Methods in drug abuse research dr barry d waterhouse, CRC press, 2003 scan

2 Methods in Drug Abuse Research: Cellular and Circuit Level Analysestransient effects on the brain, only some of which contribute to a positive drugexperience and the desire to repeat t

Published Titles

Apoptosis in Neurobiology

Yusuf A Hannun, M.D., Professor of Biomedical Research and Chairman/Department

of Biochemistry and Molecular Biology, Medical University of South Carolina

Rose-Mary Boustany, M.D., tenured Associate Professor of Pediatrics and Neurobiology,

Duke University Medical Center

Methods for Neural Ensemble Recordings

Miguel A.L Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,

Duke University Medical Center

Methods of Behavioral Analysis in Neuroscience

Jerry J Buccafusco, Ph.D., Alzheimer’s Research Center, Professor of Pharmacology and

Toxicology, Professor of Psychiatry and Health Behavior, Medical College of Georgia

Neural Prostheses for Restoration of Sensory and Motor Function

John K Chapin, Ph.D., Professor of Physiology and Pharmacology, State University of

New York Health Science Center

Karen A Moxon, Ph.D., Assistant Professor/School of Biomedical Engineering, Science,

and Health Systems, Drexel University

Computational Neuroscience: Realistic Modeling for Experimentalists

Eric DeSchutter, M.D., Ph.D., Professor/Department of Medicine, University of Antwerp

Methods in Pain Research

Lawrence Kruger, Ph.D., Professor or Neurobiology (Emeritus), UCLA School of Medicine

and Brain Research Institute

Motor Neurobiology of the Spinal Cord

Timothy C Cope, Ph.D., Professor of Physiology, Emory University School of Medicine

Nicotinic Receptors in the Nervous System

Edward D Levin, Ph.D., Associate Professor/Department of Psychiatry and Pharmacology

and Molecular Cancer Biology and Department of Psychiatry and Behavioral Sciences, Duke University School of Medicine

Methods in Genomic Neuroscience

Helmin R Chin, Ph.D., Genetics Research Branch, NIMH, NIH

Steven O Moldin, Ph.D, Genetics Research Branch, NIMH, NIH

Methods in Chemosensory Research

Sidney A Simon, Ph.D., Professor of Neurobiology, Biomedical Engineering, and

Anesthesiology, Duke University

Miguel A.L Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,

Duke University

The Somatosensory System: Deciphering the Brain’s Own Body Image

Randall J Nelson, Ph.D., Professor of Anatomy and Neurobiology,

University of Tennessee Health Sciences Center

New Concepts in Cerebral Ischemia

Rick C S Lin, Ph.D., Professor of Anatomy, University of Mississippi Medical Center

DNA Arrays: Technologies and Experimental Strategies

Elena Grigorenko, Ph.D., Technology Development Group, Millennium Pharmaceuticals

Methods for Alcohol-Related Neuroscience Research

Yuan Liu, Ph.D., National Institute of Neurological Disorders and Stroke, National Institutes

of Health

David M Lovinger, Ph.D., Laboratory of Integrative Neuroscience, NIAAA

In Vivo Optical Imaging of Brain Function

Ron Frostig, Ph.D., Associate Professor/Department of Psychobiology,

University of California, Irvine

Primate Audition: Behavior and Neurobiology

Asif A Ghazanfar, Ph.D., Primate Cognitive Neuroscience Lab, Harvard University

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Library of Congress Cataloging-in-Publication Data

Methods in drug abuse research : cellular and circuit level analyses / edited by Barry D Waterhouse

p cm (Methods & new frontiers in neuroscience) Includes bibliographical references and index.

ISBN 0-8493-2345-2 (alk paper)

1 Drugs of abuse Research Methodology 2 Drug abuse Research Methodology.

3 Neurons 4 Neural circuitry I Waterhouse, Barry D II Methods & new frontiers in neuroscience series.

RM316 M48 2002

Series Preface

Our goal in creating the Methods & New Frontiers in Neuroscience series is topresent the insights of experts on emerging experimental techniques and theoreticalconcepts that are, or will be, at the vanguard of neuroscience Books in the seriescover topics ranging from methods to investigate apoptosis, to modern techniquesfor neural ensemble recordings in behaving animals The series also covers newand exciting multidisciplinary areas of brain research, such as computational neu-roscience and neuroengineering, and describes breakthroughs in classical fields likebehavioral neuroscience We want these books to be the books every neuroscientistwill use in order to get acquainted with new methodologies in brain research Thesebooks can be given to graduate students and postdoctoral fellows when they arelooking for guidance to start a new line of research

Each book is edited by an expert and consists of chapters written by the leaders

in a particular field Books are richly illustrated and contain comprehensive ographies Chapters provide substantial background material relevant to the partic-ular subject Hence, they are not only “ methods books,” but they also containdetailed “tricks of the trade” and information as to where these methods can besafely applied In addition, they include information about where to buy equipmentand about web sites helpful in solving both practical and theoretical problems

bibli-We hope that as the volumes become available, the effort put in by us, by thepublisher, by the book editors, and by individual authors will contribute to the furtherdevelopment of brain research The extent that we achieve this goal will be deter-mined by the utility of these books

Sidney A Simon, Ph.D Miguel A.L Nicolelis, M.D., Ph.D.

Series Editors

2345_frame_FM Page 5 Wednesday, October 23, 2002 3:20 PM

About the Editor

Anat-omy and an associate dean of biomedical graduate studies at Drexel UniversityCollege of Medicine (formerly MCP-Hahnemann University School of Medicine).After receiving his B.S degree in biology in 1971 from Muhlenberg College, Dr.Waterhouse completed his Ph.D in pharmacology at Temple University in 1977.From 1977 through 1987 he worked at Southwestern Medical School, University ofTexas at Dallas, rising from postdoctoral fellow, to instructor, and then finally toassistant professor

In 1987 he was recruited to the Department of Physiology and Biophysics as

an associate professor at Hahnemann University School of Medicine, where in 1988

he developed and was subsequently appointed director of the university's graduateprogram in neuroscience, a post he held until 1994 In 1992 he was promoted toprofessor of physiology and biophysics, and in 1994, when Hahnemann Universitymerged with Medical College of Pennsylvania (MCP), Dr Waterhouse was invited

to join the Department of Neurobiology and Anatomy in the newly formed university

He continued as director of the neuroscience graduate program at MCP-Hahnemannuntil 2001 and also served as vice-chair of the Department of Neurobiology andAnatomy from 1999 to the present He was elected to the American College ofNeuropsychopharmacology in 1996 and to the College on Problems of Drug Depen-dence in 1995 Throughout his research career Dr Waterhouse has focused on theneurobiology of central monoaminergic systems and psychostimulant drug actions

2345_frame_FM Page 9 Wednesday, October 23, 2002 2:16 PM

Michael H Baumann

Medications Discovery Research

Branch

National Institute on Drug

Abuse-Intramural Research Program

National Institutes of Health

Center for Sensor Technology

University of Kentucky Chandler

Patricia H Janak

Ernest Gallo Clinic and Research CenterDepartment of NeurologyThe University of California

at San FranciscoSan Francisco, California

Laura L Peoples

Department of Psychology, Neuroscience Graduate GroupUniversity of PennsylvaniaPhiladelphia, Pennsylvania

2345_frame_FM Page 11 Wednesday, October 23, 2002 2:16 PM

MCP-Hahnemann UniversityPhiladelphia, Pennsylvania

Self-Administration of Drugs of Abuse 17

Steven I Dworkin and Dustin J Stairs

In Vivo Voltammetry in Drug Abuse Research 87

Michael F Salvatore, Alexander F Hoffman, Jason J Burmeister, and

David M Devilbiss and Barry D Waterhouse

2345_frame_FM Page 13 Wednesday, October 23, 2002 2:16 PM

Chapter 9

Pharmacological Investigations of Neural Mechanisms Underlying

Amphetamine-Like Stimulant-Induced Arousal: Involvement of Noradrenergic Systems 239

Craig W Berridge

Index 271

Overview

Barry D Waterhouse and Laura L Peoples

CONTENTS

1.1 Introduction 1

1.1.1 Theories of Drug Addiction 2

1.1.1.1 Incentive Motivation Theory of Addiction 2

1.1.1.2 Hedonic Dysregulation of Reward and Allostasis 3

1.2 Rationale for Cellular and Circuit Levels of Analysis of Drugs of Abuse 5

1.2.1 Studies of Cells within Circuits 6

1.2.2 Studies in Awake Animals 7

1.2.3 Delineation of Mechanisms That Contribute to Behavior 7

1.2.4 Mechanisms That Mediate Drug Effects on Behavior 8

1.3 Cellular and Circuit Level Analysis of Drugs of Abuse 8

1.3.1 Current Methods 8

1.3.2 Future Directions 9

Acknowledgements 9

References 10

1.1 INTRODUCTION

Drug addiction is a progressive disorder characterized by a transition from controlled

to uncontrolled and compulsive drug seeking that continues despite knowledge of adverse consequences (Hoffman and Goldfrank, 1990; Leshner, 1997; McGinnis and Foege, 1999) It is also a chronic relapse disorder Periods of successful drug abstinence for many individuals end with relapse to compulsive drug use Drug addiction is a devastating disorder that has severe health costs to both the individual and the public (McLellan et al., 2001) Although environmental variables can influ-ence an individual’s risk for developing addiction, human and animal research show that addiction is fundamentally a disorder of the brain Application of neuroscience approaches to the study of addiction is thus an integral part of efforts to understand and ultimately to treat the disorder Two issues that are central to understanding the problem of drug abuse and addiction are 1) identification of drug actions that contribute to an initially positive drug experience and 2) elucidation of the neural mechanisms underlying the progression of addiction and the development of drug craving Acute self-administration of addictive compounds produces a multitude of

1 2345_frame_MASTER.book Page 1 Wednesday, October 23, 2002 1:03 PM

2 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

transient effects on the brain, only some of which contribute to a positive drugexperience and the desire to repeat that experience in a social setting However,chronic use of these substances leads to long-lasting changes in nervous systemfunction that are thought to contribute to the development and maintenance ofcompulsive and uncontrollable drug seeking

Investigation of the acute and chronic effects of addictive drugs has led to thedevelopment of a number of formal theories of addiction Two that are particularlyinfluential today are the incentive motivation theory (DiChiara, 1998; Everitt et al.,1999; Robbins and Everitt, 1999; Robinson and Berridge, 1993; Stewart et al., 1984;Stewart, 1992) and the hedonic dysregulation theory (Koob and LeMoal, 1997).Evidence consistent with each theory can be found in the animal and human literature

on addiction; however, important predictions of each have yet to be tested fully.Although the theories differ in a number of aspects, they are not necessarily mutuallyexclusive and actually share at least two common assumptions First, it is proposedthat repeated exposure to drugs produces long-lasting changes in the brain thatcontribute to the development of compulsive and uncontrollable drug taking Second,these changes are proposed to occur in multiple regions and at several levels oforganization, from the level of molecular regulation of protein synthesis and cellularfunction to the level of local neural networks and circuits involving interactionsbetween multiple brain structures These theories, and an additional hypothesis that

is gaining increasing influence, are reviewed and critically evaluated relative toexisting data in a number of publications (Robinson and Berridge, 1993; Jentschand Taylor 1999; Wise, 1999; Koob and LeMoal, 2001) They will thus be onlybriefly summarized here because they are relevant to understanding the utility of theresearch methods described in this volume

1.1.1.1 Incentive Motivation Theory of Addiction

Incentive motivation theories of drug addiction propose that the disorder reflects apathological responsivity of individuals to the influences of drug-associated condi-tioned stimuli on behavior The theories further assert that the abnormal responsivity

to drug stimuli is caused by acute and long-lasting actions of addictive drugs onbrain Acute drug actions are proposed to amplify mechanisms that contribute tostimulus–reward learning and lead to abnormally powerful conditioning of stimuliassociated with drug administration It is also proposed that long-lasting neuroplas-ticity induced by drugs facilitates this drug-induced amplification of learning (forreview see DiChiara, 1998; Everitt et al., 1999; Robbins and Everitt, 1999; Robinsonand Berridge, 1993; Stewart et al., 1984; Stewart, 1992) These proposals are based,

in part, on evidence that stimuli associated with drugs undergo conditioning and canfacilitate drug seeking in both animals and humans (Arroyo et al., 1998; Davis andSmith, 1987; deWit and Stewart, 1981; Ehrman et al., 1992; Goldberg et al., 1976;Ranaldi and Roberts, 1996; Stewart et al., 1984; Tiffany, 1990) Additionally, animalstudies show that acute actions of addictive drugs amplify the gain of behavioral

Overview 3

responses to conditioned stimuli and may facilitate conditioning (Harmer and lips, 1998; O’Brien et al., 1998; Panililio et al., 1998; Robbins et al., 1989; Taylorand Robbins, 1986; Taylor and Horger, 1999; Weiss et al., 2000) Furthermore,repeated administration of addictive drugs sensitizes animals to various effects ofthose drugs including acute reinforcing effects and gain-amplifying effects onresponsivity to conditioned stimuli (Lorrain and Vezina, 2000; Taylor and Horger,1999; Wyvell and Berridge, 2001)

Phil-1.1.1.2 Hedonic Dysregulation of Reward and Allostasis

The hedonic dysregulation theory (Koob and LeMoal, 1997 and 2001) proposes thataddiction is a self-regulatory condition The theory is based on principles of homeo-static self-regulation and allostasis (Sterling and Eyer, 1988) and has roots in theopponent process theory of motivation described by Solomon and Corbit (1974).Homeostasis corresponds to the mechanisms that maintain stability within physio-logical systems and hold all parameters of an organism’s internal milieu withinadaptive limits Certain parameters are held at a constant set point by local negativefeedback responses to deviations from the set point Other parameters are allowed

to vary within a wide range so as to maintain balanced function within particularphysiological systems In contrast, the principle of allostasis involves the stabiliza-tion or balancing of function by a resetting of the set point in response to a chronicdemand on homeostatic mechanisms that are insufficient to fully compensate for thedeviations in the original set point These allostatic changes can compensate for thedemand, but they can also lead to an abnormal state when the demand is removedand are proposed to contribute directly to the development of drug addiction.More specifically, acute actions of addictive drugs are proposed to overactivatereward pathways in the brain This overactivation leads to a homeostatic responsethat involves down-regulation of neurochemical systems involved in mediating thedrug-induced overactivation However, the homeostatic changes in reward neu-rotransmitters are hypothesized to be insufficient to maintain balanced functionwithin the reward system This insufficiency, in conjunction with a chronic demand

on these homeostatic mechanisms, leads to the onset of an allostatic process Thisprocess involves changes that tend toward reestablishing the balanced reward func-tion by changing the reward set point that is normally guarded by homeostaticmechanisms The increase in reward set point is proposed to be mediated, in part,

by recruitment of nonreward systems, specifically brain stress systems that arenormally involved in negative emotional states The recruitment and changes in thebrain stress systems tend to counteract the drug-induced overactivation of the rewardsystem but engender a negative mood or state in the absence of drug This negativemood state is thought to set up a negative reinforcement mechanism That is,individuals begin to seek drug in order to avoid the negative mood state It is proposedthat these allostatic responses grow in magnitude with repeated drug use Thus,addiction is proposed to be a feed-forward cycle in which increases in drug intakeare followed by increases in the magnitude of the allostatic response and resetting

of the reward set point, which leads to further increases in drug intake and thereinitiation of the cycle

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4 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

This theory is based primarily on two observations First, acute withdrawal fromaddictive drugs is commonly associated with negative affective states includingdysphoria, depression, irritability, and anxiety Second, after cessation of intravenousself-administration, animals exhibit an increased threshold for intracranial self-stimulation (ICSS) reward (i.e., a higher level of stimulation is required for thestimulation to be reinforcing) Additional evidence consistent with the hypothesis isdescribed by Koob and LeMoal (2001)

1.1.1.2.1 Hypoactivity in Cortical Inhibitory Mechanisms

Neuroimaging studies in humans show that individuals addicted to drugs such ascocaine show evidence of reduced activity and lower cell density in corticalregions such as the anterior cingulate and orbitofrontal cortex (Volkow, 1991;Childress et al., 1999; Franklin et al., 2002) These and other cortical structuresare involved in willed or executive control of behavioral selection Executivecontrol of behavior involves dynamic emotional and cognitive analyses of pastand expected events and the influence of these analyses on decisions about futureactions This online willed control comes into play when automatic or habitualbehaviors controlled by subcortical brain structures are not sufficient to guideadaptive behavior Moreover, it can be important in the initiation of actions,persistence of adaptive actions in the absence of reward, and inhibition of impulses

to engage in alternative but less beneficial behaviors The anterior cingulate cortex(ACC) and orbitofrontal cortex contribute to the generation of emotion and toexecutive control of the influence of these emotions on behavior Abnormalities

in these brain regions are associated with a range of disorders involving bances in emotion and action In light of these data, it is hypothesized that thecortical abnormalities observed in addicted individual contribute to addiction(Volkow, 1991; Childress et al., 1999; Rogers et al., 1999; Volkow and Fowler,2000; London et al., 2000)

distur-Consistent with this hypothesis, addicted individuals exhibit symptoms ated with insults to the ACC and orbitofrontal cortices including anhedonia and aninability to make adaptive decisions regarding future actions (e.g., Grant et al.,1996; Rogers et al., 1999) In fact, a hallmark of drug addiction is compulsive anduncontrollable drug use, despite knowledge of adverse consequences It is thuspossible that addicted individuals, like others who suffer from hypoactivity in thesebrain regions, are unable either to experience normal affective responses to futureevents or to exert executive control over the adaptive influence of those emotions

associ-on selectiassoci-on of beneficial actiassoci-ons Perhaps most important in the case of addictiassoci-on,the deficiencies in emotional regulation may limit the ability of addicted individuals

to inhibit responses to rewards, including drug, in order to avoid harm (e.g.,incarceration or death) There is some evidence that these neural abnormalities areinduced by drug exposure, but it is also possible that some or all of the brainabnormalities are present in the individual prior to drug exposure and perhapsenhance vulnerability to addiction Finally, it has been proposed that addiction mayreflect a combination of weakened cortical inhibitory mechanisms and the overac-tive (sensitized) responses of subcortical mediated responses to drug-associatedstimuli (for review see Jentsch and Taylor, 1999)

interdis-Despite the advances that have resulted from the use of these varied experimentalapplications, they do not address the question of how addictive drugs initiate andconsolidate system-wide changes in neuronal functions that underlie drug-relatedbehavior However, combinations of electrophysiological, neurochemical, andbehavioral techniques are uniquely capable of providing information that can fillthis considerable gap in our understanding.

1.2 RATIONALE FOR CELLULAR AND CIRCUIT LEVELS

OF ANALYSIS OF DRUGS OF ABUSE

Implicit in any description of drug addiction is the fact that circuits within the brainmust be reorganized so as to generate compulsive behaviors for acquisition and self-administration of rewarding compounds Such changes are clearly long-lasting andnot readily reversible, as evidenced by the chronic relapsing character of drug addic-tion The physical and psychological symptoms that emerge during withdrawal fromchronic drug use and the sensitization that is evident with many addictive compoundsfurther underscore the likelihood that drug addiction is a behavioral manifestation offundamental changes in cellular biological processes Many studies have in fact begun

to identify neuron-specific gene products that are synthesized in response to chronic

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6 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

drug treatment and, in addition, are correlated with various behavioral dimensions ofthe addicted state (Self and Nestler, 1998; White and Kalivas, 1998) On this basisone could argue that molecular and cellular biological studies hold the greatestpromise for elucidating the underlying mechanisms of drug abuse and addiction andfor identifying genetic factors that contribute to susceptibility to substance abuse.Such reductionist approaches have, in fact, yielded many new data and have providedmany new insights regarding the intracellular targets and modes of action of abuse-potential substances Moreover, such studies may also be the most likely to identifypotential targets for therapeutic intervention and treatment of drug addiction On theother hand, this level of inquiry leaves many unanswered questions regarding themolecular and cellular actions of drugs of abuse and their impact on whole-animalbehavior For example, it is well to remember that short- and long-term molecularand cellular responses to addictive compounds are embedded within selected localcircuits and neural networks that give rise to specific behavioral responses

Drug-associated behaviors develop over time and are most likely the product ofdrug influences on sensory, motor, and associational circuits in the brain Suchalterations may result from long-lasting neuroadaptations to drug exposure or mayrepresent more fundamental physiological responses to drug administration by mem-brane receptors or synaptic release mechanisms To better understand the physio-logical bases of these dimensions of drug abuse and provide a meaningful frameworkfor more reductionist approaches studies are needed to determine the specific timecourse and outcome of drug influences on neurotransmitter levels, activity patterns,local circuit operations, and neural network interactions that are directly responsiblefor whole-animal responses to drug administration

The goal of intracellular and extracellular recording studies in tissue slice and intact,anesthetized preparations is to characterize drug actions on individual neurons underreasonably controlled conditions In many cases, particularly with in vitro tissue-slice experiments, dose-response relationships and receptor mechanisms of drugaction can be evaluated with great precision using these approaches Likewise,extracellular single-unit recording methods have been used in intact, anesthetizedanimals to monitor and measure the responses of individual neurons to local orsystemic drug administration

Such experiments have provided a useful means of assessing potential changes

in cell function over a range of drug doses In addition, in many cases neural circuitshave been well characterized in terms of cytoarchitecture and connectivity andinclude neurons that can be classified according to unique morphological and intrin-sic electrophysiological properties (Larkman and Mason, 1990; Mason and Larkman,1990; McCormick et al., 1985) Individual cells may vary substantially in theirresponsiveness to systemically administered drugs Such a view is supported byevidence (see reviews by Llinas, 1988; Nicoll et al., 1990; Schofield et al., 1990)that suggests that within a heterogeneous population of cells there are neuronalsubtypes that express different complements of membrane receptors and signaltransduction mechanisms and ion channels With both intracellular and extracellular

Overview 7

studies the morphological, electrophysiological, and pharmacological (i.e., drugeffects) properties of individual cells can be compared and sorted according torecurrent patterns that emerge This approach begins to assess the potential range

of selective responses of neurons within a complex circuit to acute or chronic drugadministration Such information is crucial for the development of future testablehypotheses concerning the impact of an exogenous agent on ensembles of function-ally related cells within awake, behaving animals

Most if not all theories of addiction share a common assumption that addiction iscaused by drug-induced changes in reward-related behavioral processes (Koob and

Le Moal, 1997; for reviews of various theories see Wise and Bozarth, 1987; Robinsonand Berridge, 1993; Koob and Le Moal, 2001) Such processes are expected to bepresent only in the awake animal and only within certain behavioral contexts It isthus possible that drug effects and neural events that are critical to the development

of addiction and relapse are observable and thus subject to investigation only underthese conditions Appreciation of this fact is an important impetus for the application

behavioral conditions relevant to drug taking and relapse in humans

The intravenous-drug self-administration model, and variations thereof, is widelyviewed as the animal model of human drug addiction and relapse that has the greatestface and predictive validity Extensive use of the paradigm has shown that the drugself-administration behavior of laboratory animals is consistent with patterns of drugtaking in humans, including that of drug-addicted individuals studied in laboratorysettings (e.g., Griffiths et al., 1980; Johanson and Balster, 1978; Pickens et al., 1978;Stewart et al., 1984) Pharmacological and neuroimaging studies in humans showthat the pharmacology- and neurobiology-mediating drug taking in animals andhumans are mediated by homologous, if not the same, neuropharmacological mech-anisms (Breiter et al., 1999; Childress et al., 1999; Grant et al., 1996; Volkow andFowler, 1999) The behavioral paradigm is thus the paradigm of choice to beemployed in in vivo investigations of mechanisms that contribute to addiction Withinthe last 15 years researchers have developed methodologies that allow for the use

of microdialysis and in vivo voltammetry recordings to characterize neurochemicalevents in animals self-administering drug (e.g., Bradberry et al., 2000; Hurd et al.,1989; Gratton and Wise, 1994; Pettit and Justice, 1989; Wise et al., 1995) In parallel,chronic extracellular recording and EEG procedures have been developed to char-acterize neurophysiological events during drug-related behavioral states (Berridge,this volume; Peoples et al., 1989, 1999; Chang et al., 1990, 1994; Carelli et al., 1993;Peoples and West, 1996)

8 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

potentially contribute to a behavior However, the method can additionally be used

to explore the mechanisms that mediate the contribution As noted by Rolls (2000),

to understand how the contribution is made it is necessary to understand what mation is represented in that region and, moreover, what information is received, howthat information is integrated, and how the information is transmitted to target sites

infor-An important aspect of characterizing this information processing is to analyze theresponses of single neurons and groups of single neurons, for it is at this level thatmuch of the information processing occurs These analyses of single-neuron responsesare most relevant when they can be made during the behavior of interest under relevanttest conditions (Rolls, 2000) Delineating the nature of information encoded and themechanics of information processing within regions important to drug seeking willultimately contribute to a blueprint of potential targets for therapeutic interventions

An effect of drug on a given behavior is mediated by drug-induced changes in theactivity of neurons that control that behavior Acute electrophysiological methodsare often used in efforts to delineate those changes in neural activity Recordings inbehaving animals can corroborate and complement these studies In slice and anes-thetized recording preparations, neurons can be stripped of normal afferent input.Anesthesia can additionally influence membrane properties Both the deafferentationand the direct effects of anesthesia on a recorded neuron can alter the impact ofdrug on the neuron The potential for this alteration is demonstrated by previousobservations of differences in the results of acute and awake animal recordings(Chapin, Waterhouse, and Woodward, 1981; Deadwyler, 1986; Moxon, 1999; West,1997) It is also emphasized by the observation that the “mere” differential activation

of a neural circuit in association with a change in behavioral state can alter theobserved effect of drug on brain (e.g., Hemby et al., 1997; Smith et al., 1980, 1982).Given the importance of the state of a neuron on the response of that neuron to drug,recordings in waking animals can be a useful complement to acute recording studies.Analysis of the comparability, as well as the differences, among the different studiescan yield a more complete and accurate picture of the drug effects on brain thatmediate a drug-induced change in behavior

1.3 CELLULAR AND CIRCUIT LEVEL ANALYSIS

OF DRUGS OF ABUSE

In this volume, we describe the rationale and methodologies for characterizing theoperation of single cells, local circuits, and neural networks before, during, and afteracute or chronic administration of drugs of abuse For each technique advantages aswell as limitations are given For example, single-unit intracellular and extracellularrecording techniques in tissue slice and anesthetized preparations (see Waterhouse,Chapter 5) offer the opportunity to obtain detailed information about drug effects oncells in intact local circuits under well-controlled conditions Knowing the identity of

Overview 9

recorded cells further enhances the interpretation of results since drug actions can beevaluated in the context of the role of selected cell types in specific neural circuitoperations Despite these advantages, drug effects in these preparations are examined

in the absence of the behavioral or physiological state in which they normally occur

By contrast, neurochemical (Baumann and Rutter, Chapter 3; Salvatore et al., Chapter4), multichannel, multineuron recording (Devilbiss and Waterhouse, Chapter 8; Janak,Chapter 6; Peoples, Chapter 7), and EEG (Berridge, Chapter 9) techniques can beapplied in the awake, behaving animal but have the added complication of introducingeffects that are secondary to drug-induced behaviors

For the most part, the experimental methods described here are used for many otherapplications in neuroscience but clearly have unique advantages for studies of drug abuseand addiction Moreover, they can be applied in combination (i.e., self-administration,Dworkin and Stairs, Chapter 2, and electrophysiological recording, Peoples, Chapter 7)

to yield new opportunities for examining physiological functions under relevant ioral conditions Finally, they can be applied in a variety of preparations and animalmodels to address specific questions about drug effects under different physiological ordrug-related conditions, e.g., evaluation of cell and circuit function in nạve vs chronicdrug-treated animals and offspring of drug-addicted mothers

In their current form, the techniques and experimental strategies described here willcontinue to provide new information about the impact of drugs of abuse on individualneurons and neural networks and the behaviors that result from these influences.However, a number of future developments that will enhance their utility can beanticipated For example, implantable probes that combine recording surfaces formultichannel, multineuron recording and in vivo voltammetry are on the horizon.This technology will provide a window on local tissue levels of endogenous trans-mitter/modulators and local spike train activity in regions of the brain that areinfluenced by drug actions and subsequently responsible for mediating drug-relatedbehaviors Other improvements in multineuron recording electrodes and proceduresmay facilitate long-term recording of spike train activity from the same neuronsacross extended time periods such that physiological changes associated with chronicdrug administration can be routinely studied Likewise, adaptation of these in vivo

voltammetry, microdialysis and multichannel, multineuron recording procedures forthe mouse brain would encourage neurochemical and electrophysiological investi-gations using a broad spectrum of genetically manipulated animals (Drouin et al.,2002) Such approaches will offer new opportunities to bridge the gap between drugactions at the cellular and molecular level in the brain and the behavioral manifes-tations of substance abuse

10 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

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Neuron., 19, 591, 1997.

6 Bozarth, M.A., Methods of Assessing the Reinforcing Properties of Abused Drugs Springer-Verlag, New York, NY, 1987.

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7831, 2001

Self-Administration of Drugs of Abuse

Steven I Dworkin and Dustin J Stairs

CONTENTS

2.1 Introduction 172.2 Three Phases of Drug Self-Administration 182.2.1 Early Research in the Area 192.3 Effects of Environmental and Behavioral Variables 212.3.1 Behavioral and Drug History 21

Administration 25

Self-Administration 272.3.4 Relapse and Reinstatement 272.4 Methodology 282.4.1 Acquisition of Drug Self-Administration 282.4.2 Maintenance of Drug Self-Administration 292.4.3 Fixed-Ratio Schedules 292.4.4 Progressive-Ratio Schedules 312.4.5 Second-Order Schedules of Drug Reinforcement 332.4.6 Subjects 332.5 Apparatus 332.5.1 Operant Chambers 332.5.2 Housing Chambers 342.5.3 Swivels 352.5.4 Catheters 352.6 Detailed Rat Catheterization Procedure 382.7 Summary and Future Directions 42Acknowledgments 42References 42

2.1 INTRODUCTION

It has been over 40 years since the technical aspects of automatic intravenous drugdelivery to rodents were described.1 The development of self-administration meth-

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18 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

odology provided a tremendous technological advance for investigating drug abuseand resulted in clear demonstrations that the behavioral actions of psychoactivesubstances could be investigated using the same methods used to evaluate thereinforcing effects of nondrug reinforcers The intravenous route of administrationhas several methodological advantages over other routes The drug is discretelypresented through an intravenous catheter so that the concentration of drug deliveredper unit time can be more accurately assessed Further, there are no problemsassociated with drug spillage, the handling of animals, or other confounding variablessuch as taste

Intravenous drug delivery is an extremely rapid procedure for delivering apharmacologically active substance Consequently, the intravenous route of drugdelivery has enabled behavioral pharmacologists to determine the behavioral effects

of a wide variety of drugs More importantly, intravenous delivery of drugs hasallowed for the characterization of behaviorally active drugs based on their ability

to maintain self-administration Intravenous self-administration procedures allow fordirect assessment of the reinforcing or abuse liability of psychoactive compounds.During the past four decades significant advances in our understanding of sub-stance abuse resulted from utilization of drug self-administration procedures Thisfueled important developments in several fields including the experimental analysis

of behavior, behavioral pharmacology, and behavioral neuroscience Since there are

a large number of reviews of the literature on rodent self-administration, this chapterwill provide only a brief history and overview of the current use of the rodentintravenous drug administration procedure Methodological and procedural consid-erations for utilizing intravenous drug delivery systems will then be discussed Fromits conception continuing through to the present, the intravenous drug deliverysystem has been utilized for drug self-administration studies While methodologicaladvances have occurred in the development of sophisticated technological enhance-ments in the areas of drug self-administration research, the basic drug deliverysystem remains relatively unchanged

2.2 THREE PHASES OF DRUG SELF-ADMINISTRATION

There are three important components of drug abuse that can be assessed usingrodent self-administration procedures These three aspects are the acquisition orengendering of self-administration, the continued maintenance of drug intake inanimals that are self-administering the drug, and the reinitiation of self-administra-tion following protracted withdrawal or extinction Evaluations of acquisition andmaintenance provide an assessment of abuse liability, whereas studies investigatingthe reacquisition of extinguished behavior are suggested to provide an assessment

of drug relapse.2 While initial studies used rodent intravenous drug delivery to assessbehavioral and pharmacologic variables that maintained self-administration, morerecent research has extended these investigations to include evaluations of the ini-tiation of drug intake and relapse of drug intake Thus, recent studies in this areahave evaluated behavioral, pharmacologic, and neurobiologic aspects of the acqui-sition, maintenance, and relapse to self-administration

Self-Administration of Drugs of Abuse 19

Initial research in the area of rodent drug self-administration was directed towardsidentification of pharmacological agents that would maintain self-administration.3–6This early work demonstrated that the rodent self-administration procedure provided

an exquisite model of human drug abuse in that all of the compounds

exceptions (e.g., hallucinogens), this research provided ample evidence that most ofthe drugs abused by humans would establish and maintain drug taking by rodents.Early studies with opiates (e.g., morphine) used experimenter-delivered injections

to make rats physically dependent on the compound before self-administration wasestablished Now many studies with heroin and other opiates have clearly shownthat this establishing procedure is not necessary to support self-administration byopiates or most other pharmacologic classes

After clearly establishing the utility of the rodent self-administration procedurefor identifying and investigating human substance abuse, research efforts weredirected towards investigating both pharmacologic and environmental determinants

of drug self-administration Some of the pharmacologic variables that were tigated were the drug, drug dose, infusion duration, effects of pharmacologic antag-onists, role of tolerance, cross-tolerance and dependence, and various drug histories.Many excellent reviews of the effects of these variables on self-administration ofseveral drug classes have been published.8–11 The data presented in Figure 2.1 depicttypical dose–response curves for responding maintained by cocaine under fixed-ratio (FR) schedules of reinforcement The lowest dose of cocaine investigated inthis study was shown to be a threshold dose for maintaining self-administration.Increasing doses of cocaine resulted in dose-related decreases in the number ofinjections self-administered during a 4-h session Furthermore, increasing doses ofcocaine resulted in dose-related increases in the time it took to complete the FR 10schedule requirement as indicated by the increase in the mean interinjection intervaland dose-related increases in the total amount of drug that was self-administeredduring the session

inves-One of the most intriguing and perhaps most studied pharmacologic aspect ofdrug self-administration is the shape of the dose–response curve The prototypicaldose–effect curve consists of a bimodal or inverted U-shaped function indicatingthat both low and high doses of a drug maintain comparable rates of response ornumber of infusions An example of this type of dose–effect curve is presented inFigure 2.2 This figure illustrates the effects of increasing the dose of heroin onresponding maintained by an FR 10 schedule of reinforcement Increasing doses of

self-administered during the 4-h session Further increases in heroin dose from 50 to

300 mg/inf resulted in dose-related decreases in the number of infusions istered during the session The total dosage of self-administered heroin increases to

self-admin-an asymptotic value as the dose per infusion increases In this case, the bitonicfunction resulted from a difference in the patterning of drug intake maintained bythe lower and higher doses However, several different explanations for the typical

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20 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

bitonic function have been proposed This issue is discussed in greater detail later

in the chapter

In addition to the considerable influence of pharmacologic variables, earlyresearch in the field elucidated the pervasive dominance of behavioral or environ-mental variables on drug taking Collectively these studies demonstrated the simi-larity of drug and nondrug reinforcers in their control over behavior (for reviewssee References 4 and 12–15) Thus, abused drugs are not unique in their ability toinfluence characteristics of behavior These characteristics include the rate and tem-poral patterns of drug intake and the persistence of drug taking

It became obvious that basic research on substance abuse could utilize theexperimental methods and procedures employed to investigate the effects of nondrugreinforcers Figure 2.3 shows how the rate and temporal patterns of cocaine intakecan be controlled by the schedule of reinforcement used to maintain self-adminis-tration Responding was maintained by the same dose of cocaine under the fourschedules shown Cocaine-maintained responding was directly influenced by sched-ule of reinforcement and was remarkably similar to patterns previously reported forfood and other nondrug reinforcers A considerable amount of research has supported

FIGURE 2.1 Dose–response curves for cocaine self-administration under an FR 10 schedule

of cocaine infusions The data are means and SD from six rats run during 4-h sessions The top panel shows the mean number of infusions maintained by the four doses investigated The bottom left and right panels show the effects of cocaine dose on the mean interinjection interval and total drug intake Increasing doses of cocaine resulted in dose-related decreases

in the number of ratios completed and increases in the mean interinjection interval (time between each injection) and amount of cocaine that was delivered during the session The large variability for the number of infusions maintained by the lowest dose of cocaine resulted from individual rats self-administering at a high rate on some days and not responding at all

on others.

100 80 60 40

0 0.1 0.3 0.5 1

0.1 0.3 0.5 1

20

COCAINE (mg/infusion)

COCAINE (mg/infusion) COCAINE (mg/infusion)

16 12 8 4 20

0

16 12 8 4 20

0 0.1 0.3 0.5 1

Self-Administration of Drugs of Abuse 21

the notion that drug taking is influenced by the same variables that control respondingmaintained by other environmental events such as food, water, and sex Althoughthe unique aspects of behavior controlled by abused drugs continued to maintainexperimental interest, recent studies using sophisticated brain-imaging techniqueshave provided confirmational support that drug and nondrug reinforcers have similarinfluences on brain systems proposed to regulate appetitive behaviors.16,17

2.3 EFFECTS OF ENVIRONMENTAL AND BEHAVIORAL

VARIABLES

Investigations of intravenous cocaine self-administration represent a major portion

of the literature on drugs as reinforcers.13 Some of the earliest reports employedunlimited access to the drug.18,19 Under these conditions, rats and rhesus monkeysexhibited cyclic patterns of responding with periods of high levels of respondingfollowed by periods of little or no drug self-administration These patterns of drug-intake would usually continue until the animals died With restricted access to thedrug, animals will self-administer cocaine during daily sessions for weeks or evenmonths without developing observable adverse health effects.20,21 The data frommore recent studies evaluating changes in the self-administration of cocaine duringextended session durations has demonstrated a dysregulation in the self-administra-tion of the drug resulting in a considerable increase in drug intake.22

FIGURE 2.2 Dose–response curves for heroin self-administration maintained under an FR

10 schedule of heroin presentations The data are means and SD from six rats allowed to administer heroin during 4-h sessions The number of infusions maintained by increasing doses of heroin resulted in a bitonic function in that the highest rate of drug intake was maintained by a moderate dose of the drug (left panel) Total drug intake followed a monotonic increasing function with an asymptote at the two largest doses Low doses of the drug maintained responding only during the first 2 h of the session, whereas larger doses maintained consistent self-administration during the 4 h with longer interreinforcement intervals com- pared to the lower doses This resulted in the equivalent number of infusions for both higher and lower doses.

3 4 5 6

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22 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

The development of intravenous self-administration has brought the investigation

of the reinforcing effects of drugs into the realm of procedures used to study otherreinforcers Thus, basic and complex operant schedules of reinforcement23 can beused to determine the conditions under which cocaine would be self-administered(for reviews see References 9 and 21) Moreover, the degree to which the results ofnonhuman laboratory self-administration studies have generalized to human drug

FIGURE 2.3 Representative cumulative response records depicting responding maintained under four different schedules of cocaine administration The completion of each schedule requirement resulted in a 5-sec injection of a 0.33-mg dose of cocaine Performance main- tained by an FR 10 schedule is shown in the top panel An injection was delivered following the completion of each ten responses The second panel displays the performance maintained under a PR 25 schedule Under this schedule the ratio value was increased by 25 responses after each infusion The third panel contains a record of responding maintained by a VI 5- min schedule Under this schedule contingency the first response after a mean interval of 5 min resulted in an injection of the drug The bottom panel depicts performance maintained under a fixed-interval 10-min schedule A 30-sec TO was scheduled following the completion

of each ratio requirement under the two ratio schedules, and a 10-min TO was presented following each injection delivered under the two interval schedules Deflections of the bottom pen in the second and fourth panels indicate the duration of the TO The motor on the cumulative recorder was stopped during the TO programmed under the VI schedule.

RESPONDING MAINTAINED BY 0.33 mg INFUSIONS OF COCAINE

PROGRESSIVE-RATIO 25

VARIABLE-INTERVAL 5 MIN

FIXED-INTERVAL 10 MIN FIXED-RATIO 10

30 MIN

Self-Administration of Drugs of Abuse 23

use and abuse suggests that many of the factors involved in modulating behaviormaintained by nonpharmacologic reinforcers may also be important determinants

of drug use.7 Several variables have been shown to alter the behavioral effects ofnonpharmacologic as well as pharmacologic agents on schedule-controlled behavior.These variables include the schedule of reinforcement, the type of reinforcer used

to maintain behavior, the behavioral and drug history of the organism, and theenvironmental context in which the behavior is studied.8

The behavioral and drug history of an organism can alter the effects of acutelyadministered drugs on schedule-controlled behavior.8,24,25 Moreover, historical vari-ables can reverse the behavioral effects of chronically administered environmentalevents Behavioral and drug history has also been shown to influence the reinforcingeffects of drugs In many situations, researchers have trained their subjects on aschedule of food presentation or engendered responding using an extremely effica-cious drug reinforcer such as cocaine before substituting a potentially less efficaciousreinforcing drug In some cases, these less efficacious drugs may not engender self-administration without this history Prior food training appears to be necessary forobtaining nicotine self-administration.26,27 Additional studies have demonstrated thatnicotine can serve as a punisher,28 negative reinforcer,29 or positive reinforcer30depending on the historical variables involved Caffeine can also maintain self-administration under conditions similar to those that support nicotine self-adminis-tration Figure 2.4 shows responding maintained by FR schedules in rats that werefirst trained to lever press using food as the reinforcer Responding was maintainedunder an FR 1–3 schedule at rates that were considerably higher than when vehiclewas substituted for caffeine These data demonstrate that even relatively weakreinforcers can maintain self-administration under the appropriate conditions.Cocaine can also have reinforcing or aversive effects depending on the history

of the organism.31,32 The rate and temporal pattern of behavior resulting in cocaineadministration can be dramatically altered in monkeys when they are exposed todifferent behavioral histories.33 Another important historical variable is previousexposure of the organism to environmental stress The involvement of stress andthe subsequent activation of the hypothalamo-pituitary-adrenal (HPA) axis in theacquisition and maintenance of intravenous cocaine self-administration has beenclearly demonstrated.34–37

A subject’s previous exposure to different pharmacologic agents can alter therate of acquisition of self-administration or affect whether or not a particular com-pound will be self-administered Schenk and colleagues have shown that preexposure

to cocaine38 or caffeine39 increases the acquisition of responding maintained bythreshold doses of cocaine The N-methyl-D-aspartate (NMDA) receptor antagonistMK-801, which is not self-administered when substituted for cocaine, will maintainresponding in the same monkeys following a history of phencyclidine self-admin-istration Evaluations of the neurobiologic mechanisms involved in the alterationsthat historical variables can exert on the behavioral effects of cocaine are limited,although caffeine preexposure was reported to increase the extracellular levels ofdopamine obtained in the ventral striatum following a cocaine challenge.39Perhaps the most frequently utilized variable in drug self-administration studies

is the level of food restriction Minimal levels of food restriction increase locomotor

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24 Methods in Drug Abuse Research: Cellular and Circuit Level Analyses

Self-Administration of Drugs of Abuse 25

activity and can enhance the acquisition of schedule-controlled behavior maintained

by both nondrug and drug reinforcers Moderate levels of food restriction increasethe acquisition and maintenance of self-administration of several different classes

of abused drugs.40 For most drugs decreasing the level of food restriction does notconsiderably attenuate drug self-administration once responding is engendered.However, several investigators have reported that moderate food restriction is nec-essary to maintain nicotine self-administration

Another important but often overlooked behavioral variable is the contingencybetween responding and the delivery of drug reinforcers This contingent relationship

is of primary importance because it provides the empirical validation that an event

is reinforcing In order for an environmental event to be considered a reinforcer theevent must increase the probability or frequency of a behavior that results in itspresentation or result in schedule-appropriate patterns of responding When an envi-ronmental event increases the behaviors it follows, the event is considered to be apositive reinforcer When the event is removed following the occurrence of a behaviorand the behavior is increased, the event is termed a negative reinforcer Thus, theterm reinforcer is used to indicate that the event has increased behavioral output.Early attempts to determine whether there are fundamental characteristics of allreinforcers led to the suggestion that perhaps the only common feature of reinforcerswas their rate-increasing effect.41 However, the reinforcing effects of a particularenvironmental event are not transsituational and must be empirically demonstrated

to reinforce behavior under any new conditions of interest Environmental events donot have inherent reinforcing or punishing properties They result in effects that can

be altered It can be very difficult to predict whether or not environmental events asdisparate as food, shock, or a drug will function as a reinforcer without information

on the current conditions and the organism’s history The findings that electric shockcould have both punishing and reinforcing effects,42,43 that electrical stimulation ofthe same brain site could serve as a positive or negative reinforcer,44 and thatpharmacologic agents (e.g., cocaine and nicotine) could serve as positive reinforcers,negative reinforcers, or punishers28–32,45,46 clearly demonstrated that environmentalevents do not have intrinsic reinforcing or punishing properties Thus, it is not alwayspossible to predict what effects an event will have on behavior The behavioral effects

of environmental events can be altered by a number of conditions that can beempirically examined These include the ongoing rate of responding, the organism’sbehavioral and drug history, and the current environmental context (for reviews seeReferences 8 and 47)

Research on potential differences between response-dependent and pendent presentation of environmental events played a pioneering role in the exper-imental analysis of behavior Some of the earliest studies in this regard investigateddifferences in the effects of electric shocks that could be postponed vs those thatcould not.48,49 Pairs of rats were studied under conditions during which responding

response-inde-by one rat postponed the delivery of electric shocks Responding response-inde-by the second rat

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