THE ROLE OF THE DOPAMINE D RECEPTORS IN CUE-INDUCED ... › bitstream › 1807 › ... · 3 RECEPTORS IN CUE-INDUCED REINSTATEMENT OF NICOTINE-SEEKING BEHAVIOUR . Maram A.T.M. Khaled

THE ROLE OF THE DOPAMINE D3 RECEPTORS IN CUE-INDUCED

REINSTATEMENT OF NICOTINE-SEEKING BEHAVIOUR

by

Maram A. T. M. Khaled

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Pharmacology and Toxicology

University of Toronto

© Copyright by Maram A. T. M. Khaled (2011)

ii

THE ROLE OF THE DOPAMINE D3 RECEPTORS IN CUE-INDUCED

REINSTATEMENT OF NICOTINE-SEEKING BEHAVIOUR

Maram A.T.M. Khaled

Degree of Master of Science

Department of Pharmacology and Toxicology

University of Toronto

2011

Dopamine D3 receptors (DRD3) are implicated in relapse to drugs. The current study

investigated the role of DRD3 in cue-induced reinstatement of nicotine-seeking in rats. Rats

were trained to lever-press for intravenous infusions of nicotine, associated with the illumination

of a cue-light, under a fixed-ratio schedule of reinforcement. Following extinction of the

behaviour, where lever pressing had no consequences, reinstatement testing was performed by

reintroduction of the cues after systemic or local administration (into discrete brain areas) of the

DRD3 selective antagonist SB277011-A. Systemic antagonism of DRD3 significantly attenuated

cue-induced reinstatement of nicotine-seeking. The same effect was observed upon infusions of

SB277011-A into the basolateral amygdala or the lateral habenula, but not the nucleus

accumbens. The current findings implicate DRD3 in cue-induced reinstatement of nicotine,

delineate some of the neural substrates underlying this role and support a potential for using

selective DRD3 antagonists for the prevention of relapse to smoking.

iii

ACKNOWLEDGEMENTS

My thanks and gratitude are greatest and foremost to Allah for giving me faith in

everything I do, strength to overcome the hardships, and will to pursue my work and to

see it to completion. I only hope the knowledge and skills I earned throughout the years

of my studies will be soon employed towards helping others whether as a doctor, a

scientist or a human being, and that I will always be contributing to knowledge in one

way or another. May Allah be pleased with my efforts and intensions and reward them

with fulfilment and satisfaction.

Next, I would like to thank my parents, to whom I am forever indebted with

everything I am and everything I will be. Thank you for raising me on principles and

morals and for planting in me the seeds of conscience, respect and perseverance. Thank

you for supporting me across the distance and guiding me when things seemed to reach a

dead end. I would’ve never been where I am today had it not been for you and your

never-fading confidence in me.

I cannot even begin to thank my incredible husband, Mohamed, who has always

been my companion and my family away from home. Thank you for being there for

every step of the way. Thank you for putting up with me through exam nights, seminars,

interviews and next day assignments. Thank you for the critique, comments and

mathematical rescues. Thank you for your support and trust and for always saying, “I told

you: you can do it”. Thank you for everything. May we always keep our pledge to push

each other to the top.

I would like to deeply thank my supervisor Dr. Bernard Le Foll. His knowledge,

support and guidance were everything that I needed in order to see this work to

completion, his insight and valuable suggestions were a great help in pulling things

together and his confidence and continuous encouragement were a motivation for me to

always do my best. I would also like to thank my co-supervisor, Dr. Anh Le, for his

support, comments and criticism and for the valuable feedback he gave me all through

iv

the course of my study. Special thanks goes to Dr. Larry Grupp, my M.Sc. advisor, for

his kind advice and support, for always having the time and patience to carefully listen to

my concerns and for providing encouragement, guidance and suggestions.

Thanks to all of my lab colleagues and lab managers (current and previous). You

have all made the lab environment very friendly and productive. We all helped and taught

each other a lot of things, and we still are. Working with you all has always been a real

pleasure.

Lastly, I would like to dedicate this thesis to my 3 year old son Zeyad. Thank you

for blessing my books and notes with your scribbling and for always pressing the “Send”

tab on my important emails. Thank you for coming to me when I am frowning and in

deep thought to say “Mommy, are you sad? What happened, mommy?” Thank you for

teaching me things no one else ever did, and for teaching me that I can push myself to the

limit and do more things than I ever thought possible.

I would also like to dedicate this thesis to my newly born baby boy Tareq who

just arrived to our world shortly after my oral defense exam. He has been with me during

the last and critical phases of my MSc studies and I am sure has endured much in the

process. May Allah bless him and his brother and grant them good health and success for

all their lives.

v

TABLE OF CONTENTS

ABSTRACT..................................................................................................................................ii ACKNOWLEDGEMENTS........................................................................................................iii TABLE OF CONTENTS.............................................................................................................v LIST OF TABLES.....................................................................................................................viii LIST OF FIGURES.....................................................................................................................ix LIST OF ABBREVIATIONS......................................................................................................x LIST OF APPENDICES.............................................................................................................xi

Chapter 1 INTRODUCTION ................................................................................................... 1

1.1 Statement of the Problem................................................................................................... 1

1.2 Overall Purpose and Objectives ........................................................................................ 2

1.3 Rationale and Hypotheses.................................................................................................. 4

1.4 Review of the Literature .................................................................................................... 7 1.4.1 Smoking and Nicotine Dependence........................................................................... 7 1.4.2 Animal Models of Drug-seeking and Drug-taking Behaviours ............................... 11 1.4.3 Neurocircuitry of Nicotine Reinforcement and Reward .......................................... 18 1.4.4 Craving and Cue-induced Relapse........................................................................... 19 1.4.5 Neurocircuitry of Cue-induced Relapse – Role of the BLA and NAcc .................. 21 1.4.6 The Mesocorticolimbic Dopamine System ............................................................. 25 1.4.7 Dopaminergic Receptors ......................................................................................... 27

1.4.7.1 The Dopamine D1 Receptor (DRD1) ................................................................ 28 1.4.7.2 The Dopamine D2 Receptor (DRD2) ............................................................... 29 1.4.7.3 The Dopamine D4 Receptor (DRD4) ............................................................... 30 1.4.7.4 The Dopamine D5 Receptor (DRD5) ............................................................... 30 1.4.7.5 The Dopamine D3 Receptor (DRD3) ............................................................... 31

1.5 Restatement of the Hypotheses ........................................................................................ 38

Chapter 2 METHODOLOGY ................................................................................................ 39

2.1 Animals ............................................................................................................................ 39

2.2 Apparatus ......................................................................................................................... 39

2.3 Experimental procedures ................................................................................................. 40 2.3.1 Experiment (1): Systemic Administration of SB 277011-A and Cue-induced Reinstatement of Nicotine-seeking Behaviour .................................................................... 40

2.3.1.1 Food Training .................................................................................................. 40 2.3.1.2 Intravenous Catheter Implantation Surgery ..................................................... 40 2.3.1.3 Nicotine Intravenous Self-administration (IVSA) Procedures ........................ 41

vi

2.3.1.4 Extinction Training and Cue-induced Reinstatement of Nicotine-seeking ..... 42 2.3.2 Experiments (2-4): Local Infusion of SB 277011-A into Discrete Brain Areas and Cue-induced Reinstatement of Nicotine-seeking Behaviour ............................................... 44

2.3.2.1 Food Training .................................................................................................. 44 2.3.2.2 Intravenous Catheter Implantation Surgery ..................................................... 44 2.3.2.3 Nicotine Self-administration Procedures ......................................................... 44 2.3.2.4 Intracranial Cannula Implantation Surgery ..................................................... 44 2.3.2.5 Procedures for Local Infusion of SB 277011-A and Cue-induced reinstatement of Nicotine-seeking .......................................................................................................... 45

2.3.3 Experiment (5): Local Infusion of SB 277011-A into the Basolateral Amygdala and Food-taking Behaviour ........................................................................................................ 48 2.3.4 Experiment (6): Responding Maintained by Light Cues ......................................... 48

2.4 Drugs ............................................................................................................................... 49

2.5 Histological Examination ................................................................................................ 50

2.6 Data Analysis ................................................................................................................... 50

Chapter 3 RESULTS ............................................................................................................... 51

3.1 Experiment (1) Effect of SB 277011-A (1, 3, 10 mg/kg i.p.) on cue-induced reinstatement of nicotine-seeking ............................................................................................ 51

3.2 Experiment (2): Effect of Local Infusion of SB 277011-A into the BLA on Cue-induced Reinstatement of Nicotine-seeking Behaviour ........................................................................ 54

3.3 Experiment (3): Effect of Local Infusion of SB 277011-A into the NAcc on Cue-induced Reinstatement of Nicotine-seeking Behaviour ........................................................................ 56

3.4 Experiment (4): Effect of Local Infusion of SB 277011-A into the LHb on Cue-induced Reinstatement of Nicotine-seeking Behaviour ........................................................................ 58

3.5 Experiment (5): Effect of Local Infusion of SB 277011-A into the BLA on Food-taking Behaviour ................................................................................................................................. 60

3.6 Histological Examination (Experiments 2-5): ................................................................. 63

3.7 Experiment (6): Responding maintained by light stimuli only ....................................... 67

3.8 Sample Size ..................................................................................................................... 68

Chapter 4 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS .................... 69

4.1 General Discussion .......................................................................................................... 69 4.1.1 Systemic Administration of SB 277011-A Attenuates Cue-induced Reinstatement of Nicotine-seeking Behaviour ............................................................................................ 70 4.1.2 Local Infusions of the DRD3 Antagonist SB 277011-A .......................................... 73

4.1.2.1 DRD3 Antagonism in the BLA, Abolishes Cue-induced Reinstatement of Nicotine-seeking Behaviour, but Has No Effect on Food Intake. ................................... 74

vii

4.1.2.2 DRD3 Antagonism in the NAcc Has No Effect on Cue-induced Reinstatement of Nicotine-seeking Behaviour ........................................................................................ 81 4.1.2.3 DRD3 Antagonism in the LHb Attenuates Cue-induced Reinstatement of Nicotine-seeking Behaviour ............................................................................................ 85

4.1.3 Cue Light per se is Unable to Maintain Active Lever Pressing Under a Fixed Ratio Schedule ............................................................................................................................... 90

4.2 Conclusions and Summary .............................................................................................. 92

4.3 Future recommendations ................................................................................................. 94 4.3.1 DRD3 Antagonism and Stress-induced Reinstatement of Nicotine-seeking Behaviour. ............................................................................................................................ 94 4.3.2 Investigating the Effect of Modulating the Function of the LHb DRD3 on Other Aspects of Nicotine-seeking Behaviour .............................................................................. 94 4.3.3 Effect of a DRD3 Agonist on Nicotine Taking and Reinstatement ......................... 95 4.3.4 Investigating the Effect of Intracranial Administration of DRD3 Selective Antagonists on Nicotine-priming-induced Reinstatement of Nicotine-seeking Behaviour. 95 4.3.5 Use of DRD3 Knock-out Mice to Confirm the Selectivity of SB 277011-A ........... 95 4.3.6 Replication of the Current Study Using Other Selective DRD3 Ligands to Confirm the Results ............................................................................................................................ 96

REFERENCES...........................................................................................................................97

LIST OF PUBLICATIONS AND ABSTRACTS ....................... ..........................................113

APPENDICES...........................................................................................................................114

viii

LIST OF TABLES

Table Title Page Table 1.1

Examples of animal models for the study of drug-seeking / drug-taking behaviours

13

Table 1.2

The differences between the dopamine receptor subtypes

27

Table 1.3

The role of DRD3 partial agonists and antagonists in different aspects of drug seeking and drug taking behaviours

34 Table 3.1

The number of rats excluded from the current study and reasons for exclusion

68

ix

LIST OF FIGURES

Table Title Page Figure 2.1

Schematic Diagram for the Experimental Procedures Performed in Experiment 1

43

Figure 2.2

Schematic Diagram for the Experimental Procedures Performed in Experiments 2-4

47

Figure 2.3

Structure of SB 277011-A

49

Figure 3.1

Acquisition / Extinction Phases of Nicotine Self-Administration

52

Figure 3.2

Systemic Administration of SB 277011-A significantly attenuates cue-induced reinstatement of nicotine-seeking behaviour

53

Figure 3.3

Intra-BLA Administration of SB 277011-A abolishes cue-induced reinstatement of nicotine-seeking behaviour

55

Figure 3.4

Intra-NAcc Administration of SB 277011-A Has No Effect on Cue-induced Reinstatement of Nicotine-seeking Behaviour

57

Figure 3.5

Intra-LHb Administration of SB 277011-A attenuates cue-induced reinstatement of nicotine-seeking behaviour

59

Figure 3.6

Acquisition Phase of Food Self-Administration

61

Figure 3.7

Intra-BLA Administration of SB 277011-A Has No Effect on Food-taking Behaviour

62

Figure 3.8

Cannulae Placement in the BLA

64

Figure 3.9

Cannulae Placement in the NAcc

65

Figure 3.10

Cannulae Placement in the LHb

66

Figure 3.11

Cue-lights per se are Unable to Maintain Stable Responding

67

x

LIST OF ABBREVIATIONS

BLA Basolateral Amygdala

BSR Brain Stimulation Reward

CeA Central Nucleus of the Amygdala

CPA Conditioned Place Aversion

CPP Conditioned Place Preference

CS Conditioned Stimulus / Stimuli

DRD1 - 5 Dopamine D1 - 5 Receptors

DS Discriminative Stimulus

DSM IV Diagnostic and Statistical Manual of Mental Disorders, 4th Edition.

FR Fixed Ratio

IC Intracranial

ICSS Intracranial Self Stimulation

i.p. Intraperitoneal

IV Intravenous

IVSA Intravenous Self-Administration

LHb Lateral Habenula

NAcc Nucleus Accumbens

NRT Nicotine Replacement Therapy

PR Progressive Ratio

SA Self-Administration

s.c. Subcutaneous

VTA Ventral Tegmental Area

WHO World Health Organisation

xi

LIST OF APPENDICES

Appendix Title Page Appendix I

Copyright Release Form

115

Appendix II

Standard Operating Procedure for Intravenous Catheterization Surgery

117

Appendix III

Standard Operating Procedure for Intracranial Cannulae Implantation Surgery

122

1

Chapter 1 INTRODUCTION

1.1 Statement of the Problem

Tobacco smoking continues to comprise a major health problem in a growing population

all over the world and is considered the world single most preventable cause of death

(WHO/WPRO, 2006). In addition, tobacco smoking is considered an addictive disorder, as per

the Diagnostic and Statistical Manual of Mental disorders, 4th edition (DSM-IV) (American

Psychiatric Association, 2000). A high percentage of the attempts to quit, whether self-directed

or treatment-guided, are reportedly followed by relapse (Hughes, Gulliver et al., 1992; Hunt,

Barnett et al., 1971). Indeed, several factors can trigger relapse to smoking including exposure

to the environmental stimuli previously associated with the smoking (drug-associated cues),

stress, or re-exposure to nicotine (Brandon, Tiffany et al., 1990; Kassel, Stroud et al., 2003;

O'Brien, Childress et al., 1992; Wikler, 1973).

Therefore, it can be concluded that currently available smoking cessation treatments

(such as nicotine replacement therapy, bupropion or varenicline) lack a satisfactory outcome in

part because they have exhibited a limited ability to prevent relapse to smoking. Consequently,

there is a strong need for a better understanding of the neural mechanisms underlying relapse

and for the development of therapies to target these specific mechanisms and pathways, in order

to prevent relapse to smoking.

Several lines of evidence implicate the dopaminergic system, and particularly the

dopamine D3 receptors (DRD3), in the processes underlying relapse to drug seeking and drug-

cue-associations (Heidbreder, Gardner et al., 2005; Le Foll, Goldberg et al., 2005). The DRD3

2

are expressed in areas in the brain strongly implicated in the mechanisms underlying drug

reinforcement and stimulus reward associations (Bouthenet, Souil et al., 1991; Diaz, Lévesque

et al., 1995; Diaz, Pilon et al., 2000; Le Foll, Diaz et al., 2003). However, no studies have

addressed the role of DRD3 in cue-induced reinstatement of nicotine seeking, or investigated the

neural pathways underlying such a role. The current availability of compounds that are highly

selective to the DRD3, such as SB 277011-A, PG 01037 and BP 897, facilitates further

investigation of the role of this receptor in different aspects of nicotine seeking behaviour

(Heidbreder and Newman, 2010; Mason, Hassan et al., 2010; Pilla, Perachon et al., 1999;

Reavill, Taylor et al., 2000).

1.2 Overall Purpose and Objectives

The purpose of the current study is to investigate the role of the DRD3 in cue-induced

reinstatement of nicotine-seeking behaviour in rats. To this end, four experiments were

conducted, using the nicotine self-administration / reinstatement paradigm, to assess the effect

of the administration of a highly selective DRD3 antagonist (SB 277011-A), both systemically

and by local infusions into discrete brain areas, on cue-induced reinstatement of nicotine-

seeking behaviour in rats. The effect of local antagonism of the DRD3, in the basolateral

amygdala on food taking behaviour was also assessed.

In a separate experiment, the ability of cue-lights per se to maintain operant responding when

un-associated with the administration of nicotine was investigated.

3

The objectives of the current study can be summarized as follows:

Primary objectives:

1. Evaluating the effect of systemically blocking the DRD3, using the selective DRD3

antagonist SB 277011-A, on cue-induced reinstatement of nicotine-seeking behaviour in

rats.

2. Delineating the neural substrates through which the above effect of DRD3 is mediated. The

following areas are investigated: the basolateral amygdala (BLA), the nucleus accumbens

(NAcc) and the lateral habenula (LHb).

Secondary Objectives:

1. Investigating the effect of blocking the DRD3 in the BLA on food-taking behaviour. This

was done to exclude non-specific effects of intra-BLA infusion of SB 277011-A on operant

behaviour.

2. Investigating the motivational / reinforcing effects of cue-lights per se, and determining

whether the cue-induced reinstatement occurs due to previous association of the cue-lights

with the infusions of nicotine, or partially due to primary reinforcing effects of the cue-

lights themselves.

4

1.3 Rationale and Hypotheses

Systemic antagonism of the DRD3 results in the attenuation of cue-induced reinstatement

of nicotine-seeking behaviour.

Hypothesis I:

Rationale:

A strong body of evidence supports a role for the DRD3 in stimulus-reward associations.

Several studies have suggested that DRD3 antagonism can be effective in decreasing the

influence of conditioned-environmental stimuli on drug-seeking behaviour, and thereby

reducing the tendency to relapse (Heidbreder, Gardner et al., 2005; Le Foll, Goldberg et al.,

2007).

The effect of the DRD3 on cue-induced reinstatement is mediated through the BLA and the

LHb, but not through the NAcc.

Hypothesis II

Rationale:

a) The BLA is clearly involved in conditioned reinforcement processes. Several studies,

clinical and behavioural, have indicated a strong role for the BLA in the processes

underlying cue-associations (Everitt, Parkinson et al., 1999). Moreover, the DRD3 in the

BLA have been found to play a role in cocaine seeking that is maintained by conditioned

stimuli (Di Ciano, 2008).

b) The NAcc is an important structure in the brain reward circuitry with an established role in

the mechanisms underlying drug seeking, reinforcement and reward (Corrigall, Franklin et

5

al., 1992; Pontieri, Tanda et al., 1996). Furthermore, the NAcc is one of the areas exhibiting

the highest density of expression of the DRD3 (Diaz, Lévesque et al., 1995). The NAcc

DRD3 have been shown be involved in stress-induced reinstatement of cocaine seeking (Xi,

Gilbert et al., 2004). However, the current study hypothesizes a limited role for the NAcc

DRD3 in the modulation of cue-induced reinstatement of nicotine-seeking. This hypothesis

is based on various studies indicating dissociation in the role the NAcc plays in primary

reinforcement processes and in cue-associations (Grimm and See, 2000).

c) The LHb has been implicated in the mechanisms underlying reward and learning

(Lecourtier and Kelly, 2007; Morissette and Boye, 2008). The role of the LHb in these

mechanisms is thought to be mediated through an interaction with the dopamine neurons in

the ventral tegmental area (VTA) (Herkenham and Nauta, 1979). The expression of the

DRD3 in the LHb (Diaz, Pilon et al., 2000) renders such interaction even more interesting.

The current study sought to uncover the role of the DRD3 in the LHb in cue-induced

reinstatement of nicotine seeking behaviour, and therefore to provide a better understanding

of the neuropharmacology of this area.

Antagonism of the DRD3 in the BLA has no effect on food taking behaviour.

Hypothesis III

Rationale:

Several factors can affect operant responding in general. A significant decrease in

operant responding is not always specific to behavioural changes as a result of administration of

a certain drug, but could be attributed to other non-specific effects such as: sedation,

hypolocomotion, anhedonia for reward in general or even an effect on learning the operant

behaviour. In order to control for such non-specific effects of the infusion of the DRD3

6

antagonist SB 277011-A, or its vehicle, into the BLA, it is crucial to examine operant responses

other than cue-induced reinstatement and reinforcers other than nicotine. Food-taking behaviour

was chosen as a behavioural response to be assessed as a measure of behavioural and operant

responses after local infusion of SB 277011-A into the BLA.

Cue lights per se, not – previously – associated with nicotine administration, have no

primary reinforcing effect that is strong enough to maintain stable responding.

Hypothesis IV

Rationale:

In drug intravenous self-administration paradigms, a conditioned stimulus (CS) (initially

neutral in itself) is often paired with the drug infusions, thereby gaining motivational salience

and becoming a conditioned (secondary) reinforcer. An important feature of such a CS is its

ability to reinforce drug-seeking response even in the absence of the primary reinforcer

(O'Brien, Childress et al., 1992; Wikler, 1973), i.e. the ability to reinstate extinguished drug-

seeking behaviour. The current experiment sought to verify that such ability of CS (cue-lights in

this case) is due to former association with the primary reinforcer (nicotine) and not due to

inherently salient or primary reinforcing properties of the cue light itself.

7

1.4 Review of the Literature

1.4.1 Smoking and Nicotine Dependence

Tobacco smoking comprises a major health problem world wide. Smoking is considered

the world’s most preventable cause of death, where around 50% of the estimated smokers (650

million out of 1.3 billion smokers estimated in 2006) are subject to premature death caused by

tobacco-related diseases. At the current rate of smoking, the number of smokers is expected to

reach 1.7 billion in 2025 (accounting for at least 10 million deaths / year) (WHO/WPRO, 2006).

In Canada, almost one fifth (17%) of Canadians aged 15 and older are current smokers,

as per Health Canada’s “Canadian Tobacco Use Monitoring Survey” (CTUMS, 2009).

Moreover, tobacco smoking comprises a disease burden as it is responsible for a large number

of fatal and disabling diseases; these include lung cancer, chronic bronchitis, emphysema and

ischemic heart diseases.

In addition to the high mortality rate and disease burden, tobacco smoking is considered

an addictive disorder when it fulfils the definition and the criteria for substance / drug

dependence as per the DSM-IV (American Psychiatric Association, 2000). According to this

definition, smoking is considered a form of drug dependence where it follows “A maladaptive

pattern of substance use, leading to clinically significant impairment or distress”. Some of the

criteria of drug dependence that are most commonly evident for nicotine include:

a) the appearance of withdrawal symptoms upon cessation of smoking (such as: irritability,

depressed mood, difficulty in concentration and anxiety); relief or avoidance of such symptoms

can be achieved by smoking (Hughes, Gust et al., 1991), b) persistent failure of trials to quit

and, c) continued use despite the knowledge of possible harm occurring due to nicotine

consumption.

8

Tobacco smoke consists of over 400 chemical components including nicotine, carbon

monoxide and tar. Many of these components have carcinogenic potential, and although they

can all contribute to the addictive properties of tobacco, nicotine is considered the primary

psychoactive as well as the main addictive component of tobacco (Dalhamn, 1972; Hoffmann,

Adams et al., 1979; Hoffmann, Rivenson et al., 1979; Stolerman and Shoaib, 1991).

Acute administration of nicotine produces of a wide range of pharmacological and

psychological effects, contributing to the addictive properties of nicotine. Nicotine produces an

increase in heart rate, blood pressure and epinephrine and cortisol levels (Stolerman and Jarvis,

1995). Smoking also produces various pleasurable pharmacological effects, such as mild

euphoria, increased concentration and heightened alertness (Etter, Bergman et al., 2000;

Pomerleau and Pomerleau, 1992; Stolerman and Jarvis, 1995). These effects give nicotine the

ability to reinforce and maintain nicotine-seeking behaviour; i.e. positive reinforcing effects.

The reinforcing properties of nicotine have been demonstrated in several studies using animal

models, where nicotine has been shown to maintain stable self-administration, induce place

preference and lower brain reward threshold (thus inducing brain stimulation reward) (Corrigall

and Coen, 1989; Fudala, Teoh et al., 1985; Goldberg and Henningfield, 1988). Negative

reinforcement is also considered an important factor in the motivational effects of nicotine, as

has been demonstrated in humans (Hughes, Gust et al., 1991) and in rats (Epping-Jordan,

Watkins et al., 1998). In humans, withdrawal from nicotine produces undesirable symptoms;

such as anxiety, irritability and frustration (Hughes, Gust et al., 1991). These negative symptoms

are likely to result in the motivation to seek nicotine and, thus, to increase nicotine seeking

behaviour (i.e. negative reinforcement). Animal studies have shown similar somatic symptoms

to occur in rats following withdrawal from nicotine, whether spontaneous or precipitated

(Epping-Jordan, Watkins et al., 1998; Malin, Lake et al., 1992). Moreover, withdrawal from

9

nicotine has been shown to markedly elevate brain reward thresholds (Epping-Jordan, Watkins

et al., 1998).

It is important to note, however, that although the reinforcing effects of nicotine are

important for the initiation and maintenance of nicotine-seeking behaviour, these factors are not

the only factors in the process of the development of nicotine dependence. Other factors, like

smoking conditioning properties, contribute to the smoking behaviour (Caggiula, Donny et al.,

2002b; Shahan, Bickel et al., 1999), as will be discussed in detail below.

Another major feature of nicotine dependence is relapse to smoking. Relapse can be

defined as the return of nicotine-seeking / nicotine-taking behaviour after a period of abstinence

(See, Fuchs et al., 2003). Although a large percentage of smokers indicate their wish to quit

smoking and actually try to quit smoking, the rates of relapse remain very high (Fiore, 2000)

(Hunt, Barnett et al., 1971), and the long-term abstinence rates (6 months) remain less than 10%

(Hughes, Gulliver et al., 1992). Several factors can predispose to the relapse to smoking,

including stress (Kassel, Stroud et al., 2003), re-exposure to nicotine (Brandon, Tiffany et al.,

1990) and exposure to the environmental stimuli (cues) that were previously associated with

smoking (O'Brien, Childress et al., 1992; Wikler, 1973). Cue-induced relapse is the main

interest in the current study and has been addressed by the work demonstrated throughout the

present thesis.

As defined by See and colleagues, craving is “the extensive desire for a specific object or

experience”, and can be considered the primary factor motivating relapse (See, Fuchs et al.,

2003). In humans, the exposure to stimuli reliably associated with different drugs of abuse has

been shown to induce cravings and increased motivation towards drug seeking (Carter and

Tiffany, 1999; Childress, 1993; O'Brien, Childress et al., 1992; O'Brien and McLellan, 1996).

Moreover, the exposure to smoking-related cues causes regional activation in the brains of

10

abstinent human smokers (Goudriaan, De Ruiter et al., 2010). Numerous studies have indicated

a similar effect in animals, where the reintroduction of conditioned stimuli previously associated

with the intake of the drug has been shown to significantly reinstate drug-seeking behaviour

after its extinction in their absence (Davis and Smith, 1976; See, Grimm et al., 1999).

Currently, a few medications are available as aids for smoking cessation. However,

several limitations exist to the effective use of these medications in smoking cessation therapy

and prevention of relapse. Nicotine replacement therapy (NRT), bupropion and varenicline

constitute the fist line of currently available smoking cessation therapies.

- NRT is available as nicotine gum, patch, spray, inhaler or lozenge. This line of treatment is

effective in alleviating withdrawal symptoms; however, its efficacy in decreasing the

reinforcing effects of nicotine remains limited (Moolchan, Robinson et al., 2005).

- Bupropion, which was originally used as an antidepressant, has been shown to decrease the

reinforcing effects of nicotine and to alleviate withdrawal symptoms. However, several side

effects of bupropion should be taken into consideration, such as increased risk of seizures and

depression (Mooney and Sofuoglu, 2006).

- Varenicline is an α4β2 nicotinic receptor partial agonist, which improves withdrawal

symptoms and mood in abstinent smokers (Rollema, Chambers et al., 2007). However, in

some cases, varenicline use has been reportedly associated with suicidal thoughts and

depression.

The fact that long term abstinence rates remain low and relapse rates remain high (as

mentioned above), despite the current availability of therapeutic agents for smoking cessation,

indicates the limited success of the currently available medications. In other words, the currently

available therapies may be either less specific or less effective than it is to be hoped for.

Investigation of novel therapeutic agents should, therefore, take into account an extensive

understanding of the neurobiological mechanisms of nicotine dependence and relapse and

11

specifically target these mechanisms and pathways. Such understanding can be reached by the

integration of different research approaches and models including preclinical models, genetic

studies and clinical trials.

In the current study, an animal model of nicotine self-administration / reinstatement has

been utilized in order to assess the role of the DRD3 in cue-induced relapse to nicotine seeking.

To better understand the parameters and utility of the paradigm used in the current study, an

overview of the animal models used in studying various aspects of drug-taking / drug-seeking

behaviour is covered in the next section. This will be followed by a review of the neurological

mechanisms of nicotine dependence and cue-induced relapse. Subsequently, a detailed literature

review will be provided highlighting the studies investigating the role of the dopaminergic

system and receptors in substance dependence in general, with a special focus on nicotine.

1.4.2 Animal Models of Drug-seeking and Drug-taking Behaviours

Before proceeding with a background on the specific mechanisms involved in nicotine

dependence and cue-induced relapse, it is important to discuss one of the established approaches

in studying drug-seeking / drug-taking behaviours, and one which was also used in the current

study, i.e. animal modelling. Although it may be difficult to mimic the entire aspects of a certain

human condition in animal models, the utility of such models remains substantial for

understanding specific mechanisms underlying this condition. For many psychiatric disorders,

as well as drug addiction, the use of animal models has long been established as a measure of

specific signs and symptoms or certain behaviours related to psychiatric disorders and / or

substance use (Markou, Weiss et al., 1993). Thus, animal models gain both construct and

predictive validity not only providing a better understanding of these complex disorders, but

12

also allowing the investigation of the potential utility of different pharmacological substances as

therapeutic agents for these disorders (Ebel, 1961; Willner, 1984).

Different methods can be used to classify animal models used for studying drug use, as

in regards of the behaviour they model, the property of the drugs of abuse they evaluate or the

type of validity they exhibit, etc.

Animal models have been developed to mimic the different stages of progression of drug

use from the stage of drug taking to withdrawal to relapse and craving. For purposes of

simplification, these stages will be used to highlight some examples of the animal models for

studying drug taking / drug seeking behaviours in Table 1.1.

13

Table 1.1 Examples of animal models for the study of drug-seeking / drug-taking behaviours

Stage of drug-taking behaviour

Examples of animal models Utility References

Models of Drug

Taking

* Drug self-administration

- Fixed ratio schedule (FR)

- Progressive ratio schedule (PR)

- Second order schedule

* Assessment of the primary reinforcing / rewarding effects of drugs. * Measures

- Primary reinforcing effects,

- Reinforcing efficacy and,

- Motivational effects of the drugs.

(Caine and Koob, 1993; Collins, Weeks et al., 1984; Schindler, Panlilio et al., 2002)

* Brain stimulation reward (BSR)

* Assessment of the reward value.

(Kornetsky and Esposito, 1979)

* Conditioned place preference (CPP)

* Assessment of primary reward and the conditioning of drug reinforcement.

(van der Kooy, 1987)

* Drug discrimination * Assessment of the interoceptive cue state produced by the drug.

(Holtzman, 1985)

Models of

Craving and

Relapse

* Resistance to extinction following self-administration

*Measures motivational effects of the drug.

(Schuster and Woods, 1968)

*Reinstatement models: - Drug-induced - Cue-induced - Stress-induced

* Used to assess the effect of different factors on drug-seeking behaviour in the absence of contingent drug administration. * Measure

- Motivational effects of the drug or the conditioned stimuli associated with the drug.

- Conditioning properties of the drug.

- The effect of stress on drug-seeking behaviour.

(Erb, Shaham et al., 1996; Gerber and Stretch, 1975; Schuster and Woods, 1968)

14

Table 1.1 (Continued) Examples of animal models for the study of drug-seeking / drug-taking behaviours

Stage of drug-taking behaviour

Examples of animal models Utility References

Models of Craving and

Relapse (Continued)

* Second order schedules of reinforcement

* Measure drug-seeking behaviour that is maintained by the presentation of drug-associated cues.

(Everitt and Robbins, 2000; Schindler, Panlilio et al., 2002)

Models of

Withdrawal

* Conditioned place aversion (CPA) * Intracranial self-stimulation (ICSS)

* Assessment of the aversive effects of withdrawal. * Assessment of the changes in the reward circuitry over the course of the addiction process. * Understanding the neurobiological mechanisms of the drugs of abuse, and the counter-adaptive mechanisms that drive the addiction process.

(Kornetsky and Esposito, 1979; Stinus, Le Moal et al., 1990) (Holtzman, 1985)

Reinstatement models have been developed primarily to assess the ability of different

factors to trigger reinstatement of an extinguished drug-seeking behaviour in abstinent animals,

and have since been used to study the neurobiological mechanisms underlying the relapse

phenomenon. Despite some procedural differences between the animal models of reinstatement

and the actual situation of relapse in humans, these models have shown a strong validity for the

study of relapse processes, over time. This validity is exhibited by the correspondence between

the outcomes of different studies assessing triggers of relapse to drug seeking after a period of

withdrawal (Breiter, Gollub et al., 1997; Shiffman, Paty et al., 1996; Sinha, 2001), also see

(Shaham, Shalev et al., 2003) for a review on reinstatement models.

15

Since the current study is mainly dealing with cue-induced relapse to nicotine seeking, it

is important to review the different types of cue-reinstatement, as well as other methods that are

used for studying the conditioning properties of drugs and conditioned reinforcement in general.

Different types of modelling of conditioned stimuli have been described in the literature.

One of the earliest types of conditioned stimuli to be developed and the most commonly

used in animal models is the discrete conditioned stimulus (CS), where the administration of

the drug is accompanied by the contingent presentation of a discrete cue, commonly an auditory

or a visual stimulus. Such association not only results in an increase in the level of responding of

the animals to the administered drug, but also imparts motivational salience onto the CS

(Lesage, Burroughs et al., 2004). The CS, therefore, becomes a conditioned reinforcer able to

reinstate extinguished drug-seeking behaviour in the absence of the drug as the primary

reinforcer (Davis and Smith, 1976). The reinforcing properties of the CS seem to be dependent

upon contingency of their presentation with the administration of the drug where non-contingent

presentation of the CS was found to have little effect on the reinstatement of drug-seeking

behaviour (de Wit and Stewart, 1981; Di Ciano, Underwood et al., 2003; Fuchs, Tran-Nguyen et

al., 1998; Grimm and See, 2000).

Discriminative stimuli (DS) are a different type of conditioned stimuli that were more

recently used in cue-reinstatement models. The presentation of the DS (which could be a tone or

a light) precedes the administration of the drug and predicts the availability of the drug

(Ettenberg, MacConell et al., 1996; Weiss, Maldonado-Vlaar et al., 2000). The reintroduction of

the DS was shown to reliably reinstate extinguished drug seeking behaviour (Gracy, Dankiewicz

et al., 2000; McFarland and Ettenberg, 1997). Interestingly, unlike discrete conditioned stimuli

(CS), contingency of DS presentation with the responding was found to be not critically

important for influencing drug-seeking behaviour (Di Ciano, Underwood et al., 2003).

16

Another method used for modelling the drug-associated-stimuli-induced reinstatement of

drug-seeking behaviour is the context-induced reinstatement, where context refers to the

physical environment of the test chambers. In this paradigm drug-seeking behaviour is

extinguished in a context that is different from the one where the drug had been administered.

Upon returning the animals to the original drug-associated context, the drug-seeking behaviour

was found to be significantly reinstated (Crombag, Grimm et al., 2002).

Cue-induced reinstatement models are not the only method used to assess the

conditioning properties of drugs or stimulus-reward associations. Another paradigm that is

commonly used for this purpose is the appetitive pavlovian conditioning paradigm. In this

paradigm behavioural autoshaping is first established where the presentation of a stimulus (for

example: visual) precedes the delivery of reward (usually food), in a different location within

the test chamber. This presentation is non-contingent upon the animal’s responding. As a result,

the animal develops a conditioned response of approaching the stimulus predictive of the

reward, prior to collecting the reward itself. Pavlovian to instrumental transfer can then be

performed where the delivery of the reward becomes contingent upon the animal’s responding,

thereby concerning active behaviour and its consequences (Bussey, Everitt et al., 1997). This

paradigm can be used, not only to assess the conditioning properties of different drugs, but also

to study the different mechanisms and pathways regulating different stages of cue-association

learning (Everitt, Parkinson et al., 1999).

Second order schedules of reinforcement have also been used to assess the

conditioning properties of drugs through measuring the level of responding to the self-

administered drug which is maintained by the presentation of drug-associated cues (Arroyo,

Markou et al., 1999; Spear, Muntaner et al., 1991). Briefly, in this paradigm, a brief (usually

visual) stimulus is presented upon completion of a component or unit of the schedule (for

17

example a certain number of lever presses or nose pokes i.e. [FRy:S]). Upon completion of the

full schedule (after a certain number of the above responses; i.e. FRx[FRy:S]), the stimulus is

introduced together with the drug (primary reinforcer). Testing can be performed in two stages;

a drug free interval (where the responding is maintained by the presentation of the stimulus

alone) and a second interval where drug is available.

It is important to note that the method of presentation of the conditioned stimuli may

vary in the above models and that the above procedures may seem to be modelling different

aspects of the stimulus-reward association process (i.e. discrete CS vs. contextual cues vs.

pavlovian conditioning, etc.). The reinstatement triggered by each type of the above stimuli may

even be mediated through different neuronal substrates within the brain stimulus-reward

circuitry. Nonetheless, the integrated knowledge of the data collected using all of these

paradigms remains crucial for further understanding the different mechanisms underlying the

conditioning process and, therefore, cue-induced relapse.

In the current study, a discrete conditioned stimulus (cue-light) was used as a

conditioned reinforcer, where self-administered nicotine infusions were accompanied by the

contingent presentation of a cue-light under a fixed ratio schedule of reinforcement. After

extinction of the nicotine self-administration behaviour (in the absence of both nicotine and the

cue-light), the cue-light was reintroduced to achieve reinstatement of nicotine-seeking

behaviour. The effect of DRD3 antagonism (systemic and local) on cue-induced reinstatement of

nicotine-seeking behaviour was then assessed.

18

1.4.3 Neurocircuitry of Nicotine Reinforcement and Reward

Although the work in the current thesis mainly addresses conditioning properties of

nicotine and cue-induced reinstatement of nicotine seeking, it remains of critical importance to

highlight the basics of the neurocircuitry underlying the primary reinforcing properties of

nicotine, and the mechanisms regulating nicotine seeking behaviour.

Nicotine exerts its actions through binding to nicotinic acetylcholine receptors (nAChR)

which are expressed peripherally as well as centrally in the brain. Specifically, activation of the

nAChR is critically involved in the reinforcing actions of nicotine, where the administration of

nAChR antagonist has been shown to block self-administration of nicotine (Corrigall, Coen et

al., 1994; Corrigall, Franklin et al., 1992). The neurocircuitry involved in mediating the

reinforcing effects of nicotine appears to be complex, and to involve integrating multiple

neurochemical systems. However, the mesolimbic dopamine system is believed to be the main

system implicated in mediating the reinforcing / rewarding effects of nicotine. This is supported

by several lines of evidence, first, the nAChR are densely expressed in the VTA, which is the

area containing the cell bodies of the dopamine neurons, as well as the NAcc, at the terminals of

the dopamine neurons (Clarke and Pert, 1985; Schwartz, Lehmann et al., 1984). Second,

nicotine administration increases the firing of dopamine neurons in the VTA (Clarke and Pert,

1985; Pidoplichko, DeBiasi et al., 1997). Third, activation of the nAChR by nicotine

(systemically administered or locally infused in the VTA or NAcc) induces dopamine release

(Nisell, Nomikos et al., 1994). This action was antagonised by concomitant intra-VTA, but not

intra-NAcc, infusions of the nicotinic antagonist mecamylamine (Nisell, Nomikos et al., 1994).

It is worth mentioning that this effect of nicotine on dopamine release is a common effect shared

by different drugs of abuse (Pontieri, Tanda et al., 1996). Furthermore, studies have shown that

19

dopaminergic lesions, by the infusion of 6-hydroxydopamine in the NAcc (Corrigall, Franklin et

al., 1992), or systemic antagonism of dopamine receptors (D1 and D2) (Corrigall and Coen,

1991) significantly reduce nicotine self-administration. In addition, nAChR antagonism in the

VTA has resulted in the attenuation of nicotine self-administration as well as nicotine-induced

potentiation of brain stimulation reward (Corrigall, Coen et al., 1994; Panagis, Kastellakis et al.,

2000).

Although the dopaminergic system is a key player in modulating the effects of nicotine,

it is not the only component in the neurocircuitry underlying these effects. A role has been

suggested for other neurochemical systems in the brain, including the cholinergic (Lanca,

Adamson et al., 2000), glutamatergic (McGehee, Heath et al., 1995), GABAergic (Kalivas,

Churchill et al., 1993), serotonergic (Ribeiro, Bettiker et al., 1993), opioid (Pierzchala, Houdi et

al., 1987) and cannabinoid systems (Cohen, Kodas et al., 2005; Forget, Hamon et al., 2005; Le

Foll, Forget et al., 2008). However, the involvement of the above systems in the mechanisms

underlying nicotine reward and reinforcement is also believed to be through an interaction with

the dopaminergic system.

1.4.4 Craving and Cue-induced Relapse

Cue-induced relapse and craving are prominent features of nicotine dependence as well

as of dependence on other addictive substances. Drug craving is a term that applies to an intense

desire to obtain and consume the drug. This desire is not only driven by the urge to gain the

pleasurable and rewarding effects of the drug, but also to avoid experiencing the negative effects

of withdrawal. Craving is commonly elicited by the exposure to the environmental stimuli that

were previously associated with the intake of the drug (Carter and Tiffany, 1999; Childress,

1993; O'Brien, Childress et al., 1992; O'Brien and McLellan, 1996) and results in an increase in

20

the motivation for drug seeking ultimately ending in relapse (See, Fuchs et al., 2003). Repeated

and effective pairing of the drug taking behaviour with certain stimuli has been shown to gain

motivational salience to these stimuli (See, Fuchs et al., 2003). Over time, the conditioned

stimuli themselves become secondary reinforcers increasing the actual drug taking behaviour

(Caggiula, Donny et al., 2002b), and inducing drug seeking behaviour in cases of abstinence;

thus gaining control over drug seeking behaviour (Everitt and Wolf, 2002).

Although the reinforcing effects of nicotine, such as euphoria, are more subtle than other

dugs, the abuse potential of nicotine and rate of relapse remain comparably high (Goldberg,

Spealman et al., 1981). A possible explanation for this discrepancy is the presence of other non-

pharmacological factors that gain incentive salience through their association with nicotine

intake, further reinforce nicotine-seeking behaviour and predispose to the high rates of relapse

encountered in human smokers. Indeed, several animal studies have indicated a strong role for

the environmental stimuli associated with nicotine intake in maintaining nicotine-seeking

behaviour (Donny, Caggiula et al., 1999; Rose and Corrigall, 1997). In 2001, Caggiula and co-

workers demonstrated an important role for nicotine-associated cues in maintaining nicotine

self-administration in rats (Caggiula, Donny et al., 2001). In their study, the rats self-

administered nicotine that was not paired with cues at a much lower rate than they self-

administered cue-paired nicotine (Caggiula, Donny et al., 2001). Moreover, following a period

of extinction, the reintroduction of nicotine together with its associated cues successfully

reinstated nicotine-seeking behaviour to a higher degree than that resulting following the

reintroduction of nicotine alone.

Taken together, a strong body of evidence supports an important role for nicotine-

associated cues in maintaining nicotine seeking, as well as in relapse.

21

1.4.5 Neurocircuitry of Cue-induced Relapse – Role of the BLA and NAcc

The neural mechanisms underlying the processes of conditioned reinforcement and its

role in relapse, and the specific areas in the brain involved in these processes have been the

focus of a large body of research.

In humans, imaging studies have revealed a relation between the dopaminergic system

and cue-induced cravings. In 2006, using PET imaging, Wong and colleagues found an increase

in dopamine receptor occupancy in the dorsal striatum of frequent cocaine users following the

exposure to cocaine-related cues (Wong, Kuwabara et al., 2006). This increase was directly

proportional to the intensity of craving experienced by the subjects as a result of exposure to the

cues (Wong, Kuwabara et al., 2006). Similarly, in the same year, another study found an

increase in extracellular dopamine in the dorsal striatum, upon exposure to cocaine-related cues

(Volkow, Wang et al., 2006). Moreover, drug-induced increase in extracellular striatal dopamine

was insufficient to produce craving, in cocaine abusers, unless it was accompanied by the

presentation of cocaine-associated cues (Volkow, Wang et al., 2008).

Animal models have also widely investigated the neurocircuitry of conditioned

reinforcement and cue-induced relapse. The amygdala and the NAcc and their dopaminergic

innervations have long been viewed as key structures underlying the processes of reinforcement

and relapse. However, the literature shows that these two areas have differential effects on

different aspects of relapse. Moreover, subdivisions of the amygdala and the NAcc have

exhibited differential functional roles in modulating conditioned reinforcement and cue-induced

relapse. Although substantial advancements have been made in the field of studying the neural

substrates mediating cue-induced relapse, the exact mechanisms regulating this process remain

to be fully elucidated. Moreover, some areas in the brain, which may have also an important role

in this process, remain understudied in this regard, such as the LHb.

22

The basolateral region (BLA) as well as the central nucleus (CeA) of the amygdala have

been shown to be involved in reward-related processes and conditioned reinforcement. The role

of the BLA in conditioned reinforcement is supported by numerous studies. First, the

presentation of drug-associated conditioned stimuli resulted in an increase in cFos expression in

the BLA. In a study by Brown and colleagues, pairing of cocaine injections with a cocaine-

paired environment produced an increase in cFos expressions in the BLA and LHb among other

areas, whereas the NAcc did not exhibit such an increase (Brown, Robertson et al., 1992). A

similar increase in cFos expression was observed in the BLA upon the reintroduction of a DS

which was previously predictive of the availability of intravenous cocaine (Ciccocioppo, Sanna

et al., 2001). The latter effect was significantly blocked by the administration of DRD1

antagonists (Ciccocioppo, Sanna et al., 2001). Secondly, BLA lesions disrupted the responding

to different types of conditioned stimuli. Bilateral excitotoxic lesions in the BLA, using

infusions of N-methyl-D-aspartate (NMDA), disrupted the responding of rats to conditioned

stimuli that were previously paired with the administration of water, and attenuated the

potentiation of conditioned reinforcement responses by intra-NAcc infusions of D-amphetamine

(Cador, Robbins et al., 1989). NMDA lesions of the BLA also disrupted responding using a

pavlovian second order conditioning. In this case, a light paired with food administration was

unable to acquire secondary reinforcing properties, in the BLA lesioned rats, and was therefore

unable to reinforce the acquisition of a second order conditioning, by the presentation of tone-

light pairings in the absence of food (Hatfield, Han et al., 1996). In another study, bilateral

NMDA lesions of the BLA resulted in the attenuation of reinstatement response of cocaine

seeking induced by the presentation of discriminative cues (Meil and See, 1997). Similarly,

quinolinic acid lesions of the BLA impaired conditioned place preference for cocaine without an

effect on cocaine-induced hyperlocomotion, indicating a role for the BLA only in stimulus-

reward conditioning rather than the unconditioned psychomotor effects of cocaine (Brown and

23

Fibiger, 1993). Moreover, BLA lesions inhibited the acquisition of a response to conditioned

stimuli, indicating that an intact BLA is important for the acquisition as well as the expression

of the reinstatement (Everitt, Parkinson et al., 1999; Kruzich and See, 2001).

The dopaminergic input to the BLA, in specific, has been strongly implicated in

conditioned reinforcement processes. In 1998, Tran-Nguyen and co-workers demonstrated an

elevation in dopamine levels in the amygdala in rats upon returning to the chambers where they

self-administered cocaine following a month of withdrawal (Tran-Nguyen, Fuchs et al., 1998).

Furthermore, intra-BLA infusions of DRD1 agonists and antagonists and DRD2-like antagonists

have been shown to modulate conditioned fear expression (Lamont and Kokkinidis, 1998) and

retention (Guarraci, Frohardt et al., 2000; Guarraci, Frohardt et al., 1999), as well as the

acquisition of cue-association learning and responses for conditioned stimuli (Berglind, Case et

al., 2006; See, Kruzich et al., 2001). More recently, in 2008, Di Ciano demonstrated an

attenuation of cocaine-seeking behaviour that is maintained by the presentation of cocaine-

associated cues, under a second order schedule of reinforcement, in rats treated with intra-BLA

infusions with the selective DRD3 antagonist SB 277011-A (Di Ciano, 2008). Infusion of the

same ligand into the NAcc or dorsal striatum was without effect on cocaine seeking under the

same second order schedule (Di Ciano, 2008).

Dissociation has been shown in the roles of the CeA versus the BLA in conditioned

learning processes. For example, Everitt and co-workers first trained the rats to associate the

presentation of a conditioned light stimulus (CS) with the delivery of food until the rats

developed a conditioned response to the presentation of the CS. In their study, bilateral lesions

of the CeA, but not the BLA, were shown to disrupt the acquisition of appetitive pavlovian

conditioning (Everitt, Parkinson et al., 1999). Conversely, whereas BLA lesions attenuated

conditioned reinforcement responses, lesions of the CeA were ineffective in this regard (Everitt,

Parkinson et al., 1999). The CeA and BLA also exhibited different roles in modulating fear

24

conditioning (Killcross, Robbins et al., 1997). Therefore, it can be concluded that each of these

subdivisions is responsible for a certain aspect of the conditioning process and stimulus-reward

association learning.

The NAcc also plays a complex role in the regulation of reward-related processes. A

marked dissociation can be observed in the roles played by the NAcc shell and the NAcc core in

the conditioning process. For example, an increase in cFos expression occurs in the NAcc shell

following the exposure to cocaine-paired stimuli, but not in the NAcc core (Neisewander, Baker

et al., 2000). On the other hand, NAcc neurons in both subregions show indistinguishable firing

rates during a DS task (Jones, Day et al., 2010b). Lesions of the NAcc core significantly

attenuated pavlovian conditioning as well as conditioned reinforcement responses, but had no

effect on the potentiation of conditioned reinforcement induced by intra-NAcc infusions of D-

amphetamine (Everitt, Parkinson et al., 1999). The opposite was true for the NAcc shell (Everitt,

Parkinson et al., 1999). The dopaminergic input to the NAcc is believed to be of critical

importance to the role played by the NAcc in reward processing. A closer investigation of the

dopaminergic innervations to the NAcc, revealed that dopamine concentrations in the NAcc

exhibit subsecond fluctuations at rest (Wightman, Heien et al., 2007), which are potentiated by

the administration of drugs of abuse (Stuber, Roitman et al., 2005). Dopaminergic depletion

from the NAcc, as a whole, prevented the formation of a pavlovian conditioning response

(Everitt, Parkinson et al., 1999). In addition, intra-NAcc infusions of D-amphetamine, an

indirect dopamine agonist, increased conditioned responses (Everitt, Parkinson et al., 1999).

Conversely, Neisewander and colleagues reported no change in the NAcc dopamine levels upon

the reintroduction of cocaine-associated cues (Neisewander, Le et al., 1996). Similarly, the

contingent presentation of cocaine-associated cues under a second order schedule was without

effect on NAcc dopamine release in rats (Ito, Dalley et al., 2000) and in rhesus monkeys

(Bradberry, Barrett-Larimore et al., 2000). Moreover, the presentation of amphetamine-

25

associated CS was found to have no effect on NAcc dopamine signal (Di Ciano, Blaha et al.,

2001).

Therefore, it can be concluded, by the preponderance of evidence to date, that the BLA

is a focal structure in the modulation of stimulus-reward-associations and conditioned

reinforcement. It can also be concluded that, although some changes have been reported in the

NAcc as a consequence of exposure to drug-associated conditioned stimuli, these changes may

not reflect a critical role for the NAcc in the regulation of stimulus-reward associations, or

conditioned reinforcement processes.

1.4.6 The Mesocorticolimbic Dopamine System

The dopaminergic system arises primarily from the dopamine neurones located in the

VTA, and the sustantia nigra pars compacta, and projecting to the NAcc, amygdala and

prefrontal cortex. These structures are in turn interconnected through glutamatergic pathways.

Moreover, cholinergic fibres connect the above structures to basal forebrain structures and

pedunculopontine nucleus and lateral dorsal tegmentum (Kelley, 2002). The dopamine neurons

also innervate other areas such as the bed nucleus of the stria terminalis and the lateral septum [a

detailed review has been presented in (Di Chiara, 1995)]. Therefore, the mesolimbic dopamine

system appears to be situated in the centre of a circuit containing the structures most implicated

in reward processing and the mechanisms underlying the regulation of reinforcements.

The dopaminergic system has long been believed to have a key role in the expression of

goal directed behaviour of drugs of abuse, including nicotine, as well as natural rewards

(Carboni, Imperato et al., 1989; Corrigall, Franklin et al., 1992; Di Ciano and Everitt, 2004).

Moreover, a clear involvement has been demonstrated for the dopaminergic system in the

26

conditioned reinforcing properties of drugs of abuse and their associated stimuli (Schultz, 1998;

Stuber, Klanker et al., 2008). Dopamine has also been implicated in the development of

behavioural sensitization, which occurs following the repeated administration of drugs of abuse

and non-drug stimuli (Kalivas and Stewart, 1991).

A large body of evidence supports the role of the dopaminergic system in cue-

associations. For example, in 1981, Miller and colleagues demonstrated, using albino rats, an

increase in the neuronal firing of the dopamine neurons in the VTA, and substantia nigra in

response to conditioning task using either a DS or a CS associated with the availability of

reward in the form of chocolate milk (Miller, Sanghera et al., 1981). Similarly, phasic neuronal

responses were recorded in the monkey dopamine neurons in response to conditioned stimuli

(Schultz, Apicella et al., 1993). Moreover, in 2008, Stuber et al. observed that CS predictive of

sucrose availability (in an appetitive pavlovian task) not only resulted in an increase in the firing

of the dopamine neurons, but also increased the synaptic strength over these neurons (Stuber,

Klanker et al., 2008). Additionally, dopaminergic neurons are believed to mediate reward-

related error signals (Ljungberg, Apicella et al., 1992). In other words, dopaminergic neuronal

firing has been shown to increase following the presentation of an unexpected reward and to

cease following the omission of an expected reward (Fiorillo, Newsome et al., 2008).

In summary, the mesocorticolimbic dopamine system is believed to be of major

importance in regulating reward processing and conditioned reinforcement. This suggests an

important role for pharmacological agents modulating the dopamine pathways and receptors, as

potential therapeutic agents for the treatment of drug addiction and relapse to drug seeking.

27

1.4.7 Dopaminergic Receptors

Dopamine exerts its action through binding to dopamine receptors (DRD1 – 5). Dopamine

receptors are a type of G-protein coupled receptors and are mainly expressed in the central

nervous system. Dopmaine receptors have been classified into two subgroups based on their

structural homology, affinities to pharmacological ligands and their downstream effects on

cyclic AMP (cAMP); namely DRD1-like and DRD2-like receptors (Seeman and Van Tol, 1994).

Table 1.2 depicts the main differences between the two subgroups. A detailed review on

different types of dopaminergic receptors can be found in (Le Foll, Gallo et al., 2009).

Table 1.2

The differences between the dopamine receptor subtypes

DRD1-like Dopamine Receptors DRD2-like Dopamine Receptors

DRD1 and DRD5 DRD2, DRD3 and DRD4

Activation of the members of this group

stimulates adenylyl cyclase and cAMP

Activation of the members of this group

inhibits adenylyl cyclase and cAMP

Show low to moderate affinity for

antipsychotic drugs

Show moderate to high affinity for

antipsychotic drugs

The main focus of the current study is the investigation of the role of the DRD3 in cue-

induced reinstatement of nicotine-seeking behaviour. However, a brief overview of the role of

other dopamine receptors in drug addiction will be presented before reviewing the available

literature on the involvement of the DRD3 in drug addiction.

28

1.4.7.1 The Dopamine D1 Receptor (DRD1)

The DRD1 was first cloned by Dearry and colleagues in 1990 (Dearry, Gringrich et al.,

1990) and is encoded by an intronless gene on chromosome 5 (Sunahara, Niznik et al., 1990). In

addition, it is the most abundantly expressed dopamine receptor. The DRD1 mRNA is expressed

in all the areas of the nigrostriatal and mesocorticolimbic dopamine system (Fremeau, Duncan et

al., 1991). The DRD1 is also found in distinct striatal areas expressing substance P, dynorphin

and co-expressing the DRD3 (Diaz, Lévesque et al., 1995; Diaz, Pilon et al., 2000)

Several studies have indicated an involvement of the DRD1 in the rewarding effects of

drugs of abuse, as well as their locomotor effects. Animal studies have shown that systemic

blockade of the DRD1 attenuates nicotine self-administration (Corrigall and Coen, 1991),

morphine self-administration (Glick and Cox, 1975), heroin intake (Gerrits, Ramsey et al.,

1994) and alcohol-seeking behaviour, and decreases the reinforcing effects of cocaine in rodents

(Koob, Le et al., 1987) and in non-human primates (Bergman, Kamien et al., 1990). Moreover,

local infusions of DRD1 antagonists into the NAcc shell blocked the acquisition of nicotine-

induced conditioned-place preference (Spina, Fenu et al., 2006), decreased the reinforcing

effects of cocaine (Bari and Pierce, 2005) and attenuated context-induced reinstatement of

heroin seeking (Bossert, Poles et al., 2007). In the BLA, DRD1 seem to be more involved in cue

and cocaine-priming induced reinstatement of cocaine seeking (Norman, Norman et al., 1999;

See, Kruzich et al., 2001).

Taken together, the aforementioned studies indicate that the DRD1 are implicated in

different aspects of drug-seeking behaviour, and can be potential targets for the treatment of

drug addiction.

29


The DRD2 was the first of the dopamine receptors to be cloned in 1988 (Bunzow, Van

Tol et al., 1988). It is also abundantly expressed in the brain in all the dopaminergic areas

(Bouthenet, Souil et al., 1991). The areas showing the highest levels of expression of the DRD2

mRNA are the dopaminergic neurons in the VTA and substantia nigra, where the DRD2 acts as

an autoreceptor (Dickinson, Sabeti et al., 1999; Mercuri, Saiardi et al., 1997). An increase in the

DRD2 expression has been found to occur following 6-hydroxy-dopamine lesions in rats

(Gerfen, Engber et al., 1990), as well as following chronic antipsychotic drug treatment

(Martres, Sokoloff et al., 1992).

The involvement of the DRD2 in drug-seeking behaviours appears to be of a complex

nature. In human smokers, DRD2 blockers increase and DRD2 agonists decrease smoking

behaviour (Caskey, Jarvik et al., 1999). Animal studies have shown that a DRD2 antagonist

decreases nicotine self-administration (Corrigall and Coen, 1991), and the intracranial

administration of a DRD2 agonist significantly reduced intracranial self-administration of

nicotine (Ikemoto, Qin et al., 2006). Similarly, discrepant effects have been observed on heroin-

seeking behaviour, where both DRD2 agonists and antagonists were shown to decrease heroin

taking (Hemby, Smith et al., 1996; Rowlett, Platt et al., 2007). On the other hand, only DRD2

antagonists were able to block morphine-induced conditioned place preference, whereas the

agonists had the opposite effect (Kuribara, 1995; Rezayof, Zarrindast et al., 2003).

Under a low ratio requirement schedule (FR5), the administration of DRD2 blockers

reduced the rewarding effect of cocaine (increasing self-administration behaviour), whereas the

DRD2 agonists produced the opposite effect (Caine and Koob, 1994; Caine, Negus et al., 2002).

However, under schedules of high response requirements (such as PR schedules and FR15)

30

DRD2 antagonists decreased cocaine self-administration which has been explained as due to a

decrease in the reinforcing effects of cocaine (Caine and Koob, 1994; Hubner and Moreton,

1991). Moreover, DRD2 knock-out mice showed a higher rate of cocaine self-administration

than their wild type controls, suggesting the DRD2 may be needed to limit the rate of self-

administration of the high doses of cocaine (Caine, Negus et al., 2002).


The DRD4 was cloned in 1991 by Van Tol et al. (Van Tol, Bunzow et al., 1991). In

comparison to other dopamine receptors, the density of the DRD4 expression in the brain is

relatively low. The areas exhibiting the highest density of expression of the DRD4 mRNA

include the retina, cerebral cortex, amygdala, pituitary, cerebellum and hypothalamus (Cohen,

Todd et al., 1992; Mei, Griffon et al., 1995; Valerio, Belloni et al., 1994).

Very few preclinical studies have been conducted investigating the role of the DRD4 in

drug addiction. A role is suggested for the DRD4 in the discriminative effects of nicotine

(Brioni, Kim et al., 1994) and morphine-induced withdrawal symptoms (Mamiya, Matsumura et

al., 2004). However, further studies are needed to uncover the exact role of the DRD4 in nicotine

dependence and drug addiction in general.


Sunahara and co-workers were the first to characterize the DRD5 in 1991 (Sunahara,

Guan et al., 1991). The expression of the DRD5 in the rat brain appears to be restricted to the

hippocampus, parafasicular thalamic nucleus and the mammillary bodies (Mamiya, Matsumura

et al., 2004; Meador-Woodruff, Mansour et al., 1992). The structural homology between the

DRD1 and the DRD5 has comprised a difficulty in assessing the exact areas of expression of the

31

DRD5. Similarly, the lack of ligands that are selective to the DRD5 has rendered it difficult to

investigate its role in drug addiction using preclinical studies.


The DRD3 was first characterized in 1990 by Pierre Sokoloff and co-workers (Sokoloff,

Giros et al., 1990). Of all the dopamine receptor subtypes, the DRD3 has the highest binding

affinity to endogenous dopamine. Unlike the DRD1 and the DRD2, the DRD3 has a restricted

pattern of expression in the brain (Bouthenet, Souil et al., 1991; Diaz, Lévesque et al., 1995;

Diaz, Pilon et al., 2000; Le Foll, Diaz et al., 2003; Le Foll, Schwartz et al., 2003). The areas

exhibiting the highest areas of expression of the DRD3 in the rat brain include the islands of

Calleja, the olfactory tubercle and the NAcc (Sokoloff, Giros et al., 1990). Nonetheless, the

DRD3 was shown to be expressed in other brain areas, with varying densities, including the

medial prefrontal cortex, ventral pallidum, BLA and LHb (Bouthenet, Souil et al., 1991). In

addition, high levels of the DRD3 mRNA are found throughout the dopaminergic system

including the VTA, bed nucleus of stria terminalis, CeA and NAcc (Diaz, Pilon et al., 2000).

Therefore, the DRD3 appears to be expressed in brain areas that are strongly implicated in the

reinforcing effects of drugs of abuse, and reward processing (Koob, 1992).

There is a growing body of evidence implicating the DRD3 in drug addiction, and

especially in the processes underlying relapse. In humans, the density of the DRD3 has been

shown to be elevated in long-term cocaine abusers, and in the brains of cocaine overdose

victims (Segal, Moraes et al., 1997; Staley and Mash, 1996). Animal studies also revealed a

selective increase in the DRD3 expression in the brains of rats and mice treated with nicotine (Le

Foll, Diaz et al., 2003; Le Foll, Schwartz et al., 2003), cocaine (Le Foll, Francès et al., 2002;

Neisewander, Fuchs et al., 2004), morphine (Spangler, Goddard et al., 2003) and alcohol

32

(Vengeliene, Leonardi-Essmann et al., 2006). Such changes were not observed in DRD1 or

DRD2 (Le Foll, Diaz et al., 2003; Le Foll, Schwartz et al., 2003).

Various pharmacological approaches have been explored to study the function of the

DRD3 and to modulate the DRD3 transmission. Different pharmacological agents have been

used as DRD3 ligands. A special interest has arisen in DRD3 partial agonists and antagonists as

potential therapeutic agents especially for reducing relapse (Heidbreder, Gardner et al., 2005;

Heidbreder and Newman, 2010). However, a lack of selectivity to the DRD3 has always

comprised an obstacle to the understanding of the functions of this receptor as most of these

ligands exhibited a partial affinity to the DRD2 as well.

Nevertheless, advances in the medicinal chemistry have recently resulted in the development of

a group of drugs that have a higher selectivity to the DRD3 than the DRD2. The first selective

ligand to be tested was the DRD3 partial agonist BP 897, which has 70 fold selectivity for DRD3

over DRD2 (Pilla, Perachon et al., 1999). This was rapidly followed by the development of the

highly selective DRD3 antagonist SB 277011-A exhibiting a 100 fold selectivity for the DRD3

over the DRD2 (Reavill, Taylor et al., 2000), which was used throughout the current study. In

addition to the higher selectivity exhibited by SB 277011-A at DRD3 over DRD2, it has no

agonist activity, and exhibits similar potencies at rat and human DRD3. SB 277011-A has good

bioavailability after oral administration and good CNS penetration and its plasma half life in rats

is two hours (Reavill, Taylor et al., 2000). Moreover, at the range of doses that are selective to

DRD3, SB 277011-A is non-cataleptogenic (up to 78.8 mg/kg), doesn’t induce

hyperprolactenemia and has no effect on locomotor activity per se, and no alteration in

stimulant-induced hyperactivity (up to 42.3 mg/kg) (Reavill, Taylor et al., 2000). In the current

study SB 277011-A was used as the selective DRD3 antagonist. The range of doses of SB 277-

11-A used in the current study was chosen based on previous studies where the same range of

33

doses has been shown to block nicotine-induced place preference and reactivity to nicotine-

associated stimuli (Le Foll, Schwartz et al., 2003)(Pak, Ashby et al., 2006).

Currently, more compounds are available that have a high potency and selectivity for the

DRD3 such as NGB 2904, PG 01037 (Mason, Hassan et al., 2010) and others (see (Heidbreder

and Newman, 2010) for a review on the advances in DRD3 antagonists as pharmacotherapeutics

for addiction). Such advancements in this field will result in better understanding of the

mechanisms through which the DRD3 are involved in drug seeking and relapse.

Numerous studies have used animal models, to investigate the role of DRD3 ligands in

different aspects of drug seeking and drug taking behaviours. The most important studies in this

regard are summarized in Table 1.3.

34

Table 1.3


Reinforcer BSR SA Reinstatement Models Second order CPP References

Nicotine

SB 277011-A, NGB 2904: Attenuation

BP 897: No effect on FR. SB 277011-A: No effect on FR, High doses of SB 277011-A decrease SA on PR

SB 277011-A: Attenuation of nicotine primed reinstatement BP 897: No effect on cue-induced reinstatement.

N/A

BP 897, ST 198 and SB 277011-A: Block the expression of CPP.

(Andreoli, Tessari et al., 2003; Khaled, Farid Araki et al., 2010; Le Foll, Sokoloff et al., 2005; Pak, Ashby et al., 2006; Ross, Corrigall et al., 2007)

Cocaine

SB 277011-A, NGB 2904: Attenuation

SB 277011-A, NGB 2904: No effect on FR, lowering PR break point

SB 277011-A, NGB 2904: Attenuation of cue-,stress- and cocaine-induced reinstatement

SB 277011-A and BP 897: Attenuation. Intra-BLA infusion of SB 277011-A: Attenuation

SB 277011-A: Block acquisition and expression.

(Cervo, Burbassi et al., 2005; Cervo, Cocco et al., 2006; Di Ciano, Underwood et al., 2003; Pilla, Perachon et al., 1999; Vorel, Ashby et al., 2002; Xi, Gilbert et al., 2004; Xi, Gilbert et al., 2005; Xi, Newman et al., 2006)

35

Table 1.3 (Continued)


Reinforcer BSR SA Reinstatement Models Second order CPP References

Methamp-hetamine

SB 277011-A, NGB 2904, BP 897 and PG01037: Attenuation

PG01037: No effect on FR CJB090 and PG01037 attenuate SA under PR

PG01037: Attenuation of cue-induced reinstatement N/A N/A

(Higley, Spiller et al., 2010; Orio, Wee et al., 2010; Xi and Gardner, 2007)

Opiates

NGB 2904: Attenuation of heroin induced BSR

N/A N/A N/A

SB 277011-A , BP 897: Block morphine, heroin induced CPP

(Ashby, Paul et al., 2003; Francès, Le Foll et al., 2004; Xi and Gardner, 2007)

Food N/A

SB 277011-A: Attenuation of food SA under FR, no effect on PR NGB 2904: No effect on FR

SB 277011-A: No effect on cue-induced reinstatement

SB 277011-A: No effect

SB 277011-A: No effect

(Cervo, Cocco et al., 2006; Di Ciano, Underwood et al., 2003; Thanos, Michaelides et al., 2008; Vorel, Ashby et al., 2002)

BSR: Brain stimulation reward, SA: Self-administration, CPP: Conditioned place preference, FR: Fixed ratio schedule, PR:

Progressive ratio schedule, SB 277011-A, NGB 2904, ST 198, PG01037 and CJB090 are selective DRD3 antagonists and BP 897 is

a selective DRD3 partial agonist. N/A: No available data.

36

It can be concluded from the above table (Table 1.3) that the DRD3 antagonists seem to

be involved in some, but not all, aspects of drug taking and drug seeking behaviour. Indeed,

selective DRD3 antagonists have a strong potential as therapeutic agents in the field of drug

addiction, and specifically for the prevention of relapse. Such potential arises from several

integrating factors, and when taken into consideration, render the DRD3 selective antagonists

promising as successful therapeutic agents in this field. These factors include:

1. The DRD3 antagonists do not produce many of the side effects that may interfere with daily

activities. For example, the DRD3 antagonists have no effect on locomotor activity, whether

spontaneous or stimulus-induced, nor on motor coordination (Reavill, Taylor et al., 2000).

Notably, the DRD3 antagonists cause no impairment in attention, learning or memory

(Millan, Di Cara et al., 2007; Sarter and Parikh, 2005). In fact, the DRD3 antagonists seem to

be involved in a mechanism that enhances cognition, attention and learning (Glickstein,

Desteno et al., 2005; Sarter and Parikh, 2005). Moreover, the selective DRD3 antagonists are

not cataleptogenic and do not produce hyperprolactenemia (Reavill, Taylor et al., 2000).

Finally, social interaction is enhanced with blocking the DRD3 (Millan, Loiseau et al., 2008),

and helplessness, anhedonia and despair were not observed in knock-out mice lacking the

DRD3 (Chourbaji, Brandwein et al., 2008).

2. The use of the DRD3 receptor antagonists to prevent relapse, triggered by different factors, in

animal models has proven to be highly successful (Table 1.3). However, this class of drugs

has been shown to have little or no effect on the actual drug taking behaviour (Table 1.3).

Therefore, it seems that the DRD3 antagonists target a specific aspect of drug addiction,

which is relapse. Indeed, the decision to stop using a certain drug depends primarily on the

readiness and motivation of the individual to quit. Although some medications have shown

some usefulness in this aspect, it remains critical to control the newly abstinent individuals’

urge to relapse. Especially at this stage, the DRD3 antagonists present an optimal choice for

37

the prevention of relapse. It is important to note, however, that there is no evidence that the

use of the DRD3 antagonists will be helpful in alleviating the withdrawal symptoms of drugs

of abuse. Further studies are needed to investigate this possibility.

3. The DRD3 antagonists appear to have no significant effect on natural reinforcers, such as

food taking behaviour. However, a potential utility for the DRD3 antagonists has been

suggested by the fact that SB 277011-A was shown to affect food taking in a rat model of

obesity (Thanos, Michaelides et al., 2008).

4. Preclinical studies have clearly indicated that DRD3 antagonists do not have an abuse

potential. When substituted for cocaine, SB 277011-A and NGB 2904 (two highly selective

DRD3 antagonists) could not maintain stable self-administration on their own.

Therefore, based on the available data from preclinical studies, it can be concluded that

the DRD3 antagonists present a relatively safe and reliable option for the prevention of relapse to

drug use. The extrapolation of the above findings to humans requires the conduction of a series

of clinical studies to validate the utility of these agents in substance dependant individuals.

38

1.5 Restatement of the Hypotheses

Hypothesis I

Systemic antagonism of the DRD3 results in the attenuation of cue-induced reinstatement of

nicotine-seeking behaviour.

Hypothesis II

The effect of the DRD3 on cue-induced reinstatement is mediated through the BLA and the LHb

but not through the NAcc.

Hypothesis III

Antagonism of the DRD3 in the BLA has no effect on food taking behaviour.

Hypothesis IV

Cue lights per se, un-associated with nicotine administration, have no primary reinforcing effect

that is strong enough to maintain stable responding.

39

Chapter 2 METHODOLOGY

2.1 Animals

Male, Long–Evans rats (Charles River, Canada) experimentally naive at the start of the

study and initially weighing 250–275 g were used for all experiments. All rats were individually

housed in a temperature controlled environment on a 12-hour reversed light/dark cycle (lights

off 07:00 am). Prior to any experimental manipulation, animals were given a minimum of 7

days to habituate to the colony room, during which time they were weighed, handled and

received unlimited access to both food and water. Subsequently, upon beginning the

experimental training, the rats were food-restricted to 20 g of chow per day, in order to maintain

their body weight throughout the experiment. All the experimental procedures described in this

thesis were carried out in compliance with the guidelines of the Canadian Council on Animal

Care (compatible with the US ‘Guide for Care and Use of Laboratory Animals’), and were

reviewed and approved by the institutional Animal Care Committee.

2.2 Apparatus

Self-administration sessions were carried out in commercially available experimental

chambers (Med Associates, USA) (see Forget et al. 2009 for details), encased in sound-

attenuating boxes and equipped with two levers, a house-light and two cue-lights, one located

above each lever. In each experiment, for half the animals the left lever was active and the right

was inactive and for the other half the opposite applied. Session start was signalled by the

illumination of the house-light and presentation of the levers. Rapid delivery of the self-

administered drug (approximately 1-s delivery time) was achieved with infusion pumps (Med

Associates Model PHM-104). The volume of infusion was adjusted according to the weight of

40

the rat, in the range of 28-45 µl. Pressing on the active lever resulted in the delivery of nicotine

(30 μg/kg per infusion) when schedule requirements were met, accompanied by extinction of the

house-light and illumination of the cue-light above the active lever. Pressing on the inactive

lever was recorded, but had no consequences.

2.3 Experimental procedures

2.3.1 Experiment (1): Systemic Administration of SB 277011-A and Cue-induced

Reinstatement of Nicotine-seeking Behaviour

The experimental procedures for the current experiment are schematically shown in Figure 2.1.

2.3.1.1 Food Training

Techniques for initial training were similar to those previously reported (Corrigall and

Coen, 1989; Forget, Coen et al., 2009; Khaled, Farid Araki et al., 2010). The training was

performed in 1-hour daily sessions for 5 days where the animals were trained to press the lever

for food reinforcement on a continuous reinforcement schedule. Under this schedule, each press

on the active lever resulted in the delivery of a 45-mg food pellet. During these training

sessions, the house-light was on, but the lever light was off.

2.3.1.2 Intravenous Catheter Implantation Surgery

Once trained, the animals were subjected to surgery for intravenous catheter

implantation. Surgical procedures for implantation of chronic intravenous catheters were

conducted using strict aseptic technique and were ethically approved by the institutional Animal

Care Committee. Surgery was performed under anaesthesia induced by xylazine (10 mg/kg i.p.)

and ketamine hydrochloride (90 mg/kg i.p.). Incision sites were infiltrated with the local

41

anaesthetic Marcaine (0.125%). Buprenorphine was given for post-operative analgesia (0.03

mg/kg s.c.). Catheters (constructed in the laboratory prior to surgery) were driven through the

scapular incision, under the skin and under the rat’s right shoulder to be implanted into the right

jugular vein. Catheters were sutured in place and further secured using cyano-acrylic glue.

Incision sites were closed, and a single dose of penicillin (30 000 U i.m.) was administered at

the completion of surgical procedures. Surgical procedures were similar to those previously

reported (Corrigall and Coen, 1989; Forget, Coen et al., 2009; Forget, Hamon et al., 2009).

Also, please see the attached standard operating procedure (SOP) for intravenous catheterization

surgery (Appendix II) for details.

Animals were allowed to recover for a 1-week period before drug self-administration sessions

were begun.

2.3.1.3 Nicotine Intravenous Self-administration (IVSA) Procedures

Acquisition of nicotine self-administration was performed under a Fixed Ratio (FR)

schedule of reinforcement at a unit dose of 30µg/kg per infusion of nicotine base. Self-

administration training was carried out in daily 1 hour sessions. Pressing on the active lever for

a fixed predetermined number of times resulted in an infusion of nicotine followed by a time-out

period of 1 minute, during which the house-light was extinguished and the cue-light above the

active lever was illuminated. Pressing on the inactive lever had no scheduled consequences.

Rats were randomly assigned the right or the left lever as the active lever. During the first week

of acquisition, response requirement was FR1, i.e., each active lever press during the time-in

period resulted in the delivery of one infusion. Response requirements were then gradually

increased to reach a final value of FR5, by which time the self-administration behaviour was

stable after 15- to 20-days history of training. Self-administration sessions occurred mostly 5

days a week. Rats were considered to have acquired stable nicotine self-administration when

42

they received a minimum of 10 infusions per 1-hour session and had <20% variation in the

number of infusions earned per session during three consecutive sessions. Moreover, by the end

of the FR5 training phase, all the rats pressed on the active lever more than twice the number of

times they pressed the inactive lever. Once stability was achieved, the rats were carried into

extinction and reinstatement testing as described below.

2.3.1.4 Extinction Training and Cue-induced Reinstatement of Nicotine-seeking

After the end of the 3- to 4-week acquisition training under the FR schedule, an

extinction phase was conducted by withholding nicotine and its associated cues (house-light

stays on and cue-light stays off throughout the session). Responses on the active or inactive

levers were recorded, but had no consequence. An extinction criterion was established for each

animal individually and was defined as total active lever responses being < 20 active lever

presses or <15% of the mean active lever responding maintained during the last 3 days of FR5

for nicotine, whichever is less. The extinction criterion had to be reached for two consecutive

days in order to conduct the testing. Nicotine-trained rats (n=8) were tested for the effect of SB

277011-A (i.p.) on cue-induced reinstatement of nicotine-seeking behaviour at the following

doses: vehicle (10% hydroxpropyl- β -cyclodextrin in sterile water), or SB 277011-A at 1, 3 and

10 mg/kg. All tests were carried out in a counter-balanced within-subject design. After each test,

extinction was re-established until the extinction criterion was obtained for at least two

consecutive days. Reinstatement tests were conducted under conditions identical to that of self-

administration, except that the responses on the active lever (on a FR5 schedule) resulted in

contingent presentation of the cues (light above the active lever on and house-light off for 60 s)

without nicotine availability (no infusions). Responses on the inactive lever were recorded

without consequences. Each testing session lasted 1 hour.

43

Schematic Diagram for the Experimental Procedures Performed in Experiment 1

IV: Intravenous, IVSA: Intravenous self-administration

Food Training

IV Surgery

Reinstatement Testing

IVSA

Extinction

Recovery

Figure 2.1

44

2.3.2 Experiments (2-4): Local Infusion of SB 277011-A into Discrete Brain Areas and

Cue-induced Reinstatement of Nicotine-seeking Behaviour

The experimental procedures for the local infusion experiments are schematically shown in

Figure 2.1.

2.3.2.1 Food Training

The food training phase was the same as that described in section 2.3.1.1.

2.3.2.2 Intravenous Catheter Implantation Surgery

Procedures for intravenous catheter implantation surgery were the same as described in section

2.3.1.2.

2.3.2.3 Nicotine Self-administration Procedures

Procedures for intravenous nicotine self-administration were the same as described in

section 2.3.1.3 with the following exceptions: A fixed ratio schedule of reinforcement was

followed for 15 days (FR1: 5 days, FR2: 3 days and FR5: 7 days). After that the rats were

subjected the intracranial cannula implantation surgeries. After a period of recovery (7-10 days),

IVSA sessions were resumed for 2-3 days, depending on catheter patency. This was done to

verify that the surgical procedure did not have an effect of the self-administration behaviour.

However, in a few rats (n=4), resumption of the IVSA sessions was not possible due to catheter

blockade during the recovery phase.

2.3.2.4 Intracranial Cannula Implantation Surgery

Following the acquisition of nicotine self-administration, surgeries were performed for

the implantation of intracranial cannulae into: the basolateral amygdala (BLA), the nucleus

accumbens (NAcc), or the lateral habenula (LHb). Stereotaxic surgical procedures were

45

conducted using the same regimen of anesthetics, analgesic, and antibiotic described in section

2.3.1.2. Strict aseptic technique was observed in all procedures, which were also ethically

approved by the institutional Animal Care Committee.

Animals were positioned in stereotaxic frame and guide cannulae (22 gauge, Plastics

One) were bilaterally implanted according to the following coordinates (being lowered to 2.0

mm above the target site); BLA: –2.5 AP, ±5 ML and –6.6 DV, NAcc: +1.6 AP, ±1.7 ML and -

4.7 DV (using a 6º off-vertical angle of approach), and LHb: –3.6 AP, ±2.5 ML and –3.1 DV

(using a 20º off-vertical angle of approach), according to the atlas of Paxinos and Watson 1998

(Paxinos and Watson, 1998). Cannulae were anchored to small screws, threaded partially into

the skull, with dental acrylic. Cannulae-occluders (28 gauge, Plastics One) were inserted into the

guide cannulae immediately after the surgery to maintain patency and restrict the entry of

foreign material into the brain in between the infusions. The occluders extended 2.0 mm lower

than then the cannulae; i.e. reaching the target site. This was done to ensure that no damage

would occur upon the introduction of the microinjectors in the first testing sessions which might

have had an effect on the responding or interpretation of the testing results. Please see the

attached standard operating procedure (SOP) for intracranial cannulae implantation surgery

(Appendix III) for details.

Following the surgery, rats were allowed to recover for 7-10 days before resuming experimental

procedures.

2.3.2.5 Procedures for Local Infusion of SB 277011-A and Cue-induced reinstatement of

Nicotine-seeking

Extinction training was carried out following recovery from the surgical procedures, and

re-stabilization of the self-administration behaviour (where applicable). Upon stabilization of

46

the extinction behaviour, according to the criterion previously mentioned (section 2.3.1.4), rats

were randomized to receive intracranial microinfusions of the vehicle or different doses of SB

277011-A, according to a counterbalanced within-subject design.

Microinjectors (28 gauge, Plastics One) were connected to 50 μl Hamilton syringes via

polyethylene tubing (inner diameter 0.38 mm; Plastics One, Roanoke, Virginia) filled with

sterile water, separated by a bubble from either vehicle or SB 277011-A. Microinjectors were

inserted into the guide cannulae, after removal of the occluders, and were 2.0 mm longer than

the guide cannulae, i.e. extending to the target site. A volume of 0.5 μl of SB 277011-A (0.01,

0.1 or 1 μg/ 0.5 μl) or vehicle (10% DMSO in 10% w/v hydroxyl propyl- β - cyclodextrin in

sterile water) was infused on each side over the course of 1 minute using a microinfusion pump

(Harvard Apparatus, Model 22, SouthNatick, Massachusetts), 5–10 minutes before a test

session. The microinjectors were left in place for 1 minute after the infusion, to allow for

diffusion, after which the microinjectors were removed, and occluders were replaced.

Reinstatement testing was conducted using the same procedures described in section 2.3.1.4,

and rats were required to maintain stable extinction levels for at least 2 consecutive days before

the next testing session. At the end of the experiment, each rat had received a total of 4

microinfusions with at least 2 days in between one testing session and the next.

47

Schematic Diagram for the Experimental Procedures Performed in Experiments 2-4

IV: Intravenous, IVSA: Intravenous self-administration, IC: Intracranial

Food Training

IV Surgery

IVSA

Recovery

Reinstatement Testing

Extinction

Re-stabilization of IVSA

Recovery

IC Surgery

Figure 2.2

48

2.3.3 Experiment (5): Local Infusion of SB 277011-A into the Basolateral Amygdala and

Food-taking Behaviour

In this experiment, the rats (n=6) were trained similarly to the nicotine self-

administration experiments, except for the following differences: Completion of the required

ratio (FR1, FR2 or FR5) was rewarded with a 45 mg sucrose pellet instead of an infusion of

nicotine; therefore, no intravenous surgery was needed in these rats. After stabilization of the

responding on the active lever on FR5 (5-7 days), the rats were subjected to intracranial surgery

for implantation of guide cannulae into the BLA, following the same procedure and the same

coordinates previously described in section 2.3.2.4. 7-10 days were allowed for recovery,

following which the rats were again trained on FR5 schedule to ensure that there was no residual

effect of surgery on the level of responding. The behaviour was considered to be stable if the

difference between any 2 consecutive days was less than 20%. In this case, the rats were ready

for testing. On the testing day, vehicle or the highest dose of SB 277011-A (1μg/0.5μl/side) was

bilaterally infused into the BLA using the same techniques described in section 2.3.2.5, 5

minutes prior the start of the testing session. The effect of SB 277011-A or vehicle on food

taking behaviour was assessed.

2.3.4 Experiment (6): Responding Maintained by Light Cues

A control experiment was performed to evaluate the responding maintained by the light-

cues presentation only. The training was identical as the nicotine self-administration

experiments, except that there was no surgery for catheter implantation and no nicotine delivery.

Rats (n=8) were first trained to respond for food (lever light off, as mentioned above) on a

continuous reinforcement schedule for 5 days. Then, responding of rats for cue presentation

only (without food delivery) was measured under a FR schedule of reinforcement. Session

duration was 60 minute, and a time-out period of 1 minute followed each ratio completion on

49

the active lever during which time the house-light was extinguished and the cue-light above the

active lever was illuminated. During the first week of acquisition, response requirement was

FR1. Response requirements were then gradually increased to reach a final value of FR5.

2.4 Drugs

Nicotine hydrogen tartrate (Sigma-Aldrich, USA) was dissolved in saline, the pH was

adjusted to 7.0 (±0.2), and the solution was filtered through a 0.22-μm syringe filter (Fisher

Scientific, USA) for sterilization purposes. All nicotine doses are reported as free base

concentrations. Nicotine was administered intravenously in a volume of 100 ml/kg per infusion.

SB 277011-A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin- 2-yl)ethyl]cyclohexyl]-4-

quinolinecarboxamide) was provided by Dr Steven Goldberg (NIH, NIDA, IRP), and

synthesized by Dr JenôVarga and Dr József Gaál (MegaPharma Kft., Budapest, Hungary), as

part of an ongoing research collaboration on involvement of dopamine D3 receptors in drug

reward mechanisms. For the systemic administration experiment (Experiment 1), SB 277011-A

was dissolved in 10% w/v hydroxypropyl- β -cyclodextrin in sterile water and sonicated for 30

minutes. The drug was administered intraperitoneally (i.p.), 30 min prior to testing in a volume

of 2 ml/kg. For the local infusion experiments (Experiments 2-5), SB 277011-A was dissolved

in 10% DMSO in 10% w/v hydroxpropyl- β –cyclodextrin in sterile water. The drug was locally

infused into the specified areas in the brain, 5 minutes prior to testing, in a volume of 0.5 μl /

side.

Figure 2.3 Structure of SB 277011-A (Reavill, Taylor et al., 2000)

50

2.5 Histological Examination

Upon completion of the behavioural testing, rats were overdosed with pentobarbital

(approximately 350 mg/kg, i.p.) and 0.5 μl of cresyl violet dye was infused bilaterally into the

target area using microinjectors inserted into the guide cannulae (procedure identical to the

procedure for microinfusion of the drug described in section 2.3.2.5). The rats were then

decapitated. Brains were dissected out, and flash-frozen in methyl butane on dry ice. Brains

were stored at –65 to –80ºC until sectioning. Serial coronal sections (25 μm-thick) were

obtained using a cryostat at –25ºC. Close to the target area, every 4th-5th section was thaw

mounted on pre-cleaned glass slides, and the sections were examined for verification of the

placement. Acceptable histology required that the tip of the injector bilaterally lay within the

target area.

2.6 Data Analysis

The number of active and inactive lever presses and the number of nicotine infusions

were recorded and analysed. A one-way analysis of variance (ANOVA) with repeated

measurements was used to analyze the effects of SB 277011-A (systemic or locally infused) or

vehicle on cue-induced reinstatement, or on food taking behaviour. Newman–Keuls post-hoc

test was used to assess the differences between individual means (baseline, vehicle and different

doses of SB 277011-A).

To assess the responding, on active and inactive levers, maintained by cues only, a two-way

ANOVA with repeated measurements (factors being time and levers), was performed followed

by LSD post-hoc test.

51

Chapter 3 RESULTS

3.1 Experiment (1) Effect of SB 277011-A (1, 3, 10 mg/kg i.p.) on cue-induced

reinstatement of nicotine-seeking

All the animals (n=8) achieved significant self-administration of nicotine over 4 weeks

of acquisition (5 d under FR1, 3 d under FR2, 12 d under FR5 schedules). Significant extinction

was also achieved after 12 sessions of extinction [the mean number of active lever presses

dropped from 132.56 (±18.67) over the last 3 days of acquisition to 15.56 (±3.23) during the last

3 days of extinction] (see Figure 3.1 which is also representative of a typical acquisition curve

obtained under the schedule of reinforcements applied in all the nicotine self-administration

experiments mentioned below). Figure 3.2 presents data related to the effects of SB 277011-A,

or vehicle, on the responding during reinstatement testing. A one-way ANOVA with repeated

measurements showed a significant group effect of treatment (SB 277011-A or vehicle)

(F4,28=8.99, p<0.01). Neuman–Keuls post-hoc tests revealed that significant reinstatement was

obtained in the vehicle-treated group as compared to the extinction baseline (average of active

lever responding on the days before the testing) (p<0.01) and that all the doses of SB 277011-A

tested (1–10 mg/kg i.p., 30 min before the session) significantly reduced reinstatement of

nicotine-seeking behaviour (p<0.01), compared to responding in the vehicle-treated group

(Figure 3.2). No significant difference was observed between the effects of the different doses of

SB 277011-A (p>0.05, Neuman–Keuls post-hoc tests). Moreover, neither the vehicle nor the

drug had a significant effect on inactive lever pressing (F4,28=1.51, p>0.1 with one-way

ANOVA) (Figure 3.2).

52

Acquisition / Extinction Phases of Nicotine Self-Administration

1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829303132330

20406080

100120140160180200

AFR1 FR2 FR5 Extinction

Day

Num

ber o

f Lev

er P

ress

es

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 1718 19200

5

10

15

20

25 FR1 FR5FR2

B

Day

Num

ber o

f Rei

nfor

cem

ents

Figure 3.1

This figure represents a typical acquisition curve in one of the groups of rats included in the

current study.

A) The level of responding during acquisition (under a fixed ratio (FR) schedule of

reinforcement) and extinction phases of nicotine self-administration. Data are expressed as mean

(±SEM) of the number of active (–●–) and inactive (–○–) lever presses. B) The mean number

(±SEM) of reinforcements (nicotine infusions) obtained during the acquisition phase (FR1-5).

53

Systemic Administration of SB 277011-A significantly attenuates cue-induced

reinstatement of nicotine-seeking behaviour

BSL 0 1 3 100

10

20

30

40

50

60

SB 277011-A (mg/kg)

Nicotine-cues††† **

Num

ber o

f Lev

er P

ress

es

Figure 3.2

This figure represents the effect of SB 277011-A (1, 3, 10 mg/kg), or vehicle (0), on the mean

number (± SEM) of active (–●–) and inactive (–○–) lever presses under conditions for cue-

induced reinstatement of nicotine-seeking. Significant reinstatement was obtained in the

vehicle-treated group (††† p<0.005 vs. baseline (BSL) with two-tailed t test). SB 277011-A

significantly reduced cue-induced reinstatement at all the doses tested (n=8) (** p<0.01 vs.

vehicle with repeated-measures ANOVA). No effect of vehicle or drug was seen on inactive

lever presses.

Figure from (Khaled, Farid Araki et al., 2010), with permission.

54

3.2 Experiment (2): Effect of Local Infusion of SB 277011-A into the BLA on Cue-

induced Reinstatement of Nicotine-seeking Behaviour

Similarly to experiment (1), animals in this group (n=7) achieved stable nicotine self-

administration after 3 weeks of training (including 7 days on FR5). Neither the intracranial

surgery nor the period of recovery had a significant residual effect on the level of responding on

the active lever. The mean number of active lever presses was 171.3 (± 1.8) during the 3 days of

FR5 prior to surgery and became 169.3 (± 17.5) during the 3 days of FR5 following the surgery

and recovery (for rats n=5). However, for 2 rats, this could not be verified due to blockade of the

intravenous catheter during the recovery period. The 2 rats were run on extinction directly after

the recovering from the surgeries. Nicotine self-administration behaviour was successfully

extinguished after 9 days of extinction (the mean number of active lever presses dropped to 13.3

(± 5.9) over the last 3 days of extinction). The mean number of lever presses exerted upon the

reintroduction of nicotine-associated cues was subjected to statistical analysis to evaluate the

effect of intra-BLA infusion of SB 277011-A on cue-induced reinstatement of nicotine-seeking

behaviour. A one-way ANOVA with repeated measurements showed a significant group effect

of treatment (SB 277011-A or vehicle) on the number of active lever pressing (F4,24=8.01,

p<0.01). Neuman–Keuls post-hoc test revealed that significant reinstatement was obtained in the

vehicle-treated group compared to the extinction baseline (average of active lever responding on

the days before the testing) (p<0.01) and that all the doses of SB 277011-A tested (0.01, 0.1 and

1 μg/μl/side infused into the BLA 5 min before the session) significantly reduced reinstatement

of nicotine-seeking behaviour (p<0.01), compared to responding in the vehicle-treated group

(Figure 3.3). No significant difference was observed between the effects of the different doses of

SB 277011-A (p>0.05). Neither the vehicle nor the drug had a significant effect on inactive

lever pressing (F4,24=1.62, p=0.2 with one-way ANOVA) (Figure 3.3).

55

Intra-BLA Administration of SB 277011-A abolishes cue-induced reinstatement of

nicotine-seeking behaviour

BSL 0 0.01 0.1 10

1020304050607080

Nicotine - cues†††

***

**

***

SB 277011-A (µg/0.5µl/side)

Num

ber o

f Lev

er P

ress

es

Figure 3.3

This figure represents the effect of SB 277011-A (0.01, 0. 1, 1 μg/μl/side), or vehicle (0), locally

infused into the BLA, on the mean number (± SEM) of active (–●–) and inactive (–○–) lever

presses under conditions for cue-induced reinstatement of nicotine-seeking. Significant

reinstatement was obtained in the vehicle-treated group (††† p<0.05 vs. baseline with two-tailed

t test). SB 277011-A significantly reduced cue-induced reinstatement at all the doses tested

(n=7) (** p<0.01 vs. vehicle with repeated-measures ANOVA). No effect of vehicle or drug

was seen on inactive lever presses.

56

3.3 Experiment (3): Effect of Local Infusion of SB 277011-A into the NAcc on Cue-


In this group of rats (n=8), stable self-administration was also achieved after 3 weeks of

training (including 7 days on FR5). No effect of intracranial surgery or recovery was noted on

the active lever responding following the recovery period. In the last 3 days prior to cannula-

implantation surgery, the level of active lever responding under FR5 schedule was 114.2 (±

14.1). Following 7-10 days of recovery, the level of mean active lever responding under FR5

schedule was 126 (± 19.2). Re-stabilization of the self-administration behaviour was conducted

for 2 sessions and in only 6 out of 8 rats, due to issues with intravenous catheter blockade.

Successful extinction of nicotine self-administration was achieved after 9 sessions of extinction

training, during the last 3 sessions of which the number of active lever responding declined to

23.5 (± 11.4).

A one-way ANOVA with repeated measurements was also used in this experiment to examine

the effect of intra-NAcc infusion of SB 277011-A on cue-induced reinstatement of nicotine

seeking. The one way repeated measures ANOVA showed a significant group effect of

treatment (SB 277011-A or vehicle) (F4,28=11.74, p<0.01). Neuman–Keuls post-hoc test

revealed that significant reinstatement was obtained in the vehicle-treated group, as well as the

groups treated by different doses of SB 277011-A (0.01-1 μg/μl/side), compared to the

extinction baseline (p<0.05). Moreover, no significant difference in the level of reinstatement

was shown between the vehicle-treated group versus the groups treated with different doses of

SB 277011-A (0.01-1μg/μl/side) (p>0.05) (Figure 3.4).

Neither the vehicle nor the drug had a significant effect on inactive lever pressing (F4,28=0.25,

p=0.95 with one-way ANOVA) (Figure 3.4).

57

Intra-NAcc Administration of SB 277011-A Has No Effect on Cue-induced Reinstatement

of Nicotine-seeking Behaviour

BSL 0 0.01 0.1 10

20

40

60

80

100

Nicotine - cues


***

ns

Num

ber o

f Lev

er P

ress

es

Figure 3.4

This figure represents the effect of SB 277011-A (0.01, 0.1, 1 μg/μl/side), or vehicle (0), locally

infused into the NAcc, on the mean number (± SEM) of active (–●–) and inactive (–○–) lever

presses under conditions for cue-induced reinstatement of nicotine-seeking. Significant

reinstatement was obtained in the vehicle-treated group as well as the group treated with SB

277011-A (*** p<0.05 vs. baseline with one way ANOVA). Moreover, the level of active lever

responding in the SB 277011-A – treated group was not significantly different from the vehicle-

treated group (ns, p>0.05 with one way ANOVA). No effect of vehicle or drug was seen on

inactive lever presses.

58

3.4 Experiment (4): Effect of Local Infusion of SB 277011-A into the LHb on Cue-


In this group of rats (n=7), stable nicotine self-administration was achieved after 14 days

of training (including 6 days of FR5). Due to technical difficulties with maintaining catheter

patency during the intracranial surgery and recovery periods, re-stabilization of responding was

carried out for one session only following the recovery from the cannulae implantation surgery.

Nonetheless, no notable change was observed in the number of active lever responding prior to

or after the surgery and recovery. The mean number of active lever presses was 124.9 (± 19.1)

during the last 3 days of FR5 preceding the surgery and 107.1 (± 13.9) after the recovery from

the surgery. Successful extinction was achieved after 7 sessions of extinction training, where the

mean number of active lever responding declined to 17.9 (± 3.3) during the last 3 days of

extinction.

To assess the effect of intra-LHb infusion of SB 277011-A on cue-induced reinstatement of

nicotine-seeking behaviour, the mean number of lever responding resulting from the

reintroduction of the cues was analysed similarly to the above experiments. A one way ANOVA

with repeated measurements revealed a significant group effect of treatment (SB 277011-A or

vehicle) on the number of active lever presses (F4,24=6.20, p<0.05). Neuman-Keuls post-hoc test

revealed that significant reinstatement was obtained in the vehicle-treated group compared to the

extinction baseline (p=0.01) and that all the doses of SB 277011-A tested (0.01, 0.1 and 1

μg/μl/side infused into the LHb, 5 min before the session) significantly reduced reinstatement of

nicotine-seeking behaviour (p<0.05), compared to responding in the vehicle-treated group

(Figure 3.5). No significant difference was observed between the effect of different doses of SB

277011-A (p>0.05). Neither the vehicle nor the drug had a significant effect on inactive lever

pressing (F4,24=0.84, p=0.51 with one-way ANOVA) (Figure 3.5).

59

Intra-LHb Administration of SB 277011-A attenuates cue-induced reinstatement of

nicotine-seeking behaviour

BSL 0 0.01 0.1 10

20

40

60

80

100

Nicotine - cues


†† **

Num

ber o

f Lev

er P

ress

es

Figure 3.5

This figure represents the effect of SB 277011-A (0.01, 0. 1, 1 μg/μl/side), or vehicle (0), locally

infused into the LHb, on the number of active (–●–) and inactive (–○–) lever presses under

conditions for cue-induced reinstatement of nicotine-seeking. Significant reinstatement was

obtained in the vehicle-treated group (†† p=0.01 vs. baseline with two-tailed t test). SB 277011-

A significantly reduced cue-induced reinstatement at all the doses tested (n=7) (** p<0.05 vs.

vehicle with repeated-measures ANOVA). No effect of vehicle or drug was seen on inactive

lever presses.

60

3.5 Experiment (5): Effect of Local Infusion of SB 277011-A into the BLA on Food-

taking Behaviour

In this group of rats (n=5), stable acquisition of food taking behaviour was achieved after

training similar to that described in the above experiments (including 5 days on FR5). No effect

of surgery or recovery was observed on the number of lever presses, or the number of

reinforcements (food pellets) obtained under FR5 schedule of reinforcements. Figure 3.6 shows

the level of responding on active and inactive levers as well as the number of reinforcements

obtained prior to and after the intracranial surgery and period of recovery.

A one way ANOVA with repeated measurements showed no effect of the infusion of SB

277011-A (1 μg / 0.5 μl / side) or vehicle, into the BLA, on the number of active (F2,17 = 2.23, p

= 0.15) or inactive (F2,17 = 0.22, p = 0.8) lever presses under FR5 schedule (Figure 3.7A and B).

Similarly, no effect of the infusion of SB 277011-A (1 μg / 0.5 μl / side) or vehicle, into the

BLA, was found on the number of reinforcements obtained by the rats under FR5 schedule (F2,17

= 0.01, p = 0.98, with one way ANOVA with repeated measurements) (Figure 3.7C).

61

Acquisition Phase of Food Self-Administration

1 2 3 4 5 6 7 8 9 100

500

1000

1500

2000

2500 Prior to Surgery Following SurgeryA

Num

ber o

f Lev

er P

ress

es

0 1 2 3 4 5 6 7 8 9 1045

50

55

60B

FR5 - Day

Rein

forc

emen

ts

Figure 3.6

This figure represents the responding of the rats for food under FR5 schedule of reinforcement.

No marked change is observed in the level of active lever responding, or the number of

reinforcements in the period following intracranial surgery and recovery as compared to the

period preceding the surgery.

A) The level of responding during the acquisition phase (under a fixed ratio 5 (FR5) schedule of

reinforcement) of food self-administration, prior to and following the cannulae implantation

surgery and recovery. Data are expressed as mean (±SEM) of the number of active (–●–) and

inactive (–○–) lever presses. B) The mean number (±SEM) of reinforcements (food pellets)

obtained during the acquisition phase (FR5).

62

Intra-BLA Administration of SB 277011-A Has No Effect on Food-taking Behaviour

0

500

1000

1500

2000

Num

ber o

f Lev

er P

ress

es

Ans

Activ

e

0100200300

ns

B

Inac

tive

BSL 0 10

20

40

60

SB 277011-A(µg/0.5µl/side)

nsC

Rein

forc

emen

ts

Figure 3.7

This figure represents the effect of intra-BLA infusion of SB 277011-A (1μg / 0.5 μl / side), or

vehicle, on food-taking behaviour. No effect is observed as a result of DRD3 antagonism on the

number of active lever responding (A) or the number of reinforcements obtained (C) as

compared to baseline responding under FR5 schedule of reinforcements. Also, no effect of

vehicle or drug is seen on inactive lever presses (B). (n=5, ns, p>0.05). Data are expressed as the

mean (±SEM) of active (A) or inactive (B) lever presses or reinforcements (Food pellets

obtained, C).

63

3.6 Histological Examination (Experiments 2-5):

The location of the tips of the microinjectors within the discrete brain areas

investigated in the current study is shown in Figure 3.8, Figure 3.9 and Figure 3.10Error!

Reference source not found.. Rats were only included in the study when the histological

analysis showed that the tips of the microinjectors, as evident by mechanical damage as

well as dye infusion, were bilaterally placed within the target site. Microscopic examination

revealed no damage, in the cellular tissue of the microinfusion field, other than the

mechanical damage caused by the penetration of the guide cannulae and microinjectors

(data not shown).

Figure 3.8 shows microinjector tips placement in the BLA, for rats used in the nicotine cue-

induced reinstatement experiment (n= 7, open circles) and for rats used in the food seeking

experiment (n=5, closed circles), Figure 3.9 shows microinjector tips placement in the

NAcc (n= 8), and Figure 3.10 shows microinjector tips placement in the LHb (n=6).

64

Cannulae Placement in the BLA

Location of the microinjector tips

in the BLA of the rats included in

the nicotine cue-induced

reinstatement experiment (–○–)

and the food taking experiment

(–●–) plotted on coronal sections

of the rat brain taken from the

atlas of (Paxinos and Watson,

1998). The numbers refer to the

anterior-posterior coordinates

relative to the bregma. The

arrows ( ) indicate the target

area.

-2.12 mm

-1.60 mm

-2.12 mm

-2.30 mm

-2.56 mm

Figure 3.8

65

1.70 mm

1.60 mm

0.70 mm

Cannulae Placement in the NAcc

Location of the microinjector tips

in the NAcc of the rats included

in the nicotine cue-induced

reinstatement experiment (–○–)

and the food taking experiment

(–●–) plotted on coronal sections

of the rat brain taken from the

atlas of (Paxinos and Watson,

1998). The numbers refer to the

anterior-posterior coordinates

relative to the bregma. The

arrows ( ) indicate the target

Figure 3.9

66

Cannulae Placement in the LHb

Location of the microinjector

tips in the LHb plotted on

coronal sections of the rat brain

taken from the atlas of (Paxinos

and Watson, 1998). The

numbers refer to the anterior-

posterior coordinates relative to

the bregma. The arrows ( )

indicate the target area.

-3.60 mm

-3.14 mm

-2.80 mm

Figure 3.10

67

3.7 Experiment (6): Responding maintained by light stimuli only

In this experiment, after the first few days of training, the responding on the active and

inactive levers became indistinguishable (Figure 3.11). A two-way ANOVA with repeated

measurements showed no significant effect of levers (F1, 14=1.01, p=0.33), a significant effect of

time (F19, 266=8.04, p<0.05) and significant lever x time interaction (F19, 266=2.49, p<0.05). Post-

hoc analysis, using LSD post-hoc test, revealed the effect of day 1 only to be approaching

significance (p=0.057).

Cue-lights per se are Unable to Maintain Stable Responding

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

20

40

60

80

100

120

140 Active Inactive

FR1 FR2 FR5

Day

Leve

r Pre

sses

Figure 3.11

This figure represents responding on the active (–●–) and inactive (–○–) levers during 1-h

sessions in which completion of the ratio requirement on the active lever resulted in illumination

of a cue-light followed by a 60-s time-out period and no delivery of nicotine. Data are expressed

as means (± S.E.M) of the number of lever presses under FR1, FR2 and FR5 schedules of

reinforcement. After a few days of training, responding on the active and inactive levers is

indistinguishable. Cues alone are unable to maintain significant responding on the active vs.

inactive lever (p=0.33).

Figure from (Khaled, Farid Araki et al., 2010), with permission.

68

3.8 Sample Size

The total number of animals used in the current study is 111 rats, 14 of which were used

for practicing the techniques of intracranial surgery and cannula placement. Table 3.1 indicates

the number of rats excluded from the study and the reasons for exclusion.

Table 3.1

The number of rats excluded from the current study and reasons for exclusion

Number of rats Reason for exclusion

11 Death: either for surgical (n=7) or other (n=4) reasons.

22 Catheter-related issues (including: blockade, failure, pain on flushing, etc.).

5 Failure of extinction

8 Cannula-related issues (including: blockade, bending, dislocation).

6 Aberrant cannula placement as evident by histological examination.

69

Chapter 4 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

4.1 General Discussion

The main objective of the current study is to investigate the involvement of the

dopamine D3 receptors in cue-induced reinstatement of nicotine-seeking behaviour. To this end,

the following stepwise approach was used:

Step 1- Evaluating the effect of systemically blocking the DRD3 on cue-induced reinstatement

of nicotine-seeking behaviour.

Step 2- Delineating the neural substrates through which the above effect of DRD3 on cue-

induced reinstatement of nicotine-seeking behaviour is mediated.

In the current study, nicotine intravenous self-administration paradigm was applied

(experiments 1-4), followed by extinction training and then reinstatement testing. In this

paradigm, the rats were trained to lever press for nicotine under gradually increasing fixed ratio

(FR) schedule of reinforcement (FR1 to FR5). Upon completion of schedule requirements, the

rats were rewarded by an infusion of nicotine (30μg/kg/infusion in a volume of 28-45μl

according to the weight of the rat), which was accompanied by the illumination of a cue light.

Such association imparts some reinforcing properties upon the cue light, which is originally

neutral in itself. Thereby, it gains secondary motivational salience and develops the ability to

reinstate extinguished nicotine-seeking behaviour in the absence of the primary reinforcer; i.e.,

nicotine in this case.

Self-administration training was carried out for a minimum of 15 sessions in the above-

mentioned experiments. During these sessions the rats obtained around 15 – 20 infusions of

nicotine / session, and accordingly the same number of cue presentations, adding up to around

225 – 300 cue-nicotine pairings during the course of the study. The length of training period and

the adequacy of the number of infusions / cue presentations earned possibly strengthened the

70

nicotine-cue association; thus, resulting in a robust reinstatement of the behaviour upon the

reintroduction of the cues after extinction.

In the current study a highly selective DRD3 antagonist (SB 277011-A) was used to

assess the effect of blocking DRD3 on cue-induced reinstatement of nicotine-seeking behaviour,

both systemically and locally. The systemic administration of SB 277011-A significantly

reduced cue-induced reinstatement of nicotine-seeking behaviour. Moreover, the same effect

was observed when SB 277011-A was infused into the BLA and LHb, but not in the NAcc.

Secondary objectives of the current study include:

1- Investigating the effect of blocking BLA DRD3 on food-taking behaviour. This was done

to exclude non-specific effects of intra-BLA infusion of SB 277011-A on operant behaviour or

general activity of the rats.

2- Investigating the motivational effects of cue-lights per se, and determining whether the

cue-induced reinstatement occurred due to previous association of the cue-lights with the

infusions of nicotine, or partially due to primary reinforcing effects of the cue-lights themselves.

4.1.1 Systemic Administration of SB 277011-A Attenuates Cue-induced Reinstatement of

Nicotine-seeking Behaviour

In the present experiment, nicotine, at the dose of 30μg/kg/infusion, supported vigorous

self-administration under a fixed ratio schedule of reinforcement in Long-Evans rats; which is

comparable to previously reported studies (Corrigall, 1999; Donny, Caggiula et al., 1995;

Forget, Coen et al., 2009; Paterson, Froestl et al., 2004; Paterson, Semenova et al., 2003;

Shoaib, Schindler et al., 1997).

71

The major finding of the present experiment is that the selective DRD3 antagonist (SB

277011-A) blocks cue-induced reinstatement of nicotine seeking (Figure 3.2), suggesting a role

for the DRD3 in the regulation of this process. Such a role is supported as the current findings

are consistent with previous studies indicating that antagonism at DRD3 attenuates reactivity to

nicotine-associated stimuli under pavlovian conditioning procedures (Le Foll, Schwartz et al.,

2003; Pak, Ashby et al., 2006), and blocks the expression of nicotine-induced place preferences

(Le Foll, Sokoloff et al., 2005; Pak, Ashby et al., 2006). It is important to note that the range of

doses producing the inhibition of reinstatement in the present experiment (i.e. 1, 3 and 10

mg/kg) is very similar to previous experiments [i.e. 1, 3 and 10 mg/kg in (Pak, Ashby et al.,

2006) and 3 and 10 mg/kg in (Le Foll, Schwartz et al., 2003)].

Interestingly, in the range of doses that are clearly DRD3 selective (such as the doses

used in the present study), SB 277011-A had no effect on nicotine self-administration under a

fixed ratio (FR) schedule of reinforcement (Andreoli, Tessari et al., 2003), or under a

progressive ratio (PR) schedule of reinforcement (Ross, Corrigall et al., 2007). Moreover, in the

range of doses tested, SB 277011-A had no effect on locomotor activity (Reavill, Taylor et al.,

2000) nor on responding for sucrose under a second order schedule of reinforcement (Di Ciano,

Underwood et al., 2003). This suggests that the effect observed in the current study is rather

specific to cue-induced reinstatement without an effect on nicotine-taking behaviour and that is

not due to other non-specific effects.

Several lines of evidence strongly suggest that at the above mentioned doses, SB

277011-A is acting at the DRD3 and not at the DRD2. First, SB 277011-A is highly selective

DRD3 antagonist, with 100 folds higher affinity to DRD3 than DRD2 (Reavill, Taylor et al.,

2000). Secondly, the doses selected and used here are far below those that produce effects

typically mediated by DRD2 blockade, such as inhibition of spontaneous locomotion or

72

stimulant-induced hyperlocomotion (up to 42.3 mg/kg SB 277011-A) and induction of

cataleptogenic effects or increase of plasma prolactin levels (up to 78. 8 mg/kg SB 277011-A)

(Reavill, Taylor et al., 2000). Therefore, the above effects appear to be mediated specifically

through the DRD3 and not through the DRD2.

It is interesting to note that a similar effect on cue-induced reinstatement of nicotine-

seeking behaviour was not obtained using the DRD3 partial agonist BP 897, which failed to

attenuate the reinstatement to nicotine seeking induced by previously associated cues (Khaled,

Farid Araki et al., 2010). This suggests that DRD3 partial agonists and antagonists are affecting

nicotine reinstatement differently. Previous studies have reported a dissociation of effects

between DRD3 partial agonists and antagonists. First, subtle differences between the effects of

BP 897 and SB 277011-A on responses maintained under second order schedule of

reinforcement for cocaine have been reported (Di Ciano, Underwood et al., 2003; Pilla,

Perachon et al., 1999). However, both ligands disrupted the responding during the first interval

(drug free), whereas only SB 277011-A decreased responding during the second-interval (after

cocaine infusion) (Di Ciano, Underwood et al., 2003; Pilla, Perachon et al., 1999). Secondly,

although, several studies found that the effects obtained with DRD3 partial agonists and

antagonists on reactivity to drug associated cues are convergent (Aujla, Sokoloff et al., 2002;

Gyertyan, Kiss et al., 2007; Heidbreder, Gardner et al., 2005), mixed findings have also been

reported with DRD3 partial agonists. A study comparing the effects of BP 897 and SB 277011-A

on cocaine-induced place conditioning found an absence of effect of BP 897 at doses ranging

from 0.3 to 3 mg/kg, whereas 3 mg/kg SB 277011-A was effective to block cocaine-induced

place conditioning (Cervo, Burbassi et al., 2005). Similarly, a discrepancy in the effects of

73

DRD3 blockade on opiate-induced place conditioning has been reported (Duarte, Lefebvre et al.,

2003; Francès, Smirnova et al., 2004; Vazquez, Weiss et al., 2007).

Another explanation is that the stimulation of the DRD3 due to the partial agonist profile may

participate in the absence of effect on reinstatement, as stimulation of dopamine receptors could

produce reinstatement of drug-seeking (Bachtell, Whisler et al., 2005) and stimulation of DRD3

potentiates DRD1-mediated responses (Le Foll, Schwartz et al., 2000; Marcellino, Ferre et al.,

2008).

Nonetheless, it appears that complete blockade of the DRD3, using an antagonist rather

than a partial agonist, is required to achieve significant reduction in the cue-induced

reinstatement of nicotine-seeking behaviour.

Taken together, the above findings indicate an important role for the DRD3 in the

modulation of cue-induced reinstatement processes. This role appears to be specific to

reinstatement and not to nicotine-taking behaviour, and is specifically mediated through the

DRD3.

The present findings, along with the findings that SB 277011-A has been shown to

significantly decrease the reinstatement of nicotine-seeking behaviour induced by nicotine

priming (Andreoli, Tessari et al., 2003), support the hypothesis that blockade of DRD3 by the

selective antagonist SB 277011-A could be effective to decrease relapse to nicotine-seeking.

4.1.2 Local Infusions of the DRD3 Antagonist SB 277011-A

The current group of experiments set out to identify some of the neural substrates

through which the effects of DRD3, on cue-induced reinstatement of nicotine-seeking behaviour,

are mediated. Based on compelling evidence of their involvement in reward-related processes

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(discussed in detail below), three areas of interest were chosen, namely; the BLA, the NAcc and

the LHb. The role of the DRD3 in each was investigated in relevance to cue-induced

reinstatement of nicotine seeking. In addition, the effect of blocking the DRD3 in the BLA on

food taking behaviour was evaluated. The findings of the current set of experiments will be

discussed in detail in the following sections.

4.1.2.1 DRD3 Antagonism in the BLA, Abolishes Cue-induced Reinstatement of Nicotine-

seeking Behaviour, but Has No Effect on Food Intake.

The major finding of the current experiments is that blockade of the DRD3, using local

infusions of the DRD3 antagonist SB 277011-A in the BLA, significantly and dose-dependently

reduced cue-induced reinstatement of nicotine-seeking behaviour (Figure 3.3). Although

significant levels of reinstatement were achieved in the vehicle treated group, shown by an

increase in the number of active lever responding relative to extinction baseline levels, upon the

infusion of SB 277011-A, dose-dependent reduction in the level of reinstatement was observed.

Such levels of reinstatement were not only significantly lower than those observed in the vehicle

treated group, but were also comparable (when the higher doses of SB 277011-A were used) to

the levels of responding during extinction. In addition, no changes were observed in the levels

of inactive lever pressing. This suggests a significant role for the DRD3 that are located in the

BLA in the modulation of the cue-induced reinstatement response. The lack of an effect of the

DRD3 blockade on inactive lever pressing suggests that the attenuation of reinstatement

observed was not due to non-specific effects of the drug.

Indeed, it is highly unlikely that the blockade of reinstatement observed in the current

experiment is attributed to motor deficits, since the infusion of SB 277011-A into the BLA, at

4μg/0.3μl/ side (i.e. 4 times higher than the highest dose used in the current study), did not have

any effect on rat spontaneous locomotor activity (Di Ciano, 2008). Moreover, neither the

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infusion of the highest dose of SB 277011-A (1μg/0.5μl/ side) nor the vehicle had an effect on

food taking behaviour under FR5 schedule of reinforcement (Figure 3.7); further discussed

below. This does not only support that, at the range of doses tested in the current study, SB

277011-A caused no locomotor deficits, but also excludes the possibility of other non-specific

effects like general anhedonia, generalized weakness or sedation which may result in alteration

in the pattern of the rats’ operant responding. This possibility is further excluded by the fact that

DRD3 antagonists enhance social interaction (Millan, Loiseau et al., 2008) and the evidence

from recent studies indicating the lack of the DRD3, in knock-out mice, show no impairments in

animal models of anhedonia or helplessness (Chourbaji, Brandwein et al., 2008).

Additionally, it is rather unlikely that the effects of the DRD3 blockade, observed in the

current study, are due to a disruption of other factors, which may have an effect on the form of

responding, tested under the current paradigm, such as an effect on cue-light perception,

memory or learning, or due to a decrease in attention. In fact, recent studies have shown that

blocking the DRD3 receptors may play an important role in enhancing some aspects of cognition

including memory, attention and learning (Millan, Di Cara et al., 2007; Sarter and Parikh, 2005).

This role is mediated through increasing the extracellular levels of acetylcholine, a

neurotransmitter critically involved in the above processes (Millan, Di Cara et al., 2007; Sarter

and Parikh, 2005), in the frontocortical regions of the brain (Lacroix, Hows et al., 2003).

Moreover, DRD3 antagonists were shown to improve learning deficits in memory impaired rats

(Laszy, Laszlovszky et al., 2005).

The effects observed in the current study are, therefore, most likely to be attributed to a

specific effect on the ability of conditioned stimuli, previously paired with the administration of

nicotine, to reinstate extinguished nicotine-seeking behaviour in the absence of nicotine

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(primary reinforcer). This result indicates the importance of DRD3 in the BLA in mediating the

effects of conditioned reinforcers.

The BLA was chosen as a primary target for the current study based on a strong and

established body of evidence implicating the BLA in the mechanisms underlying the process of

conditioned reinforcement. The amygdaloid complex is traditionally considered as an important

structure in the limbic system. It has been reported, however, that although the BLA is a

subcortical structure, it also has quasicortical properties (Carlsen and Heimer, 1988). The BLA

receives dopaminergic innervation from the VTA (Brinley-Reed and McDonald, 1999), a

connection which is of special interest in the current study. These dopaminergic inputs target

dopamine receptors expressed in the BLA (DRD1,2 or 3) (Gaspar, Bloch et al., 1995; Rosenkranz

and Grace, 1999), and contribute to the role of the BLA in the regulation of responding to

reinforcing stimuli (Nakano, Lenard et al., 1987).

Efferent projections from the BLA include dense innervation of most of the striatum, and it is

even considered that projections from the BLA constitute the main amygdalostriatal projections

(Kelley, Domesick et al., 1982). Being a corticolimbic structure and a part of the corticostriatal

system, especially in connection to the mesolimbic dopamine system, suggests a strong role for

the BLA in the motivational processes required for goal-directed behavioural responses.

Clinical studies have indicated a role for the amygdala in cue-association and cravings.

In humans, activation of the amygdala was observed upon the presentation of cocaine-related

cues, which was also associated with subjective reports of cravings (Childress, Mozley et al.,

1999). In addition, signal changes in response to drug-associated cues were observed in the

amygdala using functional magnetic resonance imaging (fMRI), as well as an increase in

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regional cerebral metabolic rate for glucose, which strongly correlated with measures of

cravings (Breiter and Rosen, 1999; Grant, London et al., 1996).

Behavioural studies, using animal models, also reported a clear involvement of the BLA

in conditioned reinforcement processes. An increase in cFos expression in the amygdala was

shown after the presentation of a cocaine-associated environment (Brown, Robertson et al.,

1992). Moreover, bilateral BLA lesions disrupted the responding of rats to conditioned

reinforcers previously associated with water (Cador, Robbins et al., 1989), second order

conditioning (Hatfield, Han et al., 1996), conditioned place preference for cocaine (Brown and

Fibiger, 1993), and abolished the reinstatement of cocaine-seeking behaviour induced by the

reintroduction of conditioned stimuli previously associated with cocaine (Meil and See, 1997).

BLA deactivation also decreased discriminative stimulus-evoked conditioned responses to

sucrose, without an effect on sucrose taking or consumption (Jones, Day et al., 2010a).

Additionally, although BLA lesions were without an effect on Pavlovian conditioning, rats with

bilateral excitotoxic lesions in the BLA were unable to acquire a new response to conditioned

reinforcement (pavlovian to instrumental transfer of control over behaviour by a conditioned

stimulus) (Everitt, Parkinson et al., 1999), or responding under a second order schedule of

cocaine self-administration (Whitelaw, Markou et al., 1996).

More specifically, the dopaminergic innervation to the BLA has been implicated in the

processes of stimulus-reward associations (Ciccocioppo, Sanna et al., 2001). Previous studies

have suggested a role for the DRD1 in the BLA in conditioned reinforcement, where the infusion

of a DRD1 antagonist in the amygdala disrupted conditioned fear expression (Lamont and

Kokkinidis, 1998), and the infusion of a DRD1 agonist in the amygdala potentiated conditioned

fear retention (Guarraci, Frohardt et al., 1999), while intra-amygdalar infusion of raclopride (a

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mixed D2/D3 antagonist) attenuated it (Guarraci, Frohardt et al., 2000). Moreover, the DRD1

antagonist SCH-23390, but not the mixed D2/D3 antagonist raclopride, attenuated conditioned

reinstatement of cocaine seeking, but not cocaine seeking per se (when unassociated with cues)

(See, Kruzich et al., 2001). This lack of effect of raclopride on cue-induced reinstatement could

be explained by its low selectivity for the DRD3. Nonetheless, a high dose of raclopride, infused

in the BLA prior to a single classical cue-association session, was able to block the acquisition

of cocaine-cue association (as evident by a failure of reinstatement induced by cues thereafter)

to a degree comparable to the DRD1 antagonist SCH-23390 (Berglind, Case et al., 2006).

In the current study, SB 277011-A was chosen due to its high selectivity to DRD3, which

facilitates the investigation of the specific roles of this receptor without being influenced by

actions on other dopamine receptors. It is therefore highly unlikely that the effects shown in the

current study are due to an action on either DRD1 or DRD2.

The present findings are in line with a recent study showing that the infusion of SB

277011-A (4μg/0.3μl/ side) into the BLA reduced cocaine seeking that is maintained by the

presentation of cocaine-associated cues, under a second order schedule of reinforcement (Di

Ciano, 2008). The discrepancy in the effective dose between the two studies can be explained by

the difference in the drug acting as a primary reinforcer (nicotine versus cocaine). A similar

difference was noticed in systemic studies, where SB 277011-A was able to block cue-induced

reinstatement of nicotine seeking at the doses of 1, 3 and 10 mg/kg (results shown above),

whereas doses of 10, 20 and 30 mg/ kg were required to block cue-induced reinstatement of

cocaine seeking or cocaine seeking under a second order schedule of reinforcement (Cervo,

Cocco et al., 2006). Another explanation could be the use of a different vehicle for dissolution

of SB 277011-A. In the current experiment, 10% DMSO in 10% w/v hydroxypropyl–β–

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cyclodextrin in sterile water was used as a vehicle for SB 277011-A, instead of 10% w/v

hydroxypropyl–β–cyclodextrin in sterile water alone. This was resorted to due to issues with the

solubility of the compound, which would have comprised difficulties in procedures for

microinfusions. A much better solubility was obtained upon adding a low concentration of

DMSO (10%) to the vehicle, which, perhaps, rendered it possible to observe an effect at such

low doses, as was seen in the current experiment. It is important to mention that careful

histological examination of the brain sections was performed to ensure that no damage has

occurred as a result of the infusion of DMSO (data not shown).

Structures adjacent to the BLA, especially the central nucleus of the amygdala (CeA),

may have an important role in processes underlying drug seeking and motivation. However, the

two areas have different roles in the processes underlying stimulus-reward associations, and the

control over the effect of conditioned stimuli on behavioural responses (as evident by

differential effects on conditioned suppression, appetitive pavlovian conditioning, conditioned

reinforcement and its potentiation by intra-NAcc D-amphetamine) (Everitt, Parkinson et al.,

1999; Killcross, Robbins et al., 1997). The effects shown in the current study are more in line

with a more evident role for the BLA than the CeA in controlling conditioned stimuli-induced

reinstatement. Furthermore, at the volume of infusion used in the present study (0.5 μl / side),

diffusion of the drug was limited to the BLA and not to the adjacent structures. This was

confirmed by localization of the dye, infused into the rat brains immediately before extraction,

to the BLA and not the adjacent structures. Therefore, it is not likely that the effects of SB

277011-A on cue-induced reinstatement of nicotine seeking, observed in the current study, are

due to its action on dopamine receptors in adjacent structures, including the CeA. Further

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studies exploring the effect of SB 277011-A infusion into the CeA will be of interest to better

elucidate the role of DRD3 in this specific area.

Importantly, the current study found no effect of intra-BLA infusion of SB 277011-A on

operant behaviour as assessed, in a separate experiment, by food taking under FR5 schedule of

reinforcement. Infusion of SB 277011-A (1 μg / 0.5μl / side), or vehicle, into the BLA resulted

in no change in the number of lever pressing (active or inactive) or, consequently, in the number

of reinforcements (food pellets) earned, as compared to baseline FR5 responding (Figure 3.7).

The fact that the rats were able to respond in an operant system for food (without noticeable

change) under the same treatment conditions, which completely abolished responding for

nicotine-associated cues, emphasizes the exclusion of general non-specific behavioural effects

of the drug (SB 277011-A) or vehicle, such as disruption in locomotion, attention or interest in

reward. Moreover, these results support the hypothesis that the blockade of cue-induced

reinstatement of nicotine seeking, shown above, is rather specific to blocking stimulus-reward

associations and the ability of conditioned stimuli to induce relapse. Consequently, the DRD3 in

the BLA appear to be involved mainly in cue-associations and processes of relapse rather than

primary reinforcement and reward processes.

In summary, the current experiments show a clear and strong inhibition of cue-induced

reinstatement of nicotine-seeking behaviour following the bilateral infusion of the selective

DRD3 antagonist, SB 277011-A, into the BLA. The present findings support the established role

of the BLA in stimulus-reward associations, enhance the understanding of the role of DRD3 in

the BLA in cue-induced reinstatement of nicotine seeking, and suggest a strong potential for the

use of selective DRD3 ligands in the prevention of relapse to smoking.

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4.1.2.2 DRD3 Antagonism in the NAcc Has No Effect on Cue-induced Reinstatement of

Nicotine-seeking Behaviour

In contrast to the above findings (Section 4.1.2.1), the present experiment showed that

intra-NAcc infusions of SB 277011-A had no effect on cue-induced reinstatement of nicotine-

seeking behaviour (Figure 3.4). Reintroduction of the nicotine-associated cues during

reinstatement testing, resulted in significant reinstatement of active lever responding in rats

treated with SB 277011-A (0.01 to 1 μg / 0.5 μl / side) to a level that was significantly higher

than their baseline level of lever pressing during extinction, and that was not significantly

different from their level of responding when treated with the vehicle. Therefore, blocking the

DRD3 in the NAcc is without effect on cue-induced reinstatement of nicotine seeking,

suggesting a lack of role for the NAcc DRD3 in this aspect.

The current data are consistent with a recent study that found no effect of blocking the

DRD3 in the NAcc shell, by microinfusions of SB 277011-A (at doses up to 4 μg / 0.3 μl / side),

on cocaine seeking that is maintained by cocaine-associated cues under a second order schedule

of reinforcement (Di Ciano, 2008). This suggests that the DRD3 in the NAcc may not be

involved in cue-association processes. The DRD3 in the NAcc, however, have been shown to

have a role in stress-induced reinstatement of cocaine seeking (Xi, Gilbert et al., 2004), which

was blocked by SB 277011-A infusions into the NAcc. The DRD3 in the NAcc, therefore, seem

to be involved in some, but not all, aspects of nicotine seeking and relapse.

Several considerations implicate the NAcc as a key structure in the drug-reward

circuitry. Anatomically, the NAcc comprises a part of the ventral striatum (Heimer, Alheid et

al., 1997), and acts as a junction / interface between cortical (allo- and periallo-cortical) and

limbic structures (Kelley, Domesick et al., 1982; Mogenson, Jones et al., 1980). Among the

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important innervations of the NAcc, which are of special interest in the current study, are: a) the

dopaminergic afferents from the VTA (especially to the shell) (Deutch and Cameron, 1992), and

b) the rich innervations from the BLA (Carlsen and Heimer, 1988). In addition, the NAcc is one

of the structures exhibiting very high densities of DRD3 expression in the brain (Diaz, Lévesque

et al., 1995).

The NAcc has an established role as an important neural substrate for drug seeking, drug

reinforcement and rewarding effects of drugs of abuse (Corrigall, Franklin et al., 1992; Pontieri,

Tanda et al., 1996). Previous studies have implicated the dopaminergic input to the NAcc in the

process underlying drug taking and self-administration, where the administration of 6-

hydroxydopamine, a dopaminergic neurotoxin, in the NAcc decreased cocaine self-

administration (Roberts, Koob et al., 1980), and NAcc DRD1 blockade attenuated the

reinforcing effects of cocaine as shown by an increase in cocaine self-administration under FR

schedules of reinforcements (Caine, Heinrichs et al., 1995; McGregor and Roberts, 1993).

Moreover, the infusion of a DRD1 or a DRD2-like antagonist (but not a DRD3 or a DRD4

selective antagonist) in the NAcc has been shown to decrease the reinforcing properties of

cocaine (Bari and Pierce, 2005). Reinstatement models have shown that the inactivation of the

NAcc inhibits cocaine-induced reinstatement of cocaine-seeking behaviour (McFarland 2001),

and that selective DRD3 antagonism by local infusions of SB 277011-A into the NAcc inhibits

stress-induced reinstatement of cocaine-seeking behaviour (Xi, Gilbert et al., 2004).

However, the involvement of the NAcc in conditioned reinforcement is more complex.

Such complexity is a result of the complex antomical structure of the NAcc. The NAcc is

differentiated into two subregions, namely; the shell and the core. This differentiation is not only

based on anatomical differences, but also, on pharmacological grounds. Whereas the NAcc core

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is considered to be more connected to the striatum, the NAcc shell seems to be leaning more

towards the limbic system (Deutch and Cameron, 1992) incorporating striatal neurons as well as

neurons of the extended amygdala (Heimer, Alheid et al., 1997). Functionally, the two

subregions have been shown to have differential effects on stimulus-reward associations.

Selective excitotoxic lesions of the core, but not the shell, disrupted pavlovian conditioning, as

well as conditioned reinforcement (Everitt, Parkinson et al., 1999). Moreover, inactivation of the

NAcc core, but not the NAcc shell, has abolished cue-induced reinstatement of cocaine seeking

(Fuchs, Evans et al., 2004). On the other hand, deactivation of the shell and not the core,

attenuated amphetamine-induced potentiation of conditioned reinforcement (Everitt, Parkinson

et al., 1999). Therefore, it can be concluded that the NAcc core plays a more important role in

cue-association processes than the NAcc shell.

As mentioned above, in 2004, Fuchs and co-workers found a decrease in cue-induced

reinstatement of cocaine-seeking behaviour following the inactivation of the NAcc core (Fuchs,

Evans et al., 2004). The fact that such an effect was not observed in the current study could be

attributed to the difference in the pharmocological agents used. Whereas a GABA-agonist was

used to deactivate the NAcc in the above study, the current experiment targeted specifically and

exclusively the DRD3 in the NAcc. It is worth mentioning, however, that a previous study,

where the undifferentiated NAcc was deactivated, also using a different pharmacological agent,

failed to show an effect on cue-induced reinstatement (Grimm and See, 2000).

The current experiment did not target each of the NAcc subregions (shell vs. core)

individually. It is possible that a different outcome would have been obtained had this been the

case. However, this is unlikely because in the current experiment the responding of the rats was

rather uniform, without remarkable variations, even though the intra-NAcc placement of the

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cannulae, and the site of infusion of the SB 277011-A, were either bordering the NAcc core and

NAcc shell or rather uniformly distributed between the two subregions (Figure 3.9).

Nevertheless, it cannot be absolutely excluded that the lack of an effect, of intra-NAcc DRD3

blockade on cue-induced reinstatement of nicotine seeking, demonstrated in the present study is

due to targeting the nucleus accumbens as a whole rather than individually targeting the DRD3

in the NAcc shell or the NAcc core. Further experiments are warranted to investigate such a

possibility.

Interestingly, it has been suggested that the amygdala is involved in the regulation of the

dopaminergic innervations of the NAcc. While the projections from the CeA to the VTA

suggest a mechanism as to how dopaminergic lesions of the CeA are reflected on dopaminergic

levels in the NAcc (Louilot, Simon et al., 1985), a recent study has shown that the BLA plays a

role in the modulation of NAcc dopamine signalling, without an effect on VTA-evoked

stimulation of dopamine release in the NAcc (Jones, Day et al., 2010a). Thus, it can be

suggested that the effect of the BLA on controlling stimulus-reward associations is mediated

through an interaction with the NAcc. However, in the current study, differential effects, on cue-

induced reinstatement of nicotine seeking, were found upon blocking the DRD3 in each of these

areas individually, where such blockade in the BLA completely abolished the response while no

effect was seen as a result of the same blockade in the NAcc. In other words, in the presence of

an intact BLA, DRD3 blockade in the NAcc per se is not enough to attenuate cue-induced

reinstatement. The case in the current study is different from the above studies in that, the

present blockade is specific to a single dopamine receptor, the DRD3. It is possible that blocking

the DRD3, the dopamine receptor exhibiting the highest binding affinity to dopamine (Sokoloff,

Giros et al., 1990), which leaves the other dopamine receptors in the NAcc (DRD2) free for the

dopamine to bind, plays a role in the reinstatement of the behaviour by the reintroduction of the

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cues. Nonetheless, it can be concluded, based on the present findings, that the DRD3 in the

NAcc are not mediators of cue-induced reinstatement processes.

In summary, the current study found no effect of DRD3 antagonism within the NAcc on

cue-induced reinstatement. These results are in line with the existing body of evidence

suggesting a role for the NAcc in aspects of drug-seeking behaviour other than cue-induced

relapse. According to the above results, the NAcc, also, serves as a neuroanatomical control

excluding that the action of SB 277011-A is extending beyond the sites it is administered. More

importantly, the present findings emphasise the regional selectivity of DRD3 in controlling cue-

induced reinstatement of nicotine-seeking behaviour.

4.1.2.3 DRD3 Antagonism in the LHb Attenuates Cue-induced Reinstatement of Nicotine-

seeking Behaviour

Another interesting and novel finding of the current study is that intra-LHb infusions of

SB 277011-A attenuate cue-induced reinstatement of nicotine-seeking behaviour (Figure 3.5). In

the present experiment, the reintroduction of the cue light previously associated with nicotine

significantly reinstated nicotine seeking behaviour in the rats upon treatment with the vehicle

only (in comparison to their baseline responding during extinction). However, when the rats

were treated with SB 277011-A (0.01-1 µg/ 0.5 µl / side), the reinstatement was significantly

reduced as compared to the vehicle treated group. It is important to note that, although the

attenuation of reinstatement in this group of rats was clear and statistically significant, the

reinstatement was not completely abolished, as was the case upon the antagonism of the DRD3

in the BLA. These findings indicate an important role for the LHb DRD3 in the regulation of

cue-induced relapse to nicotine seeking.

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The LHb is emerging as a neural site potentially involved in neuropsychiatric disorders

including depression, schizophrenia and drug addiction (Ellison, 1994; Lecourtier, Neijt et al.,

2004; Morris, Smith et al., 1999; Sartorius, Kiening et al., 2010). This region, previously

categorized as the motor subregion of the habenular complex (Herkenham and Nauta, 1977), has

been recently functionally reconsidered, and found to be additionally involved in several

processes including response to stress (Chastrette, Pfaff et al., 1991), learning (Lecourtier and

Kelly, 2007) and reward (Morissette and Boye, 2008; Vachon and Miliaressis, 1992).

Anatomically, the habenular complex comprises a part of the epithalamus (together with

the pineal body). The habenular complex is divided into two subregions; i.e. the medial (MHb)

and the lateral (LHb) habenular nuclei. In turn, the LHb is further divided into medial and lateral

subdivisions (Andres, von During et al., 1999).

The connections of the LHb play an important role in the range of functions mediated by this

structure. Afferents into the LHb arise mainly from the limbic regions and the basal ganglia

(Geisler and Trimble, 2008; Herkenham and Nauta, 1977). Among the important afferents

innervating the LHb is the strong input it receives from the ventral pallidum (Groenewegen,

Berendse et al., 1993), which is in turn densely innervated by the NAcc (Zahm, 1999).

Importantly, the main efferents of the LHb are directed towards the monoaminergic neurons-

containing nuclei, namely: the dorsal and median raphe (serotonergic neurons), lateral and

dorsal tegmental nucleus (cholinergic neurons) and the VTA (dopaminergic neurons)

(Herkenham and Nauta, 1979), all of which have been implicated in mechanisms underlying

psychiatric disorders and especially drug addiction. Through this circuit of connections, the LHb

can be considered to be acting as a converging point for the complex information coming from

the cerebral cortex, through the limbic and basal ganglia, and headed to the monoaminergic

nuclei in the brainstem. Thus, the connections of the LHb offer an explanation for the role

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suggested to be played by this structure in different psychological disorders, including drug

addiction, schizophrenia and depression.

In particular, the relationship between the LHb and the dopaminergic neurons in the

VTA is of special interest in the current study. Interestingly, it has been shown that some

dopaminergic neurons in the VTA project back to the LHb (Gruber, Kahl et al., 2007). In order

to understand the significance of such connections in the context of the current study, it is

important to briefly refer to the role of the dopamine neurons in such a context. As previously

discussed in Chapter 1, the dopamine neurons are involved in mediating reward signals and

reward prediction errors (Schultz, 1998). More specifically, the dopamine neurons have been

suggested to translate errors in prediction, and to be firing to promote behaviours that predict

reward and discourage behaviours that have negative consequences (Morris, Nevet et al., 2006;

Schultz, Apicella et al., 1993). In addition, they are involved in processing information about

novel environmental changes affecting motor behaviour (Redgrave and Gurney, 2006).

Studies have shown that stimulation of the LHb inhibits dopaminergic neurons in the

VTA as well as the in substantia nigra (Christoph, Leonzio et al., 1986), an inhibition which is

followed by delayed excitation (Ji and Shepard, 2007). Moreover, phasic inhibition of the LHb

resulted in decreased extracellular dopamine levels in the NAcc (Lecourtier, Defrancesco et al.,

2008). This effect has been hypothesized to be mediated through stimulation of GABA

interneurons in the VTA (Ji and Shepard, 2007). A recent study has also shown that

glutamatergic axons from the LHb terminate on GABAergic neurons in the VTA as well as

(although to a lesser degree) on dopaminergic neurons (Brinschwitz, Dittgen et al., 2010). These

connections offer an explanation to the tonic inhibition exhibited by the LHb on the dopamine

neurones.

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In primates, the response of the LHb was found to be opposing and preceding that of the

VTA dopamine neurons following the presentation of to reward-predictive or non-predictive

stimuli, and weak stimulation of the LHb resulted in the inhibition of the dopamine neurons

(Matsumoto and Hikosaka, 2007). In addition, recent studies have shown that electrolytic

lesions of the habenula attenuated brain stimulation reward (Morissette and Boye, 2008). In the

light of drugs of abuse, very few studies have investigated the effect of modulating the functions

of the LHb on drug taking and / or reinstatement (Ellison, 2002). In 2005, Zhang and colleagues

demonstrated that the reintroduction of heroin-associated cues, which reinstated extinguished

heroin-seeking behaviour, also increased cFos immunoreactivity in the LHb, a sign of neuronal

activation. Furthermore, a recent study, has found that deep brain stimulation (DBS), but not

lesioning, of the LHb decreased cocaine self-administration and promoted extinction behaviour

(Friedman, Lax et al., 2010). Additionally, LHb DBS was shown to attenuate cocaine-induced

reinstatement of cocaine-seeking behaviour without affecting sucrose taking, or locomotor

activity (Friedman, Lax et al., 2010). These data suggest a role for the LHb in drug conditioning

and reinstatement processes.

Taken together, the findings of the current study add to a growing body of evidence

supporting a role for the LHb-dopaminergic interaction in reward signalling, reward prediction

and drug conditioning. Indeed, the current study is the first to acknowledge and investigate the

role of DRD3 in the LHb on drug-seeking behaviour, suggesting an involvement of these

receptors in the role played by the LHb in cue-induced reinstatement of nicotine-seeking

behaviour.

It is important to mention that, a few studies have investigated the role of the MHb in

nicotine taking and withdrawal. Particularly, the nicotinic acetylcholine receptors (nAChRs) in

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the MHb have been explored for a potential role in these processes. Functional nAChR subunit

α4 have been demonstrated in the MHb (Fonck, Nashmi et al., 2009). It has also been suggested

that the MHb is involved in mediating the role of α2 and α5 subunits of the nicotinic

acetylcholine receptors in nicotine withdrawal (Salas, Sturm et al., 2009). Moreover, the MHb

nAChRs subunit α5 was suggested to be implicated in nicotine-taking behaviour, where

knocking down the MHb α5 subunit modulated brain-stimulation reward thresholds by blocking

the inhibitory effects of the higher doses of nicotine on brain reward (Fowler, Lu et al., 2010).

Since the MHb and the LHb are both relatively small structures, it could be suggested

that the microinfusions of SB 277011-A could have extended beyond the LHb to the MHb.

Although microinfusion of the dye at the infusion sites revealed localization of the infusion to

the LHb, it remains possible that some of the infused drug could have diffused into the MHb as

well. However, due to the lack of DRD3 in the MHb, and the selectivity of the DRD3 antagonist

used, it is not likely that the effect shown in the current study was mediated through the MHb as

well as the LHb.

The current study can be considered an addition to the increasing surge of interest in the

LHb in field of drug addiction in general. The current findings indicate a strong potential for the

LHb DRD3 in cue-induced reinstatement of drug seeking, and serve as a basis upon which

further studies can be directed exploring the role of the LHb DRD3 in the mechanisms

underlying drug-seeking behaviour.

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4.1.3 Cue Light per se is Unable to Maintain Active Lever Pressing Under a Fixed Ratio

Schedule

Another finding of the current study is that light stimuli had no motivational properties

of their own, under the present conditions. This was evidenced by the same level of responding

observed on the active and inactive levers after a few days of training (Figure 3.11). These

results are quite different from what has been reported by (Caggiula, Donny et al., 2002b) who

found a relatively higher level of responding on the active lever in animals pressing for saline

and cue-lights only. However, this discrepancy may be attributed to the difference in the strain

of animals used (Sprague– Dawley as opposed to the Long–Evans strain used in the present

study) or to the variability in the presentation and duration of the cue-light and/or houselight

extinctions in both studies. Further investigation is needed to outline the exact role of cue

presentation in the process of acquisition and reinstatement of drug-seeking behaviour. It has

been proposed that environmental stimuli are important contributors of nicotine self-

administration (Caggiula, Donny et al., 2002a). It could, therefore, have been expected that

blocking the DRD3 could produce a decrease of nicotine self-administration, as removal of

environmental cues could produce such a decrease in responding (Caggiula, Donny et al., 2001).

The lack of such an effect could be due to a different role of cues between different strains or

due to different experimental conditions. Further studies are required to delineate this. It is

interesting to note that in a recent study performed in naive squirrel monkeys, the responding of

nicotine-experienced animals was directed more towards delivery of nicotine than towards

presentation of the associated brief light stimuli (Le Foll, Wertheim et al., 2007). Further, after

repeated exposure to cycles of saline substitution, nicotine-associated stimuli, by themselves,

appeared unable to maintain significant self-administration behaviour under FR and PR

91

schedules (Le Foll, Wertheim et al., 2007). Those studies suggest that nicotine delivery may be

more important than cue presentation in maintaining self-administration behaviour.

92

4.2 Conclusions and Summary

The current study is the first report demonstrating the role of the DRD3 in cue-induced

reinstatement of nicotine-seeking behaviour systemically as well as locally.

The current study demonstrated that systemic administration of a selective DRD3

antagonist (SB 277011-A) results in significant attenuation of cue-induced reinstatement of

nicotine-seeking behaviour. This attenuation does not appear to be due to non-specific effects.

Furthermore, DRD3 antagonism does not affect nicotine taking. The effect observed in the

current study appears to be specifically mediated through the DRD3 and not the DRD2.

The current study also pointed out, using microinfusions of the same DRD3 antagonist into

discrete brain areas, that intra BLA and intra LHb antagonism of the DRD3 resulted in a clear

reduction in cue-induced reinstatement of nicotine seeking. These findings are consistent with

an established role for the BLA in cue-association processes and an emerging understanding of

the role of the LHb in the mechanisms underlying reward processing. Moreover, no such effect

was observed as a result of the DRD3 antgonism into the NAcc. This is further supported by the

existing body of evidence implicating the NAcc in drug reinforcement and reward rather than

stimulus-reward associations.

The main findings of the current study can be summarized as:

1. Blockade of the DRD3, using the selective DRD3 antagonist SB 277011-A, significantly

attenuates cue-induced reinstatement nicotine-seeking behaviour.

2. Antagonism of the DRD3 that are located in the BLA or the LHb results in a reduction in

cue-induced reinstatement of nicotine seeking.

3. Antagonism of the DRD3 that are located in the NAcc has no effect on cue-iduced

reinstatement of nicotine seeking.

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Taken together, the above results suggest a rather selective role for the DRD3 in the effect of

nicotine-associated cues and their importance as key factors in relapse to nicotine seeking, that

is not non-specifically mediated through all brain areas expressing the DRD3, but selective to

the BLA and LHb. This, may serve as an explanation as to why the DRD3 appear to be involved

in some aspects of nicotine-seeking behaviour and not the others.

The current study supports a body of evidence implicating the DRD3 in processes

underlying relapse to smoking, and substance use in general. Furthermore, the current findings

broaden and expand the basic understanding of the mechanisms and areas through which this

receptor exerts its role in the development of stimulus-reward associations which later on lead to

relapse in abstinent smokers.

Extrapolation of the current data to humans, suggests a strong potential for the DRD3 as a

target and for DRD3 selective antagonists as therapeutic agents for the prevention of relapse to

smoking.

94

4.3 Future recommendations

4.3.1 DRD3 Antagonism and Stress-induced Reinstatement of Nicotine-seeking

Behaviour

Stress is an important trigger of relapse to drugs of abuse, including nicotine, in abstinent

human users (Sinha, 2001). Blocking the DRD3 using SB 277011-A, significantly reduced

stress-induced reinstatement of cocaine-seeking behaviour. Interestingly, animal models have

not yet been used to explore the role of DRD3 in stress-induced reinstatement of nicotine

seeking. Such a factor cannot be ignored when studying the potential of selective DRD3 ligands

as therapeutic agents for the prevention of relapse to smoking. Such information will indeed be

needed before the current findings could be extrapolated to humans

Furthermore, the investigation of the neural substrates underlying such a role, if proven, would

be of high importance in elucidating the neurobiological circuitry of stress in regards of the

involvement of the DRD3.

4.3.2 Investigating the Effect of Modulating the Function of the LHb DRD3 on Other

Aspects of Nicotine-seeking Behaviour

The findings presented in the current study represent only a starting point highlighting

the role of the LHb DRD3 in cue-induced reinstatement of nicotine seeking. Further studies are

needed to elucidate the complete role of these receptors in this specific area in other aspects of

nicotine seeking, such as stress induced reinstatement and nicotine induced reinstatement of

nicotine seeking.

95

4.3.3 Effect of a DRD3 Agonist on Nicotine Taking and Reinstatement

The effects obtained by the selective DRD3 antagonist shown in the current study suggest

a strong potential for this category of ligands, as therapeutic agents for the prevention of relapse

to smoking. However, before such results could be extrapolated to humans, a missing piece of

information should be fulfilled; i.e., the investigation of the effect of stimulating the DRD3 on

different aspects of nicotine taking and nicotine seeking behaviour.

4.3.4 Investigating the Effect of Intracranial Administration of DRD3 Selective

Antagonists on Nicotine-priming-induced Reinstatement of Nicotine-seeking Behaviour.

DRD3 antagonism by SB 277011-A has been shown to block nicotine-induced

reinstatement of nicotine-seeking behaviour, in rats. However, the exact mechanisms underlying

this effect in the brain remain unknown. Targeting specific areas in the brain, with specific

DRD3 antagonist, is needed to assess the mechanisms underlying nicotine priming-induced

reinstatement and the role of intracranial DRD3 in such mechanisms.

4.3.5 Use of DRD3 Knock-out Mice to Confirm the Selectivity of SB 277011-A

Although the SB 277011-A is used as a highly selective DRD3 antagonist, such

selectivity can only be absolutely proven if the above findings fail to be replicated in DRD3

knock-out mice.

96

4.3.6 Replication of the Current Study Using Other Selective DRD3 Ligands to Confirm

the Results

One of the limitations of the current study is the use of only one drug as an example of

DRD3 antagonists. Future experiments should be performed using other novel DRD3 selective

ligands such as the S-22 and R-22 compounds (Newman, Grundt et al., 2009), to confirm the

results obtained in the current study, and also to expand the variety of ligands that can be used in

clinical studies at a later stage.

97

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http://www.wpro.who.int/media_centre/fact_sheets/fs_20060530.htm)�

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LIST OF PUBLICATIONS AND ABSTRACTS

Peer-reviewed Journal Publications

• Maram A.T.M. Khaled, Farid Araki, K., Li, B., Coen, K.M., Marinelli, P.W., Varga, J.,

Gaál, J., and Le Foll, B, (2010) “The Selective Dopamine D3 Receptor Antagonist SB

277011-A, but not the Partial Agonist BP 897, Blocks Cue-induced Reinstatement of

Nicotine-seeking”, International Journal of Neuropsychopharmacology, 13(2):181-190.

Conference Presentations

• Maram A.T.M. Khaled, Munmun Chatterjee, Peter W. Marinelli, Zane Qureshi and

Bernard Le Foll, “The Dopamine D3 Receptors in the Basolateral Amygdala and the Lateral

Habenula Modulate Cue-induced Reinstatement of Nicotine-seeking Behaviour” Society for

Research on Nicotine and Tobacco (Accepted – February, 2011).

Poster Presentations

• Maram A.T.M. Khaled, Keyghobad Farid Araki, Beth Li, Katheleen M. Coen, and Bernard

Le Foll, “Blockade of Cue-induced Reinstatement of Nicotine-seeking Behavior by Selective

Antagonist, but not by Partial Agonist of Dopamine D3 Receptors”, 35th Annual Harvey

Stancer Research Day (June, 2009).

APPENDICES

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APPENDIX I

Copyright Release Form

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Certainly. Good luck! Alan Alan Frazer, Ph.D. Professor and Chairman Department of Pharmacology UTHSCSA 7703 Floyd Curl Drive - MSC 7764 San Antonio, TX 78229-3900 [email protected] 210-567-4205 210-567-4300 (FAX) From: Maram Khaled [mailto:[email protected]] Sent: Tuesday, September 07, 2010 12:05 PM To: Frazer, Alan Cc: Hix, Ann W; Bernard Le Foll Subject: Permission for Reproduction of Information Dear Dr. Fraser, Editor in Chief, The International Journal of Neuropsychopharmacology, I have published a research paper in The International Journal of Neuropsychopharmacology, entitled: “The Selective Dopamine D3 Receptor Antagonist SB 277011-A, but not The Partial Agonist BP 897, Blocks Cue-Induced

Reinstatement of Nicotine-Seeking” (2010, 13(2): 181-190). Some of the work published in the above paper represents an important part of my M.Sc. project. Consequently, I needto include this work in my M.Sc. thesis, which will be submitted to the department of Pharmacology and Toxicology at theUniversity of Toronto, as a requirement for my degree completion. Therefore, I am hereby asking for your permission to reproduce some of the information from the paper (including some graphs) in my M.Sc. thesis. Please note that proper acknowledgement will be provided. I would appreciate if you can get back to me in this regards. Thanks in advance, Best regards, Maram Khaled, MD MSc / PhD Student Translational Addiction Research Laboratory Centre for Addiction and Mental Health University of Toronto 33 Russell Street, Toronto, ON, Canada M5S 2S1 Tel: 416.535.8501, ext. 4002 ______________________________________________________________________ This email has been scanned by the CAMH Email Security System. ______________________________________________________________________ ______________________________________________________________________ This email has been scanned by the CAMH Email Security System. ______________________________________________________________________

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APPENDIX II

Standard Operating Procedure for Intravenous Catheterization Surgery

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Author: M. Khaled Translational Addiction Research Lab Created: 11/06/2010 Modified from SOP# S019, Author: K. Coen, 1995

Standard Operating Procedure for

Intravenous Catheterization Surgery PURPOSE: Insertion of a catheter, in the rat’s right jugular vein, for delivery of drug/s in self-administration sessions. LAB. LOCATION: T618 INSTRUMENTS:

1. 2 syringes (1cc) fitted with blunted 22 gauge needle, 2. Small iris Scissors, 3. 2 pairs of forceps (1 straight, 1 curved), 4. Needle driver/holder, 5. Trocar (4 inch length of PE 190), 6. Catheter (please see SOP for Catheter Construction for catheter specifications), 7. Micro scissors, 8. Cyano acrylic glue (KRAZY Glue), 9. Suture needles (2-3).

All instruments should be autoclaved before the beginning of the surgeries (at the start of each day of surgery). Subsequently, in between the surgeries, the instruments can be sterilized by immersion in a cold sterilant (for e.g. Virox or Accel CS 20) for 20 minutes. The instruments must be cleaned thoroughly of blood residues before being placed in the sterilant. Everyday, at the end of the surgical procedures, the instruments should be cleaned, sonicated and left to dry then stored safely (please see SOP for Surgical Suite Disinfection for details). YOU WILL ALSO NEED:

1. 2 beakers (250 cc each) of the cold sterilant. 2. 1 beaker (250 cc) of saline, 3. 1 small vial of saline (10-20 cc).

All glassware should be autoclaved and the packs should be freshly opened on the day of surgery. SURGICAL PACKS: Each surgical pack consists of the following: • Surgical Draping, folded appropriately: 1 big square sheet (bottom liner), 1 smaller square sheet

(for moving the rat around), and 1 rectangular sheet folded length-wise in 2 (for the catheter). • Cotton-tipped Applicators (2-3) • Gauze • Suture thread (silk 4.0) around 45 cm (may vary by user) divided in 2.

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Author: M. Khaled Translational Addiction Research Lab Created: 11/06/2010 Modified from SOP# S019, Author: K. Coen, 1995 Surgical packs should be autoclaved ahead of time. Opened packs should be discarded or re-autoclaved before use. PREPARATION:

A) Catheter and surgical area preparation: • Catheters are flushed with the cold sterilant, checked for patency; pressure tested for leaks and

for length of the insertion tip (not longer than 30mm). Catheters should be immersed in the cold sterilant for 20 minutes.

• Turn the glass bead sterilizer on, and keep it ready for use in case of immediate need of sterilization during the surgery.

• Place a clean bench pad liner on the clean surgical table • Open a catheter pack and place the bottom liner draping on the bench pad liner, then the smaller

square sheet of draping on top of that one. Draping sheets should be handled only using the sterile forceps.

B) Rat Preparation: • Rats should be assessed on the day before the surgery to ensure their state of health can endure

the surgical stressor. • Rats are brought up to the surgical suite, weighed and the weight recorded on the surgical data

sheet before the procedure is started. It is recommended that the rat weighs at least 300 grams. • Adequacy of anesthesia can be confirmed by pinching the distal portion of the tail or the toes of

the hind limb and noting absence of a withdrawal reflex. • Shave the incision areas: 1- the right ventral region of the neck and 2- the dorsal area between

the scapulae. • Administer the preoperative medications as follows:

- Marcaine (1.25%) subcutaneously along the incision line (0.1 ml/site). - Ketoprofen (Anafen) subcutaneously. Please see SOP for preparation and dose. - Ringer lactate can be given for rehydration following the formula 10ml/kg/hr. For

surgeries < 45min. it’s up to the user to decide whether or not rehydration is needed. • Apply eye ointment (Lacrilube) bilaterally to avoid corneal dryness. • Clean the incision areas with betadine scrub (scrub 3 times in a circular motion starting from the

inside to the outside), rinse with 95% alcohol and wipe in the same way, then apply Betadine on the top.

• Move the rat to the surgical table and place on the prepared draping in dorsal recumbency.

C) Surgeon Preparation: • Hands should be washed thoroughly. • Sterile gloves should be opened and donned at the surgical table, immediately prior to the

beginning of the surgery. Care should be taken to minimize touching non-sterile surfaces including the body of the rat. Handling the rat thereafter should be done using the surgical draping.

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SURGICAL PROCEDURE: • Make an oblique incision of the skin in the ventral region of the right side of the neck. Clear the

superficial muscle layer by blunt dissection. • Locate the jugular vein and using finely serrated forceps, strip it of all fascia. Run a locating

suture beneath the vein to facilitate later retrieval. • Place the rat in ventral recumbence and make a transverse incision between the scapulae. By

blunt dissection, clear a subcutaneous pocket in the tissue surrounding the incision. It must be large enough to accommodate both the mesh assembly and the excess catheter tubing.

• Insert the needle driver into the dorsal incision and direct it behind the fight forearm and towards the ventral incision. In order to make this “tunnel” as superficial as possible, the tips of the driver should be pointed upwards during this process. Punch through the connective tissue to emerge at the ventral incision. Open the jaws of the driver and securely grab the trocar. Relock the jaws and pull the trocar to the dorsal incision by gently twisting the needle driver. Once the trocar has been “threaded” through both incisions, the catheter may be passed through the trocar. It must be fed in a dorsal-ventral direction; i.e. the insertion tip is introduced into the trocar from the dorsal end. Once a suitable length of catheter is visible at the ventral site, the trocar may be removed simply by pulling it out through the ventral incision. This procedure, while cumbersome, ensures that there is no stress exerted on the catheter, thereby protecting its integrity.

• Orient the catheter such that the silastic runs medial to the PE and all tubing will ultimately lie flat. Attach a 1 cc syringe with a 22 blunted needle to the catheter and flush with sterile saline. Ensure that there are no bubbles in the line.

• Lift the jugular and using micro-scissors, make an incision 2/3 of the way through the vein. Release the vein.

• Grasp the silastic tip of the catheter and insert into the jugular incision. The tubing should feed in freely, all the way to the heat shrink. It may be necessary to adjust the amount of PE tubing present at the incision site; pulling on the scapular end may do this. Once inserted, the entire assembly should lie flat and neat.

• Tie off the catheter to the jugular with two sutures, one at each end of the heat shrink connection. It is vital that the suture does not slip off onto the silastic, as it will occlude the tubing.

• Anchor the PE 10 tubing to deep muscle with a single suture. The heat shrink is then secured to underlying tissue by a single drop of Krazy glue on its underside.

• Verify the patency of the catheter by drawing back on the syringe and obtaining blood in the PE tubing. Flush back into the rat with a small amount of saline (0.1 ml)

• Close the superficial muscle layer with 1-2 sutures. This serves as additional protection should the rat scratch at his incision. Close the skin with interrupted sutures.

• Turn the rat over and disconnect the syringe from the catheter. Cap the catheter with a filled silastic plug.

• Feed the excess PE 20 tubing into the subcutaneous pocket in a looping fashion. It is quite normal for it to encircle the incision. Insert the mesh assembly and work with it until it lies flat, centered between the scapulae, and superficial to all tubing.

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• Suture the skin around the nylon bolt, making sure neither to purse the skin nor catch the mesh in the sutures.

• Move the rat to the recovery area for immediate post surgical care. IMMEDIATE POST-OPERATIVE CARE: • Upon completion of the surgery, rats should be moved to the recovery area, and placed on a

thermostatically controlled heating blanket (to avoid anaesthetic-induced hypothermia). • Rats should NEVER be left unattended during the recovery period. • Topical antiseptics could be applied to the surgical wounds. For example, Cicatrin® cutaneous

powder. • Rats should be monitored closely to ensure post-operative analgesia. • Breathing, heart beats, warmth and colour of the paws should be assessed regularly. Upon full recovery from anaesthesia, rats should be moved to a bedded cage lined with paper towels (to avoid inhalation of bedding) until transported to their respective home cage.

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APPENDIX III

Standard Operating Procedure for Intracranial Cannulae Implantation Surgery

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Author: M. Khaled Translational Addiction Research Lab Created: 26/10/2010

Standard Operating Procedure for

Intracranial Cannulation Surgery PURPOSE:

Insertion of guide cannulae into discrete areas in the rat’s brain, for guiding the insertion of mincroinjectors for the delivery of drugs intracranially. LAB. LOCATION: T618 INSTRUMENTS:

1. Stereotaxic frame mounted with cannula holders (especially if using Plastics One cannulae) and containing ear bars,

2. Scalpel and sterile scalpel blades, 3. 4 bulldog clamps for retraction of the scalp, 4. 1-2 Guide cannulae (to be inserted), 5. 1 pair of forceps (preferably curved), 6. Skull mounting screws, 7. Screwdriver, 8. Tap drill, 9. Dental drill equipped with a drill bit, 10. Dental acrylic powder and jet acrylic fluid as a solvent, 11. Spatula, 12. Petri dish / metal / porcelain dish for dissolving the dental cement.

All instruments should be autoclaved before the beginning of the surgeries (at the start of each day of surgery). Subsequently, in between the surgeries, the instruments can be sterilized by immersion in a cold sterilant (for e.g. Virox or Accel CS 20) for 20 minutes. Alternatively a hot (glass) bead sterilizer can be used for disinfection of instrument tips in between the surgeries. The instruments must be cleaned thoroughly of blood residues before being placed in the sterilant. Everyday, at the end of the surgical procedures, the instruments should be cleaned, sonicated and left to dry then stored safely (please see SOP for Surgical Suite Disinfection for details). YOU WILL ALSO NEED:

1. 2 beakers (250 cc each) of the cold sterilant. 2. 1 beaker (250 cc) of saline, 3. A calculator, 4. Data sheets with the specific insertion coordinates and, 5. A pencil

All glassware should be autoclaved and the packs should be freshly opened on the day of surgery.

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Author: M. Khaled Translational Addiction Research Lab Created: 26/10/2010 SURGICAL PACKS:

Each surgical pack consists of the following: • Surgical Draping, folded appropriately: 1 big rectangular sheet (bottom liner; between the rat

and the stereotaxic), 1 smaller rectangular sheet (blanket; covers the back of the rat), and 1 small square sheet with a circular hole in the middle (head cover; optional),

• Cotton-tipped Applicators (5-6), • Gauze, • 1-2 obturators (dummy cannulae).

Surgical packs should be autoclaved ahead of time. Opened packs should be discarded or re-autoclaved before use. PREPARATION:

A) Cannulae and surgical area preparation: • Cannulae should be autoclaved ahead of time or sterilized using the hot bead sterilizer.

Cannulae should not be sterilized using the cold sterilant. • Place the cannulae into mounting holders and secure them in place using the side screws on the

holder. • The hot bead sterilizer should be kept on and ready for use in case of immediate need of

sterilization during the surgery. • Place a clean bench pad liner on the clean surgical table and place the stereotaxic on top of it. • A “Lazy Suzan” or turning tray can be placed on top of the bench pad liner and under the

stereotaxic to facilitate moving the stereotaxic device. • Open a surgical pack and place the bottom liner draping on the stereotaxic. Draping sheets

should be handled only using the sterile forceps.

B) Rat Preparation: • Rats should be assessed on the day before the surgery to ensure their state of health can endure

the surgical stressor. • Rats are brought up to the surgical suite, weighed and the weight recorded on the surgical data

sheet before the procedure is started. It is recommended that the rat weighs at least 300 grams. • After the anaesthetic is delivered, adequacy of anaesthesia can be confirmed by pinching the

distal portion of the tail or the toes of the hind limb and noting absence of a withdrawal reflex. • Shave the incision area at the top of the skull. • Administer the preoperative medications as follows:

- Marcaine (1.25%) subcutaneously along the incision line (0.1 ml/site). - Ketoprofen (Anafen) subcutaneously. Please see SOP for preparation and dose. - Ringer lactate can be given for rehydration following the formula 10ml/kg/hr. For

surgeries < 45min. it’s up to the user to decide whether or not rehydration is needed. • Apply eye ointment (Lacrilube) bilaterally to avoid corneal dryness. • Clean the incision area with betadine scrub (scrub 3 times in a circular motion starting from the

inside to the outside), rinse with 95% alcohol and wipe in the same way, then apply Betadine on the top.

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Author: M. Khaled Translational Addiction Research Lab Created: 26/10/2010 • Move the rat to the surgical table and place on the prepared draping on the stereotaxic. Insert the

ear bars into the rat’s ear canals. Signs for successful correct insertion include blinking and / or nose pointing straight and to the midline. Tighten the ear bar enough to keep the head from moving, but taking care not to over-tighten resulting in the rupture of the rat’s ear drums.

• Place the incisors on the incisor bar and tighten it into place. • Drape the body of the rat and the skull using the autoclaved surgical drapings.

C) Surgeon Preparation: • Hands should be washed thoroughly. • Sterile gloves should be opened and donned at the surgical table, immediately prior to the

beginning of the surgery. Care should be taken to minimize touching non-sterile surfaces. Handling the rat thereafter should be done using the surgical draping.

SURGICAL PROCEDURE:

• Make a midline incision in the scalp using the scalpel blade. Retract the skin by pulling on the fascia using the bulldog clamps.

• Scrape off the periosteum using the scalpel blade and / or cotton-tipped applicator. Bregma and Lambda should be exposed and clear.

• Level the skull horizontally by taking the DV coordinates of Bregma and Lambda. Allow for 0.05 mm difference, otherwise change the nose piece position up or down to achieve a flat horizontal level of the skull.

• Three dimensional coordinates (AP, ML, and DV) of Bregma are noted and cannulae insertion coordinates are calculated accordingly, and marked. Set the stereotaxic arms to the new coordinates (AP and ML) and swing them out of the way temporarily.

• Use a tap drill to make 3-4 holes in the skull to insert the mounting screws using a screwdriver. • Use the dental drill to make a hole in the skull at the sites already marked for cannulae insertion.

Drilling is best performed perpendicularly to the skull. Use a thin sterile needle to puncture the dura. In case of excessive bleeding, use a cotton tipped applicator or gauze to ensure stoppage of the bleeding prior to cannulae insertion.

• Swing the stereotaxic arms back into place, taking care to align them at perfect zero (so their previous position is not altered). Carefully lower the cannulae to the set DV coordinates.

• Prepare the dental cement and carefully apply over the cannulae using the spatula, taking care to cover a good proportion of the cannulae protruding above the skull surface without cementing the cannulae to the cannulae holder.

• Wait for the cement to completely dry before attempting to move or unscrew the holders out of place.

• Place the obturators (dummy cannulae) in place. • Gently remove the bulldog clamps, and remove the rat from the ear and incisor bars. • Move the rat to the recovery area for immediate post surgical care. PLEASE NOTE:

- If the insertion requires angulations, the angles should be set right at the beginning of the procedure and all the coordinates should be noted and calculated accordingly.

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- For some areas, especially if the insertion is at an angle, the DV coordinates are best read at the skull surface at the target insertion point.

- Screws should not be mounted on suture lines. The depth of insertion of the screws should only be halfway into the skull to avoid widening of the holes or cracking of the bone.

- If you are inserting bilateral cannulae into a structure that is close to the midline, it may be difficult to insert both cannulae at the same time. Alternatively, you can insert the cannula on one side and cement it in place. Care should be taken, in such a case, not to occlude or cover the target site on the other side with the cement. Once the cement has completely dried and the holder is removed, the other cannula can be inserted. A thin layer of cement should then be added to cover the 2 sides homogonously.

IMMEDIATE POST-OPERATIVE CARE:

• Upon completion of the surgery, rats should be moved to the recovery area, and placed on a thermostatically controlled heating blanket (to avoid anaesthetic-induced hypothermia).

• Rats should NEVER be left unattended during the recovery period. • Topical antiseptics could be applied to the surgical wounds. For example, Cicatrin® cutaneous

powder. • Rats should be monitored closely to ensure post-operative analgesia. • Breathing, heart beats, warmth and colour of the paws should be assessed regularly. Upon full recovery from anaesthesia, rats should be moved to a bedded cage lined with paper towels (to avoid inhalation of bedding) until transported to their respective home cage. • Following intracranial surgery, rats may need soft food to avoid painful mastication resulting

from ear and incisor bars insertion (Please refer to the SOP for post operative care for details). ABBREVIATIONS:

DV: Dorsal / Ventral. AP: Antrior / Posterior. ML: Medial / Lateral.

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THE ROLE OF THE DOPAMINE D RECEPTORS IN CUE-INDUCED ... › bitstream › 1807 › ... · 3 RECEPTORS IN CUE-INDUCED REINSTATEMENT OF NICOTINE-SEEKING BEHAVIOUR . Maram A.T.M. Khaled