The Extinction of Drug Seeking: a Role for the Nucleus Accumbens Shell

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The Extinction of Drug Seeking: A Role for the Nucleus Accumbens Shell

E. Zayra Millan

Doctor of Philosophy / Masters of Psychology (Clinical) Thesis

September 2011

School of Psychology

The University of New South Wales

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Millan

First name: Zayra

Abbreviation for degree as given in the University calendar: PhD/MPsychol(Clinical)

School: Psychology Faculty: Science

Title: The Extinction of Drug Seeking: A Role for the Nucleus Accumbens Shell

Abstract 350 words maximum:

Relapse following abstinence from drug use remains the major impediment for the treatment of addiction. Major advances have been made in understanding the brain systems that promote relapse. Much less is known about the brain systems that promote abstinence. The focus of the present dissertation was to investigate the brain systems that promote abstinence by examining the neural substrates underlying the extinction of reward seeking in rats. The first series of experiments (Chapter 3) studied the role of the nucleus accumbens shell (AcbSh) and its interactions with lateral hypothalamus (LH) in mediating the extinction of alcoholic beer seeking. These experiments demonstrated that functional inactivation of the AcbSh prevented the expression of extinction and so reinstated previously extinguished alcoholic beer seeking. It had no effect on the acquisition of extinction. Moreover, the reinstatement produced by AcbSh inactivation was associated with c-Fos expression in hypothalamus, including in orexin- and CART- containing neurons in LH. It was also shown that concurrent inactivation of LH blocked reinstatement produced by AcbSh inactivation. Finally, contralateral but not ipsilateral disconnection of AcbSh from anterior LH prevented expression of extinction, suggesting that serial AcbSh→LH communication is necessary for extinction of reward seeking. The second series of experiments (Chapter 4) studied the role of AcbSh AMPA type-glutamate receptors and interactions with the basolateral amygdala (BLA) in mediating extinction of alcoholic beer seeking. These experiments demonstrated that intra-AcbSh infusions of the AMPA receptor antagonist, NBQX, attenuated the expression of extinguished responding. This effect was dose-dependent and specific to the behavioural expression of extinction; it had no effect on measures of non-extinguished responding, including context-induced reinstatement, initial extinction acquisition, and responding on a progressive ratio schedule. This chapter also reported that contralateral but not ipsilateral disconnection of the BLA from AcbSh prevented the expression of extinction, suggesting that serial BLA→AcbSh communication is necessary for the extinction of reward seeking. However, in the final experiment, reversible inactivation of the BLA failed to prevent the expression of extinction. Together, the findings from this thesis suggest that a circuit involving BLA→AcbSh and AcbSh →LH actively promotes expression of extinction of reward seeking in rats.

Declaration relating to disposition of project thesis/dissertation

I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.

I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.

……………………………………………………………

E. Zayra Millan

Signature

Table of Contents

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Originality Statement

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.

Signed ……………………………………………......

Date ……………………………………………......

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Acknowledgements

I wish to thank my supervisor, Prof. Gavan McNally. From the inception to completion of this thesis, your support and guidance has been enormous. I met with many challenges throughout the process, especially in juggling this research with clinical training. But, there was not a day when I did not look forward to running these experiments. Thank you for your patience and cheerful encouragement, even if it were just to say, “hang in there”. I have truly enjoyed working with you and learning from you, and hope that I will continue to do so in the years to come.

I also wish to thank my co-supervisor, Prof. Fred Westbrook, for your direction in seeking the early appetitive learning literature and for your ongoing support. Thank you also to the wonderful members of the McNally lab, both current and past, especially

Dr. Nathan Marchant and Dr. Teri Furlong, for your technical assistance throughout the years. I have sorely missed our casual discourses on the neuroanatomy of the accumbens and the hypothalamus. Thank you also to Lucy Choi, Christina Perry,

Andrea Willcocks, Helen Nasser, and Macy Chan, as well as to my friends and colleagues on the 6th floor of the Mathews Building.

This thesis would not have been completed so joyfully without my office mates of room 1114b: Genevra Hart, Emma Campbell-Smith, Anna McCarrey, and Adrian

Camilleri. I cannot imagine what these last three years would have been like without the four of you.

Finally, to my fiancé, Ray Wu. Thank you for everything.

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Publications

Millan, E. Z., Furlong, T. M., & McNally, G. P. (2010). Accumbens shell-hypothalamus

interactions mediate extinction of alcohol seeking. Journal of Neuroscience, 30,

4626–35. This publication forms the basis of Chapter 3 in the present

thesis.

Millan, E. Z., Marchant, N. J., & McNally, G. P. (2011). Extinction of drug seeking.

Behavioural Brain Research, 217, 454–462. This publication forms the basis

of Chapters 1 and 2 in the present thesis

Millan, E. Z. & McNally, G. P. (2011). Accumbens shell AMPA receptors mediate the

extinction of reward seeking through interactions with basolateral amygdala.

Learning and Memory, 18, 414-421. This publication forms the basis of

Chapter 4 in the present thesis.

Marchant, N. J., Millan, E. Z., & McNally, G. P. (2011). The hypothalamus and the

neurobiology of drug seeking. Cellular & Molecular Life Sciences, (in press).

Oral presentations

Millan, E. Z. & McNally, G. P. (2011). Cocaine- and amphetamine-regulated transcript

attenuates context-induced reinstatement of alcohol-seeking. European

Behavioural Pharmacology Society, Amsterdam, Netherlands.

Millan, E. Z. & McNally, G. P. (2011). Accumbens shell AMPA receptors mediate

extinction of reward seeking through interactions with basolateral amygdala. 5th

CINP Pacific-Asia Regional Meeting. Kuala Lumpur, Malaysia.

Millan, E. Z. & McNally, G. P. (2009). Accumbens shell-hypothalamus interactions

mediate extinction of alcohol seeking. Psychology Postgraduate Conference,

Sydney, Australia.

Conference Proceedings

Millan, E. Z. & McNally, G. P. (2011). Cocaine- and amphetamine-regulated transcript

in the nucleus accumbens shell attenuates context-induced reinstatement

(renewal) of reward seeking. Neurosciences, Washington, DC.

Millan, E. Z. & McNally, G. P. (2011). Accumbens shell AMPA receptors mediate

extinction of reward seeking through interactions with basolateral amygdala.

Gordon Conference on the Amygdala in Health, Portland, ME.

McGinty, V. B., Hayden, B. Y., Heilbronner, S. R., Dumont, E. C., Graves, S. M.,

Mirrione, M. M., … & Haber, S. (2011). The Reward Circuit: Emerging,

Reemerging, and Forgotten Brain Areas: Notes from the 2010 Motivational and

Neural Networks Conference. Behavioural Brain Research, (in press).

Millan, E. Z. & McNally, G. P. (2010). Accumbens shell AMPA receptors mediate

extinction of reward seeking in a rat. Neurosciences, San Diego, CA.

Millan, E. Z., Furlong, T. M., & McNally, G. P. (2010). Nucleus accumbens shell

mediates extinction of reward seeking through interactions with lateral

hypothalamus. Federation of European Neurosciences (FENS), Amsterdam,

Netherlands.

Millan, E. Z., Furlong, T. M., & McNally, G. P. (2010). Nucleus accumbens shell

mediates extinction of reward seeking through interactions with lateral

hypothalamus. Motivation Neuronal Network (MNN), Shell Island, NC.

Millan, E. Z., Furlong, T. M., & McNally, G. P. (2009). Accumbens shell-hypothalamus

interactions mediate extinction of alcohol seeking. Brain Sciences UNSW,

Sydney, Australia.

Awards

Australian Postgraduate Award (2008-2011)

Gordon Conference Travel Award (2011)

Motivation and Neuronal Networks Travel Scholarship (2010)

UNSW Brain Sciences Poster Prize (2009)

Care and Use of Animals

The experiments presented in this thesis conformed to the guidelines on the ethical use of animals maintained by the Australian Code of Practice for the Care and Use of

Animals for Scientific Purposes (7th Edition), and all procedures were approved by the

Animal Care and Ethics Committee at the University of New South Wales. All efforts were made to minimise both suffering and the number of animals used.

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List of Tables

Table Table title Page

1 Mean (SEM) counts of total c-Fos-IR, orexin-IR and CART-IR. 105

2 Mean (SEM) counts and percentages of dual orexin/c-Fos-IR and 105

CART/c-Fos-IR cells.

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List of Figures

Figure Figure title Page

1 Reinstatement of extinguished drug seeking 12

2 Context-induced reinstatement of extinguished drug seeking (renewal). 13

3 Cortico-striatal loops 23

4 Afferent and efferent connectivity of nucleus accumbens core (AcbC) 29

and shell (AcbSh)

5 Distinct preferential inputs and outputs of AcbSh subregions: dorsal 32

tip, medial AcbSh and ventrolateral AcbSh.

6 Schematic diagram illustrating connectivity between AcbSh, PVT, LH, 47

and VTA.

7 Schematic summary of spiralling organization of ventral striatal and 49

dorsolateral striatal connectivity with midbrain VTA and SN.

8 Circuit-level summary of brain regions mediating context-induced 55

reinstatement.

9 Circuit-level summary of brain regions mediating extinction of drug 80

seeking and putative junctions with structures involved in context-

induced reinstatement

10 Experiment 1: AcbSh microinfusion cannula placements. 90

11 Experiment 1: Mean ± SEM responses during extinction and test. 92

12 Experiment 2: AcbSh microinfusion cannula placements. 95

13 Experiment 2: Mean ± SEM responses during test (extinction days 1 96

and 2).

14 Experiment 3: AcbSh microinfusion cannula placements. 103

15 Experiment 3: Mean ± SEM responses during extinction and test. 104

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16 Photomicrograph of a representative coronal hypothalamus section 106

indicating approximate boundaries used for cell counts.

17 Photomicrographs of representative dual-labelled c-Fos/orexin-IR, 107

single-labelled orexin, and single-labelled Fos-IR in the hypothalamus.

18 Photomicrograph of representative dual-labelled c-Fos/CART-IR, 108

single-labelled CART-IR and single-labelled Fos-IR in the

hypothalamus.

19 Hypothalamus: Mean ± SEM counts for c-Fos-IR, dual-labelled with c- 112

Fos/orexin-IR and dual-labelled with c-Fos/CART-IR in hypothalamus

subregions.

20 Amygdala. Photomicrograph of representative coronal section and 113

representative dual-labelled c-Fos/CART-IR cells in CeA.

21 Arcuate Nucleus. Photomicrograph of representative coronal section 114

and representative dual-labelled c-Fos/CART-IR cells.

22 PVT. Photomicrograph of representative coronal section and 115

representative c-Fos -IR cells.

23 Experiment 3: Mean ± SEM number of cells expressing c-Fos-IR in 116

amygdala, PVT and Arc. Mean ± SEM number of dual-labelled CART

cells in CeA and Arc.

24 Experiment 4: AcbSh and LH microinfusion cannula placements. 121

25 Experiment 4. Mean ± SEM responses during extinction and test 122

26 Experiment 5: AcbSh and LH microinfusion cannula placements. 127

27 Experiment 5: Mean ± SEM responses during extinction and 128

subsequent test.

28 Experiment 5: Mean ± SEM responses during test for rats with anterior 131

and posterior LH infusions. Mean ± SEM responses following

unilateral bicuculline in LH.

29 Experiment 6: AcbSh microinfusion cannula placements. 150

30 Experiment 6: Mean ± SEM numbers of active and inactive responses 152

during extinction and test.

31 Experiment 7. AcbSh microinfusion cannula placements. 156

32 Experiment 7. Mean ± SEM numbers of active and inactive responses 157

during extinction and subsequent test.

33 Experiment 8. AcbSh microinfusion cannula placements. 161

34 Experiment 8: NBQX effects on extinction learning and on a 162

progressive ratio test.

35 Experiment 9: AcbSh and cBLA microinfusion cannula placements. 167

36 Experiment 9: Mean ± SEM responses during extinction and 168

subsequent test.

37 Experiment 10: cBLA microinfusion cannula placements. Mean ± 173

SEM responses during extinction and test.

38 BLA and extinction expression 198

39 Parallel circuits for reinstatement and extinction of drug seeking 201

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Abbreviations

Acb Nucleus Accumbens

AcbC Nucleus Accumbens Core

AcbSh Nucleus Accumbens Shell

AcbSh-D Nucleus Accumbens Shell (Dorsal)

AcbSh-V Nucleus Accumbens Shell (Ventral)

ACC Anterior Cingulate Cortex

AMPA D-amino- 3-hydroxy-5-methyl-4-isoxazolepropionate

Arc Arcuate nucleus

BIC Bicuculline

B/M Baclofen/Muscimol

BLA Basolateral Amygdala

CART Cocaine- and Amphetamine-Regulated Transcript cBLA Caudal Basolateral Amygdala

CeA Central Nucleus of the Amygdala

CS Conditioned Stimulus

CTb Cholera Toxin b Subunit

DAB Diaminobenzidine

DH Dorsal Hippocampus

DLS Dorsolateral Striatum

DMH Dorsomedial Hypothalamus dmPFC Dorsomedial Prefontal Cortex f Fornix

GABA γ-Aminobutyric acid

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IF interfascicular nucleus i.m. Intramuscular i.p. Intraperitoneal i.u. International Units i.v. Intravenous ilPFC Infralimbic Prefrontal Cortex

IR Immunoreactive

KOR Kappa Opioid Receptor

LH Lateral Hypothalamus lOFC Lateral Orbitofrontal Cortex

LHb Lateral Habenula

м Molar

MDT Mediodorsal Thalamic Nucleus

MDH Medial Dorsal Hypothalamus mt Mammillothalamic Tract mGluR Metabotropic Glutamate Receptor mPFC Medial Prefrontal Cortex

MSN Medium Spiny Neuron

Narp Neuronal activity-regulated pentraxin

NHS Normal Horse Serum

NMDA N-methyl-o-aspartate rBLA Rostral BLA

PB Phosphate Buffer

PBS Phosphate Buffer Saline

PeF Perifornical Hypothalamus

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PFC Prefrontal Cortex plPFC Prelimbic Prefrontal Cortex

PSD Postsynaptic density

PVT Paraventricular Thalamus rBLA Rostral Basolateral Amygdala s.c. Subcutaneous

SN Substantia Nigra

STN Subthalamic Nucleus

TTX Tetrodotoxin

US Unconditioned Stimulus vAgI Ventral Agranular Insular Cortex

VH Ventral Hippocampus vmPFC Ventromedial Prefrontal Cortex

VP Ventral Pallidum vSub Ventral Subiculum

VTA Ventral Tegmental Area

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Introduction

The World Health Report noted that 8.9% of the total burden of disease comes from the use of psychoactive substances (Rodgers, 2002). Among the disorders related to psychoactive substances, alcohol-use disorders rank as 5th leading cause of global burden of disease in high-income countries (WHO, 2008). Alcohol-use disorders are associated with increased rates of physical health problems including cardiovascular and liver disease; violence and suicide; mental health problems including depression and anxiety; economic loss related to reduced levels of productivity, as well as costs related to the utilisation of health, social welfare and criminal justice services (WHO, 2007).

Moreover, like other substance dependent disorders, alcohol dependence, is chronic and prone to relapse. Even following conventional pharmacological or psychological treatment, relapse to harmful drug and alcohol use is estimated to range from 40 to 90 per cent, depending on the definition of relapse (Finney, Moos, & Timko, 1999; Jin,

Rourke, Patterson, Taylor, & Grant, 1998; Miller, Walters, & Bennett, 2001; Moos &

Moos, 2006). In comparison to other psychopathologies, relapse rates for alcohol and other forms of substance dependence are typically higher than those of depression and anxiety-related disorders (Brandon, Vidrine, & Litvin, 2007; Finney, Hahn, & Moos,

1996). Therefore, in light of the individual and social problems associated with alcohol misuse, the vulnerability to relapse is a primary concern for treating practitioners and a focus of empirical study among researchers of addiction.

The brain systems that mediate the return to drug use following a period of abstinence is a major focus among studies of relapse. Major advances have been made to characterise the neural systems that influence behaviour in the presence of identified

Introduction

triggers of relapse including stress, environmental cues, and exposure to the drug itself

(Stewart, 2010). More recently, the brain systems that promote the ability to control drug use when exposed to sources of relapse have emerged as a focus of research. In the present thesis, the approach taken towards investigating these systems of control is to examine the neural systems that mediate extinction of drug seeking. Extinction denotes the reduction in drug seeking when the contingency between drug seeking or drug predictive stimuli and the delivery of drug reward is broken. In Pavlovian learning, extinction occurs when a conditioned stimulus (CS) that has been associated with a biologically significant event (i.e. unconditioned stimulus (US)) is subsequently presented repeatedly in the absence of the US. It is thought that this process reduces craving in the presence of drug-associated cues. In operant or instrumental learning, extinction occurs when an action or behaviour that has been associated with a reinforcer

(e.g. drug reward) is no longer reinforced.

Extinction is a fundamental process of adaptive behavioural change. It is procedurally simple, yet its underlying behavioural and neurobiological processes are not well-defined. At best, the extinction of drug seeking is understood to involve a psychological process that actively inhibits performance of drug seeking behaviour.

This will be discussed further in Chapter 1. The focus of the present thesis is to characterise the brain systems mediating this active extinction process. Understanding these neural mechanisms has implications for current neurobiological approaches to relapse. At present, little is known of how the brain systems recruited during extinction are interconnected with those that mediate relapse. This connectivity is of practical interest since processes with potential to influence relapse circuitry are possible targets for relapse intervention.

Chapter 1. Behavioural Basis of Extinction

Chapter 1.

The Behavioural Basis of Extinction

This chapter reviews the behavioural literature to provide a theoretical framework within which to study the neurobiological substrates underlying the extinction of drug seeking. It begins with an introduction to animal models of relapse to drug seeking, focusing primarily on drug-prime, stress-, cue-, and context-induced reinstatement. A key feature of these models is that reinstatement always follows the extinction of drug seeking. Thus, extinction does not erase previous learning but instead involves an active process of new learning. This concept will be elaborated further in Section 1.2, which focuses on the behavioural mechanisms underlying the extinction of drug seeking. The purpose of this chapter is to highlight the theoretical link between relapse and extinction; namely, that extinction involves an active context-dependent process that competes for behavioural expression against processes mediating relapse to drug seeking. The implication of this behavioural mechanism is that a comprehensive system for relapse involves both a facilitatory system that promotes drug seeking and also a regulatory system that is recruited during extinction, to enable control over drug seeking.

1.1. Animal models of relapse to drug seeking

Animal models of relapse are founded upon two observations from early behavioural research in rats. First is the observation that rats readily respond for (i.e. self-administer) a range of drugs of abuse, including intravenously delivered cocaine, heroin, and D- amphetamine (Ettenberg, Pettit, Bloom, & Koob, 1982; Pickens & Thompson, 1968;

Yokel & Wise, 1976). These responses are typically exercised in the form of a lever- 3

Chapter 1. Behavioural Basis of Extinction

press or nose-poke. The second observation is that although responding for food or water can be extinguished in a rat, subsequent non-contingent presentations of food or water can reinstate this lever-pressing behaviour (Skinner, 1983). The combination of these two observations is termed “reinstatement of drug seeking” (Davis & Smith, 1976;

Shaham, Shalev, Lu, de Wit, & Stewart, 2003), where “drug seeking” refers to the performance of a previously trained drug-reinforced response (e.g. lever pressing behaviour) and “reinstatement” refers to the resumption of the previously extinguished drug seeking response following non-contingent exposure to a priming dose of the drug or drug-associated stimuli (Shaham et al., 2003).

Reinstatement of drug seeking was initially demonstrated in squirrel monkeys trained to respond via lever-press for intravenous infusions of D-amphetamine or cocaine (Gerber & Stretch, 1975; Stretch, Gerber, & Wood, 1971). The authors showed that although drug seeking readily extinguished when saline was substituted for the drug reinforcer, the extinguished response was reinstated following a non-contingent intramuscular injection of D-amphetamine or cocaine (Gerber & Stretch, 1975; Stretch et al., 1971). In rats, reinstatement of extinguished drug seeking was first demonstrated in morphine-trained rats following non-contingent injections of morphine and also following exposure to a morphine-paired cue (Davis & Smith, 1976; Smith & Davis,

1973). These latter studies emphasised the implications of their findings in terms of stimulus control mechanisms that underlie relapse in drug users. However, de Wit and

Stewart (1981) were the first to propose the utility of the reinstatement preparation for the explicit purpose of modelling triggers of relapse. The reinstatement model has since been used to study the behavioural, pharmacological, and neurobiological profile of relapse to drug seeking under various conditions (e.g. in the presence of a drug reward, drug-related cues or contexts, or following an acute stressor).

Chapter 1. Behavioural Basis of Extinction

There are three experimental phases in a typical model of reinstatement: drug self-administration training, extinction, and test for reinstatement. During self- administration training, animals self-administer a drug reinforcer via a behavioural response, typically a lever-press or nosepoke. In contemporary experimental designs, two levers are present such that responses on the ‘active’ lever result in the delivery of the drug reward whereas responses on the ‘inactive’ lever are without consequence.

This inactive lever assesses the behavioural specificity of the reinstatement observed on test. Once drug-taking is stabilised, lever pressing is extinguished such that responding on either the active or inactive lever is not reinforced. This results in a decline in the frequency of responding. The rats are subsequently tested for reinstatement. If responding on the active lever on test for reinstatement is significantly higher than extinction levels of responding, reinstatement is said to have occurred. As will be discussed, reinstatement can be observed following non-contingent administration of the previously self-administered drug (drug-prime reinstatement), exposure to a stressful event, or presentation of drug associated stimuli (cue- and context-induced reinstatement.

Drug-prime reinstatement: Described as an “appetiser effect”, early studies have shown that a small priming dose of alcohol can elicit craving and drinking in detoxified alcoholics (Ludwig, Wikler, & Stark, 1974). Similarly, a small dose of cocaine elicits craving in cocaine users (Jaffe, Cascella, Kumor, & Sherer, 1989). It is thought that craving is a proximal source of relapse (Ferguson & Shiffman, 2009; O'Brien, 2005) and indeed, there is some evidence that craving is associated with relapse (O'Malley,

Jaffe, Rode, & Rounsaville, 1996).

The reinitiation of drug seeking following re-presentations of the drug reinforcer can be modelled in the rat. For example, in de Wit and Stewart (1981), rats surgically

Chapter 1. Behavioural Basis of Extinction

fitted with an intravenous catheter were trained to lever-press for infusions of cocaine, followed by extinction. The authors observed that subsequent non-contingent administration of cocaine dose dependently reinstated cocaine seeking. Reinstatement was also observed in cocaine-trained rats following non-contingent injections of amphetamine, apomorphine, and morphine, but not ethanol, heroin, or methohexital, suggesting that reinstatement is specific to the properties of the drug administered during training. Interestingly, previous studies have shown that drug-prime reinstatement interacts with the presence of drug-paired cues. For example, an external stimulus that normally accompanies drug infusion, such as a masking noise (Stretch et al., 1971), is required for reinstatement to occur. Moreover, Davis and Smith (1976) showed that in morphine-trained rats, a priming dose of morphine was able to restore responding even in the presence of a previously extinguished drug-paired cue. This finding is interesting as it suggests that a lapse in an abstinent drug user can potentially alter their response to extinguished drug-paired cues. Reinstatement has since been demonstrated in other drugs of abuse including heroin (Shaham & Stewart, 1995), methamphetamine (Reichel & See, 2010), ethanol (Backstrom, Bachteler, Koch, Hyytia,

& Spanagel, 2004), and nicotine (O’Connor, Parker, Rollema, & Mead, 2010).

Stress-induced reinstatement. Both acute and chronic stress are thought to significantly increase vulnerability to relapse in abstinent drug users (Sinha, 2001).

Similarly, in the rat, exposure to an acute stressor can reinstate a previously extinguished drug seeking response. In the earliest demonstration, Shaham and Stewart

(1995) trained then extinguished lever pressing for intravenous heroin. Reinstatement of heroin seeking was observed following exposure to 10 minutes of intermittent and inescapable 1.0 mA footshock. Moreover, the reinstatement observed was comparable to that following a priming injection of heroin and persisted despite a prolonged (4 to 6

Chapter 1. Behavioural Basis of Extinction

week) drug free period (Shaham & Stewart, 1995). A subsequent study also observed higher levels of stress-induced reinstatement following 6 and 12 days of heroin withdrawal, suggesting that heroin-withdrawal may increase vulnerability to stress- induced relapse (Shalev, Morales, Hope, Yap, & Shaham, 2001a). Footshock induced reinstatement has since been observed in rats trained to self-administer cocaine (Erb,

Shaham, & Stewart, 1996), alcohol (Martin-Fardon, Ciccocioppo, Massi, & Weiss,

2000), and nicotine (Buczek, Lê, Wang, Stewart, & Shaham, 1999), but interestingly, not in rats trained to respond for sucrose (Buczek et al., 1999; Shalev et al., 2001a).

These findings suggest that stress-induced reinstatement via footshock may be specific to drugs of abuse. A final point is that footshock is not the only stressor capable of producing reinstatement. Stress-induced reinstatement has also been observed following

24 hour food deprivation (Shalev, Yap, & Shaham, 2001b), as well as following administration of anxiolytic agents such as the α-2 adrenoreceptor antagonist, yohimbine, or the stress hormone, corticotropin releasing factor (Cippitelli et al., 2010;

Shaham et al., 1997).

Cue-induced reinstatement. Drug-associated cues have long been thought to contribute to relapse via processes of conditioned withdrawal (Lynch, Fertziger,

Teitelbaum, Cullen, & Gantt, 1973; Wikler, 1948), conditioned compensatory response

(Siegel, 1975), or by taking on the incentive properties of the drug reinforcer (Stewart, de Wit, & Eikelboom, 1984). Regardless of the process, early studies have shown that responses to cues associated with drug rewards can be classically conditioned. For example, repeated pairings of environmental cues with alcohol can elicit conditioned responses such a pulse transit time, finger temperature, and tolerance to alcohol (Dafters

& Anderson, 1982; Newlin, 1986). Moreover, in abstinent alcoholics, cues related to alcohol can elicit cravings; salivation; and instrumental alcohol-seeking behaviour, for

Chapter 1. Behavioural Basis of Extinction

example, pressing a button for alcohol (Hutchison, 2001; Ludwig et al., 1974; Monti et al., 1987). Similarly, smoking cues as well as neutral laboratory-conditioned stimuli paired with tobacco have been shown to increase the number of cigarette puffs in regular smokers (Hogarth, Dickinson, & Duka, 2010). These studies suggest an important role for drug-paired cues in the maintenance of drug dependence.

In the laboratory, the ability for discrete cues to maintain drug seeking in the absence of the drug reinforcer can be modelled in the rat using a cue-induced reinstatement preparation. In an early demonstration, Davis and Smith (1976) trained rats to lever press for an intravenous infusion of morphine. The conditions were arranged such that each lever press resulted in the presentation of an auditory buzzer concurrent with morphine infusion. Lever pressing was then extinguished in the absence of the buzzer. On test, re-presentation of the buzzer in the absence of morphine robustly reinstated the lever-pressing response. In a subsequent study, de Wit and Stewart (1981) demonstrated that cue-induced reinstatement of drug seeking was dependent on the association between the cue and the drug reward. In this study, rats were trained to self- administer cocaine (via lever-press) such that a tone was either simultaneously paired with each drug infusion (correlated group) or non-contingently presented (uncorrelated group). Rats subsequently received a period of extinction in the absence of tone presentations followed by test involving re-exposure to the tone. Although cocaine was not present during test, reinstatement of extinguished responding was observed in the correlated group but not the uncorrelated group. This suggests that the explicit pairing of a cue with a drug reward is important for observing cue-induced reinstatement of drug seeking. Together, both studies provide early evidence that stimuli associated with previous drug use might facilitate the re-initiation of drug use via processes of associative learning.

Chapter 1. Behavioural Basis of Extinction

Context-induced reinstatement (renewal). Similar to cue-induced reinstatement, background environmental stimuli or contexts paired with previous drug use can also trigger relapse. This has been studied in the context-induced reinstatement preparation, although the origins of this model are based on the “renewal” phenomenon. That is, when training and extinction occur in separate contexts, re-exposure to the original training context “renews” the extinguished response (Bouton & Bolles, 1979; Welker &

McAuley, 1978). Crombag and Shaham (2002) were the first to extend the renewal phenomenon as a model to study context-induced reinstatement of drug-seeking. These authors trained rats to lever-press for “speedball”, a heroin-cocaine mixture, in a two lever operant chamber (Context A). Responding on one ‘active’ lever was reinforced with intravenous infusions of speedball whereas responding on the other ‘inactive’ lever was not reinforced. Subsequently, rats received extinction in either the same context (A) or in a discretely different context (B). Finally, the animals were tested under extinction conditions in either the training context (A) or in the extinction context (B). The group of interest was the renewal group, ABA. These animals showed significant context- induced reinstatement when compared to control animals that were tested in the same context as extinction (groups AAA and ABB) or control animals that received test in a novel context (AAB).

Renewal is a robust behavioural phenomenon. It can be observed despite extensive extinction training. For example, in a fear conditioning preparation, Rauhut,

Thomas, and Ayres (2001) reported that the amount of renewal shown after 20 extinction trials was no different after 100 extinction trials. Similarly, Zironi, Burattini,

Aicardi, and Janak (2006) found that in ethanol-trained rats, the magnitude of renewal

(context-induced reinstatement) observed immediately after extinction training was maintained three weeks later. Bossert, Liu, Lu, and Shaham (2004) also showed that in

Chapter 1. Behavioural Basis of Extinction

heroin-trained rats, the magnitude of context-induced reinstatement is similar to animals that were tested without prior extinction training. Finally, context-induced reinstatement has been reliably demonstrated across other drug and non-drug reinforcers including heroin (Bossert, Wihbey, Pickens, Nair, & Shaham, 2009; Bossert, Gray, Lu, &

Shaham, 2006; Bossert et al., 2004; Bossert, Poles, Wihbey, Koya, & Shaham, 2007;

Bossert et al., 2011), cocaine (Crombag & Shaham, 2002; Fuchs, Eaddy, Su, & Bell,

2007; Fuchs et al., 2005; Fuchs, Ramirez, & Bell, 2008; Hamlin, Clemens, & McNally,

2008), alcohol (Burattini, Gill, Aicardi, & Janak, 2006; Hamlin, Newby, & McNally,

2007; Zironi et al., 2006), nicotine (Diergaarde, de Vries, Raasø, Schoffelmeer, & De

Vries, 2008), sucrose (Hamlin, Blatchford, & McNally, 2006), and food (Nakajima,

Tanaka, Urushihara, & Imada, 2000; Nakajima, Urushihara, & Masaki, 2002; Welker &

McAuley, 1978). This is despite differences in the route of reward administration (e.g. intravenous, oral) or differences in training parameters.

Summary

The reinstatement phenomenon has enabled researchers to model in the rat proximal sources of relapse in human drug users, including exposure to the drug reinforcer, acute stress, and stimuli paired with previous drug use. Each of these forms of reinstatement is illustrated in Figures 1 and 2. Reinstatement has been reliably demonstrated across multiple drugs of abuse, and there is some evidence that stress-induced reinstatement may be specific to drug-reinforcers. A common link between variants of the reinstatement procedure is that reinstatement is measured against a background of extinguished drug seeking. Indeed, extinction is necessary to demonstrate that there is a reduction in the motivation to acquire the drug reward prior to relapse. Although it has been argued that relapse is “rarely, if ever, preceded by the elimination of drug seeking behaviour through an extinction process” (Epstein, 2006; Katz & Higgins, 2003, p. 28), 10

Chapter 1. Behavioural Basis of Extinction

the reinstatement of drug seeking following an extinction process has important theoretical and practical implications for the study of relapse. First, it demonstrates that extinguished drug-seeking behaviour can be recovered under specific conditions. This suggests that the content of what is learned during initial drug taking is not erased following extinction. An obvious practical implication is that cue-exposure therapy, an extinction-based behavioural treatment for relapse prevention among drug users may have limited long-term efficacy. Indeed, previous research suggests that this form of treatment is ineffective in preventing relapse in the long term (Conklin & Tiffany, 2002;

Drummond & Glautier, 1994). Second, in these models of relapse, reinstatement is the inverse behavioural expression of extinction. Therefore, to the extent that the mechanisms of reinstatement in an animal model inform the mechanisms of relapse in abstinent drug users, then understanding extinction may provide insight into mechanisms that regulate relapse to drug seeking.

1.2. Behavioural basis of extinction

Several psychological explanations for the decline in responding during extinction are available. These include a reduction in the generalisability of conditions present during reinforcement versus non-reinforcement (i.e. generalisation decrement) (Capaldi, 1967); the development of a competing emotional response (e.g. frustration) that disrupts expression of the conditioned response (Amsel, 1992); a deterioration in the representation of the outcome (Rescorla & Heth, 1975); and an increase in the threshold at which the representation of the outcome can be activated (Konorski, 1948; Rescorla

& Cunningham, 1977; Rescorla, 1979). One popular approach is the view that extinction involves the weakening or loss of the originally learned response-producing

Chapter 1. Behavioural Basis of Extinction

D. Self administration training Extinction Reinstatement test

Drug-seeking

Days

Figure 1. Reinstatement of extinguished drug seeking.

There are three experimental phases in a typical model of reinstatement: self- administration training, extinction, and test for reinstatement. There is no drug delivered during extinction or reinstatement test. Reinstatement can be induced following pre-test administration of a drug-prime (A), acute stress (B), or presentation of a drug-paired cue

(C). Panel D depicts schematic behavioural data during each of the three experimental phases.

Chapter 1. Behavioural Basis of Extinction

Test for context-induced reinstatement

Self-administration training Extinction Context A

Context A Context B

Context B

Self administration training Extinction Renewal test

Context A Context B Context A

Context B Drug-seeking

Days

Figure 2. Context-induced reinstatement of extinguished drug seeking (renewal).

There are three experimental phases in a typical context-induced reinstatement

preparation: self-administration training in Context A, extinction in Context B, and test

for reinstatement in contexts A and B. There is no drug delivered during extinction or

renewal test. The bottom panel depicts schematic behavioural data during each of the

three experimental phases.

Chapter 1. Behavioural Basis of Extinction

association that was formed during initial conditioning (Estes, 1955; Rescorla &

Wagner, 1972; Rumelhart, Hinton, & Williams, 1986). This implies that extinction is a process of ‘unlearning’ or forgetting. However, that reinstatement of extinguished drug seeking occurs (as presented in the previous section) suggests that an extinguished response can be recovered; that much of the original learning actually remains intact despite the loss of responding during extinction. An alternative approach that accounts for reinstatement following extinction is the view that extinction does not erase previous learning but instead, produces a new inhibitory association that competes with and therefore prevents performance linked to the originally learned association (Konorski,

1967; Pavlov, 1927; Pearce & Hall, 1980; Wagner, 1981). Although multiple processes likely contribute to extinction, the present section focuses on this latter approach, namely, the memory-interference model proposed by Bouton and his colleagues

(Bouton & Ricker, 1994; Bouton, 1993, 1994; Bouton & Nelson, 1994). This approach is the most influential behavioural model of extinction. Moreover, in the present thesis, it serves as the behavioural basis for relating extinction with reinstatement of drug seeking.

Bouton’s memory interference model assumes that drug self-administration and subsequent extinction experiences are represented as two distinct memories: an excitatory action-outcome association formed during initial training that survives extinction, and a new inhibitory association formed during extinction between the action and a new representation, “no outcome”. A consequence of this new inhibitory association is that the meaning of the response becomes ambiguous; it has become associated with the presence and absence of the outcome, and both associations compete for control over behaviour. According to the model, behavioural performance depends on whether the excitatory or inhibitory association is retrieved at the time of testing.

Chapter 1. Behavioural Basis of Extinction

Moreover, retrieval of the extinction memory is dependent on the presence of the context in which extinction training occurred. Specifically, the model predicts that if a subject is placed outside the context of where extinction occurred, then the absence of the extinction context results in failure to retrieve the extinction memory. Instead, memory of the original training is retrieved and renewal of extinguished responding is observed. Recent data in food-seeking animals supports this model (Bouton, Todd,

Vurbic, & Winterbauer, 2011). In this study, rats were trained to lever press for food pellets in one context (Context A), and this responding was extinguished in a separate and distinct context (Context B). Not surprisingly, these animals showed low

(extinguished) levels of responding when subsequently returned to the extinction context, B. However, responding was reinstated when the rats were subsequently returned to the training context, A. This phenomenon, described as ABA renewal, has been demonstrated across other drug and non-drug reinforcers as a model of context- induced reinstatement, which was described earlier. Importantly, it suggests that extinction involves an active suppression or masking of responding and that this suppression is context-dependent. Bouton et al. (2011) provide additional evidence to support this context-dependent extinction process. They showed that when responding

(lever pressing) for food pellets is trained and also extinguished in Context A, exposure to a second and distinct context (Context B) also reinstates the previously extinguished reward seeking behaviour (i.e. AAB renewal). Similarly, when training and extinction occur in separate contexts (A and B), subsequent exposure to a third novel context

(Context C) reinstates responding (i.e. ABC renewal). Thus, as predicted by the memory interference model, in each of these cases removal from the extinction context is sufficient to unmask or recover the previously extinguished behaviour.

Chapter 1. Behavioural Basis of Extinction

There are however, several aspects relating to the memory interference model that remain unclear. First, Bouton and his colleagues (2011) observed that the effect size of renewal was larger under the ABA condition than in AAB renewal. The memory- interference model does not account for this discrepancy. One explanation is that extinction responding was lower when measured in Context B than in Context A

(Bouton et al., 2011). This might predict differences in the magnitude of subsequent renewal behaviour. However, Bouton and colleagues (2011) present two findings that speak against this possibility. First, the effect size of renewal under ABC conditions was small despite extinction occurring in a separate context, B. Second, in an AAB renewal design, facilitating extinction performance by increasing the number of extinction sessions had no effect on the magnitude of renewal expressed when animals were tested outside of the extinction context. These authors also tested the possibility that conditioning of contextual cues during training in Context A augments responding on test when animals are returned to this context. They used an ABA renewal design and subsequent to each extinction session in Context B, the animals also received exposure to Context A (no levers present) to extinguish any context-reinforcer associations.

However, this manipulation had no effect on renewal of responding. The authors suggest that Context A possibly acquires “occasion setting” properties, which, as discussed later in this section, are resistant to extinction. However, this is yet to be tested directly. Thus, the behavioural mechanisms that facilitate responding under ABA renewal relative to AAB and ABC renewal are unclear on the basis of the findings in

Bouton et al. (2011). Moreover, previous studies have failed to demonstrate renewal of drug seeking in a novel context (ABC renewal) using the following drug reinforcers: speedball (Crombag & Shaham, 2002); ethanol (Zironi et al., 2006); and heroin (Bossert et al., 2004). To date, there has been no demonstration of renewal in a novel context in

Chapter 1. Behavioural Basis of Extinction

drug seeking animals. Again, it is not known why this is the case, although an interesting possibility is that drug reinforcement might increase the reward-related properties of the training context. Nonetheless, the findings from Bouton et al. (2011) suggest that removal from the extinction context is sufficient to reinstate reward seeking and conversely, that the presence of the extinction context actively suppresses performance of reward seeking. Thus, the behavioural mechanism of extinction is intrinsically linked with that underlying renewal.

Bouton’s interference model of extinction provides a direct theoretical account of the renewal phenomenon. It also explains other post-extinction recovery phenomena by assuming that contextual information encoded during extinction is not limited to the physical environment, but also include temporal cues, the subject’s internal state, and other concurrent experimental events that form an overall background against which associative memory is encoded. For example, rapid reacquisition and drug-prime reinstatement are explained as a variant of the ABA renewal effect (Bouton &

Swartzentruber, 1991). That is, during conditioning, ‘after-effects’ of recent outcome presentations form part of the background of conditioning. These background conditions are absent during extinction since no outcomes are presented. During reacquisition of responding, resumption of response-contingent outcomes removes the animal from the extinction context and returns them to the original context of conditioning (by virtue of the background contextual features produced by the outcome). This promotes the retrieval of the original training memory over the extinction memory, and thus results in a rapid return of the originally learned response.

In the case of drug-prime reinstatement, re-exposure to the prime similarly transitions the animal from the extinction context to the original training context by virtue of the background contextual features produced by the prime (Bouton, 2011). Spontaneous

Chapter 1. Behavioural Basis of Extinction

recovery is similarly explained as a renewal effect, to the extent that temporal cues are coded as part of the extinction context. Thus, when a subject is tested immediately after extinction, much of the temporal cues associated with the extinction memory are present. However, after a long retention interval, temporal cues associated with the extinction memory have dissipated, allowing the original training memory to regain behavioural expression (spontaneous recovery). A long retention interval is therefore akin to moving the subject out of the extinction context (Bouton & Swartzentruber,

1991).

It is not immediately clear how contextual change from extinction might restore an extinguished response. One primary explanation is that contexts function as an occasion setter or ‘modulator’ (Bouton et al., 2011). From this view, contexts act as a retrieval cue in cases where the meaning of the response is ambiguous. Notably, the contexts are not directly associated with the outcome and therefore are resistant to extinction. Rather, they modulate or ‘set the occasion’ for the type of behaviour to be expressed. Although the evidence for this account is limited to studies based on

Pavlovian conditioning, the findings from Bouton and his colleagues (2011) are important because they suggest that extinction mechanisms might be similar across

Pavlovian and instrumental conditioning.

Summary

Several explanations for extinction have been proposed. Some have appealed to a mechanism of unlearning; that extinction results from the permanent loss of the originally learned association. A more influential account is that extinction reflects an active process of new learning, one that involves the formation of a new inhibitory association that competes with, and suppresses, performance of an instrumentally conditioned response. This latter account has two implications. First, learning survives 18

Chapter 1. Behavioural Basis of Extinction

extinction. Second, removal from the extinction context unmasks performance of the originally learned response. Bouton’s account for the recovery of an extinguished response is that during extinction, a new context-dependent inhibitory association is formed while leaving the originally learned response-outcome association intact. Thus removal from the extinction context recovers a previously extinguished conditioned response due to failure to retrieve the extinction memory. The model therefore provides an explanation for why recovery occurs and when to expect recovery of extinguished responding. Although explicitly an account of the renewal phenomena, the model also accounts for other post-extinction recovery phenomena (e.g. spontaneous recovery, reinstatement, rapid reacquisition) to the extent that these phenomena represent variants of renewal. Importantly, the model suggests that renewal is linked to extinction; both extinction and renewal are competing behavioural expressions of the same underlying process.

Chapter 2. Neural Mechanisms of Extinction

Chapter 2.

Neural Mechanisms Underlying Extinction of

Drug Seeking

Chapter 1 introduced the reinstatement procedure as a rat model for the study of relapse and explored the theoretical implications of the reinstatement phenomena, namely, that reinstatement provides evidence that extinction is not erasure of previous learning but a process of new learning that inhibits previous learning. Chapter 1 also introduced a theoretical account for context-induced reinstatement as a process involving two opposing influences: a facilitatory influence reflecting control over drug seeking by the original training contingency, and an inhibitory influence reflecting control over drug seeking by the extinction contingency. Therefore, a neurobiology of relapse requires an understanding of both these processes: a system that enables or unmasks drug seeking in a drug-paired context, and an extinction system that suppresses or masks drug-seeking in an extinction-paired context.

The purpose of Chapter 2 is to review the neural mechanisms underlying the extinction of drug seeking. Here, the focus is largely directed towards the nucleus accumbens because this structure, specifically its medial shell subregion, is the focus of the empirical work in this thesis. This chapter will begin with a review of the neuroanatomical and functional properties of the nucleus accumbens with respect to motivated behaviour related to drug and primary rewards. In keeping with behavioural conceptualisations of extinction as the inhibitory component of the reinstatement process, this chapter will then review the evidence for a neurobiological system of

Chapter 2. Neural Mechanisms of Extinction

context-induced reinstatement, followed by the evidence for a separate but complementary neurobiological system of extinction. In the final section, the circuit- levels mechanisms of extinction in relation to those underlying reinstatement of drug seeking are discussed.

2.1. The nucleus accumbens and control over reward and motivated behaviour

The nucleus accumbens (Acb) is the ventral extension of the striatum. In the rat forebrain, it is positioned beneath the caudate putamen and directly above the olfactory tubercle. It receives direct glutamatergic inputs from limbic sites including hippocampus, medial prefrontal cortex, entorhinal cortex, and amygdala; glutamatergic inputs from midline paraventricular thalamus (PVT); peptidergic inputs from hypothalamus; and also dopaminergic inputs from ventral mesencephalic areas including ventral tegmental area (VTA) and substantia nigra (SN) (Berendse, Graaf, &

Groenewegen, 1992). In turn, Acb has the potential to engage both limbic- and motor- related anatomical circuits via direct projections to dopaminergic cell groups in VTA and SN, and also via direct and transpallidal projections to peptidergic cell groups in lateral hypothalamus (LH). Moreover, through direct connections with ventral pallidum

(VP), the Acb has access to thalamocortical (limbic) loops involving mediodorsal thalamic nucleus (MDT) as well as motor generating sites of the basal ganglia (e.g. subthalamic nucleus (STN), entopeduncular nucleus) (Groenewegen, Wright, Beijer, &

Voorn, 1999; O’Donnell, Lavı́n, Enquist, Grace, & Card, 1997; Zahm & Brog, 1992).

The convergence of cortical, thalamic, and amygdalar inputs into the Acb are thought to recruit distinct neuronal ensembles, which via distinct patterns of output, enable accumbal control over a range of behavioural patterns (Pennartz, Dasilva, &

Groenewegen, 1994). Acb has also been proposed to function as a critical component in at least two serially-connected anatomical circuits as shown in Figure 3: the cortico- 21

Chapter 2. Neural Mechanisms of Extinction

striato-pallido-(medio)thalamic loop (O’Donnell et al., 1997; Zahm & Brog, 1992) and the cortico-striato-hypothalamo-(paraventricular) thalamic loop (Kelley, Baldo, & Pratt,

2005a; Kelley, Baldo, Pratt, & Will, 2005b; Thompson & Swanson, 2010). Through its projections to VP, the Acb has long been thought to serve as a “limbic motor interface”, anatomically positioned to translate motivational processes encoded by limbic structures to motor actions (Mogenson, Jones, & Yim, 1980; Pennartz et al., 1994). However, it has recently been suggested that an accumbal interface likely involves a series of topographically connecting spirals linking Acb with mesencephalic dopaminergic neurons that eventually reach the dorsolateral striatum (Haber, Fudge, & McFarland,

2000). Moreover, through its projections to LH, the Acb is thought to serve as a

“sensory sentinel” that regulates appetitive and visceromotor processes (Kelley et al.,

2005a; Kelley et al., 2005b). An important point is that regardless of the circuit through which the Acb functions, the Acb is anatomically positioned to influence behaviour in a manner that is closely associated with the limbic system; it receives limbic cortical processing directly, has multisynaptic influence over neocortical processing via limbic cortico-striatal loops (e.g. Figure 3), and has direct influence over limbic sites of the midbrain and hypothalamus. Accordingly, the Acb has been implicated in a variety of behaviours typically subserved by the limbic system. These include feeding

(Maldonado-Irizarry, Swanson, & Kelley, 1995; Mogenson & Wu, 1982), sexual behaviours (Barrot et al., 2005; Everitt, 1990; Liu, Sachs, & Salamone, 1998), negative motivation states (e.g. fear, stress, defensive behaviour: (Beck & Fibiger, 1995; Di

Chiara, Loddo, & Tanda, 1999; Faure, Reynolds, Richard, & Berridge, 2008; Reynolds

& Berridge, 2008); and reward (Cardinal, Parkinson, Hall, & Everitt, 2002; Everitt et al., 1999; Everitt & Robbins, 2005; Ikemoto, 2007). As this section focuses on Acb control over reward and motivated behaviour, a role for Acb in other behaviours will be

Chapter 2. Neural Mechanisms of Extinction

PVT

PFC MDT

Acb

Cortico-striato-pallido-thalamic loop Cortico-striato-hypothalamo-thalamic loop

Figure 3. Cortico-striatal-thalamic loops via pallidum (blue arrows) and hypothalamus (green arrows).

Chapter 2. Neural Mechanisms of Extinction

minimally addressed here, although this literature is extensively reviewed elsewhere

(Ikemoto, 2007; Pennartz et al., 1994).

Midbrain dopaminergic fibres heavily innervate the Acb. This provides a direct anatomical basis for Acb contributions to reward motivated behaviours. Behavioural evidence linking Acb dopamine transmission to motivation emerged from early studies showing that pharmacological manipulation of dopamine in the Acb could modulate locomotor activity. Thus, intra-Acb dopamine infusions increased locomotor activity in rats pretreated with nialamide, a monoamine oxidase inhibitor that attenuates the breakdown of dopamine (Costall & Naylor, 1975; Pijnenburg, Honig, & Rossum, 1975;

Pijnenburg & van Rossum, 1973). In non-pretreated rats, early studies similarly observed potentiated locomotor activity following stimulation of accumbal dopamine transmission via local infusions of dopamine; D-amphetamine; or cocaine, which blocks presynaptic dopamine reuptake (Delfs, Schreiber, & Kelley, 1990; Pijnenburg, Honig,

Van Der Heyden, & Van Rossum, 1976). Conversely, 6-hydroxydopamine (6-OHDA) lesions of the Acb attenuated D-amphetamine-induced increases in locomotor activity

(Kelly & Roberts, 1983) as well as spontaneous locomotor and exploratory hole-dipping behaviour (Makanjuola & Ashcroft, 1982). These early findings therefore implicated a role for Acb dopamine transmission in enabling general locomotion, while findings from Delfs et al. (1990) related this role to the psychostimulant actions of cocaine.

In contrast to the above findings, electrolytic lesions of the Acb have also been found to potentiate locomotor behaviour (Carey, 1982; Kelly & Roberts, 1983). This led to an alternative hypothesis regarding Acb contributions to motivation: that Acb has regulatory control over an “activity system” and that electrolytic destruction of the Acb releases this system from inhibition (Kelly & Roberts, 1983). However, conclusions based on electrolytic lesions are limited by their impact on fibres of passage and related

Chapter 2. Neural Mechanisms of Extinction

findings have been inconsistent. For example, fibre-sparing ibotenic acid lesions of the

Acb, which were also less extensive than those in Kelly and Roberts (1983) failed to increase locomotor activity (Annett, McGregor, & Robbins, 1989), although inhibition of Acb transmission by GABA agonist potentiated locomotor activity (Wachtel &

Anden, 1978). Nonetheless, the significance of this putative role for Acb in regulating an “activity system” has become elucidated in more recent research and I will return to discuss this concept later in this chapter.

Locomotor activity is an essential component of behavioural patterns of reward and motivation. However, more direct behavioural evidence linking Acb dopamine with reward has been demonstrated in early studies using self-administration preparations.

For example, Hoebel et al. (1983) found that rats readily self-administered intra-Acb

D-amphetamine via lever press response. More importantly, they showed for the first time that the reinforcing properties of D-amphetamine rather than its potentiating effects on locomotor activity maintained this behaviour. For example, in a two lever operant chamber where responses on only one (active) lever resulted in an injection of

D-amphetamine, the authors found that rats responded specifically on the active lever relative to the inactive lever. This was the case even when the active and inactive levers were reversed several times. That is, rats adjusted their responding to continue self- administration of D-amphetamine. In a subsequent experiment, the authors observed that rats reduced their rate of responding when the daily dose of D-amphetamine was halved, suggesting that rats were able to regulate their responding to maintain a constant concentration of intra-Acb D-amphetamine. Finally, the authors showed that intra-Acb self-administration was anatomically specific as it was not observed when injection cannulae were positioned in the lateral ventricle or caudate putamen. Together, the findings from Hoebel et al. (1983) suggest that intra-Acb D-amphetamine self-

Chapter 2. Neural Mechanisms of Extinction

administration is not likely due to hyperactivity. Rather, that Acb is a neural site for the reinforcing impact of D-amphetamine.

Consistent with a role for Acb dopamine in reward reinforcement, early studies have found that intra-Acb blockade of dopamine attenuated self-administration of electrical stimulation in the LH (Stellar, Kelley, & Corbett, 1983) or VTA (Kurumiya &

Nakajima, 1988), but did not prevent rats from altering their rate of responding following higher stimulation frequency (Kurumiya & Nakajima, 1988). This latter finding importantly suggests a role for Acb in reward reinforcement rather than general locomotion. Finally, post-training 6-OHDA Acb lesions also prevented cocaine seeking in rats previously trained to lever press for cocaine reward (Roberts, Koob, Klonoff, &

Fibiger, 1980). The authors found that while overall responding was attenuated by 6-

OHDA Acb lesions, animals with lesions initially maintained a high level of cocaine- seeking followed by cessation. This pattern of responding provides some evidence that the effects of 6-OHDA Acb lesions on cocaine seeking were not merely a consequence of non-specific motor deficits. The findings from Roberts et al. (1980) provided among the earliest pieces of evidence that the Acb is a critical substrate for drug self- administration. There has since been growing literature showing that Acb mediates the behavioural impact of natural rewards and drugs of abuse as well as the behavioural impact of their related cues and contexts. Before examining this literature, the following section introduces an added complexity to the study of the Acb – that it is a heterogeneous structure both neuroanatomically and functionally.

2.2. The Acb is a heterogeneous structure: neuroanatomy and behaviour

The neuroanatomical literature has long recognised that the organisation of the Acb is anatomically heterogeneous. For instance, Groenewegen and Russchen (1984) showed a significant difference in afferent and efferent projections of the lateral and medial 26

Chapter 2. Neural Mechanisms of Extinction

divisions of the Acb, noting especially that lateral Acb projects more directly to extrapyramidal structures while medial Acb projects back into the limbic circuitry. The authors concluded that Acb “cannot be considered a homogeneous region”

(Groenewegen & Russchen, p. 366), although the functional impact of this anatomical divide was not known at the time. Indeed, in the early behavioural literature described previously, the distinction between medial and lateral Acb compartments was not recognised. However, among more contemporary behavioural studies, which will be described later in this chapter, a functional appreciation for this anatomical divide has become increasingly evident, owing primarily to increased understanding of Acb subregions in relation to their distinct connectivity.

The Acb can be divided into rostral pole, shell (AcbSh) and core (AcbC) subregions on the basis of hodological, morphological, and histochemical criteria. The

AcbSh and AcbC are medially and laterally positioned respectively in the caudal portion of the Acb and are the most extensively studied subregions of the Acb. They have been shown to have overlapping as well as segregated patterns of afferents and efferents (Brog, Salyapongse, Deutch, & Zahm, 1993; Groenewegen et al., 1999;

Heimer, Zahm, Churchill, Kalivas, & Wohltmann, 1991; Záborszky et al., 1985; Zahm

& Heimer, 1993). For example, although core and shell subregions have common sources of afferents, including from PFC, hippocampus, BLA, and midline thalamus, the topographical organisation of these accumbal inputs varies along a dorsolateral- ventromedial gradient. PFC heavily innervates the Acb, although efferents from dorsal regions such as prelimbic prefrontal cortex (plPFC) preferentially target the laterally positioned AcbC, while more ventral regions such as ventral plPFC and infralimbic prefrontal cortex (ilPFC) preferentially target the medially positioned AcbSh. Similarly, while the subiculum region of the hippocampus innervates the Acb, its dorsal and

Chapter 2. Neural Mechanisms of Extinction

ventral regions preferentially target the AcbC and AcbSh respectively. BLA inputs into

Acb are also topographically organised such that fibres from the rostral and mid-rostral

BLA primarily terminate in AcbC, while caudal BLA projections terminate medially in

AcbSh. Even at the level of mesencephalic dopaminergic neurons, the medial VTA

(dorsal tier) preferentially terminates in the AcbSh whereas lateral VTA terminates in the AcbC (Berendse et al., 1992; Brog et al., 1993; Groenewegen et al., 1999; Ikemoto,

2007; Voorn, Vanderschuren, Groenewegen, Robbins, & Pennartz, 2004; Wright,

Beijer, & Groenewegen, 1996). Similarly, Acb efferents vary along its medial-lateral gradient such that lateral projections originating from the AcbC are heavily interconnected with extrapyramidal circuits (dorsolateral VP, STN, SN, globus pallidum entopeduncular nucleus, pedunculopontine area), while medial projections from AcbSh reach limbic regions/extended amygdala either directly via projections to VTA and LH or indirectly via projections to medial VP (Groenewegen & Russchen, 1984; Haber et al., 2000). These neuronanatomical connections are summarised in Figure 4. The relation between AcbSh and LH is a striking feature of segregation that has led to the suggestion that, although Acb is defined as a striatal structure, the AcbSh subregion may well be a part of the extended amygdala (Heimer et al., 1991). Together, the pattern of Acb inputs and outputs along its medial-lateral gradient suggest that Acb is a heterogeneous structure and that Acb-control over behaviour is likely to differ between

AcbC and AcbSh subregions.

One example demonstrating dissociative functions of AcbC and AcbSh was shown in a cue-induced reinstatement preparation (Floresco, McLaughlin, & Haluk,

2008). These authors trained rats to lever-press for food reward paired with a light-tone cue. Responding was then extinguished so that lever-presses no longer delivered food or

Chapter 2. Neural Mechanisms of Extinction

AcbC Afferents AcbSh Dorsolateral gradient Ventromedial gradient plPFC Cortical ilPFC Rostral BLA Amygdala Caudal BLA Dorsal Subiculum Hippocampus Ventral Subiculum Centromedial Midline thalamus PVT SN Midbrain VTA

Efferents VP (dorsolateral) VP (medial) Globus pallidum LH Entopeduncular Nucleus VTA Pedunculopontine Area STN, SN

Figure 4. Major afferent and efferent connectivity of AcbC and AcbSh.

Chapter 2. Neural Mechanisms of Extinction

the cue. On test, subsequent response-contingent presentations of the cue reinstated extinguished lever-pressing (food-seeking) behaviour. However, GABA agonist-induced reversible inactivation of AcbC prior to test prevented reinstatement while similar inactivation of AcbSh potentiated reinstatement of food seeking. It was therefore suggested that AcbC and AcbSh have opposing influence over behaviour; while AcbC promotes reward-seeking behaviour, AcbSh appears to regulate this behaviour. This is interesting given the early literature implicating Acb in both reward directed behaviour and behaviour regulation (Kelly & Roberts, 1983). Similar dissociations between AcbSh and AcbC have also been observed in rats trained to respond for cocaine (Di Ciano,

Robbins, & Everitt, 2008). These authors trained rats to nosepoke for cocaine paired with a light cue, and in a second phase, trained rats to lever-press for presentations of the light cue only (i.e., conditioned cue reinforcement). Lever-pressing for the conditioned (light) cue was extinguished by removal of the cue and then reacquired by lever press-contingent re-presentations of the light cue. Rats were subsequently tested for cue-induced reinstatement of cocaine seeking following nose poke-contingent re- presentations of the light cue. The authors found that reversible inactivation of the AcbC prior to test attenuated both acquisition and reacquisition of conditioned cue reinforced responding (lever-pressing), as well as cue-induced reinstatement of cocaine seeking

(nose-poke responding). In contrast, AcbSh inactivation had no impact on conditioned cue reinforced responding (lever-pressing), but enhanced cue-induced reinstatement of drug seeking. Possibly, AcbC and AcbSh process different components of reward, where

AcbC mediates more general incentive motivational properties of reward-paired stimuli while AcbSh functions to regulate drug seeking behaviour. These findings, together with those from Floresco et al. (2008) provide evidence that AcbC and AcbSh enable distinct behavioural responses in the presence of reward associated cues. However, the

Chapter 2. Neural Mechanisms of Extinction

contributions of the AcbC and AcbSh to reward seeking are not well-specified. For instance, these studies (Di Ciano et al., 2008; Floresco et al., 2008) suggest that AcbC and AcbSh mediate and inhibit reward seeking, respectively. In contrast, a role for

AcbSh in mediating rather than inhibiting context-induced and cocaine prime-induced reinstatement of drug seeking has also been shown via blockade of dopamine receptors

(Anderson, Bari, & Pierce, 2003; Bossert et al., 2007). This role will be discussed further in Section 2.3.

A final point worth noting here is that even within the AcbSh subregion itself, there is evidence for anatomical and functional heterogeneity. Anatomically, AcbSh can be segregated into separate dorsal, medial, and ventrolateral zones on the basis of neurotransmitter distribution and connectivity (Usuda, Tanaka, & Chiba, 1998; Zahm,

Williams, Krause, Welch, & Grosu, 1998), although other authors have recognised additional compartments on the basis of tyrosine hydroxylase immunoreactivity

(Todtenkopf, 2000). Findings from neuroanatomical tracing studies show that the dorsal-most tip of the AcbSh is primarily innervated by ventral subiculum, caudal BLA, interfascicular nucleus and lateral habenula (Brog et al., 1993; Hamlin, Clemens, Choi,

& McNally, 2009; McDonald, 1991; Wright et al., 1996). In contrast, posterior PVT avoids both dorsal tip and lateral AcbSh regions and instead innervates ventromedial

AcbSh (Vertes & Hoover, 2008). With regards to AcbSh efferents, the dorsomedial tip of the AcbSh projects to the VP and LH, while ventromedial and lateral AcbSh additionally targets the VTA and SN respectively (Brog et al., 1993; Ikemoto, 2007;

Thompson & Swanson, 2010). These projections are depicted schematically in Figure 5.

The functional implication for the heterogeneous patterns of AcbSh inputs and outputs are not entirely known, largely due to the difficulty of targeting these small, localized regions. For example, in one study, Stratford and Kelley (1997) showed that GABA

Chapter 2. Neural Mechanisms of Extinction

cBLA, vSub, IF, LHb

VP, LH

Dorsal tip PVT, ilPFC

VP, LH, VTA Medial

Ventrolateral

lOFC, vAgI

VP, LH, SN

Figure 5. Distinct preferential inputs and outputs of AcbSh subregions: dorsal tip, medial AcbSh and ventrolateral AcbSh.

Chapter 2. Neural Mechanisms of Extinction

agonist induced inactivation of the AcbSh induced feeding in sated rats and that this effect was observed following targeted infusion into ventromedial AcbSh sites and not dorsal AcbSh. However, inferences relating to dorsal and ventromedial AcbSh functioning is limited due to likely diffusion of the drug along the cannula track; it is possible that injections in the ventromedial AcbSh diffused into the dorsal aspect of the

AcbSh and similarly, injections in the dorsal AcbSh may have diffused into the dorsally adjacent lateral ventricle. Other studies that allow better anatomical resolution of AcbSh subregions provide correlative behavioural evidence of their functional dissociations.

For example, in a study of latent inhibition, extracellular dopamine levels in the dorsomedial AcbSh was correlated with latent inhibition of an approach response to a preexposed odour that was later paired with illness (lithium chloride), while similar measurements in the ventral AcbSh correlated with whether or not the odour stimulus was paired with illness (Jeanblanc, Hoeltzel, & Louilot, 2002). In a study of cocaine self-administration, electrophysiological recordings from dorsal, ventromedial, and ventrolateral AcbSh show distinct patterns of firing in temporal relation to an animal’s lever press response (Fabbricatore, Ghitza, Prokopenko, & West, 2010). Finally, in a study of context-induced reinstatement of drug seeking, Marchant, Hamlin and McNally

(2009) used CTb tract-tracer in combination with the activity marker, c-Fos protein, and found that projection neurons from the ventral AcbSh were activated during context induced reinstatement of drug seeking while projection neurons from dorsal AcbSh were additionally recruited in a separate extinction-paired context. This study will be discussed further in the next section.

Chapter 2. Neural Mechanisms of Extinction

Summary

Acb plays a key role in reward and motivation, owing primarily to its anatomical position in relation to limbic structures, extrapyramidal motor systems and connectivity with midbrain dopamine neurons. Early work implicated this structure in locomotor activity and reward reinforcement. In more recent studies, it has become increasingly evident that the Acb is a heterogeneous structure with distinct anatomical compartments that differentially contribute to reward behaviour. Thus, the medially positioned AcbSh and laterally positioned AcbC show distinct patterns of afferent and efferent connectivity that vary along a dorsolateral-ventromedial gradient. In particular, it was noted that the connection between AcbSh and LH is a striking feature that separates

AcbSh from AcbC; that this feature has led to the suggestion that the AcbSh may be a component of the extended amygdala. Consistent with the anatomical heterogeneity of the Acb, targeted functional inactivation studies have demonstrated dissociable roles for its AcbC and AcbSh subregions in modulating the behavioural impact of reward- associated cues. Despite these functional dissociations, the specific contributions of the

AcbC and AcbSh to reward behaviour are not fully understood. This is especially the case for the AcbSh where its contribution to reward behaviour might depend on its heterogeneously distributed patterns of input and output.

In the following section, the neurobiology of ABA renewal (here forth, context- induced reinstatement) of drug seeking is reviewed. The focus on context-induced reinstatement is presented to complement the neurobiology of extinction, which forms the basis of the present thesis. The aim is to present the evidence for a circuit-level system of context-induced reinstatement, one that is functionally organised in relation to the Acb. The section begins with the literature implicating Acb, its afferents and

Chapter 2. Neural Mechanisms of Extinction

efferents, followed by circuit-level contributions of these structures to context-induced reinstatement of drug seeking.

2.3. A neurobiological system of context-induced reinstatement

In Chapter 1, it was explained that ABA renewal provided the behavioural basis for context-induced reinstatement of drug seeking. As a reminder, ABA renewal in drug self-administering animals typically involves self-administration training in Context A, extinction in Context B, followed by test in either the drug related context (A) or the extinction context (B) (Figure 2). Contexts are always arranged to differ across multiple features (e.g. tactile, auditory, olfactory, temporal) and chambers are typically arranged to have two levers, an ‘active’ and ‘inactive’ lever (or other manipulanda, e.g. nosepokes), with the exception of one study (Zironi et al., 2006). During self- administration, responding on the ‘active’ and not ‘inactive’ lever results in the delivery of drug reward. During extinction and test, responses on either lever are not reinforced.

Context-induced reinstatement is said to occur if extinguished responding to the active lever is recovered when rats return to Context A relative to responding in Context B.

Moreover, in the studies reviewed below, a neural manipulation is typically said to have impaired reinstatement if treatment is specific to the self-administration context

(typically a treatment x context interaction), and furthermore, if treatment is specific to the active lever (e.g. by demonstrating no effect of treatment on inactive manipulandum or a 3-way treatment x context x manipulanda interaction).

2.3.i. Nucleus Accumbens (AcbSh and AcbC) and context-induced reinstatement

Several lines of evidence suggest that a role for Acb in context-induced reinstatement involves its AcbSh subdivision. The most direct evidence involves a series pharmacological inactivation studies targeting AcbSh and AcbC subregions (Bossert et

Chapter 2. Neural Mechanisms of Extinction

al., 2006; Bossert et al., 2007). In these studies, rats were trained to respond for heroin in Context A, followed by extinction in Context B. Prior to test in contexts A and B, rats received injections of LY379268 into AcbSh or AcbC (Bossert et al., 2006). LY379268 is an agonist for group II metabotropic glutamate receptors (mGluR2/3) that decreases evoked glutamate release. Therefore, if Acb glutamatergic neurotransmission is necessary for the reinstatement of heroin seeking, reducing its release prior to test should also reduce responding. Indeed, LY379268 attenuated context-induced reinstatement of heroin seeking when injected into AcbSh and AcbC. In a separate group of rats, the authors showed that LY379268 in AcbSh and AcbC was ineffective on responding for sucrose delivery, suggesting that the attenuating effects of LY379268 on reinstatement was not likely due to motor deficits. However, the effect of LY379268 was more potent in AcbSh than AcbC since the lowest dose effective in AcbSh was not effective in AcbC. Therefore, it is possible that the effects of LY379268 in AcbC were due to diffusion from AcbSh (Bossert et al., 2006). Alternatively, AcbC might be partially recruited (and to a lesser degree than AcbSh) during context-induced reinstatement owing to some overlap in AcbSh and AcbC connectivity. Therefore, these findings implicate a role for AcbSh in context-induced reinstatement, whereas the contribution of the AcbC in this preparation is less clear.

Congruent with a role for AcbSh in context-induced reinstatement, blockade of dopaminergic neurotransmission using the dopamine D1-like receptor antagonist, SCH

23390, impaired context-induced reinstatement of heroin seeking when injected into

AcbSh (medial), AcbSh (lateral), but not in AcbC (Bossert et al., 2006; Bossert et al.,

2007). In a separate group of rats, these authors showed that effects of SCH 23390 into

AcbSh (medial or lateral) were specific to context-induced reinstatement since similar infusions were ineffective on responding for 5% sucrose solution and also on a test of

Chapter 2. Neural Mechanisms of Extinction

cue-induced reinstatement in an extinction context (via re-exposure to a tone-light cue previously paired with heroin reinforcement). Interestingly, AcbC injections of SCH

23390 attenuated reinstatement triggered by a discrete heroin-paired cue. These findings therefore suggest that AcbC and AcbSh might subserve dissociable roles in cue-induced reinstatement and context-induced reinstatement of heroin seeking, respectively.

Finally, a role for AcbSh in context-induced reinstatement has been demonstrated in studies involving immunohistochemical detection of c-Fos protein. c-

Fos is an immediate early gene transcription factor that is induced in the cell nucleus following membrane depolarisation (Cole, Saffen, Baraban, & Worley, 1989). The expression of c-Fos is an index of neuronal activation. For example, in rats trained to respond for sucrose solution (Hamlin et al., 2006) and alcoholic beer (Hamlin et al.,

2009; Hamlin et al., 2007; Marchant et al., 2009), context induced reinstatement of reward seeking (ABA) was associated with greater levels of c-Fos protein expression in

AcbSh and not AcbC, relative to animals tested in the extinction context (e.g. AAA or

ABB group) and also relative to animals that were not tested but were instead transported to the laboratory and given equivalent handling (e.g. a control AA- or AB- group). These findings suggest that context-induced reinstatement was associated with neuronal activation of AcbSh, and that this activation was not likely the result of differences in training history or due to relative decreases in c-Fos induction in animals tested in the extinction context. Moreover, AcbSh c-Fos protein induction during context-induced reinstatement was equivalent to that observed in animals unable to respond during test in Context A (by blockade of the nosepoke manipulandum) (Hamlin et al., 2006; Hamlin et al., 2008). This suggests that AcbSh neuronal activation during reinstatement was not due to general motoric responses and may instead reflect motivational processes that trigger reward seeking. Finally, Hamlin et al. (2006)

Chapter 2. Neural Mechanisms of Extinction

reported that systemic injections of the dopamine D1-like receptor antagonist,

SCH23390, attenuated both context-induced reinstatement and associated increases in c-

Fos protein expression in the AcbSh. This further demonstrates the relation between

AcbSh neuronal activation and context-induced reinstatement. However, in each of these studies, induction of c-Fos protein in AcbSh during reinstatement was restricted to the ventromedial and not dorsal AcbSh. Therefore, AcbSh contributions to context- induced reinstatement appear anatomically specific. This is important given the heterogeneity of connectivity between dorsal and ventral subregions of the AcbSh, as discussed in the previous section. Moreover, it demonstrates an important advantage of measuring c-Fos protein expression – that although this method of assessment cannot be used to draw causal inferences regarding functional contributions of brain regions to behaviour, it provides greater anatomical resolution (e.g. can differentiate dorsal versus ventral AcbSh) compared to functional microinjection approaches.

Interestingly, neuronal activation of AcbSh (dorsal or ventral) during context- induced reinstatement was not observed in animals trained to respond for cocaine, despite similar training parameters (Hamlin et al., 2008). It is possible therefore that

AcbSh contribution to context-induced reinstatement does not generalise to animals with a history of cocaine-reinforcement. Moreover, in contrast to the findings from alcoholic-beer seeking rats (Hamlin et al., 2006; Hamlin et al., 2009; Hamlin et al.,

2007; Marchant et al., 2009), a study using a similar reinforcer (ethanol solution) observed no change in AcbSh c-Fos mRNA during context-induced reinstatement, using in situ hybridization (Marinelli, Funk, Juzytsch, Li, & Lê, 2007). This may be because unlike in previous studies (Hamlin et al., 2006; Hamlin et al., 2009; Hamlin et al., 2007;

Marchant et al., 2009), the AcbSh was examined as a singular structure.

Chapter 2. Neural Mechanisms of Extinction

So far, a series of pharmacological inactivation and c-Fos neuronal activation studies have implicated a role for AcbSh in context-induced reinstatement using multiple drug reinforcers (heroin, sucrose, alcoholic beer). However, findings from other studies have implicated a similarly important role for AcbC. For example in cocaine-trained animals, reversible inactivation of either AcbSh or AcbC using GABA agonists, baclofen and muscimol, attenuated context-induced reinstatement of cocaine- seeking, while having no impact on general locomotor activity or responding for food reinforcer (Fuchs et al., 2008). Similarly, Chaudhri and colleagues (2009) found that pretest microinjections of the dopamine D1-like receptor antagonist, SCH 23390, into either AcbC or AcbSh attenuated context-induced reinstatement of ethanol seeking. The effect was also more sensitive in the AcbC than in AcbSh, suggesting that AcbC may be a more critical site for context-induced reinstatement of ethanol seeking. Recall that this finding contrasts with those in Bossert et al. (2007), who reported that blockade of dopaminergic neurotransmission in AcbSh but not AcbC attenuated context-induced reinstatement of heroin-seeking (Bossert et al., 2006; Bossert et al., 2007). However,

Bossert et al. (2007) noted in their manuscript that intra-AcbSh infusions of SCH 23390 diffused into the ventricles in approximately half of their animals using a test of angiotensin-induced drinking. This diffusion was demonstrably rectified when the authors used highly angled AcbSh coordinates (angled at 25º) to avoid the ventricle

(Bossert et al. 2007). Since Chaudhri et al. (2009) did not use similarly angled coordinates, drug diffusion from AcbSh into the adjacent lateral ventricles may have occurred, resulting in reduced potency of SCH23390 in the AcbSh relative to the AcbC.

Conversely, in Fuchs et al. (2008) and Chaudhri et al. (2009) drug diffusion from AcbC to AcbSh is also possible. In line with this possibility, Fuchs et al. (2008) found that

AcbC inactivation had a (non-significant) tendency to attenuate extinction responding in

Chapter 2. Neural Mechanisms of Extinction

the extinction context whereas a more recent study found that AcbSh but not AcbC inactivation significantly attenuated extinction responding (Peters et al., 2009). This finding will be discussed further later in this chapter.

Together, there is consistent evidence demonstrating a functional role for AcbSh in context-induced reinstatement. This has been demonstrated across multiple reinforcers including heroin, ethanol, and cocaine. Moreover, this functional role for

AcbSh is supported by the upregulation of AcbSh c-Fos induction during context- induced reinstatement of alcoholic beer seeking and sucrose seeking. In contrast, a role for AcbC in context-induced reinstatement is less evident given the difficulty of ruling out drug diffusion. Nonetheless, there is a possibility that AcbC and AcbSh contributions to context-induced reinstatement might be reinforcer-dependent; AcbC and AcbSh might be recruited in cocaine- and ethanol-trained animals while AcbSh alone might be recruited in heroin-trained animals. However, this possibility is inconsistent with the findings from c-Fos studies (Hamlin et al., 2006; Hamlin et al.,

2009; Hamlin et al., 2007; Marchant et al., 2009), which implicate AcbSh and not AcbC in context-induced reinstatement of alcoholic beer- and sucrose-seeking.

In the following section, Acb contributions to context-induced reinstatement are further examined by review of the evidence implicating afferents and efferents of

AcbSh and AcbC to context-induced reinstatement of drug seeking.

2.3.ii. Accumbal afferent and efferent contributions to context-induced reinstatement

Prefrontal Cortex. The prefrontal cortex (PFC) can be partitioned into medial, orbital, and lateral (agranular insular areas) subregions. Within the medial PFC, the dorsomedial regions (dorsal prelimbic (plPFC) and dorsal anterior cingulate (ACC)) reach the rostral AcbC while ventromedial regions (ventral plPFC, infralimbic (ilPFC), 40

Chapter 2. Neural Mechanisms of Extinction

medial orbital) reach the medial caudal AcbSh. In contrast, portions of the lateral PFC

(lateral orbital and ventral agranular area) target the ventrolateral AcbSh, while the lateral PFC region and dorsal agranular area additionally targets the lateral AcbC

(Berendse et al., 1992; Reynolds & Zahm, 2005).

Given the heterogeneity of PFC projections to Acb, one might predict differential contributions of PFC subregions to context-induced reinstatement of drug seeking. However, findings implicating subregions of the medial PFC appear mixed. In one study, (Fuchs et al., 2005) implicated a role for dorsomedial PFC (dmPFC) in context-induced reinstatement of cocaine-seeking. They found that reversible inactivation of dmPFC, but not ventromedial PFC (vmPFC), using the sodium channel blocker, tetrodotoxin (TTX), attenuated context-induced reinstatement. The effect was behaviourally specific since similar infusions failed to disrupt locomotor activity in a novel context. Similarly, reversible inactivation (but not lesions) of the lateral orbital

PFC (lOFC) using GABA agonists, baclofen/muscimol, disrupts context-induced reinstatement of cocaine-seeking (Lasseter, Ramirez, Xie, & Fuchs, 2009). Thus, both dmPFC and lOFC are implicated in context-induced reinstatement of cocaine seeking and notably, both regions project preferentially to AcbC and ventrolateral AcbSh, respectively. However, inactivation of lOFC also decreased locomotor activity, raising the possibility that its effect on context-induced reinstatement may in part reflect nonspecific suppression of activity. Nonetheless, in Lasseter et al. (2009), reversible inactivation of the medial orbital PFC, which is rostrally continuous with ilPFC and typically included as part of the vmPFC (Berendse et al., 1992), had no effect on context-induced reinstatement of cocaine-seeking. This is consistent with Fuchs et al.

(2005) where TTX-induced inactivation of vmPFC had no effect on context-induced reinstatement of cocaine-seeking.

Chapter 2. Neural Mechanisms of Extinction

In contrast to the above findings, Bossert et al. (2011) reported that vmPFC, not dmPFC is critical for context-induced reinstatement of heroin-seeking. This was demonstrated in two ways. First, the authors observed that functional inactivation of vmPFC using GABA agonists, baclofen/muscimol, significantly attenuated reinstatement when rats returned to the training context A, while similar infusions in dmPFC were ineffective. Second, the authors used a Daun02 inactivation method to selectively silence activated neurons in vmPFC when rats were tested in the training context, A. This procedure, recently developed by Koya et al. (2009), uses c-Fos-lacZ transgenic rats containing a transgene with a c-Fos promoter that regulates expression of the bacterial lacZ gene, which encodes the protein E-galactosidase. This means that reinstatement-induced Fos is coexpressed in E-galactosidase nuclei. Importantly, E- galactosidase converts a microinjected prodrug, Daun02, into daunorubicin, which in turn disrupts neuronal functioning of cells expressing both E-galactosidase and Fos.

Using this procedure, vmPFC injections of Daun02 after test in Context A (to inactivate expression of Fos) attenuated subsequent context-induced reinstatement but had no effect on test in the extinction context, B. Given that only a small subset of vmPFC cells

(6.0 r 0.8%) expressed c-Fos when tested in Context A, these findings show that selective inactivation of only a small subset of neurons in the vmPFC is sufficient to attenuate context-induced reinstatement of heroin seeking.

It is possible that dmPFC and vmPFC regions differentially contribute to context-induced reinstatement of cocaine- and heroin-seeking, respectively. However, inconsistent with this possibility are findings based on c-Fos studies showing increased neuronal activation in ilPFC but not in dmPFC (plPFC and ACC) during context- induced reinstatement of cocaine seeking (Hamlin et al., 2008). Alternatively, the observed differences between the two studies (Bossert et al., 2011; Fuchs et al., 2005)

Chapter 2. Neural Mechanisms of Extinction

may be related to anatomical distinctions within mPFC. For example, within dmPFC, cannula placements in Fuchs et al. (2005) appear to cluster around the dorsal ACC whereas placements in Bossert et al. (2011) appear to cluster around plPFC. Although both dmPFC regions target AcbC, the dorsal ACC additionally targets the dorsolateral striatum (Berendse et al., 1992), a site that is also implicated in context-induced reinstatement. A role for dorsolateral striatum in context-induced reinstatement will be discussed later in this section. Similarly, vmPFC cannula in Fuchs et al. (2005) cluster rostrally in ilPFC and medial orbital PFC, while cannula in Bossert et al. (2011) cluster in caudoventral regions of ilPFC and dorsal peduncular cortex. Further work is required to investigate possible functional distinctions between mPFC subregions. Nonetheless, it is noteworthy that to date, pharmacological inactivation studies of Acb and PFC subregions show discrepant results depending on the reinforcer history of the animals.

Hippocampus. As with PFC, distinct regions of the hippocampus project preferentially to AcbC and AcbSh. For example, the dorsal hippocampus (DH; CA1 region) and dorsal subiculum preferentially target the AcbC while ventral subiculum preferentially targets the AcbSh (Brog et al., 1993). There is evidence that both dorsal and ventral hippocampal regions are important for context-induced reinstatement. For dorsal hippocampus, pharmacological inactivation of this region using TTX or the mGluR1 selective antagonist, JNJ16259685, significantly attenuated context-induced reinstatement of cocaine seeking in a manner that was anatomically specific. That is, similar injections were ineffective in the immediately dorsal somatosensory cortex.

Moreover, a role for DH in context-induced reinstatement appears behaviourally specific as these DH manipulations had no effect on general locomotor activity or food- reinforced instrumental behaviour (Fuchs et al., 2005; Xie, Ramirez, Lasseter, & Fuchs,

2010). Fuchs et al. (2005) also showed that reversible inactivation of DH failed to affect

Chapter 2. Neural Mechanisms of Extinction

reinstatement in other preparations including reinstatement induced by a cocaine-prime or exposure to a discrete light-tone cue that was previously paired with cocaine reinforcement. Finally, context-induced reinstatement of ethanol seeking has been associated with higher levels of c-Fos mRNA in the DH, CA3 region (Marinelli et al.,

2007). As with DH, there is some evidence that ventral hippocampus (VH), a posterior hippocampal region that reaches AcbSh via ventral subiculum, is also implicated in context-induced reinstatement of cocaine seeking. For example, reversible inactivation of VH was found to attenuate context-induced reinstatement of cocaine seeking in a manner that was anatomically specific and without impacting on general locomotor activity (Lasseter, Xie, Ramirez, & Fuchs, 2010).

Basolateral Amygdala (BLA). The Acb receives dense glutamatergic projections from the entire rostrocaudal gradient of the BLA, although rostral BLA preferentially innervates the AcbC and lateral AcbSh, whereas caudal-most BLA regions project to

AcbSh (Wright et al., 1996). Although the functional implication of this anatomical topography has not been explicitly investigated in preparations of context-induced reinstatement, reversible inactivation of the rostral BLA using TTX has been found to impair context-induced reinstatement of cocaine seeking (Fuchs et al., 2005). It is noteworthy that in a subsequent study from the same laboratory, Fuchs et al. (2006) showed that reversible inactivation of BLA was ineffective if context-induced reinstatement was preceded by a period of abstinence in place of extinction in Context

B. This suggests that BLA contributions to context-induced reinstatement may be specific to the recovery of an extinguished response. Finally, bilateral blockade of mu- opioid receptors in BLA using the mu-opioid antagonist, naltrexone, significantly attenuated context-induced reinstatement of ethanol-seeking (Marinelli, Funk, Juzytsch,

Chapter 2. Neural Mechanisms of Extinction

& Lê, 2010). This suggests that BLA contributions to context-induced reinstatement are mediated in part by opioid receptor neurotransmission.

Studies based on c-Fos expression also support a role for BLA in context- induced reinstatement. For example, Marinelli et al. (2007) reported increased c-Fos mRNA in the BLA associated with context-induced reinstatement of ethanol-seeking, and likewise, c-Fos protein expression is increased in BLA during test in the training context, A, relative to the extinction context, B, regardless of whether the animals received prior training with sucrose-, cocaine-, or alcoholic beer-reward (Hamlin et al.,

2006; Hamlin et al., 2009; Hamlin et al., 2007). This increase in c-Fos protein expression was also shown to be equivalent to that observed in animals unable to respond during test in the training context, A, (by blockade of the nosepoke manipulandum), suggesting that BLA neuronal activation during reinstatement is not likely related to motoric responses and may instead reflect motivational processes that trigger reward seeking (Hamlin et al., 2006; Hamlin et al., 2008). Reinstatement- associated activation of the BLA is also attenuated following systemic injections of the mu-opioid antagonist, naltrexone (Marinelli et al., 2007), which concomitantly attenuates context-induced reinstatement. Finally, Hamlin et al. (2009) found that context-induced reinstatement was associated with neuronal activation specifically in the rostral as opposed to caudal BLA. Together, there is consistent evidence implicating

BLA in context-induced reinstatement across a number of different reinforcers. There is some evidence that this involvement is anatomically specific to rostral BLA and also specific to context-induced recovery of an extinguished response.

Paraventricular thalamus (PVT), ventral tegmental area (VTA) and lateral hypothalamus (LH). Midline PVT, VTA and LH each project preferentially to AcbSh

(Brog et al., 1993; Kampe, Tschöp, Hollis, & Brian, 2009). In turn AcbSh sends

Chapter 2. Neural Mechanisms of Extinction

projections back to VTA and LH (Ikemoto, 2007). Moreover, LH is critically positioned to influence both PVT and VTA. Therefore, through LH, AcbSh has indirect access to both PVT and VTA. These projections are depicted in Figure 6. In LH, two primary projection neurons are those containing peptides, orexin and cocaine- and amphetamine- regulated transcript (CART). Orexin-containing projections from LH to PVT, VTA, and

AcbSh have been demonstrated in tract-tracer studies (Fadel & Deutch, 2002; Kirouac,

Parsons, & Li, 2005) and similarly, CART-containing projections from LH to PVT and

VTA have been observed (Parsons, Li, & Kirouac, 2006; Philpot, Dallvechia-Adams,

Smith, & Kuhar, 2005). Both orexin and CART peptides have been implicated in appetitive-motivated behaviours such as feeding (Sakurai et al., 1998; Saper, Chou, &

Elmquist, 2002; Yang, Shieh, & Li, 2005) and more recently, reinstatement of drug seeking. For example in cocaine-seeking animals, perfusion of orexin into VTA reinstates cocaine seeking behavior (Wang, You, & Wise, 2009), while systemic injections of an orexin receptor antagonist prevents cue- and stress-induced reinstatement of alcohol and cocaine seeking (Boutrel et al., 2005; Lawrence, Cowen,

Yang, Chen, & Oldfield, 2006). Harris, Wimmer, and Aston-Jones (2005) also report that conditioned place preference for a chamber previously paired with drug- (morphine, cocaine) or natural-reward (food) is correlated with activation of orexin cells in the LH and that activation of LH neurons using the Y4-receptor agonist, rat pancreatic polypeptide, reinstated an extinguished morphine place preference in an orexin- dependent manner. These findings suggest that orexin neurons contribute to the behavioural impact of reward-related cues. With regards to the CART peptide, recent studies have found that intra-cerebroventricular infusions of CART prevents context- induced reinstatement of alcohol seeking (King, Furlong, & McNally, 2010) while intra-PVT CART infusions prevents cocaine-prime reinstatement (James et al., 2010).

Chapter 2. Neural Mechanisms of Extinction

Thus, the peptide-containing LH is centrally positioned to influence reward behaviour and AcbSh can access this peptidergic influence via direct projections to LH.

To date, only few studies have implicated a functional role for PVT, VTA, and

LH in context induced reinstatement. In the case for PVT, Hamlin et al. (2009) showed that targeted excitotoxic (ibotenic acid) lesions of the PVT prior to test prevented context-induced reinstatement of alcoholic beer-seeking. In VTA, microinjection of the group II metabotropic glutamate receptor agonist, LY379268, dose-dependently attenuated context-induced reinstatement of heroin seeking, while similar manipulations in the adjacent midbrain region, SN, was ineffective (Bossert et al., 2004).

AcbSh

PVT LH VTA

CART

Orexin

Figure 6. Schematic diagram illustrating anatomical connectivity linking AcbSh, with PVT, LH, and VTA.

Finally, in LH, reversible inactivation via GABA agonists, baclofen and muscimol, attenuated context-induced reinstatement of alcoholic beer seeking and sucrose seeking (Marchant et al., 2009). This role for LH is further supported by c-Fos

Chapter 2. Neural Mechanisms of Extinction

activation studies that show that increased c-Fos activation is associated with context- induced reinstatement of sucrose-, beer-, and cocaine seeking. In these studies, c-Fos activation during context-induced reinstatement was equivalent to that observed in animals unable to respond during test in the training context, A, (by blockade of the nosepoke manipulandum), suggesting that LH neuronal activation during reinstatement is not likely related to motoric responses and may instead reflect motivational processes that trigger reward seeking (Hamlin et al., 2006; Hamlin et al., 2008).

Together, these studies provide functional evidence for PVT, VTA, and LH in context-induced reinstatement, although it is not known from these studies whether their functional contributions generalise to context-induced reinstatement of other drug reinforcers.

Dorsolateral Striatum. The limbic ventromedial accumbens system is linked to the motor dorsolateral striatum (DLS) through a series of loops that course via the midbrain. This includes descending projections from AcbSh to VTA/ventromedial SN; ascending mesoaccumbens projections reaching AcbSh and AcbC; and descending projections from AcbC to lateral VTA/SN. These loops continue until finally, ascending nigrostriatal projections reach the DLS (Haber et al., 2000). In this way, corticolimbic information projecting into Acb from structures involved in context-induced reinstatement (PFC, BLA, hippocampus, VTA, LH) can influence information from premotor and motor cortex at the level of the DLS. These pathways are summarised in

Figure 7 below.

A role for the DLS in context-induced reinstatement has been demonstrated in both cocaine- and heroin-trained animals. For example, pharmacological inactivation of the dorsolateral caudate putamen via GABA agonists, baclofen and muscimol, or via

D1-like receptor antagonist, SCH23390, attenuated context-induced reinstatement of

Chapter 2. Neural Mechanisms of Extinction

cocaine- and heroin-seeking respectively (Bossert et al., 2009; Fuchs et al., 2006).

Importantly, Bossert et al. (2009) showed that the effect of SCH23990 on reinstatement was specific to the dorsolateral and not the dorsomedial caudate putamen, thereby demonstrating the anatomical specificity of dorsal striatal contributions to context- induced reinstatement. Finally, despite the role for dopamine in locomotion, these manipulations had no observable impact on general locomotor activity or on responding for sucrose reinforcement. It is interesting that of the structures reviewed here, it is this motor output structure, DLS, that provides the only clear example of functional overlap between heroin and cocaine trained rats. This suggests that DLS may be a common output structure for enabling the behavioural expression of context-induced reinstatement.

AcbSh AcbC DLS

VTA SN

Figure 7. Schematic summary of spiralling organisation of ventral striatal (Acb) and dorsolateral striatal (DLS) connectivity with midbrain VTA and SN.

Chapter 2. Neural Mechanisms of Extinction

2.3.iii. Circuit-level mechanisms of context-induced reinstatement of drug seeking

From the studies reviewed thus far, several lines of evidence implicate the Acb and its connected structures in context-induced reinstatement of drug seeking. However, it is not known from these studies whether Acb mediates reinstatement via direct interactions with the aforementioned structures (e.g., PFC, BLA, hippocampus). To address this question, previous studies have combined immunohistochemical detection of the neuronal activity marker, c-Fos, in cells retrograde-labelled with the neuronal tract tracer cholera toxin B (CTb). This approach enables visualisation of activated projection neurons during the expression of context-induced reinstatement. For example, using CTb applied to LH, Marchant et al. (2009) identified recruitment of

AcbSh projection neurons targeting LH during context-induced reinstatement. That is, they found significantly more LH-projecting (retrograde-labelled) cells expressing c-Fos protein in the ventral AcbSh following test in the training context, A, compared to test in the extinction context, B, although a previous study failed to find similar changes in

AcbSh in animals trained to self-administer cocaine reward (Hamlin et al., 2008).

Therefore, there may be separate pathways mediating context-induced reinstatement of cocaine- and alcoholic beer seeking. More recently, Hamlin et al. (2009) examined sources of afferent projections into AcbSh recruited during context-induced reinstatement of alcohol seeking via application of CTb to the AcbSh. They observed a significant increase in the number of AcbSh-projecting cells expressing c-Fos protein associated with context-induced reinstatement in the PVT. Therefore, these findings suggest that context-induced reinstatement of alcohol seeking recruits direct projections from PVT to AcbSh, and also direct projections from ventral AcbSh to LH. However, as in other c-Fos studies, this strategy provides correlational data and cannot provide a causal role for the subcircuits recruited during context-induced reinstatement.

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Nonetheless, this strategy provides visualisation of direct projections between structures as well the directionality of these projections. This is especially important when studying structures like LH, which has afferent and efferent projections to AcbSh.

Moreover, the anatomical resolution provided by this approach enables inferences regarding the specific subregion of the Acb (i.e. ventral region of the AcbSh) that signals downstream to LH.

Other extra-Acb subcircuits mediating context-induced reinstatement have also been identified using a pharmacological disconnection approach. This involves an experimental (contralateral disconnection) group where two putatively connected structures are unilaterally inactivated in contralateral hemispheres. This manipulation disrupts their communication in both hemispheres while leaving other functions, independent of their communication, intact. In a separate control group, animals receive the same treatment except that inactivation of the two structures occurs in the same hemisphere. Therefore, communication between the two structures remains intact in one hemisphere. A final control group receives vehicle injections. Using this disconnection procedure, previous studies have investigated a role for BLA interactions with dmPFC, orbital PFC (lateral), and DH (Fuchs et al., 2007; Lasseter et al., 2010) in context- induced reinstatement of cocaine seeking. For example, disconnection of BLA with either dmPFC or lateral orbital PFC attenuated reinstatement of cocaine seeking following both contralateral and ipsilateral disconnection (Fuchs et al., 2007; Lasseter et al., 2010). The authors subsequently showed that the effect was not simply the summed effect of unilateral BLA and PFC inactivation since unilateral inactivation of BLA, dmPFC, or orbital lateral PFC was ineffective on reinstatement. Rather, unilateral inactivation of both BLA and PFC was required to disrupt context-induced reinstatement. While this study demonstrates a functional interaction between the two

Chapter 2. Neural Mechanisms of Extinction

subregions, the nature of this interaction cannot be inferred. It is possible that, as suggested by the authors, these interactions involve serial intra- and inter-hemispheric communication. However, the effect in the ipsilateral control group also suggests possible involvement of parallel BLA and PFC pathways during context-induced reinstatement. Therefore, it is not known from these findings how BLA communicates with PFC subregions. In contrast, disconnection of BLA and DH was found to attenuate context-induced reinstatement of cocaine seeking specifically in the contralateral and not ipsilateral group (Fuchs et al., 2007). This suggests that serial projections between

BLA and DH are functionally important for context-induced reinstatement. However,

BLA and DH project reciprocally to one another and the directionality of their interaction is not known using this disconnection strategy. Additionally, it is not known from this strategy whether DH-BLA interactions during reinstatement occur transynaptically or through direct anatomical projections.

Summary

This section reviewed the neurobiology of context-induced reinstatement of drug seeking with an emphasis on Acb and its connected structures. Several lines of evidence suggest that Acb plays a critical role in context-induced reinstatement of drug seeking.

However, there is inconsistency with regards to the specific subregion involved. For example, pharmacological inactivation and c-Fos activation studies implicates a specific role for AcbSh whereas other pharmacological inactivation studies implicate a role for both AcbSh and AcbC. Regardless, AcbSh is consistently implicated in context-induced reinstatement. In line with this, forebrain and midbrain structures that are preferentially linked with the AcbSh have also been shown to be involved in context-induced reinstatement. These include vmPFC (including ilPFC and medial orbital PFC), VH,

PVT, VTA, and LH. However, there is also evidence implicating forebrain structures 52

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that are preferentially linked with AcbC in context-induced reinstatement. These include dmPFC, DH, and BLA (rostral). Finally, there is functional evidence implicating the

DLS in context-induced reinstatement of both cocaine- and heroin-seeking. It is thought that through this structure, Acb modulates information from the motor cortices. A point of divergence within the literature appears to be related to the drug reinforcer. Thus pharmacological inactivation of AcbSh and vmPFC impairs reinstatement in heroin- trained rats whereas similar manipulation in AcbSh, AcbC and dmPFC impair reinstatement in non-heroin trained rats (e.g. cocaine, ethanol). However c-Fos studies implicate a specific role for AcbSh and not AcbC in other reinforcers (e.g. alcoholic beer, sucrose). Nonetheless, the possibility for specific drug-reinforcer dependent mechanisms is important with regards to current conceptual understanding of processes underlying relapse, and also practically, with regards to neuropharmacological targets of relapse. Moreover, of the structures implicated in context-induced reinstatement, functional contributions of AcbSh, DLS, and LH have been demonstrated across multiple reinforcers. Much remains to be known about the generalisability of the identified structures involved in context-induced reinstatement to other drug reinforcers

(e.g. nicotine). With regards to Acb contributions to circuit-level mechanisms of context-induced reinstatement, studies combining detection of tract tracer with c-Fos have identified two subcircuits: PVT→AcbSh and AcbSh (ventral)→LH. These circuit- level correlates provide additional evidence for the role of AcbSh in context-induced reinstatement, although this evidence is limited to a single reinforcer – alcoholic beer.

Finally, extra-Acb subcircuits mediating context-induced reinstatement have also been identified using pharmacological disconnection methods in cocaine-trained rats. These subcircuits include BLA interactions with dmPFC and orbital lateral PFC, and serial interactions between BLA and DH. It is noteworthy that these subcircuits are primarily

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Hippocampus DH

Accumbens Cortex dmPFC/lOFC AcbSh AcbC vmPFC

Amygdala rBLA

PVT VTA LH Thalamus Midbrain Hypothalamus

glutamate AcbSh afferents/efferents GABA AcbC afferents dopamine

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Figure 8. Circuit-level summary of brain regions mediating context-induced reinstatement. Solid arrows denote direction of monosynaptic projections recruited during context-induced reinstatement based on tract tracing/c-Fos data (Hamlin et al.,

2009; Marchant et al., 2009). Solid line connecting DH and BLA denotes function serial interactions contributing to context-induced reinstatement based on functional disconnection of these structures (Fuchs et al., 2007). Dotted lines denote confirmed anatomical connectivity in relation to AcbSh. Some anatomical connections have been omitted for simplicity.

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related to the AcbC. A summary of the circuit-level organisation of context-induced reinstatement of drug seeking is depicted in Figure 8.

In the following section, the neurobiology of extinction of drug seeking is reviewed, again in relation to the Acb and its circuit-level contributions. The following section begins with a review of the evidence implicating Acb and its connected structures in the extinction of drug seeking, followed by the evidence for circuit-level interactions mediating extinction of drug seeking. The review concludes by drawing upon parallel findings from the appetitive motivation literature to inform how circuit- level mechanisms of extinction might regulate those involved in reinstatement. Finally, the aims of the empirical chapters of this thesis are introduced.

2.4. A neurobiological system of extinction of drug-seeking

A neurobiology of extinction complements a neurobiology of context-induced reinstatement. Procedurally, context-induced reinstatement (as well as other forms of reinstatement) is typically measured against a background of extinction behaviour. This is theoretically relevant since extinction may be conceptually understood as the suppression of renewal behaviour. A neurobiological extension of this is that extinction might occur through inhibition of neurobiological processes mediating renewal. The previous section reviewed evidence for a connected neurobiological system underpinning context-induced reinstatement of drug seeking using a renewal preparation. The aim of the present section is to review the evidence that a parallel and similarly connected system might also mediate extinction of drug seeking. As in the previous section, this review focuses on data from drug self-administration preparations.

The reader is referred to Myers and Carlezon (2010) for a review of findings from studies using the conditioned place preference procedure. As in Myers and Carlezon

(2010), the term ‘extinction’ is used in reference to the processes underlying loss of 56

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responding in drug self-administration paradigms; ‘extinction training’ denotes the methodological procedure that promotes these losses; and ‘extinction expression’ refers to the decrement in responding observed after extinction training.

Studies of the neural mechanisms of extinction involve a three stage procedure: a period of self-administration training whereby responding (lever press or nose poke) is reinforced with a drug reward; extinction training whereby responding ceases to be reinforced; and finally, test for drug seeking under non-reinforcement. These stages typically occur within the same context. Experimental manipulations or measurements of neural function typically occur at one of three different times during this procedure.

Extinction expression studies involve manipulations or measurements prior to test.

Extinction learning studies involve manipulations or measurements prior to extinction training. Finally, studies examining consolidation of extinction learning involve manipulations or measurements in the period immediately following extinction training.

It is important to note that the study of extinction in drug self-administration preparations typically involve multiple days of extinction training in relatively long sessions (up to 2 hr). This can render empirical dissociation between extinction learning and extinction expression difficult because each day of extinction training involves the influences of both extinction learning (from that session) and extinction expression

(from previous extinction sessions as well as that session).

2.4.i. Nucleus Accumbens (AcbSh and AcbC) and extinction of drug seeking

Section 2.1 described some early literature implicating Acb control over an “activity system” (Kelly & Roberts, 1983). More recent molecular and reversible inactivation studies suggest that extinction-induced regulation over drug seeking might similarly involve a kind of inhibitory control over behavior that is Acb-dependent. Among the first pieces of evidence linking Acb to extinction of drug seeking was a series of 57

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molecular studies by Sutton and colleagues (2003), which highlighted a specific role for the AcbSh subregion. These authors trained rats to lever press for cocaine followed by extinction training. They found that extinction of cocaine-seeking was associated with up-regulated GluR1 and GluR2/3 subunits of the AMPA receptor in AcbSh compared with untreated control rats or rats that underwent withdrawal from cocaine self- administration but did not receive extinction training. The effect was specifically observed in the AcbSh and not AcbC. Moreover, two pieces of evidence suggested that increases in GluR1 but not GluR2 subunits were dependent upon extinction of cocaine seeking. First, GluR1 but not GluR2/3 failed to increase in rats that were prevented from responding in the chamber during extinction (i.e. levers retracted, extinction to the context only). Second, the magnitude of extinction that was behaviourally expressed was positively correlated with the increase in GluR1 expression, while the magnitude of cue-induced reinstatement to cocaine seeking was negatively correlated with GluR1 expression. Although these findings are based on correlational data, they are among the first demonstrations of extinction-related plasticity in an animal model of drug seeking.

However, the extinction-dependent changes in GluR1 expression were cocaine-specific; they were not observed in rats previously trained to respond for sucrose pellets. This suggests that processes underlying extinction of drug seeking may be reinforcer- dependent. In the same series of studies, Sutton et al. (2003) demonstrated a causal role for GluR1 and GluR2 subunits on extinction of cocaine-seeking. They showed that transient over expression of either GluR1 or GluR2 subunits in AcbSh using viral

(herpes simplex virus) mediated gene transfer reduced cocaine seeking during extinction and shortened the latency to achieve extinction criterion. The authors suggest that these effects were not likely due to motor deficits since animals overexpressing GluR subunits showed within- and between-session declines in responding that were typical

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of extinction. Moreover, similar manipulations were ineffective in sucrose-trained rats.

This latter finding again suggests that overexpression of AcbSh GluR1/GluR2 subunits likely impacts on cocaine-specific neuroadaptations rather than a general process of extinction learning. Congruent with this notion, the facilitated expression of extinction induced by overexpression of GluR1 and GluR2 subunits was not preserved on subsequent tests of extinction when GluR1 and GluR2 subunit levels returned to baseline. Possibly, overexpression of these AMPA receptor subunits induced a reduction in the motivation to seek cocaine rather than promoting extinction learning mechanisms (Sutton et al., 2003). Interestingly, despite GluR1 and GluR2 subunit levels returning to normal, their transient overexpression was sufficient to reduce vulnerability to stress induced reinstatement of cocaine seeking, suggesting that this manipulation may have conferred protection from reinstatement.

The findings from Sutton et al. (2003) are important because they suggest that extinction training can produce neuroadaptations in AcbSh that serve to regulate cocaine seeking during extinction and also buffer against stress-induced reinstatement.

Related studies from the same laboratory additionally suggests that extinction training actively reverses or “normalises” AcbSh neuroadaptations produced by cocaine self- administration and/or withdrawal from cocaine self-administration. For example, extinction training attenuated the reduction in tyrosine hydroxylase protein levels observed in animals that had undergone home-cage cocaine withdrawal (Schmidt et al.,

2001). Similarly, cocaine withdrawal-related reductions in the NMDA receptor subunit,

NR1, were normalised to baseline levels in rats that had undergone extinction training

(Self, Choi, Simmons, Walker, & Smagula, 2004). Finally, extinction training reversed several changes in gene expression that were observed in animals undergoing home- cage cocaine withdrawal. Among these are included extinction-induced normalisation of

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mRNA and protein levels for mu-opioid receptor 1; cannabinoid receptor CB1; and immediate early genes, Zif 268 and Narp (neuronal activity-regulated pentraxin)(Self et al., 2004). Each of these changes was specifically expressed in AcbSh and not AcbC.

Therefore, extinction training appears to produce several neuroadaptations in AcbSh that actively oppose some of the neuroadaptations otherwise produced in animals with a history of cocaine self-administration.

In contrast, more recent work suggests a role for AcbC but not AcbSh in the extinction of cocaine seeking, based on observed extinction-dependent changes in protein expression. Specifically, extinction of cocaine self-administration was associated with an increase in expression of postsynaptic density (PSD) scaffolding proteins in AcbC, including Narp, Homer1b/c, and PSD-95 (Knackstedt et al., 2010).

The extinction-induced increase in Homer 1b/c was also concomitant with a reduction in AcbC cell surface expression of the group 1 metabotropic glutamate receptor, mGluR5. This is consistent with a role for Homer 1b/c protein in the sequestration and internalisation of the mGluR5 receptor (Kammermeier, 2006). Extinction of cocaine seeking was also concomitant with impaired long-term depression (LTD) in AcbC. The authors proposed that extinction of cocaine seeking might be mediated by upregulation of Homer 1b/c, resulting in internalisation of mGluR5 and loss of LTD. In partial support of this hypothesis, adenoassociated virus (AAV)-mediated over expression of

Homer1c in AcbC impaired induction of LTD induced by low frequency stimulation of plPFC. Moreover, AAV-mediated over expression of Homer1c in AcbC prior to extinction training attenuated cue induced reinstatement of cocaine seeking (but not reinstatement in cocaine trained animals that only experienced abstinence). Notably, the upregulation of protein expression in AcbC was specific to animals with a history of cocaine self-administration and extinction training because they were not observed

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during initial extinction of cocaine self-administration nor were they observed in animals trained and extinguished using a sucrose pellet reinforcer. Therefore, as in

Sutton et al. (2003), the extinction-dependent changes in the AcbC may be cocaine- specific rather than related to a general process of extinction learning. However, contrary to Sutton et al. (2003), these changes were specific to AcbC and were not observed in AcbSh. Nonetheless, Knackstedt et al. (2010) noted a trend towards increased GluR2 expression in animals that underwent extinction, and also decreased

GluR1 and GluR2 in animals that had undergone abstinence. It is worth noting that in

Sutton et al. (2003), extinction-induced changes in GluR subunit expression were measured relative to animals that experienced home-cage abstinence in place of extinction or in untreated control rats. Therefore, changes in GluR1 and GluR2 subunit levels may have been measured against a background of lower levels of GluR protein expression. In contrast, changes in protein expression in Knackstedt et al. (2010) were measured against animals that were tested in the operant chambers (i.e., yoked saline control rats). This might account, at least partly, for the lack of AcbSh contribution in

Knackstedt et al. (2010), although it is unclear why extinction-dependent plasticity in

AcbC was not observed in the previously described studies (Schmidt et al., 2001; Self et al., 2004; Sutton et al., 2003).

Finally, findings from Ghasemzadeh et al. (2009) suggest that extinction-induced glutamatergic plasticity in the striatum of cocaine-seeking rats is broadly expressed in

AcbSh, AcbC, and DLS. In rats trained to respond for cocaine, subsequent extinction of cocaine seeking was associated with reductions in mGluR5 protein and scaffolding proteins (Homer 1 b/c and PSD95) in AcbSh, but not in AcbC or DLS. These findings contrast with the upregulation of AcbC mGluR5 observed after extinction of cocaine- seeking in Knackstedt et al. (2010). Moreover, in Ghasemzadeh et al. (2009), extinction

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was associated with increased mGluR5 protein in the DLS, and an increase in PSD-95 in the AcbC, consistent with Knackstedt et al. (2010). These changes were specific to extinction since they were not observed in animals that had undergone withdrawal in their home-cage or animals that were prevented from responding during extinction (i.e. levers retracted). However, in each of the studies reviewed above, a functional role for

Acb in extinction of drug seeking cannot be inferred. Changes in protein expression provide correlative data, while behavioural assessment of extinction following protein overexpression (Knackstedt et al., 2010; Sutton et al., 2003) demonstrates functional capacity of Acb to modulate behaviour, but not whether these Acb processes are normally recruited to mediate behavioural expression of extinction. This question can be addressed using reversible inactivation of the Acb on extinguished drug seeking.

The molecular studies described above implicate both AcbSh and AcbC in the behavioural expression of extinction in cocaine-trained rats. However, there is less evidence from functional inactivation studies that this role is shared between AcbSh and

AcbC. Recently, a functional role for the AcbSh in the extinction of cocaine seeking was demonstrated in rats that were initially trained to self-administer intravenous cocaine followed by extinction training (Peters, LaLumiere, & Kalivas, 2008). The authors found that when rats were subsequently tested under extinction conditions, reversible inactivation of AcbSh with GABA agonists, baclofen and muscimol prevented expression of extinction. In other words, pharmacological inactivation of

AcbSh reinstated extinguished reward seeking. Moreover, the effects of AcbSh manipulations were behaviourally specific to the expression rather than learning of extinction since inactivation of AcbSh did not affect responding on the first day of extinction training and did not affect the development of extinction measured on a subsequent infusion-free extinction test (Peters et al., 2008). Importantly, the effect was

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neuroanatomically specific to AcbSh and not AcbC. However, bilateral inactivation of

AcbSh significantly increased responding on both the active and inactive lever during test, and also increased locomotor activity suggesting that non-specific motor activation may contribute to the reinstatement of cocaine seeking induced by AcbSh inactivation.

A role for AcbSh in expression of extinction is consistent with earlier findings based on contextual modulation of ethanol seeking triggered by an ethanol prime

(Chaudhri, Sahuque, Cone, & Janak, 2008). These authors reported that exposure to an ethanol prime in an extinction context resulted in lower levels of responding than when presented in the original ethanol-paired context. This finding is reminiscent of an earlier demonstration showing that stress-induced reinstatement of cocaine seeking only occurred if the footshock stressor was delivered in the training and not the extinction context (Shalev, Highfield, Yap, & Shaham, 2000); that reinstatement is context- specific (Westbrook, Iordanova, McNally, Richardson, & Harris, 2002). Importantly, reversible inactivation of the AcbSh using GABA agonists, baclofen and muscimol, significantly enhanced responding following presentation of an ethanol prime in the extinction context but had no effect on responding in the ethanol-paired context. This finding is important because unlike the previous study (Peters et al., 2008), the extinction context here is not confounded by a previous history with drug reinforcement. Thus AcbSh appears to mediate the low levels of responding in the extinction context and removal of the AcbSh removes this regulation over responding.

However, a role for AcbSh in the extinction of ethanol seeking is confounded by the presence of the ethanol prime on test. That is, in Chaudhri et al. (2008), it is not known whether the extinction context alone is sufficient to recruit AcbSh control over responding. Regardless, these effects were specific to AcbSh and not observed following similar manipulations in AcbC.

Chapter 2. Neural Mechanisms of Extinction

Finally, Fuchs and colleagues (2008) demonstrated that reversible inactivation of

Acb (combined AcbSh and AcbC targets) using GABA agonists, baclofen and muscimol, prevented expression of extinction in a context-induced reinstatement preparation. However, in contrast to previous studies (Chaudhri et al., 2008; Peters et al., 2008), for cannulae restricted to the AcbSh, inactivation of AcbSh had no significant effect on extinction behaviour. Similar results were observed for cannulae restricted to the AcbC. It is notable that the pattern of results in Fuchs et al. (2008) was different to those reported by Peters et al. (2008). In the latter study, reversible inactivation of

AcbSh potentiated responding on both active and inactive manipulanda, as well as locomotor responding. In contrast, AcbSh inactivation in Fuchs et al. (2008) was ineffective on each of these measures. These discrepancies might be related to procedural differences, for example, Fuchs et al. (2008) used a renewal paradigm where extinction training (including extinction test) occurred in a context separate to the self- administration context. Alternatively, diffusion from AcbC into AcbSh may have occurred. This may explain the small (non-significant) effect of AcbC inactivation on attenuating extinction expression. Finally, the AcbSh target sites in Fuchs et al. (2008) were positioned ventrolaterally (including the olfactory tubercle transition zone), whereas the injection sites in Peters et al. (2008) were largely confined to the medial

AcbSh. This may account for the differences observed between the two studies since medial and ventrolateral AcbSh have distinct functional and anatomical properties. This evidence was reviewed in Section 2.2.

In summary, there is compelling evidence from molecular and pharmacological studies that Acb is a critical structure mediating expression of extinction. However, much of this data relies on cocaine-reinforcement and it is not known whether this role for Acb generalises to other drug reinforcers. This is especially important given that

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extinction-induced neuroadaptations in the Acb of cocaine-trained rats are not observed in rats trained with a non-drug reward. Moreover, there are several inconsistencies relating to AcbSh and AcbC contributions to extinction. For example, evidence from reversible inactivation studies implicates a functional role for AcbSh in the expression of extinction of drug seeking. This role is supported by several demonstrations of

AcbSh-specific neuroadaptations following extinction of cocaine seeking. In contrast, there is currently no functional evidence demonstrating AcbC contributions to extinction. However, that AcbC might be recruited during extinction is supported by studies demonstrating extinction-induced neuroplasticity in AcbC.

2.4.ii. Afferents of the nucleus accumbens and extinction of drug seeking

Prefrontal cortex. The most direct evidence for functional ilPFC contributions to extinction of drug seeking involves findings from a series of reversible inactivation studies (Peters et al., 2008). These authors trained rats to self-administer cocaine followed by extinction training. When subsequently tested under extinction conditions, reversible inactivation of ilPFC and not plPFC using GABA agonists, baclofen and muscimol, reinstated the previously extinguished drug seeking response. Moreover, reversible inactivation of ilPFC prior to the first day of extinction training did not affect responding during initial extinction training or on a subsequent drug-free test. This suggests that, similar to AcbSh contributions to extinction, ilPFC plays a critical role in the expression but not acquisition of extinction of cocaine seeking. However, ilPFC inactivation also produced a small but significant increase in inactive lever pressing, although, unlike similar manipulations in AcbSh, it had no significant effect on locomotor activity. Peters et al. (2008) provide a second, complementary piece of evidence supporting ilPFC contributions to the expression of extinction of cocaine seeking. They show that in a separate group of rats that received cocaine self- 65

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administration followed by extinction, cocaine-prime reinstatement was prevented by pretest infusions of the glutamate agonist AMPA into ilPFC. Thus removal of ilPFC reinstates extinguished reward seeking while pharmacological stimulation of ilPFC prevented cocaine prime-reinstatement. Finally, reinstatement induced by reversible inactivation of ilPFC was blocked by concurrent reversible inactivation of plPFC and

BLA. This suggests that removal of ilPFC reveals plPFC- and BLA-dependent reinstatement. Given the evidence that context-induced reinstatement of cocaine seeking is dependent on both plPFC and BLA (Fuchs et al., 2005), these findings suggest that, at least for cocaine-seeking rats, the behavioural expression of extinction and context- induced reinstatement might be mediated by separate circuits emerging from medial

PFC.

A role for ilPFC in extinction expression is consistent with findings from c-Fos activation studies identifying recruitment of ilPFC neurons during extinction in a context-induced reinstatement preparation. Marchant et al. (2010) observed that when animals were trained to respond for alcoholic beer in Context A followed by extinction in Context B, the return to the extinction context was associated with increased expression of c-Fos protein in ilPFC relative to animals tested in the training context.

The effect was not observed in plPFC. Therefore, a role for ilPFC in extinction expression generalises to animals trained with alcoholic beer. However, these findings are inconsistent with previous studies of context induced reinstatement of alcoholic beer- (Hamlin et al., 2007) or sucrose-seeking (Hamlin et al., 2006) where similarly pronounced levels of c-Fos protein expression in ilPFC were observed regardless of the test context. Recall also that in cocaine seeking rats, context-induced reinstatement and not extinction was associated with elevated c-Fos expression in ilPFC (Hamlin et al.,

2008). Finally, the findings from Marchant et al. (2010) contrast with recent findings

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from context-induced reinstatement of heroin seeking (Bossert et al., 2011) where c-Fos protein expression was increased in both plPFC and ilPFC when animals were returned to the self-administration context, A (group ABA), compared to animals tested in the extinction context (group AAA).

Finally, there is tentative evidence that ilPFC contributions to extinction involve the consolidation of extinction learning. For example, LaLumiere, Niehoff, and Kalivas

(2010) reported that in rats trained to self-administer cocaine followed by a series of brief (30 min / day) extinction training sessions, extinction performance on subsequent full-length (2 hr) tests was impaired in animals that received daily post-session reversible inactivation of ilPFC during extinction training relative to saline-treated animals. The effect was only observed in ilPFC and not in plPFC. Moreover, it was only observed if extinction training sessions were brief (30 min) and not relatively long (2 hr). Possibly, longer extinction sessions enabled consolidation of the extinction memory within the session and so rendered it insensitive to post-extinction inactivation manipulations of ilPFC (LaLumiere et al., 2010). However, based on these findings, a role for ilPFC in extinction consolidation is tentative. This is because for ilPFC inactivation to affect extinction consolidation, one would expect this manipulation to also impact on the development of extinction during extinction training. However, there was no evidence that the acquisition of extinction behaviour during extinction training was impaired by post-session reversible inactivation of ilPFC. Nonetheless, the authors showed that intra-ilPFC inactivation given 3 hr post session during extinction training had no effect on subsequent extinction test. This suggests that the extinction-impairing effects of ilPFC inactivation are limited to when infusions are made immediately following extinction.

Chapter 2. Neural Mechanisms of Extinction

LaLumiere and colleagues (2010) supplemented their findings with a series of additional studies implicating ilPFC in extinction consolidation. They show that daily post-session intra-ilPFC infusions of the allosteric AMPA receptor potentiator, PEPA, or the beta-adrenergic agonist, clenbuterol, during extinction training enhanced the retention of extinction learning on a subsequent (infusion-free) of extinction. However, this evidence is based on animals with a history of very brief (15 min/d) extinction training sessions and there was no evidence of extinction learning in the vehicle control group. This makes it difficult to infer that PEPA or clenbuterol is acting on processes of extinction consolidation and it is not clear from the behaviour what the animal is learning from these brief extinction sessions. Moreover, the effects of PEPA or clenbuterol on test may be due to a “hangover effect” of the drug from the previous day.

Finally, the authors show that daily pre- but not post-session intra-ilPFC infusions of the beta-adrenergic antagonist, ICI-118551, during extinction training impaired retention of extinction learning on a subsequent full-length infusion-free test of extinction. Again, the extinction-relevant processes impacted by ICI-118551 are not clear. If ICI-118551 targeted processes of extinction learning, one might expect to see differences between groups during extinction training as there was evidence of extinction acquisition.

However, there were no group differences observed during extinction training.

Conversely, if ICI-118551 targeted processes of extinction consolidation, then one might expect to see group differences at test following repeated post-session infusions of ICI-118551. However, the effect was specific to animals receiving pre-session infusions of ICI-118551 during extinction training. Therefore, inferences relating to extinction consolidation and intra-ilPFC manipulations are not clear from these findings.

Taken together, there is functional evidence implicating ilPFC in the behavioural

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expression of extinction. However, this evidence is limited to animals with a history of cocaine-reinforcement. It is not known whether a functional role for ilPFC in extinction of drug seeking is shared across multiple reinforcers, although there is preliminary evidence in alcoholic beer-trained rats showing increased neuronal activation of ilPFC concomitant with the behavioural expression of extinction (Marchant et al., 2010).

However, there are several inconsistencies among similar studies assessing extinction- related c-Fos protein expression. For example, elevated expression of ilPFC c-Fos protein was associated with the opposite behaviour, context-induced reinstatement.

Finally, a role for ilPFC in consolidation of extinction memory has also been suggested.

Together, the evidence implicating mPFC in extinction of drug seeking is largely specific to the ilPFC region and there is no functional evidence for plPFC contributions to the extinction of drug seeking. This is interesting given that plPFC may mediate context-induced reinstatement of drug seeking, as reviewed previously. Therefore, although circuits mediating extinction and context-induced reinstatement of drug seeking appear to overlap within the AcbSh, there is some evidence that these circuits diverge at the level of the mPFC.

Medial dorsal hypothalamus. The medial dorsal hypothalamus (MDH) refers to the medial part of the dorsal tuberal hypothalamus, and includes both the perifornical and dorsomedial nuclei of the hypothalamus. Anatomically, this region projects to PVT, which provides a source of glutamatergic afferents into AcbSh. Therefore, although not immediately connected with AcbSh, MDH can access AcbSh via efferent projections to

PVT.

Behaviourally, MDH has long been associated with termination of motivated behaviours such as feeding and intracranial self-stimulation (Porrino, Coons, &

MacGregor, 1983; Stellar, 1954). A role for MDH with reduction of motivated

Chapter 2. Neural Mechanisms of Extinction

behaviour is consistent with recent findings implicating MDH in extinction behaviour.

This was demonstrated in a context-induced reinstatement preparation using intra-MDH injections of the inhibitory neuropeptide, CART 55-102 (Marchant et al., 2010). Thus, in animals trained rats to respond for alcoholic beer in Context A, followed by extinction in Context B, microinjections of the peptide fragment, CART 55-102 into

MDH prevented the expression of extinction, and therefore reinstated responding, when rats were returned to the extinction context, B. However, this manipulation had no effect when rats were tested in the training context, A. The effects of CART 55-102 infusions were also dose-dependent, specific to the 55-102 CART fragment (it was not observed using the inactive CART 1-27 sequence), and anatomically specific; similar infusions into the immediately adjacent LH had no effect on the expression of extinction. Finally, similar findings were observed in animals trained to respond for sucrose reward. As

CART 55-102 neuropeptide causes inhibitory post-synaptic actions via a Gi/o containing

G-protein-coupled receptor (Rogge, Jones, Hubert, Lin, & Kuhar, 2008), it indirectly inhibits neurons in MDH. The findings from Marchant et al. (2010) therefore suggest that MDH mediates extinction across multiple reinforcers and that CART-induced neuronal inhibition removes this control over extinction.

2.4.iii. Circuit-level mechanisms of extinction expression.

As reviewed above, AcbSh and its connected structures (ilPFC, BLA and MDH) are each implicated in the expression of extinction of drug seeking. In assessing how these structures might interact to enable the behavioural expression of extinction, there are two questions of interest. The first is the nature of the connectivity between these regions – whether these connections occur directly or indirectly, serially or in parallel.

The second is how these structures interface with the circuits mediating reinstatement.

There are strong theoretical and empirical reasons for expecting such an interface. 70

Chapter 2. Neural Mechanisms of Extinction

Theoretically, the expression of extinction is viewed as an active masking or inhibition of drug seeking. This was discussed earlier in Chapter 1. Empirically, Peters et al.

(2008) reported that reinstatement produced by inactivation of ilPFC could be prevented by concurrent inactivation of plPFC or BLA, suggesting that removal of ilPFC results in activation of reinstatement circuits involving plPFC and BLA. Therefore, both theoretically and empirically, one might expect behavioural expression of extinction to result from active inhibition of circuit level mechanisms of reinstatement.

In studies that have investigated extinction circuitry, two distinct approaches have been adopted. The first involves combined immunohistochemical detection of the activity marker, c-Fos, with the neuronal tract tracer cholera toxin B (CTb) to identify activated projections during the expression of extinction. This strategy enables inferences regarding circuit-level correlates of extinction behaviour, including specificity of the direction of the connection. The second approach involves pharmacological disconnection of two putatively interacting structures. This strategy enables functional inferences regarding sub-circuits mediating extinction expression and also inferences regarding the nature of the information flow between these structures

(e.g. serial or parallel). Both these approaches were described in detail in the previous section, Section 2.2.

Using a pharmacological disconnection procedure, Peters et al. (2008) provided functional evidence that ilPFC interacts with AcbSh to mediate the extinction of cocaine seeking. In this study, the authors trained rats to self-administer cocaine then extinguished this response. On a subsequent test of extinction, they observed that simultaneous unilateral inactivation of ilPFC and AcbSh in either contralateral or ipsilateral hemispheres prevented expression of extinction of cocaine seeking. However, the effect in the ipsilateral group limits inferences regarding the serial nature of a

Chapter 2. Neural Mechanisms of Extinction

putative ilPFC→AcbSh pathway in extinction of cocaine seeking. To rule out the possibility that the disconnection effects were due to independent and additive effects of unilateral AcbSh and ilPFC inactivation, the authors confirmed that unilateral inactivation of either of these structures alone failed to reinstate cocaine seeking. It is possible that serial inter- and intra-hemispheric communication is necessary for the expression of extinction. This interpretation is consistent with known anatomical projections from ilPFC to AcbSh, which are most dense in the ipsilateral hemisphere, although they also terminate (albeit less so) in the contralateral hemisphere (Berendse et al., 1992). However, reinstatement of cocaine seeking following ipsilateral disconnection might also reflect recruitment of parallel extinction circuits involving ilPFC and AcbSh. There are two pieces of evidence from tract-tracing studies to support this possibility. First, using combined immunohistochemical detection of the neuronal tract-tracer, CTb with c-Fos protein expression, Hamlin et al. (2009) assessed retrograde labelled cells from AcbSh in animals trained to respond for alcoholic beer in an ABA context-induced reinstatement preparation. In ilPFC, neuronal activity was not expressed in AcbSh-projecting neurons, regardless of whether the animal was tested in the training context, A, or the extinction context, B. The only region showing extinction-associated increases in neuronal activity in AcbSh-projecting cells was the caudal portion of the BLA. However these projections were also recruited in rats showing context induced reinstatement. Therefore, based on this tract-tracing data, there was no evidence for an ilPFC→AcbSh pathway in extinction of alcoholic beer seeking.

Instead, there was some evidence for the recruitment of a caudal BLA→AcbSh pathway during extinction expression.

The second piece of evidence implicating parallel extinction circuits involves findings based on assessment of retrograde-labeled cells from MDH and PVT

Chapter 2. Neural Mechanisms of Extinction

(Marchant et al., 2010). In a context induced reinstatement preparation, rats were trained to respond for alcoholic beer in context A followed by extinction in context B. On test, return to the extinction context B was associated with increased c-Fos induction in

MDH-projecting cells from ilPFC. In contrast, these cells were not recruited when animals were tested in the training context, A. This provides anatomical evidence for recruitment of a direct ilPFC→MDH pathway during extinction expression. Finally,

Marchant et al. (2010) investigated recruitment of PVT-projecting cells from MDH using combined CTb/c-Fos protein detection in context-induced reinstatement of alcoholic beer seeking. PVT has previously been shown to be functionally critical for context-induced reinstatement of alcoholic beer seeking (Hamlin et al., 2009).

Moreover, it is a known efferent target of MDH (Berendse & Groenewegen, 1990; Li &

Kirouac, 2008). Importantly, Marchant et al. (2010) observed that projections from

MDH to PVT were recruited during test in the extinction context and not in the training context. The majority of these activated projection neurons also expressed the opioid peptide precursor, prodynorphin. The prodynorphin precursor encodes opioid peptides dynorphin A, dynorphin B and neoendorphin, that each shows high affinity for the receptor (KOR) (McNally & Akil, 2002). As KOR mRNA is expressed at high levels in

PVT (Mansour, Khachaturian, Lewis, Akil, & Watson, 1987), the actions of prodynorphin peptide(s) at opioid receptors in PVT may be critical for inhibiting alcoholic beer seeking thereby enabling extinction expression. Marchant et al. (2010) tested this possibility directly. They showed that microinjections of a KOR agonist into

PVT prevented context-induced reinstatement of alcoholic beer seeking. This supports the possibility that expression of extinction is due to increases in neurotransmission via a prodynorphin projection from MDH to PVT.

Together, the evidence for a functional ilPFC→AcbSh pathway mediating

Chapter 2. Neural Mechanisms of Extinction

extinction of cocaine seeking remains tentative with regards to the nature of their connectivity. It is possible that serial intra- and inter-hemispheric ilPFC→AcbSh interactions enable expression of extinction of cocaine seeking. This is consistent with the neuroanatomy of ilPFC projections to AcbSh. Alternatively, ilPFC and AcbSh might contribute to extinction via parallel pathways. In support of this possibility, there is correlative evidence from combined tract-tracing/c-Fos studies showing that extinction of alcoholic beer seeking is associated with recruitment of a direct ilPFC→MDH and

MDH→PVT pathway. There is also some evidence for an extinction-associated cBLA→AcbSh pathway, although this pathway is also recruited during context-induced reinstatement. In contrast, AcbSh-projecting cells from ilPFC were not found to be recruited during extinction. However, there are procedural differences to be considered when drawing comparisons between the findings of the functional disconnection and tract-tracing studies. These include differences in drug reinforcers (cocaine vs. alcoholic beer) as well as conditions of extinction training (i.e. extinction occurring in the training context, or in a separate distinct context). A final note is that a putative extinction ilPFC→MDH circuit might enable extinction of drug seeking through a peptidergic

MDH→PVT projection. PVT has been implicated in context-induced reinstatement and

MDH might serve to enable extinction circuit-control over reinstatement circuitry. This is an interesting possibility and as yet, there is little known of how extinction circuitry serves to interact with reinstatement circuitry.

2.5. AcbSh control over LH dependent behaviour: a putative interface of extinction and reinstatement circuits

So far, this section has presented substantial evidence implicating AcbSh as a critical component of the extinction circuitry. There is also evidence as reviewed in the previous section that LH is a functionally critical substrate for enabling context-induced 74

Chapter 2. Neural Mechanisms of Extinction

reinstatement. Moreover, assessment of retrograde labelled cells from LH shows that

LH-projecting cells from ventral AcbSh are recruited during context-induced reinstatement (Marchant et al., 2010). Interestingly, in the same study, Marchant et al.

(2010) also observed that LH-projecting cells from dorsal AcbSh are recruited during both context induced reinstatement and also during expression of extinction, relative to non-tested control rats. As in the case of MDH, AcbSh might serve to interface extinction circuitry with reinstatement circuitry. However, that dorsal AcbSh projections are recruited during both reinstatement and extinction makes it difficult to interpret the possible contribution of these projections to behaviour. Nonetheless, there are several reasons to consider that AcbSh might contribute to extinction via regulatory

(inhibitory) control over LH-dependent mechanisms of reinstatement.

Anatomically, the majority of AcbSh projection neurons are GABAergic

(Meredith, 1999). AcbSh therefore has inhibitory control over is efferent targets, including LH, which is functionally important for context-induced reinstatement

(Marchant et al., 2010). Additionally, as reviewed earlier (Section 2.1), the dorsal region of AcbSh receives projections from cBLA and there is preliminary evidence implicating cBLA→AcbSh projections in extinction of drug seeking (Hamlin et al., 2009). In contrast, ventral AcbSh receives projections from PVT, and similarly, there is evidence from c-Fos/tract-tracing studies implicating a PVT→AcbSh pathway in context-induced reinstatement of drug seeking (Hamlin et al., 2009). This anatomical arrangement is interesting in light of differential recruitment of ventral AcbSh→LH and dorsal

AcbSh→LH projections during context-induced reinstatement and extinction of drug seeking, respectively. Specifically, the dorsal AcbSh appears well-connected to an extinction circuit involving cBLA and also inhibitory GABAergic control over LH.

Behaviourally, AcbSh control over LH-dependent behaviour has been long

Chapter 2. Neural Mechanisms of Extinction

recognised within the literature of appetitive motivation. Amongst the earliest studies demonstrated that pharmacological blockade of AcbSh AMPA/Kainate receptors using antagonists, DNQX, CNQX, or NBQX, significantly and dose-dependently increased food intake in sated rats (Maldonado-Irizarry et al., 1995). The effect was specific to

AcbSh AMPA/Kainate receptors since similar manipulations in AcbC as well as blockade of AcbSh NMDA receptors were ineffective. Importantly, bilateral reversible inactivation of the LH with the GABAA agonist, muscimol, or NMDA receptor antagonist, AP-5, dose-dependently prevented feeding produced by AcbSh inactivation.

This suggests that AcbSh exerts inhibitory control over LH-dependent feeding. These findings are important because they are the first to demonstrate the functional significance of an AcbSh–LH relation; that Acb control over feeding is specific to the

AcbSh and not AcbC, and that this role for AcbSh involves inhibition over LH.

Based on these findings, Maldonado-Irizarry et al. (1995) proposed a preliminary model of AcbSh control over feeding. They proposed that AcbSh medium spiny projection neurons (MSNs) are tonically excited via non-NMDA glutamatergic afferents acting, in part, on AMPA-type receptors. Moreover, temporary removal of this excitation via blockade of AcbSh AMPA receptors disinhibits LH from AcbSh to produce feeding. Consistent with this hypothesis, Stratford and Kelley (1997) showed that in satiated rats, temporary removal of AcbSh excitation using GABA agonists, baclofen or muscimol, also produced feeding without altering water intake or locomotor activity. Although increasing local GABA via AcbSh injections of GABA uptake inhibitor, nipecotic acid was ineffective, increasing endogenous GABA by selectively inhibiting the metabolic enzyme, GABA transaminase, dose-dependently increased feeding. This suggests that control over feeding might be mediated by maintaining a level of GABAergic tone in AcbSh (Stratford & Kelley, 1997). Finally, in assessing

Chapter 2. Neural Mechanisms of Extinction

AcbSh control over LH-dependent feeding, Stratford and Kelley (1997) confirmed that reversible inactivation of LH dose-dependently prevented feeding induced by AcbSh inactivation, and showed that feeding produced by AcbSh inactivation concomitantly increased the induction of the immediate early gene product, c-Fos, in LH. Thus, consistent with the model introduced in Maldonado-Irizarry et al. (1995), the findings from Stratford and Kelley (1997) suggest that removal of AcbSh disinhibits LH thereby increasing neuronal activity in the LH. The relevance of this model is that it provides a neuroanatomical basis for the regulation of motivated behaviour at the level of the

AcbSh. Given the evidence implicating a role for AcbSh in extinction, an interesting possibility is that AcbSh similarly achieves extinction-induced regulation over drug seeking via interactions with LH.

Summary

A neurobiology of relapse spans two complementary circuits: a facilitatory circuit mediating reinstatement and an inhibitory circuit mediating extinction. A conceptual model of extinction such as the memory interference model suggests that these two processes compete for behavioural expression. Thus a putative neurobiological mechanism for the behavioural expression of extinction might similarly involve inhibition over the reinstatement circuit. In this section, the neurobiology of extinction of drug seeking was reviewed with an emphasis on Acb and its connected structures.

The circuit level mechanism of extinction was also reviewed in relation to the nature of the connectivity between structures (e.g. direct, transynaptic, serial, or parallel) and how these circuits might interface with those regulating reinstatement.

In summary of the present section, several lines of evidence suggest that AcbSh is critical for the behavioural expression of extinction. Reversible inactivation of the

AcbSh prevents expression of extinction, extinction is associated with neuroadaptive 77

Chapter 2. Neural Mechanisms of Extinction

changes in the AcbSh, and finally, overexpressing receptors that are normally upregulated by extinction training can facilitate the expression of extinction. In contrast, there is no functional evidence implicating the AcbC in the behavioural expression of extinction. Evidence implicating AcbC in extinction of drug seeking is based on extinction-associated changes in AcbC protein expression and also, overexpression of the Homer 1c protein, which is upregulated during extinction training, was found to attenuate reinstatement. In addition to AcbSh, only two other brain regions have been functionally implicated in the expression of extinction of drug seeking using functional inactivation methods. These are ilPFC and MDH. Moreover, a functional role for ilPFC in extinction is supported by evidence showing extinction-associated increases in c-Fos activation. However, there is inconsistency within the literature regarding the presence of extinction-associated c-Fos activation in ilPFC. It has also been suggested that ilPFC is involved in consolidation of extinction learning however there is not yet sufficient evidence to support this role.

In relation to circuitry, ilPFC is directly linked with both AcbSh and MDH.

Based on findings from pharmacological disconnection of ilPFC and AcbSh, recent authors have suggested that serial inter- and intra-hemispheric interactions between these structures mediate extinction of cocaine seeking. However, the findings of this study can also be explained in terms of parallel extinction circuits involving ilPFC and

AcbSh. The possibility for parallel extinction circuits is supported by several findings using combined tract-tracer with c-Fos protein expression. For example, both ilPFC→MDH and MDH→PVT pathways were recruited during expression of extinction. In contrast, there was no evidence that ilPFC→AcbSh neurons were recruited during extinction. Instead, pathways cBLA→AcbSh and AcbSh→LH were recruited during both extinction and reinstatement. Although the behavioural

Chapter 2. Neural Mechanisms of Extinction

implications of the latter pathways are difficult to interpret, several pieces of evidence implicate AcbSh control over LH as a putative mechanism enabling the extinction of drug seeking. For example, the literature on appetitive motivation provides compelling evidence for AcbSh control over LH-dependent feeding. Given the role for LH in reinstatement, AcbSh might similarly enable expression of extinction via inhibitory control over LH-dependent drug seeking.

A final point of consideration is that much of what is known about Acb and mPFC contributions to extinction is based on cocaine reinforcement. The generalisability of the contributions of these structures to extinction of responding for other drug reinforcers is unknown. This is especially important given that extinction- induced plasticity in AcbSh appears to be specific to cocaine and not sucrose reinforcement. Finally, while tract-tracing studies have identified a putative ilPFC→MDH→PVT pathway of extinction, the extinction circuitry involving AcbSh is not well-characterised. A summary of the extinction circuit as reviewed here is given in

Figure 9.

Experimental Aims

The aim of this thesis is to determine the role of the AcbSh in the extinction of alcoholic beer seeking in relation to its efferent and afferent connectivity. The first series of experiments reported in Chapter 2 examines the role of AcbSh in the extinction of alcoholic beer seeking and its functional interactions with LH. The second series of experiments reported in Chapter 3 characterises the role of AcbSh AMPA-type glutamate receptors in the extinction of alcoholic beer seeking and examines its functional interaction with the BLA.

Chapter 2. Neural Mechanisms of Extinction

Accumbens Cortex AcbSh vmPFC

PVT

Thalamus

MDH LH

Hypothalamus

glutamate Regions mediating expression of extinction GABA Regions mediating context-induced peptidergic reinstatement

Figure 9. Circuit-level summary of brain regions mediating extinction of drug seeking and putative junctions with structures involved in context-induced reinstatement. Solid arrows denote direction of monosynaptic projections recruited during extinction expression based on tract tracing/c-Fos data (Hamlin et al., 2009;

Marchant et al., 2009, 2010). Dotted line connecting vmPFC and AcbSh denotes function serial interactions contributing to extinction expression based on functional disconnection of these structures (Peters et al., 2008). Grey boxes and arrows denote putative structural interfaces between extinction and reinstatement circuitry.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Chapter 3.

Nucleus Accumbens Shell Mediates Extinction of

Reward-Seeking via Lateral Hypothalamus

Animals readily learn to self-administer drugs of abuse. In turn, self-administration can be reduced when the drug seeking behaviour no longer yields a drug reward. This decline in drug seeking is described as extinction. Extinction does not erase the original learning that mediated previous drug seeking. Rather, it actively inhibits drug seeking.

By implication, removal of this inhibition can reveal drug seeking. In Chapter 1, several studies were presented to show that this inhibition could be removed in several ways.

These include via re-presentations of the reinforcer (reinstatement; de Wit & Stewart,

1981), presentations of a drug-associated stimulus (cue-induced reinstatement; de Wit &

Stewart, 1981), or context-change between extinction and test (context-induced reinstatement; Crombag & Shaham, 2002). However, the neurobiological nature of this extinction-induced inhibition is unclear.

A possible neurobiological source of inhibition during extinction expression is the AcbSh. The evidence supporting this possibility was reviewed in Chapter 2 (Section

2.4). Notable findings were those reported in Peters et al. (2008). These authors trained rats to lever press for intravenous infusions of cocaine and subsequently extinguished cocaine seeking behaviour. AcbSh was subsequently inactivated prior to test, resulting in the reinstatement of cocaine seeking behaviour. In other words, removal of AcbSh prevented expression of extinction in cocaine-trained rats. However, the role of AcbSh in extinction of other drug and non-drug rewards, as well as the circuit-level

Chapter 3. Accumbens Shell and Lateral Hypothalamus

mechanisms for AcbSh contributions to extinction of drug seeking are not well- characterised.

Lateral hypothalamus (LH) is a potential target of AcbSh inhibition during extinction of reward seeking. Using retrograde neuronal tracer injected into LH combined with detection of c-Fos protein in the AcbSh, Marchant et al. (2009) showed that AcbSh projection neurons to LH were activated during expression of extinction in alcoholic-beer seeking rats. Functionally, LH mediates the behavioural impact of drug- associated stimuli (Harris et al., 2005) and contexts (Marchant et al., 2009). It also contains the neuropeptides orexin and CART, both of which have key roles in appetitive motivation and reinstatement of drug seeking (Dayas, McGranahan, Martin-Fardon, &

Weiss, 2008; Harris et al., 2005; James et al., 2010; King et al., 2010; Lawrence et al.,

2006; Richards et al., 2008). This role for LH and its neuropeptides CART and orexin were described previously in Chapter 2 (Section 2.3.ii).

The experiments presented in this chapter studied the role of AcbSh and its interactions with LH in the extinction of alcoholic beer seeking. There were four aims.

The first was to confirm a role for AcbSh in the extinction of drug seeking. To date, this role has been limited to rats trained to seek intravenously administered cocaine (Peters et al., 2008; Sutton et al., 2003). The second aim was to study the distribution of neuronal activation in hypothalamus associated with AcbSh inactivation-induced reinstatement of alcohol seeking. The third aim was to determine the functional contribution of LH in mediating reinstatement of alcohol seeking induced by inactivation of AcbSh. The final aim was to assess whether the expression of extinguished alcohol seeking was mediated by serial communication between AcbSh and LH.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

To address the above aims, Experiments 1 and 2 studied the effect of reversible inactivation of AcbSh on extinction expression and extinction learning respectively. In

Experiment 3, immunohistochemical detection of c-Fos (a marker of neuronal activity) was combined with immunohistochemical detection of orexin and CART peptides, to study the profile and phenotype of neural activation during reinstatement produced by

AcbSh inactivation. Experiment 4 assessed whether concurrent inactivation of LH would prevent reinstatement produced by AcbSh inactivation. Finally, Experiment 5 used a contralateral disconnection procedure to examine whether AcbSh interactions with GABAergic neurotransmission in LH enable the behavioural expression of extinction in alcoholic beer seeking rats.

Experiment 1

Experiment 1 aimed to confirm a role for AcbSh in mediating the extinction of alcoholic beer seeking. Rats received self-administration training followed by extinction. During training, rats learned to respond via nosepoke for alcoholic beer, which was delivered into a magazine from which rats were free to drink. There were two nosepoke manipulanda; responses on the ‘active’ nosepoke delivered beer into the magazine whereas responses on the ‘inactive’ nosepoke had no programmed consequences. During extinction responses on either nosepoke had no programmed consequences. Rats were subsequently tested under conditions identical to extinction.

Immediately prior to test, rats received intra-AcbSh infusions of saline or GABAB and

GABAA agonists, baclofen and muscimol (B/M). As in previous studies described in

Chapter 2, microinjections of B/M were used to pharmacologically and reversibly inhibit neuronal activity in order to study the functional contributions of the targeted neural structure (i.e. AcbSh). Given that the AcbSh is positioned ventral and adjacent to

Chapter 3. Accumbens Shell and Lateral Hypothalamus

the lateral ventricle, microinfusions in this experiment were aimed at either dorsal or ventromedial AcbSh. This was to isolate any effects of diffusion into the ventricle and to determine a sensitive site for infusions in subsequent experiments.

Method

Subjects

Thirty-nine experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed in groups of eight and maintained on a 12/12 h light/dark cycle (lights on at 7:00 A.M.).

Food and water were freely available until 1 d before behavioural training, after which rats were allowed 1 h access to food and water following daily training sessions. All procedures were approved by the Animal Care and Ethics Committee at The University of New South Wales and conducted in accordance with the National Institute of Health

(NIH) Guide for the Care and Use of Laboratory Rats (NIH Publications No. 80-23) revised 1996. The procedures were designed to minimise the number of animals used.

Surgery

Rats were injected intraperitoneally (i.p.) with the anaesthetic, ketamine (Ketapex; Apex

Laboratories; concentration of 100 mg/kg) and i.p. with 0.3 ml/kg muscle relaxant, xylazine (Rompun; Bayer; concentration of 20 mg/ml). They were shaved and then placed in a stereotaxic frame (Model 900, Kopf, Tujunga, CA) with the incisor bar maintained at ~3.3 mm below horizontal to achieve a flat skull position. Rats were implanted bilaterally with 26 gauge guide cannulae (Plastics One, Roanoke, VA) aimed at either the dorsal or ventromedial AcbSh. Flat skull coordinates relative to bregma were +1.35 mm anteroposterior (AP); ±0.75 mm mediolateral (ML); -5.6 mm or -6.5 mm dorsoventral (DV; AcbSh-D and AcbSh-V, respectively). All guide cannulae were 84

Chapter 3. Accumbens Shell and Lateral Hypothalamus

aimed 1 mm above target site, with coordinates based on the rat brain atlas of Paxinos and Watson (2005). Cannulae were secured to the skull using jeweller’s screws and acrylic cement. Obturators with dust caps were fitted to the guide cannulae to prevent occlusion. Immediately after surgery, rats were injected subcutaneously (s.c.) with 5 mg/kg carprofen, intramuscularly (i.m.) with 0.3 ml procaine penicillin (300 mg/ml) and i.m. with 0.1 ml cephazolin (100 mg/ml). Rats were given 5-7 d post-operative recovery prior to the start of behavioural procedures, during which time they were monitored and weighed daily.

Apparatus

In this and all following experiments, self-administration, extinction, and tests were conducted in eight standard Med-Associates operant chambers (St. Albans, VT), each enclosed in a sound- and light-attenuating cabinet equipped with a fan that provided constant ventilation and low-level background noise. For all chambers, front (hinged door) and rear walls were constructed of clear Perspex and end walls were made of stainless steel. Inside each chamber, two nosepoke holes containing a white cue-light were symmetrically located on one sidewall of the chamber, 3 cm above a grid floor. A recessed magazine was located behind a 4 x 4 cm opening in the centre of the same wall between the two nosepokes. Responding on one (active) nosepoke delivered beer reward to the magazine whereas responding on the other (inactive) nosepoke had no programmed consequences. There was no illumination in these chambers other than that provided by the white cue light recessed in the active nosepoke.

Microinfusion Procedure

For intracranial infusions, 33 gauge cannulae injectors (which projected 1 mm ventral to the tip of the guide cannula) were connected to a 10 μl Hamilton syringe via

Chapter 3. Accumbens Shell and Lateral Hypothalamus

polyethylene-50 tubing, which was mounted onto an infusion pump (KD Scientific,

Holliston, MA). Rats received bilateral intra-AcbSh infusions of saline or a solution containing both GABAB and GABAA agonists, baclofen (1.0 mм) and muscimol hydrobromide (0.1 mм), respectively (B/M; Sigma-Aldrich). B/M was prepared in a solution of 0.1 м phosphate buffered saline (PBS; pH 7.2). All microinjections were made in a volume of 0.5 μl per side over 2 min. These volumes and doses have been used in previous studies to inactivate the AcbSh and LH separately from their adjacent brain regions (Floresco et al., 2008; Fuchs et al., 2008; Marchant et al., 2009). After allowing an additional 2 min for diffusion, microinjectors were removed and rats were returned to their home-cage for approximately 2 min, before being tested in the self- administration chamber.

Behavioural Procedures

Self-administration training and extinction. After post-operative recovery rats were run daily in squads of eight. On the first two days, rats received 2 x 20 min magazine training sessions (per day) in the self-administration chamber. During these sessions rats received 10 non-contingent deliveries of 0.6 ml decarbonated beer

(Coopers Birrell’s Premium TM, < 0.5% w/v alcohol content) into the magazine cup at time intervals variable around a mean of 1.2 min. Pure ethanol was added to the beer so that it resembled full strength beer (adjusted to 4% v/v alcohol). From days 3 to 9, rats received daily 1 h self-administration sessions. During this phase, responses on the active nosepoke triggered a syringe pump, delivering 0.6 ml of 4% v/v alcoholic beer into the magazine cup on an FR-1 schedule of reinforcement, and extinguished the white cue light recessed in the nosepoke during a 24 s timeout. All responses on the inactive nosepoke had no programmed consequences.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Following self-administration, rats were given daily 1 h extinction sessions for 4 d. Procedures for extinction were identical to self-administration, except that syringes were removed from infusion pumps so that responses on the active nosepoke no longer resulted in the delivery of beer.

In this and all other experiments, rats were adapted to the conditions of the intracranial infusion procedure so that prior to each self-administration and extinction session, obturators were removed and the rats were placed into infusion buckets for 4 min. The obturators were then reinserted and the rats were returned to their home-cages for approximately 2 min before being placed into self-administration chambers.

Test. Following self-administration and extinction, rats were tested under conditions identical to extinction. Immediately prior to test, rats received bilateral microinfusion of saline (SAL group) or baclofen/ muscimol (B/M group) into the

AcbSh-D or AcbSh-V. An AcbSh-D group was included to determine whether reinstatement effects were related to diffusion of the drug dorsally towards the lateral ventricle. Therefore, there were four groups (SAL AcbSh-D, SAL AcbSh-V, B/M

AcbSh-D and B/M AcbSh-V), each matched according to the number of active nosepokes made over the last day of self-administration training and the first two days of extinction. Following test, rats received an additional day of extinction training (drug free, extinction day 5). This test examined whether the effect of AcbSh inactivation was specific to the presence of the drug infusion.

Histology

Rats were given an overdose of sodium pentobarbital (100 mg/kg, i.p.) at the conclusion of the study. Brains were extracted, frozen and sectioned coronally at 40 μm using a cryostat (Microm 560, Germany). All sections containing cannula tracts were collected onto glass slides, stained for Nissl substance with cresyl violet and cover-slipped with 87

Chapter 3. Accumbens Shell and Lateral Hypothalamus

the mounting agent, Entellan. Sections were examined under light microscope to determine cannula tip placements, which were then mapped onto plate sections from the atlas of Paxinos and Watson (2005).

Data analyses

The mean numbers of active and inactive responses were analysed using mixed group x

(response) factor ANOVA with orthogonal contrasts. Mean latency (minutes) to initiate the first response on the active nosepoke was analysed using planned orthogonal contrasts. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are indicated in Figure 10. Seven rats were excluded from analyses due to misplaced cannulae. Final group sizes were: SAL

AcbSh-D (n = 7), SAL AcbSh-V (n = 8), B/M AcbSh-D (n = 7) and B/M AcbSh-V (n =

10).

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

122.44 ± 12.11 and 4.03 ± 0.49, respectively. Between groups there were no overall differences in responding on the last day of training (main effect and interaction: Fs(1, 28)

< 1 ps > 0.05); all rats made significantly more active than inactive nosepokes (F(1, 28) =

182.57, p < 0.001).

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Mean ± SEM responses during extinction training are shown in Figure 11 (top panel). During extinction training, there were no significant differences between groups

(main effects and interactions Fs(1, 28) < 1.29, ps > 0.05). All rats made significantly more active than inactive nosepokes, averaged across days of extinction (F(1, 28) =

150.03, p < 0.001). Overall, responding significantly decreased across days of extinction (F(1, 28) = 126.92, p < 0.001), and this decrease was greater on the active than inactive nosepoke (F(1, 28) = 133.84, p < 0.001).

Figure 11 (middle panel) shows the mean ± SEM number of responses on test.

Testing conditions were identical to extinction training. Overall, group B/M AcbSh-V responded significantly more than all other groups (F(1, 28) = 29.25, p < 0.001) and this difference was greatest on the active than inactive nosepoke (F(1, 28) = 10.03, p < 0.001).

There were no significant differences between the other groups, SAL AcbSh-D, SAL

AcbSh-V or B/M AcbSh-D (Fs(1, 28) < 1, ps > 0.05). The latency in minutes to initiate the first active response following placement in the chamber was also analysed for

AcbSh-V groups. There was no difference in latency to first response between group

SAL AcbSh-V (mean = 4.54; SEM = 2.98) and group B/M AcbSh-V (mean = 7.23;

SEM = 4.73).

On the day following test (extinction day 5, Figure 11 bottom panel), there were no significant differences between groups (Fs(1, 28) < 2.51, ps > 0.05). Thus, the effect of AcbSh inactivation on expression of extinction was specific to the presence of B/M infused into the AcbSh-V. Together, these results show that reversible inactivation of

AcbSh-V but not AcbSh-D prevented the expression of extinction, and so reinstated responding. This confirms the finding from Peters et al. (2008) and suggests that a role for AcbSh in the expression of extinction may be shared across multiple reinforcers.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Bregma 2.04

Bregma 1.80

Bregma 1.56

Bregma 1.32

Bregma 1.20

Bregma 1.08

Figure 10. Experiment 1: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections. Groups: SAL AcbSh-D (grey dots), SAL AcbSh-V (grey triangles), B/M AcbSh-D (black dots) and B/M AcbSh-V (black triangles). In this and remaining figures, symbols represent the most ventral point of the cannula track, indicated on coronal sections adapted from Paxinos and Watson (2005).

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Extinction 100 Active 80 Inactive

Responses 20

0 1234 Day

Test 60 Active Inactive

20 Responses

0 AcbSh-D AcbSh-V AcbSh-D AcbSh-V SAL SAL BM BM

Extinction Day 5 60 Active Inactive

20 Responses

0 AcbSh-D AcbSh-V AcbSh-D AcbSh-V

SAL SAL BM BM

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Figure 11. Experiment 1: Mean ± SEM numbers of active and inactive responses during extinction (top panel), test (middle panel), and on a subsequent day of extinction

(Extinction Day 5). Before test, rats received infusions of B/M or saline. There were no microinfusions administered on Extinction Day 5.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Experiment 2

Experiment 1 examined the effects of AcbSh inactivation on extinction expression. In

Experiment 2, the effect of AcbSh inactivation on the acquisition of extinction learning was investigated. Rats were trained to self-administer beer. They then received two consecutive days of extinction training. Prior to the first day of extinction training, rats received intra-AcbSh microinjections of saline or B/M. No infusions were given on the second extinction day, which served as a test. If inactivation of AcbSh interfered with extinction learning on the first day of extinction, then animals previously treated with

B/M should show attenuated extinction behaviour (i.e. more reward seeking) on the second (infusion-free) day of extinction compared to animals previously treated with saline. In the present and subsequent experiments, intra-AcbSh microinjections were targeted at the ventromedial AcbSh because this was found to be the most effective site for reinstating the previously extinguished response.

Method

Subjects and Surgery

Twenty-one experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed and maintained as described in Experiment 1. Surgical procedures and AcbSh-V coordinates were as described in Experiment 1.

Behavioural Procedure

Rats received self-administration training followed by two days of extinction. Training and extinction procedures were as described in Experiment 1. Prior to the first day of extinction, rats received microinfusion of saline or B/M into the AcbSh. There were no infusions on the second day of extinction, which served as test. Groups were matched 93

Chapter 3. Accumbens Shell and Lateral Hypothalamus

on the average number of active nosepokes made over the last day of self- administration.

Microinfusion and Histology Procedure

Microinfusions and histology proceeded as described in Experiment 1.

Data analyses

The mean numbers of active and inactive responses were analysed using mixed group x

(response) x (day) factor ANOVA with orthogonal contrasts. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are indicated in Figure 12. Five rats were excluded from analyses due to misplaced cannulae. Final group sizes were, SAL (n = 7) and B/M

(n = 9).

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

86.69 ± 9.22 and 3.81 ± 1.02, respectively. Between groups, there were no overall differences in responding on the last day of training (main effect and interaction: Fs(1, 14)

< 1, ps > 0.05); all rats made significantly more active than inactive nosepokes (F(1, 14) =

88.71, p < 0.001).

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Bregma 2.04

Bregma 1.80

Bregma 1.56

Bregma 1.32

Bregma 1.20

Bregma 1.08

Figure 12. Experiment 2: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections. Groups SAL and B/M are indicated in grey and black dots respectively.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Extinction

Figure 13. Experiment 2: Mean ± SEM numbers of active and inactive responses during test (extinction days 1 and 2). Rats received infusions of B/M or saline into ventromedial AcbSh immediately before Extinction Day 1 and not on Extinction Day 2.

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Mean ± SEM responses during the first two days of extinction are shown in

Figure 13. There were no significant differences between groups across days or on response type (Fs(1, 14) < 1.52, ps > 0.05). All rats made significantly more active than inactive nosepokes, averaged across days (F(1, 14) = 102.41, p < 0.001). Overall, responding significantly decreased on the second day of extinction (F(1, 14) = 34.47, p <

0.001), and this decrease was greater on the active than inactive nosepoke (F(1, 14) =

40.88, p < 0.001). These results show that AcbSh inactivation has no significant effect on the acquisition of extinction; reversible inactivation of AcbSh had no effect on the first day of extinction or on the second infusion-free day of extinction. These findings are consistent with those found in cocaine-trained rats (Peters et al., 2008). Together with Experiment 1, the present findings suggest that a role for AcbSh in extinction may be specific to the behavioural expression of extinction.

Experiment 3

The results of Experiments 1 and 2 suggest that reversible inactivation of the AcbSh prevent the expression but not acquisition of extinction of alcoholic beer seeking. In

Experiment 3, the possibility that an AcbSh–hypothalamic interaction may be critical for the expression of extinction was assessed. To this end, reinstatement-associated induction of c-Fos protein expression was assessed in hypothalamic regions following

AcbSh inactivation. These hypothalamic regions included dorsomedial (DMH), perifornical (PeF) and lateral (LH) hypothalamus. The peptidergic phenotype (orexin and CART) of activated hypothalamic neurons was also assessed. To investigate the potential circuit-level mechanisms that might mediate reinstatement produced by AcbSh inactivation, Experiment 3 also studied c-Fos induction in CART neurons in the arcuate nucleus (Arc) and central nucleus of the amygdala (CeA). These structures were

Chapter 3. Accumbens Shell and Lateral Hypothalamus

selected because they are known to contain CART-containing neurons. The BLA and

PVT were also assessed based on their connectivity with AcbSh and their contributions to context-induced reinstatement of drug seeking (described in Chapter 2).

Method

Subjects and Surgery

Sixteen experimentally naive male Long-Evans rats (Monash Animal Services,

Behavioural Procedure

Rats were trained and responding was extinguished as in Experiment 1. Subsequently, all rats were tested under extinction conditions. Immediately prior to test, rats received bilateral microinfusion of saline or B/M into the AcbSh, with groups matched as in

Experiment 1. Time of testing (i.e. morning, afternoon) was counterbalanced between groups as orexin neurons in the medial and PeF hypothalamic regions show diurnal variation in c-Fos expression (Estabrooke et al., 2001). At the conclusion of testing, rats were returned to their homecage for 60 min until perfusion.

Microinfusion Procedure

Microinfusions proceeded as described in Experiment 1.

Histology and Immunohistochemistry

Rats were deeply anesthetised with sodium pentobarbital (100 mg/kg, i.p.) at 2 h from the start of testing and perfused transcardially with 50 ml of 0.9% saline, containing heparin (5000 i.u./ml), followed by 400 ml of 4% paraformaldehyde in 0.1 м phosphate 98

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buffer (PB), pH 7.4. Brains were postfixed for 1 h in the same fixative and placed in

20% sucrose solution over 2 nights. Subsequently, brains were blocked using a matrix aligned to the atlas of Paxinos and Watson (2005) and 40 μm coronal sections were cut using a cryostat (Microm 560, Germany). Four serially adjacent sets of LH sections and two serially adjacent sets of AcbSh sections were obtained from each brain and stored in

0.1% sodium azide in 0.1 м PBS, pH 7.2. To determine placement of the cannula tip, one series of sections through the AcbSh was selected from each rat and stained for

Nissl substance as described in Experiment 1.

To reveal c-Fos immunoreactivity (-IR) in combination with orexin- or CART-

IR, two separate series of hypothalamic sections were processed using two-colour peroxidase immunohistochemistry. Sections were washed in 0.1 м PB, followed by 50% ethanol, 50% ethanol with 3% H2O2, then 5% normal horse serum (NHS) in PB (30 min each). Sections were then incubated in goat antiserum against c-Fos (1:1000; c-Fos (4), s.c.-52, Santa Cruz-Biotechnology) and rabbit antiserum against orexin-A (1:20000;

Santa Cruz Biotechnology) or rabbit antiserum against CART 55-102 (1:20000; Phoenix

Pharmaceuticals), in a PB solution containing 2% NHS and 0.2% Triton X-10 (48 h at

4°C). The sections were then washed and incubated in biotinylated donkey anti-sheep

IgG for c-Fos (1:1000; Jackson Immunoresearch Laboratories, 24 h at 4°C). Finally, the sections were incubated in ABC reagent (Vector Elite kit: 6 μl/ml avidin and 6 μl/ml biotin; Vector Laboratories, 2 h at room temperature), washed in tris buffer (pH 7.6) and then incubated (15 min) in a nickel-intensified diaminobenzidine (DAB) solution containing 0.1% 3,3-diaminobenzidine, 0.8% D-glucose, 0.016% ammonium chloride and 0.032% nickel sulfate to reveal c-Fos-IR as a black immunoreactive nuclei after the addition of 0.2 μl/ml glucose oxidase (24 mg/ml, 307 units/mg, Sigma-Aldrich). Brain sections were then washed in tris/PB and re-incubated in biotinylated donkey anti-rabbit

Chapter 3. Accumbens Shell and Lateral Hypothalamus

for orexin or CART (1:1000; Jackson Immunoresearch Laboratories). The DAB reaction was repeated without nickel-intensification to localise orexin- or CART-IR, revealed as a brown reaction product. Sections were mounted onto gelatin-coated slides, dehydrated, cleared in histolene and coverslipped with Entellan. Sections were examined under light microscope to determine cannula tip placements (for Nissl-stained tissue) and distribution of c-Fos protein.

Neuronal counting

The tuberal hypothalamus was divided into three regions along the mediolateral axis based on structural landmarks (Paxinos & Watson, 2005) and in a manner similar to previous authors (Hamlin et al., 2007, 2008; Marchant et al., 2009). The DMH region was formed by the area between the third ventricle and the medial edge of the mammillothalamic tract (mt); the PeF extended from the DMH boundary to beyond the lateral edge of the fornix (approximately half the fornix width beyond the fornix, laterally); and the LH included the remaining area extending to the medial edge of the internal capsule. These boundaries are marked in the photomicrograph given in Figure

16. Two observers unaware of group allocations counted total c-Fos-positive and dual- labelled orexin/c-Fos-positive nuclei in each hypothalamic region, with inter-rater reliability of 0.9. An observer unaware of group allocations counted dual-labelled

CART/c-Fos-positive nuclei. Unilaterally matched coronal sections including the hypothalamus, amygdala, PVT and Arc were counted at six different levels, beginning at approximately -2.52 mm from bregma (Paxinos & Watson, 2005). All sections counted were approximately 160 μm apart.

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Data analyses

The mean numbers of active and inactive responses were analysed using mixed group x

(response) factor ANOVA with orthogonal contrasts. For immunohistochemical data, mean total counts of labelled neurons were analysed using ANOVA and also correlated with active responding on test using the Pearson correlation coefficient. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results

Histology

Bilateral placements of injection tips are indicated in Figure 14. Two rats were excluded from analyses; cannula tips for these rats were unverifiable due to excessive tearing of tissue along the cannula track. Final group sizes were, SAL (n = 8) and B/M (n = 6).

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

104.64 ± 9.44 and 2.64 ± 0.73, respectively. Between groups, there were no overall differences in responding on the last day of training (main effect and interaction: Fs(1, 12)

< 2.36, ps > 0.05); all rats made significantly more active than inactive responses (F(1, 12)

= 141.14, p < 0.001).

Mean ± SEM responses during extinction training are shown in Figure 15 (left panel). During extinction training, there were no significant differences between groups

(main effect and interactions: Fs(1, 12) < 1, ps > 0.05). All rats made significantly more active than inactive nosepokes, averaged across days of extinction (F(1, 12) = 98.31, p <

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0.001). Overall, responding significantly decreased across days of extinction (F(1, 12) =

73.05, p < 0.001), and this decrease was greater on the active than inactive nosepoke

(F(1, 12) = 64.69, p < 0.001).

Figure 15 (right panel) shows the mean ± SEM number of responses following infusions of either saline or B/M into AcbSh on test. Testing conditions were identical to extinction training. Overall, rats receiving B/M responded significantly more than those receiving saline (F(1, 12) = 38.86, p < 0.001). Importantly, this difference between groups was significantly greater for active than inactive nosepokes (group x response interaction: F(1, 12) = 16.59, p < 0.002). These results confirm that reversible inactivation of AcbSh reinstates extinguished responding.

Immunohistochemistry

Table 1 shows the mean ± SEM total c-Fos-IR, orexin-IR and CART-IR for each group in all brain regions examined. Table 2 shows the mean ± SEM number of cells expressing dual-IR for c-Fos/orexin, c-Fos/CART, percentage of orexin neurons expressing c-Fos-IR, and percentage of CART neurons expressing c-Fos-IR.

Hypothalamus

Figures 17 and 18 show photomicrographs of representative dual-labelled cells expressing c-Fos/orexin-IR and c-Fos/CART-IR, respectively. Figure 19 (top panel) shows the mean ± SEM total counts for c-Fos-IR in hypothalamus. The mean number of cells expressing c-Fos-IR was higher in group B/M than in group SAL in all hypothalamic regions: DMH (F(1, 12) = 38.67, p < 0.001), PeF (F(1, 12) = 199.94, p <

0.001) and LH (F(1, 12) = 76.83, p < 0.001).

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Bregma 2.04

Bregma 1.80

Bregma 1.56

Bregma 1.32

Bregma 1.20

Bregma 1.08

Figure 14. Experiment 3: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections. Groups: SAL (grey dots) and B/M (black dots).

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Extinction Test

Figure 15. Experiment 3: Mean ± SEM numbers of active and inactive responses during extinction (left panel) and test (right panel). Before test, rats received infusions of B/M or saline into AcbSh.

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Table 1. Mean (SEM) counts of total c-Fos-IR, orexin-IR and CART-IR.

c-Fos orexin CART SAL BM SAL BM SAL BM DMH 591.5 1135.5 62.9 66.8 173.4 163.8

(33.6) (101.9) (5.5) (7.2) (9.4) (19.8) PeF 855.1 2112.7 394 414 308.3 313.3

(37.1) (101.6) (19.4) (26.2) (19.7) (20.1) LH 291.6 931.7 154 171 173.1 167.3

(36.7) (78.3) (7.6) (13) (16.8) (9.6) CeA 38.8 81.5 - - 93.6 89.5

(5.5) (19.7) (5.2) (6.9) LA/BLA 200.8 360 - - - -

(23.7) (29.8) PVT 126.9 213 - - - -

(9.8) (21.7) Arc 22.3 207.8 - - 92.9 86.8

(1.0) (34.7) (5.6) (7.3)

Table 2. Mean (SEM) counts and percentages of dual-labelled orexin/c-Fos-IR and

CART/c-Fos-IR cells.

orexin / CART / CART / orexin / c-Fos c-Fos (%) c-Fos c-Fos (%) SAL BM SAL BM SAL BM SAL BM DMH 38.8 51.7 60.0 76.3 12.9 14 7.3 8.1

(5.5) (7.2) (5.2) (3.5) (2.1) (4.0) (1.0) (1.6) PeF 175.5 285.5 44.9 69.7 5.4 10.8 1.7 3.5

(17.6) (9.3) (4.5) (2.9) (1.5) (1.1) (0.5) (0.3) LH 18.9 61 12.6 35.1 2 3.7 1.1 2.1

(2.8) (12.7) (2.0) (5.5) (0.5) (1.1) (0.3) (0.6) CeA - - - - 6.9 15.5 7.0 16.8

(2.2) (4.1) (2.1) (3.9) Arc 7.4 3.8 7.6 4.0 - - - - (1.4) (0.8) (1.2) (1.0)

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LH PeF DMH

Figure 16. Photomicrograph of a representative coronal section through the hypothalamus indicating approximate boundaries used for cell counts. Abbreviations:

DMH (dorsomedial hypothalamus); f (fornix); LH (lateral hypothalamus); mt

(mammillothalamic tract); PeF (perifornical hypothalamus). Scale bar = 100 μm.

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Figure 17. Photomicrographs of representative dual-labelled c-Fos/orexin-IR (red arrow), single-labelled orexin (blue arrow) and single-labelled c-Fos-IR (black arrow) in the hypothalamus. Scale bar = 100 μm

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Figure 18. Photomicrograph of representative dual-labelled c-Fos/CART-IR (red arrow), single-labelled CART-IR (blue arrow) and single-labelled c-Fos-IR (black arrow) in the hypothalamus. Scale bar = 200 μm

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Hypothalamus: Orexin- and CART-IR. As expected, there were no significant differences between groups in the total number of orexin- or CART-expressing cells counted in DMH, PeF and LH (Fs(1, 12) < 1.52, ps > 0.05).

Figure 19 (middle panel) shows the mean ± SEM number of cells dual-labelled with c-Fos/orexin-IR in hypothalamus. In PeF and LH, there were significantly more hypothalamic cells co-expressing orexin- and c-Fos-IR in group B/M than in SAL (Fs(1,

12) > 16.56, ps < 0.002). There were no significant differences between groups in the

DMH (F(1, 12)= 2.47, p > 0.05). Analyses based on percentage of total orexin cells expressing c-Fos-IR revealed significantly higher percentages in group B/M than in

SAL for all hypothalamic regions: DMH (F(1, 12) = 6.72, p < 0.05), PeF (F(1, 12) = 21.24, p < 0.001) and LH (F(1, 12) = 22.14, p < 0.001).

For hypothalamic CART-containing cells (Figure 19, bottom panel), the mean number of cells expressing both CART- and c-Fos-IR was significantly higher for group

B/M than in group SAL in PeF (F(1, 12) = 8.36, p < 0.01), but not in DMH or LH (Fs(1, 12)

< 2.77, ps > 0.05). Analyses based on percentages of total CART cells expressing c-

Fos-IR revealed similar differences between groups in PeF (F(1, 12) = 10.25, p < 0.01), and not in DMH or LH (Fs(1, 12) < 3.15, ps > 0.05). However, very few cells expressed both CART- and c-Fos-IR, and the increase in dual-labelled CART cells was modest

(Table 2).

Amygdala, Arc and PVT

Both c-Fos-IR and CART-IR were found in the CeA and Arc, while only c-Fos-IR was found in the BLA and PVT. Figures 20, 21 and 22 (top panels) show photomicrographs of representative coronal sections through the amygdala, Arc and PVT. Bottom panels in Figures 20 and 21 depict representative dual-labelled cells in CeA and Arc respectively, while Figure 22 (bottom panel) depicts representative c-Fos-IR in PVT. 109

Chapter 3. Accumbens Shell and Lateral Hypothalamus

c-Fos-IR. Figure 23 (top panel) shows the mean ± SEM number of cells expressing c-Fos-IR in amygdala, PVT and Arc. The mean number of c-Fos-IR was higher in group B/M than in group SAL in the BLA (F(1, 12) = 21.01, p < 0.001), CeA

(F(1, 12) = 6.73, p < 0.05), PVT (F(1, 12) = 18.54, p < 0.001) and Arc (F(1, 12) = 46.9, p <

0.001).

CART-IR in CeA and Arc. Figure 23 (bottom panel) shows the mean ± SEM number of dual-labelled c-Fos/CART cells in CeA and Arc. In CeA, the mean number of cells expressing both CART-IR and c-Fos-IR was higher in group B/M than in SAL

(F(1, 12) = 4.74, p < 0.05). Analyses based on percentages of total CeA CART cells expressing c-Fos revealed similar differences between groups (F(1, 12) = 6.65, p < 0.05).

In Arc, the mean number of cells expressing both CART-IR and c-Fos-IR was higher in group SAL than in B/M (F(1, 12) = 4.84, p < 0.05). Analyses based on percentage of total

CART cells expressing c-Fos-IR approached but did not reach significance (F(1, 12) =

4.44, p = 0.057). There were no significant differences between groups in the total number of CART-expressing cells counted in CeA and Arc (Fs(1, 12) < 1, ps > 0.05).

Correlations for c-Fos-IR and dual-labelled neurons with alcohol seeking

Active nosepokes on test correlated positively with single c-Fos-IR and percentage of orexin cells labelled with c-Fos-IR across the hypothalamus region, and separately in

LH, PeF, and DMH (rs > 0.567, ps < 0.05, two-tailed). c-Fos-IR also correlated positively with active nosepokes on test in Arc, PVT, and amygdala (total amygdala as well as separately in CeA and BLA) (rs > 0.693, ps < 0.05, two-tailed). Percentage of

CART cells dual-labelled with c-Fos-IR correlated positively with active nosepokes on test in PeF (r = 0.608, p < 0.05, two tailed) and CeA (r = 0.668, p < 0.01, two tailed); negatively in Arc (r = -0.618, p < 0.05, two tailed); but not in LH or DMH (rs < 0.179, ps >0.05, two tailed). 110

Chapter 3. Accumbens Shell and Lateral Hypothalamus

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Chapter 3. Accumbens Shell and Lateral Hypothalamus

Figure 19. Experiment 3 Hypothalamus: Mean ± SEM number of cells expressing c-

Fos-IR (top panel); mean ± SEM number of cells dual-labelled with c-Fos/orexin-IR

(middle panel); and mean ± SEM number of cells dual-labelled with c-Fos/CART-IR

(bottom panel) in hypothalamus subregions.

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CeA

BLA

Figure 20. Amygdala: Photomicrograph of representative coronal section (top) and representative dual-labelled c-Fos/CART-IR cells in CeA (bottom; black arrow). Scale bars = 400 μm (top) and 100 μm (bottom).

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Figure 21. Arc: Photomicrograph of representative coronal section (top) and representative dual-labelled c-Fos/CART-IR cells (bottom; black arrow). Scale bars =

200 μm (top) and 100 μm (bottom).

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Chapter 3. Accumbens Shell and Lateral Hypothalamus

PVT

Figure 22. PVT: Photomicrograph of representative coronal section (top) and representative c-Fos -IR cells (bottom). Scale bars = 100 μm (top and bottom).

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Figure 23. Experiment 3: Mean ± SEM number of cells expressing c-Fos-IR in amygdala, PVT and Arc (top). Mean ± SEM number of dual-labelled CART cells in

CeA and Arc (bottom).

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Chapter 3. Accumbens Shell and Lateral Hypothalamus

Discussion

The findings from Experiment 3 replicate those in Experiment 1 and show that bilateral inactivation of AcbSh prevented expression of extinction and so reinstated responding.

This increase in responding occurred concomitantly with an increase in the number of cells expressing c-Fos-IR throughout the hypothalamus (DMH, PeF, LH), as well as in amygdala (CeA, BLA), PVT, and Arc. This suggests recruitment of a distributed network of structures during AcbSh-inactivation induced reinstatement of drug seeking.

Finally, within hypothalamus there was pronounced c-Fos expression in orexin-neurons in PeF, LH and DMH. This is in agreement with previous reports of c-Fos expression in

PeF/LH orexin neurons following AcbSh injections of muscimol (Baldo et al., 2004;

Zheng et al., 2003). There was also increased expression of c-Fos-IR in CART-labelled cells in PeF and CeA, and decreased c-Fos-IR in CART-labelled cells in Arc. These findings suggest that there may be possible peptidergic control over drug seeking regulated directly or indirectly by AcbSh.

Experiment 4

In the previous experiments, reversible inactivation of the AcbSh reinstated extinguished responding and also increased c-Fos protein induction in hypothalamus.

Given that the AcbSh projects preferentially to LH compared to DMH and PeF

(Marchant et al., 2009; Sano & Yokoi, 2007), and that AcbSh projection neurons targeting LH are activated during expression of extinction of reward seeking (Marchant et al., 2009), the present study investigated the functional role of LH in mediating reinstatement produced by AcbSh inactivation. To this end, rats were implanted with bilateral cannulae in both the AcbSh and LH. Rats were then trained and extinguished.

Prior to test under extinction conditions, rats received infusions of saline or B/M into

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Chapter 3. Accumbens Shell and Lateral Hypothalamus

AcbSh, concurrent with infusion of saline or B/M into LH. Thus, there were four groups, SAL–SAL, SAL–BM, BM–SAL, and BM–BM, labelled according to the type of infusion given in AcbSh and LH respectively. It was predicted that B/M-induced inactivation of AcbSh would reinstate responding (group BM-SAL) while concurrent

B/M-induced inactivation of LH (group BM-BM) would prevent this reinstatement.

Groups SAL-SAL and SAL-BM were included to isolate possible effects of LH inactivation alone on extinction responding.

Method

Subjects and Surgery

Thirty-eight experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted with four guide cannulae; bilaterally in AcbSh-V as described in Experiment 1 and bilaterally in LH.

Flat skull coordinates for LH relative to bregma were -2.3 AP, ±3.4 ML, and -7.5 DV.

These LH coordinates were selected based on a previous study showing retrograde labelled AcbSh cells from this anterior tuberal LH region (Marchant et al., 2009).

Surgery proceeded as in Experiment 1.

Behavioural Procedure

Responding was trained and then extinguished as in Experiment 1. Subsequently, all rats were tested under extinction conditions. Immediately prior to test, rats were infused with saline or B/M into the AcbSh and simultaneously infused with either saline or B/M into LH. All infusions were bilateral. Thus, there were four groups, SAL–SAL, SAL–

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BM, BM–SAL, and BM–BM, labelled according to the type of infusion given in AcbSh and LH respectively. All groups were matched as in Experiment 1.

Microinfusion and Histology Procedure

Rats received bilateral infusions of saline or B/M directly into AcbSh concurrent with bilateral infusions of saline or B/M directly into LH. All other procedures for microinfusions were as described in Experiment 1. Histology proceeded as in

Experiment 1.

Data analyses

The mean numbers of active and inactive responses were analysed using mixed group x

(response) factor ANOVA with orthogonal contrasts. Mean latency (minutes) to initiate the first response on the active nosepoke during test was analysed using planned orthogonal contrasts. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are indicated in Figure 24. Twelve rats were excluded from analyses due to misplaced cannulae or extensive tissue damage. Final group sizes were, SAL–SAL (n = 6), B/M–SAL (n = 8), SAL–B/M (n = 6), and B/M–

B/M (n = 6).

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

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96.77 ± 12.15 and 3.15 ± 0.56 respectively. Between groups there were no overall differences in responding on the last day of training (main effects and interactions: Fs(1,

11) < 2.75, ps > 0.05); all rats made significantly more active than inactive nosepokes

(F(1, 22) = 60.23, p < 0.001).

Mean ± SEM responses during extinction training are shown in Figure 25 (top panel). Across extinction training there were no significant differences between groups

(main effects and interactions: Fs(1, 22) < 1.55, ps > 0.05). All rats made significantly more active than inactive nosepokes, averaged across days of extinction (F(1, 22) =

119.67, p < 0.001). Overall, responding significantly decreased across days of extinction (F(1, 22) = 85.05, p < 0.001), and this decrease was greater on the active than inactive nosepoke (F(1, 22) = 82.86, p < 0.001).

Figure 25 (bottom panel) shows the mean ± SEM number of active and inactive responses on test following bilateral infusions of saline or B/M into AcbSh concurrent with bilateral infusions of saline or B/M into LH. Testing conditions were identical to extinction training. Overall, group B/M–SAL responded significantly more than all other groups (F(1, 22) = 13.97, p < 0.001) and this increase was greatest on the active than inactive nosepoke (F(1, 22) = 10.72, p < 0.01). To explore whether B/M infusions into LH independently suppressed responding, group SAL–SAL was compared with group

SAL–BM, and also with group BM–BM. These contrasts were not significant (main effects and interactions: Fs(1, 22) < 1, ps > 0.05). To further explore effects of B/M into either AcbSh or LH, the present study also examined latency to make first active response. There were no significant differences between groups in latency to first active nosepoke after placement in the chamber (Fs(1, 22) < 1.19, ps > 0.05).

The results from Experiment 4 show that concurrent inactivation of LH prevents reinstatement produced by inactivation of AcbSh. This finding suggests that expression

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Bregma 2.04

Bregma 1.80

Bregma 1.56

Bregma 1.32

Bregma 1.20

Bregma 1.08

Bregma - 2.28

Bregma - 2.52

Bregma - 2.76

Bregma - 3.00

Figure 24. Experiment 4: AcbSh and LH microinfusion cannula placements as verified on Nissl-stained sections (A and B respectively). All rats were infused with saline or B/M into AcbSh and concurrently with saline or B/M into LH. There were four groups: SAL-SAL (open circles), BM-BM (black circles), SAL-BM (grey circles), and

BM-SAL (grey stars); each group is labelled according to treatment received in AcbSh and LH, respectively. 121

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Extinction

100 active 80 inactive

40 Responses 20

0 1234 Day

Test

60 Active Inactive

20 Responses

0 SAL-SAL SAL-BM BM-BM BM-SAL Group

Figure 25. Experiment 4: Mean ± SEM number of active and inactive responses during extinction (top panel) and test (bottom panel). Groups are labelled according to treatment received in AcbSh and LH, respectively

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of drug seeking elicited by inactivation of AcbSh neurons is LH-dependent. It is consistent with the possibility that an AcbSh→LH pathway mediates the expression of extinction of drug seeking (Marchant et al., 2009).

Experiment 5

Experiment 4 demonstrated a functional role for AcbSh in regulating LH-dependent drug seeking during extinction. Previous studies suggest that this may occur via direct projections from AcbSh to LH since retrograde-labelled AcbSh projection neurons to

LH are activated during expression of extinction in alcoholic-beer seeking rats

(Marchant et al. 2009). However, neither findings from Experiment 4 nor Marchant et al. (2009) addressed whether serial communication from AcbSh to LH is necessary for the expression of extinction. Therefore Experiment 5 examined whether a functional serial interaction between AcbSh and LH mediated expression of extinction.

AcbSh efferents to LH are primarily GABAergic. To assess the possibility that these GABAergic AcbSh inputs into LH may be required for the expression of extinction, Experiment 5 functionally disconnected the AcbSh→LH pathway. This involved unilateral reversible inactivation of the AcbSh using B/M in combination with unilateral injection of the GABAA antagonist, bicuculline (BIC; 100 ng), into the LH of the contralateral hemisphere. A unilateral infusion of BIC ensures that GABAergic afferents to LH (including those from AcbSh) are disrupted; and in the contralateral hemisphere, reversible inactivation of the AcbSh prevents neurotransmission from

AcbSh to LH (but not from other LH afferents). Because projections from AcbSh to LH are substantially ipsilateral (Zheng, Patterson, & Berthoud, 2007), the contralateral disconnection prevents serial communication between the AcbSh and LH. However,

AcbSh is connected bidirectionally with LH. Thus, the unilateral injection of bicuculline

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into the LH provides some directional inference regarding an AcbSh→LH pathway since AcbSh projection neurons are primarily GABAergic. Disconnection of an

AcbSh→LH pathway is said to have disrupted extinction expression if contralateral but not ipsilateral disconnection reinstates previously extinguished reward seeking behaviour.

Rats were trained to respond for alcoholic beer reward followed by extinction and test, as in Experiment 1. Rats were tested in a repeated-measures design for expression of extinction under three treatment conditions: sham (contralateral saline in

AcbSh + LH); ipsilateral BM (AcbSh) + BIC (LH); and contralateral BM (AcbSh) +

BIC (LH); counterbalanced in order. There was one infusion-free extinction session between each test. Microinjections were made immediately prior to each test.

Method

Subjects and Surgery

Fifteen experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 340-390 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted with three guide cannulae; bilaterally in AcbSh-V and unilaterally in LH (counterbalanced for side). Flat skull coordinates relative to bregma were provided in Experiments 1 and 4. Surgery proceeded as in Experiment 1.

Behavioural Procedure

Responding was trained and then extinguished as in Experiment 1. Subsequently, all rats were tested in a repeated-measures design for expression of extinction under three treatment conditions: sham (contralateral saline in AcbSh + LH); ipsilateral B/M

(AcbSh) + BIC (LH); and contralateral B/M (AcbSh) + BIC (LH); counterbalanced in 124

Chapter 3. Accumbens Shell and Lateral Hypothalamus

order. All tests proceeded under extinction conditions and there was one infusion-free extinction session between each test. As in previous experiments, microinjections were made immediately prior to each test.

At the conclusion of the final test, rats remained in their homecages for 1-2 days, during which time they were maintained on food and water restriction. Subsequently, rats resumed extinction training for 2 days followed by test under extinction conditions.

Prior to test, animals received unilateral infusions of saline or BIC into LH to verify that these infusions alone had no effect on responding during extinction.

Microinfusion and Histology Procedure

Rats received unilateral infusions of B/M directly into AcbSh concurrent with unilateral infusions of saline or a solution containing the GABAA antagonist, bicuculline methiodide (BIC; 100 ng; Ascent Scientific) directly into LH. BIC was prepared in a solution of 0.1 м PBS (pH 7.2). This dose of BIC was selected based on previous studies (Stratford & Kelly, 1999). All other procedures for microinfusions were as described in Experiment 1. Histology proceeded as in Experiment 1.

Data analyses

The mean numbers of active and inactive responses were analysed using repeated measures (group) x (response) factor ANOVA with orthogonal contrasts. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are indicated in Figure 26. Five rats were excluded from analyses due to misplaced cannulae and tissue damage. 125

Chapter 3. Accumbens Shell and Lateral Hypothalamus

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

129.27 ± 12.10 and 2.63 ± 0.56 respectively. All rats made significantly more active than inactive nosepokes (F(1, 9) = 172.59, p < 0.001).

Mean ± SEM responses during extinction training are shown in Figure 27 (top panel). All rats made significantly more active than inactive nosepokes, averaged across days of extinction (F(1, 9) = 91.83, p < 0.001). Overall, responding significantly decreased across days of extinction (F(1, 9) = 62.86, p < 0.001), and this decrease was greater on the active than inactive nosepoke (F(1, 9) = 56.98, p < 0.001).

Mean ± SEM responses during test are shown in Figure 27 (bottom panel). Overall, responding was greater on the active than inactive nosepoke (F(1, 9) = 54.33; p < 0.001;).

Ipsilateral disconnection of AcbSh and LH significantly increased responding relative to group sham (F(1, 9) = 10.15; p < 0.05) and this increase was greatest on the active than inactive nosepoke (F(1, 9) = 8.97; p < 0.05). Similarly, contralateral disconnection of

AcbSh and LH significantly increased responding relative to group sham (F(1, 9) = 16.1; p < 0.01) and this increase was greatest on the active than inactive nosepoke (F(1, 9) =

11.02; p < 0.05). Therefore, it is not possible to infer from these findings that AcbSh and LH contribute to the extinction of reward seeking via serial AcbSh–LH communication. To elucidate these findings, anterior and posterior LH regions were examined separately.

Anterior and posterior LH are anatomically distinct. Unlike the posterior LH, the anterior LH receives GABAergic projections from the AcbSh (Sano & Yokoi, 2007;

Thompson & Swanson, 2010). In turn, anterior LH sends glutamatergic projections to posterior LH (Sano & Yokoi, 2007). This suggests that there are serial projections from

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Bregma 2.04

Bregma 1.56

Bregma 1.32

Bregma 1.20

Bregma 1.08

Bregma 1.80

Bregma - 2.28

Bregma - 2.52

Bregma - 2.76

Figure 26. Experiment 5: AcbSh and LH microinfusion cannula placements as verified on Nissl-stained sections. LH cannula placements were unilateral.

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Extinction

80 Active Inactive 60

Responses 20

0 1 2 3 4 Day

Test

Figure 27. Experiment 5: Mean ± SEM numbers of active and inactive responses during extinction (top panel) and subsequent test (bottom panel). Rats were tested under all treatment conditions (sham, ipsilateral, and contralateral) in a within subjects design, counterbalanced for test order.

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AcbSh to anterior LH, whereas communication from AcbSh to posterior LH occurs indirectly, possibly via anterior LH projection neurons. In the present study, the LH cannula was restricted to the anterior tuberal LH (AP relative to bregma: ≤ -2.52) for 6 rats, and restricted to the posterior tuberal LH (AP: ≥ 2.52) for 4 rats. The data was examined according to this division. Mean ± SEM responses for rats with anterior and posterior LH infusion during test are shown in Figure 28 (top and middle panels respectively). For rats receiving disconnection of AcbSh and posterior LH, ipsilateral disconnection significantly increased responding relative to group sham (F(1, 3) = 93.09; p < 0.05) and this increase was greatest on the active than inactive nosepoke (F(1, 3) =

45.03; p < 0.05). Contralateral disconnection of AcbSh and posterior LH approached significance, owing to one rat that failed to respond on test (F(1, 3) = 7.62; p > 0.05). In contrast, ipsilateral disconnection of AcbSh and anterior LH had no effect relative to sham (Fs(1, 5) < 1.96; p > 0.05). Importantly, contralateral disconnection of AcbSh and anterior LH significantly increased responding relative to group sham (F(1, 5) = 8.705; p

< 0.05) and this increase was greatest on the active than inactive nosepoke (F(1, 6) =

6.91; p < 0.05).

Finally, as described in the Methods section, the effect of unilateral bicuculline treatment in LH was examined to assess the contribution of this manipulation to the observed effects following AcbSh-LH disconnection. Mean ± SEM responses following unilateral bicuculline treatment in LH is depicted in Figure 28 (bottom panel). There were no significant differences between saline-treated rats and those receiving unilateral infusions of bicuculline in LH (Fs(1, 13) < 2.37; p > 0.05). This was regardless of injection site in anterior versus posterior LH (Fs(1, 11) < 1.85; p > 0.05).

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Test (Anterior LH)

Test (Posterior LH)

Unilateral LH

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Figure 28. Experiment 5: Mean ± SEM numbers of active and inactive responses during test for rats with anterior (top panel) and posterior (middle panel) LH infusions.

Mean ± SEM numbers of active and inactive responses following unilateral bicuculline

(BIC) treatment in LH (bottom panel).

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Together, the results of Experiment 5 show that both contralateral and ipsilateral disconnection of AcbSh from LH prevents the expression of extinction and so reinstated alcoholic beer seeking. An interesting finding however, was that contralateral but not ipsilateral disconnection of AcbSh from anterior regions of the tuberal LH prevented expression of extinction. This suggests that expression of extinction might be mediated via serial communication between AcbSh and tuberal LH. This is consistent with established anatomical projections from AcbSh to anterior LH. In contrast, both contralateral and ipsilateral disconnection of AcbSh from posterior regions of tuberal

LH impaired expression of extinction. This may be due to interhemispheric communication between AcbSh and posterior LH. Alternatively, GABA receptors in posterior LH might contribute to the expression of extinction via circuits parallel to those involving AcbSh and anterior LH. Importantly, the attenuation of extinction expression following AcbSh-LH disconnection is not likely due to the additive effects of unilateral B/M and BIC treatment in AcbSh and LH respectively. This is because neither unilateral blockade of GABA receptors in LH nor ipsilateral disconnection of

AcbSh from anterior tuberal LH affected the expression of extinguished responding.

Finally, it is important to note that the findings from this study should be interpreted with caution given the post-hoc nature of the analysis and the small sample of animals included in the anterior and posterior LH groups.

Discussion

The experiments reported in Chapter 3 suggest that AcbSh mediates the expression of extinction of alcoholic beer seeking through actions on hypothalamus. First, AcbSh inactivation prevented the expression of extinction and so reinstated previously extinguished alcoholic beer seeking. It had no effect on initial acquisition of extinction

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behaviour. Second, reinstatement produced by AcbSh inactivation was associated with pronounced c-Fos expression in hypothalamus, including in orexin- and CART- containing neurons. Third, concurrent inactivation of LH blocked reinstatement produced by AcbSh inactivation. Finally, contralateral but not ipsilateral disconnection of AcbSh from anterior regions of the tuberal LH prevented expression of extinction.

Together, these findings suggest that AcbSh mediates expression of extinction by inhibiting hypothalamic neuropeptide-containing neurons. Reversible inactivation of the

AcbSh removes this influence, thereby releasing hypothalamus from AcbSh inhibition and enabling reinstatement of reward seeking. The present results also suggest that serial AcbSh to anterior LH interactions mediates the expression of extinction.

The role of the AcbSh in mediating expression of extinction confirms previous findings based on cocaine seeking rats (Peters et al., 2008) and suggests that this role for

AcbSh might be shared across different reinforcers. Thus, there might be common neural mechanisms for extinction of reward seeking. Furthermore, reinstatement of extinguished reward seeking was observed only when microinjections were targeted at the ventromedial (rather than dorsal) region of the AcbSh. This neuroanatomical specificity might suggest that neurons in ventromedial AcbSh are especially important for extinction of reward seeking. However, Marchant et al. (2009) reported that dorsal

AcbSh projection neurons to LH were associated with expression of extinction whereas those projection neurons located ventrally in AcbSh were associated with reinstatement of alcohol seeking. This contrasts with the present findings. However, inferences regarding functional distinctions between dorsal and ventral AcbSh in the present study should be addressed with caution. It is possible that B/M in ventromedial AcbSh diffused dorsally along the 1mm protrusion of the injection cannula and into dorsomedial AcbSh, the site where projections to LH are active during extinction

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(Marchant et al., 2009). Similarly, B/M into dorsal AcbSh may have diffused dorsally and partly into the adjacent lateral ventricle, mitigating observable effects of B/M into

AcbSh. Alternatively, the differences observed between the present findings and those of Marchant et al. (2009) may be attributed to differences in test conditions. That is,

Marchant et al. (2009) used different contexts to assess extinction and reinstatement, whereas the experiments in the present chapter tested for extinction and reinstatement in the same context. Thus, in Marchant et al. (2009) the context during reinstatement test was only associated with reinforcement, whereas here the test context was associated with both reinforcement and extinction. The function of the ventral AcbSh may differ depending on the associative history of the test context. Regardless, the present findings support the general conclusion that the AcbSh mediates inhibition of drug seeking during extinction expression. Finally, consistent with Peters et al. (2008), the present investigation showed that inactivation of AcbSh immediately prior to initial extinction training had no effect on responding. This supports the behavioural specificity of the role of AcbSh on extinction expression and suggests that there are dissociable neural circuits for extinction acquisition and expression.

In the present investigation, AcbSh inactivation increased responding on test in rats with a history of extinction. Although the increase in responding was greater for the active than inactive nosepoke, there appeared to be an increase in inactive nosepoke responding on test. A similar increase in inactive nosepoke responding is typically observed during the first day of extinction training. It is reminiscent of the response enhancing effects of unexpected reward omission (Amsel, 1992) and raises the possibility that AcbSh may contribute to behavioural inhibition more generally.

Although bilateral inactivation of AcbSh has been shown to increase locomotor activity

(Peters et al., 2008), there are several reasons to suggest that the role for AcbSh in

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expression of extinction is not due simply to non-specific behavioural activation. Past research has shown that AcbSh inactivation does not increase responding in self- administration paradigms. For example, inactivation of AcbSh has no effect on lever pressing for a second-order cue (Di Ciano et al., 2008), on lever pressing in a progressive ratio schedule (Zhang et al., 2003), or on acquisition of instrumental responding for food reward (Hanlon, Baldo, Sadeghian, & Kelley, 2004). In the present experiments, there were no differences between groups in latency to initiate the first nosepoke response, although there was large variability on this measure. AcbSh inactivation also had no effect on responding when administered prior to initial extinction. Extinction training was a requirement for detecting an effect of AcbSh inactivation in the present experiments.

The present experiments suggest that a role for AcbSh in inhibiting reward seeking after extinction training depends, at least in part, on interactions with hypothalamus. AcbSh projects to LH with fewer projections to PeF and DMH (Heimer et al., 1991; Yoshida, McCormack, España, Crocker, & Scammell, 2006). Moreover,

AcbSh projections to LH, but not PeF, are active during expression of extinction

(Marchant et al., 2009). These projections are primarily GABAergic (Sano & Yokoi,

2007) and inhibition of AcbSh likely suppresses the release of GABA in LH, thereby disinhibiting LH (Stratford & Kelley, 1997, 1999). The present findings as well as others (Baldo et al., 2004; Stratford & Kelley, 1999; Zheng et al., 2003) show that inactivation of AcbSh increases c-Fos expression in LH and medial hypothalamic regions, consistent with AcbSh inactivation-induced disinhibition of LH. Indeed, it is unlikely that this increase in hypothalamic c-Fos expression simply reflects the motor component of reward seeking (nose-poke) behaviour as previous studies have shown that reversible inactivation of AcbSh alone (and in the absence of reward seeking

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behaviour) is sufficient to produce c-Fos activation throughout the hypothalamus

(Stratford, 2005). An important finding is that concurrent inactivation of LH prevented reinstatement induced by AcbSh inactivation, suggesting that the observed reinstatement effect was dependent on LH. As bilateral inactivation of LH prevents context-induced reinstatement of reward seeking and has no effect on extinction, either in a separate context (Marchant et al., 2009) or in the same context as training

(Experiment 4), it is unlikely that the attenuation of reinstatement by concurrent inactivation of AcbSh and LH was the result of non-specific motor deficits. Rather, the present findings are consistent with a role for LH in mediating reinstatement (Harris et al., 2005; Marchant et al., 2009). Finally, despite a known role for LH in feeding (as reviewed in Chapter 2), it is unlikely that the present findings implicating AcbSh inhibition over LH during extinction are due to the food restricted conditions of the rats.

Indeed, earlier studies of feeding have similarly demonstrated AcbSh inhibition over

LH-dependent behaviour in sated and non food-restricted rats (e.g. Stratford & Kelley,

1997, 1999). Together, these findings suggest that extinction is expressed via AcbSh inhibition over LH-dependent drug seeking.

A question of interest from the present investigation was whether expression of extinction was dependent on serial communication from AcbSh to LH. To address this,

AcbSh and LH were pharmacologically disconnected prior to a test of extinction expression. This involved unilateral reversible inactivation of AcbSh in combination with unilateral blockade of GABAA receptors in LH. The results showed that contralateral but not ipsilateral disconnection of the AcbSh from anterior tuberal LH prevented expression of extinction whereas both contralateral and ipsilateral disconnection of AcbSh from posterior tuberal LH prevented expression of extinction.

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These findings suggest that serial communication from AcbSh to anterior but not posterior LH mediates the expression of extinction.

There are critical anatomical differences between the anterior and posterior LH.

Genetic visualisation of striatal MSNs combined with tract-tracing procedures show that

GABAergic MSNs in the mouse accumbens terminate on glutamatergic neurons in anterior LH and separate from the posterior LH region containing the population of orexin cell bodies (Sano & Yokoi, 2007). Moreover, anatomical tract-tracing in the rat suggest that the anterior LH is the only hypothalamic target of dorsomedial AcbSh

(Thompson & Swanson, 2010), the same population of AcbSh projection neurons that show neuronal activation during extinction in alcoholic beer seeking rats (Marchant et al., 2009). Dorsomedial AcbSh also receives projections from the caudal portion of the

BLA (Hamlin et al., 2009), and as mentioned in Chapter 2, caudal BLA projection neurons targeting AcbSh are recruited during extinction expression, (Hamlin et al.,

2009). Therefore, the present disconnection study is consistent with known anatomical projections from AcbSh MSNs to anterior LH that are associated with extinction expression. They suggest that a serial AcbSh→LH interaction is necessary for mediating the expression of extinction.

The present experiments provide evidence that AcbSh regulates peptidergic mechanisms of the hypothalamus during extinction of reward seeking. First AcbSh inactivation increased neuronal activation in orexin-containing cells throughout the hypothalamus. This is in agreement with previous reports of c-Fos expression in

PeF/LH orexin neurons following AcbSh injections of muscimol (Baldo et al., 2004;

Zheng et al., 2003) and also the finding that cue-induced reinstatement of ethanol seeking significantly increased activation of orexin cells in DMH, PeF and LH (Dayas et al., 2008). Second, disconnection of AcbSh from the posterior tuberal LH prevented

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expression of extinction, although this occurred in animals receiving both contralateral and ipsilateral disconnection. Previous studies have shown that striatal MSNs terminate on glutamatergic neurons in anterior LH. In turn, anterior LH neurons terminate within the orexin peptide-containing posterior LH region (Sano & Yokoi, 2007). Thus, communication between AcbSh and orexin neurons in posterior LH might occur indirectly via anterior LH. One possibility is that bilateral inactivation of AcbSh MSNs disinhibited glutamatergic projection neurons of anterior LH, which in turn recruited activation of orexin-containing cells within posterior regions of the hypothalamus.

However, posterior LH also receives GABAergic projections from anterior LH (Gritti,

Mainville, & Jones, 1994) and disconnection of GABA neurotransmission in posterior

LH from AcbSh reinstated reward seeking. Therefore, the present findings raise the possibility that GABAergic regulation of posterior LH neurons may also be important for extinction expression. However, on the basis of the present study, this role for

GABA in posterior LH is not likely to involve serial communication with AcbSh.

Orexin neurons in LH may mediate reward-related behaviours such as reinstatement, whereas orexin neurons in DMH and PeF may mediate arousal (Aston-

Jones, Smith, Moorman, & Richardson, 2009; Harris et al., 2005; Harris, Wimmer,

Randall-Thompson, & Aston-Jones, 2007). DMH and PeF orexin neurons are activated during periods of wakefulness whereas LH neurons show no such variation (Estabrooke et al., 2001). Furthermore, c-Fos expression in LH orexin cells, but not DMH or PeF, positively correlates with the magnitude of context-induced reinstatement (Hamlin et al., 2007) and morphine conditioned place preference (Harris et al., 2005). Interestingly, in the present experiments, c-Fos induction in DMH, PeF, and LH orexin neurons were each correlated with reinstatement. Thus, inactivation of AcbSh may have recruited both arousal and reinstatement pathways. To clarify these findings it will be important

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to achieve greater pharmacological specificity when manipulating the AcbSh during extinction. With regards to CART, reversible inactivation of AcbSh modestly increased expression of c-Fos-IR in CART-labelled cells in PeF, but not in LH or DMH

(Experiment 3). However, Dayas et al. (2008) did not detect this effect during reinstatement produced by an alcohol-associated discriminative stimulus. One possibility is that GABAergic inactivation of AcbSh recruits multiple circuits involved in arousal and reward. For example, inactivation of AcbSh increases food intake

(Stratford & Kelley, 1997, 1999; Zheng et al., 2003) and food intake has been shown to be associated with increased expression of CART mRNA in medial and lateral parts of hypothalamus (Yu, South, Wang, & Huang, 2008).

Finally, orexin and CART neurons in PeF/LH project to key substrates of the reward circuit: VTA (Fadel & Deutch, 2002; Philpot et al., 2005) and PVT (Kirouac et al., 2005; Kirouac, Parsons, & Li, 2006). Both VTA and PVT have been implicated in reinstatement of extinguished reward seeking (Aston-Jones et al., 2009; Bossert et al.,

2004; Hamlin et al., 2009) and there was increased c-Fos expression in PVT during reinstatement of alcohol seeking in the present experiments. PVT is of special interest due to its projections to regions implicated in reinstatement of drug seeking, including amygdala, hippocampus and prefrontal cortex. Functionally, PVT may ‘bind’ or integrate neural circuits for reinstatement. With regards to amygdala, reinstatement of alcohol seeking following AcbSh inactivation was associated with increased c-Fos induction in BLA (Experiment 3). This supports the findings of Peters et al. (2008) that

BLA is causally involved in reinstatement after ilPFC inactivation. Although AcbSh does not project directly to amygdala (BLA or CeA), it has indirect access to amygdala via LH and PVT. Given the role of amygdala and prefrontal regions in reinstatement

(Alleweireldt, Hobbs, Taylor, & Neisewander, 2005; Feltenstein & See, 2007; Fuchs et

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al., 2005; McLaughlin & Floresco, 2007), it is possible that AcbSh inhibits drug seeking after extinction training via hypothalamic–PVT–amygdala/prefrontal pathways.

In conclusion, the findings from the studies reported in Chapter 3 suggest that

AcbSh mediates the expression of extinction by regulating hypothalamic-dependent reward seeking. Previous studies from cocaine seeking rats suggest that this regulation might be achieved via ilPFC (Peters et al., 2008). However, other corticolimbic inputs enabling AcbSh control over expression of extinction are unknown. Moreover, the receptors in the AcbSh that enable the behavioural expression of extinction are not known. These are addressed in the following chapter.

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Chapter 4.

Accumbens Shell AMPA Receptors Mediate Extinction

Through Interactions with Basolateral Amygdala

The experiments reported in this chapter continue to characterise a role for AcbSh in the behavioural expression of extinguished reward seeking. Specifically, this chapter examines a role for AcbSh glutamate-dependent AMPA receptor contributions to extinction of reward seeking, and attempts to isolate the behavioural specificity of this contribution using other reward-seeking preparations. In the previous chapter, it was concluded that expression of extinction was mediated at least partly by AcbSh inhibitory control over LH. In the final experiments of this chapter, the focus moves upstream of the AcbSh, and examines whether the basolateral amygdala (BLA) contributes to the expression of extinction through interactions with AcbSh AMPA-type receptors.

Extinction and AMPA-receptor dependent glutamatergic neurotransmission

Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. Its actions are mediated via three identified subtypes of ionotropic receptors: N- methyl-o-aspartate (NMDA), Kainate, and, D-amino- 3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA), which are primarily located on the head of striatal postsynaptic spines and also on cholinergic interneurons and presynaptic terminals

(Bernard, Somogyi, & Bolam, 1997; Gracy & Pickel, 1996; Tarazi, Campbell,

Yeghiayan, & Baldessarini, 1998). Metabotropic glutamate receptors also mediate

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glutamatergic neurotransmission. These receptors are located at pre-, post-, and perisynaptic sites (Baude et al., 1993; Manzoni, Michel, & Bockaert, 1997; Rouse et al.,

2000). Both ionotropic and metabotropic receptors have been implicated in the maintenance of drug dependence via their contribution to reinstatement of drug seeking, incubation of craving, and sensitisation to the effects of psychostimulant drugs such as cocaine. This literature is extensively reviewed in Gass and Olive (2008). Although all four types of glutamate receptors are present within the accumbens (Hu & White, 1996;

Meredith, Baldo, Andrezjewski, & Kelley, 2008; Meredith, 1999), AMPA receptors provide the primary source of depolarisation in excitatory neurotransmission (Bredt &

Nicoll, 2003) and are the primary mediators of excitatory transmission under basal conditions in the striatum (Cherubini, Herrling, Lanfumey, & Stanzione, 1988; Kita,

1996) including in the nucleus accumbens (Hu & White, 1996). For example, intra- accumbal iontophoretic administration of the glutamate receptor agonist, AMPA, produced a stronger excitatory response at lower ejection currents compared with an

NMDA receptor agonist while glutamate-induced excitatory accumbal activity was effectively suppressed with an AMPA but not NMDA receptor antagonist (Hu & White,

1996). Moreover, direct evidence linking AcbSh glutamate transmission to extinction of drug seeking has primarily implicated a role for AMPA receptor subunits (e.g. Sutton et al., 2003). For these reasons, only AMPA receptors will be reviewed here in detail.

The ionotropic AMPA receptor is a heterotetrameric protein complex that forms ligand-gated ion channels composed of several subunits (GluR subunits 1-4), each containing a binding site for glutamate. Evidence supporting a functional role for

AMPA-type receptors and extinction has been demonstrated using knockout mice lacking the GluR1 subunit. For example targeted deletion of the gene encoding GluR1 subunits of AMPA, gria1, attenuated the course of extinction during a single extended

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(17 hr) extinction session in mice previously trained to self-administer cocaine or sucrose (Mead, Zamanillo, Becker, & Stephens, 2007). Similarly, in a conditioned place preference preparation, extinction of conditioned place preference to a cocaine-paired context was dramatically impaired in conditional knockout mice lacking the AMPA

GluR1 receptor subunit selectively on midbrain dopamine neurons (Engblom et al.,

2008). The effect was specific to GluR1 and was not observed in mice lacking the

AMPA GluR2 subunit or the NMDA NR1 subunit. Extinction of conditioned place preference to a morphine-paired context was also impaired in knockout mice lacking

Narp, an immediate early gene implicated in AMPA-receptor trafficking (Crombag et al., 2008). These studies suggest that the AMPA receptor, and particularly its GluR1 subunit, is important for extinction across operant self-administration and Pavlovian- conditioning type preparations. However, these studies are limited in their inferences regarding the structural regions contributing to extinction. Although the conditional knockout mice used in Engblom et al. (2008) suggests that GluR1 knockout effects on extinction might be mediated through midbrain dopaminergic neurons, AMPA-type receptors are expressed both postsynaptically and on presynaptic terminals of dopamine neurons (Tarazi et al., 1998). Therefore, even in this study (Engblom et al., 2008) inferences regarding possible sites of effects are limited. Finally, although these studies implicate a role for the GluR1 AMPA-type receptors in the extinction of drug seeking

(Crombag et al., 2008) or conditioned place preference (Engblom et al., 2008), the permanence of the knockout manipulations used in these studies limit inferences relating to a role for GluR1 in the learning or expression of extinction.

The empirical investigations in the previous chapter demonstrated a role for

AcbSh in the expression of extinguished reward seeking. It is possible that this role depends on AMPA-type receptors. Evidence supporting this possibility includes the

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occurrence of extinction-dependent changes in glutamate receptor expression within the

AcbSh of cocaine-seeking rats (Ghasemzadeh et al., 2009; Self et al., 2004; Sutton et al., 2003). This literature was reviewed in Chapter 2. Briefly, extinction of cocaine seeking significantly increased the expression of AMPA receptor subunits (GluR1 and

GluR2/3) in AcbSh, and this expression was positively correlated with the magnitude of behavioural extinction expressed (Sutton et al., 2003). More direct evidence comes from the finding that viral-mediated overexpression of GluR1 subunits in AcbSh facilitated expression but not learning of extinction (Sutton et al., 2003). Although these findings suggest that AMPA-type glutamate receptors in AcbSh are involved in and can modulate the expression of extinction, the extinction-dependent up-regulation of AMPA receptor subunits in Sutton et al. (2003) occurred only in cocaine- but not sucrose- trained rats. Similarly, viral-mediated overexpression of GluR1 subunits inhibited seeking only in cocaine- but not sucrose-trained rats (Sutton et al., 2003). Therefore, the empirical literature is limited to evidence for a role for AcbSh AMPA receptors in mediating expression of extinction of cocaine drug seeking. To the best of the author’s knowledge, a role for AcbSh AMPA receptors in mediating the expression of extinction using drug reinforcers other than cocaine has not yet been assessed.

A role for AcbSh glutamate receptors in extinction of reward seeking is of interest given that the AcbSh receives inputs from several cortical, thalamic, and limbic sites involved in reward-related behaviour. Among them include ilPFC, BLA, and ventral hippocampus, which use glutamate and/or aspartate as their neurotransmitter

(Christie, Summers, Stephenson, Cook, & Beart, 1987; Fagg & Foster, 1983;

Groenewegen et al., 1999; Kita & Kitai, 1990; Robinson & Beart, 1988; Wright et al.,

1996). These projections were reviewed in Chapter 2. Moreover, previous studies suggest that AcbSh interacts with ilPFC to enable expression of extinguished cocaine-

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seeking (Peters et al., 2008). However, as noted in Chapter 2, this evidence is based on a disconnection study wherein both contralateral and ipsilateral disconnection of AcbSh from ilPFC prevented the expression of extinction, raising the possibility that AcbSh and ilPFC contributions to extinction occur via parallel pathways. The BLA, and primarily its caudal subregion, is another glutamatergic input to the AcbSh that might be recruited in series with AcbSh to mediate extinction of reward seeking. Consistent with this possibility, bupivacaine-induced inactivation of the caudal BLA prior to initial extinction of food-seeking retarded extinction learning as expressed on a subsequent day of extinction (McLaughlin & Floresco, 2007). Furthermore, single unit recordings show that a subpopulation of neurons primarily within the caudal BLA is recruited during extinction of sucrose-seeking and related to behavioural responding during extinction (Tye, Cone, Schairer, & Janak, 2010). Finallly, AcbSh-targeted projections primarily from caudal BLA show increased neuronal activation when rats are returned to an extinction-associated context (Hamlin et al., 2009). Therefore, AcbSh AMPA receptor contributions to extinction of reward seeking may depend on glutamatergic projections from BLA. However, the functional involvement of a BLA→AcbSh pathway in extinction remains unknown.

The experiments in this chapter had three aims. The first was to investigate the functional contribution of AcbSh AMPA-type receptors in extinction of reward seeking.

The second was to assess the behavioural specificity of this contribution. The final aim was to examine whether AcbSh AMPA-type receptors mediate extinction of reward seeking through serial interactions with the BLA. To address these aims, Experiment 6 examined the effects of the selective and competitive AMPA-receptor antagonist, 2,3-

Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f] quinoxaline -7-sulfonamide disodium salt

(NBQX), on extinction expression and reinstatement in a context-induced reinstatement

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(renewal) paradigm. Experiment 7 investigated the dose-dependent effects of NBQX on extinguished responding. Experiment 8 assessed the behavioural impact of NBQX on other measures of reward seeking and motivation via studying its effects on initial extinction learning and performance on a progressive ratio schedule of reinforcement.

Experiment 9 examined the effects of contralateral disconnection of BLA from AcbSh

AMPA receptors on the expression of extinction in alcoholic beer seeking rats. In the final empirical study of this thesis, Experiment 10 investigated the effects of BLA inactivation on expression of extinction.

Experiment 6

The experiments reported in Chapter 2 identified AcbSh as a critical substrate for the expression of extinguished reward seeking. To investigate the receptor-level mechanisms underlying this role, Experiment 6 aimed to examine whether a role for

AcbSh in extinction was AMPA-receptor dependent. An ABA context-induced reinstatement (renewal) preparation was used to assess possible effects of AMPA- receptor blockade via intra-AcbSh NBQX on both extinction expression (ABB: tested in the extinction context) and context-induced reinstatement of alcoholic beer seeking

(ABA: tested in the training context). This preparation was described in detail in

Chapter 1. The rationale for using a context-induced reinstatement preparation was two- fold. First, to implicate a behaviourally specific role for AcbSh in the expression of extinction of drug seeking, it was important to demonstrate that this role was not simply limited to conditions involving test in a context with a mixed history of reinforcement

(i.e. an ambiguous context). A role for AcbSh in mediating the expression of extinction should also be evident in an extinction context that is separate from the training context

(test ABB). Second, context-induced reinstatement provides a within-subjects 146

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assessment of the effects of intra-AcbSh NBQX on recovered responding (test ABA) compared with extinguished responding (test ABB). Thus, in the present experiment, rats underwent self-administration and extinction training in contexts A and B, respectively. They were subsequently allocated into two groups, saline or NBQX (1.0

μg), and tested in both contexts, A (the training context; ABA) and B (the extinction context; ABB), over two consecutive days counterbalanced in order. There was no beer present on test. Rats received microinjections immediately prior to each test and treatment groups were maintained across test contexts. The question of interest was whether AcbSh infusions of the AMPA antagonist, NBQX, would prevent the expression of extinction.

Method

Subjects and Surgery

Twenty-one experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted bilaterally with 26-gauge guide cannulae (Plastics One, Roanoke, VA, USA) aimed at ventromedial AcbSh

(coordinates provided in Experiment 1). Surgery proceeded as in Experiment 1.

Apparatus

The operant chambers used during self-administration, extinction and test were conducted in eight operant chambers identical to those described in Experiment 1.

For contextual manipulations, the operant chambers were arranged such that there were two distinct contexts. In one set, the chamber floors comprised stainless steel rods, and approximately 0.1 ml of dilute rose oil essence was placed in each corner of

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the bedding beneath the floor. There was no illumination in these chambers other than that provided by the white cue light recessed in the active nosepoke. In the second set of chambers, the flooring comprised of Perspex and a 100 mV white house light illuminated the chambers. Approximately 0.1 ml of dilute peppermint essence was placed in each corner of the bedding beneath these chambers. These two sets of chambers were counterbalanced to serve as Contexts A and B. These contextual manipulations were the same as those described in previous studies of context-induced reinstatement from the same laboratory (Hamlin et al., 2006; Hamlin et al., 2009;

Hamlin et al., 2008; Hamlin et al., 2007).

Microinfusion and Histology Procedure

Rats received intracranial microinfusions of saline or AMPA receptor antagonist,

NBQX (1.0 μg/0.5μl; Tocris Bioscience), directly into the AcbSh. NBQX was dissolved in 0.1 м PBS (pH 7.2). NBQX is a potent and selective antagonist for AMPA/Kainate receptors. All other procedures for microinfusions were as described in Experiment 1.

Histology proceeded as in Experiment 1.

Behavioural Procedure

Self-administration training and extinction. All procedures for magazine training were as described in Experiment 1, with the exception that magazine training sessions took place in both contexts A and B (per day) in counterbalanced order. Subsequently, responding for alcoholic beer was trained then extinguished as described in Experiment

1. Training took place in Context A and extinction in Context B.

In this and all following experiments, rats were adapted to the conditions of the intracranial infusion procedure prior to each self-administration and extinction session, as described in Experiment 1.

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Test. Following self-administration and extinction, rats were allocated into two groups, saline or NBQX (1.0 μg), each matched according to the number of active nosepokes made over the last day of self-administration training and the first two days of extinction. This dose was selected based on previous studies of NBQX on appetitive motivation (Maldonado-Irizarry et al. 1995). Rats were tested in both Context A (the training context) and Context B (the extinction context) over two consecutive days, counterbalanced in order. Both tests occurred under conditions identical to extinction.

Treatment groups were maintained across both tests. As in previous experiments, microinjections were made immediately prior to each test.

Data analyses

The mean numbers of active and inactive responses were analysed using mixed group x

(response) x (context) ANOVA with orthogonal contrasts. For all analyses, type I error rate (α) was controlled at 0.05 using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are shown in Figure 29. Three rats were excluded from analyses due to misplaced cannulae. Final group sizes were: saline (n = 8) and

NBQX (n = 10).

Behaviour

All rats acquired high levels of responding during training. On the last day of training the mean ± SEM numbers of active and inactive nosepokes were 82.78 ± 10.11 and 3.22

± 0.65, respectively. There were no significant differences between groups (Fs(1,16) < 1,

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Figure 29. Experiment 6: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections: saline (SAL, empty circles) and 1.0 μg NBQX (black-filled circles).

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ps > 0.05); all rats made significantly more active than inactive nosepokes (F(1,16) =

143.64, p < 0.001).

Mean ± SEM responses during extinction are shown in Figure 30 (top panel).

During extinction there were no differences between groups (Fs(1,16) < 1.66, ps > 0.05); all rats made significantly more active than inactive nosepokes (F(1,16) = 166.79, p <

0.001) and responding significantly decreased across days of extinction (F(1,16) = 90.30, p < 0.001). This decrease was greater on the active than inactive nosepoke (F(1,16) =

50.81, p < 0.001).

Figure 30 (bottom panel) shows the mean ± SEM number of responses on test.

Rats were tested in both contexts A and B under extinction conditions. There was no overall difference between infusions groups, averaged across context and manipulanda

(active and inactive nosepokes) (F(1,16) = 1.88, p > 0.05). There was overall greater responding in the training context (ABA) than the extinction context (ABB) (F(1,16) =

19.91, p < 0.05). There was overall more responding on the active than inactive nosepoke (F(1,16) = 72.57, p < 0.05). There was also a context x manipulanda interaction

(F(1,16) = 29.50, p < 0.05), confirming that context-induced reinstatement (ABA) was greater for the active than inactive nosepokes. Importantly, analyses of group differences showed no difference between NBQX and saline infusions on active (F(1,16)

< 1, p > 0.05) or inactive (F(1,16) = 4.28, p > 0.05) nosepokes when rats were tested in the training context, A. There was also no difference between NBQX and saline infusions on inactive nosepokes in Context B (F(1,16) < 1, p > 0.05). However, NBQX infusions significantly increased active nosepokes in Context B (F(1,16) = 5.29, p <

0.05). Therefore, AcbSh AMPA receptor antagonism attenuated the expression of extinction of alcoholic beer seeking and these same infusions did not affect the expression of context-induced reinstatement.

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Extinction

Test

Figure 30. Experiment 6: Mean ± SEM numbers of active and inactive responses during extinction (top panel) and test (bottom panel). Rats were tested in both Context

A and Context B in counterbalanced order. Saline or NBQX (1.0 μg) was administered immediately prior to test.

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Experiment 7

Experiment 6 showed that AcbSh AMPA receptor antagonism attenuated the expression of extinction. However, the size of effect was noticeably smaller compared to the reinstatement of extinguished responding following reversible inactivation of AcbSh in

Experiments 1, 3, and 4. Nonetheless, Experiment 6 is unique in that extinction training occurred in a context that was separate from the training context. Thus the relatively weaker effect of NBQX on extinction expression in the previous experiment may be due to responding being extinguished and tested in a context that was not previously paired with drug reward. Moreover, it has been noted that context-induced recovery of responding observed in the training context (ABA) is stronger compared with recovery in a separate context (AAB) (Bouton et al., 2011). Therefore, to further characterise a role for AcbSh AMPA receptors in mediating the expression of extinction, Experiment

7 aimed to examine the dose response properties of intra-AcbSh NBQX on expression of extinction, where training and extinction occurred in the same context. Rats were trained to respond for alcoholic beer followed by extinction training. They were subsequently tested in a repeated-measures design for expression of extinction under three treatment conditions: 0 μg (saline), 0.3 μg, and 1.0 μg NBQX, counterbalanced in order.

Method

Subjects and Surgery

Twelve experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted bilaterally with 26-gauge

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guide cannulae (Plastics One, Roanoke, VA, USA) aimed at ventromedial AcbSh

(coordinates provided in Experiment 1). Surgery proceeded as in Experiment 1.

Apparatus

The operant chambers used during self-administration, extinction, and test in this experiment and all following experiments were conducted in eight operant chambers identical to those described in Experiment 1.

Behavioural Procedure

Responding was trained and then extinguished as in Experiment 1. There were no contextual manipulations. Subsequently, all rats were tested under three treatment conditions: 0 μg (saline), 0.3 μg, and 1.0 μg NBQX, counterbalanced in order. Test conditions were identical to extinction, with one infusion-free extinction session between each test. Microinjections were made into the AcbSh immediately prior to each test.

Microinfusion and Histology Procedure

Rats received bilateral infusions of saline or NBQX (0.3 μg, and 1.0 μg) directly into

AcbSh using microinfusion procedures previously described in Experiment 1. Histology proceeded as in Experiment 1.

Data analyses

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Results and Discussion

Histology

Bilateral placements of injection tips are shown in Figure 31. Four rats were excluded due to misplaced or blocked cannulae. Eight rats were included in the final analyses.

Behaviour

All rats acquired high levels of responding during training. On the last day of training the mean ± SEM numbers of active and inactive nosepokes were 82.13 ± 15.11 and 3 ±

0.76, respectively. During training all rats made significantly more responses on the active than inactive nosepoke (F(1,7) = 64.23, p < 0.001).

Mean ± SEM responses during extinction training are shown in Figure 32 (top panel). During extinction training rats made significantly more active than inactive nosepokes (F(1,7) = 85.00, p < 0.001). Overall responding significantly decreased across days of extinction (F(1,7) = 84.97, p < 0.001) and this decrease was greater on the active than inactive nosepoke (F(1,7) = 103.42, p < 0.001).

Figure 32 (bottom panel) shows the mean ± SEM number of responses on test.

Testing conditions were identical to extinction training. Overall, 1.0 μg NBQX significantly increased responding relative to saline treatment (F(1,7) = 12.07, p < 0.01) and this increase was greatest on the active than inactive nosepoke (F(1,7) = 8.05, p <

0.05). There were no significant differences between 0.3 μg NBQX and saline treatment

(Fs(1,7) < 1, ps > 0.05). Averaged across treatment conditions, the number of active nosepokes was significantly greater than inactive nosepokes (F(1,7) = 13.52, p < 0.05).

Therefore, Experiment 7 confirms that intra-AcbSh NBQX attenuates the expression of extinguished reward seeking, and also shows that this effect is dose-dependent.

Moreover, as in Experiment 6, the size of effect of NBQX on extinguished responding

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Figure 31. Experiment 7: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections.

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Extinction

Test

Figure 32. Experiment 7: Mean ± SEM numbers of active and inactive responses during extinction (top panel) and subsequent test (bottom panel). Rats were tested under all NBQX treatment conditions (0 μg, 0.3 μg, and 1.0 μg) in a within subjects design, counterbalanced for test order. Rats received saline infusions when allocated to the 0 μg treatment condition.

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was noticeably smaller than the effects reported in Chapter 3 following reversible inactivation of AcbSh. Therefore, the behavioural impact of NBQX on extinguished responding does not appear to be modulated by the training history of the extinction context.

Experiment 8

Experiments 6 and 7 both show that intra-AcbSh infusions of NBQX attenuate the expression of extinction. Experiment 8 aimed to further investigate the behavioural specificity of this effect using two measures of non-extinguished reward seeking: initial extinction learning and responding on a progressive ratio schedule of reinforcement.

The former was selected to determine whether NBQX impaired extinction learning. The progressive ratio test was selected to determine whether the effects of NBQX on expression of extinction in Experiments 1 and 2 might be due to a general effect of

NBQX increasing the motivation to respond for a reward.

Method

Subjects and Surgery

Fourteen experimentally naive male Long-Evans rats (Monash Animal Services,

(coordinates provided in Experiment 1). Surgery proceeded as in Experiment 1.

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Behavioural Procedure

All sessions took place in the same context. Rats received self-administration training followed by two days of extinction, in a manner identical to Experiment 2.

Extinction acquisition test. Prior to extinction, rats were allocated to one of two groups, saline and NBQX (1.0 μg). Groups were matched on the average number of active nosepokes made over the last day of self-administration. Immediately prior to the first day of extinction, rats received microinfusion into AcbSh as per treatment group.

Rats were retested the following day under infusion-free extinction conditions. If

NBQX interfered with extinction learning on the first day of extinction, then group

NBQX should respond more than saline-treated rats on the second day of extinction.

Progressive ratio test. On the following three days, rats underwent alcoholic beer self-administration (FR-1) to re-establish responding. They were then tested on the following day on a progressive ratio schedule, where the number of responses required to obtain the alcoholic beer reward increased exponentially with each reinforcement.

The schedule used in this experiment was derived from the function described in

Richardson and Roberts (1996) where response ratio equals to [5e(reinforcer number x 0.2)] –

5, rounded to nearest integer (i.e., 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50 ... ). During this

90 min test, animals were assessed for their break point, which was defined as the final completed response ratio that was reinforced with alcoholic beer. Critically, break point is considered an index of reinforcement efficacy (Richardson & Roberts, 1996).

Immediately prior to the progressive ratio test, rats received microinfusions of either saline or 1.0 μg NBQX using a crossover design for drug infusion. Therefore, rats were tested twice – those receiving saline treatment during the extinction learning test subsequently received NBQX treatment in the progressive ratio test, and vice versa.

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Microinfusion and Histology Procedure

Rats received bilateral infusions of saline or NBQX (1.0 μg) directly into AcbSh using microinfusion procedures previously described in Experiment 1. Histology proceeded as in Experiment 1.

Data analyses

The mean numbers of active and inactive responses were analysed using ANOVA with orthogonal contrasts. For the progressive ratio study, the last completed ratio (i.e. breakpoint) was also compared by ANOVA between groups. For all analyses, Type I error rate (α) was controlled at 0.05 for each contrast tested using the procedure described by Hays (1972).

Results and Discussion

Histology

Bilateral placements of injection tips are shown in Figure 33. Two rats were excluded due to misplaced cannulae. Twelve rats were included in the final analysis. Final group sizes: n = 5 and n = 7.

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

60.42 ± 11.84 and 2.08 ± 0.51, respectively. During training, there were no differences between groups (Fs(1,10) < 1, ps > 0.05); all rats made significantly more active than inactive nosepokes (F(1,10) = 141.70, p < 0.001).

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Figure 33. Experiment 8: AcbSh microinfusion cannula placements as verified on

Nissl-stained sections: saline (SAL, empty circles) and NBQX (black circles).

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Extinction

Progressive Ratio Test

Figure 34. Experiment 8: NBQX effects on extinction learning (top panel): Mean ±

SEM numbers of active and inactive responses during test (extinction days 1 and 2).

Rats received infusions of saline or NBQX into AcbSh immediately before test

(extinction day 1) and not on extinction day 2. NBQX effects on progressive ratio test

(bottom panel): Mean ± SEM numbers of total active and inactive responses during test

(left panel) and total number of infusions received on test (i.e. break point; right panel).

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Mean ± SEM responses on the two days of extinction test are shown in Figure

34 (top panel). Across groups, rats made significantly more active than inactive nosepokes (F(1, 10) = 55.46, p < 0.001) and responding significantly decreased from Day

1 to Day 2 of extinction (F(1, 10) = 11.99, p < 0.01), confirming the acquisition of extinction. This decrease was greater on the active than inactive nosepoke (F(1, 10) =

12.60, p < 0.01). There was no main effect of group (F(1, 10) < 1, p > 0.05) or an effect of

NBQX on the decrease in responding from Day 1 to Day 2 of extinction (group x day interaction, F(1,10) < 1, p > 0.05). In addition, there was no effect of AcbSh infusions of

NBQX on either Day 1 (the day of infusion) or Day 2 (the infusion free test) of extinction (Fs (1,10) < 1, ps > 0.05). These results show that in contrast to their effects on expression of extinction, AcbSh infusions of NBQX have no significant effect on the acquisition of extinction learning. That is, AcbSh infusions of NBQX did not prevent the decrement in responding observed between Day 1 and Day 2 of extinction training.

Mean ± SEM responses and break point (number of completed ratios) during the progressive ratio test are shown in Figure 34 (bottom left and right panels, respectively).

There was no significant difference between groups on break point (F(1,10) < 1, p > 0.05).

Moreover, there were no significant differences between groups on total active and inactive responding (Fs (1,10) < 1, ps > 0.05).

The findings from Experiment 8 show that blockade of AcbSh AMPA receptors have no effect on non-extinguished behaviour as measured by responding during initial extinction learning or on overall performance on a progressive ratio test. It is unlikely that a null effect of NBQX on overall responding or on break point during the progressive ratio test is due to a ceiling effect since NBQX had no effect on renewed responding (Experiment 5) or on the first day of extinction (present experiment), where overall levels of responding were lower.

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Experiment 9

Experiments 6 and 7 found that AcbSh AMPA receptors are necessary for the expression of extinction. The AcbSh receives glutamatergic inputs from BLA

(Robinson & Beart, 1988), a structure shown to be recruited during extinction of reward seeking (Hamlin et al., 2009; McLaughlin & Floresco, 2007; Tye et al., 2010).

Anatomically, the caudal portion of the BLA projects to the caudomedial portion and the rostro-dorsomedial corner of the AcbSh (French & Totterdell, 2003; Kita & Kitai,

1990), forming asymmetric synapses primarily with dendritic spines (Kita & Kitai,

1990). Previous studies have demonstrated a role for functional serial interactions between BLA and AcbSh in other appetitive preparations (e.g. Pavlovian-instrumental transfer) using a disconnection design wherein contralateral but not ipsilateral disconnection prevented the studied behaviour (Shiflett & Balleine, 2010). This suggests that the study of putative functional BLA→AcbSh interactions maybe amenable to a disconnection procedure. Thus, to assess the possibility that glutamatergic BLA inputs into AcbSh may be required for the expression of extinction,

Experiment 9 conducted a functional disconnection of the BLA →AcbSh pathway.

The functional disconnection procedure involved a unilateral injection of NBQX

(1.0 μg) into the AcbSh in combination with unilateral reversible inactivation of the

BLA using GABAB and GABAA agonists, baclofen (1.0 mм) and muscimol hydrobromide (0.1 mм) (B/M), in the contralateral hemisphere. A unilateral infusion of

NBQX ensures that glutamatergic afferents to the AcbSh (including those from BLA) are disrupted, and in the contralateral hemisphere, reversible inactivation of the BLA prevents transmission from BLA (but not from other AcbSh afferents). Therefore, on the basis that projections from BLA to AcbSh are both unidirectional and ipsilateral, the

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contralateral disconnection procedure used here disrupts AMPA-dependent communication between the BLA and AcbSh.

BLA + AcbSh); ipsilateral BM (BLA) + NBQX (AcbSh); and contralateral BM (BLA)

+ NBQX (AcbSh); counterbalanced in order. There was one infusion-free extinction session between each test. Microinjections were made immediately prior to each test.

Method

Subjects and Surgery

Twenty-one experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 310-330 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted with three guide cannulae; bilaterally in AcbSh-V (coordinates as in Experiment 1) and unilaterally in

BLA (counterbalanced for side). Flat skull coordinates for BLA relative to bregma were: -3.0 mm AP; ±5.0 mm ML; -7.65 mm DV, Surgery proceeded as in Experiment 1.

Behavioural Procedure

Responding was trained and then extinguished as in Experiment 1. There were no contextual manipulations. Subsequently, all rats were tested under three treatment conditions in a repeated-measures design: saline (contralateral saline in AcbSh + BLA); ipsilateral NBQX (AcbSh) + B/M (BLA); and contralateral NBQX (AcbSh) + B/M

(BLA); counterbalanced in order. Test conditions were identical to extinction, with one

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infusion-free extinction session between each test. Microinjections were made into the

AcbSh immediately prior to each test.

Microinfusion and Histology Procedure

Rats received unilateral infusions of NBQX (1.0 Pg) directly into AcbSh concurrent with unilateral infusions of saline or B/M (1.0 /0/1 mм) directly into BLA. All other procedures for microinfusions were as described in Experiments 1 and 6. Histology proceeded as in Experiment 1.

Data analyses

Results and Discussion

Histology

Bilateral placement of injection tips are shown in Figures 35A and B for AcbSh and

BLA, respectively. Seven rats were excluded due to misplaced cannulae. Fourteen rats were included in the final analysis.

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

64.86 ± 14.14 and 3.57 ± 1.14, respectively. During training rats made significantly

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Figure 35. Experiment 9: Microinfusion cannula placements in AcbSh (A.) and

BLA (B.) as verified on Nissl-stained sections. BLA cannula placements were unilateral.

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Extinction

Test

Figure 36. Experiment 9: Mean ± SEM numbers of active and inactive responses during extinction (top panel) and subsequent test. Rats were tested under all treatment conditions (saline, ipsilateral, and contralateral) in a within subjects design, counterbalanced for test order.

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more responses on the active than inactive nosepoke (F(1, 13) = 131.63; p < 0.001).

Mean ± SEM responses during extinction training are shown in Figure 36 (top panel). During extinction rats responded significantly more on the active than inactive nosepokes (F(1, 13) = 61.54; p < 0.001). Overall responding significantly decreased across days of extinction (F(1, 13) = 34.27, p < 0.001) and this decrease was greater on the active than inactive nosepoke (F(1, 13) = 41.86, p < 0.001). Figure 36 (bottom panel) shows the mean ± SEM number of responses on test. Testing conditions were identical to extinction training. Overall, responding was greater on the active than inactive nosepoke (F(1, 13) = 65.36; p < 0.001). There were no significant differences between saline and ipsilateral treatment groups (F(1, 13) = 2.05; p values > 0.05). In contrast, responding was significantly greater following contralateral disconnection of BLA and

AcbSh relative to saline treatment (F(1, 13) = 9.07; p < 0.01) and this effect was greatest on the active than inactive nosepoke (F(1, 13) = 9.31; p < 0.01). Therefore, contralateral but not ipsilateral disconnection of BLA and AcbSh attenuated the expression of extinguished reward seeking suggesting that serial communication between BLA and

AcbSh is required for the expression of extinction.

Experiment 10

Experiment 9 showed that serial caudal BLA→AcbSh interactions are important for the expression of extinction. One interpretation of this finding is that glutamatergic transmission from caudal BLA recruits AcbSh MSNs, which in turn inhibits responding during extinction. Findings from Chapter 2 suggest that this inhibition may occur via

AcbSh control over LH. A question of interest is whether bilateral inactivation of caudal

BLA would disinhibit extinguished reward seeking. In support of this possibility are early studies demonstrating a role for the amygdala in the regulation over various 169

Chapter 4. Accumbens Shell and Basolateral Amygdala

motivated behaviours. For example, lesions of the amygdala increase exploratory behaviour in the rat (Burns, Annett, Kelly, Everitt, & Robbins, 1996; Corman, Meyer,

& Meyer, 1967; White & Weingarten, 1976) while NMDA-induced excitation of the

BLA (0.2-0.4 Pg) dose-dependently suppress exploratory behaviour (Chi Yiu &

Mogenson, 1989). BLA lesions have also been shown to disinhibit neophobia, thereby potentiating feeding of novel foods (Burns et al., 1996). More recently, previous authors have observed that reversible inactivation of the caudal BLA using the sodium channel blocker, lidocaine or TTX, potentiated cue-induced reinstatement of cocaine- (Kantak,

Black, Valencia, Green-Jordan, & Eichenbaum, 2002) and food- (McLaughlin &

Floresco, 2007) seeking. However, these effects may have been mediated by inactivation of fibres of passage running through the BLA since lidocaine and TTX have been noted to prevent potentiation and conduction in cell bodies as well as axons

(Lomber, 1999; Martin & Ghez, 1999). Nonetheless, Fuchs, Weber, Rice, and

Neisewander (2002) showed that post-training fibre-sparing NMDA-induced lesions of the BLA attenuated the progression of extinction in cocaine-seeking rats, supporting the possibility that BLA is actively involved in inhibiting reward seeking during extinction, although this effect may reflect a role for the BLA in either extinction expression or learning. Conversely, as reviewed in Chapter 2, studies based on context-induced reinstatement of drug seeking suggest that a role for BLA is specific to mediating reinstatement in the drug-associated context (Context A) and not extinction expression

(Context B). However, these reinstatement studies appear to typically target the rostral

BLA, which primarily projects to AcbC and lateral AcbSh. In contrast, it is caudal BLA that projects to the medial AcbSh and has been implicated in extinction in the abovementioned studies.

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Experiment 10 aimed to examine a role for caudal BLA on the expression of extinction in reward-seeking rats. Rats were trained to respond for alcoholic beer followed by extinction training. They were subsequently tested under conditions identical to extinction. Immediately prior to test, rats received infusions of saline or

GABAB and GABAA agonists, baclofen and muscimol (B/M) into cBLA.

Method

Subjects and Surgery

Fifteen experimentally naive male Long-Evans rats (Monash Animal Services,

Gippsland, Victoria, Australia) weighing 340-360 g prior to surgery were housed and maintained as described in Experiment 1. Rats were implanted with cannulae bilaterally in BLA using flat skull coordinates presented in Experiment 9.

Behavioural Procedure

Responding was trained and then extinguished as in Experiment 1. There were no contextual manipulations. Subsequently, all rats were tested under extinction conditions.

Immediately prior to test, rats received bilateral microinfusion of saline or B/M into caudal BLA, with groups matched as in Experiment 1.

Microinfusion and Histology Procedure

Procedures for microinfusions were as described in Experiment 1. Histology proceeded as in Experiment 1.

Data analyses

The mean numbers of active and inactive responses were analysed using group x

(response) factor ANOVA with orthogonal contrasts. For all analyses, Type I error rate

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(α) was controlled at 0.05 for each contrast tested using the procedure described by

Hays (1972).

Results and Discussion

Histology

Bilateral placement of injection tips are shown in Figure 37A. Two rats were excluded due to misplaced cannulae. Thirteen rats were included in the final analysis.

Behaviour

All rats acquired high levels of responding during training. On the last day of acquisition training the mean ± SEM numbers of active and inactive nosepokes were

79.62 ± 8.06 and 5.85 ± 1.50, respectively. During training rats made significantly more responses on the active than inactive nosepoke (F(1, 11) = 225.04; p < 0.001). There were no differences between groups (Fs(1, 11) < 1, ps > 0.05).

Mean ± SEM responses during extinction training are shown in Figure 37B.

Some data was lost during extinction day 2 due to software-related problems. Therefore, data for extinction day 2 was excluded from the following analyses. During extinction training, there were no significant differences between groups (main effect and interactions: Fs(1, 11) < 4.0, ps > 0.05). All rats made significantly more active than inactive nosepokes, averaged across days of extinction (F(1, 11) = 99.06, p < 0.001).

Overall, responding significantly decreased across days of extinction (F(1, 11) = 51.41, p < 0.001), and this decrease was greatest on the active than inactive nosepoke (F(1, 11) =

50.31, p < 0.001).

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A..

B. Extinction C. Test

Figure 37. Experiment 10: Microinfusion cannula placements in cBLA respectively as verified on Nissl-stained sections (A). Mean ± SEM numbers of active and inactive responses during extinction (B). Some data was lost during extinction day 2 due to software-related problems. Therefore, average data from only 6 of 13 rats are presented on extinction day 2. (C), Mean ± SEM numbers of active and inactive responses during test.

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Mean ± SEM responses during test are shown in Figure 37C. Responding was significantly greater on the active than inactive nosepoke (F(1, 11) = 10.12, p < 0.01).

However, there were no differences between groups on test (main effect and interaction:

Fs(1, 11) < 1, ps > 0.05). Therefore, reversible inactivation of cBLA did not attenuate the expression of extinction.

Discussion

Chapter 4 assessed a role for AcbSh AMPA receptors and their interaction with BLA on the expression of extinction of reward seeking. The findings from this chapter suggest that AcbSh AMPA receptors mediate expression of extinguished reward seeking, and this is mediated at least partly via glutamatergic interactions with the BLA. Specifically, this chapter showed that intra-AcbSh infusions of the AMPA receptor antagonist,

NBQX, dose-dependently attenuated the expression of extinguished responding. This chapter additionally showed that AcbSh infusions of NBQX was ineffective on measures of non-extinguished responding, including context-induced reinstatement, initial extinction acquisition, and responding on a progressive ratio schedule. In

Experiment 9, contralateral but not ipsilateral disconnection of the BLA from AcbSh prevented the expression of extinction, suggesting that serial communication from BLA to AcbSh is necessary for extinction of reward seeking. However, in the final experiment, reversible inactivation of the BLA failed to prevent the expression of extinction.

A role for AcbSh AMPA receptors in mediating expression of extinction is consistent with previous studies that implicate the involvement of AMPA GluR1 receptor subunits in the extinction of cocaine seeking in rats (Crombag et al., 2008;

Engblom et al., 2008; Mead et al., 2007; Sutton et al., 2003) and suggest that AcbSh

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AMPA receptors may mediate the expression of extinguished reward seeking across different reinforcers. The studies of the present chapter are unable to assess the contribution of specific AMPA-receptor subunits to the expression of extinction (e.g.

GluR1 versus GluR2) and achieving subunit specificity may reveal reinforcer- dependent contributions of AMPA to extinction of drug seeking, as reported by Sutton et al. (2003). Nonetheless, the pharmacological manipulations of the present study enable direct assessment of the behavioural role of AcbSh AMPA receptors on extinction of drug seeking – whether they are involved in the acquisition or the expression of extinction. The studies from this chapter found that similar to the effect of

AcbSh reversible inactivation on the extinction of alcoholic beer seeking (Chapter 3) or cocaine seeking (Peters et al., 2008), blockade of AcbSh AMPA receptors attenuated the expression but not acquisition of extinction. However, it is notable that the recovery of extinguished responding by NBQX was less pronounced than that observed following reversible inactivation of AcbSh using B/M. This may be attributed to the pharmacokinetics of NBQX, the dosage used in the present study, or more importantly, may suggest that neurotransmitters/peptides other than AMPA receptor-dependent mechanisms in the AcbSh are also important for the expression of extinguished reward seeking. Examination of these possibilities is worthy of further investigation.

In addressing the behavioural specificity of AcbSh contributions to extinction, several findings from the present chapter suggest that blockade of AcbSh AMPA receptors specifically impacts on the behavioural expression of extinction in reward seeking rats. First, NBQX attenuated the expression of extinction in rats, regardless of whether extinction occurred in a context that was identical (i.e. an AAA design) or distinct (i.e. an ABB design) from the self-administration context. Moreover, it is noteworthy that the attenuation of extinction responding on test following intra-AcbSh

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NBQX infusions appeared similar in magnitude regardless of the test context. As previously noted in Chapter 1, Bouton et al. (2011) showed that context-induced reinstatement of extinguished instrumental responding was stronger when an animal was returned to a context that was previously associated with reward than when tested in a context separate from the original training context. This is important as it suggests that although contexts can modulate recovery of an extinguished response, the function of AcbSh AMPA receptors during extinction expression is not likely influenced by the contextual elements on test. The results support a behaviourally specific role for AcbSh

AMPA receptors in the masking process involved in the expression of extinction.

Additionally, the effects of NBQX on extinction expression are not likely due to nonspecific behavioural activation or a general effect on the motivation to work for reward since similar infusions were ineffective on measures of initial extinction learning and on overall measures of responding on a progressive ratio schedule respectively.

This is consistent with previous findings that reversible inactivation of AcbSh using the

GABAA agonist muscimol was similarly ineffective on breakpoint for food reinforcement (Zhang, Balmadrid, & Kelley, 2003). This suggests that the impact of

NBQX on extinguished responding is not due to a general invigoration of responding.

Finally, it is unlikely that NBQX impacted the animals’ level of food deprivation since both overall responding and breakpoint on a progressive ratio schedule are increased by food deprivation (Jewett, Cleary, Levine, Schaal, & Thompson, 1995) and in the present study there was no effect of NBQX on either of these measures. Finally, intra-AcbSh infusions of NBQX were ineffective on responding under context-induced reinstatement

(ABA test, Experiment 6), suggesting that a history of extinction training alone is insufficient to detect an effect of NBQX on test. Rather, the findings from the present

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chapter suggest that under conditions of an extinction test, NBQX appears to specifically disrupt mechanisms that mediate the expression of extinction.

Possibly, intra-AcbSh NBQX exerts its effects on behaviour in a manner similar to GABA agonist-induced reversible inactivation; that is, via a reduction in AcbSh neuronal activity. Consistent with this, locally applied AMPA receptor antagonist,

CNQX, in the dorsal striatum blocks spontaneously active as well as glutamate-induced neuronal activity (Sandstrom & Rebec, 2003). Moreover, on the basis that accumbal neurons typically have hyperpolarised resting potentials and little spontaneous firing activity, owing primarily to inwardly rectifying potassium currents (Hu & White, 1996;

O'Donnell & Grace, 1995), it has been suggested that pharmacological blockade of accumbal AMPA receptors potentially “turn off” neurons (Tzschentke & Schmidt,

2003; Yun, Nicola, & Fields, 2004). This raises an interesting possibility that a role for

AcbSh in the expression of extinction may be to gate reward seeking behaviour and this gating mechanism may be specifically disrupted following AcbSh AMPA receptor blockade. I will return to discuss this further in the General Discussion.

A major finding of the present chapter is that pharmacological disconnection of

BLA from AcbSh AMPA receptors attenuated the expression of extinction. This finding supports the conclusion that expression of extinction might be mediated by BLA-driven

AMPA receptor signalling in AcbSh. Indeed it is inferred that the direction of inter- structural interaction mediating extinction expression involves serial BLA→AcbSh communication since the disconnection study of Experiment 9 applied an AMPA receptor antagonist in the AcbSh. Although it is well-established that the BLA is a major source of glutamatergic input into the AcbSh, possible recruitment of a direct monosynaptic BLA→AcbSh pathway during extinction cannot be fully inferred on the basis of the disconnection study used in this thesis. However, as reviewed previously,

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BLA neurons projecting to AcbSh have previously been shown to be recruited during the expression of extinction using combined c-Fos/tract-tracing methods (Hamlin et al.,

2009), supporting the possibility that BLA-AcbSh interactions during extinction may involve, at least in part, recruitment of direct projections between these structures.

Nonetheless, BLA-driven glutamatergic signalling in AcbSh might also be mediated indirectly via BLA projections to mPFC, which in turn projects glutamatergically to

AcbSh. However, the findings from the present chapter suggest that an indirect

BLA→mPFC→AcbSh pathway for extinction is unlikely since mPFC projection neurons target the accumbens inter-hemispherically (Berendse et al., 1992), which yields the prediction that both contralateral and ipsilateral disconnection of BLA from

AcbSh would disrupt the expression of extinction. Indeed, disconnection studies involving mPFC and AcbSh typically show this pattern of results (Fuchs et al., 2007;

Peters et al., 2008). However, in the present thesis, only contralateral and not ipsilateral disconnection of BLA from AcbSh attenuated the expression of extinction. Finally, it is not clear from the present disconnection study whether BLA→AcbSh interactions contributing to the expression of extinction are mediated via AMPA receptor-dependent actions on post-synaptic sites (e.g. medium spiny projection neurons, cholinergic or parvalbumin interneurons), presynaptic sites (e.g. on dopaminergic terminals) or a combination of these.

As reviewed earlier in this chapter, previous findings have suggested that the

BLA, and primarily its caudal region, is critical for the initial learning involved in extinguishing drug seeking (McLaughlin & Floresco, 2007). In the present thesis, a

BLA→AcbSh pathway was shown to be involved in the expression of extinction. This finding implies that in addition to its suggested role in extinction learning, the BLA critically contributes to the expression of extinction. However, the findings from

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Experiment 10 suggest that this contribution is likely to be complex and dissimilar to

AcbSh contributions to the expression of extinction; whereas reversible inactivation of

AcbSh disinhibits extinguished reward seeking, reversible inactivation of the BLA had no observable effects on extinguished reward seeking. This is despite targeting the caudal portion of the BLA, the region most implicated in extinction of reward seeking

(Hamlin, 2009; McLaughlin & Floresco, 2007). Nonetheless, the absence of an effect following reversible inactivation of cBLA on extinction expression is consistent with several findings showing that reversible inactivation of the BLA is similarly ineffective on performance of an extinguished cocaine seeking response, regardless of whether extinction occurred in a context identical to or distinct from the training context (Fuchs et al., 2005; Peters et al., 2008). Instead, in these studies, BLA inactivation prevented the reinstatement of drug and reward seeking (e.g., Fuchs et al., 2005; Peters et al.,

2008). One possibility is that there may be a dual role for BLA in inhibiting and enabling reward seeking and this may be mediated by distinct projections from BLA.

The present data show a role for a BLA→AcbSh pathway in the inhibition of reward seeking during extinction expression. However, rostral BLA neurons are selectively activated, as indexed by expression of the c-Fos during reinstatement (Hamlin et al.,

2008, 2009) and these neurons project to PFC (Hoover & Vertes, 2007). Studies based on pharmacological disconnection of the rostral BLA from PFC (Fuchs et al., 2007;

Mashhoon, Wells, & Kantak, 2010) suggest that BLA–PFC interactions may be critical for reinstatement of reward seeking. BLA might contribute to both extinction and reinstatement via projections to AcbSh and PFC respectively. However, as pharmacological reversible inactivation of the BLA non-selectively inhibits neuronal activity, it is possible that BLA contributions to extinction expression may not be

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detectable under conditions where BLA is bilaterally compromised. This will be addressed further in the General Discussion.

In conclusion, Chapter 3 shows that AcbSh infusion of the AMPA receptor antagonist, NBQX, attenuates the expression of extinction of alcoholic beer seeking in rats. This effect of NBQX was dose-dependent and behaviourally specific to the expression of extinction. This chapter also shows that pharmacological disconnection of a BLA→AcbSh pathway involving AcbSh infusion of NBQX in combination with reversible inactivation of the contralateral BLA attenuates expression of extinction of alcoholic beer seeking in rats. Therefore, it is concluded that AcbSh AMPA receptors are critical for the expression of extinction of drug and reward seeking behaviour and that this role for AcbSh AMPA receptors is dependent on serial communication from the BLA.

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Chapter 5. General Discussion

Chapter 5.

General Discussion

This thesis examined the neurobiological mechanisms underlying the extinction of drug seeking. The experiments reported here addressed two aims. The first was to confirm a role for the AcbSh in suppressing drug seeking behaviour during extinction. The second was to examine the circuit-level mechanisms that enable AcbSh control over drug seeking during extinction. Behavioural learning theories of extinction have long argued that extinction is an active process that inhibits drug seeking behaviour. Accordingly, it was suggested in the General Introduction (Chapter 2) that the neurobiological substrates underlying extinction expression similarly exerts inhibitory control over those promoting the reinstatement of drug seeking. In this final chapter, the empirical findings reported in Chapters 3 and 4 are summarised and discussed in relation to the circuit- level mechanisms of extinction and context-induced reinstatement as reviewed in

Chapter 2. Finally, the implications, future directions and limitations of the findings in this thesis are discussed.

5.1. Summary of empirical results and their implications

Chapter 3 investigated the functional contribution of AcbSh in the extinction of alcoholic beer seeking and asked whether this AcbSh contribution involved regulation over the efferent target, LH. Experiment 1 showed that reversible inactivation of the

AcbSh prevented the expression of extinction and so reinstated previously extinguished alcoholic beer seeking. In Experiment 2, reversible inactivation of the AcbSh had no effect on the acquisition of extinction, suggesting that AcbSh contributions to extinction

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are likely specific to its behavioural expression. Experiment 3 combined functional inactivation of the AcbSh with immunohistochemical visualisation of c-Fos protein expression and showed that the reinstatement of alcoholic beer seeking following

AcbSh inactivation was associated with increased levels of c-Fos induction throughout the hypothalamus, including in hypothalamic orexin- and CART-containing neurons of the PeF/LH region. Experiment 4 showed that reinstatement induced by AcbSh inactivation was prevented if LH was simultaneously inactivated. Finally, Experiment 5 showed that pharmacological disconnection of an AcbSh→LH pathway via unilateral

GABA receptor blockade in LH combined with reversible inactivation of the contralateral AcbSh attenuated expression of extinction. This effect was specific to contralateral and not ipsilateral disconnection of AcbSh from more anterior regions of the LH. However disconnection of AcbSh from posterior LH disrupted extinction in both contralateral and ipsilateral groups, raising the possibility that parallel pathways mediate the expression of extinction. Together, the findings from this chapter suggest that: 1) AcbSh mediates expression of extinction by inhibiting hypothalamic neuropeptidergic neurons and reversible inactivation of the AcbSh removes this influence, thereby disinhibiting LH-dependent reinstatement of reward seeking; 2) this

AcbSh inhibition over hypothalamus might occur serially via GABAergic projections from AcbSh to anterior LH.

Chapter 4 examined a role for AcbSh AMPA receptors in mediating the expression of extinction and characterised the behavioural specificity of this role.

AMPA receptors were studied because the majority of thalamic, cortical, and amygdaloid afferents to AcbSh use glutamate as a neurotransmitter. This chapter also examined whether AcbSh AMPA-receptor contributions to extinction occurred via serial interactions with its afferent structure, BLA. In Experiment 6, intra-AcbSh

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blockade of AMPA-type glutamate receptors via local infusion of the AMPA receptor antagonist, NBQX, attenuated the expression of extinction of alcoholic beer seeking.

The effect was dose dependent (Experiment 7) and behaviourally specific to the expression of extinction. That is, similar infusions had no effect on context-induced reinstatement, initial acquisition of extinction, or responding on a progressive ratio schedule (Experiments 6 and 8). These results therefore suggest that one mechanism through which AcbSh contributes to the behavioural expression of extinction is via glutamatergic AMPA receptor signalling and that this contribution involves recruitment of extinction-specific processes. As reviewed in Chapter 2, AcbSh receives glutamatergic signalling from multiple sources, one of which is from the caudal portion of the BLA. Experiment 9 showed that pharmacological disconnection of a caudal

BLA→AcbSh pathway via unilateral AMPA receptor blockade in AcbSh combined with reversible inactivation of the contralateral BLA attenuated expression of extinction. While this finding implicates caudal BLA as an important component in circuits mediating the expression of extinction, Experiment 10 found no effect of bilateral reversible inactivation of caudal BLA on the expression of extinguished responding (Experiment 10). Together, the empirical findings of Chapter 4 suggest that:

1) extinction expression is mediated, at least in part, via AcbSh AMPA receptors and 2) that the expression of extinction is mediated via serial communication between BLA and AcbSh AMPA receptors.

Taken together, the implications of the findings from this thesis can be summarised as follows:

1. The AcbSh is a functionally critical component of the neural circuitry underlying

the behavioural expression of extinction in alcohol seeking rats. This role for

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AcbSh has previously been demonstrated in rats that were initially trained to

respond for cocaine (Peters et al., 2008). Thus, AcbSh mediates extinction

expression across different reinforcers. This notion of a common mechanism

underlying extinction expression is important because much of the earlier

evidence implicating AcbSh in extinction is based on cocaine-trained rats and, in

some instances, the evidence linking AcbSh to extinction was specific to cocaine

and not sucrose reinforcement (Sutton et al., 2003). Thus, on the basis of earlier

findings, it has been proposed that AcbSh was recruited during extinction to

reverse cocaine-induced neuroadaptations (Schmidt et al., 2001; Sutton et al.,

2003). While this proposition remains tenable, the present findings from alcohol-

trained rats support a more general role for the AcbSh in mediating the

performance of extinction behaviour, independent of reinforcer-type. Moreover,

there was no indication from the present findings or others (Peters et al., 2008) to

suggest a role for AcbSh in extinction learning.

2. AcbSh contributes to the expression of extinction via inhibition over drug seeking

circuitry involving LH. Inactivation of AcbSh reinstated extinguished reward

seeking and this reinstatement was 1) associated with an increase in c-Fos

expression in hypothalamic neurons, including in LH orexin neurons and 2)

blocked following concurrent inactivation of LH. Given the role for LH in

reinstatement of drug seeking (Marchant et al., 2009), this finding suggests that

extinction circuits involving AcbSh interface with circuitry mediating the

reinstatement of drug seeking. Psychological theories of relapse have long

proposed that relapse is regulated by processes mediating the expression of

extinction. The present findings provide a neurobiological basis for this regulation

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Chapter 5. General Discussion

involving AcbSh inhibition of LH-dependent drug seeking.

3. AcbSh mediates the expression of extinction via serial interactions with anterior

but not posterior tuberal LH. This is consistent with known anatomical

projections from AcbSh to anterior LH and suggests that serial AcbSh→LH

communication mediates the behavioural expression of extinction. Moreover, it is

consistent with an earlier finding showing that recruitment of a direct AcbSh→LH

pathway was associated with the behavioural expression of extinction (Marchant

et al., 2009). Thus, together with the present findings, it was suggested that a

direct AcbSh→LH pathway might function to mediate the behavioural expression

of extinction. The disconnection finding however does not exclude the possibility

that AcbSh-LH interactions may also occur via parallel circuits. Consistent with

this, the expression of extinction was disrupted following contralateral and

ipsilateral disconnection of AcbSh from more posterior LH regions. This finding

suggests that other pathways involving posterior LH might also be implicated in

the expression of extinction.

4. Glutamatergic AMPA receptor-dependent neurotransmission in the AcbSh is

critical for the expression of extinguished drug seeking. There was no evidence

that blockade of AcbSh AMPA receptors interrupted behaviour on other measures

of reward including context-induced reinstatement, initial extinction behaviour or

responding on a progressive ratio. Importantly, blockade of AcbSh AMPA

receptors attenuated the expression of extinction regardless of whether the

extinction context was different or identical with the context previously associated

with drug reward. The present findings therefore suggest that AcbSh inactivation

removes the contextual mask imposed by extinction training over drug seeking 185

Chapter 5. General Discussion

behaviour and confirms a specific role for AcbSh in the behavioural expression of

extinction. A functional role for other glutamate-type receptors (e.g. NMDA or

metabotropic receptor-types) or specific AMPA receptor subunits (e.g. GluR1 and

GluR2) in mediating the expression of extinction is yet to be demonstrated.

5. AcbSh contributes to the expression of extinction via serial communication with

caudal BLA. The present finding suggests that extinction performance is

dependent, at least partly, on glutamatergic projections from caudal BLA to

AcbSh. Previous studies have identified ilPFC as another source of glutamatergic

signalling enabling AcbSh control over extinction (Peters et al., 2008). Although

multiple sources might engage AcbSh control over extinction expression, the

evidence implicating ilPFC is based on findings that both ipsilateral and

contralateral disconnection of ilPFC from AcbSh attenuated the expression of

extinction in cocaine-trained rats (Peters et al. 2008). Thus there remains a

possibility that ilPFC and AcbSh sit in separate yet parallel circuits mediating

extinction. This possibility was discussed in detail in Chapter 2. In contrast, the

present findings directly suggest a serial BLA→AcbSh pathway in the expression

of extinction because contralateral but not ipsilateral disconnection of AcbSh

attenuated expression of extinction of reward seeking.

6. A role for caudal BLA contributions to extinction expression is not revealed

following reversible inactivation of caudal BLA on a test of extinction expression.

Despite previous findings implicating a role for caudal BLA in the initial

acquisition of extinction (McLaughlin & Floresco, 2007), several studies

involving general inactivation of the BLA confirm that BLA inactivation has no

effect on a test of extinction expression (Fuchs et al., 2005; Peters et al., 2010). It 186

Chapter 5. General Discussion

was suggested in this thesis that distinct neuronal populations within BLA might

contribute differentially to extinction expression and reinstatement of drug

seeking and that BLA contributions to extinction expression may not be

detectable when BLA is bilaterally compromised using non-selective

pharmacological inhibition as done in the present thesis (Chapter 4, Discussion).

This will be discussed further in Section 5.2.3.

5.2. The neural circuitry underlying extinction of drug seeking

5.2.1. A role for AcbSh in the behavioural expression of extinction.

As reviewed in Chapter 2, previous findings have implicated AcbSh in the extinction of drug-seeking behaviour. These findings include the occurrence of extinction-associated neuroplasticity (Ghasemzadeh et al., 2009; Schmidt et al., 2001; Sutton et al., 2003) and facilitation of extinction behaviour via overexpression of AcbSh GluR1 AMPA receptor subunits (Sutton et al., 2003). AcbSh reversible inactivation studies from the present thesis (Chapter 3) as well as others (Peters et al., 2008) also implicate a functional role for AcbSh in mediating the expression of extinction in drug seeking animals. Similarly, blockade of AcbSh AMPA receptors disrupted the expression of extinction (Chapter 4).

It was suggested here (Chapter 4, Discussion) that AcbSh AMPA receptor blockade and reversible inactivation similarly disrupted the expression of extinction via a reduction in

AcbSh neuronal activity. This importantly suggests that AcbSh neuronal activity inhibits reward seeking during extinction and that an overall reduction in AcbSh neuronal activity disinhibits reward seeking. Thus, a role for AcbSh in the expression of extinction may be to gate reward seeking behaviour.

A role for AcbSh in gating reward-directed behaviour has been proposed and demonstrated in previous studies involving appetitive behaviour (Ambroggi, 187

Chapter 5. General Discussion

Ghazizadeh, Nicola, & Fields, 2011; Krause, German, Taha, & Fields, 2010; Taha &

Fields, 2005, 2006). Studies of single unit recordings show that a subpopulation of accumbal neurons abruptly reduce their spike firing during reward seeking and appetitive responding, including in a sucrose-licking task (Krause et al., 2010; Taha &

Fields, 2005) or in a task where rats performed a sustained nosepoke response for sucrose reinforcement (Taha & Fields, 2006). Moreover, Krause et al. (2010) showed that electrical stimulation of these cells suppressed consummatory behaviour. Thus, they suggested that a pause in Acb firing is necessary for feeding behaviour to occur

(Krause et al., 2010). Similarly, a pause in AcbSh firing may be necessary for reward seeking to occur.

It is noteworthy that in the electrophysiological studies described above, suppression of neuronal firing was observed in both AcbSh and AcbC subregions

(Krause et al., 2010; Taha & Fields, 2005, 2006). Moreover, Krause et al. (2010) showed that a transient suppression of licking behaviour could be achieved by electrical stimulation of either AcbSh or AcbC subregions. However, as reviewed in Chapter 2, the available evidence from functional inactivation studies suggests that AcbSh, not

AcbC, is critical for extinction. Possibly, AcbSh and AcbC neurons encode similar information, but may mediate different components of behavioural performance due to the divergence in their efferent targets (Ambroggi et al., 2011). In partial support of this,

Ambroggi et al. (2011) found that reversible inactivation of either AcbC or AcbSh produced disinhibition of lever pressing. Intriguingly, disinhibition of responding following AcbC inactivation was restricted to lever-pressing immediately following receipt or consumption of the reward. In contrast, disinhibition of responding following

AcbSh inactivation was largely associated with the presentation of the cue signalling reward omission and also following receipt of the reward. AcbSh inactivation also

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Chapter 5. General Discussion

disinhibited to a lesser degree, responding during the interstimulus interval. The authors proposed that the disinhibition of responding following AcbSh inactivation reflects the combined increase in general arousal and also the removal of a specific inhibition over responding driven by a cue signalling the omission of reward (Ambroggi et al., 2011).

The findings from Ambroggi et al. (2011) are interesting for two reasons. First, they suggest that an increase in responding following AcbSh inactivation is not wholly due to an increase in general arousal; it involves the release of active inhibition over non-reinforced reward seeking behaviour in the presence of a cue signalling reward omission. Second, single unit recordings from Ambroggi et al. (2011) implicate a role for AcbSh in gating consummatory behaviour specifically under conditions of non- reinforcement: AcbSh neurons responded more selectively to the presentation of the cue signalling reward omission and rewarded lever presses were most frequently associated with inhibition of AcbSh neuronal activity. There is good reason to suggest that AcbSh- mediated inhibition of responding during cued-reward omission in Ambroggi et al.

(2011) and expression of extinction in the present thesis may involve similar mechanisms; in both cases, the animal is required to learn that responding is non- reinforced. Moreover, they involve similar patterns of behaviour; in Ambroggi et al.

(2011), rats initially responded to the cue signalling reward omission and subsequently exhibited a decrement in responding to the cue. Similarly, during extinction training animals initially exhibit high levels of responding followed by a marked decrease in responding across days of training. Therefore, the expression of extinguished reward seeking may be mediated by a distinct population of neurons in AcbSh that gate reward seeking specifically under conditions when this behaviour is non-reinforced.

Pennartz et al. (1994) proposed that the behavioural impact of accumbal neuronal activity depends on the synchronous firing of specific neuronal “ensembles”,

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defined as a group of neurons with similar afferent or efferent relations as well as closely-related behavioural functions. The output of these ensembles is thought to communicate with a specific set of target structures to produce specific actions from an animal’s behavioural repertoire (Pennartz et al., 1994). Holding the notion that a specific ensemble of AcbSh neurons enables expression of extinction in reward seeking animals, two questions arise: First, what are the efferent targets of the AcbSh ensemble that mediates extinction-induced suppression of reward seeking? Second, how is this ensemble recruited in relation to its afferent structures? A major implication of the present thesis is that the behavioural expression of extinction is mediated at least partly via serial communication from BLA→AcbSh, and from AcbSh→LH. These extinction- related subcircuits are separately discussed in the subsequent sections.

5.2.2. AcbSh interfaces extinction circuitry with reinstatement circuitry: AcbSh- hypothalamus interactions.

The medium-spiny GABAergic projection neurons of the AcbSh directly target the LH.

AcbSh neurons can therefore exert direct inhibitory control over the LH (Sano & Yokoi,

2007). Reductions in the activity of AcbSh projection neurons to LH may disinhibit LH neurons, thereby promoting reward seeking. Conversely, increases in the activity of these AcbSh projecting neurons may reduce reward seeking. There are several findings consistent with this possibility: extinction training upregulates AMPA receptor subunit expression in AcbSh (Sutton et al., 2003); activation of AcbSh projection neurons to LH is associated with the behavioural expression of extinction (Marchant et al., 2009); the reinstatement of extinguished responding induced by general inactivation of AcbSh depends on LH neuronal activation (Chapter 2 results); and disconnection of the AcbSh from LH prevented expression of extinction (Chapter 2 results). Thus, extinction may

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involve upregulation of AMPA receptor signalling in AcbSh neurons projecting to LH.

Inhibition of AcbSh might, in turn, reduce this signalling thereby disinhibiting LH and reinstating reward seeking (Maldonado-Irizarry et al. 1995; Stratford and Kelley 1999).

An important question of connectivity is how AcbSh exerts its influence over

LH. The formulation described above implies that this influence occurs directly and monosynaptially. This is consistent with well-established anatomical connectivity between AcbSh MSNs and LH (Sano & Yokoi, 2007). Moreover, a subset of dorsomedial AcbSh neurons that project to LH are recruited in association with the behavioural expression of extinction (Marchant et al., 2009), suggesting a monosynaptic

AcbSh→LH pathway. However, recruitment of these LH-projecting AcbSh neurons represented only approximately 16% of the total neurons recruited in association with extinction (Marchant et al., 2009). Possibly, AcbSh-LH interactions during the expression of extinction also involve an intervening structure. In Experiment 5, disconnection of AcbSh from LH was achieved via GABA receptor antagonist in the

LH. Therefore, a possible interposing structure mediating AcbSh-LH interactions is limited to one that is: ipsilaterally connected with both structures, an efferent target of the AcbSh, and projects GABAergically to LH. There are at least two candidate structures that fit these criteria: medial VP and the mesopontine rostromedial tegmental nucleus (RMTg).

The medial VP receives direct and primarily ipsilateral projections from AcbSh and in turn sends GABAergic projections to LH (Bevan, Clarke & Bolam, 1997;

Groenewegen & Russchen, 1984; Stratford, 2005; Usuda, 1998). Reversible inactivation studies suggest that like LH, VP is also important for reinstatement of drug-seeking

(McFarland & Kalivas, 2001). Moreover, blockade of GABA receptors in mVP potentiates feeding in sated rats, supporting the possibility that GABA release into mVP

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from AcbSh is necessary for regulating appetitive behaviour (Stratford, Kelley, &

Simansky 1999). However, it remains to be tested whether similar blockade of GABA receptors in mVP will prevent the expression of extinction or whether AcbSh projection neurons to mVP are recruited during extinction expression. Importantly, previous authors have hypothesised that an AcbSh→VP→LH pathway might mediate regulation over feeding (Stratford et al., 1999). A similar pathway may also underlie the expression of extinction. However, it is not clear how GABAergic VP projections to LH are recruited by similarly GABAergic AcbSh neurons to mediate extinction.

In addition to mVP, RMTg is similarly interposed to relay information between

AcbSh and LH, and is closely interconnected with corticolimbic-striatal- hypothalamic/pallidal loops (Jhou, Geisler, Marinelli, Degarmo & Zahm, 2009).

However, to the best of the author’s knowledge, behavioural evidence implicating

RMTg interactions with either AcbSh or LH is lacking. Thus, while there is some evidence to support a direct AcbSh→LH pathway mediating extinction, further work is due to examine possible transynaptic AcbSh control over LH-mediated reward seeking.

This is especially important as these pathways may reflect parallel pathways through which AcbSh accomplishes complex control over distinct aspects of reward seeking behaviour (e.g. regulation over general arousal, motor coordination, hedonic properties of the reward).

5.3. A BLA→AcbSh→ LH subcircuit mediates extinction of reward seeking

Glutamatergic afferents from the BLA directly and monosynaptically target accumbal medium spiny projection neurons (Kirouac & Ganguly, 1995; Kita & Kitai, 1990;

Robinson & Beart, 1988). Specifically, projections to AcbSh are largely derived from caudal portions of the BLA (Hamlin et al., 2009; Heimer et al., 1997). The

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microinjection cannulae in Experiment 9 were targeted to the caudal BLA to increase the probability of affecting these AcbSh projection neurons. Importantly, BLA afferents terminate proximally to AcbSh cells that project to the LH, with BLA fibre terminals appearing to contact LH-projecting AcbSh cells (Kirouac & Ganguly, 1995). Based on this pattern of connectivity and the findings of the present thesis, it is possible that extinction is expressed, at least in part, via an amygdalo-ventral striato-hypothalamic circuit. Therefore AcbSh holds a pivotal role as an interface between BLA-mediated extinction circuitry and LH-dependent reinstatement circuitry. However, AcbSh receives converging inputs from multiple corticolimbic structures including BLA, ilPFC, and hippocampus. This literature was reviewed earlier in Chapter 2. How these inputs interact to modulate AcbSh-contributions to the expression of extinction is not known, although, there is evidence to support their interaction at the level of AcbSh. For example, ilPFC gates BLA stimulation-induced dopamine efflux in AcbSh (Jackson &

Moghaddam, 2001) and hippocampus gates ilPFC-induced activity in AcbSh

(O’Donnell & Grace, 1995). Most likely, BLA contributions to the expression of extinction will need to be considered within the complexity of converging corticolimbic inputs into the AcbSh.

Within the broader context of reward circuitry, the BLA, AcbSh, and LH each represent distinct and interconnected components of the motivational system: the limbic-cortical system, the mesostriatal dopaminergic system, and an action/arousal system, respectively (Ikemoto, 2007). As a key component of the limbic-cortical system, the BLA is thought to have a role in forming and maintaining representations of outcomes to guide goal-directed behaviour. Thus, bilateral lesions impair an animal’s ability to modify instrumental responding based on changes in the value of an outcome

(reward devaluation); changes in the contingency between reward and outcome

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(degradation of the instrumental contingency, extinction learning); or in the presence of cues previously paired with reward (Pavlovian-instrumental transfer) (Balleine,

Killcross, & Dickinson, 2003; Corbit & Balleine, 2005; Johnson, Gallagher, & Holland,

2009). Moreover, the behaviour expressed depends on BLA interactions with the Acb, a major component of the mesostriatal dopaminergic system. For example, Shiflett and

Balleine (2010) recently showed that contralateral disconnection of the BLA from

AcbSh disrupted the ability of reward-related cues to potentiate reward seeking (i.e.

Pavlovian-instrumental transfer) but had no impact on the animal’s ability to appropriately modify behaviour following satiety-induced outcome devaluation.

Conversely, contralateral disconnection of the BLA from AcbC resulted in the reverse pattern of behaviour. This suggests that BLA guides behaviour via differential projections to the mesostriatal system.

Within the mesostriatal system, it has been proposed that dopamine release in the Acb gates the input from the limbic cortical system to promote the most appropriate behavioural strategy (Yun et al., 2004). Appropriate action selection also implies inhibition of inconsequential actions, and AcbSh may contribute to this inhibition

(Ambroggi et al., 2011). Importantly, the influence of corticolimbic and mesostriatal processing on behaviour is integrated and coordinated via an action arousal system, mediated in part by the LH, either directly from AcbSh, or indirectly via Acb projections to VP (Ikemoto, 2007). LH contains the neuropeptide, orexin. As previously mentioned in the General Introduction, this neuropeptide plays a critical role in arousal and reward, and consistent with this role, reinstatement of reward seeking was associated with increased activation of orexin-containing peptides (Experiment 3). LH also contains isodendritic neurons, which are morphologically suited to receive extensive afferent input (Leontovich & Zhukova, 1963), and has broad-ranging

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influence over the autonomic nervous system via projections to the nucleus of the solitary tract and parabrachial nucleus; endocrine secretion via projections to more medial hypothalamic neuropeptide-dense sites; emotional motor effector sites via projections to central amygdala and periaqueductal gray; and reward pathways via projections to the VTA (Ikemoto, 2007; Saper et al., 1979). Thus, LH regulates control over behaviour via an extensive network mediating general arousal (e.g. locomotion, foraging), motor patterns, and reward, and this control is influenced by limbic cortical and mesostriatal dopamine systems. It is within the interaction of these systems that extinction behaviour, which involves modifying behaviour in the absence of an expected outcome, is likely acquired and expressed.

5.4. Dual contributions of BLA to extinction and reinstatement?

Of interest to the present findings is the specific BLA contributions to the expression of extinction via interactions with the AcbSh. The BLA has long been implicated in reward-related behaviours and there is compelling evidence to suggest that this structure is a functionally critical node within the reinstatement circuitry. This literature was reviewed earlier in Chapter 2. The findings from the present thesis suggest that additionally, the BLA is critically placed within the extinction circuitry through its interactions with the AcbSh. This is consistent with previous findings based on reversible inactivation and electrophysiological recordings implicating a role for BLA in the extinction of reward seeking (McLaughlin & Floresco, 2007; Tye et al., 2010). Thus

BLA contributes to both reinstatement and extinction of reward seeking.

Consistent with a dual role for BLA, there is compelling evidence that BLA comprises at least two distinct neuronal populations, one of which is recruited in the presence of reward-associated cues or contexts, while the other is recruited specifically

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under conditions of extinction or reward omission (Ambroggi, Ishikawa, Fields, &

Nicola, 2008; Hamlin et al., 2009; Herry et al., 2008; Tye et al., 2010). Importantly, recent findings from single unit recordings in the BLA during fear extinction suggest that it is the relative recruitment of distinct neuronal populations (ensembles) within

BLA that predicts expressed behaviour (Herry et al., 2008). That is, there was increased recruitment of putative “extinction neurons” immediately preceding the behavioural expression of extinction and this co-varied with a reduction in the activity of neurons that were recruited during initial fear learning prior to extinction. Moreover, the authors showed that in an ABA renewal fear conditioning preparation, return to the training context re-recruited neurons that responded during initial training whereas putative

“extinction neurons” were re-recruited when animals were returned to the extinction context. Thus, differential recruitment of these distinct populations of neurons is not a transient phenomenon (Herry et al., 2008). Consistent with behavioural learning theories, they are actively recruited during expression of extinction and renewal.

The information communicated from BLA to AcbSh during extinction expression remains elusive. An interesting hypothesis is borrowed from the fear conditioning literature: that the BLA contributes to the expression of either initial learning or extinction by promoting the behavioural transition or switching between these two behavioural states (Herry et al., 2008). This hypothesis was based on the observation that functional inactivation of the BLA prevents both initial fear conditioning and extinction learning. Thus, animals with inactivated BLA are able to express high and low (extinction) levels of freezing behaviour; they are instead impaired in their ability to switch between these high and low fear states (Herry et al., 2008).

Likewise, in the context of drug seeking, BLA might promote the behavioural switching between initially learned drug seeking and extinction behaviour. Indeed, functional

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inactivation of the BLA prevents acquisition of extinction of reward seeking

(McLaughlin & Floresco) and also the expression of reinstatement (Fuchs et al., 2005).

Thus, as in the case of fear conditioning, animals with inactivated BLA are able to express high and low (extinction) levels of reward seeking. Importantly, this hypothesis predicts that post-extinction training inactivation of the BLA should have no impact on extinction expression, as there is no change in behavioural state. This is consistent with the null finding in Experiment 10, and similarly observed in previous studies of drug seeking (Fuchs et al., 2005; Peters et al., 2008) and extinction of fear conditioning

(Herry et al., 2008).

A tentative role for BLA in promoting the behavioural transition between reward seeking and extinction is also consistent with the finding that disconnection of AcbSh from BLA prevented the expression of extinction (Experiment 9). A basic assumption however is that since bilateral AcbSh inactivation promotes reward seeking, unilateral inactivation of AcbSh might similarly induce a reward seeking state. Consistent with this possibility, muscimol-induced unilateral AcbSh inactivation promotes c-Fos induction throughout a distributed network of structures within the forebrain including in the ipsilateral LH; and bilaterally in structures typically involved in reward-seeking including PFC, BLA, and VTA (Stratford, 2005). It also increases feeding in sated rats

(Maldonado-Irizarry et al., 1995). Although unilateral inactivation of AcbSh alone is not sufficient to promote drug seeking (Peters et al., 2008), it is speculated that this is due to an intact BLA→AcbSh pathway in the opposite hemisphere, where BLA acts to promote an extinction state. Accordingly, extinction expression should be maintained following ipsilateral disconnection of BLA from AcbSh. In contrast, reward seeking is predicted to result from contralateral BLA-AcbSh disconnection as unilateral inactivation of AcbSh promotes a state of reward seeking and BLA inactivation in the

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A. B.

BLA BLA BLA BLA

AcbSh AcbSh AcbSh AcbSh

= extinction = extinction

C. D.

BLA BLA BLA BLA

AcbSh AcbSh AcbSh AcbSh

= extinction = reward seeking

Figure 38. BLA and extinction expression. A role for BLA in promoting the transition between extinction and reward seeking predicts that bilateral BLA inactivation has no effect on already acquired extinction behaviour (A.). It is proposed that under extinction conditions, unilateral inactivation of the AcbSh (in B., C., and D.) produces a state of reward seeking. Concomitantly, BLA is bilaterally recruited, which serves to inhibit responding via AcbSh. Thus, unilateral inactivation of AcbSh (B.) or ipsilateral BLA-AcbSh disconnection (C.) produces extinction expression via an intact

BLA→AcbSh pathway. In contrast, contralateral BLA-AcbSh disconnection promotes reward seeking as BLA is unable to suppress reward seeking through its interactions with AcbSh.

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contralateral hemisphere prevents the animal from switching to the correct behavioural state of extinction via communication with AcbSh. Thus a dual role for BLA in extinction and reinstatement may reflect a role for this structure in transitioning between extinction and reward seeking states. This is summarised in Figure 38.

5.3. Parallel circuits of extinction and reinstatement.

A highly distributed meso-corticolimbic-striatal-thalamic-hypothalamic network of neural structures mediate context-induced relapse to drug seeking. As reviewed in

Chapter 2, this circuit involves interconnections between mesolimbic (VTA), cortical

(mPFC, OFC), sub-corticolimbic (hippocampus, BLA), striatal (DS, Acb), thalamic

(PVT), and hypothalamic (LH) control over drug seeking. This distribution is thought to reflect neural processes of reward reinforcement via PFC, Acb, VTA, and hypothalamus; learning and conditioning via amygdala, hippocampus, DS, and thalamus; and inhibitory control via dlPFC and lateral OFC. Moreover, dysregulation of these processes is thought to maintain the addiction pathology (Volkow, 2010).

Anatomical connectivity between these structures has long been established and more recent work reviewed in Chapter 2 has begun to characterise the functional connectivity between them including BLA-mPFC and hippocampal-mPFC interactions (Fuchs et al.,

2007). Intriguingly, a recently emerging circuit for the extinction of drug seeking suggests that the extinction circuit closely follows a similar corticolimbic-striatal- hypothalamic network of structures. Thus, as reviewed in Chapter 2, this extinction circuit involves, at least in part, cortical (ilPFC), striatal (Acb), and hypothalamic

(MDH) control over drug seeking. The findings from the present thesis add BLA to this circuitry. This overlap between reinstatement and extinction circuitry raises the intriguing possibility that parallel cortico-striatal and/or cortico-striato-hypothalalmic- thalamic pathways play a critical role in initiating as well as inhibiting drug and reward 199

Chapter 5. General Discussion

seeking. The behavioral studies reviewed in Chapter 1 have shown that after extinction training, the memory from original training promoting relapse and the extinction memory promoting abstinence co-exist and compete for control over motivation and behavior (Bouton, 2002; Delamater, 2004; Rescorla, 2001). This competition may be determined by competition between these parallel circuits. Critically, expression of extinction might be achieved at inter-structural junctions involving MDH-inhibition over PVT-dependent reward seeking (Marchant et al., 2010). The present thesis suggests that an AcbSh→LH pathway similarly mediates inhibition over the reinstatement circuitry. Thus, as summarised in Figure 39, there may be parallel circuits mediating extinction via AcbSh→LH and MDH→PVT pathways. A final point is that although an emphasis has been placed throughout this thesis on projections surrounding the Acb, it is important to note that the circuitry of extinction is very likely complex, involving structures with projections that extend beyond those of the Acb.

5.4. Theoretical implications and future directions

It is interesting to consider the findings of the present thesis in relation to the overlap between the neural mechanisms that mediate both drug and appetitive reward-related behaviour. There are several instances of this overlap. For example, food deprivation increases sensitivity to the rewarding effects of LH electrical stimulation (Carr &

Simon, 1984), augments drug self-administration (Comer et al., 1995), and reinstates extinguished drug seeking (Carroll, 1985; Shalev et al., 2000). Moreover, reinstatement of drug seeking depends on the orexin neuropeptide (e.g., Harris et al., 2005; Aston-

Jones et al., 2009). Thus, endogenous orexigenic mechanisms mediate drug seeking.

Conversely, there is considerable overlap between mechanisms of extinction of drug seeking and satiety. AcbSh inactivation reinstates extinguished responding for drug rewards and in the present thesis, it was shown that this reinstatement of responding was 200

Chapter 5. General Discussion

Figure 39. Circuitry mediating context-induced reinstatement and expression of extinction of drug seeking are shown in green arrows (A.) and red arrows (B.), respectively. Note that the LH and PVT are two sites of convergence of the circuits for extinction and reinstatement expression. Depicted circuitry is based on studies using c-

Fos/tract-tracing or contralateral disconnection methods, including data from the present thesis and those reviewed in Chapter 2.

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Chapter 5. General Discussion

LH-dependent. Similarly AcbSh inactivation induces feeding behaviour in satiated rats in an LH-dependent manner (Maldonado-Irizarry et al., 1995; Stratford and Kelley,

1999). At the receptor level, AcbSh AMPA receptors are important for expression of extinction of drug seeking (present findings; Sutton et al., 2003) and are likewise important for satiety (Kelley & Swanson, 1997; Maldonado-Irizarry et al., 1995).

Together, these findings suggest that extinction of drug seeking and satiety share common mechanisms involving AcbSh control over motivated behaviour. Importantly, the common link between extinction of drug seeking and satiety does not appear to be due to the inhibition of an orally consumed reward since the role for AcbSh in extinction is also observed in studies using intravenously administered cocaine (Sutton et al., 2003; Peters et al., 2008). Rather, extinction of drug seeking may be achieved by co-opting an endogenous ventral striatal-hypothalamic circuit mediating satiety. This overlap between extinction and satiety processes is important as it lends itself to a general working hypothesis: that extinction expression might be promoted via anorexigenic manipulations including agonists of satiety-related peptides and antagonists of orexigenic peptides. Conversely, that extinction expression might be disrupted via orexigenic manipulations including antagonists of satiety-related peptides and agonists of orexigenic peptides.

Finally, much remains to be learned about the mechanisms of extinction. The available evidence suggests that mechanisms for extinction are common across different drug reinforcers. Thus, ilPFC has been implicated in expression of extinction in animals trained on cocaine, heroin, and alcoholic beer. Likewise, AcbSh has been implicated in the expression of extinction based on cocaine and alcoholic beer. These findings raise the possibility that there may be common mechanisms for extinction across different drug reinforcers, at least at the level of brain region. Whether these commonalities

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extend to other drug reinforcers and whether they extend to the receptor, intracellular, and molecular mechanisms within these regions remains to be determined. In addition, although it is clear that both LH and PVT are two sites of convergence between circuits controlling expression of extinction versus reinstatement, the precise nature of the interactions between these circuits in LH and PVT is unclear. Moreover, it is not known whether there are other sites of convergence between these circuits. Finally, the research presented here has focussed on the mechanisms for expression of extinction. Little is known of the mechanisms underlying other behavioural components of extinction including initial extinction learning and consolidation of this learning, as well as how they interact with those mediating the expression of extinction.

5.5. Methodological limitations

5.5.1. Use of pharmacological inactivation strategies. The primary method of investigation in the present thesis involved pharmacological manipulation of structural sites. However, a major issue with this strategy is the anatomical specificity of the observed drug effect, since pharmacological agents diffuse away from the injection site.

To address the anatomical specificity of the effects involving AcbSh microinjections, the present thesis (Experiment 1) included an anatomical control involving microinjections that targeted the dorsal AcbSh. This assessed for any effects relating to diffusion of the drug dorsally along the cannula track. However, anatomical controls were not used to assess the possibility that microinjections into the ventromedial AcbSh diffused medially or laterally into adjacent sites including the medial septal nucleus and

AcbC, respectively. It is unlikely that drug diffusion into the AcbC promoted the behavioural effects observed in the present study since in cocaine-seeking rats, neuronal inhibition of the AcbC had no effect on extinguished responding (Peters et al., 2008).

However, the possibility that drug diffusion into the medial septal nucleus impacted on 203

Chapter 5. General Discussion

the observed results cannot be discounted. A related caveat is that embedded within the ventromedial AcbSh are other nuclei, including the Islands of Calleja and rostral extensions of the VP. Thus the present experiments cannot rule out the possibility that the behavioural effects are at least partly related to the effect of drug on these nuclei.

However, due to the size and sporadic spatial distribution of VP and Islands of Calleja at the coronal level of the AcbSh, it is difficult to isolate the functional contribution of these nuclei using pharmacological microinjection techniques. Additionally, as the

AcbSh is crescent-shaped, it is not possible to selectively manipulate the entire AcbSh region in a single microinjection. Thus, the effects reported in the present experiment are specific to the medial and not lateral AcbSh. Finally, it is important to note that the present thesis did not use anatomical controls for microinjections targeting either the

LH or the BLA. However, caudal BLA and LH are anatomically larger structures relative to AcbSh, and diffusion outside of these structures is thought to minimal.

Although anatomical controls directly assess the anatomical specificity of drug effects, this mode of assessment can be supplemented using radioactive or fluorescent ligands to facilitate visualisation of the extent of drug diffusion. In combination with iontophoretic application of the ligand to limit drug diffusion, the anatomical specificity of the drug effect can be inferred with greater confidence. This is especially important for future studies given the anatomically specific recruitment of dorsal and ventral

AcbSh, and rostral and caudal BLA subregions during extinction and context-induced reinstatement of reward seeking (Marchant et al., 2009; McLaughlin & Floresco, 2007).

5.5.2. Use of NBQX as an AMPA receptor antagonist. NBQX is a competitive

AMPA/Kainate receptor antagonist, although it is commonly used as an AMPA receptor antagonist given that it has greater potency at AMPA than Kainate receptor sites. It is also more selective to the AMPA receptor site compared with other

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competitive non-NMDA receptor antagonists (e.g. DNQX, CNQX) (Sheardown et al.,

1990). Nonetheless, the present experiments cannot rule out the possibility that Kainate receptor antagonism may have contributed to the observed effects of NBQX on extinguished responding. To examine more selectively the contribution of AMPA receptors to the extinction of reward seeking, future studies may consider using the compounds, GYKI 52466 or GYKI 53655, both selective allosteric non-competitive

AMPA/Kainate receptor antagonists. These compounds have been reported to have greater selectivity for the AMPA than Kainate receptor (Paternain, Morales, & Lerma,

1995; Wilding & Huettner, 1995). Alternatively, possible contributions of Kainate receptors to the expression of extinction may be assessed using a more selective Kainate receptor antagonist such as NS-102. Finally, NBQX acts broadly on AMPA-type receptors and is unable to discriminate between AMPA receptor subunits. However the functional contributions of these specific subunits may be of interest in understanding reinforcer-dependent mechanisms of extinction given that the GluR1 AMPA receptor subunit has previously been implicated in the extinction of cocaine but not sucrose seeking (Sutton et al., 2003). Future studies may consider subunit-specific pharmacological manipulation of AMPA GluR1 subunits, which is partly possible using the selective polyamine antagonist of Ca2+-permeable AMPA receptors (i.e., GluR2- lacking), 1-naphthylacetyl spermine (Takazawa et al., 1996).

5.5.3. Use of pharmacological disconnection. Finally, in the present thesis, circuit-level contributions of AcbSh→LH and BLA→AcbSh in the expression of extinction was assessed using pharmacological disconnection of these structures.

However, as mentioned previously, it is not possible to infer from this disconnection design whether these contributions were mediated via mono- and or poly-synaptically connected pathways. A direct assessment of whether monosynaptic pathways mediate

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expression of extinction is achievable using optogenetic technology. For example, viral vector-mediated gene transfer of the channelrhodopsin gene targeted at the BLA, driven by the Camk2α promoter, combined with either optical stimulation or inhibition of transduced projection fibres in AcbSh has been used in previous studies to examine a role for BLA→Acb projections in reward seeking (Stuber et al., 2011). A similar strategy may be used to test involvement of a BLA→AcbSh pathway (using a Camk2α promoter) or an AcbSh→ LH pathway (using a tetracycline transactivator (tTA)– tetracycline operator (tetO) promoter strategy) (Chuhma, Tanaka, Hen, & Rayport,

2011) during extinction and reinstatement of reward seeking.

Concluding Remarks

Since the inception of the reinstatement model of drug seeking as an animal model of relapse, progress has been made towards understanding the broadly distributed organisation of the neurobiological systems that promote the return to drug seeking.

One approach towards understanding this organisation is influenced by behavioural theories of reinstatement; namely, that following extinction training, an extinction process actively masks or inhibits drug seeking, and removal of this extinction process

(either by contextual modulation or neurobiological manipulation) reveals or reinstates drug seeking. The organisation of the neurobiological systems that promote control over drug seeking – viz. the extinction system – was the focus of the present thesis.

The AcbSh is anatomically well-positioned to receive reward-related corticolimbic inputs and to modulate reward motivated behaviour via key substrates involved in action generation, arousal, and reward. In the present thesis, I have shown that the AcbSh is an integral substrate for mediating the suppression of drug seeking during extinction. This role appears to be behaviourally specific to the expression of

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extinction and is mediated in part by local AMPA receptors. Moreover, a role for

AcbSh in the expression of extinction involves its inhibitory control over LH-dependent drug seeking. Finally, I have demonstrated that a serial BLA→AcbSh interaction is necessary for the expression of extinguished drug seeking. Together, this thesis implicates a role for a cBLA→AcbSh→LH pathway in the extinction of drug seeking.

Importantly, this pathway suggests that the organisation of the extinction circuitry interfaces with the neurobiological systems involved in the reinstatement of drug seeking at the level of the LH. Finally, the findings from this thesis suggest that the neurobiological systems underlying extinction of drug seeking overlap with the basic organisation of an endogenous circuit involved in satiety.

Vulnerability to relapse is a primary concern in the treatment of substance dependence. This is especially important given the weight of the individual and social problems associated with this psychopathology. Thus, understanding processes such as extinction that have potential to influence relapse circuitry, may yield important targets for the improvement of current relapse interventions.

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