 00- -0- 0-0

a-TUBULIN 0 , , , --- v--

Figure 4.4 Representative autoradiograms of hybridization signals from Westem blot using antibodies specific for syntaxin 1 and a-tubulin performed on brain tissue samples isolated from the core region of nucleus accumbens. Syntaxin 1 and a-tubulin expression was determined for three groups of animals, one group administered repeated intermittent doses of d-amphetamine (d-amph), a second group administered repeated intermittent saline injections (saline) and the third group consisting of untreated animals (control). Data from three representative samples from each group is shown.

0 Control animals

Saline-treated animals

EE3 Amphetamine-treated animals

Control Saline Amph

Figure 4.5 Comparison of syntaxin 1 expression in the core region of nucleus accumbens between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline vehicle (Saline) and untreated control animals (Control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Syntaxin 1 expression was determined as the percentage of the optical density measurements of syntaxin 1 to a-tubulin labeling. A one way analysis of miance (ANOVA) was used to determine statistical significance with a Newman Keul's post hoc test for significance betwen the individual groups. Syntaxin 1 expression was significantly reduced in chronic d-amphetamine-treated animals compared to animals treated with saline ( + indicates p<0.05) and untreated control animals ( X indicates pc0.01).

CONTROL SALINE I-AMPH

SYNTAXIN 1 I - 1-0 - 0 -

Figure 4.6 Representative autondiograms of hybridization signals from Western blot using antibodies specific for syntaxin I and a-tubulin performed on brain tissue samples isolated from the shell region of nucleus accumbens. Syntaxin I and a-tubulin expression was determined for three groups of animals, one group administered repeated intermittent doses of d-amphetamine (d-amph), a second group administered repeated intermittent saline injections (saline) and the third group consisting of untreated animals (control). Data from three representative samples from each group is shown.

0 Control animals

I......I Saline-treated animals

EZEl Amphetamine-treated animals

Control Saline Amph

Figure 4.7 Comparison of syntaxin 1 expression in the shell region of nucleus accumbens between three groups, 2 treatment groups of animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline chicle (Saline) and one no-treatment group of untreated control animals (Control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Syntaxin 1 expression was determined as the percentage of the optical density moasurments of syntaxin 1 to a-tubulin labeling. A one way analysis of variance (ANOVA) was used to determine statistical significance with a Newman Keul's post hoc test for significance betwen the indiddual groups. Syntaxin 1 expression was significantly increased in chronic d-amphetamine-treated animals compared to animals treated with saline ( + indicates pe0.05) and untreated control animals ( X indicates pc0.05).

CONTROL SALINE d-AMPH

SYNTAXIN 1 - - - r * . . ---

a-TUBULIN - , - - - , - - - Figure 4.8 Rcprcscntativc autoradiograms of hybridization signals from Wcstcm blot using antibodies specific for syntaxin I and a-tubulin pcrformed on brain tissuc samples isolatcd from the ventral tcgmcntal area. Syntaxin 1 and a-tubulin cxprcssion was determined for thrcc groups of animals, one group administered rcpcatcd intermittent doscs of d-amphetamine (d-amph), a sccond group administcrcd rcpcatcd intcrmittcnt salinc injections (saline) and the third group consisting of untreated animals (control). Data from thrcc rcprescntativc samples from cach group is shown.

0 Control animals

EEl Saline-treated animals

EEiZl Amphetamine-treated animals

Control Saline Amph

Figure 4.9 Comparison of syntaxin expression in the ventral tegmental area between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (d-arnph) or saline vehicle (saline) and untreated control animals (control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Syntaxin 1 expression was determined as the percentage of the optical density measurments of syntaxin 1 to a-tubulin labeling. A one way analysis of mriance (ANOVA) was used to determine statistical significance. There was no statistically significant difference in syntaxin expression in the ventral tegrnental area between the three groups.

48 VTA) are illustrated in Figures 4.4,4.6 and 4.8 respectively. The expression of syntaxin is

expressed as the percentage of the optical density of hydrization signals corresponding to

syntaxin 1 to that of a-tubulin labeling for each sample. The mean values for the

percentage of syntaxin 1 to a-tubulin optical density for the three groups for each of the

three regions (core shell and VTA) are illustnted in Figures 4.5,4.7 and 4.9 respectively. A

one-way analysis of variance (ANOVA) was used to compare the levels of expression of

syntaxin 1 in each of the three groups. Post-hoc tests were performed using Newman-Keuls test

to compare significance between the individual groups.

The percentage of syntaxin 1 to a-tubulin labeling in the core region of the nucleus accumbens

was found to be significantly reduced (Figure 4.5) in animals administered repeated intermittent

doses of d-amphetamine compared to repeated-saline-treated animals w0.01) as well as

untreated control animals w0.05). Syntaxin 1 expression in the nucleus accumbens core of d-

amphetamine-sensitized animals ranged from approximately 22% (compared to saline-treated

animals) and 34% (compared to untreated control animals). Though the percentage of syntaxin

I to a-tubulin labeling was reduced in saline-treated animals compared to untreated controls,

this difference was not statistically significant. (Figure 4.5).

In contrast to the expression levels of syntaxin 1 in the nucleus accumbens core, syntaxin 1

expression was found to be significantly increased (Figure 4.7) in the nucleus accumbens shell

of animals treated with repeated injections of d-amphetamine compared to saline-treated

(~1~0.05) and untreated control animals @<0.05). Syntaxin 1 expression in chronic d-

49 amphemmine-administered animals was increased approximately 22% compared to saline-

treated animals and 3 1% compared to untreated controls (Fiyre 4.7).

Comparison of the levels of expression of syntaxin 1 in the VTA between d-amphetamine-

treated, saline-h.eated and untreated control animals revealed a lack of significant difference in

syntaxin 1 expression in the VTA between the three groups (Fiyre 4.9). Though d-

amphetamine-treated animals showed reduced syntaxin l expression compared to saline-treated

and untreated control animals in the VTA, it failed to attain statistical significance.

4.3.2 Synaplophysin expression in the nrrcleus accrot~bens core, shell and VTA

Synaptophysin expression in the nucleus accumbens core and shell as well as the ventral

tegmental area was assessed by western blot hybridization using a monoclonal antibody

specific to synaptophysin. Autoradiographic signals of synaptophysin and a-tubulin

labeling in representative samples from each of the three groups for the three regions (

nucleus accumbens core, shell and VTA) are illustrated in Figures 4.10,4.12 and 4.14. The

expression of synaptophysin is expressed as the percentage of the optical density of

hybridization signals corresponding to synaptophysin to that of a-tubulin for each sample.

The mean values for the percentage of synaptophysin to a-tubulin optical density for the

three groups for each of the three regions (nucleus accumbens core, shell and VTA) are

illustrated in Figures 4.1 1, 4.13 and 4.15. A one-way analysis of variance (ANOVA) was

used to compare the levels of expression of synaptophysin in each of the three groups. Post-hoc

tests were performed using Newman-Keuls test to compare significance between individual

groups.

CONTROL SALINE d-AMPH

Figure 4.10 Rcprcscntativc automdiograms of hybridization signals from Western blot using antibodies specific for synaptophysin and a-tubulin pcrformed on brain tissue samples isolated from the core region of nucleus accumbcns. Synaptophysin and a-tubulin cxprcssion was determined for three groups of animals, one group administered repeated intermittent doses of d- amphetamine (d-amph), a second group administered repeated intermittent salinc injections (saline) and the third group consisting of untreated animals (control). Data from thrcc rcprcsentative samples from each group is shown.

0 Control animals

CZl Saline-treated animals

EEE! Amphetamine-treated animals

Control Saline Arnph

Figure 4.11 Comparison of synaptophysin expression in the core region of nucleus accumbens between three groups of animals (animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline vehicle (Saline) and untreated control animals (Control)). Each of the three groups consisted of I 0 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Synaptophysin expression was determined as the percentage of the optical density measurements of synaptophysin to a-tubulin labeling. A one way analysis of variance (ANOVA) was used to determine statistical significance with a Newman Keul's post hoc test for significance betwen the indiidual groups. Synaptophysin expression was significantly reduced in chronic d-amphetamine treated animals compared to animals treated with saline ( + indicates pC0.05) and untreated control animals (X indicates p<0.05).

CONTROL SALINE d-AMPH

Figure 4.12 Representative autondiograms of hybridization signals from Western blot using antibodies specific for synaptophysin and a-tubulin performed on brain tissue samples isolated from the shell region of nucleus accumbens. Synaptophysin and a-tubulin expression was determined for thrce groups of animals, one group administcrcd repeated intcrmittcnt doses of d- amphetamine (d-amph), a second group administcrcd repeated intcrmittcnt saline injections (saline) and the third group consisting of untreated animals (control). Data from thrce representative samples from each group is shown.

I Control animals

EZ3 Saline-treated animals

EiiEEl Amphetamine-treated animals

Control Saline Arnph

Figure 4.13 Comparison of synaptophysin expression in the shell region of nucleus accumbens between three groups, 2 treatment groups of animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline vehicle (Saline) and one no-treatment group of untreated control animals (Control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Synaptophysin expression was determined as the percentage of the optical density measurments of synaptophysin to a-tubulin labeling. A one way analysis of ~ r i a n c e (ANOVA) was used to determine statistical significance. The percentage of synaptophysin to a-tubulin expression in the d-amphetamine-treated animals was greater than that for both saline-treated as well as control animals. However, this increase was not sufficient to attain statistical significance.

CONTROL SALINE d-AMPH

SYNAPTOPHYSIN 0 - m v a-c .---

a-TUBULIN a - r C - a-- - 0

Figure 4.14 Rcprcscntativc autoradiograms of hybridization signals from Western blot using antibodies specific for synaptophysin and a-tubulin performcd on brain tissue samplcs isolated from the ventral tcgmcntal area. Synaptophysin and a-tubulin cxprcssion was determined for thrcc groups of animals, one group administcrcd rcpeatcd intcrmittcnt doses of d-amphetamine (d- amph), a second group administcrcd repeated intcrmittcnt salinc injections (saline) and the third group consisting of untrcatcd animals (control). Data from thrcc rcprcscntativc samples from each group is shown.

0 Control animals

1......1 Saline-treated animals

EZEZl Amphetamine-treated animals

Control Saline Amph

Figure 4.15 Comparison of synaptophysin expression in the wntral tegmental area between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (d-amph) or saline whicle (saline) and untreated control animals (control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to giw a total of 5 samples per group (N=15). Synaptophysin expression was determined as the percentage of the optical density measurments of synaptophysin to a-tubulin labeling. A one way analysis of wriance (ANOVA) was used to determine statistical significance. There was no statistically significant difference in synaptophysin expression in the wntral tegmental area between the three groups. Though d-amphetamine-sensitized animals showed decreased synaptophysin expression compared to saline-treated and untreated control animals, this difference was not statistically significant.

56 The percentage of synaptophysin to a-tubulin labeling in the core region of the nucleus

accumbens was found to be significantly reduced (Figure 4.1 1) in animals administered

repeated intermittent doses of d-amphetamine compared to repeated-saline-treated animals

6 0 . 0 5 ) as well as untreated control animals 60.05) . Animals administered repeated

intermittent d-amphetamine showed a reduction in Synaptophysin expression of approximately

24% compared to saline-treated animals and 38% compared to untreated control animals.

Synaptophysin expression wes found to be increased in the nucleus accumbens shell of

animals treated with repeated d-amphetamine (Figure 4.13) compared to saline-treated

animals (1 1%) and untreated controls (19%) although this increase was not statistically

significant.

Comparison of the levels of expression of synaptophysin in the VTA between d-amphetamine-

treated, saline-treated and untreated control animals revealed a lack of significant difference in

synaptophysin expression in the VTA between the three groups (Figure 4.15).

4.3.3 Synapsin I erpression in the nucleus accumbens core, shell and VTA

Synapsin 1 expression in the nucleus accumbens core and shell as well as the ventral tegmental

area was assessed by westem blot I~ybridition using a polyclonal antibody specific for

synapsin 1. Autoradiographic signals of synapsin 1 and a-tubulin labeling in representative

samples from each of the three groups for the three regions tested are illustrated in Figures 4.16,

4.18 and 4.20. The expression of synapsin is expressed as the percentage of the optical density

ofhybridization signals corresponding to synapsin 1 to that ofa-tubulin for each sample.

CONTROL SALINE d-AMPH

Figure4.16 Representative autoradiograms ofhybridization signals from Western blot using antibodies specific for synapsin 1 and a-tubulin performed on brain tissue samples isolated from the core region of nucleus accumbens. Synapsin 1 and a-tubulin expression was determined for three groups of animals, one group administered repeated intermittent doses of d-amphetamine (d-amph), a second group administered repeated intermittent saline injections (saline) and the third group consisting of untreated animals (control). Data from three representative samples from each group is shown.

0 Control animals

I......I Saline-treated animals

EEETI Amphetamine-treated animals

Control Saline Amph

Figure 4.17 Comparison of synapsin 1 expression in the core region of nucleus accumbens between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline ~ h i c l e (Saline) and untreated control animals (Control). Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). Synapsin 1 expression was determined as the percentage of the optical density measurments of synapsin I to a-tubulin labeling. The optical density measurements for synapsin I and a-tubulin labeling for one sample fmm the saline-treated group was excluded. A one way analysis of wriance (ANOVA) was used to determine statistical significance.There was no statistically significant difference in the percentage for synapsin I to a-tubulin labeling between the three groups.

CONTROL SALINE d-AMPH

Figure 4.18 Rcprcscntativc nutoradiograms of hybridization signals from Wcstcrn blot using antibodies spccific for synapsin I and a-tubulin performed on brain tissue samplcs isolated from thc shcll region of nuclcus accumbcns. Synapsin 1 and a-tubulin exprcssion was dctcrmincd for thrcc groups of animals, onc group administcrcd rcpcatcd intcrmittcnt doscs of d-amphctaminc (d- amph), a sccond group administcrcd rcpcatcd intcrmittcnt salinc injections (salinc) and thc third group consisting of untrcatcd animals (control). Data from thrcc rcprcscntativc samplcs from cach group is shown.

0 Control animals

I.......I Saline-treated animals

DEiZl Amphetamine-treated animals

Control Saline Amph

Figure 4.19 Comparison of synapsin 1 expression in the shell region of nucleus accumbens between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (Amph) or saline vehicle (Saline) and untreated control animals (Control). Each of the three groups consisted of 10 animals. For each group. tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N45). Synapsin 1 expression was determined as the percentage of the optical density measurements of synapsin 1 to a-tubulin labeling. A one way analysis of mriance (ANOVA) was used to determine statistical significance with a Newman Keul's test for significance betwen the individual groups. There was no significant difference in synapsin 1 expression between the three tested groups.

CONTROL SALINE d-AMPH

Figure 4.20 Representative autoradiograms of hybridization signals from Western blot using antibodies specific for synapsin 1 and a-tubulin performed on brain tissue samples isolated from the ventral tegmental area. Synapsin 1 and a-tubulin expression was determined for three groups of animcls, one group administered repeated intermittent doses of d-amphetamine (d-amph), a second group administered repeated intermittent saline injections (saline) and the third group consisting of untreated animals (control). Data from three representative samples from each group is shown.

0 Control animals

Saline-treated animals

EEEl Amphetamine-treated animals

Control Saline Arnph

Figure 4.21 Comparison of synapsin expression in the ventral tegmental area between three groups, animals treated repeated intermittent injections of either d-amphetamine sulphate (d-amph) or saline vehicle (saline) and untreated control animals (control). Synapsin expression was determined as the percentage of the optical density measurments of synapsin to a-tubulin labeling. Each of the three groups consisted of 10 animals. For each group, tissue samples isolated from two animals was pooled, to give a total of 5 samples per group (N=15). A one way analysis of ~ r i a n c e (ANOVA) was used to determine statistical significance. There was no statistically significant difference in syntaxin expression in the ventral tegmental area between the three groups.

63 The mean values for the percentage of synapsin 1 to a-tubulin optical density for the three

groups for each of the three regions are illustrated in Figures 4.17,4.19 and 4.21. A one-way

analysis of variance (ANOVA) was used to compare the levels of expression of synapsin 1 in

each of the three groups.

For the three regions tested (accumbens core, shell and VTA), the percentage of synapsin 1 to

a-tubulin labeling was found to be unaltered in d-amphetamine treated animals compared to

both saline-treated and untreated control animals (Figures 4.17,4.19 and 4.21).

4.4 Expression of glutamate NMDA, kainate receptors and dopamine D3 receptor

4.4.1 NMDA receptor auloradiography

NMDA receptor expression was evaluated with receptor autondiognphy technique

employing ['251]-~K-80 I in subregions of prefrontal cortex, striatum, nucleus accumbens,

hippocampus, substantia nigra and VTA (Figures 4.22, 4.23, 4.24). For each region,

NMDA receptor levels were compared between 3 groups, amphetamine-pretreated, saline-

pretreated and untreated control animals. A one way analysis of variance (ANOVA) was

used to compare NMDA receptor densities between the three groups for each brain region

tested.

In the prefrontal cortical region, NMDA receptor levels were found to be higher in the

frontal cortex compared to other cortical regions for all three groups (Figure 4.22). At the

0 striatal level, NMDA receptor levels were higher in the nucleus accumbens (both shell

0 Control animals

Saline-treated animals

d-amphetamine-treated animals

C S A C S A C S A C S A C S A Fr.C C1ng.C IL MONO LO

Figure 4.22 Quantitative analysis data of NMDA receptor densities in prefrontal cortical regions following receptor autoradiography using [I1?-MK-801. NMDA receptor densities were compared between 3 groups of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; Frontal cortex (Fr.C), Cingulate cortex (Cing.C), lnfralimbic cortex (IL). Medial orbital 1 Ventral orbital cortex (MONO) and Lateral orbital cortex (LO). A one way analysis of mriance (ANOVA) was used to test for statistical significance.

0 control animals

EZZd Saline-treated animals

d-amphetamine-treated animals

CSA CSA CSA CSA CSA CSA NA-CORE NA-SHELL CPU-DL CPU-DM CPU-VM CPU-M.

Figure 4.23 Quantitatiw analysis data of NMDA receptor densities in striatallaccumbal regions following receptor autoradiography using [I1?-MK-801. NMDA receptor densities were compared between 3 groups of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; Nucleus accumbens Core (NA-CORE) and Shell (NA-SHELL) and the Dorsolateral (CPu-DL), Dorsomedial (CPu-DM), Ventrolateral (CPu-VL) and Ventromedial (CPu-VM) divisions of the Caudate Putamen. A one way analysis of mriance (ANOVA) was used to test for statistical significance.

I Control animals

A i??zz?d Saline-treated animals

d-amphetamine-treated animals

C S A C S A CAI CA2

C S A C S A CA3 DG

Figure 4.24 Quantitative analysis data of NMDA receptor densities at the hippocampus ( A) and nigral level (6) following receptor autoradiography using [I ' 2 5 ] - ~ ~ - 8 0 1 . NMDA receptor densities were compared between 3 groups of animals: chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; CAI-3 fields, dentate gyrus (DG), ventral tegmental area (VTA) and substantia nigra (SN). A one way analysis of variance (ANOVA) was used to test for statistical significance.

67 and core) compared to the striatal subregions (Figure 4.23). Although, striatal regions from

amphetamine-pretreated animals showed a higher level of NMDA receptor binding than

saline-pretreated and control animals (Figure 4.23), this increase was not sufficient to attain

statistical significance. NMDA receptor binding in the dorsal hippocampus showed

increased binding in the CAI followed by the dentate gyms, CA2 and CA3 for the three

groups (Figure 4.24,A). Analysis of NMDA receptor binding in the VTA and substantia

nigra revealed significantly lower levels of NMDA receptor densities in these areas

compared to all other brain regions tested (Figure 4.24,B).

For all the brain regions analyzed, NMDA receptor expression was found to be unaltered

between amphetamine-pretreated, saline-pretreated and control animals.

4.4.2 Kainate receptor autoradiography

Kainate receptor expression was determined with receptor autoradiography technique

employing ['HI-Kainic acid in subregions of prefrontal cortex, striatum, nucleus

accumbens, hippocampus, substantia nigra and VTA (Figures 4.25, 4.26,4.27). For each

region, Kainate receptor levels were compared between 3 groups, amphetamine-pretreated,

saline-pretreated and untreated control animals. A one way analysis of variance (ANOVA)

was used to compare kainate receptor densities between the three groups for each brain

region tested.

0 Control animals

EZZd Saline-treated animals

l?EEl d-amphetamine-treated animals

- C S A C S A C S A C S A C S A

Fr.C Cing.C IL MONO LO

Figure 4.25 Quantitative analysis data of kainate receptor densities in prefrontal cortical regions following receptor autoradiography using [3~-ka in ic acid. Kainate receptor densities were compared between 3 groups of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; Frontal cortex (Fr.C), Cingulate cortex (Cing.C), lnfralimbic cortex (IL), Medial orbital I Ventral orbital cortex (MONO) and Lateral orbital cortex (LO). A one way analysis of wriance (ANOVA) was used to test for statistical significance.

0 Control animals

FZZd Saline-treated animals

I loo1 E d-amphetamine-treated animals

C S A C S A C S A C S A C S A C S A NA-CORE NA-SHELL CPU-DL CPU-DM CPU-VM CPU-M

Figure 4.26 Quant i ta t i~ analysis data of kainate receptor densities in striatallaccumbal regions following receptor autoradiography using [3H]-kainic acid.Kainate receptor densities were compared between 3

of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; Nucleus accumbens Core (NA-CORE) and Shell (NA-SHELL) and the Dorsolateral (CPu-DL), Dorsomedial (CPu-DM), Ventrolateral (CPu-VL) and Ventromedial (CPu-VM) divisions of the Caudate Putamen. A one way analysis of mriance (ANOVA) was used to test for statistical significance.

0 Control animals EZZl Saline-treated animals

d-amphetamine-treated animals

CAI CA2 CA3 DG

0 Conlml animals

EZZJ Salinetreated animals d-amphelaminbtreated animals

C S A C S A

Figure 4.27 Quantitative analysis data of kainate receptor densities at the hippocampus ( A) and nigral ( B) levels following receptor autoradiography using [3~-kainic acid. Kainate receptor densities were compared between 3 groups of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; CAI-3 fields, Dentate gyrus (DG), Ventral tegmental area (VTA) and Substantia nigra (SN). A one way analysis of variance (ANOVA) was used to test for statistical significance.

7 1 In contrast to NMDA receptor expression in the PFC, the frontal cortical areas showed

lower kainate receptor binding than other cortical areas with the cingulate cortex showing

the highest level of kainate receptor binding (Figure 4.25). Kainate receptor binding at the

striatal level showed a higher level of kainate receptor expression in the subregions of the

caudate putamen compared to the accumbens (both core and shell) (Figure 4.26). Similar to

the expression of NMDA receptors in nigral regions, V T A and substantia n ign also

showed significantly lower levels of kainate receptor densities compared to all other brain

regions (Figure 4.27).

There was no significant difference in kainate receptor levels between amphetamine-

pretreated, saline-pretreated and control animals for all the regions analyzed.

4.4.3 Dopamine D3 receptor autoradiography

Expression of dopamine D3 receptor in striatal and nucleus accumbens subregions was

determined by receptor automdiognphy using ['HI-7-OH DPAT. D3 receptor binding in the

striaturn and nucleus accumbens was compared between three groups, amphetamine-pretreated,

saline-pretreated and untreated control animals. D3 receptor binding was found to be

significantly higher in the nucleus accumbens compared to the subregions of the caudate

putamen with the shell subregion of accumbens showing highest level of D3 receptor level

(Figure 4.28). The three groups showed no statistically significant difference in D3 receptor

levels for the regions tested (Figure 4.28).

0 Control animals

E Z d Saline-treated animals

d-amphetamine-treated animals

C S A C S A C S A C S A C S A C S A NA-CORE NASHEU CPU-DL CPU-DM CPU-W CPU-VL

Figure 4.28 Quantitatiw analysis data of dopamine D3 receptor densities in striatailaccumbal regions following receptor autoradiography using [ 3H]-7-OH-DPAT. Dopmaine D3 receptor densities were compared between 3 groups of animals; chronic amphetamine-treated (A), saline-treated (S) and untreated animals (C) for the following brain areas; Nucleus accumbens Core (NA-CORE) and Shell (NA-SHELL) and the Dorsolateral (CPU-DL), Dorsomedial (CPu-DM), Ventrolateral (CPu-VL) and Ventromedial (CPu-VM) didsions of the Caudate Putarnen. A one way analysis of mriance (ANOVA) was used to test for statistical significance.

5. DISCUSSION

5.1, Estnblishment of the beliavloral sensitization parndigm

Several factors influence the behavioral outcome ofan amphetamine-induced animal model

of sensitization including dosage, treatment schedule and withdrawal period. A paradigm

involving the repeated intermittent administration of a relatively low dose (1-2 mdkg) of

d-amphetamine followed by an extended period of withdrawal (10-14 days) has been

shown to induce long-lasting behavioral sensitization that closely mimics the persistent

changes evident in amphetamine psychosis. A similar paradigm has been utilized in this

study and the behavioral effects observed following the administration of a subsequent

amphetamine challenge are a clear indication of long lasting behavioral sensitization

resulting from this paradigm. Animals advinistered repeated d-amphetamine displayed

progressively increasing higher levels of locomotor activity compared to saline-treated and

untreated control animals on each day of treatment. More importantly, amphetamine-

pretreated animals showed significantly higher levels of locomotor activity compared to

saline and control animals upon administration of amphetamine challenge following 14

days of withdrawal. Thus this paradigm involving the repeated intermittent (5 injections on

alternate days) administration of a low dose amphetamine (lSmg/kg, IP) followed by 14

days of withdrawal as employed in this study was sufficient to induce long-lasting

behavioral sensitization to d-amphetamine and therefore a valid paradigm to investigate

underlying synaptic molecular mechanisms.

Repeated exposure to stressful stimuli has been shown to cross-sensitize animals to

subsequent psychstimulant administration. Thus it was surprising to note that saline-treated

74 animals did not display a sensitized response to amphetamine challenge. One explanation

for this could be the magnitude of the stressful stimulus. The majority of studies reporting

cross-sensitization between chronic stress and psychostimulant challenge involved the

repeated administration of footshock or tail-pinch (Herman et al., 1984; Robinson et al.,

1985; Hahn et al., 1986; Snyder-Keller, 1990; Badiani et al., 1992; Henry et al., 1995)

which are more potent stressors compared to saline injection-induced stress. Further,

behavioral sensitization induced by repeated saline injections could be better revealed by

the adminstration of a challenge stressor (foot shock, tail pinch or saline-injection)

compared to psychostimulant challenge.

To investigate neurochemical alterations, the same paradigm was repeated with two

separate cohorts of animals with the exception of the challenge administration. A challenge

amphetamine dose was not administered in these two cohorts since the neurochemical

changes directly responsible for long-lasting behavioral sensitization are believed to occur

independent of the challenge administration. Studies have shown that even a single

exposure to amphetamine is sufficient to produce behavioral and neurochemical changes

(Vanderschuren et al., 1999). Hence administration of a challenge dose could possibly

affect, alter or even mask some of the changes directly responsible for amphetamine

sensitization.

The analysis of the behavioral data from the two cohorts of amphetamine pretreated

animals revealed a progressively increasing behavioral response to indicating the induction

of long-term behavioral sensitization.

75 5.2 Synaptic protein expression and amphetamine sensitizntlon

One of the primary aims of this study was to investigate the expression of synaptic proteins

involved in vesicle-mediated dopamine release following sensitization to repeated

amphetamine administration. Since the increased dopamine release in response to acute

amphetamine has been shown to be persistently augmented following repeated exposure to

amphetamine, it was postulated that chronic amphetamine-induced alteration in the

expression levels of synaptic proteins directly involved in dopamine release could mediate

the long-lasting effects on dopamine release evident in amphetamine-sensitized animals.

The major results of this study were:

I . A significant reduction in the expression of the synaptic proteins, syntaxin I and

synaptophysin at the level of the core subregion of the nucleus accumbens in animals

sensitized with repeated amphetamine compared to saline-treated and untreated controls.

2. A significant increase in syntaxin 1 expression and a trend toward increase in

synaptophysin expression in the shell subregion of the nucleus accumbens in amphetamine-

sensitized animals compared to saline-treated and untreated controls.

The differential expression of synaptic proteins between the core and shell subregions of

nucleus accumbens observed in this study is consistent with growing evidence supporting

a distinct dichotomy in morphological and functional characteristics between the two

subregions of the nucleus accumbens (For review refer Zahm and Brog, 1992).

76 5.2. I Di//^erential properlies of the core und shell subregions of Nucleus accumbens

Besides receiving afferents from the VTA, the nucleus accumbens core also receives major

projections from the anterior cingulate and dorsocaudal prelimbic cortices also referred to

as the dorsal prefrontal cortex (Zahm and Brog, 1992; Brog et al., 1993). The core projects

via the ventral pallidum to the substantia nigra pars reticulata and subthalamic nucleus and

in turn to the premotor cortex via the ventromedial-ventrolateral complex of the thalamus

(Zahm and Brog, 1992; Brog et al., 1993). In contrast to the core, the shell, in addition to

dopamine afferents from the VTA, receives main afferents from the ventral prefrontal

cortex, the basolateral amygdala and the ventral subiculum of the hippocampus (Zahm and

Brog, 1992; Zahm and Heimer, 1993). The shell projects to the medial ventral pallidum

which projects to the mediodorsal thalamus and then to the ventral tegmental area (Zahm

and Brog, 1992; Zahm and Heimer, 1993). The core and shell subregions also differ in the

morphology of their medium spiny neurons with neurons in the shell having fewer primary

dendrites, fewer dendritic segments and lower spine densities than those in the core

(Meredith et al., 1992; 1995).

Several studies have demonstrated a differential dopamine response between the core and

shell subregions in response to drugs of abuse (Di Chiara, 1995), atypical neuroleptics

(Graybiel et al., 1990;Marcus et al., 1996), as well as restrain/pharmacological stress

(Deutch and Cameron, 1992); Kalivas and Durn, 1995). As well, the two subregions

display differential effects in the behavioral response to dopamine agonists (Swanson et al.,

1997), excitotoxic lesions (Maldonado-Irizany and Kelley, 1995) as well as to

77 amphetamine administration (Heidbreder and Feldon, 1998; Weiner et al., 1996; Bernstein

and Beninger, 2000).

In addition to the above mentioned changes, the core and shell subregions of nucleus

accumbens display differential changes in dopamine release in response to both acute and

chronic administration of psychostimulants (Pontieri et al., 1995; Nisell et al., 1997;

Cadoni and Di Chiara, 1999; 2000; Cadoni et al., 2000). Acute administration of

amphetamine, cocaine, morphine and nicotine resulted in enhanced dopamine release

preferentially in the shell subregion of accumbens (Pontieri et al., 1995; Nisell et al., 1997).

Repeated exposure to amphetamine, cocaine, morphine and nicotine, on the other hand,

induced sensitized dopamine release preferentially in the core subregion of nucleus

accumbens (Cadoni and Di Chiara, 1999; 2000; Cadoni et al., 2000).

5.2.2 Sensitized dopamine release and chronic atnphefatnine adtninisfrafion

Since chronic amphetamine-induced augmented dopamine release has been postulated to

play a crucial role in behavioral sensitization (Robinson et al., 1988, Kalivas and Stewart,

1991) and the synaptic proteins investigated in this study are significantly involved in

neurotransmitter release, an attempt has been made to discuss the results of this study in

relation to sensitized dopamine release. Though this study did not measure dopamine

release in response to chronic amphetamine, several studies employing similar paradigms

have consistently reported a persistent enhancement in dopamine release at dopaminergic

terminal fields. Hence, it is very likely that a long lasting enhancement in dopamine release

78 would also have been induced in the two cohorts of animals treated repeated amphetamine,

used in this study.

5.2.3 Synapsin I expression and sensitized doparnine release

Western blot analysis of synapsin I expression in this study revealed no significant

alteration in synapsin I expression in all brain regions tested following repeated exposure

to amphetamine. Synapsin 1 is a phosphoprotein shown to play a major role in the

regulation of neurotransmitter release (Greengard et al., 1993). The unphosphorylated form

of synapsin is involved in the immobilization of synaptic vesicles to the reserve pool by

attaching to the actin-cytoskeleton. The phosphorylation of synapsin 1 by

calcium/calmodulin-dependent protein kinase I1 has been shown to disrupt these

interactions allowing the vesicles to migrate to the releasable pool at close proximity to the

terminal (Greengard et a1.,1993).

The results of this study showing no changes in total synapsin I level between

amphetamine-sensitized and control animals concur with earlier studies that report similar

lack of altered total synapsin 1 expression following chronic amphetamine administration

(Iwata et al., 1996; 1997). However, repeated amphetamine administration has been shown

to induce increased Ca2'-calmodulin dependent protein kinase II-dependent phosphorylation

of synapsin 1 (Iwata et al., 1996; 1997). Although there is no direct evidence indicating

increased vesicle migration following chronic amphetamine administration, enhanced

synapsin 1 phosphorylation in response to chronic amphetamine could cause an increase in

the number of vesicles detaching from the reserve pool and migrating to the releasable

79 pool. Thus the phosphorylation state ofsynapsinl rather than total amount may be involved

in vesicle-mediated dopamine release in sensitized animals. Alternatively, other synapsin

isoforms such as synapsin I1 and synapsin I11 may be involved and further experiments are

necessary to entirely comprehend the involvement of synapsin in dopamine release.

5.2.4 Syntaxin I /Synaptophysin expression and sensitized dopantine release

The other candidate proteins investigated in this study were syntaxin I and synaptophysin,

synaptic proteins involved in the docking and fusion steps of calcium-dependent vesicular

exocytotic dopamine release. During the docking and fusion steps, vesicles become

attached to the active zone of the terminal and are then primed for neurotransmitter release.

Syntaxin 1, a synaptic terminal plasma membrane-bound protein and synaptophysin, a

vesicle-bound protein interact with other synaptic proteins to form what is called the

SNARE complex. Calcium influx into the terminal activates the disruption of the SNARE

complex and induces neurotransmitter release by fusion of the vesicles with the plasma

membrane.

Western blot analysis of syntaxin 1 and synaptophysin expression revealed a significant

reduction in the expression of these proteins at the level of the core subregion of the

nucleus accumbens, in animals sensitized with repeated amphetamine. The significance of

syntaxin 1 and synaptophysin in the SNARE complex suggests that reduction in syntaxin 1

and synaptophysin expression could result in a possible downregulation of the SNARE

complex formation. Thus the significantly reduced levels of syntaxin 1 and synaptophysin

in the nucleus accumbens core of sensitized animals, could result in a possible

80 disruption/downregulation of amphetamine-induced dopamine release by inhibiting

vesicular exocytosis.

However, such a disruption in vesicular machinery does not necessarily imply decreased

dopamine release occurring from the nucleus accumbens core in amphetamine-sensitized

animals. In fact, repeated exposure to amphetamine has been shown to induce sensitized

dopamine release preferentially in the nucleus accumbens core compared to the shell

(Cadoni et al., 1000). Thus our results raise a paradox as to how amphetamine-induced

dopamine release could be sensitized in the nucleus accumbens core with a concurrent

disruption in the vesicular release mechanism.

As described earlier, amphetamine induces the release of dopamine either by vesicle-

mediated exocytosis or by its action on the dopamine transporter. Amphetamine has been

shown to bind to the dopamine transporter and promote reverse transport of dopamine into

the synaptic cleft. Thus one possible explanation for sensitized dopamine release occurring

in the nucleus accumbens core of amphetamine-sensitized animals could be an

enhancement in amphetamine-induced dopamine transporter-mediated reverse transport of

dopamine in contrast to vesicular release.

Support for the idea of enhanced transporter-mediated dopamine release occurring at the

nucleus accumbens core in response to chronic amphetamine comes from studies reporting

upregulation of dopamine transporter mRNA at the level of the ventral tegmental area in

amphetamine sensitized animals (Shilling et al., 1997; Lu and Wolf, 1997). The ventral

tegmental area has been shown to play a major role in the initiation of behavioral

81 sensitization to amphetamine. Enhanced dopamine transporter mRNA expression at the

VTA following chronic amphetamine administration could result in the enhancement of

dopamine transporter levels at the dopaminergic terminal fields.

In contrast to the reduced syntaxin I and synaptophysin expression observed in the core

subregion of nucleus accumbens, repeated exposure to amphetamine resulted in enhanced

syntaxin 1 expression at the level of nucleus accumbens shell. However, the relevance of

this increased syntaxin I in amphetamine-sensitized dopamine release is unclear. As

mentioned earlier, syntaxin I plays a major role in the docking and fusion steps of vesicular

neurotransmitter release. Hence it is possible that the enhanced expression of syntaxin 1, as

observed in this study could indicate an upregulation of vesicular exocytosis-mediated

dopamine release in the nucleus accumbens shell ofchronic-amphetamine-treaizd animals.

However, chronic amphetamine treatment has been shown to sensitize amphetamine-

induced dopamine release selectively in the core compared to the shell. Hence a direct role

for enhanced syntaxin 1 expression in sensitized dopamine release in the nucleus

accumbens shell seems unlikely.

Another possible role for the observed alteration in syntaxin 1 expression in accumbens

shell could be in mediating behavioral /neurochemical changes associated with cross-

sensitization. Cross-sensitization is characterized by the interchangeability of behaviorall

neurochemical alterations resulting from repeated administration of cocaine or

amphetamine (Robinson et al., 1985, Kalivas and Weber, 1988). Repeated exposure to

amphetamine followed by amphetamine challenge as well as repeated exposure to cocaine

82 with a subsequent administration of cocaine challenge have both been shown to induce

sensitized increases in dopamine release preferentially in the nucleus accumbens core

compared to shell (Cadoni et al., 2000). In contrast, administration of challenge

amphetamine following repeated exposure to cocaine has been shown to result in sensitized

dopamine release preferentially at the level of the shell compared to core (Pierce and

Kalivas,1995). This suggests that the locus (nucleus accumbens core or shell) for sensitized

dopamine release resulting from exposure to repeated administration of amphetamine or

ccicaine could be dependent on the psychostimulant used for challenge.

This raises the interesting possibility that concurrent but reciprocal neurochemical changes

could be occurring in the nucleus accumbens core and shell in response to repeated

amphetamine, with core-related changes mediating the sensitized dopamine response to

subsequent amphetamine challenge and shell-related changes being more relevant in cross-

sensitization. Since syntaxin I expression was selectively enhanced in the nucleus

accumbens shell in response to chronic amphetamine administration, this change could be

related to cross-sensitization. However, a specific role for alteration in syntaxin 1

expression in the nucleus accumbens shell in cross-sensitization is not known since most

studies investigating amphetamine-induced behavioral sensitization/cross-sensitization

have not considered differential neurochemical changes occumng in sensitization and

cross-sensitization.

Though the changes in synaptic protein expression have been discussed so far on the basis

of sensitized dopamine release, chroni:: amphetamine administration has been shown to

83 also alter the release of other neurotransmitters (Glutamate, GABA and acetylcholine) in

the nucleus accumbens. Repeated exposure to amphetamine has been shown to induce

enhanced glutamate efflux at the level of the nucleus accumbens (Abekawa et al., 1994,

Xue et al., 1996). Repeated administration of amphetamine has also been reported to alter

the expression of the AMPA receptor subunit, GluRl selectively in the nucleus accumbens

shell (Lu and Wolf, 1999). Thus the subregion-selective changes in synaptic protein

expression, observed in this study could be related to amphetamine-induced sensitized

glutamate release in the nucleus accumbens. So also, prior experience with amphetamine

has been shown to reduce GABA release and induce acetylcholine release at the nucleus

accumbens (Lindefors et a1.,1992). Since glutamatergic neurons project to, and GABA-

ergic and cholinergic neurons are localized at, the nucleus accumbens, the observed

a synaptic protein changes could be related to alterations occurring within the terminals of

one or more of these neurons in the nucleus accumbens in response to chronic

amphetamine administration.

Interactions between the different synaptic proteins involved in the SNARE complex have

been shown to mediate the insulin-activated transport of glucose from subcellular regions

to the plasma membrane (Foster et al., 1999, St.Denis et al., 1998). SNARE-related proteins

such as syntaxin, SNAP-23 and synaptophysin have been found to co-localize with the

glucose transporters (GLUT 1 & 4) and promote insulin-dependent translocation of these

transporters (Macaulay et al., 1997, Foran et a]., 1999). Synaptic proteins syntaxin, SNAP-

25 and synaptobrevin co-localize with GABA receptors and syntaxin 1 and synaptophysin

have been shown to induce the Protein Kinase C-mediated functional regulation of the

84

@ GABA transporter (Kame et al.,l997, Quick et al.,l997). Takcn together, there is growing

evidence to demonstrate that synaptic proteins are involved in the localization, organization

and regulation o f transporters, receptors and other membrane-associated proteins.

Though a direct i nhznce of synaptic proteins on the dopamine transporter has not been

reported, it is interesting to speculate on the possibility of SNARE-DAT interactions in

relation to the results of this study. Reduction in synaptophysin and syntaxin 1 in the

nucleus accumbens core in response to chronic amphetamine administration has already

been discussed to possibly indicate a downregulation in vesicular dopamine release in the

core occurring along with an upregulation of DAT to compensate for the loss of vesicular

dopamine release. In contrast to the changes occurring in the core, repeated amphetamine

administration was shown to induce elevated levels of syntaxin 1 in the nucleus accumbens

shell. In addition, there are rcports of a reduction in DAT transporter levels in the nucleus

accumbens shell following eLposure to repeated psychostimulant administration (Shape et

al., 1991, Boulay et al., 1996). Taken together, these results suggest the possibility of a

reciprocal relationship between synaptic protein expression and DAT activity. Synaptic

proteins could be interacting with the dopamine transporter so as to induce an inhibitory

influence on DAT activity. Thus the reduced expression o f synaptic proteins in the core

following repeated amphetamine administration results in sensitized dopamine release in

the core possibly by disinhibition of DAT activity and the subsequent enhanced DAT-

mediated reverse transport of dopamine.

85 Repeated exposure to amphetamine has been shown to result in a significant enhancement

in the expression of astrocytic basic fibroblast growth factor (bFGF) at the level of the

VTA (Flores et al., 1998). Increased bFGF in the VTA is mediated by the enhanced

glutamate release occurring at the VTA following repeated exposure to amphetamine

(Flores et al., 1998). bFGF has been shown to enhance the phosphorylation state of

neuromodulin (GAP-43) and selectively alters the expression of AMPA receptor GluRl

both of which are changes evident in amphetamine-sensitized rats (Meiri et al., 1998,

Cheng et al., 1995, Iwata et al., 1997, Gnegy et al., 1993, Lu and Wolf,1999). Further,

increased levels of bFGF has been reported to result in altered synaptophysin expression

(Gargini et a1.,1999). These results suggest the possibility that, enhanced bFGF production

occurring at the leve! of the VTA following chronic amphetamine administration may

induce the synaptic protein changes observed in this study.

5.2.5 Synaptic proteins, disease and plasticity

Several studies report alteration in synaptic protein expression as an index of synaptic

plasticity as well as a marker for changes in synaptic density (Eastwood and Hamson,

1995, Eastwood et al., 1995). Altered synaptophysin expression in the nucleus accuml ms,

as observed in this study, could represent modifications in the morphology of neurons in

this region following repeated administration of amphetamine. This is consistent with other

studies that demonstrate chronic amphetamine administration resulting in alterations to

neuronal morphology in the nucleus accumbens (Robinson and Kolb, 1997, 1999).

Syntaxin 1 and synaptophysin expression have been reported altered in hippocampal

86 kindling models of epileptogenesis (Kamphius et al., 1995, Mahata et al., 1992, Helme-

Guizon et al., 1998). Further, changes in syntaxin I, SNAP-25, and synaptophysin

expression, occur in cortical brain areas of subjects with schizophrenia ( Eastwood and

Harrison, 1995, Eastwood et al., 1995, Gabriel et al., 1997, Glantz et al., 1997).

Synaptophysin in particular, has been repoited to be significantly altered in schizophrenia

with evidence for modified synapt~physin gene (Eastwood et al., 2000). mRNA (Eastwooo

and Harrison, 1999) and protein (Landen et al., 1999, Davidsson et al., 1999) expression in

schizophrenic brains.

Thus the alterations in synaptic protein expression observed in this study could indicate

persistent neurochemical adaptations occurring at the level of the nucleus accumbens in

response to chronic amphetamine administration which mirror similar presynaptic protein

changes observed in schizophrenia as well as in kindling models of synaptic plasticity.

Since the VTA plays a significant role in the initiation of amphetamine sensitization

(Kalivas and Weber, 1988; Vezina and Stewart, 1990; Kalivas and Stewart, 1991), this study

investigated the expression of synaptic proteins in the VTA following chronic

amphetamine administration. Western blot analysis revealed that the expression levels of

synapsin 1, syntaxin 1 and synaptophysin were unaltered in the ventral tegmental area of

amphetamine-sensitized animals compared to saline-treated and untreated control animals.

Future experiments will aim to measure the mRNA levels o f these proteins in the ventral

tegmental area. Changes evident in the expression of these proteins at the level of the

nucleus accumbens suggest that parallel alterations could be occurring in the mRNA levels

87 of these synaptic proteins within the VTA. Future experiments that include analysis of

mRNA lcvels of these as well as other candidate proteins in the VTA via in silu

hybridization techniques could enable the identification of target proteins with altered

expression in amphetamine sensitization.

5.3 Glutamatergic 1 dopaminergic receptors and amphetamine sensitization

5.3.1 NMDA/Kainale receptors and sensirization

Analysis of NMDA receptor expression using ['2SI]-labeled MK-801 revealed no

significant alteration in NMDA levels between amphetamine-pretreated, saline-pretreated

and untreated control animals. Lack of significant changes in NMDA receptor expression

in the VTA and Substantia nigra observed in this study is consistent with other studies that

report no significant alteration in NMDA expression in these brain regions (Wolf, 1998,

White et al., 1995, Zhang et al., 1997).

No change in NMDA receptor expression in the PFC with a trend toward decreased NMDA

levels in the nucleus accumbens was observed in this study. Other studies have reported

significantly reduced levels of the NMDA receptor in the nucleus accumbens as well as in

the prefrontal cortex after 14 days of withdrawal following repeated exposure to

amphetamine (Lu et al., 1996, Lu and Wolf, 1997, Wolf, 1998). The behavioral paradigms

used, however, were different from that used in this study. These studies employed a higher

dose (5mgkg) of amphetamine compared to the present study (1.5mgkg). Further,

repeated amphetamine was administered on consecutive days compared to intermittent

88 injections as employed in this study. As described earlier, the characteristics of the drug-

injectio;: paradigm influences not only the behavioral outcome, but also the neurochemical

changes resulting from the administration of the paradigm. Hence, the differences between

the behavioral paradigms could explain the discrepancy in results.

Another possible explanation for the discrepancy could be differences in the variables

measured and the techniques used. The present study-measured [1251]-MK801 labeling of

the binding site within the NMDA channel as an index of NMDA receptor expression. Lu

and Wolf (1997), on the other hand, measured mRNA levels of the NRI subunit of the

NMDA receptor. Since the MK-801 binding site is located within the NMDA channel,

access to the binding site is restricted by the functional state of the receptor. Hence, binding

of [1251]-MK-801 occurs only when the channel is open or activated. Thus, measurement of

[125~] -~~801- l abe l ing seems to be a more appropriate technique to determine NMDA

receptor expression as it requires the functional activation of the receptor and could thus be

a possible measure of the functional state of the receptor. However, the integral role for the

NRI subunit in the NMDA receptor suggests that measurement o f NRl subunit expression

could also be used to determine NMDA receptor levels.

Under the experimental conditions of this study, no significant difference in kainate

receptor binding was evident in any o f the brain regions tested. Very few studies have

investigated kainate receptor expression in relation to psychostimulant-induced

sensitization. The results of this study are consistent with Fitzgerald et al., (1996) who

reported no alteration in Kainate receptor densities following cocaine sensitization).

89 @ Kashiwabara et al (1984) showed decreased Kainate receptor expression through ['HI-

Kainic acid binding in the cortex following sensitization to methamphetamine. However,

this study used a shorter period of withdrawal (7 days) compared to the present study.

Moreover, methamphetamine has been shown to be more toxic to neurons than d-

amphetamine. Hence, the reduced Kainate receptor expression could be related to repeated

methamphetamine-induced neurotoxicity rather than sensitization.

5.3.2 Dopantine 0 3 receprors and sensitization

Though dopamine D3 receptors have been shown to be altered following behavioral

sensitization to the indirect dopamine agonist levodopa (Bordet et al., 1997) as well as in

prenatally stressed rats that display an increased responsivity to the sensitizing properties of

amphetamine (Henry et a1.,1995), studies to date have reported no significant alteration in

D3 receptor expression following amphetamine sensitization. The results of this study

indicating lack of significant alteration in D3 receptor expression in the striaturn and

nucleus accumbens is consistent with at least one study (Hondo et a1.,1999) reporting no

change in D3 receptor mRNA levels in the PFC, nucleus accumbens and striatum following

amphetamine sensitization.

In summary, the results of the present study suggest synaptic changes in the nucleus

accumbens following amphetamine sensitization with no alteration in glutamate NNMDA,

kainate and dopamine D3 receptors. The synaptic changes could possibly mediate altered

dopamine or other neurotransmitter responses in the sensitized animals. Importantly,

a reciprocal changes in the synaptic protein syntaxin I in the nucleus accumbens core and

90 shell subregions of sensitized animals underscores the differential role of these structures in

the sensitized response of animals to repeated amphetamine.

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