THE EFFECTS OF CHRONIC INTERMITTENT ETHANOL …...impact you have made on me has taught me scores beyond science. All of these lessons will stay with me well beyond my time in graduate

1

THE EFFECTS OF CHRONIC INTERMITTENT ETHANOL EXPOSURE ON

WITHDRAWAL-RELATED BEHAVIORS, DOPAMINE TERMINAL

FUNCTION AND KAPPA OPIOID SYSTEM SENSITIVITY IN THE NUCLEUS

ACCUMBENS CORE

BY

JAMIE HANNAH ROSE

A Doctoral Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Program in Neuroscience

December, 2015

Winston-Salem, North Carolina

Approved By:

Sara R. Jones, PhD, Advisor

T. Jeffrey Martin, PhD, Committee Chair

Rong Chen, PhD

Brian A. McCool, PhD

Carolanne E. Milligan, PhD

Jeffrey L. Weiner, PhD

ii

DEDICATION AND ACKNOWLEDGEMENTS

My family is nothing less than my foundation. Without the unconditional love and

support of my family, I would have never left the comforts and security of home to start

my educational journey and achieve all that I have thus far.

This is for them.

Mom and Dad.

First: thank you, I love you. Second, know that this dedication could never

appropriately convey the gratitude I have for you both. I feel incredibly fortunate to

have parents like you two. You have built a beautiful life, an enviable fruit and

vegetable garden, a model-worthy marriage, a farm’s worth of pets and have raised

three (awesome/fabulous/outgoing/smart/funny/beautiful) kids from the ground-up.

Thank you for developing and maintaining a secure and loving home where learning

and play were equally important. The emphasis you placed on individuality, self–

reliability, persistence and perseverance has been the platform from which I built my

personal and professional portfolios. Thank you for your example, your guidance and

understanding, for your sage advice when I needed it most and for pushing me forward

when I didn’t have the foresight to continue. Further, thank you for your unconditional

support of my decision to continue with my post-post-secondary education. For better

or worse, I am definitely your eternal student, and I promise to have a wing of my

house set-aside for you when I’m all said and done. You two are my very best friends.

Thank you for everything – you mean the world to me! I love you!

iii

Katelyn.

I am so proud to not only call you my sister, but one of my very best friends. Thank

you for being my rock and one of my biggest supporters through graduate school. You

have given me insightful and realistic advice when I found myself in a pickle and

readily became my sounding board when things need to be discussed. I admire and

envy your ability to manage life like a champion – school, work and motherhood, all at

the same time and with a clear head. You have always performed well in work and

school, and on top of that you are a five-star mom. Thank you for being my play dough-

baking partner and my morning Skype call. You are beautiful, inside and out, smart and

incredibly funny. I can’t wait to see what your future holds. I love you!

Tommy.

You are an awesome combination of terrifying and terrific. You’ve gotten hurt

more times than can be counted, and it amazes me how you’ve grown from these

incidences – I’m sorry I couldn’t be around to see more of those changes. Thank you

for being one of the most selflessly devoted, dedicated, loyal and noble people I know.

Everyone knows that you are smart, funny and talented, but your courage and

perseverance through adversity is your most enamoring trait. Thank you for

entertaining all the crazy birthday and Christmas plans I come up with – they would’ve

(literally) never happened without you. You are awesome, underestimated, and capable

of everything you put your mind to. Thank you for everything! I love you!

- - - - - - -

iv

During my time in Wake Forest University’s Graduate School of the Arts and Sciences’

Program in Neuroscience, I have encountered many notable people and outstanding

scientists that have enriched my experience and made my time here successful and

enjoyable, and I want to extend my sincere gratitude to each.

My Mentor, Dr. Sara Jones

You deserve more acknowledgement and thanks than I could ever express. Your

consistent enthusiasm and support of my endeavors have been the driving forces behind

my efforts and studies at Wake Forest. I admire your tireless work ethic and the

dedication you have to your students and craft. With your guidance, I have developed

the depth and dynamic of thought that has provided me with ability to delve into

advanced studies. Thank you for advising my experiments, the countless manuscript

revisions and your thoughtful feedback regarding my data and presentation skills.

Thank you for helping me see the forest through the trees and pointing me in an

appropriate direction when I went off-course. You pushed me to do more and think

deeper than ever before, and I am a better scientist for it. Your example will last beyond

my time in academia. Thank you!

Jones Laboratory members, present and past.

Dr. Anushree Karkhanis

You are a spectacular scientist and one of my dearest friends. Despite being the size

of a gerbil, your witty and sarcastic (but diplomatic) nature, has added an un-matched

amount of levity to graduate school. Without you, the time I spent earning my PhD

v

would have been considerably more stressful and not nearly as entertaining. Thank you

for charging me to think deeper and challenging me to be more concise. Thank you for

reading nearly every word I wrote down, for being the person who knew when I needed

to be kicked around, and for not holding back when I could’ve been better. The work

you have accomplished during your time at Wake Forest so far is astounding – not

many people can claim half of your achievements in double the time. I can not wait to

what your career has in store for you! Thank you for everything!

Drs. Erin S. Calipari, Mark J. Ferris and Jordan T. Yorgason

I would like to thank you three for assisting in my training, particularly during my

early time in the lab. I am incredibly grateful for all I have learned from talking and

working with each of you. Together, you have set the (very, very high) standard in the

Jones Lab. This has challenged incoming students, including myself, and showed them

what needs to be done to be a successful graduate student in the Jones Lab. All three of

you are spectacular investigators, and I am privileged to have had the opportunity to

collaborate with you. Thank you for all you’ve done and continue to do for me, the

Jones Lab and the scientific community at large.

James Melchior and Drs. Steve Fordahl, and Cody Siciliano

You guys are awesome members of the Jones Lab team. Thank you for the fun,

sarcastic and intelligent conversations we shared. Your willingness to assist me in

anything I needed – from thinking through problems to helping me with experiments –

was greatly appreciated. Your impressive publication records and positive contributions

vi

to the lab environment are continuously moving the Jones Lab forward at an impressive

rate. You are all fantastic scientists, deep and thorough thinkers and really great guys. I

will forever be grateful for your unconditional support and friendship throughout all of

my time here at Wake. Thank you!

Joanne Konstantopoulos and Jason Locke

The life-line of the lab. You two are completely irreplaceable. There is considerably

more on your daily task list than any one of us could imagine, and you handle it all with

a cool head. Thank you for keeping everything under-wraps - the lab would’ve

crumbled without you a long time ago! Thank you for your excellent technical

contributions to the laboratory and your assistance in my own training. Your patience

and expertise has helped me and the other students on a daily basis. You two have

taught me so much, and even after I’ve learned, you have saved me more times than I

can remember. Thank you for everything!

My Dissertation Committee: Drs. T. Jeff Martin, Sara Jones, Rong Chen,

Brian McCool, Carol Milligan and Jeff Weiner

In a nutshell, thank you for your constant and enthusiastic support of my scientific

endeavors and career goals. Thank you for the time you took out of your busy

schedules to attend my committee meetings and for meeting with me one-on-one. Our

enlightening discussions and your willingness to help me think through my scientific

misunderstandings were incredibly helpful and are sincerely appreciated. Furthermore,

thank you for all of the recommendation letters and collaborations. Most of all, thank

vii

you for believing in me. You all are not only spectacular scientists, but also really

fantastic people. I understand that my trajectory through graduate school was slightly

unorthodox, and I do not take for granted what you have done to help me along. The

impact you have made on me has taught me scores beyond science. All of these lessons

will stay with me well beyond my time in graduate school. Thank you!

Friends.

I consider myself incredibly blessed to have such honest and loyal friends. You

helped me stay sane over the long months of late nights and helped me take a break

when I really needed one. Although you may not have understood what I was doing,

you supported me nonetheless. Thank you for providing me with non-scientific

conversations, an escape from the isolation of dissertation writing and the stress of

deadlines. Thank you for your countless encouraging and comical text messages,

endless hours of conversation, your interest in, and support of, my graduate work and

(occasionally) your editing skills. No matter where we each end up in life, industry,

academia, professional dog walker or interpretive dancer, next-door neighbors or

friends on opposite coasts, whether we talk every day or completely lose touch, I hope

you know that each of you mean more to me than I can express. Thank you for

everything. My home is – and always will be – yours.

- - - - - -

viii

TABLE OF CONTENTS

LIST OF FIGURES….…………………………….…………………..………………x

LIST OF ABBREVIATIONS..…………………………………………………......xiv

ABSTRACT…………..……………………………………………………………….xvi

PREFACE…………..………………………………………………………………..xviii

CHAPTER I

THE MODULATORY ROLE OF ETHANOL EXPOSURE ON AFFECT, THE

NEUROCIRCUITRYOF BEHAVIOR, DOPAMINE TERMINAL AND KAPPA

OPIOID RECEPTOR-SYSTEM FUNCTION……………..…………....…………..….1

CHAPTER II:

SUPERSENSITIVE KAPPA OPIOID RECEPTORS PROMOTE ETHANOL

WITHDRAWAL-RELATED BEHAVIORS AND REDUCE

DOPAMINE SIGNALING IN THE NUCLEUS ACCUMBENS..…...........................68

CHAPTER III:

FUNCTIONAL INTERACTION BETWEEN DOPAMINE D3 AND KAPPA

OPIOID RECEPTORS ON DOPAMINE TERMINALS AND PARALLEL

INCREASES IN RECEPTOR SENSITIVITY FOLLOWING

CHRONIC ETHANOL EXPOSURE.………………………..…………………..…..113

CHAPTER IV:

DISTINCT EFFECTS OF NALMEFENE ON DOPAMINE TERMINAL

FUNCTION AND KAPPA OPIOID RECEPTOR ACTIVITY IN THE

NUCLEUS ACCUMBENS FOLLOWING CHRONIC INTERMITTENT

ETHANOL EXPOSURE….…………………………………...…………………......143

CHAPTER V

DISCUSSION..……………………………………………...……………………..…171

ix

APPENDIX I:

GREATER ETHANOL-INDUCED LOCOMOTOR ACTIVATION IN

DBA/2J VERSUS C57BL/6 MICE IS NOT PREDICTED BY

PRESYNAPTIC STRIATAL DOPAMINE DYNAMICS……………….………...207

APPENDIX II:

DEVELOPMENT OF THE CURRENT CHRONIC INTERMITTENT

ETHANOL EXPOSURE PROCEDURE…………………………………………..233

APPENDIX III:

GENERAL METHODOLOGY…………………………………………………….239

CHAPTER VI

CURRICULUM VITAE………………………………...…………………………247

x

LIST OF FIGURES

CHAPTER I

1 A visual representation of the neuronal connections

within the nucleus accumbens core…..……………………………6

2 Direct and indirect motor pathways through the basal ganglia……8

3 The role of opioid receptors in the opponent process theory

of motivation and repeated cycles of ethanol exposure and

withdrawal.…………………………………………..…………...16

4 Downstream signaling of KORs…………………...…………….19

5 Ethanol-induced increases in KOR and D3R function

may be due to alterations in RACK1and BDNF/TrkB

systems…………………………………………….………….…30

CHAPTER II

1 Ethanol exposure protocols…………………………………...…75

2 Chronic intermittent ethanol-exposed mice demonstrated

increased ethanol consumption and preference………………….80

3 Systemic norBNI administration reduced ethanol intake

and preference CIE-treated mice which was replicated with

KOR activation.………………………………..………….……..83

xi

4 CIE exposure–induced increases in marble burying

behavior are reduced with KOR blockade and replicated

with KOR agonist administration……..…………………………85

5 CIE exposure attenuated accumbal dopamine terminal

Function and increased KOR sensitivity…….…………………...88

S1 CIE increased ethanol intake and preference with

inter-cycle ethanol drinking……………………………….……107

S2 Blood ethanol concentration for back-to-back protocol………..109

S3 Post- norBNI drinking was stable over the five day test……….111

S4 NorBNI does not change locomotor activity…………….….….112

CHAPTER III

1 Increased accumbal D2R/D3R sensitivity in CIE-exposed

mice was driven by D3Rs.………….………………………….123

2 Augmented KOR function in the NAc of CIE-exposed

mice compared to air-exposed controls.…………………..……125

3 D2R/D3R blockade attenuated the dopamine-decreasing

effects of KOR activation ...…………………………………....127

4 D3 blockade reduced the dopamine-decreasing effects

of KOR activation …………………………………….………..129

xii

CHAPTER IV

1 Chronic intermittent ethanol-exposure reduced dopamine

transmission in the nucleus accumbens core…………………...151

2 Nalmefene slowed the uptake rates more in brain slices

from chronic intermittent ethanol-exposed mice……………….152

3 Nalmefene reversed the dopamine-decreasing effects of

U50,488 in chronic intermittent ethanol-exposed mice………...155

CHAPTER V

1 Direct and indirect motor pathways are altered following

chronic ethanol exposure………….……………………………176

2 Effects of chronic ethanol exposure on dopamine

terminal function……………………………………………..…179

APPENDIX I

1 DBA mice exhibited enhanced ethanol-induced

locomotor responses…………………………………………….219

2 DBA mice exhibited reduced responses to a novel

environment…………………………………………………….220

3 DBA and C57 mice exhibited similar ethanol elimination

time courses…………………………………………………….221

4 Dopamine release and clearance in the nucleus accumbens

core of DBA and C57 mice………………………………….….223

xiii

5 Dopamine release and clearance in the caudate-putamen

of DBA and C57 mice………………………………………….226

6 The effects of ethanol on dopamine terminals in the NAc core

and CPu were similar between strains…………………………228

APPENDIX III

1 Ethanol vapor exposure maintains elevated BECs…………..…245

xiv

LIST OF ABBREVIATIONS

BDNF – Brain derived neurotrophic factor

BEC – Blood ethanol concentration

cAMP - Cyclic adenosine monophosphate

CIE/air – Chronic intermittent ethanol/air (exposure)

CPP – Conditioned place preference

CRE - cAMP response element

CREB - cAMP response element-binding protein

D1R – Dopamine D1 receptor



DOR – Delta opioid receptor

DSM-4 – Diagonistic and Statistical Manual, version 4

DSM-5 – Diagonistic and Statistical Manual, version 5

GABA - Gamma amino butyric acid

GPCR – G-protein coupled receptor

GRK – G-protein coupled receptor kinases

GTPγS - guanosine 5'-O-[gamma-thio]triphosphate (assay)

ICSS - Intracranial self-stimulation

JNK - c-Jun N-terminal kinase

KOR – Kappa opioid receptor

MAPK - Mitogen-activated protein kinase

MOR – Mu opioid receptor

xv

norBNI – Nor-binaltorphimine

PFC – Prefrontal cortex

PKA – Protein kinase A

PKB - Protein kinase B

RACK1 - Receptor for activated C kinase 1 scaffolding protein

SAMHSA- The Substance Abuse and Mental Health Services Administration

TrkB - Tropomyosin-related kinase B

VTA- Ventral tegmental area

xvi

ABSTRACT

THE EFFECTS OF CHRONIC INTERMITTENT ETHANOL EXPOSURE ON

WITHDRAWAL-RELATED BEHAVIORS, DOPAMINE TERMINAL FUNCTION

AND KAPPA OPIOID SYSTEM SENSITIVITY IN THE NUCLEUS ACCUMBENS

CORE

Jamie H. Rose

Dissertation under the direction of Sara R. Jones, Ph.D., Tenured Professor of

Physiology and Pharmacology

xvii

Alcohol use disorders are a rampant economic and health concern in the United

States. Chronic alcohol use and abstinence is often followed by relapse to ethanol

drinking occurs in response to the withdrawal symptoms of the drug. However, the

neurobiological underpinnings of this disorder are not well understood. Previous work

has shown that chronic ethanol exposure and withdrawal downregulates dopamine

transmission in the nucleus accumbens (NAc). Reduced dopamine terminal function may

be due to upregulated presynaptic receptor proteins such as kappa opioid receptors

(KOR) and dopamine D2/D3 autoreceptors (D2R/D3R), and changes in receptor-system

function may, in part, drive symptoms of ethanol withdrawal and subsequent relapse

drinking behavior. To this end, the present body of work identified some of the effects of

five weeks of chronic intermittent ethanol (CIE) exposure on ethanol withdrawal

behavioral phenotypes, including ethanol drinking parameters and anxiety/compulsive-

like behavior, as well as dopamine transmission and presynaptic receptor function in the

NAc of C57BL/6J mice. We found that CIE increased ethanol drinking and

anxiety/compulsive-like marble burying compared to air-exposed mice, phenotypes that

were reduced by KOR blockade and replicated with KOR activation. Neurobiological

examination of accumbal dopamine transmission with fast scan cyclic voltammetry in

brain slices showed reduced dopamine release, augmented uptake and increased

sensitivity of KORs and D3Rs following five weeks of CIE exposure. A subsequent

examination of interactions between KORs and dopamine autoreceptors showed that

D3Rs and KORs are functionally linked. Finally, the effects nalmefene, a high-affinity

KOR partial agonist used in some European countries to treat alcohol use disorders, on

dopamine terminal and KOR function were examined in efforts to understand its clinical

xviii

efficacy. Nalmefene reduced dopamine uptake rates and reversed the dopamine-

decreasing effects of KOR activation selectively in brain slices from CIE-exposed mice.

These effects are credited to the increased affinity and distinct effects of this compound

on KORs. We hope that this work supports the development of KOR-specific lignads to

reduce ethanol withdrawal symptoms and relapse drinking. A discussion of the present

data, future directions, clinical implications of this work and experimental caveats are

considered.

xix

PREFACE

The following dissertation contains work examining the functional consequenses of

chronic intermittent ethanol exposure on ethanol drinking and anxiety/compulsive

behavior, kappa opioid and autoreceptor responsivity to agonist, as well as a functional

interaction between receptor systems, and the effects of nalmefene on dopamine terminal

and kappa opioid receptor function. This document was prepared in partial fulfillment of

the requirements for the degree of doctor of philosophy in biomedical science at Wake

Forest University Graduate School of Arts and Sciences. Some of this work has been, or

will be, submitted for publication, and has been reformatted per the Graduate School

requirements for thesis preparation; however, journal requirements dictate small stylistic

variations between chapters.

1

CHAPTER I

THE MODULATORY ROLE OF ETHANOL EXPOSURE ON AFFECT, THE

NEUROCIRCUITRYOF BEHAVIOR, ACCUMBAL DOPAMINE TRANSMISSION

AND KAPPA OPIOID SYSTEM FUNCTION

Jamie H. Rose

2

1.0. Opening

Alcohol use disorders are a major economic and financial burden in the United States,

affecting approximately 18 million Americans and costing taxpayers over $223.5 billion

annually (Centers for Disease Control and Prevention, 2014; Substance Abuse and

Mental Health Services Administration, 2014). The magnitude of this financial burden is

linked to healthcare expenses, law enforcement, criminal justice and incarceration costs,

motor vehicle accidents, and lost work productivity (Centers for Disease Control and

Prevention, 2014). Despite being a significant public health issue, only three

pharmacotherapies are currently approved by the United States’ Food and Drug

Administration (FDA) for alcohol use disorders: acamprosate, disulfiram and naltrexone

(Pettinati & Rabinowitz, 2006). Regrettably, none of these drugs are completely

efficacious in reducing relapse to alcohol drinking after a period of abstinence (Latt et al.,

2002; Mann et al., 2004; Jørgensen et al., 2011; Koob, 2013; Yoshimura et al., 2014;

Higuchi et al., 2015). In fact, resumption of ethanol drinking in alcoholics following

treatment is as high as 86% (Moos & Moos, 2006); thus, there is clinical and scientific

consensus over the need for novel pharmacotherapies and receptor targets that confer

greater abstinence from alcohol use in this population. The present document reviews the

current literature regarding alcohol use disorders in humans and animal models and

provides an investigation in to a promising pharmacotherapeutic target for this disease.

1.1. Alcohol use disorders in the United States: Diagnosis and treatment

The development of an alcohol use disorder is invariably preceded by recreational

alcohol intake that is characteristically driven by the positive, euphoric effects of the drug

3

(Avery et al., 1982; van Hemel-Ruiter et al., 2015). Alcohol users with an inclination for

substance abuse disorders, due to a combination of genetic and environmental factors

(Bierut et al., 1998; Chassin et al., 2004; Cservenka et al., 2015), transition from

consuming alcohol for its positive reinforcing effects to drinking in an attempt to relieve

the symptoms of withdrawal (Rousseau et al., 2011; Cohn et al., 2012; Kiselica &

Borders, 2013), a hallmark of alcohol use disorders. Contemporary diagnosis of an

alcohol use disorder is based on criteria described in the recently published version of the

Diagnostic and Statistical Manual of Mental Disorders, Version 5 (DSM-5; American

Psychological Association, 2013). Unlike the previous edition, the DSM-4 (American

Psychological Association, 2000), alcohol use disorders are now diagnosed based on a

continuum of criteria (American Psychological Association, 2013), and the distinction

between “alcohol abuse” and “alcohol dependence” no longer exists (American

Psychological Association, 2000, 2013). To be diagnosed as having an alcohol use

disorder per the DSM-5 (American Psychological Association, 2013), patients must meet

between two and six (or more) out of eleven descriptors that identify specific elements of

a mild, moderate or severe alcohol use disorder. These criteria include, but are not limited

to: drinking in spite of deleterious consequences, increasing consumption over time (due

to tolerance to the physicological and psychological effects of ethanol), loss of

friendships, romantic and familial relationships, as well as physical or psychological

withdrawal symptoms and relapse drinking behavior (American Psychological

Association, 2013).

Clinical diagnosis of an alcohol use disorder is often followed by psychological and,

or pharmacological therapy. For example, alcoholics may participate in cognitive and/or

4

behavioral therapy with a trained therapist or group-based 12-step program such as

Alcoholics Anonymous (Alcoholics Anonymous World Services, Inc, 2001), in

conjunction with pharmacological treatment. The pharmacotherapies currently approved

by the United States’ FDA are disulfiram (Antabuse®), acamprosate (Campral®) and

naltrexone (Revia®). These compounds will be reviewed in-depth later in this document

(Section 6.0). Unfortunately, decades of evidence show that abstinent alcoholics relapse

multiple times prior to complete abstinence, even when consistently treated with one or

both methods of intervention (Ballenger & Post, 1978; Lechtenberg & Worner, 1990,

1992; Worner, 1996). These clinical observations have prompted a number of laboratory

models of alcohol use disorders in monkeys and rodents that replicate alcohol exposure

and withdrawal symptoms per the human condition which will be discussed later in this

document (Appendix II).

Withdrawal symptoms underlie relapse-drinking behavior after a period of chronic,

heavy alcohol consumption and abstinence (Zywiak et al., 1996; Wetterling et al., 2006;

American Psychological Association, 2000, 2013; Meyerhoff et al., 2013). These

symptoms include augmented feelings of anxiety (Wetterling & Junghanns, 2000),

compulsive behaviors (Suzuki et al., 2002), sleep disturbances and sleeplessness (Perney

et al., 2012; de Zambotti et al., 2014), depression (Johnson & Perry, 1986; Schoonover et

al., 2015), dysphoria (Hayne & Louks, 1991; Locke & Newcomb, 2003), and in severe

cases, seizures and delirium tremens (Eyer et al., 2011; Kim et al., 2015). Alleviation of

the ethanol withdrawal symptoms by ethanol intake negatively reinforces continued

drinking behavior; thus, relief of these symptoms with innovative pharmacotherapies and

identification of novel targets is of clinical and scientific interest.

5

2.0. The nucleus accumbens core

The region of interest for the upcoming studies is the nucleus accumbens core, a

ventral portion of the striatum that is highly involved in establishing the reinforcing

effects of drugs of abuse (Alderson et al., 2001; Gill et al., 2010; du Hoffman & Nicola,

2014) and ethanol (Bull et al., 2014; Pina & Cunningham, 2014; Young et al., 2014). The

primary neurotransmitter in this region is the catecholamine, dopamine. Dopaminergic

transmission in this region arises primarily from ventral tegmental area (VTA) dopamine

neurons, and a smaller contribution of dopamine projections from the substantia nigra

pars compacta (Benivoglio et al., 1979; Phillips et al., 1992; Forster & Blaha, 2000;

Owesson-White et al., 2012). VTA-area dopamine cells display tonic firing patterns that

provide dopamine tone in the nucleus accumbens that is present in awake animals (Grace

& Bunney, 1983). Phasic or burst firing (Grace & Bunney, 1983) or electrical stimulation

of VTA dopamine neurons causes a time-locked increase in dopamine levels in the

nucleus accumbens, while ablation of VTA-area dopamine neurons reduces dopamine

release in this region (Phillips et al., 1992; Forster & Blaha, 2000; Owesson-White et al.,

2012).

The nucleus accumbens core is a region of intense and diverse neurotransmitter

activity. Figure 1 depicts a representation of accumbal neurocircuitry. In addition to

dopamine projections from the VTA, other inputs include norepinephrine from the locus

coeruleus (Berridge et al., 1997), serotonin from the dorsal raphe nucleus (Yoshimoto &

McBride, 1992) and glutamatergic inputs from the prefrontal cortex (Del Arco & Mora,

6

Figure 1: A visual representation of the neuronal connections in the nucleus

accumbens core.

The nucleus accumbens is a highly heterogenous and complex region. Red terminal

boutons: dopaminergic inputs from the ventral tegmental area (VTA). Pink, blue and

purple terminal boutons: glutamatergic afferents from the amygdala, prefrontal cortex

(PFC) and hippocampus, respectively. Orange terminal bouton: serotonergic projection

neuron from the dorsal raphe nucleus (DR). Periwinkle terminal bouton: noradrenergic

input from the locus coeruleus (LC). Maroon interneurons: GABAergic. Orange

interneurons: acetylcholinergic. Green neurons: D1/D2-containing (unspecified)

GABAergic medium spiny projection neurons.

FROMVTA

GABAergicinterneurons

Acetylcholineinterneurons

Mediumspinyneurons

Dopamineterminals

FROMHIPPOCAMPUS

FROMPFC

FROMAMYGDALA

Glutamatergicafferents

TODIRECT/INDIRECTPATHWAYS

FROMLC

FROMDR

Serotonergicafferents

Noradrenergicafferents

7

2008), hippocampus (Blaha et al., 1997) and amygdala (Floresco et al., 1998). All

afferents converge on to dopamine terminals, cholinergic (Bluth et al., 1985) and gamma

amino butyric acid (GABA)-ergic (Tepper & Bolam, 2004) interneurons and GABAergic

medium spiny projection neurons (Huang et al., 2008; Lobo & Nestler, 2011; MacAskill

et al., 2012; Gangarossa et al., 2013; Grueter et al., 2013).

Medium spiny neurons, the primary output neurons of the nucleus accumbens

(Huang et al., 2008; Lobo & Nestler, 2011; MacAskill et al., 2012; Gangarossa et al.,

2013; Grueter et al., 2013) comprise over 95% of the accumbal milieu (O’Donnell &

Grace, 1993). These neurons are classified as either D2 receptor (D2R)- or D1 (D1R)-

containing (Surmeier et al., 1996; Cepeda et al., 2008) which activate distinct signaling

pathways. In fact, D2-containing medium spiny neurons project to the globus pallidus

externa, through the striatopallidal indirect pathway, while D1-containing medium spiny

neurons provide a feedback signal to the VTA and mediate neurotransmission by the

striatonigral direct pathway (Figure 2; Surmeier et al., 1996; Cepeda et al., 2008;

Perreault et al., 2013). In sum, dopaminergic projections from the ventral midbrain

converge in the accumbal milieu (Figure 1), mediating the downstream signaling of

GABAergic medium spiny neurons. D1-containing medium spiny neurons project to non-

dopaminergic neurons in the substantia nigra pars reticulata while D2-containing medium

spiny neurons project through the globus pallidus externa. Inhibitory projections from the

globus pallidus externa extend to the subthalamic nucleus and substantia nigra pars

reticulata. The subthalamic nucleus sends glutamatergic projections to the substantia

nigra pars reticulata. The substantia nigra pars reticulata receives projections from the

direct and indirect pathways, sending a net GABAergic signal to the thalamus. Excitatory

8

Figure 2: Direct and indirect motor pathways through the basal ganglia.

Dopaminergic projections (red arrows) from the ventral midbrain (substantia nigra

pars compacta and ventral tegmental area) converge in the accumbal milieu (Figure 1),

mediating the signaling of post-synaptically located GABAergic medium spiny neurons.

Medium spiny neurons are classified based on the presence of excitatory D1 (+, yellow

circle) or inhibitory D2 (-, purple circle) receptors. The GABAergic medium spiny

neurons project through distinct motor pathways through the basal ganglia and release

specific opioids. D1-containing medium spiny neurons project to non-dopaminergic

neurons in the substantia nigra pars reticulata whereas D2-containing medium spiny

neurons project through the globus pallidus externa. In this pathway. inhibitory

projections from the globus pallidus externa extend to the subthalamic nucleus and

substantia nigra pars reticulata. The subthalamic nucleus sends excitatory (glutamatergic)

projections to the substantia nigra pars reticulata. The substantia nigra pars reticulata

receives projections from the direct and indirect pathways, sending a GABAergic signal

to the thalamus. Excitatory signals from the thalamus are sent to the nucleus accumbens

and cortex. Glutamatergic signaling from the cortex extends back to the striatum.

9

signals from the thalamus are sent to the nucleus accumbens and cortex, and

glutamatergic signaling from the cortex extends back to the striatum. Recent work,

however, suggests that the direct and indirect pathways through the basal ganglia may not

be as clear as traditionally considered (Kupchik et al., 2015).

Notably, D1R- and D2R-expressing medium spiny neurons release distinct opioid

peptides (Shuster et al., 2000; Rutter & Tsuboi, 2004). Specifically, D2-containing

medium spiny neurons release enkephalin (Surmeier et al., 1996) which activate

postsynaptically located delta opioid receptors (DOR; Dilts & Kalivas, 1990), while D1-

containing medium spiny neurons release dynorphin (Curran & Watson, 1995) which

activates presynaptic kappa opioid receptors (KOR) and inhibit dopamine transmission

(Spanagel et al., 1990, 1992). See Section 4.2 for more information regarding

dynorphins.

2.1. The effects of ethanol exposure on mesolimbic dopamine function

Dopamine transmission in the nucleus accumbens drives reward-learning behavior,

including those associated with the rewarding effects of ethanol. For example, dopamine

receptor blockade in the nucleus accumbens disrupts the acquisition or expression ethanol

conditioned place preference (Young et al., 2014), a measure of ethanol reward.

Furthermore, acute ethanol reduces intra-cranial self-stimulation (ICSS) brain reward

thresholds (Boutros et al., 2014), suggesting that ethanol augments dopamine

transmission in the mesolimbic dopamine system. Voluntary ethanol consumption in non-

ethanol dependent rats (Melendez et al., 2002), and experimenter-administered ethanol in

non-dependent humans (Aalto et al., 2015) induces dopamine release that is time-locked

to ethanol exposure and not present with sucrose consumption, indicating a direct

10

pharmacological effect of ethanol on dopamine transmission, and points to this effect as a

driver of continued ethanol consumption. Although the positive reinforcing effects of

ethanol are mediated by accumbal dopamine transmission, chronic ethanol exposure

produces adaptations in this system that fundamentally reduces its function (Volkow et

al., 1992, 2006; Carroll et al., 2006; Budygin et al., 2007; Karkhanis et al., 2015). The

effects of chronic ethanol exposure on dopamine terminal and presynaptic receptor

function are of particular points of interest to the present body of work and will be

discussed in Section 2.2.

Acute ethanol augments dopamine transmission through multiple mechanisms. For

example, ethanol increases the hyperpolarization-activated cation current (Ih) of VTA

dopamine neurons, thereby augmenting the classic pacemaker-firing rate of these cells

and dopamine release in projection areas (Okamoto et al., 2006; Tateno & Robinson,

2011; Chen et al., 2012). Additionally, ethanol reduces GABA interneuron firing in the

VTA, an effect that disinhibits dopamine cell body firing and increases in dopamine

release in projection regions (Chefer et al., 2009; Niikura et al., 2010; Jalabert et al.,

2011; Margolis et al., 2012).

The effects of ethanol on dopamine transmission in vivo appear to be biphasic. For

example, systemic ethanol administration up to 2.0g/kg increased dopamine neuron

firing, while doses at and above 4.0g/kg caused a marked reduction in dopamine neuron

firing rate (Mereu et al., 1984). A loss of dopamine neuron firing at high doses of the

drug may explain why voluntary ethanol consumption in non-dependent animals is rarely

above 2.0g/kg (Becker & Lopez, 2004; Lopez & Becker, 2005; Griffin et al., 2009).

Ehanol intake up to 2.0g/kg produces pharmacologically relevant brain concentrations of

11

the drug. For example, systemic administration of 2.0g/kg ethanol augmented striatal

levels of ethanol to 10mM (Tang et al., 2003) while a 3.0g/kg of ethanol consumed by

mice provide a brain level of ~12mM ethanol (Griffin et al., 2007). Notably, the

dopamine-releasing properties of ethanol do not appear to directly modify dopamine

terminal function (to be discussed below). Although physiologically relevant to an intact

animal, brain concentrations of ethanol in these studies are lower than the concentrations

used in ex vivo preparations when dopamine cell bodies are severed from terminal

regions (20-200mM; Budygin et al., 2001, 2005; Rose et al., 2013; Yorgason et al.,

2014). In fact, ethanol concentration-dependently reduced dopamine release in brain

slices containing the nucleus accumbens at 100-200mM of the drug, while having no

effect on dopamine release or uptake at low concentrations (Budygin et al., 2001, 2005;

Rose et al., 2013; Yorgason et al., 2014). These data suggest that the excitatory effects of

ethanol on dopamine signaling are limited to modulation of dopamine neuron firing

(Mereu et al., 1984; Gessa et al., 1985; Brodie & Appel, 2000) rather than dopamine

terminals (Budygin et al., 2001, 2005; Rose et al., 2013; Yorgason et al., 2014).

2.2. Chronic ethanol exposure reduces dopamine terminal function

Chronic ethanol exposure and withdrawal reduces dopamine system function, an

effect that may (Shen & Chiodo, 1993; Shen et al., 2007) or may not (Diana et al., 1995;

Brodie, 2002) be due to attenuated dopamine cell firing of ventral tegmental area-located

dopamine cell bodies or deficient terminal function (Carroll et al., 2006; Budygin et al.,

2007; Karkhanis et al., 2015). In fact, recent work from our laboratory has shown

augmented dopamine uptake and reduced dopamine release in mice (Karkhanis et al.,

2015) and rats (Budygin et al., 2007), and positron emission tomography scanning of

12

human alcoholics shows reduced dopamine receptor binding in the basal ganglia

(Volkow et al., 1996, 2002). Despite these consistent cross-species findings (Hietala et

al., 1994; Laine et al., 1999; Budygin et al., 2007; Volkow et al., 1996, 2002; Karkhanis

et al., 2015), the precise mechanism(s) underlying ethanol-induced changes in dopamine

transmission are not well understood.

Of particular interest are chronic ethanol-induced changes in kappa opioid receptor

(KOR) and dopamine D2 (D2R) and D3 (D3R) autoreceptor function. These receptors

may be presynaptic release-inhibiting receptors (Spanagel et al., 1990, 1992; Maina &

Mathews, 2010) that are hypersensitive following chronic ethanol, and chronic

intermittent ethanol (CIE) exposure (Perra et al., 2011; Kissler et al., 2014; Kivell et al.,

2014; Karkhanis et al., 2015; Siciliano et al., 2015). These data suggest that D2Rs and

D3Rs and KORs may be driving chronic ethanol-induced reductions in dopamine

transmission.

3.0. The opioid system and ethanol

The three classical opioid receptors: mu (MOR), DOR and KOR are Gαi/o-coupled G-

protein coupled receptors (GPCR) with specific endogenous ligands: endorphins,

enkephalins and dynorphins, respectively (Kosterlitz & Paterson, 1980; Chavkin et al.,

1982; Raynor et al., 1994; Le Merrer et al., 2009). All ligands share limited affinity for

non-primary opioid receptor due to a conserved enkephalin pentapeptide sequence

(tyrosine-glycine-glycine-phenylalanine-methionine/leucine) on the amino terminus of

each endogenous neuropeptide (Kosterlitz & Paterson, 1980; Chavkin et al., 1982;

Raynor et al., 1994). The opioid receptors and their ligands are located ubiquitously

throughout the brain to modulate transmission of various neurotransmitters (Werling et

13

al., 1987; Hjelmstad & Fields, 2003; Zhang & Pan, 2012; Le Merrer et al., 2009; Pu et

al., 2012; Ponterio et al., 2013; Al-Hasani et al., 2013), including dopamine (Spanagel et

al., 1992; Chefer et al., 2005; Hipólito et al., 2008). Notably, the anatomical positions of

MORs within the VTA and KORs on nucleus accumbens dopamine terminals modulate

dopamine transmission in opposition, whereby activation of MORs and KORs positively

and negatively modulate dopamine transmission, respectively (Spanagel et al., 1992;

Devine et al., 1993; Mitrovic & Napie, 2002).

Based on their ability to modulate dopamine release, MORs and KORs may

contribute to some of the positive, euphoric and negative withdrawal symptoms

associated with abstinence following chronic ethanol exposure. For example, acute

ethanol activates MORs on GABAergic interneurons in the VTA, disinhibiting dopamine

neurons in this area, thereby increasing dopamine release in terminal fields (Chefer et al.,

2009; Guan & Ye, 2010; Niikura et al., 2010; Jalabert et al., 2011; Margolis et al., 2012;

Tseng et al., 2013) and in part, driving positive hedonic states (Figure 3, green section;

Méndez et al., 2001; Hipólito et al., 2011). Conversely, activation of KORs on a

subpopulation of dopamine cell bodies in the VTA reduces dopamine transmission in the

prefrontal cortex (Margolis et al., 2006), and of interest to the present body of work, local

activation of KORs in the nucleus accumbens attenuates dopamine transmission

specifically in this region (Spanagel et al., 1992, Chefer et al., 2005; Niikura et al.,

2010). Notably, these effects may engender negative affective states, particularly those

experienced following ethanol exposure, during withdrawal states (Figure 3, red section).

To separate the positive and withdrawal effects of ethanol into signaling by MORs

and KORs, respectively, would be an oversimplification of the system. Rather, the sum

14

effects of ethanol exposure are due to a balance of multiple neurotransmitter and

neuromodulator systems, including MORs and KORs, and not due to increased function

of only one of these receptors. In fact, it has been widely accepted that chronic ethanol

exposure has distinct effects on multiple neurotransmitter and neuromodulator systems,

including corticotropin releasing factor and neuropeptide Y (Thorsell et al., 2005; Criado

et al., 2011), serotonin (Esteban et al., 2002; Smith et al., 2008), glutamate (Christian et

al., 2012; Griffin et al., 2014, 2015), GABA (Mhatre & Ticku, 1994; Kumar et al., 2002)

and norepinephrine (Smith et al., 2008; Karkhanis et al., 2015b), all of which likely

influence ethanol withdrawal symptoms. With respect to KORs, specifically, the

rewarding effects of ethanol can be obliterated with KOR activation (Logrip et al., 2009)

and prodynorphin knock-out mice and mice treated with a KOR antagonist show higher

levels of ethanol-conditioned place preference than wild-type and non-treated mice

(Nguyen et al., 2012). Moreover, acute ethanol not only induces dopamine release

(Chefer et al., 2009; Guan & Ye, 2010; Niikura et al., 2010; Jalabert et al., 2011;

Margolis et al., 2012; Tseng et al., 2013), but also increases dynorphin release and, by

extension, causes KOR activation (Lindholm et al., 2000; Marinelli et al., 2006). These

data suggest that the KOR system may be involved in dampening the rewarding and

reinforcing effects of ethanol on an acute timescale. Despite this complexity, the

separation of the effects of ethanol into changes in MOR and KOR signaling systems in

the present body of work is for example purposes only.

15

3.1. A theoretical framework for the development of alcohol use disorders as

diseases of chronic relapse

The chronic relapsing pattern of alcohol use disorders is driven by both positive and

negative reinforcement (Koob & Le Moal, 1997, 2008), a pattern that can be described

with the opponent process theory of motivation (Figure 3, Solomon & Corbit, 1973,

1974). The opponent process theory posits that psychological challenges and drugs of

abuse would alter the hedonic state of the subject in a predictable manner. Essentially, the

positive affect associated with exposure to drugs of abuse would be countered with

compensatory withdrawal symptoms and negative affect (Figure 3; Solomon & Corbit,

1973; Koob & Le Moal, 1997, 2008). The affective state of this subject would return to

its normal, homeostatic set-point (Figure 3, solid black line) following drug exposure and

remain stable until a subsequent drug challenge (Figure 3, yellow arrow). With chronic,

repeated drug use and withdrawal, this pattern of affect trends downward: the positive

hedonic state associated with drug exposure is attenuated and followed by a significant

increase in the withdrawal symptoms (Figure 3; Koob & Le Moal, 1997, 2008). Further,

the drug abusers’ normal affective state in between drug uses is also reduced, whereby

normal processes do not return to the homeostasitic set-point, rather they settle at a new,

more negative hedonic state known as an “allostatic set-point” (Figure 3, dotted lines;

Solomon & Corbit, 1973; Koob & Le Moal., 2008).

Notably, this pattern is strikingly similar to dopamine system modulation by opioid

receptors following acute and chronic ethanol exposure and withdrawal. In fact, chronic

ethanol exposure reduces MOR activity (Turchan et al., 1999; Chen & Lawrence, 2000;

Saland et al., 2005) and increases KOR/dynorphin system activity (Figure 3, red section;

16

Figure 3: The role of opioid receptors in the opponent process theory of motivation

and repeated cycles of ethanol exposure and withdrawal.

Acute ethanol exposure augments affect (following first yellow arrows), in part due to

increased MOR signaling. After acute ethanol exposure, affect returns to the normal

homeostatic set-point (solid black line) until another there is a subsequent ethanol

challenge (second yellow arrow). After repeated heavy exposure to ethanol, the euphoric

effects of the compound, and associated MOR signaling, is reduced (following second

yellow arrow). With regard to acute and chronic ethanol exposure, the positive

reinforcing effects of ethanol exposure are followed by the withdrawal effects of ethanol,

KOR activation (red section). Over time and continued exposure to ethanol, the affective

state of the subject does not return to the homeostatic set-point. Instead, their affective

state adjusts, known as an allostatic set-point, to a reduced level (black dotted lines).

Reductions in affect and the homeostatic set-point are marked with the withdrawal effects

of ethanol that drive patients to volitionally re-expose themselves to the compound (pink

arrows).

17

rat: Kissler et al., 2014; monkey: Siciliano et al., 2015). This pattern of opioid signaling

may be one of the neurobiological substrates that modulate the positive and negative

motivational aspects of chronic drug exposure.

4.0. KORs and dynorphin system function

4.1. KOR pharmacology

The primary interest of the present body of work centers on the modulatory effects of

accumbal KORs on dopamine transmission. KORs modulate dopamine transmission via

intracellular signaling pathways and modulation of ion channels. For example, KOR

activation with agonists such as its endogenous ligand, dynorphin, or (trans)-3, 4-

dichloro – N – methyl – N - [2 -(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide

methanesulfonate (U50,488H), results in liberation of the Gαi/o-protein from the βγ

subunit. The βγ subunits of the heterotrimeric G-protein block voltage-dependent calcium

channels (Gross & Macdonald, 1987; Hjelmstad & Fields, 2003; Hassan & Ruiz-Velasco,

2013), and augments potassium efflux (Grudt & Williams, 1993; Ma et al., 1995), which

reduces the probability of cell firing and exocytotic release at the level of the dopamine

terminal (Figure 4.1). Downstream signaling pathways such as p38 mitogen-activated

protein kinase (MAPK), protein kinase B (PKB), c-Jun N-terminal kinase (JNK) β-

arrestin have all been reported to be engaged following KOR activation (Figure 4.1;

Lemos et al., 2011, 2012; Cunha et al., 2012; Melief et al., 2011; Kumar & Rai, 2011).

Further, KOR activation attenuates adenylyl cyclase activity. Reduced adenylyl cyclase

activity precludes the metabolism of adenosine triphosphate to cyclic adenosine

monophosphate (cAMP) production, reducing protein kinase A (PKA) activity and

18

cAMP response element-binding protein (CREB) phosphorylation in the nucleus (Figure

4.1; Konkoy & Childers, 1989; Avidor-Reiss et al., 1995). Attenuated CREB

phosphorylation subsequently reduces tyrosine hydroxylase transcription (Vié et al.,

1999; Lewis-Tuffin et al., 2004), the rate-limiting enzyme responsible for dopamine

productionthus decreasing the concentration of dopamine available for release over time,

while dephosphorylation of this enzyme by phosphatases attenuates its activity, reducing

tyrosine hydroxylase activity on a shorter time-scale (Arbogast & Voogt, 2002; Fehér et

al., 2010; Saraf et al., 2010). Finally, prolonged KOR activation drives receptor

phosphorylation by G-protein coupled receptor kinases (GRK) and β-arrestin recruitment

prior to receptor internalization (Figure 4.2; Li et al., 1999; Tian et al., 2014).

Interestingly, KOR blockade by commonly used long-lasting KOR antagonists, such as

17,17'-(Dicyclopropylmethyl)-6,6',7,7'-6,6'-imino-7,7'-binorphinan-3,4',14,14'-tetrol

dihydrochloride (nor-binaltorphimine hydrochloride, norBNI) and (3R)-7-hydroxy-N-

[(2S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]-3-methylbutan-2-yl]-

1,2,3,4-tetrahydroisoquinoline-3-carboxamide dihydrochloride (JDTic) activate the JNK

pathway (Figure 4.3; Bruchas et al., 2007; Bruchas & Chavkin, 2010), indicating that

these compounds are inverse agonists activating intracellular signaling pathways of their

own (Khilnani & Khilnani, 2011; Kenakin & Williams, 2014). It is hypothesized that this

is due to a putative linker protein associated with JNK signaling (Figure 4.3; Bruchas &

Chavkin, 2010). Development of novel, clinically relevant and short-acting KOR

antagonists that do not modulate KORs via inverse agonism appear to be promising

pharmacotherapeutics for alcohol use and mood disorders (Cerecor, 2015; reviewed in

the Discussion Section).

19

20

Figure 4: Downstream signaling of KORs

(4.1) KOR activation with endogenous or exogenous ligands (closed teal circles), results

in liberation of the Gαi/o-protein from the βγ subunit. The βγ subunits of the

heterotrimeric G-protein reduce influx from voltage-dependent calcium channels and

increase potassium outflux, thus attenuating the probability of dopamine release. The Gα

subunit reduces adenylyl cyclase activity, thereby attenuating the conversion of

adenosine triphosphate to cyclic adenosine monophosphate (cAMP). This effect

subsequently decreases protein kinase A (PKA) activity, its translocation into the

nucleus, phosphorylation/activation of cAMP response element-binding protein (CREB)

and the transcription of downstream gene products. Additional downstream signaling

cascades of the KOR include p38 mitogen-activated protein kinase (MAPK), protein

kinase B (PKB), c-Jun N-terminal kinase (JNK) and β-arrestin. (4.2) Prolonged KOR

activation drives receptor phosphorylation by PKA and G-protein coupled receptor

kinases (GRK) and subsequent β-arrestin recruitment prior to receptor internalization.

Internalized receptors can be dephosphorylated and the β-arrestin molecule removed

before reinsertion into the membrane. (4.3) KOR blockade (red X) by commonly used

long-lasting KOR antagonists reportedly activate the JNK pathway, which is mediated by

an unknown linker protein.

21

Not only do KORs reduce dopamine transmission by attenuating dopamine release,

some reports suggest that KORs increase the rate of dopamine uptake (Thompson et al.,

2000; Kivell et al., 2014). For example, in EM4 cells, KOR activation produced a

norBNI-sensitive increase in dopamine transporter activity and presence at the cell

membrane (Kivell et al., 2014). One of these studies suggests that a functional interaction

between KORs and dopamine transporters is mediating this effect, such as the ERK1/2

pathway (Kivell et al., 2014). Augmented dopamine uptake rates would also reduce

dopamine transmission by decreasing the concentration of dopamine in the synapse

available to activate postsynaptic receptors. Thus, KOR activation reduces dopamine

transmission by two distinct mechanisms: reduction in release and augmented rates of

uptake.

4.2. Dynorphin activity

As noted earlier, dynorphins are a class of neuropeptides that are the endogenous

ligands of KORs (Chavkin et al., 1982; Merg et al., 2006; Le Merrer et al., 2009). In the

nucleus accumbens, activation of receptors on D1R-containing medium spiny neurons,

such as D1Rs (Engber et al., 1992; Xu et al., 1994; Hara et al., 2006; Muschamp &

Carlezon, 2013) and tyrosine-related kinase B (TrkB) receptors (Strömberg & Humpel,

1995; Logrip et al., 2008) results in downstream signaling and CREB phosphorylation in

the nucleus. Binding of CREB to cAMP response element (CRE) in the promotor region

of the prodynorphin gene sequence transcribes prodynorphin (Muschamp & Carlezon,

2013). There are several subtypes of dynorphin derived from the prodynorphin precursor

peptide (Day et al., 1998; Berman et al., 2000; Hauser et al., 2005). These include

dynorphin A (1-8, 1-13, 1-17), dynorphin B, big dynorphin, leumorphin and α- and β-

22

neoendorphin (Goldstein et al., 1979; Vaughn & Taylor, 1989; Ukai et al., 1992; Fischli

et al., 1982; Kilpatrick et al., 1982; Kakidani et al., 1982). Dynorphins are stored in

dense core vesicles until release in a calcium-dependent mechanism (Pickel et al., 1995;

Yakovlena et al., 2006). Once released, dynorphins activate KORs (Chavkin et al., 1982;

Merg et al., 2006), and are metabolized by carboxypeptidases, aminopeptidases and

endopeptidases into several metabolites within minutes of release (Müller & Hochhaus,

1995; Müller et al., 1996).

All dynorphins have selectivity for KORs over the other classical opioid receptors

(MORs, DORs), although the affinity of each for KORs varies. For example, some of the

dynorphin A subtypes, particularly dynorphin A 1-13, appear to have the highest affinity

for KORs (Chavkin & Goldstein, 1981), followed by dynorphin B and α-neoendorphin,

and lastly dynorphin A 1-8 and β-neoendorphin (Chavkin & Goldstein, 1981; James et

al., 1984; Goldstein et al., 1979). The dynorphin A subtypes (Khachaturian et al., 1982;

Jamensky & Gianoulakis, 1997; Marinelli et al., 2006) and dynorphin B (Lindholm et al.,

2000) have been found in the nucleus accumbens, thus these compounds will be referred

to as “dynorphin” for the remainder of this document.

4.3. Changes in KOR/dynorphin system function in response to acute and

chronic ethanol exposure

Data regarding the effects of acute ethanol on dynorphin levels are limited, largely

due to the multiple subtypes of this peptide and the disparate ethanol exposure and

withdrawal procedures used across laboratories. For example, using in vivo microdialysis,

extracellular levels of dynorphin A (1-8) were significantly elevated for the first 30

minutes after administration of 1.6 g/kg and 3.2 g/kg ethanol in rats, returning to baseline

23

60 minutes following administration (Marinelli et al., 2006). Notably, this effect was

more pronounced at the 3.2 g/kg dose, indicating that ethanol-induced changes in

dynorphin levels may be dose-dependent (Marinelli et al., 2006). Similarly, a treatment

regimen of twice per day ethanol (2.0 g/kg; i.p.) for 13 days showed increased levels of

dynorphin B in the nucleus accumbens 30 minutes following administration compared to

control animals (Lindholm et al., 2000). In this study, levels of dynorphin B were

augmented for at least 21 days after the final ethanol injection (Lindholm et al., 2000),

demonstrating a long-lasting effect of repeated ethanol exposure on dynorphin levels.

Conversely, five days after four weeks of ethanol drinking in C57BL/6 (C57) mice,

dynorphin B levels were reduced in the amygdala and substantia nigra (Ploj et al., 2000).

However, in this study, levels of dynorphin B were elevated above control levels 21 days

following ethanol exposure (Ploj et al., 2000). Further, recent work has shown that four

weeks of CIE in rats increases dynorphin A levels in the central nucleus of the amygdala,

6 to 8 hours following the cessation of ethanol exposure (Kissler et al., 2014). The effects

of ethanol on dynorphin levels are inconclusive and may depend on the rodent species,

ethanol exposure procedure used and withdrawal time, and dynorphin subtype examined.

Insight into the effects of CIE exposure on KOR activity is just emerging. For

example, recent work in monkeys has shown an increase in KOR sensitivity to agonist

using fast scan cyclic voltammetry following chronic oral ethanol consumption (3.5-6.5

hours following the cesstion of ethanol exposure; Siciliano et al., 2015). Furthermore,

data from the Walker laboratory has shown that four weeks of chronic ethanol exposure

augments KOR activity in the central nucleus of the amygdala as measured with GTPγS

assays, although this is shortly into withdrawal (6-8 hours; Kissler et al., 2014). As noted

24

earlier, this study also reported increased dynorphin A levels in the central nucleus of the

amygdala (Kissler et al., 2014). These data (Kissler et al., 2014) are contradictory based

on the normal behavior of GPCRs, whereby increased ligand binding promotes a

functional downregulation and receptor internalization (Zhang et al., 2002; Li et al.,

2003). Based on previous evidence suggesting a biphasic time-course of dynorphin

transmission (Ploj et al., 2000; Marinelli et al., 2006), it is plausible that the duration of

increased KOR activation and augmented dynorphin levels overlap; however, based on

the rapid metabolism of dynorphin (Gambús et al., 1998), it is plausible that the source of

the dynorphin may be due to reduced enzymatic activity rather than increased dynorphin

release. Experiments that test dynorphin levels, enzymatic activity and KOR function at

different time points following chronic ethanol exposure and withdrawal will shed light

on this discrepancy.

Although additional work may be necessary to fully elucidate the precise effects of

chronic ethanol exposure and withdrawal on KOR/dynorphin system function, it is likely

that chronic ethanol exposure and withdrawal increases the sensitivity of this system. For

example, rats treated with chronic ethanol exposure demonstrate augmented

anxiety/compulsive-like behavior that is reduced to control levels with KOR blockade

(Schank et al., 2012; Valdez & Harshberger, 2012). Moreover, consistent and significant

increases in ethanol consumption after two and four weeks of CIE exposure are

attenuated with systemic and intracerebroventricular KOR blockade (Walker & Koob,

2008; Walker et al., 2011). Notably, CIE-induced increases in ethanol consumption were

also reduced with intra-accumbal administration of norbinaltorphimine (norBNI), a KOR

antagonist (Nealey et al., 2011), demonstrating an important role of the nucleus

25

accumbens in ethanol drinking behavior. In all of these experiments, control animal

behavior is not altered with norBNI administration, demonstrating an upregulation in

KOR system function selectively in animals following chronic ethanol exposure and

withdrawal.

In summation of the evidence presented thus far, reduced accumbal dopamine

transmission following chronic ethanol exposure and withdrawal (Budygin et al., 2007;

Karkhanis et al., 2015) may be due, in part, to accumbal KORs (Kissler et al., 2015;

Siciliano et al., 2015), which may be driving the negative symptoms associated with

chronic ethanol exposure and withdrawal that drive ethanol consumption during a period

of abstinence (Walker & Koob, 2008; Nealey et al., 2011; Walker et al., 2011; Kissler et

al., 2014). However, changes in KOR function in the nucleus accumbens at the level of

the dopamine terminal following CIE exposure have not been explored, and are the focus

of Chapter II.

5.0. Functional alterations in dopamine autoreceptors following chronic

ethanol exposure

In addition to functional changes in KORs following chronic ethanol exposure, it is

possible that CIE also functionally alters dopamine D2 (D2R)-type autoreceptors

(Karkhanis et al., 2015). Similar to KORs, the D2-family of dopamine receptors,

comprising D2R, D3 (D3R) and D4 (D4R), are Gαi/o-coupled receptors that reduce

dopamine release upon activation (Maina & Mathews, 2010; Karkhanis et al., 2015).

D2Rs and D3Rs are encoded by DRD2 and DRD3 gene sequences, respectively, (Platania

et al., 2012; NCBI Genes and Expression DRD2, 2015; NCBI Genes and Expression

26

DRD3, 2015) and are structurally and functionally similar, and act as autoreceptors on

dopamine terminals. Unlike D2R/D3Rs, D4Rs do not act as autoreceptors and are located

primarily in brain regions outside the basal ganglia (Van Tol et al., 1991; Cohen et al.,

1992). With respect to D2Rs and D3Rs, anatomical differences between extracellular

amino acid residues and helices I, II and VII alter the electrostatic surface of the receptors

and confer specificity between D2R and D3R exogenous ligand binding, although the

actual binding pockets of D2Rs and D3Rs are nearly identical (Chien et al., 2010).

Additionally, D2Rs have both long (D2R-Long) and short (D2R-Short) subtypes

(Itokawa et al., 1996; Khan et al., 1998). Although functionally similar, D2Rs-Long are

considered primarily a post-synaptic protein that mediate signaling through the striatal

indirect pathway, whereas D2Rs-Short are located presynaptically and function as

autoreceptors (Khan et al., 1998; Lindgren et al., 2003). The present work focuses on

presynaptic D2Rs and D3Rs.

Due to the structural and functional similarity between D2Rs and D3Rs, many

experiments examining ethanol-induced changes in autoreceptor function investigate

combined D2Rs/D3Rs, making an interpretation of receptor-specific alterations nearly

impossible. For example, previous work from our laboratory demonstrated increased

D2R/D3R system responsivity to agonist following CIE in the nucleus accumbens using

ex vivo fast scan cyclic voltammetry (Karkhanis et al., 2015). Notably, chronic ethanol

intake increased D2R-type expression in the mesolimbic dopamine system (Kim et al.,

1997), which may underscore increased receptor sensitivity reported earlier (Karkhanis et

al., 2015). Although these data (Kim et al., 1997; Karkhanis et al., 2015) suggest D2R-

type upregulation following chronic ethanol exposure, some data shows chronic ethanol-

27

induced alters each receptor, independent of the other. For example, one study found that

D2Rs in the high-affinity, functional state are elevated in ethanol-withdrawn animals

(Seeman et al., 2004) while another found an upregulation of D3Rs following chronic

ethanol consumption (Vengeliene et al., 2006). More evidence suggests regulation of

ethanol withdrawal-associated phenotypes specifically by D2Rs or D3Rs. For example,

D2R knockdown increases ethanol drinking and preference, while augmented D2R levels

reduces ethanol intake (Thanos et al., 2001, 2004, 2005; but see: Silvestre et al., 1996).

Similarly, over-expression of D2R-Short in mice increases the ethanol consumption

compared to wild-type control animals (Bulwa et al., 2011). With regards to D3Rs, D3R

overexpression increases ethanol intake and conditioned place preference while D3R

antagonists and genetic knockdown reduces these measures (Bahi & Dreyer, 2014). It

appears as if both D2Rs and D3Rs mediate ethanol intake and are functionally changed

by chronic ethanol exposure. An examination of CIE-induced alterations in receptor

function at the level of the dopamine terminal would assist in a further understanding of

these changes (Chapter III).

5.1. Neurobiological links between autoreceptors and KORs

Some work suggests that D2R/D3Rs and KORs are functionally interactive. For

example repeated KOR activation reduced pre- and post-synaptic D2R/D3R expression

(Izenwasser et al., 1998) and pretreatment with a KOR agonist reduces the dopamine-

decreasing efficacy of D2R/D3R activation (Acri et al., 2011). Notably, pharmacological

co-activation of D2R/D3Rs and KORs drive anxiety/compulsive-like ethanol withdrawal

phenotypes noted in rodents (Perreault et al., 2006, 2007; Dvorkin et al., 2010; Ballester-

Gonzales et al., 2015). A functional interaction between these receptors, particularly at

28

the dopamine terminal is not known, and may provide evidence to support and novel

target for pharmacotherapeutic development. As previous work from our laboratory

showed augments D2R-type autoreceptor system sensitivity following CIE exposure, it is

also of interest whether alterations in the sensitivity of one or both autoreceptor subtypes

mediate oberseved changes in autoreceptor function (Karkhanis et al., 2015).

5.1a. KORs and D3Rs are upregulated via a similar pathway: Potential role of receptor

for activated C kinase 1 (RACK1) and the brain derived neurotrophic factor

(BDNF)/tropomysosin receptor kinase B (TrkB) system

Previous work has noted that RACK1 and BDNF/TrkB systems link the

KOR/dynorphin systems and D3Rs following ethanol exposure (Yaka et al., 2003;

McGough et al., 2004; Jeanblanc et al., 2006; Logrip et al., 2008). For example, acute

ethanol increases protein kinase C (PKC) and RACK1 binding, and translocation of

RACK1 to the nucleus of the neuron, subsequently augmenting both D3Rs and BDNF

levels (Ron et al., 2000; Yaka et al., 2003; McGough et al., 2004; Jeanblanc et al., 2006).

D3Rs are inserted in presynaptic dopamine neurons and function as autoreceptors while

BDNF is stored and released to activate TrkB receptors, particularly those located in

D1R-containing medium spiny neurons (Easton et al., 1999; Yoshii & Constantine-Paton,

2010; Koo et al., 2014). Effectors downstream of TrkBs, including MAPK and

phospholipase C (PLC; Logrip et al., 2008; Iwakura et al., 2008) results in augmented

phosphorylation of CREB, its binding to CRE, and the transcription of prodynorphin and

BDNF (Logrip et al., 2008; Tropea et al., 2011). Metabolism of prodynorphin into

dynorphin (Day et al., 1998; Berman et al., 2000; Hauser et al., 2005) results in KOR

activation and reductions in dopamine release. The effects of chronic ethanol exposure on

29

this trans-synaptic circuit are still under investigation. Hypotheses developed by Logrip

and colleagues (2008) suggest that sustained high levels of BDNF, due to continued

ethanol exposure, would reduce TrkB sensitivity to this endogenous ligand. Reductions in

presynaptic dopamine release due to CIE exposure and withdrawal (Karkhanis et al.,

2015) would also attenuate D1R activation, and, in conjunction with reduced TrkB

signaling, downstream products of these receptors, such as prodynorphin. Decreases in

dynorphin, through this process would augment KOR sensitivity (Logrip et al., 2008). It

is therefore hypothesized that KORs may be functionally upregulated concomitantly with

D3Rs (Jeanblanc et al., 2006; Vengeliene et al., 2006; Kissler et al., 2014; Siciliano et

al., 2015). This proposed circuit is depicted in Figure 5. Experiments that specifically

investigate ethanol-induced alterations in KOR and D3R sensitivity with a measurement

of their endogenous ligands and RACK1 in the same brain region may provide insight

into this hypothesis.

6.0. Pharmacotherapies indicated for alcohol use disorders

6.1. Disulfiram

Disulfiram (Antabuse®) was approved by the United States’ FDA in 1951 for the

treatment of alcoholism (FDA Center for Drug Evaluation and Research, 1951).

Disulfiram is an acetaldehyde dehydrogenase inhibitor that reduces the conversion of

acetaldehyde in to acetate, both metabolites of ethanol (Heit et al., 2013), following

ethanol consumption, thereby elevating plasma and brain levels of acetaldehyde (Gaval-

Cruz & Weinshenker, 2009). The rapid increase in plasma acetaldehyde causes negative

physiological responses in patients, often referred to as the “Antabuse reaction”, which

30

31

Figure 5: Ethanol-induced increases in KOR and D3R function may be due to

alterations in RACK1and BDNF/TrkB systems

(A1) Acute ethanol augments dopamine neuron firing at the level of the soma and

augments RACK1 translocation to the nucleus. (A2) These actions augment dopamine

release and D3R expression in dopamine terminal boutons, respectively. (A3) Increased

levels of dopamine and BDNF increases D1R and TrkB receptor signaling. D1R

activation increases adenylyl cyclase activation, cyclic adenosine monophosphate

production from adenosine triphosphate, augmented protein kinase A activity and CREB

phosphorylation in the nucleus. Activation of TrkB receptors increases phospholipase C,

which metabolizes phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate

(IP3) and diacylglycerol. IP3 increases calcium release from intracellular stores and when

paired with diacylglycerol, activates protein kinase C (PKC). PKC

phosphorylates/activates CREB after its translocation to the nucleus. (A4) CREB binding

to cAMP response element (CRE) on specific promotor regions of the genome augments

prodynorphin (PDYN) and BDNF transcription. Once translated into protein,

prodynorphin is metabolized by proprotein convertase 2 into dynorphin and further

refined via post-translational processing, and stored in dense core vesicles until release.

(A5) Upon release, dynorphin and BDNF are released to act on their respective receptors

(A6).

32

includes symptoms such as flushing and sweating, dyspnea, blurred vision, headache,

cardiac arrhythmia, vertigo and physical weakness (Substance Abuse and Mental Health

Services Administration, Center for Substance Abuse Treatment, 2009). Some studies

suggest that ethanol drinking is reduced with disulfiram treatment; however, this effect is

observed largely under strict clinical observation (Kristenson, 1992, 1995), and primarily

successful in those with strong social support and mild alcohol use disorders (Schuckit,

1985). Although long-term, consistent use of disulfiram appears to be helpful in

maintaining ethanol abstinence over placebo controls (Elbreder et al., 2010), the negative

symptoms and potentially serious health risks associated with disulfiram treatment in

conjunction with relapse drinking behavior lead to poor patient compliance (Suh et al.,

2006).

6.2. Acamprosate

Acamprosate (Campral®) was FDA approved for the treatment of alcoholism in 2004

(FDA, 2004). Although the precise mechanism of action of acamprosate is unknown, it is

hypothesized that this compound regulates aberrant GABAergic (Daoust et al., 1992;

Dahchour & De Witte, 1999) and glutamergic (Dahchour et al., 1998; Dahchour & De

Witte, 1999; Hinton et al., 2012) signaling that exist during ethanol withdrawal.

Alcoholics who take acamprosate for alcohol use disorders often report severe gastro-

intestinal disturbances (Paille et al., 1995; Bouza et al., 2004). Acamprosate has been

reported as superior in maintaining abstinence in alcoholics (18-61% of patients)

compared to placebo-controlled group (4-45%; Boothby & Doering, 2005; Jonas et al.,

2014; Higuchi et al., 2015). However, the variability of abstinence success in treatment

groups calls into question the precise efficacy of the drug and suggest there may be

33

individual variability in clinical responses (Boothby & Doering, 2005; Jonas et al., 2014;

Higuchi et al., 2015).

6.3. Naltrexone

Of the current pharmacotherapies indicated for alcohol use disorders, naltrexone

(Revia®, Vivitrol®) is of particular interest based on its ability to pharmacologically

inhibit all three classical opioid receptor subtypes, MORs, DORs and KORs (Bart et al.,

2005). As reviewed earlier, clear evidence points to the opposing functional and region-

specific effects of MORs and KORs on dopamine transmission, making it unclear what

aspects of its pharmacology are important for clinical efficacy (Turchan et al., 1999;

Chen & Lawrence, 2000; Saland et al., 2005; Kivell et al., 2014). Notably, naltrexone

reduces the reinforcing potency of natural rewards, such as sucrose, a finding that has

been demonstrated in monkeys (Williams et al., 1998) and humans (Fantino et al., 1986;

Langleben et al., 2012) and has been credited to its antagonistic effects on MORs.

Although naltrexone reduces ethanol craving in humans (Kruse et al., 2012), over one-

third of all patients taking this drug relapse to alcoholism (Latt et al., 2002). It is

therefore possible that a compound that differentially modulates MORs and KORs

(Section 4.3) and preserves the reinforcing salience of natural rewards may be a more

efficacious pharmacotherapeutic agent to combat alcohol use disorders. Notably, Chapter

IV of the present body of work addresses a potential compound, nalmefene, an alcohol

drinking harm reduction agent and high-affinity KOR partial agonist (Bart et al., 2005;

European Medicines Agency’s Committee for Medicinal Products for Human Use, 2012)

on KOR function and accumbal dopamine transmission for this indication.

34

7.0. Conclusion

Alcohol use disorders are a major health concern in the United States (Centers for

Disease Control and Prevention, 2014; Substance Abuse and Mental Health Services

Administration, 2014). Current pharmacotherapeutic treatment is relegated to three FDA-

approved compounds that have limited efficacy in reducing relapse drinking (Latt et al.,

2002; Mann et al., 2004; Jørgensen et al., 2011; Yoshimura et al., 2014; Higuchi et al.,

2015). As such, there is a need for novel pharmacotherapeutics and targets to promote the

development of agents that reduce the withdrawal effects of ethanol and subsequent

relapse drinking behavior. Considerable evidence strongly suggests that increased KOR

function may be driving reductions in dopamine signaling in the nucleus accumbens and

some of the withdrawal symptoms of ethanol following repeated exposure to ethanol that

drive ethanol consumption (Walker & Koob, 2008; Nealey et al., 2011; Walker et al.,

2011; Kissler et al., 2014; Kivell et al., 2014). Additional work has suggested that CIE

exposure also induces functional alterations in dopamine autoreceptors (Karkhanis et al.,

2015). Notably, D3Rs and KORs may be functionally connected (Izenwasser et al., 1998;

Acri et al., 2011) via similar upregulation mechanisms (Jeanblanc et al., 2006; Logrip et

al., 2008), demonstrating a novel and unexplored relationship between these receptors.

In short, the present body of work aimed to identify the effects of five weeks of CIE

exposure on ethanol drinking and anxiety/compulsive-like phenotypes and

neurochemistry in male C57BL/6J mice. Behavioral work included an examination of

behavior following CIE exposure and KOR blockade. These measures were replicated by

KOR activation in naïve mice. Using ex vivo fast scan cyclic voltammetry, dopamine

terminal, KOR and autoreceptor function were assessed in slices containing the nucleus

35

accumbens core following five weeks of CIE. Experiments investigating a functional

interaction between KORs and autoreceptors were conducted in naïve mice. The final

study shifted the focus of this dissertation from elucidating the functional changes of

dopamine terminal and KORs following CIE to examining the effects nalmefene on

dopamine terminal and KOR function in efforts to understand its clinical efficacy. In

short, data showing a functional link between KORs and D3Rs deserves additional

investigation and may provide a novel target for pharmacotherapeutic development. We

hope that these data provide evidence to support a role for KOR antagonists and partial

agonists in the clinic, specifically to ameliorate the withdrawal effects of alcohol use

disorders and reduce relapse drinking in treatment-seeking alcoholics.

36

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68

CHAPTER II

SUPERSENSITIVE KAPPA OPIOID RECEPTORS PROMOTE ETHANOL

WITHDRAWAL-RELATED BEHAVIORS AND REDUCE DOPAMINE SIGNALING

IN THE NUCLEUS ACCUMBENS

Jamie H. Rose, Anushree N. Karkhanis, Rong Chen, Dominic Gioia, Marcelo F. Lopez,

Howard C. Becker, Brian A. McCool, Sara R. Jones

Accepted for publication to The International Journal of Neuropharmacology,

November, 2015

69

Abstract:

Background: Chronic ethanol exposure reduces dopamine transmission in the nucleus

accumbens (NAc), which may contribute to the negative affective symptoms associated

with ethanol withdrawal. Kappa opioid receptors (KORs) have been implicated in

withdrawal-induced excessive drinking and anxiety-like behaviors, and are known to

inhibit dopamine release in the NAc. The effects of chronic ethanol exposure on KOR-

mediated changes in dopamine transmission at the level of the dopamine terminal and

withdrawal-related behaviors were examined.

Methods: Five weeks of chronic intermittent ethanol (CIE) exposure in male C57BL/6

mice was used to examine the role of KORs in chronic ethanol-induced increases in

ethanol intake and also marble burying, a measure of anxiety/compulsive-like behavior.

Drinking and marble burying were evaluated before and after CIE exposure, with and

without KOR blockade by nor-binaltorphimine (norBNI, 10mg/kg i.p.). Functional

alterations in KORs were assessed using fast scan cyclic voltammetry (FSCV) in brain

slices containing the NAc.

Results: CIE-exposed mice showed increased ethanol drinking and marble burying

compared to controls, which was attenuated with KOR blockade. CIE-induced increases

in behavior were replicated with KOR activation in naïve mice. FSCV revealed that CIE

reduced accumbal dopamine release and increased uptake rates, promoting a

hypodopaminergic state of this region. KOR activation with U50,488H concentration-

dependently decreased dopamine release in both groups; however, this effect was greater

in CIE-treated mice, indicating KOR supersensitivity in this group.

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Conclusions: These data suggest that the CIE-induced increase in ethanol intake and

anxiety/compulsive-like behaviors may be driven by greater KOR sensitivity and a

hypodopaminergic state of the NAc.

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Introduction

Alcoholism is a disease of chronic relapse, consisting of repeated bouts of excessive

intake and abstinence, resulting in withdrawal symptoms that contribute to the

resumption of heavy drinking. For example, human alcoholics report increased anxiety

(Wetterling & Junghanns, 2000) and compulsive behaviors (Suzuki et al., 2002) during

abstinence periods, which correlate with escalated ethanol consumption (Wetterling et

al., 2006; American Psychological Association, 2013). Examination of these symptoms

in a mouse model of alcohol use disorders showed that C57BL/6 (C57) mice exposed to

ethanol vapor in a chronic, intermittent (chronic intermittent ethanol, CIE) pattern

demonstrate escalated ethanol intake during abstinence (Griffin et al., 2009). Traditional

measures of anxiety-like behavior, such as the elevated plus maze and light-dark box,

have not found ethanol withdrawal-induced anxiogenic phenotypes in C57 mice

(Ghozland et al., 2005; McCool & Chappell, 2015). However, data supporting ethanol

withdrawal-related increases in nontraditional anxiety/compulsive-like behaviors, such as

marble burying, are emerging. For example, recent work utilizing C57 mice exposed to

chronic ethanol injections or an ethanol-containing liquid diet (Perez & De Biasi, 2015)

showed increased marble burying during ethanol withdrawal. Marble burying is used to

model compulsive-like and anxiety-like behaviors in rodents, and pharmacotherapies for

anxiety and obsessive-compulsive disorder reduce this behavior (Nicolas et al., 2006).

Thus, marble burying may provide a useful behavioral measure of ethanol withdrawal-

associated negative affect in this mouse strain.

Dopamine transmission is markedly attenuated after chronic ethanol exposure

(Karkhanis et al., 2015), which may be driving CIE-induced changes in behavior. A

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previous study reported reduced dopamine terminal function in the nucleus accumbens

(NAc) after three cycles of CIE and 72 hours of abstinence (Karkhanis et al., 2015),

indicating a hypodopaminergic state of this region during abstinence. The time-course of

these findings are similar to CIE-induced increases in ethanol drinking using a similar

protocol (Griffin et al., 2009), suggesting a link between attenuated accumbal function

and augmented ethanol intake. As the dopamine system has been implicated as an

important regulator of ethanol drinking (Nealey et al., 2011) and anxiety/compulsive-like

behaviors (Ballester-Gonzáles et al., 2015), it is possible that dysregulated dopamine

transmission may underlie chronic ethanol-induced increases in these behaviors.

Although dopamine terminal function is attenuated after CIE, the precise

mechanism(s) underlying this change are not well understood. Dopamine transmission in

the NAc is regulated by a variety of receptors, including kappa opioid receptors (KORs).

Intra-accumbal pharmacological activation of KORs reduces dopamine release in this

region, while blockade transiently increases extracellular levels of dopamine (Spanagel et

al., 1992). Consistent with these data, administration of a KOR agonist in naïve rats

(Todtenkopf et al., 2004) increases brain reward thresholds, as measured by intra-cranial

self-stimulation, suggesting that KOR activation reduces mesocorticolimbic signaling and

produces negative affect. Notably, KORs on mesolimbic dopamine neurons mediate

place aversion to KOR agonists (Chefer et al., 2012), supporting a modulatory role of

KORs on mesolimbic dopamine system function that influences hedonic state. Further,

withdrawal from ethanol (Schulteis et al., 1995) and cocaine (Chartoff et al., 2012)

increases brain reward thresholds, an effect which is blocked with KOR antagonists

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(Chartoff et al., 2012). Thus, KOR blockade may attenuate the withdrawal symptoms of

ethanol by regulating dopamine transmission.

Of interest to this particular study are data demonstrating the utility of KORs as a

potential pharmacotherapeutic target to alleviate the symptoms of ethanol withdrawal.

CIE-exposed rats routinely demonstrate increased ethanol self-administration after a

period of abstinence compared to air-exposed controls, an effect that is reduced to control

levels with systemic (Walker et al., 2011), intracerebroventricular (Walker & Koob,

2008) or intra-NAc (Nealey et al., 2011) administration of nor-binaltorphimine (norBNI),

a KOR-specific antagonist. As KOR manipulation alters dopamine system function

(Todtenkopf et al., 2004; Zapata & Shippenberg, 2006; Chartoff et al., 2012), and KOR

blockade reduces ethanol self-administration in dependent rats (Walker & Koob, 2008;

Walker et al, 2011; Nealey et al., 2011), it is possible that changes in KOR function and

dopamine transmission drive CIE-induced behavioral changes observed during ethanol

abstinence. The present study used repeated cycles of CIE and air exposure to examine

the role of KORs in ethanol consumption and marble burying in C57 mice. Additionally,

ex vivo fast scan cyclic voltammetry (FSCV) in brain slices of the NAc was used to

identify CIE-induced changes in dopamine terminal and KOR function.

Methods and Materials

Subjects

Male C57 mice (65-75 days old, Jackson Laboratories, Bar Harbor, ME) were used

for all experiments. Mice were individually housed and maintained on a 12:12 hour light

cycle (lights off at 14:00), with a red room light illuminated during the dark cycle. Mice

74

habituated to housing conditions for one week before experiment start and were provided

with standard chow and water ad libitum, unless otherwise noted. The Institutional

Animal Care and Use Committee at Wake Forest School of Medicine approved all

experimental protocols. Animals were cared for according to National Institutes of Health

guidelines in Association for Assessment and Accreditation of Laboratory Animal Care

accredited facilities.

CIE Exposure: Back-to-Back CIE and Back-to-Back CIE+Drinking Protocol

Mice were exposed to ethanol vapor or room air for four days (16 hours of exposure +

8 hours of abstinence), followed by 72 hours of room air exposure (one cycle). Cycles

were repeated five times. Mice were treated (i.p.) with a solution containing 1.6g/kg

ethanol (CIE) or saline (air) and 1.0mmol pyrazole (Sigma Aldrich, St. Louis, MO), an

ethanol dehydrogenase inhibitor, 30 minutes prior to inhalation treatment. Blood ethanol

concentrations (BEC) were tested after the first and fourth inhalation exposure to ensure

physiologically relevant BECs (Griffin et al., 2009; Supplementary Figure 2). The Back-

to-Back CIE Protocol (Figure 1A) was modified (Back-to-Back CIE+Drinking Protocol,

Figure 1B) to examine CIE-induced changes in ethanol drinking.

Ethanol Drinking Tests

Animals in ethanol drinking groups underwent a two bottle choice procedure that

included two weeks of sucrose fade (Samson, 1986) consisting of two days each: 10%

ethanol/5% sucrose, 12%/5%, 15%/5%, 15%/2%, and 15%/1% (w/v), followed by four

weeks of access to 15% (v/v) ethanol. Experimental solutions were provided for two

hours per day (13:30-15:30), five days per week, and paired with an identical

75

Figure 1. Back-to-Back chronic intermittent ethanol (CIE) exposure protocols.

(A) Visual depiction of the Back-to-Back CIE Protocol. Mice were exposed to five cycles

of ethanol vapor or room air (green bars). Each cycle consisted of four days of 16 hours

of exposure (red bars), 8 hours of abstinence (dark blue bars), followed by 72 hours of

room air exposure (orange bar). (B) Visual depiction of the Back-to-Back CIE + Drinking

Protocol. Mice underwent a two bottle choice drinking procedure (two weeks of sucrose

fade (blue bar) followed by four additional weeks of access to 15% (v/v) ethanol (left

purple bar). Both CIE and air groups underwent five cycles of inhalation exposure (green

bars) and ethanol drinking was examined following all CIE exposure cycles (right purple

bar).

76

bottle of water. Bottle placement was switched daily. Mice were assigned to inhalation

groups, counterbalanced for intake. Ethanol drinking was examined for five days after all

cycles (Figure 1B). Separate groups of animals were injected with norBNI (10mg/kg, i.p.;

NIDA, Bethesda, MD) or vehicle (injectable water) 24 hours prior to the post-inhalation

ethanol drinking test. A separate group of mice was not exposed to the inhalation

treatment, but during the final (fourth) week of baseline (15% vs water) drinking, they

were injected with saline (0.1mL, i.p.) 30 minutes prior to drinking start time (13:30).

Across the following several weeks, mice were injected with saline (0.1mL) on Mondays,

Wednesdays and Fridays. In a latin-square design, mice were injected i.p. with 1.0, 3.0,

6.0, and 10mg/kg U50,488 on Tuesdays and Thursdays. Drinking data were collapsed

across saline and each dose of U50,488.

Marble Burying

Pre-inhalation marble burying tests occurred on the morning of the first day of CIE

cycle 1. Mice were placed in a standard polycarbonate cage lined with 4cm of cob

bedding (The Andersons, Inc., Maumee, OH) for 120 minutes before the addition of 20

clean, black glass marbles (14mm, Rainbow Turtle, Portland, OR). A template was used

to ensure marble placement (Figure 4A, left panel). After the 30-minute testing session,

the number of marbles over 75% buried was counted. Water was provided during

habituation, but both food and water were unavailable during testing. Animals were

placed into inhalation groups, counterbalanced for marbles buried during the pre-test.

CIE-induced changes in marble burying were assessed 72 hours after the fifth cycle.

Separate groups of mice were systemically injected with norBNI (10mg/kg) or vehicle 24

hours prior to post-inhalation testing. A separate group of mice was injected with saline

77

(0.1mL) or 3.0mg/kg U50,488, 30 minutes prior to testing, approximately 90 minutes into

their habituation session.

Ex Vivo FSCV

Ex vivo FSCV was used to characterize dopamine kinetics in the NAc core

(Karkhanis et al., 2015). Mice were sacrificed 72 hours after the fifth inhalation exposure

to coincide with behavioral testing. Mice were anesthetized with isoflurane, rapidly

decapitated and brains removed. Brain slices containing the NAc (300µm thick) were

prepared with a vibrating tissue slicer and incubated in oxygenated artificial

cerebrospinal fluid (aCSF, 32OC). A carbon fiber recording electrode (≈50-100µm length,

7µM radius; Goodfellow Corporation, Berwyn, PA), and a bipolar stimulating electrode

(Plastics One, Roanoke, VA) were placed (~100µm apart) on the surface of the slice.

Dopamine efflux was induced with a single, rectangular, 4.0ms duration electrical pulse

(350µA, monophasic, inter-stimulus interval: 180sec). To detect dopamine release, a

triangular waveform (-0.4 to +1.2 to -0.4V vs. silver/silver chloride, 400V/sec) was

applied every 100ms to the recording electrode. To assess the effects of inhalation

treatment on KOR function, the KOR agonist, U50,488 (0.01-1.0µM, Tocris Bioscience,

Minneapolis, MN), was added cumulatively to aCSF after baseline collections were

stable. To obtain clear current versus time plots, background current subtraction methods

were utilized. Electrode calibration was performed following each experiment using a

flow-injection system.

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Data Analysis

Ethanol consumption was calculated by weighing ethanol and water bottles before

and after drinking tests. Preference was calculated as the volume of ethanol consumed

divided by total intake (ethanol+water) consumed in each session. Marble burying was

scored by visual inspection of marbles.

Demon Voltammetry and Analysis software (Yorgason et al., 2011) was used to

collect and analyze all voltammetry data. Representative signals were analyzed before

drug application to determine dopamine release per electrical stimulation (μM) and

uptake (Vmax, μM/sec). Representative traces after drug application were similarly

analyzed. The effects of U50,488 on dopamine release across the concentration response

curve are presented as a percent of pre-drug dopamine release.

Graphs were created and statistical tests were applied using GraphPad Prism, Version

5 (La Jolla, CA). Two-way repeated measures (RM) analysis of variance (ANOVA) were

used to compare the effects of CIE exposure and drug treatment (norBNI vs. vehicle) on

ethanol drinking, preference and marble burying as well as the U50,488 concentration

response curve, with drug concentration and inhalation treatment as factors. All drinking

data are collapsed across the five-day exposure or drug dose (saline or U50,488). When a

significant main effect was found, Bonferroni post hoc analysis was performed. A one-

way ANOVA was used to analyze the effects of acute U50,488 on ethanol drinking and

preference. When a significant main effect was detected, Tukey’s post-hoc analysis was

applied. Two-tailed Student’s t-tests were used to reveal pre-inhalation ethanol drinking

and preference differences between inhalation groups, the effects of CIE exposure on

79

baseline dopamine kinetics, the effect of 3.0mg/kg U50,488 on marble burying and

U50,488 IC50.

Results

CIE increased ethanol intake and preference without inter-cycle ethanol drinking.

To confirm that the CIE exposure protocol used here increased ethanol drinking

similar to previously used protocols, separate groups of mice underwent a CIE+Drinking

protocol, (Griffin et al.,2009; Supplementary Figure 1). BECs were collapsed across CIE

exposure protocols and were within the behaviorally relevant range (Griffin et al., 2009;

Supplementary Figure 2, 204.9±58.47mg/dl). Two-way ANOVA revealed a main effect

of inhalation exposure (Figure 2A, F1,23=6.599, p<0.05) and time (pre vs. post inhalation

exposure, F1,23=11.16, p<0.01). Post hoc analysis confirmed similar pre-inhalation

ethanol consumption between groups (Air: 2.53±0.33, CIE: 2.60±0.28g/kg, p>0.05), but

an increase in ethanol intake in CIE-exposed mice was observed (p<0.001). An

interaction between inhalation exposure and time was also detected (F1,23=5.543, p<0.05).

As hypothesized, CIE-exposed mice exhibited increased ethanol intake, as a percent of

pre-exposure intake levels, compared to controls (Figure 2B: Air: 112.00±14.30%, CIE:

180.30±13.15%, t10=4.49, p<0.01). The amount of ethanol consumed on each day is

depicted in Supplementary Figure 3.

Analysis of ethanol preference revealed a main effect of inhalation treatment (Figure

2C, F1,22=9.38, p<0.05) and time (pre vs. post inhalation, F1,22=7.03, p<0.05). Augmented

ethanol preference in CIE-exposed mice compared to pre-CIE (p<0.01) and air-exposed

animals (p<0.01) were observed with post hoc analysis levels (Air: 0.81±0.02, CIE:

0.83±0.02). A two-tailed Student’s t-test revealed increased ethanol preference in CIE-

80

Figure 2: CIE-exposed mice demonstrated increased ethanol consumption and

preference without inter-cycle ethanol drinking tests.

(A) Pre-inhalation ethanol intake was similar between groups. CIE significantly

augmented ethanol drinking in CIE-exposed mice compared to air-exposed control

animals and pre-CIE exposure ethanol intake. (B) Ethanol intake was significantly

greater in CIE-exposed compared to air-exposed mice when expressed as a percent of

pre-inhalation intake. (C) CIE increased ethanol preference in CIE-exposed mice

compared to air-exposed control mice and pre-inhalation ethanol preference. (D) Ethanol

preference was significantly greater in CIE-exposed mice compared to air-exposed

animals. n(air)=6; n(CIE)=7. *p<0.05; **p<0.01; ***p<0.001.

Air CIE0

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exposed mice compared to controls when expressed as a percent of pre-inhalation

preference (Figure 2D: Air: 103.00±4.69%, CIE: 113.30±2.30%, t11=3.51, p<0.05).

KOR blockade reduced ethanol consumption and preference in CIE-treated mice.

To examine the role of KORs in CIE-induced changes in ethanol drinking, norBNI or

vehicle was systemically administered 24 hours prior to the five-day drinking test

(Broadbear et al., 1994). Two-way ANOVA revealed a main effect of inhalation (Figure

3A; F1,28=9.20, p<0.01) and drug treatment (norBNI vs. vehicle) on ethanol intake

(F1,28=28.60, p<0.001). An interaction between inhalation and drug treatment was also

found (F1,28=10.01, p<0.01). Post-hoc analysis confirmed a significant increase in ethanol

drinking in CIE-exposed mice treated with vehicle (4.57±0.40g/kg, p<0.001) compared to

CIE-exposed mice treated with norBNI (2.23±0.19g/kg). Ethanol drinking in norBNI

treated/CIE-exposed mice was similar to intake of norBNI treated/air-exposed

(2.27±0.19g/kg, p>0.05).

Examination of ethanol preference after CIE/air exposure revealed a main effect of

norBNI treatment (Figure 3B; F1,27=26.21, p<0.001). Although no main effect of

inhalation treatment was detected (F1,27=1.22, p=0.28), potentially reflecting a ceiling

effect due to high baseline preference, an interaction between inhalation and drug

treatment (norBNI vs. vehicle) was observed (F1,27=11.25, p<0.01). Reduced ethanol

preference in CIE-exposed mice treated with norBNI (preference ratio: 0.70±0.03)

compared to CIE-exposed vehicle-treated animals (0.92±0.015, p<0.001) was found

using post hoc analysis, but norBNI treated/CIE exposed mice had similar preferences to

air-exposed mice treated with vehicle (0.79±0.02, p>0.05) or norBNI (0.76±0.02,

82

p>0.03). The effects of inhalation and drug treatment were maintained over the five-day

drinking test (Supplementary Figure 3).

U50,488 increased ethanol consumption and preference

NorBNI reduced ethanol intake in CIE-exposed animals (Figure 3A, 3B), suggesting

that KORs positively modulate drinking. Thus, we hypothesized that KOR activation

would augment ethanol consumption and preference in naïve mice. A one-way ANOVA

revealed a significant effect of U50,488 on ethanol intake (Figure 3C, F5,46=18.86,

p<0.0001) and preference (Figure 3D, F5,46=5.903, p<0.001). Tukey’s post-hoc analysis

revealed a significant difference increase at the 1.0 (p<0.001), 3.0 (p<0.001) and 6.0

(p<0.5) mg/kg doses of U50,488 in ethanol drinking compared to saline drinking.

Further, Tukey’s post hoc analysis revealed a significant increase in ethanol preference at

the 1.0 (p<0.01), 3.0 (p<0.05) and 6.0 (p<0.5) mg/kg doses of U50,488 compared to

control (saline). In both datasets (Figure 3C, 3D), post hoc analysis showed that drinking

in mice given saline or the 10mg/kg dose of U50,488 was similar (p>0.05).

CIE increased marble burying behavior.

Marble burying was used to examine the effects of CIE or air exposure on

anxiety/compulsive-like behaviors. Representative pictures of testing chambers are

shown in Figure 4A: before testing (left panel), after five cycles of air exposure (middle

panel), and CIE exposure (right panel). A two-way RM ANOVA revealed a main effect

of time (pre vs. post inhalation; Figure 4B; F1,14=4.69, p<0.05). Post-hoc analysis showed

an increase in marble burying behavior between pre- (9.44±2.32 marbles buried) and post

(16.67±0.73 marbles buried) CIE exposure (p<0.01). A significant increase in marble

burying between post-CIE and post-air exposure inhalation groups (8.86±1.82 marbles

83

Figure 3: Systemic norBNI administration reduced ethanol intake and preference

CIE-treated mice which was replicated with KOR activation.

(A) CIE significantly augmented ethanol drinking compared to air-exposed control mice,

which was reduced with norBNI treatment. (B) Ethanol preference of CIE-exposed mice

was significantly greater than air-exposed controls, which was reduced to control levels

with norBNI treatment. A+B: n(air/vehicle)=6; n(air/norBNI)=9; n(CIE/vehicle)=7

n(CIE/norBNI)=10. (C) U50,488 increased ethanol intake and (D) preference, which

returned to baseline at 10mg/kg. C+D: n(saline)=14; n(1.0mg/kg U50,488)=9;

n(3.0mg/kg U50,488)=10; n(6.0mg/kg U50,488)=9; n(10.0mg/kg U50,488)=9. *p<0.05;

**p<0.1 ***p<0.001

Air CIE 0.0

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1.0

Inhalation Exposure

****

Eth

an

ol P

refe

ren

ce

R

atio

Saline 1 3 6 100.0

1.0

2.0

3.0

4.0

5.0

*******

Dose U50,488 (mg/kg)

Eth

an

ol In

take (

g/k

g)

Saline 1 3 6 100.0

0.5

1.0

1.5

***

*

Dose U50,488 (mg/kg)

Eth

an

ol P

refe

ren

ce

Ra

tio

C D

Post-inhalation, vehicle treatment

Post-inhalation, norBNI treatment

84

buried, p<0.05) and an interaction between inhalation exposure and time (F1,28=5.42,

p<0.05) were observed.

KOR blockade reduced CIE-induced increases in marble burying.

Mice were systemically injected with norBNI or vehicle 24 hours prior to the post-

inhalation marble burying test. Two-way ANOVA revealed a main effect of inhalation

condition (Figure 4C, F1,26=7.35, p<0.05) and drug treatment (norBNI vs. vehicle,

F1,26=21.74, p<0.001) on marble burying. Post-hoc analysis confirmed an increase in

marble burying in vehicle treated CIE- vs. air-exposed mice (p<0.01). Additionally,

marble burying behavior of vehicle treated/CIE-exposed mice was greater than that of

CIE-exposed mice treated with norBNI (p<0.001). An interaction between CIE exposure

and drug treatment was detected (F1,16=8.59, p<0.001). NorBNI reduced marble burying

in CIE-exposed mice (6.13±1.22 marbles buried) to norBNI-treated air (6.43±1.60

marbles buried) and pre-CIE levels (Figure 4C; F1,26=0.01, p=0.93). Additional

experiments in naïve mice show that the effects of norBNI on ethanol drinking and

marble burying are likely not attributable to changes in locomotion (Supplementary

Figure 5).

To confirm that KOR activation would also augment marble burying, naive mice

were injected with 3.0mg/kg U50,488, 30 minutes prior to testing. A two-tailed Student’s

t-test revealed augmented marbles buried in response to U50,488 administration (Figure

4E, Saline: 12.57±0.649 marbles buried; 3.0mg/kg U50, 488: 16.00±0.723 marbles

buried; t12=3.526, p<0.01).

85

86

Figure 4: CIE exposure increased marble burying behavior which was norBNI-

sensitive and was replicated with KOR agonist administration.

(A) Representative pictures of marble configuration: before testing (left panel), after five

cycles of air (middle panel) and CIE (right panel) exposure. (B) CIE exposure

significantly augmented marble burying compared to air-exposed mice and pre-inhalation

marble burying behavior. (C) CIE augmented marble burying in CIE-exposed compared

to air-exposed mice, which was reduced with norBNI treatment. (D) Marble burying

following norBNI treatment is similar to pre-inhalation exposure marble burying

behavior. A-D: n(air)=7; n(CIE)=9 (E) KOR activation with a 3.0mg/kg dose of U50,488

significantly augmented marble burying behavior in naïve mice compared to mice treated

with a saline challenge. n(saline)=7; n(3.0mg/kg U50,488)=7. **p<0.01; ***p<0.001.

87

CIE attenuated dopamine terminal function via reduced dopamine release and increased

uptake.

To examine changes in dopamine terminal function after CIE exposure, ex vivo FSCV

was used. Figure 5A shows representative dopamine release and uptake traces with false

color plots (Air: left panel; CIE: right panel). A two-tailed Student’s t-test revealed

decreased dopamine release (Figure 5B; Air: 1.15±0.12µM; CIE: 0.80±0.07µM,

t18=2.687, p<0.05) and augmented uptake rate (Figure 5C; Air: 2.02±0.07µM/sec; CIE:

2.42±0.17µM/sec, t21=2.20, p<0.05) in CIE-exposed mice vs. controls.

CIE increases KOR sensitivity in the NAc core.

Ex vivo FSCV was used to examine CIE-induced changes in KOR sensitivity. Two-

way RM ANOVA revealed a main effect of CIE treatment on KOR sensitivity,

suggesting increased KOR function in CIE-exposed mice compared to controls (Figure

5D; F1,11=5.00, p<0.05). A main effect of U50,488 on dopamine release (F4,11=80.87,

p<0.001) and an interaction between inhalation treatment and U50,488 concentration

were detected (F4,11=3.22, p<0.05). A reduction in the U50,488 IC50 in CIE- compared to

air-exposed mice was found (Figure 5D inset: Air: 3.38±0.27µM, CIE: 2.10±0.22µM,

t7=3.72, p<0.01).

88

89

Figure 5: CIE exposure attenuated accumbal dopamine terminal function and

increased KOR sensitivity.

(A) Representative voltammetric traces after electrical stimulation of dopamine and

associated false color plots (air, left, green; CIE, right, purple). False color plots

demonstrate changes in current over time and in response to electrically evoked

dopamine release (X-axis: time, Y-axis: command voltage (−0.4 to +1.2 to -0.4V), Z-

axis: current). Cyclic voltammograms (insets) identify the peak oxidation (~+0.6V) and

reduction voltages (~−0.2V) of dopamine. (B) CIE reduced electrically evoked dopamine

release compared to air-exposed mice. (C) CIE augmented dopamine uptake in brain

compared to controls. B,C n(air)=9; n(CIE)=11. (D) U50,488 dose-dependently reduced

dopamine release across increasing concentrations, an effect was more pronounced in

CIE-treated brain slices, reducing its IC50 in CIE-exposed mice (inset). n(air)=6;

n(CIE)=7. *p<0.05; **p<0.01.

90

Discussion

The present work showed that CIE-induced behavioral changes in C57 mice were

associated with augmented sensitivity of KORs on dopamine terminals and reduced

dopamine transmission in the NAc. Ethanol drinking and preference were increased

following a five-week CIE protocol which also increased anxiety/compulsive-like marble

burying behavior. FSCV in the NAc showed that CIE reduced dopamine release,

increased rates of dopamine uptake, and augmented the inhibitory effects of KORs on

dopamine release, promoting a hypodopaminergic state of the NAc. These data suggest

that accumbal hypodopaminergia may be partially driven by increased KOR function,

leading, at least in part, to the potentiation of ethanol intake and anxiety/compulsive-like

behaviors during ethanol abstinence.

CIE exposure increased ethanol consumption, preference and marble burying

behavior.

To examine the effects of CIE exposure on ethanol intake, both ethanol consumption

and preference were examined using two distinct CIE exposure protocols. Experiments

that included five days of ethanol drinking between each CIE cycle (Supplementary

Figure 1) replicated previous findings that showed escalated ethanol consumption with

sequential bouts of CIE and abstinence (Griffin et al., 2009). These data were similar to

the abbreviated Back-to-Back CIE+Drinking protocol, suggesting that CIE exposure

alone is sufficient to increase ethanol drinking and preference. Thus, the Back-to-Back

CIE exposure protocol was utilized for the remainder of the experiments.

Escalation of ethanol intake is a hallmark of chronic alcohol exposure that has been

documented in humans (Wetterling et al., 2006; American Psychological Association,

91

2013), as well as in monkey (Grant et al., 2008), rat (Walker & Koob, 2008) and C57

mouse (Griffin et al., 2009; present work) models of alcoholism. Because abstinent

alcoholics also report increased anxiety (Wetterling & Junghanns, 2000) and compulsive

behaviors (Suzuki et al., 2002), a marble burying assay was used to examine CIE-induced

changes in anxiety/compulsive-like behaviors in C57 mice. This task is a nontraditional

test for anxiety-like behavior, and appears to measure some of the negative affective

components of ethanol withdrawal, including anxiety-like and compulsive-like behaviors

(Nicolas et al., 2006; Perez & De Biasi, 2015). During ethanol abstinence, CIE-exposed

mice exhibited increased marble burying compared to air-exposed controls. Since this

CIE procedure also augmented ethanol drinking and preference, it is possible that CIE-

exposed mice increase their ethanol intake to reduce the negative reinforcing effects of

CIE exposure and withdrawal.

KOR blockade attenuated CIE-induced increases in ethanol consumption, preference

and marble burying.

After finding CIE-induced elevations in ethanol intake and marble burying, the role

of KORs in both behaviors was tested. A single systemic administration of norBNI

ameliorated CIE-induced increases in ethanol consumption and preference. This effect

was maintained over the five-day test, consistent with reports documenting a long-lasting

effect of norBNI (Kishioka et al, 2013). Further, norBNI treatment attenuated CIE-

induced increases in marble burying. Additional work in the present study showed that

KOR activation with U50,488 increased ethanol drinking and preference in CIE-naïve

mice. Moreover, a single dose of U50,488 increased the number of marbles buried,

showing increased anxiety/compulsive-like behavior following KOR activation.

92

Together, these data suggest that KORs play a role in ethanol withdrawal-associated

negative affective symptoms and augmented drinking, and suggest that pharmacological

blockade of KORs could potentially serve a therapeutic role in withdrawn alcoholics.

Increased anxiety/compulsive-like behavior, opioid receptor signaling and dopamine

transmission following prolonged ethanol exposure are strikingly similar to studies

examining different sources of negative affective states. In particular, negative affective

states engendered by chronic stress or drug exposure have been tied to elevated KOR

activity. For example, symptoms of withdrawal from ethanol (Walker et al., 2011; Nealey

et al., 2011) or cocaine (Chartoff et al., 2012), social isolation rearing (Karkhanis et al.,

submitted), social defeat stress (McLaughlin et al., 2006) and separation of pair-bonded

prairie voles (Resendez et al., 2012) are rescued with KOR blockade. It is likely that

KOR activity may be a ubiquitous mediator of negative affect and stress, although

elevated KOR activity is a part of an extensive stress response system that also includes

elevated levels of cortocotropin releasing factor (CRF, Britton et al., 2000). In fact, the

CRF and KOR systems often play similar roles in stress responses (Britton et al., 2000;

Lu et al., 2003), which has led to the hypothesis that the CRF- and KOR-systems are

functionally linked (Van’t Veer et al., 2012).

Ethanol and opioids

Acute ethanol administration increases both endorphin (Olive et al., 2001) and

dynorphin levels (Marinelli et al., 2006; Lindholm et al., 2000), activating both mu

opioid receptors (MOR) and KORs simultaneously. This would result in opposing effects

on the mesolimbic dopamine system, as MOR activation increases, and KOR activation

reduces, accumbal dopamine transmission (Spanagel et al., 1992, current study).

93

Augmented accumbal dopamine levels in response to acute ethanol are due, in part, to

increased inhibitory MOR activity on ventral tegmental area GABAergic interneurons,

which causes a disinhibition of dopaminergic neurons in this region (Bergevin et al.,

2002). Conversely, chronic ethanol exposure reduces MOR activity (Chen & Lawrence,

2000) while augmenting KOR signaling (Kissler et al., 2014, present work), promoting

an overall deficiency in reward system function (Koob et al., 2013). The neurochemical

changes associated with chronic ethanol exposure may produce negative affective

symptoms specifically by increasing KOR sensitivity and reducing dopamine system

function. The present data support this hypothesis, as reduced dopamine transmission and

increased KOR signaling are associated with augmented anxiety/compulsive-like

behavior and ethanol intake after a period of abstinence, and further because KOR

blockade reduces CIE-induced changes in behavior to control levels.

Currently, only one opioid receptor antagonist, naltrexone, is approved for alcohol

use disorders by the Food and Drug Administration (Center for Substance Abuse

Treatment, 2009). Naltrexone is an antagonist of all three classical opioid receptors

(MOR, KOR, DOR). Because of the opposing functional effects of MORs, DORs and

KORs, the usefulness of a pan-opioid antagonist in treating alcoholics may be reduced, as

inhibition of all opioid receptors may decrease the positive reinforcing effects of natural

rewards as well as ethanol, thus decreasing drug-taking compliance. For example, in

humans, naltrexone decreased reward-related brain activation in response to palatable

foods (Murray et al., 2014) as well as overall food (Yeomans & Wright, 1991) and

sucrose (Fantino et al., 1986) intake. Additionally, naltrexone produces an aversive state

in monkeys (Williams & Woods, 1999), healthy humans and alcoholics (Swift et al.,

94

1994). Since the present data show that KOR blockade is sufficient to reduce

anxiety/compulsive-like behavior and ethanol drinking in CIE-exposed mice to non-

dependent levels, a KOR-specific antagonist may be fully efficacious at reducing relapse

drinking in the human population while sparing the positive reinforcing salience of

natural rewards (Fantino et al., 1986; Murray et al., 2014).

The effects of CIE exposure on accumbal dopamine transmission

Chronic ethanol exposure consistently reduces dopamine system function. For

example, previous work from our laboratory showed that CIE exposure of mice and rats

attenuates dopamine transmission by reducing dopamine release and increasing the rate

of dopamine uptake (Budygin et al., 2007; Karkhanis et al., 2015; current work), which is

congruent with findings of reduced limbic system function in human alcoholics compared

to control subjects (Volkow et al., 2002). These functional changes in dopamine

transmission may reflect changes in the expression levels of proteins such as dopamine

transporters, tyrosine hydroxylase, vesicular monoamine transporters or other

components of the machinery regulating dopamine release and reuptake. Further studies

will be needed to define the underlying mechanisms of the functional changes

documented here.

Chronic ethanol exposure and dynorphin levels

Despite consistent results across laboratories showing increased KOR-system

function after chronic ethanol exposure, and the present work demonstrating augmented

KOR function at the level of the dopamine terminal, there is controversy regarding the

status of dynorphin levels after ethanol exposure. Some studies suggest that acute ethanol

95

increases dynorphin in the NAc (Marinelli et al., 2006; Lindholm et al., 2000) and that

these effects may be long-lasting (Lindholm et al., 2000). Consistent with reduced

dopamine system function following CIE, it has been hypothesized that extended

exposure to ethanol may desensitize TrkB receptors on D1-containing accumbal

GABAergic medium spiny neurons (Logrip et al., 2008), one of the postsynaptic

receptors that regulate dynorphin release. This would result in reduced dynorphin levels

and supersensitive KORs, as reported here. However, one manuscript demonstrated a

transient elevation of prodynorphin levels in the NAc after chronic ethanol exposure

(Przewlocka et al., 1997) and another suggested that CIE exposure increased KOR

sensitivity and dynorphin levels simultaneously in the central nucleus of the amygdala

(Kissler et al., 2014). These data show that the effects of ethanol exposure and

withdrawal on dynorphin levels are time-dependent, as Kissler and colleagues (2014)

examined dynorphin and KOR levels six hours into withdrawal in rats while Przewlocka

et al (1997) found elevated prodynorphin levels 24-48 hours after the final ethanol

exposure that returned to control levels by 96 hours. We report increased KOR function

72 hours into ethanol withdrawal in mice, which may or may not coincide with

augmented dynorphin levels. These findings suggest that the temporal profile of

dynorphin and KOR changes may be important in understanding how chronic ethanol

alters KOR signaling.

Conclusions

Human alcoholics report relapse to alcohol drinking in an effort to reduce withdrawal

symptoms experienced during abstinence (Wetterling et al., 2006). As mounting evidence

supports the involvement of accumbal KORs in the development of anxiety/compulsive-

96

like behaviors and dependence-induced ethanol drinking (Nealey et al., 2011; Ballester-

Gonzáles et al., 2015), chronic ethanol-induced reductions in dopamine transmission and

increased KOR sensitivity in the NAc may promote the development of negative

affective behavioral phenotypes associated with ethanol withdrawal. As such, our work

supports a growing body of literature suggesting the potential utility of a KOR-antagonist

to rescue chronic ethanol-induced changes in behavior and neurobiology.

Funding

This work was supported by the National Institutes of Health (grant numbers: F31

DA03558 to JHR, T32 AA007565 21 to DG and ANK, DA006634 to RC, UO1

AA020929 to MFL, P50AA010761 to HCB P01 AA021099 to BAM and SRJ, R01

AA014445 and U01 AA020942 to BAM and U01 AA014091 to SRJ).

Acknowledgements

Nor-binaltorphimine was generously provided by the National Institute of Drug Abuse.

The Statement of Interest

None.

Author Contributions

JHR, SRJ, BAM, RC, HCB and MFL developed experiments. JHR and DG executed

experiments. JHR analyzed and graphed all data. JHR, ANK and SRJ wrote the paper.

All authors listed sufficiently contributed to and edited the manuscript before submission.

97

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CHAPTER II: SUPPLEMENTARY MATERIALS

Methods:

Ethanol Drinking Tests

Baseline drinking procedures were established prior to chronic intermittent ethanol

(CIE) exposure followed that described in-text. Ethanol drinking was examined for five

days between each CIE cycle (“CIE/Air+Drinking Protocol”; Figure S1A). Blood

samples for blood ethanol concentration (BEC) determination were collected twice per

CIE cycle (see below).

Blood collection and analysis for BEC determination

Blood samples of CIE-exposed mice were obtained immediately after removal from

the vapor chamber on the mornings after the first and final exposure per cycle. A

submandibular venipuncture was performed to collect each blood sample. Samples were

collected in microtainer tubes lined with lithium heparin (Becton Dickinson & Company,

Franklin Lakes, NJ) before processing. Blood collection volumes did not reach the

maximum set forth by the Institutional Animal Care and Use Committee at Wake Forest

University School of Medicine. For BEC measurement, standards and samples were

prepared using a commercially available alcohol dehydrogenase assay (Carolina Liquid

Chemistries Corporation, Brea, CA). Five microliters of each blood collection was placed

in a clean eppendorf tube with 45μl of trichlorocetic acid solution (Sigma Aldrich, St.

Louis, MO), and centrifuged at room temperature for ten minutes (10,000 rev/min).

Thirty microliters of the supernatant from each sample was removed and placed into a

105

separate eppendorf tube with the buffer and enzymatic reagent provided in the alcohol

dehydrogenase assay. Each standard and sample was loaded in triplicate into a 96 well

pre-read plate before 15-minute incubation at 37OC. Immediately following incubation,

the plate was analyzed with SoftMax Pro Software, Version 5 (Molecular Devices

Corporation, Sunnyvale, CA).

Locomotor Analysis

Changes in locomotor activity after systemic norBNI administration was examined as

an additional control to ensure the pharmacological effects of norBNI did not inhibit

locomotion. Locomotor activity was assessed via infrared beam breaks in automated

locomotor activity monitors (20 cm × 20 cm × 20 cm; Med Associates, St. Albans, VT).

(Day 1) To allow for chamber habituation, naïve mice were placed into activity chambers

and monitored for 120 minutes. To desensitize mice to injection stress, mice were

injected with saline (0.1 mL) twice, and activity was recorded for 60 minutes after each.

Mice were injected with norBNI (10mg/kg) immediately after the second 60 minute

recording session. (Day 2) Twenty-four hours following, mice were placed in the same

chambers for 120 minutes followed by two saline injections. Locomotor activity was

measured as distance traveled (in centimeters) and as a percent of each animal’s pre-

norBNI second saline injection. The second saline injection on Day 2 (open circles) was

plotted relative to the second saline injection on Day 1 (closed circles).

Data analysis

Graph Pad Prism (version 5, La Jolla, CA) was used to create figures and analyze all

data. Column statistics were run on group BEC data. Two-way repeated measures (RM)

106

analysis of variance (ANOVA) was used to examine ethanol drinking and preference

between inhalation groups over time, and analyze pre- versus post- norBNI effects on

locomotion. One way ANOVA was used to detect any effect of time on ethanol drinking

in inhalation and drug treatment groups. Student’s t-tests were used to analyze percent

pre-norBNI distance traveled.

Results:

CIE Exposure: CIE/Air+Drinking Protocol

CIE exposure followed the in-text description with the addition of a two-bottle choice

procedure (15% ethanol and water) for five days after each cycle. Each CIE/Air exposure

cycle was followed by one-five-day drinking test (1 cycle). Each cycle was repeated four

times. An additional (fifth) inhalation exposure and 72-hour withdrawal period, without

the following drinking test, was added for neurobiological testing (Figure S1A). Blood

samples were collected twice per inhalation cycle to examine BECs (BEC=200.4 ± 62.76

mg/dL, Figure S1B).

Two-tailed Student’s t-tests confirmed similar pre-inhalation ethanol consumption

between groups (Figure S1C: t14=0.4556, p>0.05). RM two-way ANOVA revealed

increased ethanol consumption across drinking tests (Figure S1D: F4, 13=11.90, p<0.001),

and as a consequence of inhalation treatment (F1, 13=18.64, p<0.001). An interaction

between inhalation treatment and drinking test was also detected (F4, 13=2.981, p<0.05).

Bonferroni post-hoc analysis revealed increased ethanol intake in CIE-treated mice at the

fourth ethanol drinking test (p<0.001).

107

108

Supplementary Figure 1: CIE/Air+Drinking Protocol

(1A) Mice underwent a two bottle choice drinking procedure: two weeks of sucrose fade

(Samson, 1986; [blue bar] two days each: 10% ethanol/5% sucrose, 12% ethanol/5%

sucrose, 15% ethanol/5% sucrose, 15% ethanol/2%, and 15% ethanol/1% sucrose [w/v]),

followed by four additional weeks of access to 15% (v/v) ethanol [left purple bar]. Mice

were exposed to ethanol vapor or room air (green bars) for four days (16 hours of

exposure [red bars]/8 hours of abstinence [dark blue bars]), followed by 72 hours of room

air exposure [orange bar]. Following each inhalation cycle, animals were given two-

bottle choice (15% ethanol and water) for five days [purple bars]. The ethanol and air

inhalation exposure and five-day drinking test schedule was repeated four times, with a

fifth inhalation exposure and 72 hour withdrawal period added for neurobiological

testing. (1B) BECs of CIE-treated mice. (1C) Pre-inhalation ethanol consumption was

similar between groups, but CIE exposure (1D) increased ethanol consumption across

drinking tests, and as a consequence of inhalation treatment. Post-hoc analysis revealed

increased ethanol intake in CIE-treated mice at the fourth ethanol drinking test. (S1E)

Ethanol preference was similar between inhalation groups, but (1F) increased over

weeks, particularly at the fourth drinking test.

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Analysis of ethanol preference revealed similarities between inhalation groups prior

to treatment (Figure S1E, t14=1.574, p>0.05). RM two-way ANOVA showed increased

preference over weeks in CIE-treated compared to air-treated mice (Figure S1F:

F4,14=10.19, p<0.0001) and a main effect of inhalation treatment (F1,14=14.67, p<0.01).

Bonferroni Post-hoc analysis revealed increased ethanol preference between inhalation

groups at the fourth drinking test (p<0.001).

Blood ethanol concentrations reached physiologically relevant levels

Column statistics revealed physiologically relevant BECs in CIE-exposed mice in the

Back-to-back CIE/Air exposure protocols (204.9 ± 58.47 mg/dL).

Supplementary Figure 2: Back-to-back CIE protocol blood ethanol concentrations

BECs in mice CIE-exposed were physiologically and behaviorally relevant.

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Supplementary Figure 3: CIE-induced increases in ethanol drinking and post-norBNI

ethanol drinking was stable over the five day drinking test.

To ensure a consistent effect of inhalation exposure and norBNI administration over

the five day drinking test, drinking levels from animals systemically administered with

norBNI were analyzed. Two-way RM ANOVA revealed a main effect of inhalation and

drug treatment (F3,28=20.92, p<0.0001), as well as an effect of time (F4,28=3.591, p<0.01)

on ethanol consumption over the five day test. An interaction between inhalation/drug

treatment and time was also detected (F12,28=2.007, p<0.05). Bonferroni post hoc analysis

revealed a significant difference in ethanol intake between CIE-exposed mice treated

with vehicle and CIE-exposed mice treated with nor-BNI ([denoted with asterisks (*)]

Day 1: p<0.01; Day 2: p<0.001; Day 3: p<0.001; Day 4: p<0.01). A significant

difference between CIE-exposed mice treated with vehicle and air-exposed animals

treated with vehicle ([denoted with ampersands (&)] Days 2 and 3: p<0.001; Day 4:

p<0.01), and air-exposed mice treated with norBNI ([denoted with carrots (^)] Days 1-3:

p<0.001; Day 4: p<0.01; Day 5: p<0.05) were also detected. Using one-way ANOVAs,

the effects of time within each treatment group were examined. Data showed that ethanol

consumption did not escalate over the five day drinking test in air (F4,25=0.064, p>0.05)

or CIE (F4,30=2.680, p>0.05) exposed, vehicle treated animals. Similarly, no effect of

time was detected in norBNI treated air (F4,40=0.0212, p>0.05) or CIE (F4,45=0.0617,

p>0.05) exposed animals.

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Supplementary Figure 3: CIE-induced increases in ethanol drinking and post-

norBNI ethanol drinking was stable over the five day drinking test.

Analysis of inhalation and drug treatment over time revealed increased ethanol intake

following CIE-exposeure and saline treatment compared to and CIE-exposed mice treated

with norBNI (*), differences between CIE-exposed mice treated with saline and air-

exposed animals treated with saline (&), and differences between CIE-exposed mice

treated with saline and air-exposed mice treated with norBNI (^). Ethanol consumption

did not escalate over the five day drinking test. ^,&

p<0.05; **,^^ p<0.01; ***, &&&

, ^^^

p<0.001.

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NorBNI does not alter locomotor behavior.

To ensure the effect of norBNI on ethanol drinking and marble burying was not due

to absolute changes in locomotion, mice were systemically injected with norBNI

(10.0mg/kg) and tested 24 hours for changes in locomotion to a systemic saline injection.

Raw data are presented after the second saline injection (see above). Two-way RM

ANOVA revealed no difference between pre and post nor-BNI locomotor behavior in

response to a saline injection (F1,13=0.7964, p>0.05). However, an effect of time was

detected (F5,13=37.63, p<0.0001), indicating a habituation of the mice to the locomotor

chambers after injection on both days. Data were transformed to analyze within-subject

changes in locomotion, and a two-tailed Student’s t-test detected no change in locomotor

behavior (inset: t13=0.6525, p>0.05).

Supplementary Figure 4: norBNI does not affect locomotor behavior.

Raw data are presented after the second saline injection on Day 1 (closed circles) and

Day 2 (open circles). There was no difference between pre- and post- norBNI locomotion

to a saline injection, although an effect of time was detected. (Inset) Histogram

demonstrating similarities between pre- and post-norBNI injection.

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CHAPTER III

FUNCTIONAL INTERACTION BETWEEN DOPAMINE D3 AND KAPPA OPIOID

RECEPTORS ON DOPAMINE TERMINALS AND PARALLEL INCREASES IN

RECEPTOR SENSITIVITY FOLLOWING CHRONIC ETHANOL EXPOSURE

Jamie H. Rose, Anushree N. Karkhanis, Sara R. Jones

This manuscript is in preparation for submission to the

Journal of Neurochemistry

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Abstract

Background: Chronic intermittent ethanol (CIE) exposure reduces dopamine

transmission in the nucleus accumbens (NAc) core. D2-type dopamine autoreceptors

(D2R, D3R) and kappa opioid receptors (KOR), located on dopamine terminals, inhibit

dopamine release, and are supersensitive following CIE, likely contributing to low

dopamine activity. D2Rs and D3Rs are differentially modulated by chronic ethanol

exposure, although differences in autoreceptor function are not well understood. Further,

dopamine autoreceptors have been suggested to functionally associate with KORs,

although the location of this link is unknown. Thus, CIE-induced alterations in

autoreceptor and KOR system sensitivity, and interactions between these receptor

systems were explored.

Methods: Male C57BL/6 mice were sacrificed following five weeks of CIE- or air-

exposure for NAc slice voltammetry experiments. Concentration response curves (CRC)

of the agonists quinpirole (D2R/D3R), sumanirole (D2R) and (+)-PD128907 (D3R) were

used to assess CIE-induced alterations in autoreceptor function. KOR sensitivity was

measured using U50,488. To probe D2R/D3R and KOR interactions, brain slices from

naïve mice were pretreated with the antagonists raclopride (D2R/D3R), L-741,626

(D2R), U99-194 (D3R), SB-02771A (D3R) prior to CRCs of U50,488. Nor-

binaltorphimine was used to block KORs before a (+)-PD128907 CRC.

Results: CIE reduced dopamine neurotransmission and augmented KOR and D2R/D3R

function. Increased autoreceptor sensitivity was driven selectively by D3Rs. Investigation

into autoreceptor-KOR interactions revealed that D3R blockade occluded the dopamine-

decreasing effects of KOR activation, without reciprocal effects of the KOR antagonist.

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Interpretation: As CIE-induced increases in D3R function and D3R blockade reduced

the dopamine-decreasing effects of KOR activation, D3Rs may be a promising

pharmacotherapeutic target for alcohol use disorders.

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Introduction

Considerable evidence points to a marked disruption in dopamine neurotransmission

following chronic ethanol exposure, particularly in the nucleus accumbens (Zapata et al.,

2006; Zapata & Shippenberg, 2006; Budygin et al., 2007; Karkhanis et al., 2015; Rose et

al., 2015). Chronic ethanol-induced reductions in dopamine transmission in this region

may be due to attenuated dopamine cell firing (Shen & Chiodo, 1993; Shen et al., 2007,

but see: Diana et al., 1995; Brodie, 2002) or deficient terminal function (Carroll et al.,

2006; Budygin et al., 2007; Karkhanis et al., 2015; Rose et al., 2015). Several inhibitory

receptors found on presynaptic dopamine nerve terminals have been shown to have

increased activity following chronic ethanol exposure, including D2-type autoreceptors

(D2Rs and D3Rs) and kappa opioid receptors (KOR; Perra et al., 2011; Kivell et al.,

2014; Karkhanis et al., 2015; Rose et al., 2015; Siciliano et al., 2015). These data suggest

that presynaptic dopamine autoreceptors and, or KORs may be driving chronic ethanol-

induced reductions in dopamine transmission following chronic ethanol exposure and

withdrawal.

Although CIE exposure functionally augments D2R-type autoreceptor activity (Perra

et al., 2011; Karkhanis et al., 2015), it is not known whether alterations in the sensitivity

of one or both autoreceptor subtypes mediate this change. For example, one

autoradiography study found that D2Rs in the high-affinity, functional state were

elevated for at least eight days following cessation of 10 days of twice-daily injections of

ethanol in Sprague-Dawley rats (2.0 g/kg; Seeman et al., 2004); however, this study used

sulpiride, a D2R/D3R antagonist, for binding studies, indicating that D3Rs may also be

upregulated during ethanol withdrawal. Some evidence points to a specific involvement

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of D3Rs in alcohol use disorders. For example, upregulated D3R mRNA levels following

chronic ethanol consumption following one year of oral ethanol consumption in Wistar

rats (Vengeliene et al., 2006), and increased D3Rs have been observed in abstinent

alcoholics (Erritzoe et al., 2014). As these studies are variable in their ethanol exposure

paradigms and receptor measures, an examination of CIE-induced alterations in both

D2R and D3R function at the level of the dopamine terminal using a single protocol

would assist in a further understanding of these changes.

Similar to chronic ethanol-induced alterations in D3R levels (Vengeliene et al., 2006;

Erritzoe et al., 2014), some studies suggest augmented KOR sensitivity following chronic

ethanol exposure and withdrawal (Kivell et al., 2014; Rose et al., 2015; Siciliano et al.,

2015), and some evidence suggests a functional relationship between dopamine

autoreceptors and KORs. In fact, repeated KOR activation reduced pre- and post-synaptic

D2R/D3R expression (Izenwasser et al., 1998) and function (Acri et al., 2011). However,

it is possible that this effect is a post-synaptic phenomenon or mediated by D2Rs or

D3Rs. Notably, pharmacological co-activation of D2R/D3Rs and KORs drive

anxiety/compulsive-like ethanol withdrawal phenotypes noted in rodents (Perreault et al.,

2006, 2007; Dvorkin et al., 2010; Ballester-Gonzales et al., 2015). In sum, these findings

suggest that simultaneous activation of autoreceptors and KORs may produce unique

alterations in dopamine signaling via a functional connection. However, it is not known

which of the dopamine autoreceptors, D2Rs or D3Rs, may be involved in KOR

interactions.

The present study used ex vivo fast scan cyclic voltammetry (FSCV) to identify

specific changes in D2R, D3R and KOR sensitivity following five weeks of CIE or air

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exposure in C57BL/6J (C57) mice. To identify potential functional interactions between

D2-type dopamine autoreceptors and KORs, experiments using blockade of one receptor

and activation of others were executed in naïve mice. Overall, these investigations

revealed that D3Rs, but not D2Rs, and KORs are functionally upregulated following CIE,

and that D3Rs and KORs on presynaptic dopamine terminals are functionally linked in

the nucleus accumbens core of C57 mice.

Methods

Subjects

Male C57 mice (65-75 days old; Jackson Laboratories, Bar Harbor, ME) were used


cycle (lights on: 02:00) with a red room light illuminated during the dark hours. Animals

were allowed at least one week to acclimate to housing conditions before experiment

start. Mice were provided with standard rodent chow and water ad libitum. Chow from

CIE-exposed cages was replaced the morning following exposure. All experimental

protocols were approved by the Institutional Animal Care and Use Committee at Wake

Forest University School of Medicine. Mice were cared for according to the National

Institutes of Health guidelines in Association for Assessment and Accreditation of

Laboratory Animal Care.

CIE exposure protocol

CIE exposure procedures followed those published previously (Rose et al., 2015).

Briefly, C57 mice were exposed to five cycles of ethanol vapor (CIE) or air, each

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consisting of four days (16 hours of exposure/8 hours of abstinence) and 72 hours of

room air (one cycle). Cycles were repeated five times. All CIE-exposed animals were

treated with 1.6g/kg ethanol (saline in air-exposed mice) and 1.0mmol pyrazole (Sigma

Aldrich, St. Louis, MO), an ethanol dehydrogenase inhibitor, 30 minutes prior to

chamber start time (17:00). Blood ethanol concentrations (BEC) were tested the mornings

after the first and fourth night of ethanol exposure per cycle, at chamber stop time

(09:00).

Blood collection and BEC analysis

A submandibular vein blood draw used to collect no more than 15µL of blood from

one to two mice per BEC collection day. BECs were determined with a commercially

available alcohol dehydrogenase assay, derived from a standard calibration curve that

was made fresh from reagents provided in the kit (Carolina Liquid Chemistries

Corporation, Brea, CA).

Ex vivo FSCV concentration response curves

The use of ex vivo FSCV allowed for examination of dopamine terminal function and

receptor sensitivity on a sub-second timescale (Ferris et al., 2013). Using this technique,

pre-drug dopamine release and uptake kinetics, as well as effects of D2R, D3R and KOR

activation and blockade on dopamine transmission in the nucleus accumbens, were

examined 72 hours after the cessation of five cycles of CIE or air exposure. Mice were

anesthetized with isoflurane until unresponsive and rapidly decapitated. Brains were

removed and placed into ice-cold oxygenated artificial cerebrospinal fluid (aCSF)

containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), CaCl2 (2.4), MgCl2 (1.2),

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NaHCO3 (25), glucose (11), L-ascorbic acid (0.4) and pH was adjusted to 7.4. Brain

slices containing the nucleus accumbens core were prepared (300µm thick) with a

vibrating tissue slicer and incubated in oxygenated aCSF, heated to 32oC, until

experiment start. A carbon fiber recording electrode (≈30-50µm length, 7µm radius;

Goodfellow Corporation, Berwyn, PA), and a bipolar stimulating electrode (Plastics One,

Roanoke, VA) were placed in close proximity (≤100µm) on the surface of the slice. A

single, rectangular, 4.0ms long electrical pulse (350µA, monophasic; inter-stimulus

interval: 180sec) was used to elicit endogenous dopamine release. The concentration of

dopamine released was detected by applying a triangular waveform (-0.4 to +1.2 to -0.4V

vs. silver/silver chloride, 400 V/sec) every 100ms to the recording electrode. Background

current subtraction methods were utilized to obtain clear current versus time plots.

Calibration of recording electrodes was performed following each experiment using a

flow-injection system and a known concentration of dopamine (3.0µM).

When pre-drug collections were stable for three consecutive stimulations, quinpirole

(0.01-0.3µM), a D2R/D3R agonist, sumanirole (0.01-1.0µM), a D2R-specific agonist,

(+)-PD 128907 (0.01-1.0µM), a D3R-specific agonist or U50,488 (0.01-1.0µM), a KOR

agonist was added cumulatively to bath solution (aCSF). To explore the possibility of a

functional interaction between D2R/D3Rs and KORs, accumbal brain slices from naïve

mice were incubated in raclopride (100nM), a D2R/D3R antagonist, L-741,626 (30nM), a

D2R-specific antagonist, U99-194 (2,3-Dihydro-5,6-dimethoxy-N, N-dipropyl-1H-inden-

2-amine maleate, 1.0µM) or SB-02771A (N-[trans-4-[2-(6-Cyano-3,4-dihydro-2(1H)-

isoquinolinyl)ethyl]cyclohexyl]-4-quinolinecarboxamide dihydrochloride, 300nM), both

D3R antagonists, or nor-binaltorphimine (norBNI, generously provided by NIDA,

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Bethesda, MD 1.0µM), a KOR antagonist, for 60 minutes prior to the addition of

cumulative concentrations of either U50,488 (0.01-1.0µM), on D2R or DR3 antagonist-

treated slices or (+)-PD 128907 (0.01-1.0µM), on norBNI pretreated slices. All

compounds were obtained at the highest quality from Tocris Bioscience (Bristol, UK),

unless otherwise noted.

Data Analysis

Collection and analysis of all FSCV data was completed with Demon Voltammetry

and Analysis software (Yorgason et al., 2011). To determine CIE exposure-induced

changes in dopamine release and uptake, representative signals were analyzed before

drug application. Dopamine release (μM) was quantitated as the amount of dopamine

released per electrical stimulation and dopamine clearance was calculated as the maximal

rate of uptake at the dopamine transporter (Vmax; μM/sec). The effect of each receptor on

dopamine release was calculated as a percent pre-drug dopamine release. In experiments

where brain slices were pretreated with an antagonist, effects of subsequent agonist

treatment were analyzed as a percent of dopamine release after antagonist, and the effects

of antagonist on dopamine release were quantitated and compared to pre-drug dopamine

release.

All graphs were created and statistical tests were applied using GraphPad Prism,

Version 5 (La Jolla, CA). Effects of CIE exposure on dopamine release, U50,488 IC50

and antagonist-induced alterations in dopamine release were analyzed with two-tailed

Student’s t-tests. All concentration response curves were examined with repeated

measures (RM) two-way analysis of variance (ANOVA), with inhalation exposure or

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antagonist pre-treatment and drug concentration as factors. When a significant main

effect was found, Bonferroni post-hoc analysis was applied.

Results

CIE exposure promotes a hypodopaminergic state of the nucleus accumbens

core.

Ex vivo FSCV was used to examine the effects of CIE and air exposure on dopamine

release and uptake in the nucleus accumbens core. Analysis of group data with two-tailed

Student’s t-test replicated previous work from our lab (Karkhanis et al., 2015; Rose et al.,

2015), showing attenuated dopamine release (t9=3.487, p<0.01) and increased uptake

rates (t9=3.252, p<0.01) in brain slices collected from CIE-exposed mice compared to

those from air-treated controls (data not shown).

CIE exposure selectively increased the sensitivity of D3Rs to agonist

Functional changes in D2R and D3R autoreceptors following five cycles of CIE exposure

were first investigated with quinpirole, a D2R/D3R agonist. A two-way RM ANOVA,

with inhalation treatment and quinpirole concentration as factors, revealed a significant

effect of quinpirole (Figure 1A, F3,8=45.63, p<0.0001) and inhalation treatment

(F1,8=7.377, p<0.05) on dopamine release, although no interaction between these factors

was detected (F3,8=0.596 p>0.05).

Sumanirole, a D2R-specific agonist, and (+)-PD 128907, a D3R-specific agonist,

were used to examine the function of each receptor after CIE exposure. A two-way RM

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Figure 1: Increased accumbal D2R/D3R sensitivity in CIE-exposed mice was driven

by D3Rs.

(A) Autoreceptor sensitivity is greater in CIE exposed mice compared to air-treated

controls. Receptor-specific ligands were used to discern the receptor(s) driving this

effect. (B) Increased autoreceptor sensitivity was not driven by D2Rs, but (C) likely

driven by D3Rs, as evidenced by augmented sensitivity of this autoreceptor subtype

following CIE. *p<0.05

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ANOVA examining the effects of D2R activation on dopamine release showed that

sumanirole concentration-dependently reduced dopamine release (Figure 1B, F4,8=54.74,

p<0.0001), although this effect was similar between inhalation groups (F1,9=0.078,

p>0.05). Similarly, a two-way RM ANOVA revealed significant dopamine-decreasing

effects of D3R activation (Figure 1C, F4,9=34.51, p<0.0001), although this effect was

greater in brain slices from CIE exposed mice (F1,9=16.20, p<0.01). An interaction

between these factors was not detected (F4,9=0.57, p>0.05). Bonferroni post hoc analysis

revealed a significant difference between CIE and air-treated mice at the 0.03µM

concentration of (+)-PD 128907 (p<0.05).

CIE increased accumbal KOR sensitivity to agonist treatment compared to air-

exposed controls

To examine the effects of CIE exposure on KOR function, increasing concentrations

of U50,488, a KOR-specific agonist, were added to brain slices containing the nucleus

accumbens core. A two-way RM ANOVA, with drug concentration and inhalation

treatment as factors, revealed the concentration-dependent reduction of dopamine release

by U50,488 in brain slices from both groups (Figure 2A, F4,10=76.56, p<0.0001), which

was significantly greater in the CIE exposed mice (F1,10=75.89, p<0.0001). An interaction

between inhalation treatment and U50,488 concentration was also detected (F4,10=3.910,

p<0.01). Bonferroni post hoc analysis revealed a significant difference in dopamine

release between inhalation groups at the 0.03µM (p<0.05), 0.1µM, 0.3µM and 1.0µM

(p<0.0001) U50,488 concentrations. Two-tailed Student’s t-test of the IC50 of U50,488

revealed a significant reduction in the IC50 of CIE-treated mice (Figure 2B, t9=3.065,

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Figure 2: Augmented KOR function in the NAc of CIE-exposed mice compared to

air-exposed controls.

(A) KOR activation more effectively reduces dopamine release in brain slices harvested

from CIE-exposed mice compared to air-exposed controls. (B) IC50 analysis of the effects

of KOR activation on dopamine release showed an increased potency of U50,488 in brain

slices from CIE-exposed animals, underscoring the augmented efficacy of KOR

activation on dopamine release in this group. *p<0.05; ***p<0.001.

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p<0.05), underscoring the increased potency of the drug in this group observed in Figure

2A.

D2R/D3R blockade attenuated the dopamine-decreasing effects of KOR

activation

To investigate the possibility of a functional interaction between autoreceptors and

KORs, a separate group of brain slices from naïve mice were incubated in raclopride,

D2R/D3R antagonist, for 60 minutes prior to probing KOR function with increasing

concentrations of U50,488. Raclopride had no effect on baseline dopamine release, as

confirmed by a Student’s t-test between these two points (t9=0.474, p>0.05, data not

shown). A two-way RM ANOVA revealed the dopamine-decreasing effects of U50,488

(Figure 3A, F5,9=8.730, p<0.0001) which were blocked in slices pretreated with

raclopride (F1,9=8.368, p<0.05). An interaction between pretreatment (raclopride vs

aCSF) and U50,488 concentration was also detected (F5,9=4.060, p<0.01). Bonferroni

post hoc analysis revealed a significant difference between brain slices pretreated with

raclopride and non-pretreated brain slices at the 0.3µM (p<0.05) and 1.0µM (p<0.001)

U50,488 concentrations. These data suggest a functional interaction between

autoreceptors and KORs.

Reduced effect of KOR activation by D2R/D3R blockade was not mediated by

D2Rs.

To identify whether the raclopride-induced attenuation of U50,488 effects on

dopamine release were specifically driven by D2Rs, additional brain slices were

incubated in L-741,626, a D2R-specific antagonist, for 60 minutes prior to the U50,488

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Figure 3: D2R/D3R blockade attenuated the dopamine-decreasing effects of KOR

activation

To investigate a functional relationship between KORs and autoreceptors, occlusion

experiments targeting each receptor were executed. (A) Raclopride attenuated the

dopamine-decreasing effects of U50,488. (B) L-741,626 did not influence the effects of

U50,488 on dopamine release. *p<0.05; ***p<0.001.

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concentration response curve. Per a Student’s t-test, L-741,626 had no effect on pre-drug

dopamine release (t10=0338, p>0.05, data not shown). U50,488 significantly decreased

dopamine release across the concentration response curve in both groups (Figure 3B,

F5,10=49.11, p<0.0001), which was not influenced by L-741,626 pretreatment

(F1,10=0.023, p>0.05), suggesting that the attenuation of KOR-activation on dopamine

release observed following raclopride pretreatment was not mediated via D2Rs.

D3 blockade reduced the dopamine-decreasing effects of KOR activation

To investigate a functional interaction between KORs and D3Rs, U99-194, a D3R-

specific antagonist, was applied to slices for 60 minutes prior to the U50,488

concentration response curve. U99-194 did not alter baseline dopamine release

(t10=1.440, p>0.05, data not shown). A two way RM ANOVA showed that U50,488

reduced dopamine release in both pretreatment groups (Figure 4A, F5,10=40.99,

p<0.0001), although this effect was significantly attenuated in brain slices pretreated with

U99-194 (F1,10=36.92, p<0.0001). A significant interaction between these factors was

also detected (F5,10=3.152, p<0.05). Bonferroni post hoc analysis revealed significant

differences between U99-194 pretreated and non-pretreated brain slices at the 0.03µM,

0.1µM, 0.3µM and 1.0µM (p<0.001) concentrations.

Data shown in Figure 4A suggest a functional interaction between D3Rs and KORs.

In confirmation, a separate group of brain slices were incubated in SB-02771A, a

molecularly dissimilar D3R-specific antagonist from U99-194. SB-02771A had no effect

on baseline dopamine release (t10=0.887, p>0.05, data not shown). A two-way RM

ANOVA revealed U50,488 concentration-dependent reductions in dopamine release

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Figure 4: D3 blockade reduced the dopamine-decreasing effects of KOR activation

To identify the effects of D3R blockade on KOR function, two molecularly dissimilar

D3R antagonists were bath applied to brain slices prior to KOR probe. (A) U99-194

reduced the dopamine-decreasing effects of U50,488. (B) SB-02771A also reduced the

dopamine-decreasing effects of KOR activation. (C) KOR blockade also did not alter the

dopamine-decreasing effects of D3R activation. *p<0.05; ***p<0.001.

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(Figure 4B, F5,10=71.35, p<0.0001), which was significantly attenuated by SB-02771A

pretreatment (F1,10=7.174, p<0.05). An interaction between drug pretreatment and

U50,488 concentration was also detected (F5,10=3.839, p<0.01). Bonferroni post-hoc

analysis revealed significant differences in dopamine release between pretreatment

groups at the 0.03µM and 1.0µM (p<0.05) U50,488 concentrations.

KOR blockade did not attenuate the effects of D3R activation on dopamine

release.

As D3R blockade attenuated the dopamine-decreasing effects of KOR activation, we

next blocked KORs across a D3R agonist concentration response curve. KOR blockade

had no effect on baseline dopamine release (t7=1.092, p>0.05, data not shown). Although

(+)-PD 128907 reduced dopamine release in both groups (Figure 4C, F5,7=49.75,

p<0.0001), there was no effect of norBNI pretreatment on D3R activation (F1,7=4.951,

p>0.05).

Discussion

Although chronic ethanol exposure and withdrawal functionally downregulate the

mesolimbic dopamine system in mice (Zapata et al, 2006; Karkhanis et al., 2015), rats

(Carroll et al., 2006; Budygin et al., 2007) and humans (Volkow et al., 1996, 2007), the

underlying causes that produce this change are not fully understood. The present series of

experiments sought to identify specific changes in D2R, D3R and KOR sensitivity to

agonist following five weeks of CIE exposure in C57 mice and explore a functional

relationship between these receptors. We showed that D3Rs, but not D2Rs, and KORs

were more sensitive in brain slices from CIE exposed animals compared to control mice.

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An investigation into whether autoreceptors and KORs were functionally associated

revealed that D3R blockade attenuated the dopamine-decreasing effects of KOR

activation, however the converse, blocking KORs, did not reduce the inhibitory effects of

D3R agonists on dopamine release. As D3Rs are functionally hypersensitive to agonist

following CIE, and D3R blockade obliterates the dopamine-decreasing effects of KOR

activation, it is plausible that D3Rs may be a promising target in alcohol use disorders.

CIE-induced increases in D3R and KOR sensitivity

Recently, we showed that quinpirole, a non-selective D2/D3 dopamine receptor

agonist, reduced dopamine release to a greater extent in brain slices from CIE-exposed

mice than their air-exposed counterparts (Karkhanis et al., 2015). To ascertain whether

this effect was driven by D2Rs or D3Rs, we probed each receptor with a more selective

agonist. We found augmented sensitivity of D3Rs, but not D2Rs, after CIE exposure,

suggesting that CIE exposure-induced increases in autoreceptor function (Karkhanis et

al., 2015; present work) are driven exclusively by D3Rs. Increased D3R sensitivity may

reflect an increase in the number of receptors on dopamine terminals. Consistent with this

hypothesis, chronic ethanol consumption produced an increase in D3R mRNA

(Vengeliene et al.,2006) and receptor (Jeanblanc et al., 2006) levels in the striatum of

mice. Notably, systemic treatment with a D3R antagonist reduced ethanol intake in a

model of alcohol relapse (Vengeliene et al., 2006), and genetic D3R deletion (Bahi &

Dreyer, 2014; Leggio et al., 2014) or pharmacological blockade (Leggio et al., 2014)

reduced ethanol intake in naïve mice. Together, these data suggest that increased D3R

activity may contribute to excessive ethanol drinking, and indicate that D3R antagonists

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may provide a promising pharmacotherapeutic target to treat alcohol use disorders,

although the influence of D3R ligands specifically on alcohol use disorders in the human

condition has not been directly explored in humans (Takaki & Ujike, 2013).

A functional association between D3Rs and KORs

Some evidence suggests a functional relationship between autoreceptors and KORs.

In fact, repeated KOR activation reduces expression (Izenwasser et al., 1998) and

function (Acri et al., 2011) of D2R/D3Rs. Further, pharmacological co-activation of

D2R/D3Rs and KORs promotes the development of anxiety/compulsive-like checking

behavior in rats (Perreault et al., 2006, 2007; Dvorkin et al., 2010; Ballester-Gonzales et

al., 2015). Therefore, a functional connection between receptors may exist and alter

accumbal dopamine signaling to drive this behavioral phenotype (Dvorkin et al., 2010;

Ballester-Gonzales et al., 2015). To explore a functional interaction between D3Rs and

KORs, we used two pharmacologically distinct D3R antagonists, and found that D3R

blockade occluded the dopamine-decreasing effects of KOR activation. We hypothesize

that KOR blockade did not reduce D3R sensitivity to agonist for two primary reasons:

First, the D3R/KOR interaction may be an ordered effect, such that pharmacological

manipulation of D3Rs must occur before there is a measurable effect on KOR function.

This hypothesis posits that D3Rs and KORs may be physically interacting, and an

allosteric change in D3Rs due to antagonist binding (Lane et al., 2013) may cause a

subsequent conformational shift in the associated KORs that could occlude proper KOR

agonist binding. Second, it is possible that D3Rs and KORs do not physically interact,

but their signaling cascades may interact. As both receptors are Gαi/o-coupled, D3Rs and

KORs share similar intracellular signaling molecules (Ahlgren-Beckendorf & Levant,

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2004; Bruchas & Chavkin, 2010). Ligands that target specific downstream pathways of

G-protein coupled receptors are informative experimental tools and may be helpful in

additional investigation of this hypothesis (Zhou et al., 2013; Lovell et al., 2015).

D3R-KORs may be simultaneously upregulated via receptor for activated C

kinase 1 (RACK1)

Our voltammetric data show augmented sensitivity of both D3Rs and KORs to

agonist following CIE exposure. It is possible that this parallel increase in receptor

sensitivity is due to an intracellular protein kinase C (PKC) scaffolding protein, RACK1.

In an elegant series of papers, Dorit Ron’s laboratory has described the activating effects

of ethanol administration on RACK1 and the results of this activation on D3R regulation

and brain derived neurotrophic factor (BDNF)/tyrosine receptor kinase B (TrkB)

signaling (McGough et al., 2004; Jeanblanc et al., 2006; Logrip et al., 2008). Briefly,

acute ethanol exposure increases RACK1 levels and translocation to the nucleus (Ron et

al., 2000; McGough et al., 2004). In the nucleus, RACK1 phosphorylates cAMP response

element-binding protein (CREB; Wan et al., 2009) and increases the transcription of

downstream genetic products. One of the results of this cascade is augmented D3R

expression and BDNF levels in the striatum (Jeanblanc et al., 2006; Logrip et al., 2008).

Activation of TrkB receptors by BDNF, particularly on postsynaptic dopamine D1

receptor-containing medium spiny neurons (Strömberg & Humpel, 1995; Koo et al.,

2014), drives increased prodynorphin production. Further, while acute ethanol exposure

increases dynorphin levels (Lindholm et al., 2000; Marinelli et al., 2006), Logrip and

colleagues (2008) suggested that a sustained increase in BDNF levels, due repeated

increases in RACK1 over continued ethanol exposures, would reduce TrkB sensitivity. In

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conjunction with reduced D1R activation from attenuated dopamine transmission

following CIE exposure (Karkhanis et al., 2015; Rose et al., 2015), this would attenuate

subsequent downstream signaling and dynorphin release (Logrip et al., 2008), resulting in

a compensatory increase in KOR sensitivity as seen here (present work).

To evaluate one aspect of this hypothesis, we measured the levels of dynorphin in

CIE treated mice, our laboratory utilized a dynorphin enzyme-linked immunosorbent

assay (ELISA) of accumbal tissues from C57 mice exposed to five weeks of CIE. Levels

of dynorphin in nearly all CIE-exposed animals were below the detectable range of the

kit (>1.0 picogram), whereas air-exposed samples were largely within range (data not

shown). As such, a statistical assessment of CIE-induced alterations in dynorphin was not

possible, although we anecdotally suggest that five weeks of CIE exposure reduces

dynorphin levels in the nucleus accumbens of C57 mice. Taken together, we hypothesize

that the changes shown here may be due to ethanol-induced upregulation of RACK1

signaling that drives increased D3R expression and BDNF production, and ultimately

reduces dynorphin levels and increases the sensitivity of KORs on dopamine terminals

(McGough et al., 2004; Jeanblanc et al., 2006; Logrip et al., 2008).

Summary and Conclusions

Converging cross-species evidence has shown that chronic ethanol exposure and

withdrawal attenuates dopamine terminal function (Volkow et al., 1996, 2007; Carroll et

al., 2006; Budygin et al., 2007; Karkhanis et al., 2015; present work), and increased D2-

type receptor (Karkhanis et al., 2015; present work) and KOR sensitivity to agonist

(Kissler et al., 2004; Rose et al., 2015; Siciliano et al., 2015; present work). Evidence

presented here has identified CIE-induced increases in D3R function, in addition to

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augmented KOR sensitivity, furthering our current understanding of autoreceptor changes

following chronic ethanol and withdrawal. Moreover, we have identified a functional

interaction between these receptors, which may be due to a physical coupling between

receptors or connection between downstream signaling pathways. Based on the present

observation that D3Rs are upregulated following CIE exposure and blockade of these

receptors obliterates the dopamine-decreasing effects of KOR agonists,

pharmacotherapeutics targeting these receptors, namely D3Rs, may restore appropriate

dopamine transmission and alleviate some of the negative reinforcing effects of chronic

ethanol use.

Acknowledgements

Nor-binaltorphimine was generously donated by the National Institute of Drug Abuse.

Funding: F31 DA035558 (JHR); T32 AA007565 (ANK); P01 AA017056, U01

AA014091 (SRJ).

Conflict of Interest

The authors declare no conflict of interest.

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CHAPTER IV

DISTINCT EFFECTS OF NALMEFENE ON DOPAMINE TERMINAL FUNCTION

AND KAPPA OPIOID RECEPTOR ACTIVITY IN THE NUCLEUS ACCUMBENS

FOLLOWING CHRONIC INTERMITTENT ETHANOL EXPOSURE

Jamie H. Rose, Anushree K. Karkhanis, Björn Steiniger-Brach, Sara R. Jones

This manuscript is in preparation for submission to the

European Journal of Neuroscience

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Abstract

The development of pharmacotherapies aimed at reducing relapse to ethanol drinking in

alcoholics is of considerable research interest. Preclinical data support a role for nucleus

accumbens (NAc) kappa opioid receptors (KOR) in chronic intermittent ethanol (CIE)

exposure-induced increases in ethanol intake. Nalmefene, a high-affinity KOR partial

agonist reduces heavy alcohol drinking in at-risk patients, potentially due to its effects on

accumbal KORs; however, the effects of nalmefene on dopamine transmission and KOR

function in the NAc are not well understood. The present study investigated the effects of

nalmefene on dopamine transmission and KOR function in accumbal brain slices from

male C57BL/6J mice following five weeks of CIE or air exposure using fast scan cyclic

voltammetry. Nalmefene concentration-dependently reduced dopamine release similarly

in both inhalation groups, but attenuated dopamine uptake rates to a greater extent in

slices from CIE-exposed mice compared to controls. Similarities in nalmefene-induced

reductions in dopamine release suggest that dyno7rphin tone is not present in brain slices,

and the distinct effects of nalmefene on uptake rates suggest that dopamine transporter

interactions with KORs may be fundamentally altered following CIE. Further, we found

that nalmefene reversed the dopamine-decreasing effects of a maximal concentration of a

KOR agonist selectively in brain slices of CIE-exposed mice, suggesting that CIE-

exposure may alter the number or activity of KORs in the presence of an agonist.

Modulation of dopamine transmission via KORs by nalmefene may attenuate CIE-

induced reductions in dopamine transmission in vivo, and attenuate withdrawal-induced

increases in ethanol consumption.

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1.0 Introduction

Chronic alcohol (ethanol) use disorders are an enormous economic and financial

burden in the United States (Substance Abuse and Mental Health Services

Administration, 2014). A large body of literature across many species has shown that

chronic alcohol exposure downregulates dopamine transmission in the nucleus

accumbens (mice: Karkhanis et al., 2015; rats: Carroll et al., 2006; Budygin et al., 2007;

humans: Volkow et al., 1992, 2006), potentially leading to the negative affective states

experienced during withdrawal (Schulteis et al., 1995; Boutros et al., 2014). One of the

potential candidates that underlies attenuated dopamine neurotransmission following

chronic ethanol exposure and withdrawal is increased function of inhibitory receptors on

dopamine terminals in the nucleus accumbens (Karkhanis et al., 2015; Rose et al., 2015;

Siciliano et al., 2015), namely kappa opioid receptors (KOR; Kissler et al., 2014; Rose et

al., 2015; Siciliano et al., 2015). Augmented accumbal KOR function may mediate CIE-

induced reductions in dopamine terminal function and upregulated ethanol consumption

following ethanol exposure and withdrawal (Walker & Koob, 2008; Nealey et al., 2011).

In fact, intra-accumbal KOR blockade reduces relapse-drinking behavior in rodents

(Nealey et al., 2011). These data suggest that KORs may be a promising

pharmacotherapeutic target to reduce relapse drinking in human alcoholics. With no

highly efficacious pharmacotherapeutic agent for alcoholism (Jørgensen et al., 2011;

Yoshimura et al., 2014; Mann et al., 2004; Higuchi et al., 2015; Latt et al., 2002;

O’Malley et al., 2015; Garbutt et al., 2005), considerable research efforts have been put

forth to gather evidence to support novel pharmacotherapies for this indication,

146

particularly those that target KORs (Walker & Koob, 2008; Nealey et al., 2011; Walker

et al., 2014).

Nalmefene (6-methylene naltrexone) is a pharmacotherapeutic agent recently

approved (March, 2013) in some European countries to combat heavy alcohol drinking in

at-risk patients (European Medicines Agency’s Committee for Medicinal Products for

Human Use, 2012). Preclinical work using intra-accumbal infusions of nalmefene

showed that this compound reduced ethanol intake at lower doses in ethanol-dependent

ethanol self-administering rats than non-dependent animals (Nealey et al., 2011), an

effect that was credited to the partial agonist activity and high affinity of nalmefene at

KORs (Bart et al., 2005; Nealey et al., 2011). Despite this behavioral evidence, the

effects of nalmefene on dopamine terminal and accumbal KOR function following

chronic ethanol exposure are poorly understood, and elucidation of its effects may aid in

understanding the basis of its clinical efficacy. To this end, ex vivo fast scan cyclic

voltammetry (FSCV) was used to evaluate the effects of nalmefene on dopamine

transmission and KOR function in the nucleus accumbens core, 72 hours following five

weeks of chronic intermittent ethanol (CIE) exposure in male C57BL/6 (C57) mice.

2.0 Experimental Procedures

2.1 Subjects

Male C57 mice (6-8 weeks old, Jackson Labs, Bar Harbor, ME) were used for all

experiments. Animals were allowed at least one week of habituation to the housing

environment before CIE procedures began. All mice were individually housed and

maintained on a 12-hr light-dark cycle (lights off at 14:00), with a red-room light

147

illuminated during the animals’ dark cycle. Standard rodent chow and water were

available ad libitum, and replaced daily in the CIE exposed group. Animals were cared

for according to the National Institutes of Health guidelines in Association for

Assessment and Accreditation of Laboratory Animal Care, and all experimental protocols

were approved by the Institutional Animal Care and Use Committee at Wake Forest

University School of Medicine.

2.2 CIE exposure

Mice were exposed to ethanol vapor (CIE) or room air for 16 hours/day, followed by

8 hours of room air. This procedure was repeated four times before a 72-hour abstinence

period (one cycle). Cycles were repeated five times. Approximately 30 minutes prior to

chamber start time (17:00), mice in both groups were systemically administered with

1.0mmol/kg pyrazole (Sigma-Aldrich, St. Louis, MO), an alcohol dehydrogenase

inhibitor, mixed with 1.6g/kg ethanol (CIE exposure group) or saline (air inhalation

group). Blood ethanol concentrations (BEC) were verified the mornings after the first and

final inhalation exposure per each cycle.

2.3 BEC measurement

To ensure proper ethanol chamber function and physiologically relevant BECs, a

submandibular vein blood draw was performed. Less than 15µL of blood was collected in

BD microtainer tubes lined with lithium heparin (Becton Dickinson & Company,

Franklin Lakes, NJ). For BEC measurement, standards and samples were prepared with a

commercially available alcohol dehydrogenase assay (Carolina Liquid Chemistries

148

Corporation, Brea, CA), carefully pipetted into a 96 well plate and analyzed with

SoftMax Pro Software version 5 (Molecular Devices Corporation, Sunnyvale, CA).

2.4 Ex vivo FSCV

FSCV was used to detect CIE-induced changes in dopamine release and uptake, as

well as the effects of nalmefene, U50,488, and nor-binaltorphimine (norBNI), a KOR-

specific agonist and antagonist, respectively, on these measures. Briefly, 300µm thick

coronal brain slices containing the nucleus accumbens core were prepared using a

vibrating tissue slicer. Slices were incubated in oxygenated artificial cerebrospinal fluid

(aCSF), heated to 32oC for approximately 60 minutes prior to experiment start. A bipolar

stimulating electrode (Plastics One, Roanoke, VA) and carbon fiber microelectrode

(≈50µm length, 7µm radius (Goodfellow Corporation, Berwyn, PA) were placed within

100µm of each other on the surface of the slice. Dopamine efflux was induced by a

single, rectangular, electrical pulse (4.0ms; 350µA, monophasic; interstimulus interval:

180sec), and detected by applying a triangular waveform every 100ms to the recording

electrode (-0.4 to +1.2 to -0.4 V vs. Ag/AgCl, 400V/sec). When baseline collections were

stable for three consecutive stimulations, nalmefene (1.0-100.0µM, generously provided

by H. Lundbeck A/S), norBNI (1.0µM, graciously provided by NIDA) U50,488 (0.01-

0.3µM, generously provided by NIDA) were cumulatively added to the bath. Following

the maximal concentration of U50,488 (0.3µM), a challenge concentration of nalmefene

(10.0μM) was added to the bath solution. In a separate set of experiments, a single dose

of norBNI (1.0µM) was applied to brain slices to identify the effects of KOR blockade on

dopamine release and uptake measures. Clear current versus time plots were obtained

using background current subtraction methods. Electrodes were calibrated immediately

149

after experiments by recording their response (in nA) to a known concentration of

dopamine (3.0μM) using a flow-injection system.

2.5 Data analysis

Representative pre-drug traces of electrically-stimulated dopamine release were

individually modeled and data were collapsed across inhalation groups to obtain baseline

dopamine release and uptake measures. Dopamine release was calculated as the amount

of electrically-evoked dopamine released per stimulation, and Vmax was calculated as the

maximal rate of uptake at the dopamine transporter. The apparent affinity of dopamine

for the dopamine transporter (apparent Km) remained constant at 160nM throughout

analysis (Wu et al., 2001). The effects of KOR ligands on dopamine release and uptake

parameters were similarly analyzed. Demon Voltammetry and Analysis software

(Yorgason et al., 2011) was used to collect and analyze all data.

Graphs were created and statistical analyses were computed with GraphPad Prism

(version 5, La Jolla, CA). Student’s t-tests were used to analyze the effects of inhalation

exposure on dopamine release and uptake rates. Repeated measures (RM) two-way

analysis of variance (ANOVA) was used to determine the effects of increasing

concentrations of U50,488 and nalmefene on dopamine release and uptake, with

inhalation exposure and drug concentration as factors. Additionally, non-RM two-way

ANOVAs, with inhalation exposure and drug concentration as factors, were used to

determine the ability of nalmefene to reverse the dopamine-decreasing effects of the

0.3µM U50,488 concentration, the effects of nalmefene on the maximal rate of uptake

following 0.3µM U50,488, as well as the effects of 1.0µM norBNI on dopamine release

150

and uptake parameters. When a significant main effect was detected, Bonferroni post-hoc

analysis was applied.

3.0 Results

3.1 CIE exposure reduced dopamine transmission in the nucleus accumbens

core

CIE exposed mice had behaviorally and physiologically relevant BECs

(219.50±39.31mg/dL; Griffin et al., 2009, data not shown). Representative FSCV traces

are overlaid in Figure 1A (Air: blue trace; CIE: red trace). Consistent with previous work

from our laboratory (Karkhanis et al., 2015), CIE reduced dopamine release (Figure 1B,

t18=2.38 p<0.05; Air: 1.06±0.26 µM; CIE: 0.71±0.40µM) and increased rates of

dopamine uptake (Figure 1C, t17=3.80, p<0.05; Air: 1.52±0.32µM/sec; CIE:

2.46±0.68µM/sec).

3.2 Nalmefene slowed dopamine uptake rates more in brain slices from CIE-

exposed mice than controls

To examine the effects of nalmefene on dopamine release and uptake, increasing

concentrations of this compound were bath-applied to accumbal brain slices. Two-way

RM ANOVA revealed a main effect of nalmefene concentration on dopamine release

(Figure 2A, F4,9=48.34, p<0.001), which was similar between inhalation groups

(F1,9=7.27, p>0.05). Additionally, nalmefene dose-dependently reduced uptake rates in

both groups (Figure 2B, F4,8=5.29, p<0.01), although this effect was greater in CIE

151

Figure 1: CIE exposure reduced dopamine signaling in the nucleus accumbens core

Representative FSCV traces are overlaid in Figure 1A (Air: blue trace; CIE: red trace).

(1B) CIE reduced dopamine release in accumbal slices from CIE-exposed mice compared

to their air-exposed counterparts. (1C) CIE increased dopamine uptake rates (Vmax) in

brain slices from CIE-exposed mice compared to controls. *p<0.05, **p<0.05

152

153

Figure 2: Nalmefene attenuates the uptake rates only in brain slices from CIE-

exposed mice

(2A) Nalmefene concentration-dependently decreased dopamine release, which was

similar between inhalation groups. (2B) Similarly, nalmefene dose-dependently

attenuated Vmax in both groups, although this effect was greater in brain slices from CIE

exposed animals compared to air-exposed mice. (2C) The KOR antagonist,

norbinaltorphimine (norBNI) did not alter dopamine release. (2D) NorBNI reduced

uptake rates in brain slices from CIE-exposed mice compared to controls. *p<0.05.

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exposed animals (F1,8=7.94 p<0.05). No interaction between these factors was detected

(F4,8=5.40, p>0.05).

A separate group of brain slices were incubated in norBNI for 60 minutes to assess

the effects of KOR blockade on dopamine transmission. Two-way ANOVA revealed no

effect of norBNI on dopamine release (Figure 2C, F1,24=0.12, p>0.05); however, a main

effect of inhalation treatment on uptake rates (Figure 2D, F1,26=4.40, p<0.05) was

detected. Bonferroni post-hoc analysis revealed a significant difference between the

effects of norBNI on CIE-exposed mice compared to air-exposed controls (p<0.05).

3.3 Nalmefene reversed the dopamine-decreasing effects of U50,488 in CIE-

exposed mice

Representative traces showing the effects of 0.3µM U50,488 on dopamine release and

uptake (Figure 3A, blue, air; Figure 3B, red, CIE) and 10.0µM nalmefene reversal (black

line, overlaid). The effects of CIE on KOR function were examined with increasing

concentrations of U50,488. A RM two-way ANOVA revealed a main effect of KOR

activation on dopamine release that was dose-dependent (Figure 3C, F3,10=31.69,

p<0.001), which was greater in brain slices from mice exposed to CIE (F1,10=6.26,

p<0.05). An interaction between these factors was also detected (F3,10=5.51, p<0.01).

Bonferroni post hoc analysis revealed a significant difference between inhalation groups

at the 0.1µM (p<0.01) and 0.3µM (p<0.05) U50,488 concentrations.

As nalmefene is a partial KOR agonist, it competes for the ligand binding site on the

receptor (Bart et al., 2005; Zhu et al., 2005). To determine the ability of nalmefene to

reverse the dopamine-decreasing effects of KOR activation, 10.0µM of the compound

was added to the bath solution following the 0.3µM concentration of U50,488. A two-

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Figure 3: Nalmefene reversed the dopamine-decreasing effects of U50,488 in

CIE-exposed mice

(3A) Representative traces of the effects of 0.3µM U50,488 on dopamine release in brain

slices from air (solid blue) and (3B) CIE (solid red) exposed mice. The black line in 3A

and 3B (overlaid) represents the effect of 10.0µM nalmefene on these signals. (3C) KOR

activation with U50,488 dose-dependently reduced dopamine release greater in brain

slices from CIE-exposed mice compared to controls. (3D) 10µM nalmefene restored

dopamine release following a maximal concentration of U50,488 in brain slices from

CIE-exposed mice to control levels. (3E, 3F) U50,488 did not alter dopamine uptake rates

across the concentration response curve, and nalmefene had no additional effect on

uptake rates in either inhalation group. *p<0.05

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way ANOVA, with U50,488 and nalmefene as factors revealed a main effect of drug

(U50,488 vs nalmefene; Figure 3D, F1,10=5.67, p<0.05), as well as an interaction between

drug and inhalation treatment (F1,10=6.19, p<0.05). An effect of inhalation treatment was

not detected (F1,10=2.46, p>0.05). Bonferroni post hoc analysis revealed a significant

increase in dopamine release due to nalmefene reversal in the CIE-exposed group

compared to the 0.3µM U50,488 concentration (p<0.01).

As nalmefene reduced dopamine uptake rates to a greater extent in brain slices from

CIE-exposed mice compared to air-exposed controls, we examined the effects of a

nalmefene challenge on U50,488-induced alterations in dopamine uptake rates.

Surprisingly, a two way RM ANOVA revealed no effect of U50,488 on dopamine uptake

rates (Figure 3E, F3,8=1.26, p>0.05) in either inhalation group (F1,8=0.17, p>0.05).

Further, a two-way RM ANOVA revealed that nalmefene had no effect on dopamine

uptake rates following the maximal (0.3µM) concentration of U50,488 (Figure 3F,

F1,16=0.04, p>0.05).

4.0 Discussion

The present study aimed to discern the pharmacological effects of nalmefene on

dopamine terminal and KOR function following CIE exposure in mice. Congruent with

previous work (Carroll et al., 2006; Budygin et al., 2007; Karkhanis et al., 2015; Rose et

al., 2015), CIE exposure reduced dopamine release, augmented rates of dopamine uptake

and KOR sensitivity to agonist, which is consistent with a hypodopaminergic state of the

nucleus accumbens. Nalmefene concentration-dependently reduced dopamine release

similarly between inhalation conditions, but attenuated uptake rates more in brain slices

from CIE-exposed mice compared to controls. Additionally, we found that a single

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concentration of nalmefene reversed the dopamine-decreasing effects of a maximal

concentration of U50,488 selectively in brain slices from CIE-exposed mice. These data

are the first to demonstrate dopamine terminal modulation by nalmefene and point to

mechanisms that may underlie nalmefene-induced reductions in ethanol intake following

CIE exposure (Walker & Koob, 2008; Nealey et al., 2011).

4.1 Nalmefene reduced dopamine release equally in both inhalation groups, but

attenuated dopamine uptake rates more in brain slices of CIE-exposed mice

Our data show similar dopamine-decreasing effects of nalmefene between inhalation

groups, suggesting two interpretations. First, these data indicate that dynorphin tone may

not be present in striatal brain slices. If dynorphin tone were present, nalmefene would

compete with the endogenous ligand for receptor occupancy (Calvey & Williams, 2009),

resulting in antagonist-like effects. This effect would be measured voltammetrically as an

increase in stimulated dopamine release (Spanagel et al., 1992). Notably, control

experiments using norBNI in this study showed no effect of KOR blockade on dopamine

release in either inhalation group, further suggesting that dynorphin tone is not

measurable in brain slices. As such, it is possible that nalmefene was acting as an agonist

at KORs to reduce dopamine release (Bart et al., 2005).

In addition to being a partial KOR agonist, nalmefene is also a MOR and DOR

antagonist (Bart et al., 2005), making definitive designation of its effects to any one

opioid receptor difficult. However, DORs are thought to specifically regulate

postsynaptic signaling the nucleus accumbens (Dilts & Kalivas, 1990), thus DOR-

modulation of dopamine transmission is unlikely. MORs are primarily localized to

GABAergic interneurons that feed onto dopamine terminals (Svingos et al., 1997).

159

Therefore, it is also unlikely that MOR blockade drove the dopamine-decreasing effects

of nalmefene observed here, as disinhibition of dopamine terminals by GABAergic

interneurons (Svingos et al., 1997) would most likely result in an increase in stimulated

dopamine release. Even so the influence of nalmefene modulation of DORs/MORs on

dopamine transmission is not known in this context and some contribution of these

receptors to the present findings cannot be entirely ruled out.

Although the effects of nalmefene on dopamine release were similar between

inhalation groups, this compound concentration-dependently reduced dopamine uptake

rates more in brain slices from CIE-exposed mice compared to controls. In this instance,

it appears as if nalmefene is acting to reduce dopamine release and uptake through KORs.

Notably, dopamine transporters have been consistently reported to be functionally

upregulated following CIE exposure (Carroll et al., 2006; Budygin et al., 2007;

Karkhanis et al., 2015; present work). Here, we found that KOR blockade slowed uptake

rates in brain slices from CIE-exposed mice compared to air-exposed controls using a

single concentration of norBNI, which may be due to its effects on KOR signaling

cascades (Bruchas & Chavkin, 2010). A recent study showed that KORs exist both

independently and in complex with dopamine transporters, and regulate dopamine

transporter function via an ERK1/2-dependent pathway (Kivell et al., 2014). Therefore,

we hypothesize that the physical or functional connection between dopamine transporters

and KORs (Kivell et al., 2014) is fundamentally altered following CIE, driving KOR

antagonist-mediated reductions in uptake rates in the present experiments. However, we

also showed that KOR activation did not augment (Thompson et al., 2000; Kivell et al.,

2014; Simonson et al., 2015) or attenuate (Das et al., 1994; Chudapongse et al., 2003)

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dopamine uptake rates as reported previously, but eliminated nalmefene-induced

reductions in dopamine uptake. Additional investigations into the interplay of U50,488

and nalmefene on KOR-induced alterations in dopamine uptake rates are needed to fully

elucidate these findings.

4.2 Dopamine release is restored by nalmefene following KOR activation in

brain slices from CIE-exposed mice

To better understand the distinct effects of nalmefene on KOR function between the

inhalation groups, a middle concentration of nalmefene was applied to brain slices

following a maximal concentration of U50,488. As earlier experiments suggested that

dynorphin tone is undetectable in brain slices, the addition of U50,488 was necessary to

examine any antagonistic effects of nalmefene on KORs. We found that nalmefene

reversed the dopamine-decreasing effects of U50,488 in brain slices from CIE-exposed

mice, suggesting that nalmefene competed with U50,488 for KOR occupancy. It is

possible that this effect is due to CIE-induced receptor upregulation or functional

supersensitivity of KORs compared to air-exposed animals. In other work, CIE exposure

increased levels of KOR binding in seizure-resistant mice (Beadles-Bohling & Wiren,

2005) and augmented dynorphin-stimulated KOR activity in Wistar rats (Kissler et al.,

2014) compared to controls. These data indicate that the observed reversal of the

dopamine-decreasing effects of U50,488 in brain slices from CIE-exposed mice could be

due to physical or functional upregulation of KORs. Experiments that examine these

possibilities with the present model would be helpful in discerning this change.

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4.3 Behavioral implications of the effects of nalmefene on dopamine terminal

function

Intra-cerebroventricular (Walker & Koob, 2008) and intra-accumbal (Nealey et al.,

2011) nalmefene reduces ethanol intake at lower doses in ethanol dependent animals,

compared to non-dependent animals, in part due to its high affinity and the unique action

of this compound on KORs. It is plausible that nalmefene-induced reductions in ethanol

drinking may be due to increased dopamine transmission via attenuated uptake rates and

increased dopamine release, as noted selectively in brain slices from CIE-exposed mice

ex vivo. In fact, dopamine transporter knockout mice, with inherently reduced rates of

dopamine clearance compared to wild-type mice (Jones et al., 1998) consume less

ethanol than their wild-type and heterozygous counterparts (Mittleman et al., 2011),

providing evidence to support this hypothesis.

The time-course of nalmefene-induced alterations in neurobiology is rapid and

beneficial in the therapeutic application of this compound. In fact, ethanol intake in

ethanol dependent rats at lower doses than non-dependent animals (Walker & Koob,

2008) was reduced only 30 minutes prior to ethanol self-administration testing. Similarly,

alcoholics who are prescribed nalmefene take the compound orally when they predict

they will encounter a high-risk situation (i.e. social environments where alcohol may be

present), and consistently report reductions in overall ethanol consumption (Karhuvaara

et al., 2007; Gual et al., 2013; Mann et al., 2013). Use of nalmefene on a continuous

schedule dose-dependently reduces ethanol intake over time (Mason et al., 1994, 1999)

and attenuates relapse to heavy drinking (Mason et al., 1999; Karhuvaara et al., 2007) in

treatment-seeking alcoholics. Together, preclinical and clinical evidence strongly suggest

162

that nalmefene reduces ethanol intake on a rapid timescale, and data presented here

suggest that this may be due to KOR modulation of dopamine transmission.

4.4 Conclusions

Due to the high rate of recidivism to alcoholism, the need for effective

pharmacotherapies for this disorder is high. Modulation of KORs to restore dopamine

system function during alcohol withdrawal is of interest therapeutically (Lichtigfeld &

Gillman, 1996). Overwhelming preclinical behavioral evidence (Walker & Koob, 2008;

Nealey et al., 2011) and clinical work with alcoholics (Mason et al., 1994, 1999;

Karhuvaara et al., 2007; Gual et al., 2013; Mann et al., 2013) indicate favorable

therapeutic effects of nalmefene on ethanol drinking. Notably, the effects of nalmefene

on drinking are due, in part, to its high affinity and partial agonist activity of this

compound on KORs, particularly in the nucleus accumbens (Nealey et al., 2011). We

report that nalmefene reduced dopamine uptake rates and reversed the dopamine-

decreasing effects of KOR activation, suggesting that nalmefene may augment dopamine

transmission in vivo. Increased accumbal dopamine transmission by nalmefene would

attenuate the hypodopaminergic state of this region, namely through reductions in KOR

activity and dopamine uptake rates. These mechanisms may underlie nalmefene-induced

reductions in ethanol intake in ethanol-dependent rodents (Walker & Koob, 2008; Nealey

et al., 2011) and humans (Mason et al., 1994, 1999; Karhuvaara et al., 2007; Gual et al.,

2013; Mann et al., 2013). These data provide an understanding of the pharmacological

effects of nalmefene, and hope to promote understanding of its clinical efficacy.

163

Acknowledgements

Lundbeck A/S, AA014091 (SRJ). F31DA035558 (JHR), T32AA007565 (ANK)

Conflict of Interest

Part of this work was funded by Lundbeck A/S. This corporation provided nalmefene that

was utilized in the present work.

164

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CHAPTER V: DISCUSSION

Overview

The present set of studies aimed to discern the behavioral and neurobiological effects

of five weeks of CIE or air exposure in male C57 mice. To begin, we explored the effects

of CIE exposure on withdrawal-associated behaviors, dopamine terminal and presynaptic

receptor-system function. These experiments revealed CIE-induced increases in ethanol

drinking and anxiety/compulsive-like behavior in the marble burying task, which were

reduced selectively in norBNI-treated, CIE exposed mice. Notably, KOR activation in

naïve mice increased ethanol drinking, preference and marble burying behavior. Data

collected with ex vivo FSCV showed reduced dopamine release, increased dopamine

uptake rates and augmented KOR sensitivity to agonist following CIE exposure. These

data suggest that CIE-induced increases in ethanol drinking and anxiety/compulsive-like

behavior may be due to accumbal KORs.

Based on the heterogeneous nature of the nucleus accumbens, and the variety of

presynaptic dopamine-regulating receptors (Yoshimoto & McBride, 1992; Berridge et al.,

1997; Blaha et al., 1997; Floresco et al., 1998; Del Arco & Mora, 2008), it is likely that

KORs are not the only presynaptic receptor system that is altered following chronic

ethanol exposure. In fact, previous work from our laboratory showed similar reductions

in dopamine release and uptake, in addition to increased autoreceptor system function

following CIE exposure (Karkhanis et al., 2015). In this study, however, a D2R/D3R

agonist, quinpirole, was utilized, making dissociation of CIE-induced changes in D2R

and D3R function difficult. By using receptor-specific agonists, we found a selective

172

upregulation in D3R sensitivity following CIE exposure, with no effect of CIE on D2R

sensitivity to agonist. Additional experiments in this chapter examining CIE-induced

functional alterations in KORs confirmed Chapter II findings showing this receptor’s

supersensitivity to agonist. These data suggest that D3Rs and KORs are both functionally

hyper-responsive following CIE exposure. This effect may be due to a third-party

neuromodulator system, namely RACK1 and BDNF/TrkB signaling (see Section 5.1a).

Investigation into a functional relationship between KORs and autoreceptors showed a

D3R-KOR, but not D2R-KOR, interaction, whereby D3R blockade reduced the

dopamine-decreasing effects of KOR activation. These data are some of the first to show

specific hypersensitivity of D3Rs following CIE, and a functional relationship between

D3Rs and KORs.

The fourth chapter of the present body of work examined the effects of nalmefene, a

partial KOR agonist and MOR/DOR antagonist (Bart et al., 2005), on accumbal

dopamine transmission and KOR activity. We found that nalmefene concentration-

dependently reduced dopamine release similarly in both inhalation groups, suggesting

that dynorphin levels are undetectable in brain slices using fast scan cyclic voltammetry.

Nalmefene also attenuated dopamine uptake rates across the concentration response

curve, and therefore, was acting as an agonist in this assay, although whether or not this

effect was via KORs, DATs or an off-target mechanism is unknown. Notably, nalmefene-

induced reductions in dopamine uptake rates were greater in brain slices collected from

CIE-exposed compared to air-exposed mice. It is possible that these findings are due to a

CIE-induced fundamental alteration in the functional connection between KORs and

dopamine transporters (Kivell et al., 2014). Additional experiments showed that

173

nalmefene reversed the dopamine-decreasing effects of KOR activation in brain slices

from CIE-exposed mice, suggesting that nalmefene competes with the agonist for

receptor occupancy to augment dopamine transmission to control levels (Calvey &

Williams, 2009). The ability of nalmefene to reverse CIE-induced attenuations in

dopamine transmission via increased release and reduced rates of uptake may be

mechanisms by which this compound alters ethanol consumption in CIE-exposed rodents

(Walker & Koob, 2008; Nealey et al., 2011) and humans (Karhuvaara et al., 2007; Gual

et al., 2013; Mann et al., 2013).

In short, the present body of work furthers our understanding of accumbal dopamine

terminal receptor function following CIE exposure in mice, and some of the behavioral

implications of these changes. We hope that this information promotes the development

of new pharmacological agents to combat the withdrawal effects of ethanol exposure,

particularly those that target KORs. Future work should focus on expanding the present

knowledge to use a combination of whole animal studies and in vitro experiments to

rigorously evaluate KORs as a target for the treatment of alcohol use disorders.

Systemic administration of KOR ligands in ethanol drinking and marble burying

experiments: The need for neuroanatomical specificity.

Behavioral and neurobiological data in Chapter II suggest that CIE exposure

augments KOR sensitivity, and that this effect is behaviorally relevant. In fact,

voltammetric experiments revealed an increase in KOR sensitivity to agonist, and we

found norBNI-sensitive CIE-induced increases in ethanol drinking and marble burying

behaviors that were replicated with KOR activation in naïve mice. These studies also

included findings that show specific changes in accumbal KOR function; however, the

174

systemic nature of drug administration during our behavioral studies makes identification

of the precise brain location of CIE-induced alterations of KOR function, as they relate to

CIE exposure-induced behaviors, difficult. Although the present work and additional

evidence from monkeys chronically exposed to ethanol show increased KOR sensitivity

(Siciliano et al., 2015), it is possible that CIE-upregulation of KORs in regions outside of

the nucleus accumbens drive these behavioral phenotypes. In fact, recent work showed an

increase in dynorphin-stimulated KOR activation in the central nucleus of the amygdala

following four weeks of CIE exposure (Kissler et al., 2014). Based on CIE exposure-

induced alterations in the direct and indirect motor pathways (discussed below), it is

hypothesized that intra-nucleus accumbens core administration of norBNI would reduce

ethanol drinking and marble burying behavior in C57 mice following five weeks of CIE,

as this treatment would increase dopamine transmission and regulate downstream

signaling. In fact, some studies have shown that deep brain stimulation of the nucleus

accumbens (Kuhn et al., 2009, 2011) and transcranial magnetic stimulation of the

mesolimbic dopamine system (Ceccanti et al., 2015) both augment dopamine

transmission and reduce ethanol craving and consumption in alcoholics, supporting this

hypothesis.

Neurocircuitry associated with chronic ethanol exposure

The nucleus accumbens is a portion of both the direct and indirect motor pathways,

which are distinguished by D1R and D2R-containing medium spiny projection neurons

that modulate regions within this circuit (Figure 2). Neurotransmission through the

“direct” (D1R), and “indirect” (D2R), or “go” and “no-go” pathways, respectively, may

be fundamentally altered following CIE exposure (Figure 1). Following chronic drugs of

175

abuse, our data suggest that stimulated dopamine release is reduced. Attenuated

dopamine transmission attenuates the activity of D1R direct pathway while increasing

downstream activity of D2R medium spiny neurons (Volkow & Morales, 2015).

Increased D2R medium spiny neuron signaling following chronic alcohol exposure

would reduce GABAergic signaling from the globus pallidus (Sano et al., 2013) and

increase glutamaterigic signaling from subthalamic nucleus, thus augmenting

transmission from GABAergic projections from the ventral midbrain to thalamic nuclei

(Di Chiara et al., 1979; Haegelen et al., 2009; Kase et al., 2015). As such, inhibition of

thalamic signaling to the cortex would reduce glutamatergic signaling from cortical

regions to the striatum. It is likely that low glutamatergic transmission during ethanol

abstinence may confer increased withdrawal symptoms and dysregulated behavioral

outputs. See Figure 1 for a representation of the proposed circuit.

Several pharmacotherapeutics that reduce the withdrawal effects of ethanol regulate

aberrant neurotransmitter signaling in this circuit. For example, alcoholics can be treated

with chlorodiazepoxide (Librium®) and lorazepam (Ativan®), both benzodiazepines,

which augment GABAergic signaling though the basal ganglia (Kuman et al., 2009;

Rajmohan et al., 2013) to reduce some of the symptoms of alcohol withdrawal. These

compounds would reduce the anxiety (Baskin & Esdale, 1982; Meibach et al., 1987;

Diaper et al., 2012) and depression (Lindemann et al., 2015) often associated with

alcohol withdrawal. Similarly, the work presented here showed that behaviors altered by

CIE, ethanol drinking and marble burying, returned to control levels with KOR blockade.

By reducing the inhibitory signaling of KORs on dopamine terminals, dopamine release

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Figure 1: Direct and indirect motor pathways are alteredfollowing chronic ethanol

exposure

Chronic ethanol exposure alters signaling through the direct and indirect motor pathways.

Dopaminergic projections (red arrows) from the ventral midbrain (substantia nigra pars

compacta and ventral tegmental area) are downregulated following chronic ethanol

exposure. These signaling reduce signaling downstream of D1-containing medium spiny

neurons and disinhibits D2R medium spiny neuron signaling through the globus pallidus

externa. Increased GABAergic signaling to the globus pallidus externa reduces that

regions’ output to the substantia nigra pars reticulata and subthalamic nucleus. An

augmentation in the excitatory projection from the subthalamic nucleus increases the

GABAergic output of the substantia nigra pars reticulata to the thalamus. Augmented

GABA signaling to the thalamus from the ventral midbrain reduces the glutamatergic

signaling that projects from this region. Attenuated glutamatergic signaling from the

thalamus to the cortex reduces excitatory input into the striatum.

177

is increased (Spanagel et al., 1992), thus augmenting signaling through the D1R medium

spiny neurons and reducing signaling through the D2R medium spiny neurons, thus

increasing the inhibition on to the ventral midbrain, reducing thalamic output and

attenuating the behavioral manifestations of aberrant basal ganglia function.

In stark contrast to the above hypothetical neurocircuit, a considerable body of

literature suggests that glutamatergic signaling is upregulated following chronic ethanol

exposure. For example, chronic ethanol drinking increases extracellular glutamate levels

and reductions in glutamate clearance in the nucleus accumbens (Dahchour & De Witte,

2003; Ding et al., 2013). Behaviorally, increasing glutamate transmission in the nucleus

accumbens of naïve mice increases ethanol drinking to the levels of their CIE-exposed

counterparts (Griffin et al., 2014). Notably, pharmacotherapeutics that augment GABA

signaling, as noted above, also reduce glutamatergic transmission (Smith et al., 2007),

thus regulating aberrant neurotransmission through the basal ganglia following chronic

ethanol exposure and withdrawal.

Chronic ethanol-induced reductions in dopamine transmission may be due to

attenuated dopamine terminal function

Chronic alcohol exposure in humans (Hietala et al., 1994; Laine et al., 1999; Volkow

et al., 1996, 2002, 2007, 2013) and rodents (Carroll et al., 2006; Budygin et al., 2007;

Karkhanis et al., 2015) reduces dopamine transmission in the basal ganglia, although the

neurobiological loci of this effect are unknown. Chronic ethanol-induced attenuation in

dopamine transmission in these regions may be due to reduced dopamine cell firing

(Shen & Chiodo, 1993; Shen et al., 2007, but see: Diana et al., 1995; Brodie, 2002) or

deficient terminal function (Carroll et al., 2006; Budygin et al., 2007; Karkhanis et al.,

178

2015). In support of reduced dopamine terminal function, we found increased KOR

sensitivity to agonist, which may, in part, drive the reductions in dopamine transmission

observed in the current project. However, dopamine-regulating presynaptic receptors are

not limited to KORs. Thus, to investigate the effects of chronic ethanol exposure

specifically on autoreceptor function, CIE-induced alterations in D2R and D3R

sensitivity to agonist were examined in concert and individually. In support of previous

work (Karkhanis et al., 2015), we found upregulated D2R-type sensitivity to agonist

following CIE exposure, and furthered this finding by demonstrating that this increased

autoreceptor function was driven by selectively by D3Rs. As both dopamine D3Rs

(Maina & Mathews, 2006) and KORs (Spanagel et al., 1992) reduce dopamine

transmission following activation, it is plausible that a CIE-induced increase in the

sensitivity of these receptors could reduce dopamine transmission with low

concentrations of the endogenous ligand. Experiments that expand upon the mechanism

of this change would provide insight CIE-induced alterations in presynaptic receptor

sensitivity.

D3Rs and KORs are hypersensitive following CIE: Hypothetical upregulation

through a third-party neuromodulator

As reviewed in Section 5.1a, ethanol exposure increases the translocation of RACK1

to the nucleus (Yaka et al., 2003; McGough et al., 2004). This movement drives an

increase in D3R gene transcription and membrane expression (Vengeliene et al., 2006) in

addition to an upregulation in BDNF levels (McGough et al., 2004). Release of BDNF

179

180

Figure 2: Effects of chronic ethanol exposure on dopamine terminal function

(A1) Ethanol-induced increases in RACK1 augments D3Rs in presynaptic terminal

boutons and levels of BDNF in the nucleus accumbens. (A2) Increased D3R sensitivity

reduces dopamine release. (A3) Consistently high levels of BDNF reduces the sensitivity

of TrkB receptors and downstream signaling from these receptors. Further, reduced

dopamine release attenuates D1R activation. Together, postsynaptic neuron signaling and

gene transcription is attenuated. (A4) Reductions in CREB signaling due to

downregulated receptor signaling attenuates the transcription of gene products. (A5)

Reduced dynorphin levels drive an upregulation in KOR sensitivity which then reduces

dopamine release.

181

into the extrasynaptic space activates TrkBs on D1R-containing medium spiny neurons

(Strömberg & Humpel, 1995; Logrip et al., 2008). Activation of TrkBs results in the

movement of PKA into the nucleus and the phosphorylation of CREB, driving the

transcription of prodynorphin and other gene products (Muschamp & Carlezon, 2013).

Metabolism of prodynorphin to dynorphin, and subsequent release of the endogenous

KOR ligand results in presynaptic KOR activation (Day et al., 1998; Berman et al., 2000;

Hauser et al., 2005). In this way, both D3Rs and KORs on presynaptic dopaminergic

terminals would be upregulated and mediating dopamine transmission. See figure 2 for a

hypothetical schematic of the effects of chronic ethanol on dopamine terminals.

A hypothetical scenario developed by scientists of Dorit Ron’s laboratory is supported by

some of our data. Logrip and colleagues (2008) suggest that repeated bouts of ethanol

exposure and withdrawal drives consistently high levels of BDNF, subsequently reducing

TrKB sensitivity to its endogenous ligand and downstream signaling, including

dynorphin production (Figure 2; Logrip et al., 2008). In an effort to examine this

possibility, we utilized a dynorphin enzyme-linked immunosorbent assay (ELISA) to

measure levels of this peptide in accumbal tissues from C57 mice following five weeks of

CIE or air exposure. This experiment revealed that levels of dynorphin in nearly all CIE-

exposed animals were lower than air-exposed samples; however, dynorphin levels in

brain samples from CIE-exposed mice were largely below the detectable range of the kit

(>1.0 picogram), whereas nearly all the air-exposed samples were within range. As such,

a statistical assessment of CIE-induced alterations in accumbal dynorphin was not

possible (data not shown). It is suggested that this particular experiment is repeated in

order to draw more conclusive findings. Recent work from the Walker laboratory (Kissler

182

et al., 2014) showed a concomitant increase in KOR sensitivity and dynorphin levels in

the central nucleus of the amygdala following CIE exposure in rats. It is possible that

differences in species (rat vs mouse), ethanol exposure (four weeks vs five weeks) or

length of abstinence before ex vivo experimentation (6 to 8 hours vs 72 hours) underlies

the potential disparate findings in dynorphin levels (Kissler et al., 2014; present study).

The effects of nalmefene on dopamine transmission: Modulation of accumbal

KORs and its clinical utility

To promote the clinical use of nalmefene in the United States and understand the

clinical efficacy of this compound, the fourth chapter of the present document examined

effects of nalmefene on dopamine transmission and KORs. We found that nalmefene

reduced dopamine release similarly between inhalation groups across increasing

concentrations of the compound, while reducing dopamine uptake rates differentially

between inhalation groups. It is hypothesized that differential effects of nalmefene on

maximal rates of uptake between inhalation groups are mediated by a CIE-induced

alteration in the fundamental relationship between KORs and dopamine transporters

(Kivell et al., 2014). Additional work examining the effects of nalmefene following KOR

activation showed that a single concentration of this compound reversed the dopamine

decreasing effects of U50,488 in brain slices from CIE exposed mice to control levels.

These data suggest that nalmefene competed against the ligand (U50,488) for receptor

occupancy, thus reversing dopamine release to control levels. It is possible that this

reversal may be due to upregulated KOR expression or receptor function, although the

precise mechanism cannot be determined by ex vivo voltammetry. Using a GTPγS assays,

one study reported increased dynorphin-stimulated KOR activity in the central nucleus of

183

the amygdala following four weeks of CIE exposure in rats (Kissler et al., 2014).

However, extension of those findings to the present body of work must be done with

caution based on the distinct species, region and ethanol exposure protocols used between

studies.

Nalmefene reduces ethanol drinking in chronic ethanol exposed rodents (Walker &

Koob, 2008; Nealey et al., 2011) and humans (Mason et al., 1999; Karhuvaara et al.,

2007). The rapid timescale by which nalmefene reduces ethanol consumption is

consistent with the effects of pharmacological KOR manipulation on extracellular

dopamine levels (Spanagel et al., 1992). In fact, administration of nalmefene shortly (30

minutes) before an ethanol drinking experiment reduced intake in rats (Walker & Koob,

2008; Nealey et al., 2011) and humans, who often take the compound shortly before

encountering a situation where alcohol may be present, show attenuated ethanol intake

compared to control subjects (Karhuvaara et al., 2007; Gual et al., 2013; Mann et al.,

2013). Our findings show that nalmefene reduced dopamine uptake and reversed the

dopamine-decreasing effects of KOR activation in the presence of an agonist, suggesting

that this compound may also increase dopamine transmission in vivo. It is hypothesized

that augmented dopamine transmission by nalmefene may underlie reductions in ethanol

intake in rodents (Walker & Koob, 2008; Nealey et al., 2011), and humans (Karhuvaara

et al., 2007; Gual et al., 2013; Mann et al., 2013). To this end, experiments that examine

the effects of nalmefene on dopamine transmission in an in-tact animal during ethanol

drinking experiments following chronic ethanol exposure would be informative.

184

The pharmacology of nalmefene versus naltrexone

As reviewed earlier (Section 6.3), naltrexone is a pan-opioid receptor (Bart et al.,

2005), FDA approved antagonist used to reduce alcohol craving and consumption in

alcoholics (Pettinati & Rabinowitz, 2006) following chronic ethanol exposure. However,

naltrexone has been shown to induce negative affective state (Swift et al., 1994; Williams

& Woods, 1999), and decrease the reinforcing efficacy of natural rewards such as

palatable food (Fantino et al., 1986; Yeomans & Wright, 1991; Murray et al., 2014), in

addition to reducing ethanol consumption. These effects may be due to MOR blockade.

As some work suggests that KOR-specific blockade reduces ethanol intake (Walker &

Koob, 2008; Nealey et al., 2011; Walker et al., 2011; present work) and

anxiety/compulsive-like behavior (Valdez & Harshberger, 2012; present work)

specifically in ethanol-dependent animals, without alterations in control animal behavior,

it is plausible that a compound with a greater affinity for KORs would reduce ethanol

intake and withdrawal symptoms more efficaciously than naltrexone.

Nalmefene, a structural congener of naltrexone, has augmented affinity and partial

agonist activity at KORs and reduced affinity and antagonistic activity at MORs and

DORs (Bart et al., 2005). Nalmefene reduced ethanol consumption in ethanol dependent

rodents at lower doses of nalmefene than naltrexone (Walker & Koob, 2008; Nealey et

al., 2011), and has been observed as a direct result of intra-accumbal administration of

the compound (Nealey et al., 2011). The increased ability of nalmefene to reduce ethanol

intake over naltrexone was hypothesized to be due to its high affinity and partial agonist

activity at KORs (Walker & Koob, 2008). Despite the distinct pharmacology of

naltrexone and nalmefene, clinical studies comparing these compounds showed similar

185

effects of on ethanol drinking in human subjects (Drobes et al., 2003, 2004). These data

suggest that the pharmacological differences between nalmefene and naltrexone may

have similar collective effects on neurotransmission in humans. Although the high

affinity and partial agonist effects of nalmefene on KORs are not required to reduce

ethanol intake in humans, nalmefene may reduce the symptoms of ethanol withdrawal

and intake in a subset of alcoholics that do not positively respond to naltrexone.

CERC-501: A novel KOR antagonist in clinical trials for mood and substance

abuse disorders

Preclinical work, including some of the findings shown here, is bolstering the

development of KOR ligands for clinical use. In fact, all rights to a short-acting (Zheng et

al., 2013), orally bioavailable (Rorick-Khan et al., 2014; Jackson et al., 2015) compound

developed by Eli Lilly and Company has been recently sold to Cerecor (February, 2015),

a clinical-stage pharmaceutical company, to develop and commercialize LY2456302

(CERC-501), a KOR antagonist, to combat substance abuse and alcohol use and mood

disorders (Cerecor, 2015). As opposed to the long-lasting effects of common KOR

antagonists used in laboratories today and in the present set of studies, such as norBNI

(Endoh et al., 1992; Broadbear et al., 1994, Kishioka et al., 2013), CERC-501

pharmacologically blocks KORs for less than seven days in rats (Rorick-Kehn et al.,

2014). One study examining the effects of CERC-501 on nicotine withdrawal revealed a

reduction in anxiety-like behavior, hyperalgesia and conditioned place aversion to

withdrawal compared to vehicle treated mice (Jackson et al., 2015), although additional

work is needed to examine the effects of this compound on other drugs of abuse,

including ethanol. Unlike naltrexone and nalmefene, CERC-501 does not display

186

significant activity at MORs and DORs (Lowe et al., 2014; Rorick-Khen et al., 2014),

suggesting that it may not reduce the reinforcing salience of natural rewards (Fantino et

al., 1986; Yeomans & Wright, 1991; Murray et al., 2014) or cause an aversive state in

humans (Swift et al., 1994; Williams & Woods, 1999). In fact, in healthy humans, this

compound appears to be well tolerated, without significant adverse events (Lowe et al.,

2014). Additional work is needed in healthy and diseased populations to fully understand

the pharmacokinetics and pharmacodynamics of this compound in and out of the

presence of ethanol before a large-scale public release is executed.

Ex vivo examination of accumbal dopamine transmission with optogenetic

techniques may improve dopamine release specificity

Ex vivo FSCV in accumbal brain slices allows for presynaptic measurement of

dopamine transmission and investigation of receptor function (Ferris et al., 2013).

However, the nucleus accumbens core is a heterogeneous region with multiple inputs that

converge on medium spiny output neurons, interneurons and dopamine terminals

(Cachope et al., 2012; Laplante et al., 2012; Steinberg et al., 2014), suggesting that

neurotransmitters such as glutamate (Stuber et al., 2010; Fortin et al., 2012), adenosine

(Quarta et al., 2004; Adamah-Biassi et al., 2015) acetylcholine (Bluth et al., 1985) and

GABA (Tepper & Bolam, 2004) may be influencing dopamine transmission and the

dopamine release parameters measured with FSCV.

The effects of electrically-induced neurotransmitter release on dopamine terminal

function are not completely understood. Recent work from our laboratory showed that

selective optogenetic activation of dopamine terminals in the nucleus accumbens released

a greater concentration of dopamine compared to electrical stimulation of the same

187

location (Melchior et al., 2015). It was hypothesized that augmented dopamine release

with optogenetic stimulation may be due to a removal of the inhibitory feedback of

GABAergic interneurons that synapse onto dopamine terminals (Melchior et al., 2015). If

this is the case, it is plausible that selective stimulation of dopamine terminals may reveal

a dopamine signal that is larger than those recorded using electrical stimulation. Other

work has shown that optogenetically-activated acetylcholine interneurons in this region

induce stimulation-independent dopamine release (Cachope et al., 2012). Electrical

stimulation likely induces release of these and other neurotransmitters and

neuromodulators, which may also be clouding the dopamine signals measured with the

present techniques.

Although it is possible that relative differences between electrically and optically

evoked dopamine release may be equal across treatment groups, it is more likely that CIE

also alters interneuron function, thereby altering feedback on dopamine terminals and

dopamine transmission. For example, some work suggests that MORs are physically and

functionally downregulated following CIE exposure (Turchan et al., 1999; Chen &

Lawrence, 2000; Saland et al., 2005). As MORs are expressed in GABAergic

interneurons (Svingos et al., 1997) that typically attenuate dopamine terminal function,

reductions in MOR function may augment GABAergic interneuron firing, thus further

reducing dopamine terminal function. In the case of acetylcholine interneurons, one study

showed reductions in the varicosities of these interneurons in the nucleus accumbens

following chronic ethanol exposure and withdrawal (Pereira et al., 2014). If this change is

functionally relevant, electrical stimulation of the nucleus accumbens may not evoke a

concentration of acetylcholine that influences dopamine transmission. Replication of

188

these studies using voltammetric techniques and negative optogenetic modulators, such

as halorhodopsin in GABAergic and acetycholinergic interneurons, following CIE

exposure would provide insight into the changes associated with each component of the

accumbal milieu and how it affects dopamine terminal function.

Future directions

Molecular underpinnings of CIE-induced alterations in KOR and D3R function are

poorly understood

Our studies clearly demonstrate that CIE reduced dopamine transmission and

increased KOR- and D3R-system sensitivity to agonist. We surmise that reduced

dopamine terminal function may be due to upregulated KOR and D3R function, although

the link between CIE-induced alterations in these receptors and reductions in dopamine

terminal function following chronic ethanol exposure and withdrawal has yet to be

conclusively demonstrated. Examination of CIE-induced changes in each receptor, their

downstream pathways and modulation of calcium and potassium channels may provide

valuable information regarding the effects of CIE on dopamine release. With respect to

CIE-induced alterations in dopamine uptake rates, experiments that examine the

relationship between KORs or D3Rs and dopamine transporters following CIE exposure

are warranted. As one study found that KORs regulate dopamine transporter function via

an ERK1/2-dependent mechanism, and also proposed a physical link between KORs and

dopamine transporters (Kivell et al., 2014), it is plausible that CIE alters downstream

signaling or physical interactions between receptors and dopamine transporters. These

189

experiments may also inform on the effects that nalmefene has on this system and may

shed light on the mechanism underlying nalmefene-induced reductions in uptake rates.

Tandem functional upregulation of KORs and D3Rs following CIE exposure

Similar functional upregulation in KORs and D3Rs following CIE exposure is

hypothesized to be through a third-party neuromodulator, namely RACK1 and the

BDNF/TrkB signaling pathway (Section 5.1a). However, it should be noted that a

considerable portion of this hypothesis is yet to be tested. For example, although it is

hypothesized that high levels of BDNF, due to continued intermittent ethanol exposure,

would reduce TrkB sensitivity to this endogenous ligand, it is unknown whether or not

CIE drives augmented levels of BDNF and reductions in TrkB sensitivity. Furthermore,

attenuated presynaptic dopamine release, as measured presently, should attenuate D1R

activation, which, together with reduced TrkB signaling, would reduce the downstream

genetic products of these receptors, such as prodynorphin. However, it is unknown

whether or not dynorphin levels are reduced following CIE exposure. In an attempt to

discern levels of the KOR ligand, dynorphin, we utilized an ELISA for this neuropeptide

following five weeks of CIE exposure. Regrettably, results from this experiment remain

inconclusive. We suggest that this particular experiment should be repeated with higher

concentrations of protein per sample, likely by combining tissue within treatment groups

and increasing the total number of mice used in the study. Additional examination of the

effects of CIE on RACK1, TrkBs and KORs, as well as their endogenous ligands, would

assist in the understanding of the mechanistic link between these neuromodulators.

190

The functional association between D3Rs and KORs requires more in-depth investigation

We report that D3R blockade reduces the dopamine-decreasing effects of KOR

activation, indicating that these receptors interact. As these data were collected with

FSCV, it is unknown whether or not there is a physical or functional interaction between

D3Rs and KORs. To examine the possibility of a physical interaction between receptors,

seminal experiments should include those that examine the likelihood of a physical

connection, as well as similarities in receptor-attached candidate linker proteins.

Alternatively, it is possible that D3Rs and KORs are associated by interactions within

these receptors’ respective signaling cascades or the same population of calcium and

potassium channels. Identification of the specific downstream pathway that links these

two receptors may be difficult to determine, as bath (ex vivo) or site-specific (in vivo)-

application of pharmacological manipulators of downstream effectors involved in

receptor signaling would likely disrupt the signaling cascades of other receptors, in both

pre- and post-synaptic neurons. As considerable efforts are being made to develop

ligands that target specific downstream pathways of these receptors (Zhou et al., 2013;

Lovell et al., 2015), development of receptor- and signaling-specific ligands may aid in

the understanding of the D3R-KOR connection.

Caveats

Questions regarding the construct validity of the marble burying assay remain

Identification of anxiety-like behaviors in C57 mice is a topic of controversy in the

ethanol field. Many traditional measures of anxiety-like behaviors are augmented in mice

that exhibit high levels of anxiety-like behaviors, such as DBA/2J mice compared to

191

C57BL/6 mice, including elevated plus maze, light-dark box (Kulesskaya & Voikar,

2014; McCool & Chappell, 2015) and elevated zero maze (Flanigan & Cook, 2011). This

is not to say that C57 mice do not show anxiety-like behavior, but that specific measures,

such as marble burying, are needed for its demonstration (Nicolas et al., 2006; Perez &

De Biasi, 2015; Pleil et al., 2015).

Measurement of phenotypic changes in rodent models is based on the construct

validity of this measure. For example, the use of a forced swim assay to measure

depressive-like behavior in rodents is due to the ability of antidepressants to reduce that

behavior (Ushijima et al., 2005; Chung et al., 2014; Olivares-Nazario et al., 2015), just as

the use of an elevated plus assay measures anxiety-like behaviors in rodents because

benzodiazepines increase time spent in the open arms (Lister, 1987; File & Zangrossi,

1993; Kilic et al., 2014), an anxiogenic environment for rodents.

Rodents are known to bury aversive and anxiogenic objects, such as electrified

probes, a behavior known as defensive burying (Korte et al., 1992, 1994; Dringenberg et

al., 2008). As C57 mice avoid the marble-containing side in a two-compartment chamber,

it is likely that marbles are aversive to these animals (Nicolas et al., 2006). Some work

has shown that defensive burying is done largely with the forepaws, while hind limb

burying indicates a normal digging behavior (Deacon, 2009). Anectdotally, mice in the

present set of studies performed both actions, indicating that CIE-induced increases in

marble burying were due, at least in part, to augmented defensive burying (Chapter II).

Marble burying is used to measure anxiety/compulsive-like behaviors based on the

ability of pharmacotherapies for this indication to reduce this behavior. For example,

benzodiazepines such as alprazolam, diazepam, fenobam and chlorodiazepoxide reduce

192

marble burying without affecting locomotor behavior at low doses (Nicholas et al.,

2006), suggesting that marble burying is a valid measure of anxiety/compulsive-like

behavior. However, miscellaneous compounds such as atropine, d-amphetamine and

morphine also reduce marble burying while preserving locomotor activity (Nicholas et

al., 2006), calling into question the construct validity of marble burying as a strict

measure of anxiety/compulsive-like behavior. Additionally, antidepressants also reduce

the number of marbles buried compared to control animals, suggesting a depressant-like

phenotype, although this effect is largely due to the attenuating effects that these

compounds have on locomotion (Nicholas et al., 2006). As marble burying behavior is

reduced by compounds outside of the benzodiazepine class, an exact interpretation of its

etiology is difficult. However, as marble burying has been published primarily as a

measure of anxiety or compulsive-like behavior (Nicolas et al., 2006; Perez & De Biasi,

2015; Pleil et al., 2015), this claim that has been maintained through the present body of

work.

The potential for D3Rs as a pharmacotherapeutic target

The behavioral relevance of D3R upregulation following CIE exposure is not known.

A body of evidence showed that D3R blockade reduced ethanol intake in a model of

alcohol relapse (Vengeliene et al., 2006), supporting the notion that chronic ethanol

exposure and withdrawal upregulates ethanol drinking in a D3R-dependent mechanism.

Further, genetic deletion of D3Rs (Bahi & Dreyer, 2014; Leggio et al., 2014) and D3R

blockade (Leggio et al., 2014) reduces ethanol intake in drug-naïve mice. It has been

hypothesized that compounds that reduce ethanol drinking in both dependent and non-

dependent animals may also reduce the rewarding and reinforcing effects of natural

193

reinforcers such as food (Fantino et al., 1986; Yeomans & Wright, 1991; Murray et al.,

2014). Although additional work is needed to elucidate the effects of D3R antagonists on

the reinforcing efficacy of natural and drug rewards, this evidence may reduce the

pharmacotherapeutic appeal of this compound.

194

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APPENDIX I

GREATER ETHANOL-INDUCED LOCOMOTOR ACTIVATION IN DBA/2J

VERSUS C57BL/6J MICE IS NOT PREDICTED BY PRESYNAPTIC STRIATAL

DOPAMINE DYNAMICS

Jamie H. Rose, Erin S. Calipari, Tiffany A. Mathews, Sara R. Jones

The following manuscript was published in PLoS ONE, 8(12):e83852, 2013 and is reprinted

with permission. Stylistic variations are due to the requirements of the journal. Jamie H. Rose

performed all experiments, data analysis and prepared the manuscript. Drs. Erin S. Calipari,

Tiffany A. Mathews and Sara R. Jones acted in an advisory and editorial capacity.

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Abstract:

A large body of research has aimed to determine the neurochemical factors driving

differential sensitivity to ethanol between individuals in an attempt to find predictors of

ethanol abuse vulnerability. Here we find that the locomotor activating effects of ethanol

are markedly greater in DBA/2J compared to C57BL/6J mice, although it is unclear as to

what neurochemical differences between strains mediate this behavior. Dopamine

elevations in the nucleus accumbens and caudate-putamen regulate locomotor behavior

for most drugs, including ethanol; thus, we aimed to determine if differences in these

regions predict strain differences in ethanol-induced locomotor activity. Previous studies

suggest that ethanol interacts with the dopamine transporter, potentially mediating its

locomotor activating effects; however, we found that ethanol had no effects on dopamine

uptake in either strain. Ex vivo voltammetry allows for the determination of ethanol

effects on presynaptic dopamine terminals, independent of drug-induced changes in firing

rates of afferent inputs from either dopamine neurons or other neurotransmitter systems.

However, differences in striatal dopamine dynamics did not predict the locomotor-

activating effects of ethanol, since the inhibitory effects of ethanol on dopamine release

were similar between strains. There were differences in presynaptic dopamine function

between strains, with faster dopamine clearance in the caudate-putamen of DBA/2J mice;

however, it is unclear how this difference relates to locomotor behavior. Because of the

role of the dopamine system in reinforcement and reward learning, differences in

dopamine signaling between the strains could have implications for addiction-related

behaviors that extend beyond ethanol effects in the striatum.

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Introduction

DBA/2J (DBA) and C57BL/6J (C57) mice are two inbred strains that show disparate

phenotypes with respect to ethanol preference, drinking, and reward, among many other

ethanol-mediated behaviors (1-6). The strains’ differential responses to ethanol exposure

are often thought to model behaviors associated with alcohol abuse vulnerability, abuse

and dependence. For example, C57 mice demonstrate high levels of voluntary ethanol

intake, but little conditioned place preference (CPP) to ethanol, a measure of reward,

while DBA mice voluntarily consume little ethanol but exhibit robust CPP for an ethanol-

paired environment (1-3). Because DBA and C57 mice demonstrate differential

responses to ethanol-mediated behaviors, these strains have become valuable tools for

examining the individual differences that predict ethanol abuse vulnerability.

In addition to the differences in drinking behavior and ethanol reward between DBA

and C57 mice, DBA mice are more sensitive to the locomotor-activating effects of

ethanol (7, 8), although the neurochemical differences that are driving these behavioral

disparities are unclear. Many studies have demonstrated that increases in dopamine in the

ventral (nucleus accumbens, NAc) and dorsal (caudate-putamen, CPu) striatum mediate

locomotor responses to drugs of abuse, including ethanol (9-12). Although it has been

shown that ethanol significantly increases striatal dopamine levels, the precise

mechanisms by which ethanol enhances locomotor activity is unclear. Striatal dopamine

increases have been attributed to a number of factors including increases in ventral

tegmental area (VTA) dopamine cell firing (13-15) and ethanol effects directly on striatal

dopamine terminals (16-18). Previous work has demonstrated that the locomotor-

enhancing effects of stimulant drugs such as cocaine and amphetamine are due to their

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specific actions on presynaptic dopamine terminals, where they inhibit the dopamine

transporter (DAT) to cause increases in synaptic dopamine levels (9, 19, 20). It has been

argued previously that ethanol has direct actions on the DAT (16-18), and differences

between ethanol-DAT interactions could underlie strain differences in the locomotor-

activating properties of ethanol. Here, we aimed to determine if ethanol has direct effects

on dopamine terminals that could in part explain the disparities in ethanol-induced

locomotion between strains.

The primary focus of the current research was to assess ethanol-induced locomotor

activity in DBA and C57 mice, and whether or not this activity was mediated by altered

dopamine dynamics at the level of striatal terminals. DBA mice exhibited much greater

locomotor activation by ethanol, an effect that was specific to ethanol, since other

locomotor stimulating events elicited opposite responses. For example, DBA mice exhibit

reduced locomotor activation in a novel environment compared to C57 mice, as shown

here and by others (21). In order to determine whether these effects originated at the

dopamine terminals, we utilized fast scan cyclic voltammetry to examine dopamine

release and clearance at baseline, and in the presence of ethanol, to determine if

differences in these measures predict behavioral outcomes. An advantage of ex vivo

voltammetry is that it allows for the determination of the effects of ethanol on striatal

terminals, independent of afferent inputs from both the dopamine system and other

neurotransmitter systems. This technique is particularly useful as many pharmacological

approaches to developing treatments for psychiatric disorders rely on an understanding of

the region-specific effects of drugs. Because it has been demonstrated that ethanol’s

ability to increase dopamine levels is due to a balance of its actions on dopamine

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terminals (16) and modulation of VTA cell firing (13-15), this study determined if the

actions of ethanol on dopamine terminals in the NAc core and CPu were predictive of the

strain differences in the locomotor-activating effects of the drug. Our data indicate that

ethanol does not change dopamine uptake, suggesting that increases in dopamine levels

are via another mechanism. Further, DBA and C57 mice have similar presynaptic

dopamine responses to ethanol in both striatal areas in regards to both release and

dopamine uptake via the DAT, indicating that the increased sensitivity of DBA mice to

the locomotor-activating effects of ethanol are likely not due to the effects of ethanol at

dopamine terminals.

Methods:

Subjects:

Male DBA and C57 mice (6 weeks old; Jackson Laboratory, Bar Harbor, ME) were

used for all experiments. Animals were group housed in polycarbonate cages and

maintained on a 12:12 light-dark cycle (7:00 pm lights off) with standard rodent chow

and water ad libitum. Brain slices from both strains were obtained from naïve animals

after at least one full week of habituation to the housing colony. The Institutional Animal

Care and Use Committee at Wake Forest University School of Medicine approved the

experimental protocol (Protocol Number: A10-177). All mice were cared for according to

the National Institutes of Health guidelines in Association for Assessment and

Accreditation of Laboratory Animal Care accredited facilities.

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Locomotor Analysis:

Locomotor activity was assessed via infrared beam breaks in automated locomotor

activity monitors (20 cm × 20 cm × 20 cm; Med Associates, St. Albans, VT). Mice (n=9-

10 per strain) were first placed into activity monitor chambers for 120 minutes to record

response to novelty and to allow for habituation to the chamber. Twenty-four hours

following habituation, animals were placed in the same chambers for 30 minutes before

administration of two injections of saline, 60 minutes apart, to desensitize the animals to

injection stress and allow for a within-subject control. Every day thereafter, animals

received ethanol injections (0.125, 0.25, 0.5, 1.0, 2.0 g/kg i.p.) in the locomotor chambers

after 60 minutes of habituation. Activity in response to each injection was reported for

the first 30 minutes. Locomotor activity was measured as distance traveled (in

centimeters) and as a percent of each animal’s second saline injection.

In Vitro Voltammetry:

Fast scan cyclic voltammetry in brain slices was used to characterize pre-drug striatal

dopamine kinetics, as well as the effects of quinpirole and ethanol on single pulse evoked

dopamine release. Briefly, a vibrating tissue slicer was used to prepare 400 µm thick

coronal brain slices containing the striatum. Slices were incubated in oxygenated

artificial cerebrospinal fluid and heated to 32oC. A carbon fiber microelectrode (≈150 µM

length, 7 µM radius (Goodfellow Corporation, Berwyn, PA) and a bipolar stimulating

electrode (Plastics One, Roanoke VA) were placed in close proximity (≈100 µM) on the

surface of the slice, in either the NAc core or CPu. Endogenous dopamine efflux was

induced by a single, rectangular, electrical pulse applied every five minutes for four

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milliseconds (350 µA, monophasic). Dopamine released was detected by applying a

triangular waveform (-0.4 to +1.2 to -0.4 V vs. silver/silver chloride, 400 V/sec) every

100 milliseconds to the recording electrode. Background current subtraction methods

were applied to obtain clear current versus time plots. When baseline collections were

stable for three consecutive stimulations, quinpirole (0.01, 0.03, 0.1, 0.3, 1.0 µM; n = 7

per strain) or ethanol (25, 100, 150, 200 mM; n = 9 per strain) was bath applied

cumulatively to brain slices. Electrodes were calibrated immediately following

experiments by recording their response (in nA) to 3μM dopamine using a flow-injection

system.

To determine dopamine release and clearance, representative signals were analyzed

before bath application of quinpirole or ethanol. Dopamine release (µM) was calculated

as the amount of dopamine released per electrical stimulation whereas clearance was

determined using the rate constant, tau. All data was collected and analyzed with Demon

Voltammetry and Analysis software (22).

Blood Collection and Analysis:

Blood ethanol concentrations (BEC) from DBA and C57 mice were obtained at 5, 15,

30, 45, 60, 90 and 120 minutes after a 0.5 g/kg i.p. dose of ethanol (n=5-9 per strain, per

time point). After injection, a submandibular vein blood draw was performed at each of

the respective time points and collected in BD microtainer tubes lined with lithium

heparin (Becton Dickinson & Company, Franklin Lakes, NJ). Each animal had no more

than two bilateral blood draws per day and blood collection volumes did not exceed the

maximum set forth by the institutional Animal Care and Use Committee. For

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determination of BECs, standards and samples were prepared with a commercially

available alcohol dehydrogenase assay (Carolina Liquid Chemistries Corporation, Brea,

CA). Briefly, five microliters of each blood collection was placed in a container with

45μl of trichlorocetic acid solution (Sigma Aldrich, St. Louis, MO), and centrifuged at

10,000 revolutions per minute at room temperature for ten minutes. 30μl of the

supernatant of each sample was removed and placed into a separate container with 300μl

of buffer and 112.5μl of enzymatic solution provided in the alcohol dehydrogenase assay.

100μl of each standard and sample was loaded, in triplicate, into a 96 well plate, covered

and incubated at 37o-38

oC for 15 minutes. Immediately following incubation, the plate

was analyzed with SoftMax Pro Software version 5 (Molecular Devices Corporation,

Sunnyvale, CA).

Statistical Analysis:

Statistical analyses and graphs were prepared using Graph Pad Prism (version 5, La

Jolla, CA, USA). Summed data for the groups’ locomotor response to novelty and saline

injection as well as baseline release and tau were compared across groups using a two-

tailed Student’s t-test. Locomotor data (in time bins) and voltammetric data (quinpirole

and ethanol concentration response curves) were compared using a two-way analysis of

variance (ANOVA) with strain and dose as the factors. When a significant main effect

was obtained (p < 0.05), Bonferroni post-hoc analysis was used to determine significant

effects.

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Results

DBA mice exhibited enhanced locomotor responses to ethanol.

To study the behavioral sensitivity of DBA and C57 mice to ethanol, we determined

ethanol-induced locomotor activity over a range of ethanol doses (0.125-2.0 g/kg). Two-

way ANOVA revealed a main effect of strain on ethanol-induced locomotor behavior (F1,

19 = 14.68, p < 0.001; Figure 1). Bonferroni post hoc analysis revealed a significantly

greater locomotor response of DBA mice at the 0.25 (p < 0.05) and 0.5 (p < 0.01) g/kg

doses. Analysis revealed no difference between DBA and C57 mice, with respect to their

locomotor response to a saline injection, indicating that basal locomotor activity did not

differ between the two strains. Because DBA and C57 do not differ in their responses to

a saline injection, their differential behavioral responses to ethanol cannot be attributed to

disparate baseline locomotor activity levels.

DBA mice had reduced locomotor responses to a novel environment.

To determine if the enhanced response to ethanol in DBA mice was specific to

ethanol or was due to an enhanced response to all locomotor-activating stimuli, we

examined locomotor responses to a novel environment in DBA and C57 mice. Two-way

ANOVA revealed a main effect of strain on response to novelty (F1, 39 = 13.45, p < 0.01;

Figure 2A). Bonferroni post hoc analysis revealed a reduced response to novelty in DBA

mice, compared to C57 mice during the first 10 (t = 6.972, p < 0.01), 55 (t = 3.876, p <

0.01), 60 (t = 3.878, p < 0.01) 65 (t = 3.600, p < 0.01), 70 (t = 3.132, p < 0.05), 75 (t =

3.599, p < 0.01), 80 (t = 3.943, p < 0.01) and 85 (t = 3.602, p < 0.01) minute time points.

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Figure 1. DBA mice exhibited enhanced ethanol-induced locomotor responses.

DBA mice exhibited an enhanced locomotor response over a dose response curve for

ethanol, as compared to C57 mice. Data is summed over the first 30 minutes post-ethanol

or saline injection. *p< 0.05; **p< 0.01

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Figure 2. DBA mice exhibited reduced responses to a novel environment.

(A) DBA mice showed a reduced response to a novel environment compared to C57 mice

over a 120-minute locomotor session. (B) Summed data from the 120-minute locomotor

session. *, p < 0.05; **, p < 0.01; ***, p < 0.001; min, minute.

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DBA and C57 mice did not differ in blood ethanol concentrations following ethanol

administration.

Because it is possible that strain differences in blood ethanol elimination time could

be driving differences in locomotor responses to ethanol, we examined the time course of

BECs after a 0.5 g/kg i.p. injection in DBA and C57 mice. We found a significant effect

of time on BECs (two-way ANOVA: F6,20 = 32.55, p < 0.001). However, we found no

differences between strains with respect to blood ethanol elimination rate at any time

point tested (Figure 3).

Figure 3. DBA and C57 mice exhibited similar ethanol elimination time courses.

The time course of ethanol clearance, as measured by blood ethanol concentrations

(BECs) over time, was determined in C57 and DBA mice following a 0.5 g/kg ethanol

challenge. There were no significant differences in ethanol clearance between the strains.

Min, minute; i.p., intraperitoneal; BEC, blood ethanol concentration.

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DBA and C57 mice had similar presynaptic dopamine dynamics and autoreceptor

sensitivity in the NAc core.

Because we found robust differences in dopamine-mediated behaviors, we aimed to

determine if there were differences in striatal dopamine system functioning between

strains at baseline. To do this, we examined evoked dopamine release and tau, a measure

of dopamine clearance, in the NAc core. We found no differences between strains in

regards to dopamine release (Figure 4B, left) or clearance in this region (Figure 4B,

right).

Additionally, we assayed D2-like autoreceptor activity in the NAc core by conducting

dose-response curves for the D2/D3 agonist, quinpirole. A repeated measures two-way

ANOVA revealed a significant effect of quinpirole concentration on dopamine release

(F4,19 = 57.81, p < 0.001), where quinpirole dose-dependently reduced evoked dopamine

release. The effects of quinpirole on dopamine release were similar between strains,

demonstrating that D2-like autoreceptor function was not different (Figure 4C).

DBA mice have faster dopamine clearance in the CPu.

Next, we aimed to determine if differences between strains were present in the CPu.

We found that DBA and C57 mice have similar dopamine release in the CPu (Figure 5B,

left); however, a Student’s t-test revealed that DBA mice had a faster rate of dopamine

clearance in this region (Figure 5B, right; t13 = 9.43, p < 0.05).

In order to determine the D2-like autoreceptor function between the strains, we ran

concentration-response curves for quinpirole. A repeated measures two-way ANOVA

223

224

Figure 4. Dopamine release and clearance in the nucleus accumbens (NAc) core of

DBA and C57 mice.

DBA and C57 mice have similar presynaptic dopamine dynamics in the NAc core. (A)

Raw dopamine traces from the NAc core of C57 (left; red) and DBA (right; blue) mice.

(B) Electrically evoked dopamine release (left) and tau (dopamine clearance, right) were

similar between strains. (C) Quinpirole, a D2-like autoreceptor agonist, was applied to

brain slices containing the NAc core to determine autoreceptor sensitivity. There were no

differences between strains with respect to autoreceptor sensitivity. DA, dopamine; Stim,

stimulation.

225

revealed a significant effect of quinpirole concentration on dopamine release (F4,13 =

41.17, p < 0.001), where quinpirole significantly decreased dopamine release over

increasing concentrations of the compound. We found no strain differences in

autoreceptor function as both strains had similar sensitivity to the effects of quinpirole

(Figure 5C) in the CPu.

The effects of ethanol on evoked dopamine release striatal subregions were not different

between DBA and C57 mice.

To determine if increased ethanol-induced locomotion in DBA mice is mediated by

ethanol effects on striatal dopamine terminals, we examined the effects of ethanol at the

terminal by bath application of increasing doses of the drug over brain slices. A two-way

repeated measures ANOVA revealed a significant effect of ethanol in both the NAc core

(F3,15 = 14.91, p < 0.001) and CPu (F3,12 = 16.23, p < 0.001), where increasing

concentrations of ethanol significantly reduced evoked dopamine release. We found that

DBA and C57 mice have similar responses to bath-applied ethanol in both the NAc core

(Figure 6A) and CPu (Figure 6B).

In addition to determining the effects of ethanol on evoked dopamine release, we also

determined the effects of ethanol on dopamine clearance. Dopamine clearance is

mediated by the DAT, thus changes in the clearance of dopamine following bath

application of ethanol can give information as to how ethanol alters DAT function.

Contrary to previously published reports, we showed no effect of ethanol on dopamine

clearance. Further, DBA and C57 mice have similar synaptic dopamine clearance

226

227

Figure 5. Dopamine release and clearance in the caudate-putamen (CPu) of DBA

and C57 mice.

(A) Raw dopamine traces from the CPu of C57 (left; red) and DBA (right; blue) mice.

(B) Strains were similar in electrically evoked dopamine release (left), however DBA

mice had a faster tau, indicating increased dopamine clearance (right). (C) The sensitivity

of D2-like autoreceptors in the CPu was not different between the two strains. *, p <

0.05; DA, dopamine; Stim, stimulation.

228

Figure 6. The effects of ethanol on dopamine terminals in the NAc core and CPu

were similar between strains.

DBA and C57 mice had similar dopamine responses to bath applied ethanol in both the

NAc core (A) and CPu (B). Furthermore, brain slices from DBA and C57 mice

demonstrated similar dopamine clearance rates (tau) in both the NAc core (C) and CPu

(D) in the presence of increasing concentrations of ethanol. DA, dopamine.

229

measures in the NAc core (Figure 6C) and CPu (Figure 6D) in the presence of increasing

concentrations of ethanol.

Discussion

Here we show that, although ethanol-induced locomotor activity is enhanced in DBA

versus C57 mice, this difference is not due to ethanol’s effects on dopamine release or

DAT activity. The ability of ethanol to reduce stimulated dopamine release in the NAc

core and CPu was similar between the two strains of mice. Also, evoked dopamine

release and uptake were comparable between the two strains, except in the CPu, where

DBA mice exhibited faster clearance. Although some work has suggested that ethanol

has direct effects at the DAT, here we show that ethanol does not influence dopamine

uptake via the DAT in either the NAc core or CPu. Previous microdialysis work has

shown that ethanol increases dopamine levels in the striatum to a greater extent in DBA

mice (23), and this likely mediates the enhanced locomotor activity in this strain.

Additionally. our data suggest that differences in ethanol-mediated increases in

dopamine levels observed previously between the two strains may not be due to

differential pharmacokinetic effects of ethanol, because elimination rates were the same.

Previous work has suggested that ethanol-induced increases in dopamine levels are due to

both increased dopamine cell firing in the VTA (13, 14) as well as ethanol effects on

striatal dopamine terminals (16). We suggest that the elevations in dopamine levels

observed in in vivo models following ethanol administration are most likely not due to the

effects of ethanol on dopamine terminals.

The enhanced response to ethanol in DBA mice as compared to C57 mice is not due

to an enhanced response to all locomotor-activating stimuli, as DBA mice showed a

230

reduced response to novelty. Responses to novelty have been shown previously to

correlate with acquisition of stimulant self-administration and addiction vulnerability for

these compounds (24-26). Accordingly, C57 mice, which have enhanced novelty

responses, are also more sensitive to the locomotor activating effects of psychostimulants

(27, 28). However, while DBA mice are less sensitive to the behavioral activating effects

of stimulants, they are more sensitive to ethanol, as highlighted by enhanced ethanol-

induced CPP (2, 3) and locomotor activity (7, 8). These data, combined with previous

work, highlight the unique effects of ethanol, as DBA mice do not exhibit an increased

sensitivity for all drugs of abuse. Additionally, these data suggest that there is not an

overall hyperactivity of the dopamine system in DBA mice, but rather, drug-specific

behavioral differences between strains.

Although differences in locomotor activity point to differential striatal dopamine

system functioning between DBA and C57 mice, our data indicate that these effects do

not occur at the level of the dopamine terminal. It has been shown previously that DBA

mice have enhanced ethanol-induced dopamine overflow, an effect that is likely

mediating the enhanced ethanol-induced locomotion in this strain (23).

Electrophysiological reports using brain slices containing VTA dopaminergic cell bodies

suggest that DBA mice have an enhanced firing rate in response to bath applied ethanol,

as compared to C57 mice (13, 14). The enhanced firing could be responsible for in vivo

increases in dopamine overflow, and could explain the locomotor differences between the

two strains. In addition, postsynaptic dopamine receptors, which have been shown to

have differential expression levels between the two strains, may play a role in the

behavioral disparities (29).

231

Although previous research has pointed to the DAT as being altered by acute ethanol

exposure (16-18), it remained uncertain as to whether ethanol increases, decreases or

does not alter the function of dopamine transporters in the striatum. Here we show that

ethanol does not change dopamine clearance. Voltammetric analyses of the effects of

ethanol on DAT function in an in vivo preparation have found ethanol-induced decreases

in dopamine uptake in the olfactory tubercle, an effect that could lead to increased

dopamine levels following ethanol administration (16). However, because this work was

conducted in vivo, it is possible that ethanol effects on other neurotransmitters systems

are involved in modulating dopamine dynamics, including uptake. Ex vivo voltammetry

allows for the isolation of dopamine terminals separate from afferent inputs, which

allows for the determination of ethanol effects directly at the DAT. Here we show that

ethanol does not affect dopamine clearance by direct interactions with the DAT.

Here we demonstrate that ethanol-induced modulations of dopamine release and

clearance at the level of the striatum are not mediating ethanol-induced locomotor

activity. We demonstrate that the dopamine release inhibiting effects of ethanol do not

differ between strains, and that ethanol does not have any direct effects at the DAT.

Furthermore, our data adds to a body of literature showing that the effects of ethanol on

the dopamine system are a balance of its inhibitory and excitatory effects. We show here

that ethanol, when applied to the dopamine terminal, results in reduced stimulated

release, while previous work has shown that ethanol, when bath applied to VTA cell

bodies, results in enhanced firing. It is likely that these effects converge to result in the

behavioral outputs that are observed following ethanol administration.

232

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236

APPENDIX II

DEVELOPMENT OF THE CURRENT CHRONIC INTERMITTENT ETHANOL

EXPOSURE PROCEDURE

Jamie H. Rose

237

Seminal work examining alcohol use disorders in humans prompted the development

of alcohol exposure paradigms of laboratory animals that model this chronic relapsing

disorder. Largely, these procedures are considered to have construct validity via the

expression of withdrawal symptoms following a period of ethanol abstinence, namely

escalated ethanol drinking compared to control animals (Becker & Lopez, 2004; Lopez &

Becker, 2005; Griffin et al., 2009). Some of the more popular paradigms that induce

ethanol dependence-like symptoms include an ethanol liquid diet, whereby animals are

given only a nutritionally balanced ethanol-containing liquid diet to consume for an

experimenter-determined length of time (Pilström et al., 1972; Umathe et al., 2008; Perez

& De Biasi, 2015), chronic ethanol injections, often several times a day over a series of

days or weeks (Griffin et al., 2009; Iwaniec & Turner, 2013; Perez & De Biasi, 2015),

and exposure to ethanol vapor for several hours per day, commonly over several weeks

(Becker & Hale, 1993; Walker & Koob, 2008; Griffin et al., 2009; Nealey et al., 2011;

Walker et al., 2011; present work). Some of these procedures engender increased ethanol

drinking (Walker & Koob, 2008; Griffin et al., 2009; Nealey et al., 2011; Walker et al.,

2011; present work), anxiety/compulsive-like (Valdez et al., 2002, 2004; Umathe et al.,

2008; Valdez & Harshberger, 2012; Perez & De Biasi, 2015; present work) and

depressive-like symptoms (Schulteis et al., 1995; Boutros et al., 2014) in rodents.

However, not all procedures produce the classic negative reinforcing effects of ethanol,

such as those listed. For example, extended exposure to oral ethanol and chronic ethanol

injections do not cause an increase in ethanol intake after a period of abstinence (Griffin

et al., 2009), while CIE exposure models alcohol use disorders as a disease of chronic

relapse by providing animals with prolonged bouts of intense ethanol vapor exposure and

238

abstinence periods, similar to the human condition (Dahlgren, 1978; Becker & Lopez,

2004; Lopez & Becker, 2005; Griffin et al., 2009). In fact, this procedure reliably

increases ethanol drinking (Becker & Lopez, 2004; Lopez & Becker, 2005; Griffin et al.,

2009; present work) and anxiety/compulsive-like behavior (Pleil et al., 2015; present

work) over repeated cycles of vapor exposure. As such, a pattern of CIE exposure was

used in the present body of work.

Both mice and rats are used to examine CIE-induced changes in behavior and

neurobiology, although these alterations are examined at distinct time-points due to

inherent differences in procedure. For example, mice are treated with repeated cycles

(four days) of ethanol vapor or air exposure (16 hours of exposure, 8 hours of room air)

followed by three complete days of abstinence (Becker & Lopez, 2004; Lopez & Becker,

2005; Griffin et al., 2009; present work) while rats undergo several weeks of ethanol or

air inhalation (14 hours) with daily abstinence sessions (10 hours) for multiple weeks

without a prolonged abstinence period (Walker & Koob, 2008; Walker et al., 2011;

Nealey et al., 2011). As such, inhalation-induced changes in ethanol drinking in mouse

models is typically examined between each CIE cycle (Becker & Lopez, 2004; Griffin et

al., 2009) while the effects of CIE exposure on ethanol consumption in rats occurs after

the inhalation exposure treatment (Walker et al., 2008; Nealey et al., 2011; Walker et al.,

2011). The ethanol exposure protocol used in the current work is a hybrid of these

procedures. Our experimental procedure used C57 mice that established ethanol drinking

prior to CIE exposure, underwent five weeks of CIE or air exposure (four days inhalation

treatment/three days room air exposure) followed by a five-day post-CIE exposure

drinking test or neurobiological examination. Notably, we report increased ethanol

239

consumption, preference and anxiety/compulsive-like behavior when tested 72 hours

following the final CIE or air exposure.

240

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241





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242

APPENDIX III

GENERAL METHODOLOGY

Jamie H. Rose

243

1. Subjects

Male C57 mice (65-75 days old; Jackson Laboratories, Bar Harbor, ME) were used


cycle (off 14:00), with a red room light illuminated during the animals’ dark cycle

(14:00-02:00). Mice habituated to housing conditions for at least one week before

experiment start and were provided with standard rodent chow and water ad libitum,

unless otherwise noted. Chow exposed to ethanol vapor was replaced each morning. The

Institutional Animal Care and Use Committee at Wake Forest University School of

Medicine approved all experimental protocols. All animals were cared for according to

National Institutes of Health guidelines in Association for Assessment and Accreditation

of Laboratory Animal Care accredited facilities.

2. Chronic intermittent ethanol/air exposure

The basis for all ethanol exposure protocols is adapted from a well-established

protocol developed by the Becker Laboratory (Becker & Hale, 1993; Becker & Lopez,

2004; Lopez & Becker, 2005; Griffin et al., 2009). In short, mice were exposed to ethanol

vapor or room air for four days (16 hours of exposure/8 hours of abstinence), followed by

72 hours of room air exposure (one cycle). Cycles were repeated five times. All CIE-

exposed animals were treated with 1.6 g/kg ethanol (CIE exposure) or saline (air

exposure) and 1.0mM pyrazole (Sigma Aldrich, St. Louis, MO) an ethanol

dehydrogenase inhibitor, approximately 30 minutes prior to chamber start time. Blood

ethanol concentrations (BEC) were tested after the first and fourth inhalation exposure to

ensure proper chamber function and physiologically relevant BECs (Figure 8; Griffin et

al., 2009).

244

3. Blood collection and analysis for BEC determination

Blood samples of CIE-exposed mice were obtained immediately after removal from the

vapor chamber on the mornings after the first and final exposure per cycle. A

submandibular vein puncture was performed to collect no more than 15µL of blood from

no more than two animals per collection day. Samples were held in microtainer tubes

lined with lithium heparin (Becton Dickinson & Company, Franklin Lakes, NJ) before

processing. Blood collection volumes did not reach the maximum set forth by the

Institutional Animal Care and Use Committee at Wake Forest University School of

Medicine. For BEC measurement, standards and samples were prepared using a

commercially available alcohol dehydrogenase assay (Carolina Liquid Chemistries

Corporation, Brea, CA). Briefly, five microliters of each blood collection was placed in a

container with 45μl of trichlorocetic acid solution (Sigma Aldrich, St. Louis, MO), and

centrifuged at 10,000 revolutions per minute at room temperature for ten minutes. 30μl of

the supernatant of each sample was removed and placed into a separate container with

300μl of buffer and 112.5μl of enzymatic solution provided in the alcohol dehydrogenase

assay. 100μl of each standard and sample was loaded, in triplicate, into a 96 well plate,

covered and incubated at 37o-38

oC for 15 minutes. Immediately following incubation, the

plate was analyzed with SoftMax Pro Software version 5 (Molecular Devices

Corporation, Sunnyvale, CA).

245

Figure 8: Ethanol vapor exposure maintains elevated BECs

Mice in CIE-exposure groups are systemically injected with 1.6g/kg ethanol +

1.0mmol/kg pyrazole at approximately 16:30, 30 minutes prior to chamber start-time

(17:00). Administration of this solution raises BECs (blue dotted line), which are then

maintained by ethanol vapor exposure (green dashed line) until the vapor chambers are

stopped by the experimenter (09:00). Dark portion: dark cycle. Ordinate: Ethanol

concentration. Abscissa: time.

246

4. Ex vivo fast scan cyclic voltammetry

Ex vivo fast scan cyclic voltammetry is a neurobiological a technique that allows for

spatiotemporal-specific examination of dopamine kinetics (Ferris et al., 2013). Using this

technique, pre-drug accumbal dopamine release and uptake kinetics, as well as changes in

presynaptic receptor function, were examined in the NAc core after five cycles of CIE or

air-exposure. In all experiments, mice were sacrificed 72 hours after the fifth inhalation

exposure to coincide with the onset of all behavioral testing. Mice were anesthetized with

isoflurane, rapidly decapitated and brains removed. Brain slices containing the NAc core

(300µm thick) were prepared with a vibrating tissue slicer and incubated in oxygenated

artificial cerebrospinal fluid (aCSF, 32OC). A carbon fiber recording electrode (≈50-

100µM length, 7µM radius; Goodfellow Corporation, Berwyn, PA), and a bipolar

stimulating electrode (Plastics One, Roanoke, VA) were placed (≈100µM apart) on the

surface of the slice. Dopamine efflux was induced with a single, rectangular, 4.0ms long

electrical pulse (350µA, monophasic; inter-stimulus interval: 180sec). To detect

dopamine release, a triangular waveform (-0.4 to +1.2 to -0.4V vs. silver/silver chloride,

400 V/sec) was applied every 100ms to the recording electrode. Proper identification of

electroactive neurotransmitters in voltammetry is ensured via a species-specific cyclic

voltammogram, which identifies the peak oxidation and reduction potentials of the

neurotransmitter, as dopamine is oxidized at to dopamine-o-quinone at +0.6V, and

reduced at -02V (Heien et al., 2003). To obtain clear current versus time plots,

background current subtraction methods were utilized.. To assess dopamine terminal

function, all compounds were added cumulatively to aCSF when baseline collections

247

were stable. All compounds were obtained at the highest quality from Tocris Bioscience

(Bristol, UK) unless otherwise noted.

All graphs were created and statistical tests were applied using GraphPad Prism,

Version 5 (La Jolla, CA). Baseline apparent Km was set to 160nM for each slice in both

regions, based on the known affinity of dopamine for the DAT (Wu et al., 2001) while

pre-drug Vmax values were allowed to vary. To ensure to ensure proper detection of

dopamine release, specific experimental measures, notably the cyclic voltammogram, are

used. Cyclic voltammograms allows experimenters to specifically identify the

electroactive species being examined, particularly dopamine. Electrode calibration was

performed immediately following each experiment using a flow-injection system. Traces

that did not return to baseline were minimally corrected using baseline correction

techniques within the Demon software.

5. Ethanol drinking procedures

Animals in ethanol drinking groups began a two bottle choice procedure after housing

habituation (two weeks of sucrose fade [Samson, 1986]; two days each: 10% ethanol/5%

sucrose, 12% /5%, 15% /5%, 15% /2%, and 15% /1% (w/v), followed by four additional

weeks of access to 15% [v/v] ethanol). Experimental solutions were provided for two

hours per day (13:30-15:30), five days per week, and paired with an identical bottle of

water. Bottle placement was switched daily. Mice were assigned to CIE or air inhalation

groups immediately prior to five cycles of CIE/Air exposure (below), counterbalanced for

intake. Ethanol drinking was examined for five days after all cycles (Fig. 1B). Separate

groups of animals were systemically injected with norBNI (10mg/kg i.p.; NIDA,

Bethesda, MD) or vehicle (saline) 24 hours prior to the ethanol drinking test.

248

6. Marble burying behavior

Pre-inhalation marble burying tests occurred on the morning of Cycle 1. Mice were

placed in a testing chamber, a polycarbonate cage lined with 3.5 cm of cob bedding (The

Andersons, Inc., Maumee, OH) for 120 minutes (Rose et al., 2013) before the addition of

20 clean, black glass marbles (14mm, Rainbow Turtle, Portland, OR). A template was

used to ensure consistent placement of marbles (Fig. 4A, left panel). After the 30-minute

testing session, mice were returned to their home cages. The number of marbles over

75% buried was counted in analysis. Water was provided during the habituation session

while both food and water were restricted during the test session. Animals were placed

into inhalation groups, counterbalanced for the number of marbles buried during the pre-

test. CIE/Air-induced changes in marble burying were assessed 72 hours after the fifth

cycle. Separate groups of mice were systemically injected with norBNI (10mg/kg) or

saline 24 hours prior to post-inhalation treatment testing.

249

REFERENCES

Becker HC, Hale RL (1993) Repeated episodes of ethanol withdrawal potentiate the

severity of subsequent withdrawal seizures: an animal model of alcohol withdrawal

"kindling". Alcohol Clin Exp Res 17:94-8.



12:1829-1838.









688-696.

Rose JH, Calipari ES, Mathews TA, Jones SR (2013) Greater ethanol-induced locomotor

activation in DBA/2J versus C57BL/6J mice is not predicted by presynaptic striatal

dopamine dynamics. PLoS One 8:e83852.

Wu Q, Reith ME, Wightman RM, Kawagoe KT, Garris PA. 2001. Determination of

release and uptake parameters from electrically evoked dopamine dynamics measured

by real-time voltammetry. J Neurosci Methods. 112(2):119-33.

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APPENDIX IV:

CURRICULUM VITAE

JAMIE HANNAH ROSE

Department of Physiology and Pharmacology


Wake Forest School of Medicine

PERSONAL INFORMATION

Business Address Department of Physiology and Pharmacology


Medical Center Blvd.

Winston-Salem, NC 27157

Phone (336) 716-5504

Fax (336) 716-8501

E-mail [email protected]

EDUCATION

Jan 2016-Mar 2018 Elon University Physician Assistant Program

Elon, NC

Aug, 2010-Present Wake Forest School of Medicine

Winston-Salem, NC

Advisor, Sara R. Jones, Ph.D.

Ph.D. in Neuroscience

Aug, 2006- High Point University

Dec, 2010 High Point, NC

Advisor, Dinene Crater, Ph.D.

Bachelor’s of Science Degree acquired, Biology

Graduated Summa Cum Laude

Aug, 2006- High Point University

Dec, 2010 High Point, NC

Advisor, Deborah Danzis, Ph.D.

Bachelor’s of Science Degree acquired, Psychology

Minor concentration, Chemistry

Graduated Summa Cum Laude

251

RESEARCH EMPLOYMENT HISTORY

Graduate

Aug, 2010-Present Doctoral Candidate

Department of Physiology and Pharmacology



Laboratory of Sara R. Jones, PhD

Dec-Aug, 2010 Clinical Research Assistant

Mendenhall Clinical Research Center

High Point, NC

Melanie Fein, MD

Undergraduate

June-Aug, 2009 Research Assistant

Behavioral Pharmacology

Emory University

Laboratory of Leonard Howell, PhD

Mentor, Heather Kimmel, PhD

HONORS AND AWARDS

Dec, 2014 Awarded 3rd

place in poster and presentation during the

Wake Forest University Health Sciences Neuroscience

Poster Day

Feb, 2014 Alumni Student Travel Award to attend the Gordon

Research Conference: Alcohol and the Nervous System

Meeting in Galveston, TX

Oct, 2013 Individual Ruth L. Kirschstein National Research Service

Award (Graduate NRSA; F31 DA035558), Wake Forest

School of Medicine

Jun, 2013 Research Society on Alcoholism (RSA) Student Merit

Award from the RSA to attend the 36th Annual RSA

Scientific Meeting in Orlando, FL

Jun, 2013 Alumni Student Travel Award to attend the 36th Annual

Research Society on Alcoholism Scientific Meeting in

Orlando, FL

252

Aug, 2012 Ruth L. Kirschstein Institutional National Research Service

Award (Graduate NRSA; T32 AA07565), Wake Forest

School of Medicine

Mar, 2007-Dec, 2010 Alpha Chi – Academic Honor Society President

PROFESSIONAL MEMBERSHIPS

Mar, 2013 - Present Research Society on Alcoholism

Aug, 2012 - Present Western North Carolina Society for Neuroscience

Jan, 2008 - Present Beta Beta Beta Biology Honor Society

Apr, 2007 - Present Psi Chi Psychology Honor Society

Mar, 2007 - Present Alpha Chi, Academic Honor Society,

Ad hoc manuscript reviewer for Neuropharmacology

SERVICE: VOLUNTEERING AND OUTREACH

Institutional

July, 2015 National Youth Leadership Forum organizer

June, 2015 SciTech Institute Outreach presenter

Feb, 2012 - Present Authorship contributor, Western North Carolina Society for

Neuroscience newsletter, The Neurotransmitter

Mar, 2011 - Present Brain Awareness Council Chair for Brain Awareness Week

Dec, 2010 - Present Presenter, Kernersville Cares for Kids Initiative

Nov, 2010 - Present Presenter, Brain Awareness Council School Visits

Student Mentorship / Training

July, 2014-Present Sarah Ewin, Wake Forest University, Integrated Physiology

and Pharmacology Graduate Program

Feb, 2014 - Present Deborah Luessen, Wake Forest University, Integrated

Physiology and Pharmacology Graduate Program

Jan-Aug, 2015 Molly McGinnis, Wake Forest University, Neuroscience

Graduate Program

253

Aug-Dec, 2014 Josh Seideman, Wake Forest University, Neuroscience

Graduate Program

Mar-Aug, 2014 Stephanie Saadeh, Vanderbilt University, Excellence in

Cardiovascular Sciences Research Program

Jun-Oct, 2012 Crystal Bolden, Wake Forest School of Medicine, Post-

Baccalaureate Program

PUBLICATIONS

Karkhanis A, Rose JH, Weiner JL, Jones SR (2015) Early-life social isolation

sensitizes kappa opioid receptors and downregulates dopamine in the nucleus

accumbens. Submitted to Neuropsychopharmacology, October, 2015.

Rose JH, Karkhanis A, Chen R, Gioia D, Lopez MF, Becker HC, McCool BA, Jones

SR (2015) Supersensitive kappa opioid receptors promote ethanol withdrawal-related

behaviors and reduce dopamine signaling in the nucleus accumbens. Accepted, The

International Journal of Neuropsychopharmacology, November 2015.


NM, Maldonado-Devincci1AM, Morrow L, Jones SR, Kash TL (2015) Effects of

chronic ethanol exposure on neuronal function in the prefrontal cortex and extended



intermittent ethanol exposure reduces presynaptic dopamine system function in the

mouse nucleus accumbens. Drug and Alcohol Dependence 150:24-30.

Ferris MJ, Calipari ES, Rose JH, Siciliano CA, Chen R, Jones SR (2015) A single

amphetamine infusion reverses deficits in dopamine nerve-terminal function caused by

a history of cocaine self-administration. Neuropsychopharmacology 40: 1826-1836.

* Editor’s choice, July, 2015 issue.

254

Ferris MJ, España RA, Locke JL, Konstantopoulos JK, Rose JH, Chen R, Jones SR

(2014) The dopamine transporter governs diurnal variation in extracellular dopamine

tone. Proc Natl Acad Sci USA 111(26):E2751-9.

Yorgason JT*, Rose JH*, McIntosh MJ, Ferris MJ, Jones SR (2014) Greater Ethanol

Inhibition of Presynaptic Dopamine Release in C57BL/6J than DBA/2J Mice: Role of

Nicotinic Acetylcholine Receptors. Neuroscience. 284:854-64.

*Authors contributed equally to work

Rose JH, Calipari ES, Matthews TJ, Jones SR (2013) Greater ethanol-induced

locomotor activation in DBA/2J versus C57BL/6J mice is not predicted by presynaptic

striatal dopamine dynamics. PLOS One 8(12): e83852.

Published Abstracts

Martin TJ, Kim SA, Rose JH, Jones SR, Aubé J, Bohn LM (May, 2016) A novel G-

protein biased kappa opioid receptor that displays analgesic but not dysphoric actions in

the rat. Abstract for poster presentation. American Pain Society 35th

Annual Scientific

Meeting. Austin, TX.

Karkhanis AN, Rose JH, Weiner JK, Jones SR (June, 2015) Social isolation stress

increases Κ-opioid receptor activity in the nucleus accumbens shell and core. Abstract

for poster presentation. The 38th

Annual RSA Scientific Meeting. San Antonio, TX.

Rose JH, Karkhanis, A, Gioia DA, McCool BA, Jones SR (2014, December). C57BL/6

mice demonstrate increased compulsive behavior and decreased dopamine system

signaling after chronic intermittent ethanol exposure. Abstract for poster presentation.

Wake Forest University Health Sciences Neuroscience Poster Day. Winston-Salem,

NC. *Poster and presentation awarded 3rd

place in contest.

255

Rose JH, Karkhanis, A, Gioia DA, McCool BA, Jones SR (2014, November).

C57BL/6 mice demonstrate anxiety-like behavior and decreased dopamine system

signaling after chronic intermittent ethanol exposure. Abstract for poster presentation.

Society for Neuroscience Annual Meeting. Washington, DC.

Karkhanis AN, Rose JH, McCool BA, Weiner JL, Jones SR (2014, November) Social

isolation stress affects dopamine signaling and kappa opioid receptor system in the

nucleus accumbens and the basolateral amygdala. Abstract for poster presentation.


Severino AL, Wagoner AL, Haynes K, Skelly MJ, Rose JH, Constantinidis C, Godwin

DW (2014, November). Wake Forest University's Brain Awareness Council: Evolution

of Outreach to the Piedmont Triad Comminuty. Abstract for poster presentation.


Rose JH, Gioia DA, McCool BA, Jones SR (2014, February). Increased dopamine

transporter and ΔFosB expression in striatal subregions of C57BL/6J mice after

repeated chronic intermittent ethanol vapor exposure and ethanol consumption tests.

Abstract for poster presentation. Alcohol and the Nervous System Gordon Research

Conference. Galveston, TX.

Rose JH, Calipari ES, Mathews TA, Jones SR (2013, June). Ethanol-induced

hyperlocomotion of DBA/2J mice is not mediated by striatal dopamine. Abstract for

poster presentation. The 36th

Annual RSA Scientific Meeting. Orlando, FL.

Calipari ES, Ferris MJ, Rose JH, Roberts DCS, Jones SR (2013, May). Cocaine self-

administration results in neurochemical tolerance and dopamine transporter complex

formation which is reversed by a single amphetamine bolus. Abstract for poster

presentation. Dopamine. Alghero, Italy.

Rose JH, Calipari ES, Mathews TA, Jones SR. (2013, March) Differential dopamine

system dynamics in mouse models of alcohol vulnerability. Poster for presentation.

Graduate School Research Day. Winston-Salem, NC

256

Rose JH, Calipari ES, Mathews TA, Jones SR. (2012, December) Differential

dopamine system dynamics in mouse models of alcohol vulnerability. Poster Presented

at Western North Carolina Society for Neuroscience Research Day. Winston-Salem,

NC.

LP Pattison, MJ Skelly, ER Aston, MA Riegle, K Haynes, TE Spence, JH Rose, HQ

Shan, SV Bach, L Nattkemper, W Johnston, AM Peiffer, DP Friedman, RE Hampson,

RW Oppenheim, DW Godwin. (2012, October) A Graduate Course in Scientific

Outreach Initiated by the Wake Forest University Brain Awareness Council. Poster for

presentation. Society for Neuroscience Meeting. New Orleans, LA.

Kimmel HL, Manvich DF, Zhou M, Rose JH, Howell LL (2010, June) Effects of

repeated disulfiram on cocaine-induced increases in locomotor activity in the squirrel

monkey. College on Problems of Drug Dependence Annual Meeting. Scottsdale, AZ.

PRESENTATIONS

Institutional / Departmental

Rose, JR (2015, March). Increased kappa opioid receptor function may underline

ethanol-induced increased compulsive behaviors and decreases in dopamine signaling.

Program in Neuroscience Seminar, Wake Forest School of Medicine, Winston-Salem,

NC.

Rose, JR (2013, November). Chronic Intermittent Ethanol Treatment Results in

Increased Ethanol Consumption and Preference in C57BL/6J Mice. Program in

Neuroscience Seminar, Wake Forest School of Medicine, Winston-Salem, NC.

Rose, JR (2013, April). Dopamine Dynamics and Psychomotor Activation in a Mouse

Model of Ethanol Use Vulnerability. Program in Neuroscience Seminar, Wake Forest

School of Medicine, Winston-Salem, NC.

257

Rose, JR (2013, February). Differential Dopamine System Dynamics and Behavioral

Responses in Mouse Models of Alcohol Vulnerability. Department of Physiology and

Pharmacology Seminar, Wake Forest School of Medicine, Winston-Salem, NC.

Rose, JR (2012, August). Amphetamine Reverses Cocaine-Induced Changes in the

Dopamine Transporter. Neuroscience Graduate Student Summer Seminar, Wake Forest

School of Medicine, Winston-Salem, NC.

Invited Presentations

Rose, JR (July, August, 2015) Drugs and the brain: Neurochemistry and consequences.

National Youth Leadership Forum hosted by Wake Forest University. Winston Salem,

NC.

Rose, JR (January, 2014). Chronic intermittent ethanol treatment: Ethanol consumption

and preference in C57BL/6J mice. Winter Retreat for the Integrative Neuroscience

Initiative on Alcoholism Consortium. Rockville, MD.

Rose, JR (2013, December). The brain: a look inside. Private outreach-based seminar

for advanced high school students. Red Bank Regional High School. Little Silver, NJ.

DISSERTATION PROJECT

Title: The effects of chronic intermittent ethanol exposure on withdrawal phenotypes,

dopamine terminal function and kappa opioid receptor sensitivity in the nucleus

accumens

258

CURRENT RESEARCH SUPPORT

F31DA035558 Rose Mentor: Sara. R. Jones, Ph.D.

Recurrent

NIDA

Role: Principal Investigator/Graduate Student.

Project Title: Cocaine-Induced Dopamine Transporter Changes are Reversed by

Amphetamine

Project Summary/Abstract:

Cocaine dependence in the United States is a major health problem with no

pharmacotherapeutic treatment. To study this condition in the laboratory, rats self-

administer compounds, particularly cocaine, intravenously. We have used this model

to show that extended exposure to cocaine (1.5mg/kg/infusion, 40 infusions/day, 5

days) results in a pharmacodynamic tolerance of the dopamine transporter to cocaine

(Ferris et al., 2015). Notably, we also found that administration of a single

amphetamine bolus (0.56 mg/kg IV) rapidly and completely reversed

pharmacodynamic tolerance of the dopamine transporter to cocaine, and even

augmenting the potency of this compound above control levels. The specific aims of

this grant include experiments that characterize and expand on this discovery. In

Specific Aim 1, we will explore the lowest effective dose of amphetamine that can

cause a reversal of dopamine transporter plasticity and the time course of this effect.

Although this characterization is important, we are primarily interested in discovering

the structural and functional changes in the dopamine transporter that are responsible

for this effect. Thus, in Specific Aim 2, we will use structurally different dopamine

releasers and blockers with different dopamine transporter affinities in place of an

amphetamine bolus and use slice voltammetry to explore cocaine potency and

presynaptic dopamine function (release and uptake) in response to these compounds.

Additionally, we are interested in the behavioral implications for our neurochemical

data. In Specific Aim 3 we will test animals before and after self-administration and

an amphetamine bolus with locomotor assessments while another group will be

assessed on a progressive ratio schedule of cocaine self-administration. While our

259

interest is primarily in the basic pharmacology driving the dopamine transporter

changes, this may be relevant to the literature proposing dopamine transporter

releasers such as amphetamine as putative cocaine addiction pharmacotherapies, as

agonist therapies are commonly used for indications such as heroin (methadone) and

cigarettes (nicotine patches, gum). Although agonist therapies for cocaine addiction

are controversial, we may be able to define a putative mechanism for some of their

therapeutic effects which could potentially drive more rationale design of such

therapies.

COMPLETED REASEARCH SUPPORT

T32 AA07565 Rose Mentor: Sara. R. Jones, Ph.D.

2012-2013

NIAAA

Role: Graduate student.

Project Title: Differential dopamine system dynamics in model systems for alcohol

vulnerability: Comparison of C57BL/6 and DBA/2 neurochemical and behavioral

measures.

Project Summary/Abstract:

A large body of research has aimed to determine the neurochemical factors driving

differential sensitivity to ethanol between individuals, in an attempt to find predictors

of ethanol abuse vulnerability. Here, we find that the locomotor activating effects of

ethanol are markedly greater in DBA/2J compared to C57BL/6J mice, although it is

unclear as to what neurochemical differences between strains mediate this behavior.

Dopamine elevations in the nucleus accumbens and caudate-putamen regulate

locomotor behavior for most drugs, including ethanol; thus, we aimed to determine if

differences in these regions predict strain differences in ethanol-induced locomotor

activity. Ex vivo voltammetry allows for the determination of ethanol effects on

presynaptic dopamine terminals, independent of drug-induced changes in firing rates

of afferent inputs from either dopamine neurons or other neurotransmitter systems.

Previous studies suggest that ethanol interacts with the dopamine transporter,

260

potentially mediating its locomotor activating effects; however, using fast scan cyclic

voltammetry, we found that ethanol had no effects on dopamine uptake in either

strain. However, differences in striatal dopamine dynamics did not predict the

locomotor-activating effects of ethanol, since the inhibitory effects of ethanol on

dopamine release were similar between strains. There were differences in presynaptic

dopamine function between strains, with faster dopamine clearance in the caudate-

putamen of DBA/2J mice; however, it is unclear how this difference relates to

locomotor behavior, although this finding may have implications for the augmented

anxiety that this strain displays. Because of the role of the dopamine system in

reinforcement and reward learning, differences in dopamine signaling between the

strains could have implications for addiction-related behaviors that extend beyond

ethanol effects in the striatum.

TECHNICAL PROFICIENCY

Since beginning research in neuroscience in August, 2011, I have acquired

proficiency in a number of techniques used in mice and rats. Training received in the

laboratories of Rong Chen (WFU), Sara Jones (WFU), and Dave Roberts (WFU)

include biochemical assays for western blot hybridization, in vitro voltammetry,

various behavioral assays (e.g., locomotor assessment, conditioned place preference,

forced swim test, marble burying assays), ethanol drinking and vapor chamber

procedures, blood ethanol concentration calculation as well as operant procedures such

as rodent self-administration of psychostimulants. Data rendered by projects are

graphing and analyzed using statistical packages, particularly GraphPad Prism.

CURRENT RESEARCH INTERTESTS

Although alcoholism is a rampant social, economic and financial burden in the

United States, there are only three Food and Drug Administration-approved

pharmacotherapies for this indication, and none are completely efficacious. Previous

work from our laboratory indicates that striatal dopamine system signaling decreases

with chronic ethanol exposure. It has been speculated that chronic ethanol exposure-

induced reductions in dopamine system function in the nucleus accumbens may drive

261

withdrawal phenotypes, namely increased withdrawal symptoms that lead to relapse

drinking. Because opioid receptors are known to modulate dopamine

neurotransmission, it is plausible that ethanol-induced changes in kappa opioid receptor

function may underlie aberrant dopamine system signaling during ethanol withdrawal.

Considerable research points to the utility of the kappa opioid receptor as a

pharmacotherapeutic target to relieve the symptoms of alcohol withdrawal, which often

drive relapse drinking in the human alcoholic. As such, I am interested in how changes

in kappa opioid receptor function affects mesolimbic dopamine/reward system function

and withdrawal-mediated phenotypes, namely ethanol drinking parameters and marble

burying, a measure of anxiety/compulsive behavior, following five weeks of chronic

ethanol exposure. Notably, kappa opioid receptors are not the only presynaptic

dopamine regulating protein, thus, my interests also expand to D2 and D3

autoreceptors, and interactions between kappa opioid receptors and autoreceptors.

Because the striatal dopamine system is a critical regulator of reward, restoration of

proper striatal dopamine signaling may alleviate some of the withdrawal symptoms of

chronic ethanol exposure and withdrawal, and curtail ethanol drinking during

abstinence.

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THE EFFECTS OF CHRONIC INTERMITTENT ETHANOL …...impact you have made on me has taught me scores beyond science. All of these lessons will stay with me well beyond my time in graduate