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Full text of "Review of NIAAA's neuroscience and behavioral research portfolio"

National Institute on Alcohol Abuse and Alcoholism 

RESEARCH MONOGRAPH - 34 




Review of NIAAA's 

Neuroscience and Behavioral 

Research Portfolio 



4 



U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Public Health Service 
National Institutes of Health 



NIAAA Research Monograph No. 34 



REVIEW OF NIAAA'S 

NEUROSCIENCE 

AND BEHAVIORAL 

RESEARCH PORTFOLIO 



Edited by: 

Antonio Noronha, Ph.D. 

Michael Eckardt, Ph.D. 

Kenneth Warren, Ph.D. 



NAT! OMAL I NSTITf ? 7F5s r 

U.S. DEPARTMENT OF HEALTH ANt) HUMAN SERVICES 

Public Health Service 

National Institutes of Health 

National Institute on Alcohol Abu&e.and Alcoholism 

6000 Executive BouWrd 4 ZllOO 

Bethesda, MD 20892 



Pi 



n ■■-•? 



43 



About the Editors: Antonio Noronha, Ph.D., is chief of the Neurosciences 
and Behavioral Research Branch of the National Institute on Alcohol Abuse 
and Alcoholism (NIAAA); Michael Eckardt, Ph.D., is senior science advisor to 
the Office of Scientific Affairs, NIAAA; and Kenneth Warren, Ph.D., is director 
of the Office of Scientific Affairs, NIAAA. 

NIAAA has obtained permission from the copyright holders to reproduce fig- 
ures and tables throughout this monograph. Further reproduction of these 
materials is prohibited without specific permission from the copyright holders. 
All other material contained in this monograph, except quoted passages from 
copyrighted sources, is in the public domain and may be reproduced without 
permission from NIAAA or the authors. Citation of the source is appreciated. 

The U.S. Government does not endorse or favor any specific commercial prod- 
uct (or commodity, service, or company). Trade or proprietary names (or com- 
pany names) that appear in this publication are used only because they are 
considered essential in the context of the studies reported herein. 

The opinions expressed herein are those of the authors and do not necessarily 
reflect the official position of NIAAA or any other part of the National Insti- 
tutes of Health. 

Key words are included in the beginning of each article. These descriptors are 
drawn from The Alcohol and Other Drug Thesaurus: A Guide to Concepts and 
Terminology in Substance Abuse and Addiction, Second Edition, 1995 and may 
be used to retrieve this monograph in the Alcohol and Alcohol Problems Sci- 
ence Database (commonly referred to as ETOH). 



NIH Publication No. 00-4520 
Printed 2000 



CONTENTS 



Foreword v 

Preface vii 

Abbreviations and Acronyms xi 

ACUTE ETHANOL ACTIONS ON SPECIFIC 
NEURAL TARGETS 

1 Emerging Areas of Research on Neural Proteins Involved in Acute 
Alcohol Actions 

David M. Lovinger 3 

2 Lipid Involvement in the Acute Actions of Alcohol in the Nervous System 
Steven N. Treistman 45 

3 Effects of Alcohol on the Neuroendocrine System 

Catherine Rivier 61 



MOLECULAR AND CELLULAR RESPONSES 
TO CHRONIC ETHANOL EXPOSURE 

4 Neuroadaption to Ethanol at the Molecular and Cellular Levels 
Paula L. Hoffman, A. Leslie Morrow, TamaraJ. Phillips, 

and George R. Siggins 85 

5 Neurotoxicity of Alcohol: Excitotoxicity, Oxidative Stress, 
Neurotrophic Factors, Apoptosis, and Cell Adhesion Molecules 

Pulton T. Crews 189 



ADDICTION AND OTHER BEHAVIORS 
IN ANIMAL MODELS 

6 Basic Behavioral Effects and Underlying Neurocircui tries of Alcohol 

Kathleen A. Grant 209 



7 Neuroadaptive Changes in Neurotransmitter Systems Mediating 
Ethanol-Induced Behaviors 

Friedbert Weiss 261 

8 Adolescent Period: Biological Basis of Vulnerability To Develop 
Alcoholism and Other Ethanol-Mediated Behaviors 

Linda Patia Spear 315 

STUDIES OF ACUTE AND CHRONIC EFFECTS 
OF ALCOHOL IN HUMANS 

9 Acute Effects of Alcohol on Cognition and Impulsive - 
Disinhibited Behavior 

Peter R. Finn 337 

10 Clinical Neuroscience Studies of Behaviors Associated With Alcohol 
Consumption in Alcoholism 

John H. Krystal, Ismene L. Petrakis, Louis Trevisan, 

and Neill Epperson 357 

1 1 The Hypothalamic- Pituitary- Adrenal Axis: Changes and Risk 
for Alcoholism 

Gary Wand 397 

12 Alcohol and Sleep 

Cindy L. Ehlers 417 



STUDIES OF COGNITIVE/BEHAVIORAL/STRUCTURAL 
DEFICITS IN HUMANS 

13 Neuropsychological Vulnerabilities in Chronic Alcoholism 

Marlene Oscar-Berman 437 

14 Human Brain Vulnerability to Alcoholism: Evidence 
from Neuroimaging Studies 

Edith V. Sullivan 473 



15 Human Brain Dysfunction Secondary to Alcohol Abuse: 
Suggestions for New Research Initiatives 

George Fein, Daniel Fletcher, and Victoria Di Sclafani 509 

SUBCOMMITTEE REPORT 

16 Report of a Subcommittee of the National Advisory Council on Alcohol 
Abuse and Alcoholism on the Review of the Extramural Research 
Portfolio for Neuroscience and Behavior 523 



IV 



FOREWORD 



The National Institute on Alcohol Abuse and Alcoholism (NIAAA) is charged 
with the important mission of stimulating research on the causes, consequences, 
prevention, and treatment of alcohol-related problems. One aspect of this mis- 
sion that we find increasingly satisfying is sharing the results of our research 
efforts with the scientific community, policymakers, program officials, and the 
general public. This monograph is based on a review of NIAAA's neuroscience 
and behavioral research portfolio by a subcommittee of the National Advisory 
Council on Alcohol Abuse and Alcoholism. It contains reviews of the breadth 
and depth of our current neuroscience and behavioral research portfolio, looks 
at areas that are ripe for research stimulation, and serves as the mechanism by 
which this knowledge can be shared with a wider audience. 

The progress made in the neurosciences over the last two decades has been 
nothing short of spectacular. NIAAA has taken full advantage of this progress 
to help stimulate — and provide support for — the application of neuroscience 
techniques to the study of alcohol use problems. As a result, our understanding 
of the neural processes that underlie alcohol-seeking behavior and of how alco- 
hol's actions in the brain are related to the phenomenon of addiction has 
grown dramatically. Alcohol neuroscience has led, among other things, to the 
development of new pharmacotherapies for alcoholism treatment, such as nal- 
trexone and acamprosate,, and to the possibility of developing "designer" med- 
ications targeted at specific alcohol actions. 

Recently, I was asked to predict where the alcohol research field would be by the 
year 2020. With regard to neuroscience research, one could almost say the sky's 
the limit. Often prediction turns out to be far from reality. However, based on 
the tremendous progress that has been made in a relatively short period of time 
in the neurosciences, I believe that by 2020 we will have advanced far beyond 
our current grasp of individual neural connections in animals and in humans to 
an understanding of how circuits in the brain actually operate in terms of 
appetite, affect, and cognition, and that even subjective states, such as volition 
and consciousness, will yield to scientific investigation. Based on the findings of 
the NIAAA neuroscience and behavioral research portfolio review, I am certain 
that we will be prepared for the challenges and opportunities ahead. 

I commend the members of the Subcommittee of the National Advisory Coun- 
cil on Alcohol Abuse and Alcoholism on the Review of the Extramural 



Research Portfolio for Neuroscience and Behavior, the staff of the NIAAA Divi- 
sion of Basic Research and the Neurosciences and Behavioral Research Branch, 
and the grantee representatives whose work is reflected in this compilation for 
their efforts to make certain that NIAAA-supported neuroscience and behav- 
ioral research continues to represent the best science for today and for the 
future. I especially commend the efforts of Dr. Antonio Noronha, Chief, Neu- 
rosciences and Behavioral Research Branch, Division of Basic Research, 
NIAAA, for his efforts in seeing this manuscript to completion. 

Enoch Gordis, M.D. 

Director 

National Institute on Alcohol Abuse and Alcoholism 



VI 



PREFACE 



The actions of alcohol that cause intoxication, initiate and maintain excessive 
drinking behavior, and promote relapse during abstinence occur primarily in 
the brain. The specific mental processes thought to underlie the development 
of alcoholism involve normal brain functions such as learning, attention, emo- 
tion, and cognition. A thorough understanding of the biochemical mechanisms 
of brain function and their response to alcohol is essential to develop and 
improve alcoholism prevention and treatment strategies. Basic neuroscience 
research sponsored by the National Institute on Alcohol Abuse and Alcoholism 
(NIAAA) has contributed significantly to this goal. 

NIAAA's neuroscience and behavioral research portfolio is broad and diverse, 
reflecting the cooperative efforts of the Institute and the alcohol research com- 
munity over many years. Most of NIAAA's research portfolios have been 
recently reviewed by subcommittees of the National Advisory Council on Alco- 
hol Abuse and Alcoholism, with the goal of evaluating the appropriateness, 
breadth, coverage, and balance of each portfolio and identifying areas that 
require greater attention. Subcommittees were also asked to provide specific 
advice and guidance on the scope and direction of the Institute's extramural 
research activities. 

The Subcommittee for the Review of the Extramural Research Portfolio for 
Neuroscience and Behavior consisted of an advisory group of individuals with 
demonstrated expertise in both alcohol-and non-alcohol-related areas. The 
review process was initiated by having experts in alcohol neurosciences and 
behavior prepare written assessments of the state of knowledge, gaps in knowl- 
edge, and research opportunities in specific areas. NIAAA program staff 
presented the current extramural portfolio, categorized into the areas of basic 
neuroscience and behavioral research, and also included training and career 
development activities. All information was shared with experts, selected 
NIAAA staff, and the subcommittee before the meeting. The proceedings and 
recommendations of the subcommittee were conveyed to and endorsed by the 
National Advisory Council. NIAAA has begun to implement these recommen- 
dations. We hope that this monograph will serve as a valuable reference as well 
as a guide to future research questions. 

The total scope of NIAAA's neuroscience research program extends beyond 
the topics covered in this monograph. For example, studies on the neuroscience 



and behavioral aspects of fetal alcohol syndrome (FAS), neurogenetics, and 
medications development were reviewed as part of the FAS, genetics, and 
prevention and treatment research portfolios, respectively. The research 
covered in this monograph ranges from molecular aspects of neuronal commu- 
nication to the integrated activity of multiple brain regions. Brief descriptions of 
each chapter are presented below, grouped by theme rather than in the order in 
which they appear. These themes include neurobiological mechanisms of 
alcoholism development, alcohol's effects on brain function, and factors that 
influence vulnerability to alcohol's effects. 



MECHANISMS OF ADDICTION 

A key concept in alcoholism research is neuroadaptation, the development 
of persistent alterations in brain function at the molecular, cellular, and systems 
level in response to chronic alcohol exposure. Chapter 4 explains how 
neuroadaptation associated with alcohol dependence can induce craving and 
relapse in response to alcohol-related cues or stress even after long periods 
of abstinence. 

Alcohol exposure can alter the structure and function of both the lipid and 
protein constituents of neuronal membranes. Chapter 1 discusses alcohol's 
effects on proteins at the molecular level and their influence on the overall 
physiology of specific brain regions. Chapter 2 emphasizes the importance 
of lipid-protein interactions in assessing alcohol's effects on the function of 
ion channels. 

Interactions among multiple neurotransmitter systems help mediate both the 
acute reinforcing actions of alcohol and the persistent neuroadaptive changes 
that may motivate relapse. Key neurotransmitters involved in these processes 
include dopamine, serotonin, and gamma- aminobutyric acid. These findings 
are based on studies using laboratory animals (chapter 7) as well as clinical 
research on humans (chapter 10). 

A knowledge of the integrated activity of neural circuits is required to 
provide the link between molecular events and behavior. Chapter 6 explores 
mechanisms by which ethanoPs effects on cognitive processes such as learning 



vui 



and memory can lead to reinforcement. Exposure to stress may affect the 
development of dependence and may help trigger relapse following recovery. 
According to chapter 11, alcohol consumption can potentially induce 
both underactivity and overactivity of the body's primary stress response system 
(i.e., the hypothalamic-pituitary- adrenal [HPA] axis). 



ALCOHOL'S EFFECTS ON BRAIN FUNCTION 

Alcohol-induced alteration of brain function can itself influence alcohol con- 
sumption patterns. Alcohol's effects on hormonal balance are discussed in chap- 
ter 3. For example, in addition to inhibiting the release of reproductive 
hormones, alcohol administration leads to increased synthesis of a key compo- 
nent of the HPA axis, leading to dysregulation of the stress response. Chapter 9 
reviews alcohol-induced impairment of cognition and impulse control, which 
can lead to aggressive behavior and decreased caution in decisionmaking. The 
prefrontal cortex, one of the brain regions most closely associated with higher 
cognitive functions, such as decisionmaking, is particularly vulnerable to alco- 
hol-induced dysfunction. As discussed in chapter 5, this dysfunction may be 
attributable to cell death caused by various metabolic mechanisms. Finally, 
chapter 12 illustrates that both acute and chronic alcohol consumption alter the 
normal sleeping pattern, potentially influencing other body functions. 



VULNERABILITY TO ALCOHOL'S EFFECTS 

Vulnerability to the harmful consequences of chronic alcohol consumption is 
affected by factors such as aging, nutrition, and gender, as discussed in chapter 
13. Adolescence may be a time of enhanced vulnerability. Research presented in 
chapter 8 suggests that adolescents may develop tolerance to alcohol's sedative 
effects and its effects on coordination more readily than do adults, perhaps con- 
tributing to greater levels of alcohol use later in life. Chapter 14 describes inno- 
vative neuroimaging techniques that may help identify structural and functional 
brain abnormalities associated with increased vulnerability to alcohol's psycho- 
logical and behavioral effects. Finally, chapter 15 suggests multidisciplinary 
approaches to investigating some of the most important questions regarding 
alcohol-related brain dysfunction. 



IX 



ACKNOWLEDGMENTS 

The contributors to this monograph are recognized experts in their respective 
disciplines. Their time and effort in preparing their presentations for the review 
and for these monograph chapters are truly appreciated. We thank the program 
staff of the Neurosciences and Behavioral Research Branch, especially Drs. Wal- 
ter Hunt, Robert Karp, Yuan Liu, and Ellen Witt. Their efforts, together with 
those of the advisory group, were largely responsible for the success of the 
review and the ensuing recommendations. We also wish to thank Diana 
O'Donovan of the Scientific Communication Branch, Office of Scientific 
Affairs, NIAAA, and Dianne Welsh and her staff at CSR, Incorporated, includ- 
ing John Doria and Pat Freedman, for their valued efforts in completing this 
monograph. 



Antonio Noronha, Ph.D. 

Chief 

Neurosciences and Behavioral Research Branch 

Division of Basic Research 

National Institute on Alcohol Abuse and Alcoholism 

Michael Eckardt, Ph.D. 

Senior Science Advisor 

Office of Scientific Affairs 

National Institute on Alcohol Abuse and Alcoholism 

Kenneth Warren, Ph.D. 

Director 

Office of Scientific Affairs 

National Institute on Alcohol Abuse and Alcoholism 



ABBREVIATIONS AND ACRONYMS 



A 


angstrom(s) 


AC 


adenylyl cyclase 


ACh 


acetylcholine 


ACTH 


adrenocorticotropic hormone (Chapters 3, 8, 12, 16) or 




adrenocorticotropin (Chapter 11) 


AD 


Alzheimer's disease 


ADH 


alcohol dehydrogenase 


AIDS 


acquired immunodeficiency syndrome 


AMPA 


L-a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (Chapter 




4) or a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 




(Chapter 6) 


AOD 


alcohol and other drug 


ATP 


adenosine triphosphate 


AVP 


arginine vasopressin 


BAL 


blood alcohol level 


BDNF 


brain-derived neurotrophic factor 


BOLD 


blood oxygen level dependent 


BZD 


benzodiazepine 


cAMP 


cyclic adenosine monophosphate 


CCK 


cholecystokinin 


cDNA 


complementary deoxyribonucleic acid 


CeA 


central nucleus of the amygdala 


Cho 


choline 


CIE 


chronic intermittent ethanol 


CNS 


central nervous system 


CPT 


continuous performance task 


Cr 


creatine 


CRE 


3',5'-cyclic adenosine monophosphate response element 


CREB 


3',5'-cyclic adenosine monophosphate response element binding 


CRP 


corticotropin-releasing factor 


CRH 


corticotropin-releasing hormone 


CSF 


cerebrospinal fluid 


CT 


computed tomography 


d 


day 


DA 


dopamine 


DHEA 


dehydroepiandrosterone 


DHT 


dihydrotestosterone 



XI 



dL 


deciliter(s) 


DNA 


deoxyribonucleic acid 


DSM-IV 


Diagnostic and Statistical Manual of Mental Disorders, 4th edition 


DTs 


delirium tremens 


DZ 


dizygotic 


EC 50 


median effective concentration 


ECF 


executive cognitive function 


ED 50 


median effective dose 


EDE 


ethanol delayed effect 


EEG 


electroencephalographic 


EPR 


electron paramagnetic resonance 


EPSP 


excitatory postsynaptic potential 


ERP 


event-related potential 


ES 


embryonic stem [cells] 


FAS 


fetal alcohol syndrome 


FDG 


fluorodeoxyglucose (Chapter 6) or 18 fluorine- labeled 




deoxy glucose (Chapter 14) 


FHP 


family history positive 


FHN 


family history negative 


fMRI 


functional magnetic resonance imaging 


FRET 


fluorescence resonance energy transfer 


FY97 


fiscal year 1997 


g 


grams(s) 


GABA 


gamma- aminobutyric acid 


GABA A 


gamma-aminobutyric acid type A 


GC 


glucocorticoids 


GHB 


y-hydroxybutyrate 


GnRH 


gonadotropin-releasing hormone 


GRF 


growth hormone-releasing factor 


h 


hour(s) 


5-HIAA 


5-hydroxyindoleacetic acid 


HIV 


human immunodeficiency virus 


HPA 


hypothalamic-pituitary- adrenal 


HPG 


hypothalamic-pituitary-gonadal 


HPS 


hypothalamic-pituitary-somatotropic 


5-HT 


5 - hy droxytryptamine ( serotonin ) 


Hz 


hertz 



Xll 



ICSS 


ntracranial self- stimulation 


icv 


ntracerebroventricularly 


IEG 


mmediate early gene 


IGF-1 


msulin-like growth factor- 1 


IL-lp 


nterleukin-ip 


123j M p 


odoamphetamine 123 


ip 


ntraperitoneal 


IPSP 


nhibitory postsynaptic potential 


IQ 


ntelligence quotient 


ISI 


nterstimulus interval 


kd 


tilodalton(s) 


kg 


tilogram(s) 


KS 


Korsakoff s syndrome 


LD 50 


median lethal dose 


LH 


uteinizing hormone 


LS 


ong sleep [mice] 


LTD 


ong-term depression 


LTP 


ong-term potentiation 


MAP 


mitogen-activated protein [kinase] 


mCPP 


w-chlorophenylpiperazine 


MDMA 


3,4-methylenedioxymethamphetamine 


mg 


milligram(s) 


mg% 


milligrams percent 


mL 


milliliter(s) 


mm 


millimeter(s) 


mM 


millimolar 


MRI 


magnetic resonance imaging 


mRNA 


messenger ribonucleic acid 


MRS 


magnetic resonance spectroscopy 


MRSI 


magnetic resonance spectroscopic imag 


MT 


magnetization transfer 


^g 


microgram(s) 


uL 


microliters ) 


[xm 


micrometer(s) 


MZ 


monozygotic 


Nac 


N- acetyl compounds 


nACh 


nicotinic acetylcholine 



xm 



nAChR nicotinic acetylcholine receptor 

NCAM nerve cell adhesion molecule 

NF-kB nuclear regulatory factor-KB 

NGF nerve growth factor 

NIAAA National Institute on Alcohol Abuse and Alcoholism 

NIH National Institutes of Health 

NK natural killer [cells] 

NMDA N-methyl-D-aspartate 

NMR nuclear magnetic resonance 

NO nitric oxide 

6-OHDA 6-hydroxydopamine 

8-OH-DPAT 8-hydroxy-2-(di-»-propylamino)tetralin 

PCP phencyclidine 

PCR polymerase chain reaction 

PE phosphatidylethanolamine 

PEA prenatal exposure to alcohol 

PET positron emission tomography 

PFC prefrontal cortex 

PKA protein kinase A 

PKC protein kinase C 

POMC pro-opiomelanocortin 

PS phosphatidylserine 

PTZ pentylenetetrazol 

PVN paraventricular nucleus 

QTL quantitative trait loci 

REM rapid eye movement 

RIP rapid information processing 

RNA ribonucleic acid 

RT reaction time 

RT-PCR reverse transcriptase-polymerase chain reaction (Chapter 4) or 

reverse transcription-polymerase chain reaction (Chapter 16) 

SCAM substituted cysteine scanning mutagenesis [technique] 

SPECT single photon emission computed tomography 

SS short sleep [mice] 

T testosterone 

TE echo-time 

TFMPP m- trifluoromethylphenylpiperazine 



xiv 



THDOC 3a,5a-tetrahydrodeoxycorticosterone 

THIP 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol 

THP tetrahydroprogesterone 

THDOC tetrahydrodeoxycorticosterone 

VF visual field 

VGCC voltage-gated calcium channel 

VTA ventral tegmental area 

v/v volume to volume [ratio] 

WAIS-R Wechsler Adult Intelligence Scale — Revised 

WSP withdrawal seizure prone 

WSR withdrawal seizure resistant 

w/v weight per volume 



xv 



ACUTE ETHANOL 

ACTIONS ON SPECIFIC 

NEURAL TARGETS 



Chapter 1 

Emerging Areas of Research on Neural 
Proteins Involved in Acute Alcohol Actions 

David M. Lovinger, Ph.D. 



KEY WORDS: acute AODE (effects ofAOD [alcohol or other drug] use, abuse, 
and dependence); proteins; neurotransmission; drug binding; molecular struc- 
ture; spectroscopy; brain function; research and evaluation method; AOD use be- 
havior; AOD sensitivity; gene expression; animal model; literature review 



Characterizing the primary sites of 
alcohol action and the mechanisms of 
alcohol effects at these sites is an 
important goal of basic alcohol 
research. The understanding gained 
from this research will lead to the 
development of pharmacological and, 
perhaps, genetic treatments aimed at 
reducing alcohol abuse, alcoholism, 
and alcohol-related brain damage. 
This research should make it possible 
to target therapeutic approaches not 
only to specific molecules within the 
brain, but also to specific molecular 
substructures within these molecules. In 
addition, with a better understanding 
of alcohol effects in brain regions that 
play important roles in alcohol-related 



behaviors, appropriate therapeutic 
treatments can be focused on these 
brain regions. 

This chapter begins with a discus- 
sion of research examining effects of 
alcohol at the molecular level, fol- 
lowed by a discussion of research 
examining roles of different molecules 
in alcohol effects in more intact sys- 
tems, up to and including the intact 
organism. The chapter outlines exper- 
imental approaches to the examina- 
tion of acute effects of alcohol that are 
likely to be at the heart of the most 
productive research in this area. Some 
of these approaches are already being 
applied to studies of acute alcohol 
actions, while others are novel and 



D.M. Lovinger, Ph.D., is a professor in the Department of Molecular Physiology and Biophysics and the 
Department of Pharmacology, Vanderbilt University Medical School, 702 Light Hall, Nashville, TN 
37232-0615. 



NIAAA's Neuroscience and Behavioral Research Portfolio 



powerful techniques that are just 
emerging in the field of neuroscience as 
a whole and should be applied to 
alcohol research within the next 5 to 10 
years. Each section in the chapter 
begins with a statement of the goals 
of a particular research area, followed by 
a discussion of approaches to achieving 
these goals and, finally, a brief discus- 
sion of the significance of the research 
to understanding and treatment of 
alcohol abuse and alcoholism. The 
chapter concludes with a discussion of 
techniques for discovery of novel targets 
of alcohol actions and a special note 
on the use of mouse genetic models 
in future alcohol research. 

STRUCTURAL BIOLOGICAL 
ANALYSIS OF ALCOHOL 
TARGETS 

Goals 

There are two major goals of this line 
of research. The first goal is to 
describe alcohol-induced alterations 
in the molecular structure and 
dynamics of proteins that are sensitive 
to alcohol and are believed to partici- 
pate in the neural effects of acute 
alcohol. This type of information will 
follow from studies of the structures 
of the proteins themselves. Once 
some information about protein 
structure and dynamics has been 
obtained, then analysis of alcohol 
effects can be undertaken. The sec- 
ond goal is to define the molecular 
structure of potential sites of alcohol 
interactions (alcohol binding sites?) 
within these proteins. This analysis 
will take the field to the next stage in 



understanding the molecular basis of 
alcohol actions by allowing investiga- 
tors to truly understand the sites of 
alcohol action and the molecular 
changes that take place upon acute 
exposure to alcohol. 

A number of sophisticated tech- 
niques for examination of protein 
structure have been devised within the 
last 25 years. It is now possible to gain 
information about protein secondary 
and tertiary structure as well as pro- 
tein dynamics using an array of bio- 
physical techniques. These techniques 
are applicable not only to cytosolic 
proteins, but also to membrane pro- 
teins. Several strategies to employ 
these structural biology techniques in 
alcohol research are described in the 
following section. This discussion will 
concentrate mainly on techniques that 
can provide information about struc- 
ture at several levels (i.e., primary, sec- 
ondary, and tertiary structure). 
Biochemical techniques that provide 
information about molecular topology 
and solvent exposure of amino acid 
residues (e.g., protease-based assays, 
in situ phosphorylation, and antibody- 
based assays) will not be reviewed in 
this chapter, but they are important 
additional approaches to understand- 
ing protein structure. 

Approaches 

The most definitive information 
about protein structure can be 
obtained from x-ray crystallography. 
Exact molecular coordinates of each 
amino acid residue can be firmly 
established with this approach. 
Furthermore, crystallization of pro- 
teins with small interacting molecules 



Neural Proteins 



can sometimes be achieved, providing 
information about the sites of interac- 
tion between a small molecule and 
the protein of interest. Thus, the 
structure of a protein could be exam- 
ined in the presence and absence of 
alcohol, and alcohol binding sites on 
the protein could be localized and 
understood in detail. This analysis has 
been performed for some proteins, 
such as alcohol dehydrogenase 
(Cedergren-Zeppezauer et al. 1982; 
Eklund et al. 1982). Two conditions 
must be met in order to apply this 
technique: (1) purified protein must 
be available in sufficient quantity to 
allow for growth of crystals, and (2) 
crystallization must be feasible. It has 
proved difficult to crystallize integral 
membrane proteins, because high 
concentrations of lipid or detergent 
are needed to maintain the structure 
of these proteins. Thus, this tech- 
nique has proved useful mainly for 
determining the structure of more 
hydrophilic cytosolic or nuclear pro- 
teins. However, reports of crystalliza- 
tion of Escherichia coli porins (Cowan 
et al. 1992) and a more recent study 
of a prokaryotic potassium channel 
indicate that the technology necessary 
to examine some membrane proteins 
is rapidly evolving (Doyle et al. 
1998). In addition, it may be possible 
to obtain crystals of the non-mem- 
brane-spanning portions of some 
integral membrane proteins (e.g., the 
ligand binding domains of ligand- 
gated ion channels). However, it is 
not clear that the soluble parts of the 
proteins retain their native configura- 
tion in the absence of the membrane- 
spanning domains. It must also be 



kept in mind that a crystallized pro- 
tein is not a functional protein. X-ray 
crystallography can only provide 
information about the static structure 
of a protein in one particular configu- 
ration. Information about protein 
dynamics (e.g., during acute alcohol 
exposure) must be obtained with 
other approaches. 

Techniques that can be used to 
gain structural information from 
intact proteins in a more native envi- 
ronment include nuclear magnetic 
resonance (NMR) spectroscopy, elec- 
tron paramagnetic resonance (EPR) 
spectroscopy, and fluorescence spec- 
troscopy. These techniques can be 
applied to integral membrane proteins 
as well as other proteins. 

Nuclear magnetic resonance spec- 
troscopy can provide information 
about both the static and dynamic 
structures of proteins. By examining 
the behavior of atomic nuclei under a 
magnetic field, it is possible to gain 
information about the relative loca- 
tion and motion of particular amino 
acid residues. This technique has the 
advantage that proteins can be exam- 
ined in solutions or lipid environ- 
ments that more closely mimic the 
physiological situation than a crys- 
talline array. Like crystallography, 
NMR can potentially provide infor- 
mation about the entire structure of 
the protein at one time. When exam- 
ining small- to medium-sized cytoso- 
lic proteins (up to 35 kd) or small 
stretches of membrane proteins, solu- 
tion NMR can be used to provide the 
entire protein structure at one time. 
However, limitations on this tech- 
nique prevent investigators from 



NIAAA's Neuroscience and Behavioral Research Portfolio 



examining particularly large proteins; 
these limitations mostly result from 
die slower motion of these proteins, 
which leads to broadening of spectral 
features. Examination of integral 
membrane protein structure with 
NMR is even more limited (Opella 
1994, 1997). One can use solid-state 
NMR to examine proteins in a phos- 
pholipid environment, but the 
motion of these proteins is limited, 
and thus limited information about 
the movement of particular amino 
acids can be obtained. This technique 
can be combined with multidimen- 
sional solution NMR of proteins in 
nonpolar solvent mixtures, but the 
slow motions in these solutions do 
not lead to the sorts of optimal spec- 
tra that one obtains from more 
hydrophilic proteins in a polar solvent 
environment. The purity and identity 
of the detergents used in micelle for- 
mation is also a major consideration 
in multidimensional solution NMR 
studies. Despite these limitations, 
investigators have been able to deter- 
mine the structures of small mem- 
brane-spanning peptides such as the 
M2 domain of the nicotinic acetyl- 
choline (ACh) receptor (Bechinger et 
al. 1991) and to gain some structural 
information about the G protein- 
coupled family of receptors by exami- 
nation of bacteriorhodopsin (Keniry 
et al. 1984; Sobol et al. 1992). 

An additional limitation of this 
technique is the large amount of pure 
protein needed to obtain a reasonable 
NMR spectrum (up to milligrams, 
rather than micrograms, of protein are 
needed in some cases). The clearest 
spectral features are often obtained 



with isotopically labeled proteins, and 
thus it is best to be able to incorpo- 
rate 15N or another isotope into the 
protein prior to purification. This 
problem may be solved with the use 
of techniques that allow for protein 
overexpression in cells that can be 
grown in large quantities. Two systems 
that are useful in this regard are the 
baculovirus/SF9 insect cell system (as 
in Green et al. 1995) and the Semliki 
Forest Virus/BHK cell system (as in 
Hovius et al. 1998). Both of these 
techniques allow for infection of a cell 
leading to overexpression of the 
desired protein in cells that can be 
grown in large-volume suspension 
cultures. Given a suitable protein sol- 
ubilization and purification strategy, a 
large amount of relatively pure protein 
can be obtained from such cells. 
However, it is still a daunting task to 
produce sufficient quantities of pro- 
tein for NMR analysis. 

The theoretical basis of EPR is sim- 
ilar to that of NMR with the exception 
that spin of unpaired electrons, rather 
than protons, is measured in a mag- 
netic field. The unpaired electrons are 
part of a "spin-labeled" functional 
group that can be attached to a mole- 
cule that interacts with or is part of a 
protein. Hubbell and colleagues have 
pioneered the use of "site-directed 
spin-labeling" in which the spin label is 
covalently attached to a cysteine 
residue within a protein (Hubbell and 
Altenbach 1994). This technique 
allows the investigator to gain informa- 
tion about the molecular environment 
of a particular amino acid residue 
within a protein as well as information 
about distances between different 



Neural Proteins 



amino acid residues. The information 
can be used to gain some idea about 
the secondary and tertiary structure of 
a protein. This technique can be 
applied to proteins in solution or in a 
lipid environment such that functional 
proteins can be analyzed. This allows 
the investigator to examine dynamic 
changes in protein structure when a 
particular protein is undergoing a 
conformational change, such as upon 
activation by a ligand or in the pres- 
ence of alcohol. For example, this 
technique has been used to measure 
the structural changes in rhodopsin 
upon exposure to light (Altenbach et 
al. 1996). Application of this tech- 
nique requires a lesser amount of 
purified protein than is required for 
NMR or crystallization (microgram 
quantities are needed for EPR analysis 
of most proteins). However, examina- 
tion of several labeled amino acids is 
rather time-consuming. Furthermore, 
the mutations needed to insert the 
cysteine residues that are labeled may 
disrupt protein function. Still, this is a 
promising method for examining 
membrane protein structure and 
dynamic effects of alcohol. 

Several techniques have been 
developed for measurement of protein 
structure and structural dynamics 
using fluorescence spectroscopy. 
These techniques range from the use 
of intrinsically fluorescent amino acids 
such as tryptophan to reactions with 
fluorescently labeled ligands. Amino 
acids such as cysteine can also be 
modified to incorporate a fluorescent 
label (Wu and Kaback 1994; Stratikos 
and Gettins 1997; Sahoo et al. 1998). 
Fluorescence spectroscopy, like EPR, 



has often been applied to functional 
proteins in a more or less native envi- 
ronment, and thus information about 
conformational changes in relation to 
protein function can be obtained 
using fluorescence techniques. Fluo- 
rescence spectroscopy can have quite 
favorable signal/noise characteristics 
with the proper fluorophore, and this 
means that the amounts of protein 
needed to apply these techniques can 
often be smaller than those needed for 
EPR analysis. Through the use of time- 
resolved laser fluorescence spectro- 
scopy, information about molecular 
conformational changes can be 
acquired in the picosecond to second 
temporal domains (Millar 1996; 
Beechem 1997). This technique 
allows for observation of very fast 
molecular transitions within a protein. 
With the use of two fluorophores that 
can emit and absorb photons in an 
interactive manner, one can apply flu- 
orescence resonance energy transfer 
(FRET) techniques to determine dis- 
tances between fluorophores (dos 
Remedios and Moens 1995). This 
technique can be used to gain informa- 
tion about molecular distances within 
a protein. Fluorescence spectroscopy has 
drawbacks similar to EPR, since gener- 
ally only one or two fluorescent groups 
can be examined at one time. However, 
investigators using fluorescence-based 
techniques are making increasingly 
important contributions to our under- 
standing of biomolecular structure, 
and these techniques should be quite 
useful in studying alcohol effects on 
protein structure and dynamics. 

Information about protein structure 
can also be obtained using photoaffinity 



NIAAA's Neuroscience and Behavioral Research Portfolio 



labeling techniques. If a suitable probe 
molecule with a photolabile functional 
group can be produced, then interac- 
tions between this molecule and spe- 
cific amino acids or groups of amino 
acids can be examined. Using photo- 
affinity probes that act via different 
pathways of access to the protein (e.g., 
hydrophobic access vs. access through 
the open channel), it is possible to 
obtain information about the relative 
positions of different residues on a 
membrane-bound protein such as an 
ion channel or transporter. Photo- 
affinity techniques have already been 
applied to examination of the structure 
of membrane proteins such as the 
nicotinic ACh receptor (Galzi et al. 
1990; Blanton and Cohen 1994; 
Czajkowski and Karlin 1995; Hucho 
et al. 1996). Photoaffinity labeling 
and subsequent determination of sites 
of labeling does not require a great 
deal of protein, and the protein does 
not necessarily have to be completely 
pure if the label has sufficiently high 
affinity; thus, this technique is applica- 
ble to many purified and even partially 
purified proteins. Examination of 
alcohol-induced alterations in photo- 
affinity labeling may help to elucidate 
structural changes in proteins in the 
presence of alcohol. 

Another approach that uses muta- 
genesis techniques to gain information 
about protein structure is the substi- 
tuted cysteine scanning mutagenesis 
(SCAM) technique. In this approach, 
cysteines are inserted into different 
amino acid positions within a protein, 
and effects of modification by thiol 
compounds on protein function are 
examined. Thiol-modifying agents 



with different relative hydro- and 
lipophilicities can be used to determine 
the molecular mode of access to dif- 
ferent sites of cysteine modification 
within the protein. This technique has 
been used to gain information about 
which residues are exposed to the ion- 
conducting pore within ion channels 
(Akabas et al. 1994, 1995). The tech- 
nique has several advantages over the 
techniques discussed previously, since 
it does not require protein purifica- 
tion. Functional studies can be per- 
formed on proteins expressed at the 
sorts of levels usually achieved with 
standard heterologous expression sys- 
tems. Furthermore, the technique 
involves assaying protein function in 
ways that are normally used by investi- 
gators to examine their proteins of 
interest. The major drawbacks of the 
approach include (1) the possibility 
that cysteine substitution may greatly 
alter protein structure or function and 
(2) direct effects of the thiol reagents 
on the unmodified protein that may 
preclude the use of this technique. It 
may be possible to use this technique 
to examine alcohol effects on accessi- 
bility of particular amino acid residues 
within proteins. 

All of the data pertaining to protein 
structure and structural dynamics are 
difficult to comprehend without 
proper models of protein structure and 
possible conformational changes within 
proteins. This type of modeling can be 
achieved using many molecular mod- 
eling programs, such as INSIGHT. 
Using software that allows molecular 
dynamics simulations can greatly aid 
investigators in understanding changes 
in molecular structure and can help 



Neural Proteins 



describe conformational changes in 
proteins. Hypotheses about the effects 
of particular alterations in a protein 
and molecular interactions can also be 
generated using these approaches. 
This sort of molecular modeling will 
also be an integral part of structural 
biological analysis of alcohol targets. 

The most likely impact of this 
research will be on the development 
of pharmacotherapeutic approaches to 
the treatment of alcohol abuse and 
alcoholism. A more exact definition of 
the sites of action and allosteric effects 
of alcohol at the molecular level will 
aid in application of rational drug 
design to the development of such 
pharmacotherapies. If particular alco- 
hol binding sites can be identified, it 
may lead to development of drugs 
that have very specific actions at dif- 
ferent molecular sites of alcohol inter- 
action. This could lead to selective 
alterations of some, but not all, of the 
effects of alcohol, while minimizing 
the side effects of the treatment. 

MOLECULAR BIOLOGICAL 
ANALYSIS OF ALCOHOL 
TARGETS 

Goals 

After discovery of protein targets of 
acute alcohol actions, it becomes pos- 
sible to examine in more detail the 
molecular basis of alcohol actions on 
these proteins. This analysis should 
proceed at several levels. Many of the 
holoproteins that are alcohol sensitive 
are made up of multiple subunits. 
Examination of the relationship 
between the subunit composition of 
these holoproteins and their alcohol 



sensitivity is thus an important area for 
future research. In-depth analysis of 
the secondary structure of proteins has 
revealed several different domain struc- 
tures or local regions that can exist 
within a protein. This domain structure 
can be important in forming hydro- 
phobic pockets that could be sites of 
direct alcohol-protein interactions. 
Thus, analysis of the relationship 
between alcohol sensitivity and protein 
domain structure will make up an 
important facet of future research 
designed to characterize the molecu- 
lar sites of alcohol actions. Of course, 
all of these structural properties of 
proteins are conferred by the amino 
acid sequence, or primary structure, 
of the protein. A complete under- 
standing of the molecular makeup of 
an alcohol-sensitive site on a protein 
will require an understanding of the 
role of individual amino acids in 
determining protein domain structure 
and in conferring alcohol sensitivity. 
Individual amino acids can also be sites 
for posttranslational protein modifica- 
tion, and analysis of this aspect of the 
relationship between primary struc- 
ture and alcohol sensitivity will also be 
a part of future alcohol research. 

Protein-protein interactions are 
emerging as a major theme in molecular 
biology and will no doubt be important 
determinants of the effects of alcohol 
on molecules within a given neuron. 
Interactions between two proteins can 
determine subcellular localization of a 
given protein and can affect protein 
function and enzyme-substrate inter- 
actions. Recent studies have provided 
an enormous body of information on 
interactions between proteins that can 



NIAAA's Neuroscience and Behavioral Research Portfolio 



be explored in relation to alcohol 
actions. Some of the questions that 
need to be addressed are as follows: 
Does alcohol alter subcellular localiza- 
tion of proteins by altering protein- 
protein interactions? Does alcohol 
alter function of particular proteins via 
indirect effects on partner proteins? 
Does the alcohol sensitivity of a single 
molecule differ in different subcellular 
regions due to differences in protein- 
protein interactions? Acute alcohol 
actions on some target proteins are 
known to vary depending on the cellular 
and experimental context in which the 
alcohol effects are examined. Some of 
this variability might arise from differ- 
ent interactions of these targets with 
other proteins in different cellular 
contexts or under different experi- 
mental conditions. These possibilities 
need to be explored to fully under- 
stand the molecular determinants of 
acute alcohol actions. 

Approaches 

Molecular biological analysis of alcohol- 
sensitive proteins has already been 
undertaken in several laboratories. 
Two approaches that have proved 
quite powerful are the use of chimeric 
and point-mutated receptors to exam- 
ine the relationship between receptor 
secondary/primary structure and alco- 
hol sensitivity. Experiments examining 
both voltage-gated and ligand-gated 
ion channels have revealed particular 
regions of channels that confer alcohol 
sensitivity, or in some cases account 
for small differences in alcohol sensi- 
tivity (Covarrubias et al. 1995; Mascia 
et al. 1996; Yu et al. 1996; Mihic et al. 
1997). The construction of chimeric 



proteins is a good starting point for 
this sort of analysis. Using recombi- 
nant DNA technology, researchers 
can create proteins in which large or 
small stretches of amino acids from 
one protein are combined with those 
from another protein (Mihic et al. 
1997). If the two proteins differ in 
some important characteristic, such as 
alcohol sensitivity, this approach can 
help the investigator to determine the 
importance of protein domains in 
conferring this characteristic, and it 
can also help to pinpoint regions of the 
protein where amino acids reside that 
have important roles in the function 
of interest. Amino acids that differ 
within the important regions of the 
two proteins can then be altered by 
point mutation, and the effects of these 
alterations on protein function and 
alcohol sensitivity can be examined. 
Single amino acids that play important 
roles in the action of alcohol can be 
identified in this way. By examining 
mutations of a single site to several 
different amino acids, one can begin to 
appreciate what molecular attributes are 
needed at that site (e.g., side chain bulk, 
hydrophobicity) for alcohol sensitivity. 
For particular alcohol targets it may 
not be possible to use the chimeric 
protein approach, since a highly 
homologous protein that differs 
greatly in alcohol sensitivity has not 
been identified. In this case, the muta- 
genesis approach should be consid- 
ered, but it must proceed in a manner 
that is as logical as possible based on 
existing information. For example, we 
know that most molecular target sites 
for alcohol actions are hydrophobic in 
character. Thus, it would make sense 



10 



Neural Proteins 



to target mutations to hydrophobic 
regions of proteins. Investigators can 
also use information from studies of 
structurally related proteins, if available, 
to help pinpoint regions conferring 
alcohol sensitivity. With the advent of 
software for prediction of protein sec- 
ondary structure based on primary 
sequence information and analysis of 
related proteins, it may become possi- 
ble for investigators to compare the 
structure of an alcohol-sensitive pro- 
tein with that of a protein that has 
already been characterized with 
respect to molecular determinants of 
alcohol sensitivity. This might allow 
investigators to identify "alcohol- 
responsive" domains within proteins 
and proceed to examine effects of muta- 
genesis in these domains on alcohol 
sensitivity. More will be said about 
this possible approach later in this 
chapter (see the section Searching for 
Molecular Targets With High Sensi- 
tivity to Acute Alcohol). 

The chimera/mutagenesis approach 
can also help to localize sites of poten- 
tial alcohol-protein interactions that 
can be examined more closely with 
direct physical measurements. This 
molecular biological approach is 
already being applied to alcohol 
research in several laboratories, and it 
should continue to be a major focus 
of research. In particular, extending 
this analysis to newly identified alcohol- 
sensitive proteins should be a priority 
for the coming years. 

In light of newly reported evidence 
for roles of protein phosphorylation in 
modulating alcohol sensitivity of par- 
ticular proteins, it will be important to 
use site-directed mutagenesis to alter 



potential sites of protein phosphoryla- 
tion on alcohol target molecules (as in 
Coultrap and Machu 1997). This will 
allow investigators to determine if 
phosphorylation at a particular site on 
a protein plays a role in determining 
alcohol effects on the function of that 
protein. Mutations can be made that 
prevent and/or mimic the addition of 
a phosphate group to a particular 
amino acid residue, thus allowing 
investigators to look at both gain and 
loss of function. This technique may 
also lead investigators to examine 
alcohol effects on the activity of pro- 
tein kinases that phosphorylate impor- 
tant amino acid residues, since these 
enzymes may be actual primary targets 
of alcohol actions. The effects of 
mutagenesis on phosphorylation of 
the substrate protein can be directly 
assessed, and this sort of analysis will 
be facilitated by development of anti- 
bodies to target proteins that can be 
used to identify particular phosphory- 
lated proteins. This line of research 
should benefit greatly from the recent 
initiative to produce antibodies spe- 
cific for alcohol target proteins. 

Use of genetically engineered mice 
is also likely to play a big role in the 
future of molecular analysis of alcohol 
effects on target proteins. Homologous 
recombination procedures are now in 
use that make it possible to create mice 
in which point mutations have been 
introduced in one key protein (Askew 
et al. 1993; MacMillan et al. 1996). If 
successful, the protein is expressed as in 
the wild-type animal but contains the 
point mutation. This approach has the 
potential to allow an investigator to 
examine the importance of a particular 



11 



NIAAA's Neuroscience and Behavioral Research Portfolio 



amino acid in a particular protein 
within the context of a whole animal 
or an individual neuron. For studies of 
acute neural actions of alcohol, these 
animals can be examined for alcohol- 
related behavior. In addition, more 
reduced neuronal preparations can be 
used to examine alcohol effects on 
neuronal function and protein function. 
In this way, information gathered in 
molecular biological studies of recom- 
binant proteins can be used to test 
predictions about the importance of 
particular alcohol-sensitive sites on a 
protein in intact neurons and the 
intact brain. 

The alcohol sensitivity of a protein 
may also differ as a function of the 
subunit composition of the holoprotein, 
or it may depend on crucial interac- 
tions with other proteins. Indeed, dif- 
ferences in protein assembly and 
interactions might underlie some of 
the variability in alcohol actions. In 
addition, closely related proteins, such 
as different kinase isoforms, may differ 
in alcohol sensitivity, and it will be 
important to determine which protein 
subtypes are most sensitive to alcohol 
and contribute to acute alcohol effects 
on the brain. 

I will begin with a discussion of 
multimeric proteins. The first step in 
this line of research is to determine 
the subunits that can contribute to the 
formation of multimeric proteins by 
cloning homologous proteins and to 
examine subunit expression in neurons 
as well as co-assembly in heterologous 
expression systems. This has already 
been done for a number of potential 
alcohol targets, including ligand- and 
voltage-gated ion channels (for reviews, 



see Sanna and Harris 1993; Seeburg 
et al. 1995; Diamond and Gordon 
1997; Lovinger 1997). Determining 
subunit composition and stoichiometry 
of assembled, functional proteins is 
more difficult. Techniques such as co- 
immunoprecipitation can be used to 
provide evidence for subunit co-assembly 
(Khan et al. 1994; Ruano et al. 1994; 
Sheng et al. 1994), and this line of 
research will benefit from development 
of subunit-specific antibodies. Pharma- 
cological approaches that can identify 
certain subclasses of heteromeric pro- 
teins might also be of use. 

Single-cell polymerase chain reac- 
tion (PCR) identification of subunit 
expression in neurons, combined with 
analysis of alcohol effects and pharma- 
cological analysis, is likely to be useful 
(Criswell et al. 1996; Sapp and Yeh 
1997). However, this technique cannot 
provide any information about protein 
expression or co-assembly. The develop- 
ment of techniques for more rapid 
and complete identification of protein 
expression in a single neuron would 
be a significant advance, but no suitable 
approach has been developed as yet. 

Co-expression of protein subunits in 
a heterologous expression system can 
also yield valuable information about 
the relationship between protein subunit 
composition and acute alcohol sensi- 
tivity. This approach has now been 
widely used to examine alcohol effects 
on ion channels and other proteins 
expressed in Xenopus oocytes and 
mammalian cells ( Wafford and Whit- 
ing 1992; Kuner et al. 1993; Lovinger 
1993; Sigel et al. 1993; Masood et al. 
1994; Dildy-Mayfield and Harris 
1995; Lovinger 1995; Mihic et al. 



12 



Neural Proteins 



1997). It is best to use this approach 
in conjunction with techniques that 
can allow the investigator to be sure 
that all of the desired subunits are 
expressed. This sort of analysis might 
include pharmacological characteriza- 
tion of receptors and immunological 
detection of subunit protein expression. 
Alcohol effects on proteins in heterol- 
ogous expression systems may not 
always agree with effects observed in 
neurons expressing native receptors 
(e.g., Lovinger 1993, 1995). Likewise, 
results obtained using different heterol- 
ogous systems and methods of assaying 
receptor function may not always agree 
( Wafford and Whiting 1992; Kuner et 
al. 1993; Sigel et al. 1993; Marszalec 
et al. 1994; Masood et al. 1994; 
Lovinger 1995; Mihic et al. 1997). It 
will be important to document these 
differences, since differences in alco- 
hol sensitivity of a protein in different 
cellular contexts may provide informa- 
tion that will be useful in identifying 
the cellular constituents that deter- 
mine differential alcohol sensitivity. 

Preventing expression of a particular 
subunit protein followed by analysis 
of changes in protein function, phar- 
macology, and alcohol sensitivity is an 
experimental approach that could 
yield a wealth of information about 
subunit structure of proteins and the 
importance of particular subunits in 
conferring alcohol sensitivity. Anti- 
sense RNA technology can be used to 
reduce expression of a particular pro- 
tein subunit (for reviews, see Baertschi 
1994; Bennett 1998). However, this 
technique is not always applicable to 
proteins with a slow turnover rate in 
cells, and antisense knockout is often 



incomplete. Still, there may be specific 
cases where antisense knockout of a 
particular protein subunit is feasible and 
can yield information about the impor- 
tance of that subunit in alcohol actions. 

Production of subunit knockout 
mice is likely to be a more fruitful 
approach to the problem. Indeed, sev- 
eral animals with receptor subunit 
knockouts have already been produced, 
and some of these animals have been 
tested for acute alcohol sensitivity 
(Homanics et al. 1997). Other knock- 
out mouse lines have been made that 
would be quite useful in alcohol 
research, but are not presently avail- 
able to alcohol researchers. A crucial 
direction for future research on acute 
alcohol actions will be to foster the 
creation and use in alcohol studies of 
knockout mice that lack proteins 
thought to be critical for acute alco- 
hol actions in the brain. The standard 
mouse gene knockout approach is not 
without problems, however. The gene 
is usually engineered such that no 
expression of the protein occurs 
throughout the lifespan of the mouse. 
This can lead to problems of develop- 
ment and compensation that may 
affect analysis of acute alcohol actions 
in the mature animal. Thus, it will be 
important to use alternative knockout 
strategies, such as inducible knockouts, 
that allow for removal of protein expres- 
sion at defined times in mouse devel- 
opment. More will be said about these 
powerful alternative approaches, and the 
need to use them in alcohol research, 
in the last section of this chapter. 

Many of the experimental strategies 
discussed in the last few paragraphs 
can also be applied to the study of 



13 



NIAAA's Neuroscience and Behavioral Research Portfolio 



different protein subtypes even in 
homomeric proteins. For example, in 
examining alcohol effects on protein 
kinase activity it will be important to 
identify kinase subtypes within neu- 
rons that are expressed and are alco- 
hol sensitive. Likewise, examination of 
acute alcohol effects in kinase knock- 
out mice will be an important step in 
determining the role of these enzyme 
subtypes in alcohol sensitivity. A few 
kinase knockout mice have already 
been found to exhibit differences in 
alcohol sensitivity and acute tolerance 
(Harris et al. 1995; Miyakawa et al. 
1997). One additional strategy that 
should be valuable in the study of 
alcohol effects on enzyme function is 
the examination of the activity of 
purified enzyme subtypes. Many of 
the enzymes that are of interest to 
alcohol researchers can be purified to 
homogeneity, and their activity can be 
assayed directly. This approach is use- 
ful in examining acute alcohol effects 
on particular enzyme subtypes. 

As discussed earlier, interactions 
between proteins can regulate the 
subcellular distribution and function 
of alcohol target proteins. Analysis of 
the importance of such interactions in 
acute alcohol sensitivity is thus a key 
area for future research. The first step 
in this line of research is the identifica- 
tion of proteins that interact with 
alcohol target proteins. This identifi- 
cation can proceed in a number of 
ways. Interacting proteins can be 
identified using gel overlay assays 
(Carr and Scott 1992), solution bind- 
ing assays such as surface plasmon res- 
onance (Faux and Scott 1997), and 
genetic screens for binding such as the 



yeast two-hybrid screen (Fields and 
Stern glanz 1994). Most of the binding 
assays require having at least one puri- 
fied protein, such as a GST-fusion 
protein, that can be used to assay 
binding to the unknown interacting 
protein. The yeast two-hybrid screen 
requires only that DNA for the pro- 
tein of interest can be successfully 
transfected into yeast and will yield 
protein. This protein is used as "bait" 
to catch other proteins from brain 
DNA libraries that are expressed in 
yeast (Fields and Sternglanz 1994). 
The DNA sequences for these proteins 
can then be determined and the pro- 
teins, if unknown, can be cloned and 
characterized. All of these techniques 
have been used to identify interacting 
proteins with great success in recent 
years. One other way to search for 
potential protein-protein interactions 
is to examine the sequences of puta- 
tive alcohol target proteins for motifs 
known to be involved in protein bind- 
ing (e.g., PDZ domains [Kornau et al. 
1997; Ranganathan and Ross 1997]). 
This approach can help direct the 
search toward proteins that are known 
to interact with such motifs. 

Once interacting proteins are iden- 
tified, then the impact of expressing 
or removing these proteins on alcohol 
sensitivity of the putative alcohol tar- 
get protein can be examined. These 
sorts of analyses can be performed in 
neurons and in heterologous expres- 
sion systems. Knockout animals that 
lack an interacting protein of interest 
can also be produced, and their alco- 
hol sensitivity can then be examined. 
This work should include examination 
of alcohol effects in the intact animal 



14 



Neural Proteins 



using behavioral-pharmacological 
approaches, as well as examination of 
changes in alcohol effects on the recep- 
tor, channel, transporter, or signaling 
enzyme of interest in cells or brain 
slices. Strategies aimed at preventing 
specific protein-protein interactions in 
intact cells can also be employed. Pep- 
tides that disrupt interactions by bind- 
ing to the interaction domain can be 
overexpressed in cells by cDNA trans - 
fection (Hundle et al. 1997) or injected 
into cells via a patch-clamp pipette 
(Rosenmund et al. 1994). These strate- 
gies allow investigators to disrupt 
interactions and examine the alcohol 
sensitivity of the target protein with 
and without this protein interaction. 

In some cases direct alcohol effects 
on protein -protein interactions can be 
examined. If the proteins are sufficiently 
pure to allow performance of a solution 
binding assay, then alcohol effects on 
binding can be quantified using this 
approach. Semiquantitative measure- 
ments of binding and alcohol effects 
can be obtained using a gel overlay 
assay. A less direct approach, but one 
that can yield important preliminary data, 
is immunocytochemical examination of 
alcohol effects on target protein subcel- 
lular localization (Gordon et al. 1997). 
This technique allows researchers to 
determine if alcohol alters interaction of 
one protein with a target protein that 
participates in localization. Powerful 
techniques for subcellular imaging, such 
as confocal and multiphoton excitation 
laser microscopy, will be needed to carry 
out these studies. This sort of experiment 
might be a good first step in identifying 
protein-protein interactions that are 
affected by alcohol in the intact cell. 



The significance of this research will 
be mosdy in the area of development of 
pharmacotherapies for treatment of alco- 
hol abuse and alcoholism. More exact 
definition of the sites of action and 
allosteric effects of alcohol at the level of 
single molecules, molecular interactions, 
and subcellular compartments should 
aid in application of rational drug design 
to the development of such pharma- 
cotherapies. However, understanding 
targets of alcohol action at the molecular 
level may also lead to clinical applica- 
tions such as enhanced diagnosis of 
susceptibility to alcoholism. 

ANALYSIS OF PROTEIN 
FUNCTION 

Goals 

An important part of understanding 
acute alcohol effects on key neuromol- 
ecular targets is determining the changes 
in molecular function produced by 
alcohol. However, describing functional 
effects should not be the ultimate goal 
of investigators examining functional 
effects of alcohol. Alcohol effects on 
protein function should be undertaken 
with an eye to identifying molecular 
characteristics that impart alcohol sen- 
sitivity. With a wealth of information 
emerging about the relationship 
between protein structure and func- 
tion, functional information can be 
used to home in on regions of proteins 
that impart alcohol sensitivity. 

Approaches 

Different subtypes of ion channels 
appear to be sensitive to pharmaco- 
logically relevant concentrations of 



15 



NIAAA's Ncuroscicncc and Behavioral Research Portfolio 



alcohol (Sanna and Harris 1993; Dia- 
mond and Gordon 1997; Lovinger 
1997). A great deal of information 
about the perturbation of ion channel 
function can be gained by kinetic 
analyses using data collected at the 
whole-cell, and particularly at the sin- 
gle-channel, electrophysiological 
recording level. These techniques 
have already been applied to research- 
ing alcohol effects on several ion 
channel subtypes (Mullikin-Kilpatrick 
and Treistman 1995; Nagata et al. 
1996; Wright et al. 1996; Zhou et al. 
1998). Studies have revealed effects 
on probability of channel opening, 
ligand dissociation at ligand-gated 
channels, and interactions with G pro- 
tein modulation of voltage-gated 
channels (Mullikin-Kilpatrick and 
Treistman 1995; Mullikin-Kilpatrick 
et al. 1995; Nagata et al. 1996; 
Wright et al. 1996; Zhou et al. 1998). 
This sort of information, along with 
information about alcohol interactions 
with agonists, antagonists, and 
allosteric modulators, can help focus 
further studies designed to identify 
molecular sites of alcohol action. 
Since these approaches are well known 
in the alcohol field, there is no need 
for further description of this research 
area. However, it will be important to 
apply these kinetic analysis techniques 
to examination of alcohol effects on 
recombinant receptors of known 
structure, since this might help to 
avoid problems with mixed alcohol 
effects observed in neurons containing 
many channel subtypes. 

Kinetic analysis can also be per- 
formed on alcohol-sensitive nonchannel 
receptors as well as alcohol-sensitive 



enzymes and even nuclear factors. 
This information can be combined with 
thermodynamic analysis to provide 
some idea of the energetic changes 
taking place within target proteins 
during exposure to alcohol. 

Transporter proteins for neuro- 
transmitters and other molecules can 
function as ion channels (Parent and 
Wright 1993; DeFelice and Galli 
1998). The channel mode of activity 
may or may not play a role in trans- 
port, but it is likely to have an impact 
on neuronal physiology (Bruns et al. 
1993). It will be important for investi- 
gators to examine alcohol effects on 
channel activity of these neuromole- 
cules. This will be particularly impor- 
tant in examination of transporters 
that are found to display high alcohol 
sensitivity when assayed for transport 
activity. Transporters for neurotrans- 
mitters suspected to play a role in 
acute alcohol actions (adenosine, 
dopamine, gamma-aminobutyric acid 
[GABA], glutamate, serotonin) might 
also be examined for changes in trans- 
porter/channel function. 

The body of evidence implicating 
protein phosphorylation/dephosphory- 
lation in the neural effects of alcohol 
continues to grow, although examina- 
tion of phosphorylation and kinases 
has greatly outpaced study of dephos- 
phorylation and phosphatases. In par- 
allel, our understanding of the 
mechanisms regulating protein kinases 
and phosphatases has also greatly 
expanded. For example, it is now well 
known that these enzymes are regu- 
lated by intracellular targeting, and 
that this targeting involves interac- 
tions with "anchoring" proteins or 



16 



Neural Proteins 



scaffolding proteins (Mochly- Rosen 
1995; Dell'Aqua and Scott 1997). 
This is an area that is just beginning 
to receive attention in the alcohol 
research field, and was discussed in 
more detail earlier in this chapter. In 
the context of differential kinase/ 
phosphatase localization, it is also 
important to examine enzyme activity at 
different substrates, since differential 
enzyme localization will bring 
kinases/phosphatases into contact 
with different substrates. Future studies 
of kinase and phosphatase function 
should include examination of phos- 
phorylation and dephosphorylation of 
different substrates and activation of 
the kinase by different cofactors. Sub- 
strate and cofactor identity has already 
proved important in studies of protein 
kinase C (PKC), since alcohol and 
anesthetic effects on this kinase appear 
to be different under different sub- 
strate and cofactor conditions (Slater 
et al. 1993, 1997). Examination of 
activation of enzymes by different 
activators and cofactors will also be 
helpful in identifying potential sites of 
alcohol action on the enzyme molecule. 
Likewise, examination of holoen- 
zymes and enzyme activity in the 
presence of interacting proteins will 
be important, since these studies 
might reveal actions of alcohol that 
involve other proteins in addition to 
the enzyme itself. 

The importance of this line of 
research is mainly to help guide analysis 
of molecular structure. Understanding 
the functional effects of alcohol on a 
protein should help investigators to 
discover the parts of the protein that 
are involved in the actions of alcohol. 



Understanding the functional effects 
of alcohol on individual proteins can also 
aid in the design and evaluation of 
potential pharmacotherapies. Preclini- 
cal screens for the efficacy of potential 
therapeutic agents can be designed 
using assays of protein function. 

ALCOHOL EFFECTS 
ON TRANSMISSION 
AT INTACT SYNAPSES 

Goals 

Another important goal of alcohol 
research is to understand the way in 
which alcohol alters communication 
between neurons at intact synapses. It 
has now become apparent that alcohol 
has potent actions on synaptic transmis- 
sion. To some extent, these actions 
can be accounted for by alterations in 
synaptic proteins that are known to be 
alcohol sensitive (e.g., neurotransmitter 
receptors and ion channels). However, it 
is not clear that all of the actions of alco- 
hol at intact synapses can be explained 
by effects on these proteins. For exam- 
ple, there is emerging evidence that 
alcohol enhances inhibitory transmission 
and inhibits excitatory neurotransmission 
by altering presynaptic mechanisms of 
neurotransmitter secretion (Thomas 
and Morrisett 1997#; Weiner et al. 
1997 a). Furthermore, past studies have 
suggested alcohol effects on release of 
neuromodulators (Wang et al. 1991; 
Wozniak et al. 1991). Many of these 
actions cannot be fully explained by 
our current knowledge of alcohol 
effects on synaptic targets. The extent 
to which known alcohol-sensitive target 
proteins contribute to alcohol actions 



17 



NIAAA's Ncurosciencc and Behavioral Research Portfolio 



on synaptic transmission also needs to 
be examined in greater detail. For these 
reasons, it is important to examine 
alcohol effects on synaptic transmission 
using techniques that allow the inves- 
tigator to infer the pre- or postsynap- 
tic locus of the effects and to assay the 
involvement of particular synaptic 
proteins in the actions of alcohol. 

Approaches 

The quantal nature of neurotransmit- 
ter release has long been known from 
studies of the neuromuscular junction. 
However, it had been quite difficult 
to apply analyses based on quantal 
theory to examination of central 
synapses (Redman 1990; Korn and 
Faber 1991). Recent innovations, 
including development of tight-seal 
whole-cell recording from neurons 
visualized in brain slices, have made it 
possible to analyze quantal transmis- 
sion at central nervous system (CNS) 
synapses with greater accuracy 
(Clements 1990; Bekkers and Stevens 
1994; von Kitzing et al. 1994; Isaac 
et al. 1996). The most promising 
techniques are those that allow the 
investigator to examine spontaneous 
synaptic responses at excitatory and 
inhibitory synapses. Information 
about the pre- versus postsynaptic 
locus of changes in transmission can 
be gained from analysis of the fre- 
quency, amplitude, and time course of 
these responses. Furthermore, one can 
directly examine quantal responses by 
examining spontaneous "miniature" 
synaptic currents under conditions in 
which calcium-dependent secretion 
has been blocked. This technique pro- 
vides even more powerful determina- 



tion of the locus of changes at individ- 
ual synapses, and allows the investiga- 
tor to separate effects on presynaptic 
neuronal firing from effects on the 
presynaptic terminal itself. Using vari- 
ations on this basic experimental 
approach, the involvement of presy- 
naptic calcium entry and specific post- 
synaptic receptors and signaling 
enzymes in effects on transmission can 
also be examined. These techniques are 
just beginning to be applied to exami- 
nation of alcohol effects at glutamater- 
gic and GABAergic synapses, and 
preliminary studies indicate that 
important information about hereto- 
fore-overlooked presynaptic effects of 
alcohol will be forthcoming from this 
line of research (Thomas and Mor- 
risett 1997^; Weiner et al. 1997^). 

More sophisticated methods for 
analysis of transmission at single CNS 
synapses have been developed in 
recent years. Investigators can use 
modifications of basic techniques for 
examination of stimulus-evoked 
transmission to investigate changes in 
quantal release when afferent fibers 
are stimulated (e.g., Sr 2+ -induced 
asynchronous transmitter release 
[Oliet et al. 1996; Choi and Lovinger 
1997]). In addition, the possibility 
that CNS synapses deviate from 
quantal behavior observed at periph- 
eral synapses can now be assessed 
using techniques to examine "silent 
synapses" (Malenka and Nicoll 1997) 
and variations in neurotransmitter 
release at single synapses (Liu and 
Tsien 1997). Thus, detailed investiga- 
tion of alcohol effects on quantal 
synaptic transmission can now be car- 
ried out at CNS synapses. 



18 



Neural Proteins 



If, indeed, presynaptic mechanisms 
contribute to the actions of alcohol at 
central synapses, then it will be neces- 
sary to more closely examine alcohol 
effects on physiological and neuro- 
chemical events taking place within 
axon terminals. Calcium has a key role 
in neurotransmitter secretion, and 
examination of axon terminal calcium 
dynamics will likely be a prominent 
area of future investigation. Although 
alcohol effects on calcium flux have 
been examined in numerous studies, 
most of these studies have measured 
calcium flux into synaptosomes or 
other reduced preparations, and cal- 
cium flux has been evoked by stimuli 
of quite long duration (seconds) in 
comparison with the timing of influx 
during excitation/secretion coupling 
at CNS synapses (a few milliseconds). 
Thus, it cannot be determined from 
these studies if alcohol predominantly 
affects calcium influx related to neu- 
rotransmitter secretion or calcium 
increases that take place at later times 
and may not be so crucial for release. A 
large body of literature on alcohol 
effects on voltage -gated calcium chan- 
nels also exists, and some of these stud- 
ies have focused on channels, such as 
the N-channel, that play a role in exci- 
tation/secretion coupling (Solem et al. 
1997). However, these examinations 
have been limited to measuring chan- 
nel function in neuronal somata and 
not at synaptic terminals, and it is quite 
possible that channels within terminals 
are regulated differently than channels 
in the cell body. Thus, further investi- 
gation of alcohol effects on calcium 
transients and calcium channel func- 
tion in axon terminals is warranted. 



The most direct way to examine 
calcium channel function in presynaptic 
terminals is to use voltage-clamp tech- 
niques to measure pharmacologically 
isolated calcium currents. Studies indi- 
cate that simultaneous voltage-clamp 
recording from both pre- and postsy- 
naptic elements of the synapse can be 
carried out at brainstem calyx synapses 
such as the synapse of Held (Borst 
and Sakmann 1996). This approach 
will allow investigators to examine 
alcohol effects on presynaptic physiol- 
ogy, including calcium channel func- 
tion, and to relate those effects to 
alcohol-induced changes in synaptic 
transmission at an excitatory synapse. 

However, most presynaptic terminals 
are too small to be examined with patch- 
clamp recording. Thus, presynaptic 
terminal calcium dynamics must be 
examined in other ways at these 
synapses. Newer and more sophisti- 
cated techniques for measurement 
and imaging of presynaptic calcium 
dynamics have been developed in 
recent years. Most of these techniques 
involve the use of calcium-sensitive 
fluorescent dyes, such as fura-2, that 
are reliable indicators of changes in 
intracellular calcium. Using a photo- 
multiplier tube, it is possible to detect 
photon emission excitation from a flu- 
orescent dye in real time and thus 
measure intracellular calcium dynamics 
without the need for sophisticated 
imaging hardware. Saggau and col- 
leagues have elegantly demonstrated 
that one can use such an approach to 
measure pre- and postsynaptic calcium 
transients during synaptic transmission 
evoked by a single presynaptic stimu- 
lus at a population of CA3-CA1 



19 



NIAAA's Neuroscience and Behavioral Research Portfolio 



synapses in die hippocampal slice (Wu 
and Saggau 1997). Through the use 
of fura-2 loading only in the synaptic 
region and pharmacological separation 
of pre- and postsynaptic transients, 
these investigators have demonstrated 
modulation of presynaptic calcium 
transients by G protein-coupled 
receptors. This method for analysis 
could easily be applied to examination 
of alcohol effects on synapses in the 
hippocampus and other brain regions. 
Direct visualization of presynaptic 
terminal calcium transients can be car- 
ried out using confocal and two-pho- 
ton excitation laser microscopy. These 
techniques allow one to examine 
regions as small as 0.5 um, making 
presynaptic terminal visualization pos- 
sible. Indeed, these techniques have 
already been used to image calcium 
transients in single dendritic spines 
(Denk et al. 1996), and this technol- 
ogy should be applicable to axon ter- 
minals as well. Using techniques 
designed to load dyes into terminals 
in combination with vital dyes that 
can label terminals (such as FM-143), 
it should be possible to unequivocally 
identify terminals that are loaded with 
calcium indicator dye and examine 
calcium transients and the effects of 
alcohol on these transients in single 
axon terminals. Improvements in 
microscopy hardware should make it 
possible to measure such transients at 
high scan and digitization rates, and 
thus it should be possible to measure 
the transients that are directly related 
to neurotransmitter release. Combining 
this analysis with pharmacological 
approaches to assess the involvement 
of calcium channels, intracellular calcium 



release processes, and calcium buffering/ 
extrusion mechanisms will allow inves- 
tigators to pinpoint particular aspects 
of the calcium/secretion relationship 
that are altered by alcohol. This will 
stimulate further analysis of alcohol 
effects on potential target proteins 
involved in these processes. Combining 
calcium measurement techniques with 
sophisticated electrophysiological 
analysis of synaptic transmission will 
provide detailed information about the 
role of the physiological consequences 
of alterations in calcium dynamics. 

Imaging techniques can also be 
applied to obtain real-time measure- 
ments of neurotransmitter release 
processes in addition to calcium 
dynamics. For example, one can mea- 
sure vesicle release and recycling using 
the fluorescent membrane probe FM- 
143 (Ryan et al. 1993, 1997). This 
dye incorporates into vesicular mem- 
branes and will remain incorporated 
until vesicle fusion is stimulated. Mea- 
surements of secretion can be made 
using this technique in combination 
with confocal microscopy. Similar 
approaches can also be taken using 
fluorescent antibody detection of 
synaptic vesicle-associated proteins 
(Malgaroli et al. 1995). This approach 
can provide information about alcohol 
effects on secretion that can be com- 
pared with measurement of calcium 
dynamics in order to help investiga- 
tors determine the most probable 
locus of alcohol effects on neurotrans- 
mitter secretion. 

Monoamines, such as dopamine, and 
neuropeptides, such as opioids, may 
have significant roles in acute alcohol 
actions related to the reinforcing 



20 



Neural Proteins 



actions of the drug. Studies performed 
in vivo indicate that alcohol can alter 
extracellular dopamine concentrations 
in the nucleus accumbens ( Wozniak et 
al. 1991; Samson and Hodge 1993; 
Weiss et al. 1993). These studies have 
been performed using neurotransmitter 
turnover, microdialysis, and in vivo 
voltammetry techniques. However, it 
appears that alcohol does not act directly 
on dopaminergic terminals and may 
increase extracellular dopamine in vivo 
by actions in the ventral tegmental area 
(VTA) (Samson et al. 1997; Yim et al. 
1997). Thus, it is not clear that further 
examination of alcohol effects on 
dopaminergic axon terminals is war- 
ranted at this time. However, exami- 
nation of mechanisms of release of 
other monoaminergic transmitters 
might be warranted. 

Little emphasis has been placed on 
examining alcohol effects on presy- 
naptic proteins involved in the secretion 
process. However, many of the pro- 
teins that have been implicated in 
alcohol actions reside in presynaptic 
terminals as well as in postsynaptic 
elements. Notable among these pro- 
teins are modulatory neurotransmitter 
receptors (e.g., adenosine and opiate 
receptors), neurotransmitter trans- 
porters, voltage-gated calcium chan- 
nels, and protein kinases, such as 
PKC, that have been implicated in 
altering the secretion process. In addi- 
tion, there has been substantial 
progress in recent years in the identifi- 
cation of proteins that make up the 
neurotransmitter release machinery 
(see Sudhof 1995 for review), and as 
yet there has been little effort to 
examine these proteins in relation to 



alcohol effects on synaptic transmission. 
This is obviously another area that 
needs to be emphasized more strongly 
in future studies of alcohol effects on 
synaptic transmission. 

Plastic changes in the efficacy of syn- 
aptic transmission have been suggested 
as a major mechanism of information 
storage in the nervous system (Bliss and 
Collingridge 1993; Goda and Stevens 
1996). Alcohol is known to have 
amnestic effects and to disrupt other 
aspects of cognitive and motor function 
that may involve such plastic changes. 
However, studies of alcohol effects on 
synaptic plasticity have been limited 
mainly to examination of long-term 
potentiation (LTP) in the hippocampal 
formation (Blitzer et al. 1990; Mor- 
risett and Swartzwelder 1993; Criado et 
al. 1996; Schummers et al. 1997). Long- 
term potentiation and long-term depres- 
sion (LTD), as well as shorter lasting 
changes in synaptic efficacy, have been 
observed at a number of synapses in 
the CNS (Bear and Malenka 1994; 
Linden and Connor 1995). There is no 
compelling reason to examine the alcohol 
sensitivity of every one of these forms of 
plasticity. It is known, for example, that 
LTP in the cortex involves mechanisms 
similar to those involved in LTP at the 
hippocampal Schaffer collateral-CAl 
synapses. Thus, it may not be necessary 
to examine alcohol effects on cortical LTP 
in too much depth. However, LTP at 
mossy fiber-CA3 synapses in hippocam- 
pus and LTP at parallel fiber-Purkinje 
neurons in cerebellum are forms of 
plasticity that involve mechanisms dif- 
ferent from "classical" NMDA receptor- 
dependent LTP (Nicoll and Malenka 
1995; Salin et al. 1996). It may well be 



21 



NIAAA's Neuroscience and Behavioral Research Portfolio 



important to examine alcohol effects 
on these forms of plasticity. In particular, 
cerebellar LTD should be examined 
since alcohol produces ataxic effects 
and cerebellar LTD has been impli- 
cated in motor learning and is related 
to ataxic phenotypes in mutant mice 
(Linden 1994). Examination of these 
forms of plasticity may help investigators 
to pinpoint molecular mechanisms by 
which alcohol disrupts plasticity and to 
identify new molecular targets of alcohol 
action. It will also be important to 
examine forms of plasticity that involve 
molecules identified as alcohol sensitive 
since these forms of plasticity should 
be altered by alcohol. Ultimately, infor- 
mation about alcohol effects on trans- 
mission and synaptic plasticity may be 
combined at certain synapses to provide 
more detailed information about alcohol 
effects on neuronal communication. 

The use of mutant, knockout, and 
transgenic mouse models will also be 
an important component of future 
studies of alcohol effects on synaptic 
transmission. Brain slices as well as cul- 
tured and acutely isolated neurons and 
subneuronal preparations (e.g., 
microsacs and synaptoneurosomes) can 
easily be prepared from mouse brain. 
Studies of alcohol effects on synaptic 
transmission and ligand-gated ion 
channel function have already been per- 
formed in preparations from mutant mice, 
highlighting the utility of this pre- 
paration (Harris et al. 1995; Miyakawa 
et al. 1997). Combining analysis of neu- 
rophysiology and synaptic transmission 
with the new generation of mouse 
genetic alterations, as described later in 
this chapter, is a crucial future direction 
for research on alcohol mechanisms. 



It is known that alcohol has potent 
effects on synaptic transmission. 
Understanding the mechanisms under- 
lying these effects will require exami- 
nation of intact synapses to determine 
the relative contribution of pre- and 
postsynaptic mechanisms in the effects 
of alcohol. This will lead to identifica- 
tion of additional important molecular 
targets of alcohol actions and will pro- 
vide assays for preclinical tests of drugs 
that may be developed for treatment of 
alcohol abuse and alcoholism. 

EXAMINATION OF 
ALCOHOL EFFECTS 
ON NEUROPHYSIOLOGY 
IN KEY BRAIN REGIONS 

Goal 

The aim of this line of research is to 
determine the relationship between 
alcohol effects on neuronal activity and 
behavior in brain regions, such as the 
VTA, amygdala, hypothalamus, cere- 
bellum, and prefrontal cortex, thought 
to be important in alcohol-related 
behaviors. This research will help inves- 
tigators to evaluate the potential role of 
particular alcohol targets in alcohol 
effects on key brain regions; it will also 
aid in the search for previously undis- 
covered targets of alcohol action that 
play key roles in alcohol effects in these 
brain regions. 

Approaches 

Relationship of Neuronal 
Activity to Behavior 

Brain regions that appear to play 
important roles in different aspects of 



22 



Neural Proteins 



drug- seeking behavior and different 
behavioral consequences of alcohol 
intake have been defined using experi- 
mental approaches such as behavioral 
pharmacology (see Koob and Nestler 
1997 for review) and gross measures 
of neuronal activity in defined brain 
regions (2-deoxyglucose, c-Fos) 
(Ryabinin et al. 1997; Williams- 
Hemby and Porrino 1997). In vivo 
neurophysiological approaches have 
also been widely used (Gessa et al. 
1985; Mereu and Gessa 1985; Criado 
et al. 1995; Lee et al. 1995; Ludvig et 
al. 1995; Givens 1996; Matthews et 
al. 1996; Wang et al. 1996; Wood- 
ward 1996). Studies designed to 
examine the relationship between the 
activity of identified neurons within 
these regions and the behavioral 
effects of acute alcohol have begun to 
appear in the last few years (Givens 
1996; Woodward 1996). These stud- 
ies are of particular importance 
because understanding the pattern of 
changes in the activity of these neu- 
rons will help to guide examination of 
the specific alcohol-induced alter- 
ations in ion channel function and 
membrane properties that underlie 
the in vivo activity changes. Put 
another way, it will be easier to search 
for the most relevant cellular and mol- 
ecular targets of alcohol in these neu- 
rons once we know how their activity 
is altered in vivo by alcohol. 

While it is true that a number of 
laboratories have examined alcohol 
effects on the activity of single neu- 
rons in vivo over the years, many of 
these investigators have not been able 
to take advantage of newly developed 
techniques for neurophysiological 



recording and simultaneous behavioral 
analysis. These techniques can also be 
combined with pharmacological analy- 
sis to provide an even stronger experi- 
mental approach. Several techniques 
should be applied to this analysis. Multi- 
unit recording allows the investigator 
to sample several neurons within a 
brain region and gain a more complete 
picture of activity in a given brain 
region in a shorter time period (Wilson 
and McNaughton 1994; Woodward 
1996). Identification of neuronal sub- 
types, while often performed in studies 
of in vivo alcohol effects, is not uni- 
versally performed. This technique is 
a necessity, given the idea that this 
analysis will guide future studies of 
alcohol effects in vitro, and investigators 
will need to know which neurons to 
target for in vitro analysis. Video analy- 
sis of animal behavior correlated in time 
with the activity of single neurons 
allows investigators to determine the 
temporal relationship between neu- 
ronal activity and different aspects of a 
complex behavior pattern. This tech- 
nique is beginning to be applied to 
studies of alcohol actions in the nucleus 
accumbens and striatum (Woodward 
1996). Patterns of activity of neurons 
that have well-known behavioral cor- 
relates, such as place cell activity and 
activity during learning and memory 
tasks of hippocampal formation neu- 
rons, are also being examined in rela- 
tion to alcohol effects (Givens 1996; 
Ludvig et al. 1995; Matthews et al. 
1996). Investigators can now combine 
the physiological/behavioral analysis 
with pharmacological manipulation of 
the local neurons by in vivo microdial- 
ysis and other techniques (Ludvig et 



23 



NIAAA's Neuroscience and Behavioral Research Portfolio 



al. 1995; Yang et al. 1996). These 
techniques will allow investigators to 
examine localized actions of alcohol 
and interactions between alcohol and 
pharmacological agents acting at sus- 
pected alcohol target sites. This analy- 
sis will help to guide future studies by 
providing information about which 
brain regions are directly affected by 
alcohol and what targets may be 
important in these alcohol actions. 

Brain regions that need to be 
examined include the VTA, nucleus 
accumbens, amygdala, and prefrontal 
cortex, which are part of the brain 
"reward" circuitry (Koob and Nestler 
1997). The amygdala also has impor- 
tant roles in anxiety and may play an 
important role in the anxiolytic 
actions of alcohol. The cerebellum is 
known to participate in alcohol- 
induced ataxia, and thus it is impor- 
tant to examine this brain region in 
more detail. Continuing examination 
of the hippocampal formation is war- 
ranted, but this brain region need not 
receive the majority of attention as it 
has in the past. Finally, the prefrontal 
cortex has important cognitive roles 
that may also be affected by alcohol. 
Thus, the different types of neurons in 
this brain region require examination 
as well. 

Cellular/Molecular 
Mechanisms That Underlie 
In Vivo Alcohol Effects 

Neurons suspected to be alcohol sen- 
sitive from in vivo studies need to be 
studied in vitro to determine alcohol 
effects on intrinsic membrane proper- 
ties or synaptic transmission that 
underlie the in vivo alcohol effects. 



These studies should not take the 
form of surveys of all neuronal 
responses, but should instead be tar- 
geted at mechanisms that are likely to 
underlie the types of changes in activ- 
ity observed in vivo. For example, if a 
rhythmically firing neuron shows 
decreased activity in the presence of 
alcohol, then ion conductances 
known to contribute to that rhythmic 
activity should be examined. If an 
alcohol-sensitive neuron is generally 
quiescent in the absence of synaptic 
input, then synaptic transmission 
might be the logical target for exami- 
nation. Where possible, these studies 
should utilize information from in 
vivo pharmacological studies in 
designing experiments. 

The techniques that can be used 
for examination of alcohol effects on 
intrinsic neuronal responses and 
synaptic transmission are numerous. 
Neuronal isolation techniques used in 
combination with patch-clamp elec- 
trophysiological approaches have 
advanced significantly in recent years, 
such that healthy neurons can be 
acutely dissociated from many brain 
regions in relatively mature rodents. 
This is the preferred preparation for 
studying ion channel modulation by 
alcohol, because isolated neurons are 
free of the influences of neighboring 
cells, and voltage-clamp is easily 
achieved in these preparations. Ion 
currents of different types can also be 
easily isolated and pharmacologically 
manipulated in this preparation. 

Brain slices and primary neuronal 
cultures allow investigators to exam- 
ine synaptic transmission in reduced, 
semi-intact preparations. This allows 



24 



Neural Proteins 



for sophisticated analysis of the pre- or 
postsynaptic locus of alcohol effects as 
described earlier in this chapter. Phar- 
macological analysis of ligand- gated ion 
channels under voltage -clamp can also 
be performed in cultured neurons. 
This can best be achieved by blocking 
action potential firing and using local, 
rapid application of receptor agonists 
and modulatory drugs with a system 
that can completely superfuse neurons 
with a known concentration of drug 
(Lovinger 1995). Detailed analysis of 
postsynaptic receptors in neurons in 
brain slices is not recommended since 
it is difficult to achieve application of 
known concentrations of drugs to the 
entire cell in this preparation. Further- 
more, it is not possible to definitively 
rule out presynaptic effects in slice 
preparations. However, local pressure 
or iontophoretic application of drugs 
to these cells may be applied to neurons 
in culture to examine alcohol effects on 
receptors or transporters in different 
parts of a given neuron. In addition, 
techniques for laser uncaging of agonists 
in spatially defined extracellular regions 
might help investigators to pinpoint 
regions of neurons where receptors and 
transporters are particularly sensitive 
to alcohol (Pettit et al. 1997). 

Examination of voltage-gated ion 
channel function in brain slices and cul- 
tured neurons is unlikely to be produc- 
tive given the space-clamp problems 
encountered in these studies. However, 
some channels with slow kinetics, such 
as the M-current and inwardly rectify- 
ing K + channels activated by G protein- 
coupled receptors, can be studied with 
single-electrode voltage-clamp or 
patch-clamp techniques in brain slices 



(Dutar and Nicoll 1988; Moore et 
al. 1990). 

Given the emerging evidence for 
modulation of alcohol effects on recep- 
tors involving protein phosphorylation 
(Mirshahi and Woodward 1997; Weiner 
1997&), it will be especially important 
for investigators to take precautions to 
prevent alteration of phosphoryla- 
tion/dephosphorylation pathways 
during whole-cell recording experi- 
ments. This can be accomplished 
using the perforated patch technique, 
in which macroscopic current record- 
ing can be performed without dialysis 
of intracellular molecules larger than 
small ions. 

One problem that may be encoun- 
tered when comparing alcohol effects 
in vitro with those observed in the 
same neurons in vivo is that synaptic 
connections that participate in alcohol 
effects might be severed during brain 
slice preparation. Indeed, recent stud- 
ies in dopaminergic VTA neurons 
suggest that addition of neurotrans- 
mitters in vitro can enhance alcohol 
sensitivity of neuronal activity (Brodie 
et al. 1995). This sort of result sug- 
gests the possibility that a neurotrans- 
mitter that is known to have strong 
effects on a neuron in vivo might not 
be active at the neuron in the slice 
preparation. If this neurotransmitter is 
a key component in conferring alco- 
hol sensitivity on the neuron, then its 
absence may reduce or eliminate alco- 
hol effects. 

One way to attempt to overcome 
such problems in vitro will be through 
the use of organotypic brain slice cul- 
ture preparations such as those cur- 
rently in use for studies of chronic 



25 



NIAAA's Neuroscience and Behavioral Research Portfolio 



alcohol actions (Thomas and Mor- 
risett 1997b). Complex brain circuitry 
can be reconstituted in such a prepa- 
ration (Plenz and Kitai 1998). The 
advantages of in vitro preparations, 
such as the ability to examine synaptic 
transmission in detail and apply drugs 
to defined regions of a neuron, are 
retained using such techniques. 

One additional advantage of in 
vitro slice and slice culture prepara- 
tions is the ability to perform experi- 
ments using intracellular imaging 
techniques. These approaches allow 
the investigator to examine membrane 
potential or intracellular calcium 
changes in large arrays of neurons 
within a given brain region. Examina- 
tion of intracellular changes within 
small subregions of neurons (e.g., 
dendritic spines or axon terminals) is 
also possible with these techniques. 
The use of such techniques will also 
allow investigators to examine physio- 
logical changes and effects on intracel- 
lular signaling in different subcellular 
compartments. 

Examination of alcohol effects in 
knockout, knockin, transgenic, and 
other mutant mice will be an impor- 
tant component of future studies of 
alcohol actions in defined brain 
regions. Information from combined 
physiological and pharmacological 
studies can be used to determine what 
specific neuronal proteins should be 
overexpressed, knocked out, or subtiy 
altered in the intact animal. These ani- 
mals can then be examined using in 
vivo electrophysiological and behav- 
ioral approaches, as well as in vitro 
electrophysiological techniques. This 
will allow investigators to determine 



the importance of particular proteins 
in the effects of alcohol on more inte- 
grated neural systems. 

These studies will guide preclinical 
investigation of potential pharmaco- 
therapeutic agents by providing infor- 
mation about which molecular targets 
are affected by alcohol in a given brain 
region. Understanding alcohol effects 
in a given brain region will aid in the 
development of diagnostic tools for 
use in humans examined with nonin- 
vasive techniques. Development of 
brain region-specific therapeutic 
approaches might also be a useful 
product of this research. 

SEARCHING FOR 
MOLECULAR TARGETS 
WITH HIGH SENSITIVITY 
TO ACUTE ALCOHOL 

A number of neurotransmitter recep- 
tors, ion channels, and signaling mol- 
ecules have been identified that are 
sensitive to effects of alcohol in the 
25-100 mM concentration range. 
Certainly, effects of these concentra- 
tions are relevant to in vivo actions 
after ingestion of moderate to high 
doses of alcohol. However, alcohol 
has effects on neural function and 
behavior at much lower concentra- 
tions (5-10 mM and lower), and these 
effects may be especially relevant to 
the behavioral activation and rein- 
forcement produced by acute alcohol. 
To date, very few neuromolecules 
have been identified that show consistent 
functional alterations in the presence 
of such low alcohol concentrations. 
Identification of molecules that are 
extremely sensitive to alcohol actions 



26 



Neural Proteins 



should thus be a priority in future 
alcohol research. 

The search for such molecular targets 
can proceed in a number of ways. The 
first approach is essentially the "top- 
down" experimental strategy 
described in the preceding section of 
this chapter. This approach involves 
identification of changes in neuronal 
activity in vivo that are produced by 
low concentrations of alcohol. For 
example, it is known that the firing of 
dopaminergic VTA and substantia 
nigra reticulata neurons is altered after 
low-dose alcohol administration 
(Gessa et al. 1985; Mereu and Gessa 
1985). Strategies for identifying the 
molecular targets that underlie these 
effects using a reductionist approach 
have already been discussed in this 
chapter. However, it is possible that 
these techniques will not meet with 
success if in vivo actions are secondary 
to actions on other neurons or involve 
alcohol interactions with agents or 
neuronal pathways that are not pre- 
served in a particular brain slice prepa- 
ration. If this is the case, then other 
screening strategies may be needed. 

The use of genetic screening meth- 
ods is already established in the alco- 
hol field. Animals with differential 
responses to low-dose alcohol and dif- 
ferential alcohol-drinking behavior 
have been identified and selectively 
bred. The use of F2 intercross breed- 
ing strategies combined with genetic 
analyses such as quantitative trait loci 
(QTL) screening has the potential to 
provide information about which 
gene products are likely to be 
involved in these differential responses 
to alcohol (Belknap et al. 1997; Buck 



et al. 1997). The proteins identified in 
this manner can then be tested for 
alcohol sensitivity, and many of the 
molecular and genetic strategies for 
examination of these proteins that are 
outlined in this chapter can be applied 
to these gene products. The major 
drawbacks to this approach are the 
time and expense needed to identify 
the genes within QTL that are impor- 
tant, and the possibility that the prod- 
ucts of these genes are secondarily 
influencing alcohol sensitivity and are 
not primary targets of alcohol action. 
However, this experimental approach 
is likely to provide information that is 
relevant to understanding the neural 
basis of differentially acute alcohol 
effects in humans. 

An alternative genetic screening strat- 
egy involves the generation of mutant 
animals that can then be screened for 
altered alcohol sensitivity. This 
approach can most readily be applied 
to invertebrates such as Drosophila 
melanogaster and Caenorhabditis ele- 
gans at present, because single-gene 
mutations and propagation of mutant 
animals can be rapidly achieved in these 
organisms. Of the two models, 
Drosophila may hold more promise 
since these animals have a somewhat 
larger behavioral repertoire than C. ele- 
gans. Studies by the Heberlein and 
Nash laboratories are aimed at reveal- 
ing mutations in Drosophila that alter 
effects of alcohol and general anesthet- 
ics (Krishnan and Nash 1990; Lei- 
bovitch et al. 1995; Lin and Nash 
1996; Scholz et al. 1997; Moore et al. 
1998). However, most of the alcohol 
effects being examined in Drosophila 
at present are responses to fairly high 



27 



NIAAA's Neurosciencc and Behavioral Research Portfolio 



doses of alcohol, and one challenge in 
future studies will be to develop behav- 
ioral assays of lower dose alcohol effects 
and alcohol preference in Drosophila. 
The differences in molecular complexity 
of potential alcohol targets in 
Drosophila and mammals are also a 
concern. The diversity of subtypes/ 
subunits of a particular molecule may 
be much greater in mammals than in 
Drosophila, and the alcohol sensitivity 
of these different molecules may differ 
as well. However, there is great poten- 
tial for initial identification of classes 
of molecules that contribute to acute 
alcohol sensitivity using this approach. 
Techniques for production of "ran- 
dom" mutations in rodents are also 
being developed, and this approach 
could potentially be used to screen for 
mutations that would alter acute 
behavioral and neuronal sensitivity to 
alcohol. One such approach is called 
gene trapping (Hicks et al. 1997). 
This involves insertion of a gene trap 
retroviral shuttle vector into embry- 
onic stem (ES) cells. Clonal lines of 
these cells are then grown to generate 
a library of ES clones, each of which 
contains a single gene that has incor- 
porated the retroviral DNA. The 
incorporation of this DNA will usually 
disrupt proper expression of that 
gene. The expression vector codes for 
a Neo resistance gene that can be 
used to select for expressed genes, and 
it also contains a PST sequence tag 
that allows the disrupted gene to be 
identified in the ES cells prior to 
insertion into an animal. The ES cells 
can be inserted into a blastocyst and 
implanted into mice; if germ line trans- 
mission is achieved, then mice that 



lack the disrupted gene can be gener- 
ated. This approach has already been 
used to identify a number of genes 
that are disrupted in the ES cell 
libraries. In addition, several mutations 
have been introduced into the germ line, 
and many of these have identifiable 
phenotypes. This approach is much 
more costly and time-consuming than 
mutagenesis and screening of Dro- 
sophila. Furthermore, not too many 
laboratories are currently using this 
technique. An additional consideration 
is that the genes targeted for disrup- 
tion using this technique are those 
expressed in ES cells, and thus the tech- 
nique may select for genes involved in 
early development. This may lead to 
several embryonic lethal phenotypes 
and may not allow disruption of genes 
that play key roles in alcohol effects in 
the adult animal. However, the use of 
rodents would allow investigators to 
examine a wide variety of behavioral 
effects of acute alcohol in mutant 
animals and to concentrate on 
subtle alterations in responses to low 
alcohol doses. 

Screening of available mutant, trans- 
genic, and knockout mice for alter- 
ations in low- dose alcohol sensitivity is 
also a possibility. However, a survey of 
alcohol effects on genetically altered 
animals is not advisable. Ultimately, 
this research will best proceed from 
hypotheses generated from pharmaco- 
logical and physiological studies. 

One alternative possibility would 
be to search for alcohol- sensitive protein 
motifs within known proteins with 
high alcohol sensitivity. Such proteins 
might include alcohol dehydrogenase 
(Cedergren-Zeppezauer et al. 1982; 



Neural Proteins 



Eklund et al. 1982; Xie et al. 1997) 
and olfactory receptor molecules (Buck 
and Axel 1991; Raming et al. 1993), 
some of which respond to relatively low 
concentrations of airborne alcohol (Sato 
et al. 1994). A combination of struc- 
tural biological, molecular biological 
(e.g., chimera production, site-directed 
mutagenesis), and three-dimensional 
molecular modeling approaches can be 
used by investigators to examine the 
molecular features of the regions of 
these proteins that interact with alco- 
hol. This analysis can lead to a search 
for similar motifs in CNS proteins 
that may help to identify proteins that 
are particularly sensitive to alcohol. 
This approach may fail because of the 
weak interactions of alcohol with even 
the most alcohol-sensitive protein 
sites. There may be several hydropho- 
bic molecular sites that interact with 
alcohol with approximately equal 
"affinity," and thus it may not be pos- 
sible to identify a single alcohol-sensitive 
protein motif. However, searching 
highly alcohol-sensitive molecules has 
the potential to yield information 
about the general features of alcohol 
target sites. 

SPECIAL NOTE ON MOUSE 
GENETIC MODELS 

As can be seen from the foregoing 
discussion, mouse genetic models are 
likely to play an increasingly impor- 
tant role in studies of acute alcohol 
actions over the next few years. It will 
be important for alcohol researchers 
to have access to mice with desired 
genetic alterations, to be able to take 
advantage of significant advances in 



the development of these mouse 
models. In addition, researchers need 
to be cognizant of problems associated 
with the use of genetically engineered 
mice. In this section I briefly discuss 
these issues and present some ideas 
about how the National Institute on 
Alcohol Abuse and Alcoholism can 
encourage and support the use of these 
powerful techniques by investigators 
interested in acute alcohol actions. 

Several techniques are now widely 
in use for the production of mice with 
altered expression of particular genes. 
Transgenic mice are usually engi- 
neered to overexpress a particular gene 
and its gene product through insertion 
of a genetic sequence into a mouse 
blastocyst, which is then implanted into 
a pseudopregnant female (Faerman and 
Shard 1997). The gene of interest is 
generally linked to a mammalian pro- 
moter of some type that controls gene 
expression. Levels of expression of the 
desired gene as well as the cellular 
locus of expression will be controlled 
by factors including the number of 
copies of the transgene expressed in a 
particular mouse line and the size and 
identity of the promoter chosen to drive 
expression. Thus, two lines of mice 
designed to overexpress the same gene 
may differ considerably in locus and 
amount of actual protein expression and 
hence in behavioral and cellular pheno- 
types. These factors must be taken 
into consideration when embarking 
on production of transgenic mice, 
since the investigator most often 
wishes to produce overexpression in 
brain regions and at times when the 
gene is normally expressed. Temporal 
control of gene expression can be 



29 



NIAAA's Neuroscience and Behavioral Research Portfolio 



gained by fusing the gene of interest 
to a promoter that is inducible (e.g., 
by steroids or antibiotics). One advan- 
tage of the transgenic approach is that 
the investigator can choose to produce 
transgenic lines on any one of a variety 
of mouse strain genetic backgrounds. 
Thus, a strain with a known phenotype 
can be chosen, and differences in that 
phenotype produced by transgene 
expression can be examined. 

A powerful technique for examina- 
tion of the role of a particular protein 
in a set of cellular and behavioral pro- 
cesses is to produce a line of knockout 
mice that do not express the gene cod- 
ing for that protein (see Tonegawa et 
al. 1995 for a review). Production of 
such a line is achieved by the gene- 
targeting technique using ES cells, 
and takes advantage of homologous 
recombination events that take place 
during cell division. This technique 
allows an altered gene to be inserted 
into the ES cell genome in place of 
the wild-type gene (Capecchi 1989). 
This substitute gene is engineered to 
contain a marker driven by a promoter 
that can help to select for the knockout 
in the cells. In some cases the wild-type 
gene is replaced with a part of that gene 
that lacks a region needed for proper 
expression. The stem cell containing 
this altered gene is then inserted into 
a blastocyst and implanted into a 
pseudopregnant female to give rise to 
production of genetically altered off- 
spring. If the altered gene makes its 
way into the germ line of animals after 
breeding the progeny of the female, 
then the replacement gene can be 
propagated through breeding and 
eventually mice that are homo- and 



heterozygotic for the gene knockout 
can be produced. This technique has 
already been used by several investigators 
in the alcohol research field (Harris et 
al. 1995; Crabbe et al. 1996; Homanics 
et al. 1997; Miyakawa et al. 1997; 
Rubinstein et al. 1997). For more 
information on these techniques and 
their application to neuroscience 
research, I refer the reader to a review 
by Chen and Tonegawa (1997). 

Having an animal that does not 
express a particular protein is, of course, 
valuable to a researcher with an interest 
in determining the role of that protein 
in a behavior such as alcohol intoxica- 
tion or a cellular response such as 
synaptic inhibition by alcohol. How- 
ever, the standard mouse gene knock- 
out approach is not without its 
drawbacks. The fact that most homo- 
2ygotic knockout animals never express 
the gene of interest at any point dur- 
ing development can lead to develop- 
mental abnormalities. Thus, altered 
phenotypes may be a secondary or even 
tertiary consequence of the absence of 
the gene of interest. In addition, homo- 
logous proteins that can compensate 
for the missing protein may become 
overexpressed in the animal during 
development. This can lead to a 
"false-negative" lack of phenotype 
that can be too easily interpreted as 
showing no important role for the 
protein of interest. These problems 
have been discussed in reviews of the 
subject (e.g., Joyner 1994). 

To avoid these problems and the 
large numbers of control experiments 
necessitated by them, investigators have 
begun to develop approaches that will 
allow gene knockout at any time during 



30 



Neural Proteins 



development. This inducible knockout 
technique potentially allows the animals 
to develop normally until the time at 
which the investigator decides to remove 
the gene of interest. The gene can then be 
eliminated and the animals can be exam- 
ined shortly thereafter to determine the 
effects of losing gene expression prior 
to any substantial compensation. The 
most popular current technique for pro- 
ducing such mice is the cre/lox tech- 
nique (Marth 1996). This technique 
takes advantage of the fact that the ere 
recombinase will excise genes that are 
flanked by the loxV signal sequence. Thus, 
one can create mice with the loxV 
sequences surrounding a gene by homo- 
logous recombination, and then breed 
these animals with transgenic mice that 
express the ere gene driven by a suitable 
promoter. If the ere gene is fused with a 
hormone or antibiotic binding domain 
such the human estrogen receptor (Feil 
et al. 1996), then ere expression can be 
induced in the cre/lox animals, leading 
to temporally controlled gene knockout. 
This technique can also help to elimi- 
nate another problem with standard 
knockout technology, namely, the fact 
that gene expression is eliminated 
throughout the brain and maybe even 
throughout the entire body. By driving 
ere expression with a tissue- or cell-specific 
promoter, the cre/lox technique can be 
adapted to generate tissue- or brain 
region-specific knockouts by breeding 
"floxed" mice with those expressing 
the ere transgene only in a specific set 
of neurons (Tsien et al. 1996). 

An additional problem that arises 
in examining knockout mice is the 
problem of strain differences in pheno- 
type (Gerlai 1996; Crawley et al. 1997). 



As we in the alcohol research field know 
all too well, different mouse strains 
exhibit different behavioral pheno- 
types, and these differences can also 
extend to physiological processes. 
This factor must be taken into account 
when designing experiments to examine 
phenotypes of knockout mice. The stem 
cells currently available for creation of 
knockouts are derived from the 129/ 
Sv mouse line, and thus the initial 
knockout animals will necessarily have 
this genetic background. These animals 
are known to drink alcohol and to 
show normal sensitivity to acute alco- 
hol actions (Crabbe et al. 1996), and 
these behaviors can be altered by 
knockouts on this genetic background. 
However, the factors that control 
alcohol consumption in this strain of 
animals in comparison with better char- 
acterized strains are relatively unclear. 
An additional problem with this mouse 
strain is the relatively small average lit- 
ter size, which may make it difficult to 
perform within-litter comparisons. A 
strategy that can be used to overcome 
this problem is to outbreed the initial 
knockout mice with another strain of 
mouse with a more desirable back- 
ground phenotype until mice are 
obtained that are congenic with the 
desired strain and still lack the wild-type 
gene. These animals can then be com- 
pared with wild-type animals of the 
same strain. However, this can take sev- 
eral generations of breeding. Misinter- 
pretation of phenotypic characteristics 
is quite possible if the knockout animals 
are not genetically homogeneous, in 
that a wild-type strain with suitable 
genetic background for comparison 
with the knockout mice may not be 



31 



NIAAA's Neuroscience and Behavioral Research Portfolio 



available. More attention needs to be 
paid to the genetic background of the 
wild-type and mutant mice in future 
studies of alcohol effects using mouse 
genetic models. In the future it may 
be possible to use stem cells that can be 
implanted into a more commonly used 
mouse strain. However, these cells are 
not yet widely available. A recent 
report suggests that interactions 
between genetics and laboratory envi- 
ronment can also influence alcohol's 
effects in mice (Crabbe et al. 1999). 

An alternative to production of true 
knockout mice is production of mice 
that express a particular protein that 
has been subtly mutated in a region 
that is thought to be important for a 
particular function. For example, an 
amino acid residue thought to be 
important for alcohol sensitivity of a 
particular protein might be mutated, 
and a receptor containing this mutation 
could be expressed in a genetically 
engineered mouse. This is possible 
using techniques that take advantage 
of homologous recombination, such 
as the "tag-and-exchange" (Askew et 
al. 1993) and "hit-and-run" 
(Ramirez-Solis et al. 1993, MacMillan 
et al. 1996) techniques. Since the 
mutated receptor coding region can be 
substituted for that of the wild-type 
receptor, this technique theoretically 
allows the investigator to produce 
mice that retain expression of a protein, 
but with a single-point mutation. This 
technique should allow researchers to 
determine the importance of a region 
of a protein or a single amino acid in 
an alcohol response within the context 
of an intact neuron or an intact animal. 
This is a potentially powerful approach 



that may be free of many of the unde- 
sirable consequences of true knockout 
approaches. For example, removing 
expression of some genes and inser- 
tion of a novel promoter in the stan- 
dard knockout strategy can affect 
expression of other genes, and this 
can be avoided by substitution of a 
gene that has only a subtie mutation. 
Furthermore, the tag-and-exchange 
approach allows the mutated gene to 
be substituted into a traditional 
knockout animal. This allows for 
investigation of both the knockout 
and subtle mutation phenotypes, and 
may allow for gene rescue in knock- 
outs. However, care must be taken to 
ensure that the mutation of the pro- 
tein does not disrupt its expression 
such that the animal inadvertently 
becomes a "functional knockout" 
(e.g., as in Lakhlani et al. 1997). 

It will also be important for investi- 
gators using these techniques to 
attempt to use approaches to rescue 
the knockout phenotype by reintro- 
ducing the gene of interest in the 
knockout mice. This could be accom- 
plished by the tag-and-exchange tech- 
nique or by overexpression with a 
transgene (Iwasato et al. 1997). Tech- 
niques for inducing expression of a 
rescue gene should also become avail- 
able in the not- too -distant future. 

Researchers interested in acute 
actions of alcohol must be poised not 
only to use mouse genetic models but 
also to take advantage of the next 
generation of approaches to creation 
of these mouse models. Developing 
research strategies for the use of these 
mice that avoid the confounds dis- 
cussed earlier will help prevent the 



32 



Neural Proteins 



field from becoming bogged down 
with issues of compensation and 
genetic background when interpreting 
data from knockout mice. 

An additional problem faced by 
alcohol researchers anxious to use 
genetically altered mice is the limited 
availability of mice that were originally 
created for other research purposes. 
These mice often cannot be obtained 
by researchers with an interest in 
acute alcohol effects. One important 
step in solving this problem would be 
to encourage more investigators with 
experience in the creation of geneti- 
cally engineered mice to enter the 
alcohol research field. This could be 
done by a request for applications 
(RFA) or other grant submission 
process that would call for proposals 
aimed at the creation of mouse lines 
for alcohol research, or the use of 
existing mouse lines for alcohol 
research. One possible structure of 
such a program would be to encour- 
age investigators with the skills to cre- 
ate the mouse lines to team up with 
investigators who can assess behav- 
ioral and cellular actions of alcohol in 
the mice. Encouraging the use of 
genetically engineered mice in alcohol 
research should be a high priority in 
coming years. 

In addition to mice in which the 
genome is explicitly altered to express or 
not express a particular protein, alcohol 
researchers can take advantage of 
recombinant inbreeding approaches 
to create animals in which small areas 
of the genome differ that give rise to 
differences in alcohol -related behav- 
ioral phenotypes as discussed earlier. 
These animals can be examined at the 



cellular and molecular level to gain 
more information about the differ- 
ences that underlie the differing 
behavioral phenotypes. Proteins that 
are coded for by candidate genes 
identified by genetic analyses, such as 
QTL screening, may be targets for 
examination in these analyses. 

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Elevation of basal protein kinase C activity 
increases ethanol sensitivity of GABA( A) 
receptors in rat hippocampal CA1 pyrami- 
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1959, 1997b. 

Weiss, F.; Lorang, M.T.; Bloom, F.E.; 
and Koob, G.F. Oral alcohol self- adminis- 
tration stimulates dopamine release in the 
rat nucleus accumbens: Genetic and moti- 
vational determinants. J Pharmacol Exp 
77kt267(1):250-258, 1993. 

Williams-Hemby, L., and Porrino, L.J. 
Functional consequences of intragastri- 
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sured by the 2-[14C]deoxyglucose method: 
The contribution of dopamine. Alcohol 
Clin Exp Res 21(9): 158 1-1 591, 1997. 

Wilson, M.A., and McNaughton, B.L. 
Reactivation of hippocampal ensemble 
memories during sleep. Science 265(5172): 
676-679, 1994. 

Woodward, D.J. Behavioral neurophysiol- 
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hol research. Alcohol Clin Exp Res2§(% 
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Wozniak, K.M.; Pert, A.; Mele, A.; and 
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540(l-2):31-40, 1991. 

Wright, J.M.; Peoples, R.W.; and Weight, 
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42 



Neural Proteins 



and hippocampal neurons. Brain Res 
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Wu, J., and Kaback, H.R. Cysteine 148 in 
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Wu, L.G., and Saggau, P. Presynaptic inhi- 
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Xie, P.; Parsons, S.H.; Speckhard, D.C.; 
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43 



Chapter 2 

Lipid Involvement in the Acute Actions 
of Alcohol in the Nervous System 

Steven N. Treistman, Ph.D. 



KEY WORDS: acute AODE (effects ofAOD [alcohol or other drug] use, abuse, 
and dependence); lipid metabolism; nervous system; lipids; membrane proteins; 
calcium; potassium; voltage gated channel; biochemical mechanism; cholesterol; 
literature review 



In this chapter I address the effects of 
alcohol (ethanol) on membrane lipids 
(in particular in the nervous system) 
and the influence of the lipid environ- 
ment on the functioning of membrane 
proteins, and I propose some recom- 
mendations for the conduct of research 
in this area. Although many of the 
studies of lipids and alcohol have been 
framed and interpreted to determine 
whether the primary target of alcohol 
action is the lipid or the protein, I 
would argue that this is not the most 
productive approach. Rather, it is nec- 
essary to consider functioning mem- 
brane proteins and their lipid 
environment (as well as the various 
interfaces, such as lipid-protein, lipid- 



protein-water, protein-lipid-protein, 
etc.) as a dynamic system, in which the 
small amphiphilic alcohol molecule will 
interact with a number of targets. 
Although alcohol is sometimes referred 
to as a lipophilic molecule, it is impor- 
tant to keep in mind that it is about 10 
times more "comfortable" in the aque- 
ous compartment (Goldstein 1983). 

The volume of literature on lipids 
and alcohol is so great that an exhaus- 
tive summary is impossible within the 
space limitations of this chapter. 
Moreover, a summary of this research 
would produce a bewildering mass of 
data, with little indication of the 
import of each of the pieces on our 
ultimate understanding of alcohol 



S.N. Treistman, Ph.D., is professor of pharmacology and director of the Neuroscience Program at the 
Department of Pharmacology, University of Massachusetts Medical Center, 55 Lake Ave. North, 
Worcester, MA 01655. 



45 



NIAAA's Neuroscience and Behavioral Research Portfolio 



action. In fact, this observation sug- 
gests one of the problems that have 
accompanied much of the work on 
lipids and alcohol. It is certainly fair 
to say that, driven by the results of 
classic studies dating from the turn of 
the century, membrane lipids were 
the primary focus of biochemical and 
biophysical research on alcohol's 
actions until recently. However, the 
need to use very high alcohol concen- 
trations to get reliably measurable 
perturbations, frequently coupled 
with an absence of the physiological 
consequence (e.g., the functioning of 
membrane channels and receptors) of 
the reported perturbations, makes it 
extremely difficult to assess the 
importance of each of the reports. In 
this chapter, therefore, I have chosen 
only a small subset of the experiments 
reported in the literature, focusing on 
some of those that offer the greatest 
promise for pinpointing those aspects 
of lipids and alcohol action that are 
physiologically relevant. The data dis- 
cussed are chosen to provide some 
background for the approaches that 
will be advocated at the conclusion of 
this chapter. Unavoidably, many 
exciting results from the literature 
have been ignored, because they did 
not fit this criterion. 

I will explore the role of lipids in 
alcohol action from three primary 
perspectives. First, I will look at some 
of the lipid perturbations produced 
by acute alcohol, highlighting a shift 
in emphasis from bulk lipid effects to 
more subtle effects on different lipid 
compartments. Second, I will exam- 
ine the manner in which lipid envi- 
ronment affects the functioning of 



ligand- and voltage-gated channels 
and might affect the manner in which 
the channels respond to alcohol. 
Finally, I will discuss some approaches 
that combine our knowledge of lipid 
effects on channel function with our 
knowledge of alcohol's actions on 
membrane lipids. Because so much of 
the impetus for questioning the role 
of lipids in alcohol action derives 
from a large body of data document- 
ing changes in membrane lipid com- 
position as a function of chronic 
exposure of the cells or the animal to 
the drug, some discussion will be 
devoted to these effects, even though 
the primary focus of this paper is on 
the acute effects of alcohol. 

The remarkable shift in emphasis 
from lipids to proteins as the targets 
of acute alcohol action is well 
founded. A significant contributor to 
this trend has been the development 
of cloned channels and receptors, as 
well as the use of mutagenesis to 
relate protein sequence to physiology 
and pharmacology. It is arguable, 
based upon data already collected, 
that alcohol can interact directly with 
membrane proteins to produce alter- 
ations in function. However, this 
should not distract us from the 
important role that lipid environment 
may play in the interaction between 
the drug and the protein. That lipids 
may play an important role is sug- 
gested by the great diversity of lipids 
in the membrane, by the strong influ- 
ence of lipid composition on channel 
protein function, and by the fact that 
apparent compensatory changes in 
lipids occur as a function of chronic 
drug exposure. 



46 



Lipid Involvement in Acute Alcohol Actions 



EVIDENCE THAT 
ALCOHOL AFFECTS THE 
LIPID ENVIRONMENT 

Much of the extensive literature on 
alcohol and lipids represents a body 
of sophisticated biophysical measure- 
ments documenting the alteration of 
various parameters of the lipid phase 
of the membrane. Although many of 
these studies provide potential mech- 
anisms for the actions of alcohol in 
the nervous system, it is important to 
point out that many of these studies 
use exceedingly high concentrations 
of alcohol and that there remains a 
question of whether the perturbations 
seen translate into altered protein 
function. In recent years, it has 
become apparent that it is necessary 
to think of the membrane as com- 
posed of various compartments, such 
as the separate leaflets, the annular 
versus the bulk lipid, the acyl chain 
region versus the headgroup region, 
interfacial regions, and a number of 
others. The effects of alcohol may be 
significantly more potent on individual 
components than on the bulk proper- 
ties of the membrane taken as a whole. 
Various functions of proteins such as 
membrane channels are far more sen- 
sitive to influences from some of these 
compartments than others. 

Biological membranes are highly 
organized structures with nonrandom 
distribution of lipids (Gennis 1989). 
For example, some lipid species pref- 
erentially distribute into the extracel- 
lular leaflet of the membrane, while 
others are found predominantly in 
the intracellular leaflet, forming verti- 
cal "transbilayer" domains (Gennis 



1989; Devaux and Zachowski 1994). 
Lipids also preferentially cluster 
within a bilayer leaflet to form lateral 
domains (Welti and Glaser 1994). 
Formation of lateral domains can 
result from the juxtaposition of coex- 
isting areas of gel and fluid phase 
lipids, the nonrandom mixing 
between different lipid species, or the 
presence of cholesterol, Ca 2+ , or pro- 
teins. A number of studies have 
demonstrated that alcohols have 
selective actions on vertical and lateral 
domains. For example, ethanol selec- 
tively increases the fluidity of the 
extracellular leaflet in synaptic plasma 
membranes, an effect attributable to 
differences in transbilayer cholesterol 
distribution. Using fluorescence pho- 
tobleaching recovery techniques in 
Aplysia neurons, ethanol was shown 
to increase the diffusion of the probe 
rhodamine-phosphatidyl-ethanolamine 
more than the probe l-acyl-2-(6-[?v r - 
(7-nitrobenz-2-oxa-l,3-diazol-4-yl)] 
aminohexanoyl ) phosphatidylcholine , 
suggesting that ethanol's actions on 
membrane proteins, such as gated ion 
channels, might be dependent upon the 
existence of dissimilar lateral domains 
(Treistman et al. 1987). 

More detailed studies with respect 
to alcohol action on lateral domains 
have been conducted in model mem- 
branes. For example, alcohol's ability 
to disorder model membranes is 
enhanced by gangliosides (Harris et al. 
1984) and phospholipid polyunsatura- 
tion (Ho et al. 1994) but is antagonized 
by cholesterol (Chin and Goldstein 
1981). Consequently, native mem- 
brane domains rich in gangliosides 
and polyunsaturated phospholipids, but 



47 



NIAAA's Neuroscience and Behavioral Research Portfolio 



low in cholesterol, would presumably 
be particularly sensitive to perturbation 
by alcohol (Deitrich et al. 1989). 
Computer modeling studies also sug- 
gest that ethanol preferentially accu- 
mulates in domains with special 
packing properties favoring the inter- 
calation of alcohols (Jorgensen et al. 
1993). Membrane proteins in these 
domains would be exposed to concen- 
trations of ethanol higher than that in 
the bulk membrane (Goldstein 1984). 

One argument that has received 
significant attention is that lipids are 
unlikely to be a primary player in the 
actions of alcohol, since a small tem- 
perature change will produce changes 
in, for example, probe mobility, 
greater than the changes measured in 
the presence of alcohol. However, 
this argument must be taken with 
caution, given the existence of lipid 
compartments within the membrane. 
For example, in our Aplysia studies, 
temperature and alcohols had decid- 
edly different and selective effects on 
each of the probes used, as well as on 
the kinetics of channel function 
(Treistman and Wilson 1987&, 
1987b; Treistman et al. 1987), and 
similar discrepancies between temper- 
ature and alcohol effects have been 
reported for a number of other pro- 
teins (Wood et al. 1996). 

One of the earlier attempts to 
assign alcohol's effects on lipids to 
membrane compartments examined 
the role of gangliosides in alcohol's 
actions. Harris, Hitzemann, and col- 
leagues (Harris et al. 1984) noted that 
the signal obtained from a fluorescent 
probe intercalated into most artificial 
vesicles was not affected by alcohol, 



whereas the fluorescence signal from 
natural membranes was. They examined 
whether the presence of gangliosides 
might be contributing to this differ- 
ence, and they examined three probes, 
thought to selectively sample different 
depths and environments of the 
bilayer. They concluded that the outer 
leaflet was most sensitive to alcohol, 
in the presence of the gangliosides, 
and that phosphatidylcholine was par- 
ticularly important for the effect. 

Interfacial surfaces are proving par- 
ticularly attractive as sites of alcohol 
action. Gawrisch and Barry have ques- 
tioned the prevailing belief that protein 
hydrophobic pockets are the site of 
alcohol action, noting that the ampho- 
philic nature of alcohols favors an inter- 
facial location, and interactions are 
driven by both the opportunity for 
hydrogen bonding and hydrophobic 
interactions (Holte and Gawrisch 
1997). Using nuclear magnetic reso- 
nance (NMR) spectroscopy of artifi- 
cial phosphatidylcholine membranes, 
they probed the lipid-water interface 
and found evidence for alcohol acting 
in this region, noting that the disor- 
dering influence of the drug was 
enhanced by gangliosides and inhib- 
ited by cholesterol. 

Wood and colleagues (1996) made 
the argument that the membrane 
must be treated as a complex system, 
in which properties such as dielectric 
constant, interdigitation, lipid domains, 
and lipid-protein interactions are con- 
sidered in discussions of alcohol action. 
The dielectric constant can be viewed 
as a measure of the access of water to 
the membrane interior. At 20 °C the 
dielectric constant of water is 80, 



48 



Lipid Involvement in Acute Alcohol Actions 



while that of oleic acid is 2.5. Alcohol 
appears to increase the dielectric con- 
stant of membranes, weakening hydro- 
gen bonding and allowing water to 
infiltrate the hydrocarbon core (Orme 
et al. 1988; Rottenberg 1992; Wood 
et al. 1996). Increased interdigitation 
of the leaflets induced by alcohol may 
have consequences for protein function 
by, for example, changing the protein 
conformation, allowing more hydro- 
phobic portions to become available 
to the aqueous medium (Wood et al. 
1996). Wood and colleagues stressed 
the fact that measurements of bulk 
lipid properties may be misleading in 
the determination of lipid involve- 
ment in alcohol actions. For example, 
cholesterol is not distributed evenly 
between the two membrane leaflets, 
and a change in the concentration 
ratio between the exo- and cytofacial 
leaflets could have significant conse- 
quences for protein function, even if 
the change in overall cholesterol con- 
tent is minimal. 

Rubin, Janes, Taraschi, and collea- 
gues (Janes et al. 1992; Channareddy 
et al. 1996) have put forth the idea 
that configurational entropy is the 
driving force for alcohol action on 
membrane architecture. Because the 
membrane is quite different at different 
levels, particular characteristics of indi- 
vidual drugs, such as charge, result in 
actions specific to particular regions of 
the membrane. These authors used 
data obtained with NMR techniques 
in artificial membranes and thermody- 
namic analysis to demonstrate that 
partitioned alcohols perturb the proper- 
ties of the entire membrane, both sur- 
face and core, by altering the energetic 



balance among membrane structures. 
Their analysis precludes the need to 
provide a specific mechanism of surface 
adsorption to account for alcohol- 
induced alterations at the membrane 
surface. Partitioning differences 
between membrane structures are a 
prerequisite for alcohol action via con- 
figurational entropy. Barry and Gawrisch 
(1994) presented data using NMR 
spectroscopy that suggest that alcohol 
binds at the lipid-water interface of 
phospholipid bilayers, disorienting the 
headgroups and, through interfacial 
interaction, causing significant disor- 
dering along the entire hydrocarbon 
acyl chain. 

Although the focus of this chapter is 
to assess lipid involvement in the acute 
actions of alcohol, it is difficult to 
completely ignore the vast body of 
work addressing the influence of chronic 
alcohol exposure on membrane lipid 
composition and function. As pointed 
out by Salem (1989), the number of 
variables that enter into these studies 
makes it unsurprising that results from 
different laboratories, such as reports 
of alterations in membrane cholesterol 
levels, are often at odds. However, as 
indicated later in this chapter, the 
influence of cholesterol on the function- 
ing of some membrane receptors and 
channels is well established, making 
such alterations particularly important. 
Some reported changes, such as a 
decrease in 22:6u)3 fatty acids in 
retina (Pawlosky and Salem 1995), 
may be associated with functional con- 
sequences, such as visual pathologies. 
Some of the chronic studies suggest 
that bulk lipid properties, such as 
membrane disordering induced by 



49 



NIAAA's Ncurosciencc and Behavioral Research Portfolio 



alcohol, are significantly reduced in 
animals that have been chronically 
exposed to the drug (Ellingson et al. 
1988; Rubin 1990). 

HOW DOES THE LIPID 
ENVIRONMENT AFFECT 
THE FUNCTIONING OF 
LIGAND- AND VOLTAGE- 
GATED CHANNELS? 

As already discussed, there is abundant 
evidence that exposure to alcohol has 
measurable effects on the lipid matrix 
of the cell membrane. However, the 
mechanisms by which these effects 
translate into altered functioning of 
the ion channels and receptors that 
are the ultimate arbiters of neuronal 
activity have been difficult to determine. 
It is especially difficult to make sense 
of this relationship when studying 
intact cells, with their complex intracell- 
ular milieu and membrane. However, 
I believe that it is imperative that we 
understand the manner in which lipids 
modulate protein activity in the absence 
of alcohol, before we can reasonably 
expect to understand the basis for the 
influence of lipid environment on 
alcohol's actions on those proteins. 

In this section, I will first describe 
data that focus on the large conductance 
calcium-activated potassium channel 
(BK channel). This is a widely present 
channel in the nervous system and, 
because it is activated by intracellular cal- 
cium as well as transmembrane voltage, 
it serves to link different channel types, 
in addition to intracellular metabolic 
processes. Functionally, it serves impor- 
tant functions both in shaping individual 
action potentials and in controlling 



complex firing patterns in neurons. It 
has been studied in situ and in a very 
reduced preparation, the planar lipid 
bilayer, over the last 12 years. The effects 
of the lipid microenvironment on chan- 
nel activity have been described, and 
some hypotheses for the biophysical basis 
of these effects have been put forward. 

In their elucidation of lipid effects 
on this channel, Moczydlowski and 
colleagues (1985) noted that one of the 
first suggestions that the lipid micro- 
environment has significant effects on 
the functioning of ion channels was 
put forward by Frankenhauser and 
Hodgkin (1957), who invoked the 
presence of membrane surface charge 
to explain the shift in activation of Na 
and K currents in squid axon to more 
depolarized values (i.e., less excitable) 
when the external Ca concentration was 
increased. Frankenhauser and Hodgkin 
suggested that Ca effectively screened 
the negative charge associated with 
membrane lipids. However, this 
hypothesis is difficult to prove in a 
complex system. In the experiments 
conducted by Moczydlowski and col- 
leagues, the BK channel was studied 
in a very simplified system, in which the 
isolated channel protein was reconsti- 
tuted into an artificial planar lipid 
bilayer. This technique has proved to 
be a very powerful method to elucidate 
the role of lipids in channel modulation. 
The functioning of an ion channel can 
be broken down into a number of 
separate components, including the 
gating component, which shifts the 
channel confirmation between con- 
ducting and nonconducting states; the 
permeation component, which is 
determined by the ion-passing pore; and 



50 



Lipid Involvement in Acute Alcohol Actions 



a number of other components, such 
as kinetic aspects of function, including 
the inactivation of the channel after it 
activates. Single-channel recording 
techniques allow the examination of 
each of these parameters. Considerable 
power is added to this technique by the 
emerging database from mutagenesis 
studies relating protein sequence to 
function. In relating these functional 
parameters to lipid environment, single- 
channel recording is coupled with the 
planar bilayer technique, in which the 
protein of interest is biochemically 
removed from the native membrane and 
reconstituted into an artificial bilayer. 
Using single -channel recording and 
planar bilayer techniques, Moczydlowski 
and colleagues (1985) reexamined the 
effect of membrane lipid surface charge 
on ion channel function. By manipu- 
lating the composition of the bilayer, 
they were able to use Gouy-Chapman- 
Stern double layer theory to assess the 
effect of lipid surface charge on channel 
conduction properties. Bilayers were 
composed of either phosphatidyleth- 
anolamine (PE) or phosphatidylserine 
(PS), allowing comparison of a neutral 
versus a negatively charged lipid envi- 
ronment. The conductance of the chan- 
nel for K was significantly increased in 
the PS bilayer, compared with the PE 
bilayer. The simplest interpretation of 
this finding is that the K and Ca concen- 
trations near the surface of a negatively 
charged membrane are greater than 
the concentration of these ions in the 
bulk solution. This would both shift 
the gating of the channel toward the 
open state (because of the higher con- 
centrations of the "agonist," Ca) and 
provide for a greater K flux through 



the open channel (because of the 
increased driving force resulting from 
the increased concentration of K). A 
series of calculations determined that 
the mouth of the channel did not "see" 
the negative charge in the PS bilayer 
immediately adjacent to the channel, 
but rather at a distance of approxi- 
mately 8-10 A. They interpreted this 
to represent the "insulation," presum- 
ably part of the channel protein, of the 
mouth of the channel from the lipid 
charge. This is consistent with the size 
of channel proteins, which are as large 
as 85 A for the nicotinic acetylcholine 
receptor (nAChR), whereas the thick- 
ness of the membrane hydrocarbon 
layer is 40 A. The gating of the chan- 
nel was also "potentiated" by the 
presence of the negative charge, with 
the current- voltage relationship indi- 
cating that less depolarization was 
necessary to shift the channel to the 
open state in the PS bilayer, compared 
with the PE bilayer. Once again, the 
protein structure appeared to insulate 
the voltage -sensing site from the sur- 
rounding charge by a value < 10 A. 

The interpretations of these results 
are predicated upon a number of 
assumptions. First, the native lipid car- 
ried with the protein must exchange 
with the bilayer lipid. The authors 
present convincing arguments that this 
is the case (Moczydlowski et al. 1985). 
Less easy to show is that the differ- 
ences are due solely to charge and do 
not reflect either lipid-specific lipid- 
protein interactions or differences in 
dipole potential between the two lipids, 
which cannot be definitively ruled out 
on the basis of the experiments per- 
formed. The results obtained in these 



51 



NIAAA's Neuroscience and Behavioral Research Portfolio 



experiments demonstrate "indirect" 
effects of the lipid environment on 
protein function, as contrasted with 
other effects of lipids, in which they 
influence the conformation or integral 
properties of the protein. 

I will next describe in situ studies of 
the BK channel, which provide further 
insights into the lipid modulation of 
this channel, and I will follow that 
with a description of subsequent planar 
bilayer studies that help to illuminate 
the results obtained in the natural 
membranes. In addition to their role in 
nerve cells, BK channels are critical 
players in vascular tissue. Bregestovski 
and colleagues studied the role of 
membrane cholesterol and membrane 
fluidity on the kinetic properties of BK 
channels in cultured vascular smooth 
muscle cells, using a combination of 
fluorescence techniques and patch- 
clamping (Bolotina et al. 1989). They 
manipulated the cholesterol content 
of the plasma membrane of these cells 
and found that depletion of cholesterol 
caused an increase in D, the rotational 
diffusion coefficient of a fluorescent 
probe, concurrent with a ninefold 
increase in P , the probability of the 
channel being in the open state. Con- 
versely, treatments that led to increased 
membrane cholesterol produced a 
twofold decrease in D and a twofold 
decrease in P . Alterations in P could 
be explained by a redistribution of open 
and closed times of the channel, reflect- 
ing the thermodynamic stability of the 
channel in each of these states. These 
changes in channel gating were unac- 
companied by any change in the unitary 
conductance of the channel, high- 
lighting the fact that all regions of the 



protein are not similarly responsive to 
the putative change in membrane fluid- 
ity. Since these experiments were per- 
formed with "ripped-off patches in 
the inside-out configuration, the 
influence of intracellular milieu and 
cytoskeleton is minimized. There is 
reasonable evidence that the influence 
of cholesterol on channel gating does 
not result from direct interaction of 
the lipid with the channel, or with a 
lipid annulus associated with the 
channel, but rather reflects changes in 
the bulk (or some subbulk compart- 
ment) membrane lipid properties. 

Work from Gruener and colleagues 
has provided a mechanistic framework 
for the actions of cholesterol on the 
gating of the BK channel, and a model 
for a class of lipid-protein interaction 
(Chang et al. 1995). This group, using 
brain BK channels reconstituted in 
PE/PS lipid bilayers, confirmed the 
finding made in natural membranes, 
that increased membrane cholesterol 
decreased the open probability of the 
channel. They also tested the idea that 
since cholesterol is known to change 
the order state and the modulus of 
compressibility of bilayers, altered pro- 
tein conformation may result from an 
overall increase in the lateral stress in 
the bilayer, transmitted to the channel 
and biasing it into the closed state. It 
was found that there was a relatively 
sharp transition of the P , with short- 
ened openings, at cholesterol concen- 
trations around 10 percent. Below 
and above this concentration of choles- 
terol, there was little concentration- 
dependent effect on channel function. 
Data obtained from small angle x-ray 
diffraction of the bilayers indicated 



52 



Lipid Involvement in Acute Alcohol Actions 



that at cholesterol concentrations that 
alter channel activity the liquid crys- 
talline/gel phase composition of the 
membrane was altered, and the gel phase 
was no longer present. The authors also 
constructed Arrhenius plots, using temp- 
erature as a modulator of channel 
activity, and determined the thermo- 
dynamic properties of the BK channel 
open-to-closed transition as a function 
of temperature. They concluded that 
the calculated reduction in activation 
energy required to move the channel 
from the open to the closed state is 
consistent with the hypothesis that 
cholesterol destabilizes the open state 
of the channel, causing it to close 
sooner than in the absence of choles- 
terol. Estimates of the lateral elastic 
stress energy produced by cholesterol 
are higher than estimates of the activa- 
tion energy required to move the 
channel from the open to the closed 
state, consistent with the bias into the 
closed state by lateral elastic stress. In 
other words, the conformational change 
from the closed to the open state 
involves an increase in protein volume, 
generating lateral stress force, gener- 
ating, in turn, a counterforce deflected 
back on the channel. The magnitude of 
this force would depend on the mod- 
ulus of compressibility of the mem- 
brane, and would facilitate the return 
of the channel to the closed state at 
different rates. The authors go on to 
describe values of enthalpy, entropy, 
and free energy calculated from their 
data, consistent with this interpretation 
(Chang et al. 1995). While this type 
of analysis is far from foolproof, the 
composite work done with this channel 
and lipid modulation provides some 



insights into approaches that will 
likely be the most productive. 

The previous discussion focused on 
the Ca-activated potassium channel, 
but a similar type of analysis of lipid 
influence on channel function has also 
been done for other channels, includ- 
ing ligand-gated channels, such as the 
nAChR. Barrantes (1993) explored 
the role of the motionally restricted 
shell of lipids typically referred to as the 
"annulus lipids," immediately adjacent 
to the nAChR protein and other pro- 
teins. These lipids do exchange with 
adjacent membrane lipids, but at a 
slower rate than those lipid molecules 
that are not part of the annulus. Bar- 
rantes outlined a number of influences 
of the lipid environment on the func- 
tioning of the nAChR The findings 
include the fact that cholesterol and 
negatively charged phospholipids are 
necessary for receptor function, and 
that fatty acids can block the activity 
of the receptor. Although most head- 
group modifications were without sig- 
nificant effect, exposure of the inner 
face of the membrane to PE (in 
ripped-off patches) affected gating by 
reducing the channel open time. 
Other phospholipids did not have this 
effect. Barrantes suggested that this 
PE effect may reflect the perturbation 
of the normal membrane asymmetry, 
in which PE is predominant in the 
exofacial leaflet. 

Miller and colleagues have also 
provided information on the influence 
of membrane lipids in nAChR func- 
tion. They have used ethidium to 
measure nACh channel activity, allow- 
ing fast kinetic analysis, necessary to 
avoid complications from receptor 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



desensitization (Rankin et al. 1997). 
Both in natural membrane preparations 
and in artificial bilayers, these investi- 
gators confirmed that cholesterol is 
necessary for receptor function, but 
that the action of cholesterol is specific 
to particular aspects of receptor func- 
tion. While the baseline activity of the 
channel was relatively similar in differ- 
ent lipid environments, the ability to 
reach the open state from the resting 
state was cholesterol dependent, but 
the transition from the open to the fast 
desensitized state was not. Miller and 
colleagues have proposed a new form 
of posttranslational processing, stating 
that nAChR channels are not primed by 
cholesterol until they are inserted into 
the membrane (Rankin et al. 1997). 
Work is proceeding in Miller's and 
collaborators' laboratories to determine 
the nature of the cholesterol influence, 
although they present evidence sug- 
gesting that a simple alteration of 
membrane fluidity is unlikely to be 
the determinant. In addition, work is 
ongoing to determine the influence of 
lipids such as cholesterol on alcohol 
sensitivity of the receptor channel. 

In my laboratory, we have been 
focusing on the function and alcohol 
modulation of proteins involved in the 
secretion of vasopressin from neuro- 
hypophysial terminals. One of these is 
the BK channel described in detail 
above, and for which a significant 
body of data describing the influence 
of membrane lipids exists. We have 
amassed quite a bit of understanding 
of the actions of alcohol on the BK 
channel in situ, using both macroscopic 
and single-channel recording tech- 
niques (Dopico et al. 1996, 1998) in 



nerve terminals of hypothalamic neu- 
rons. Reasonable concentrations of 
alcohol potentiate channel activity, 
primarily by altering the gating of the 
channel. Other parameters, such as 
voltage dependency and ion selectiv- 
ity, appear unaffected by the drug. In 
addition, we are able to monitor the 
activity of cloned BK channels in 
expression systems and to assess alco- 
hol action in this situation. These 
studies have allowed us to observe the 
manner in which alcohol interacts 
with the channel, leading to some 
specific hypotheses, such as that alco- 
hol acts as a partial agonist at the Ca- 
binding site within the channel protein 
(Dopico et al. 1998). The large body 
of data that we have collected on this 
channel in situ and in expression systems 
allows us to now explore the role of 
lipid environment on alcohol's actions 
on the channel. To this end, we have 
begun studies on the BK channel recon- 
stituted into planar lipid bilayers (Chu 
et al. 1998). These studies are still at an 
early stage, but we have found that 
the reconstituted channel is potentiated 
by alcohol in a manner qualitatively 
similar to the channel in situ and in 
expression systems. The potentiation is 
not significantly different in PE versus 
PE/PS bilayers, although baseline char- 
acteristics of the channel are altered in a 
manner consistent with exchange of the 
native lipid with the lipid constituents 
of the artificial bilayer. We are encour- 
aged by these early results, and hopeful 
that this approach will yield insights 
not possible without integrating results 
focused on a single protein. 

In addition, we have discovered that 
acute alcohol suppression of vasopressin 



54 



Lipid Involvement in Acute Alcohol Actions 



release from the terminals taken from 
rats that have been chronically exposed 
to alcohol is lessened in comparison to 
the suppression seen in terminals from 
alcohol-naive rats. Of course, the ter- 
minal Ca and BK channels discussed 
above are an important part of the 
release machinery. The ability to 
explore the activity of the BK channel 
both in situ and in artificial bilayers 
will allow a full examination of both 
protein and lipid alterations in the 
mechanisms of this tolerance. 

CONCLUSIONS AND 
RECOMMENDATIONS 

The complexity of natural membranes 
and the numerous and interlinked 
lipid metabolism pathways make a rea- 
sonable analysis of lipid involvement 
in alcohol's actions in intact animals 
and tissue problematic. Contrasting the 
complexity of the situation with lipids, 
recent developments in the study of 
ligand- and voltage-gated channels 
have made the study of alcohol effects 
on proteins significantly more direct. 
The advent of cloning, expression, 
and mutagenesis of putative target 
proteins allows the testing of specific 
hypotheses regarding the relationship 
between protein structure and alcohol 
action. However, we should not for- 
get that these expressed proteins are 
operating in a lipid environment, and 
as already shown, protein function is 
strongly influenced by this environ- 
ment. Since, ultimately, our under- 
standing of alcohol's effects on nervous 
system function will derive from alter- 
ations of protein function, our explo- 
ration of the role of lipids in alcohol's 



actions must not be done in isolation 
from the impact on protein function. 
Once again, I would stress a basic tenet 
of my commentary, which is to say that 
phrasing the question as whether alco- 
hol acts primarily on the lipid or the 
protein is unproductive; rather, we must 
ask how the interactions between pro- 
tein subunits, lipids, and water are 
affected by alcohol. A few suggestions 
for how to accomplish this follow: 

1 . The function of a protein should 
serve as the readout of the lipid 
perturbations. Measurements of 
lipid perturbations without a pro- 
tein "sensor," even when employ- 
ing elegant biophysics, are not 
going to be as productive as those 
studies that approach the system as 
a dynamic interaction between the 
various components. 

2. The operation of the protein 
should be well understood before 
attempts are made to assess the 
perturbation of function by alco- 
hol. Ideally, this understanding will 
include information in the native 
membrane, as well as in artificial 
bilayers. The functioning of mem- 
brane proteins is becoming under- 
stood at a very sophisticated level. 
For ligand- and voltage-gated 
channels, this knowledge includes 
an understanding of the modular 
nature of individual proteins, with 
conserved stretches of amino acids 
controlling discrete functions, such 
as voltage dependency, permeation, 
inactivation, desensitization, and so 
on. This type of knowledge will 
greatly facilitate the testing of 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



hypotheses regarding the interac- 
tion of alcohol, lipids, and proteins. 
Similar structure -function relation- 
ships are also becoming available 
for nonchannel proteins important 
in nervous system function. 

3. Reasonable concentrations of alcohol 
should be used. This is a difficult 
problem, since an argument is often 
made that our physical measurement 
techniques are not sophisticated 
enough (e.g., may not be sampling a 
subcompartment where the effects 
are larger) to detect important 
changes occurring at relevant con- 
centrations. Although this argument 
may be reasonable, there are many 
processes that can be successfully 
studied at reasonable concentrations 
of alcohol with current technology. 
The alcohol field should wait for 
the appropriate technology before 
attacking problems that require 
very high drug concentrations to see 
measurable effects. Parenthetically, 
a case can be made that when tech- 
nology development is critical for the 
testing of an important hypothesis 
regarding alcohol perturbation of 
the lipid environment, such studies 
should be supported enthusiastically, 
even if they will not involve alcohol 
at the early stages of the research. 

4. Studies should be integrated when- 
ever possible. The ideal system will 
have certain attributes and will 
allow data collection at multiple 
levels: (a) a target protein of 
known behavioral or physiological 
relevance to alcohol action; (b) the 
effects of lipid perturbation and 



possibly modulation of lipid com- 
position on the protein in its native 
environments; (c) a protein that can 
be reconstituted into a simplified 
lipid environment for examining 
alcohol's effects; and (d) a protein 
that has been cloned, allowing for 
expression in a variety of native 
membranes, and for mutagenesis 
studies. Also, as it is becoming 
apparent that the function of most 
membrane channels and receptors 
actually reflects communities of 
coupled proteins, ideally we will be 
able to co-express combinations of, 
for example, channel subunits, to 
explore the role of lipids in their 
interactions and the actions of alco- 
hol on these interactions, resulting 
in altered function. 

5. We should not lose sight of the fact 
that lipids appear to play a major role 
in the compensatory responses of the 
cell to chronic exposure to the drug, 
and these responses should continue 
to be explored. However, whenever 
possible, these studies should also be 
performed within the framework 
described in the preceding sugges- 
tion. The response to alcohol is likely 
to be both short term and long term. 
In the short term, we might expect 
posttranslational changes in protein, 
such as phosphorylation and/or 
changes in acyl chains of phospho- 
lipids. In the longer term, we might 
predict shifts in the subunit com- 
position of the channel protein or in 
the phospholipid headgroup popu- 
lation, with changes in (e.g.) 
charge characteristics or the com- 
partmentalization of cholesterol. 



56 



Lipid Involvement in Acute Alcohol Actions 



Of course, these are just a few of 
very many possibilities. 

Each of the separate approaches, 
taken alone, has shortcomings. For 
example, for the reductionist approach, 
I realize that proteins do not exist in 
one- or two-lipid environments (prob- 
ably). However, the information 
obtained from these simple experiments 
can be used to derive hypotheses 
testable in more complicated environ- 
ments. Of course, information will 
flow the other way as well, with infor- 
mation (some already present in the 
literature) obtained in complex mem- 
brane environments leading to experi- 
ments performed in the simplified 
bilayer. There is reasonable evidence 
that lipids play a role in the actions of 
alcohol on proteins. The explosion of 
information on the functioning of 
neural membrane channels, as well as 
other proteins, presents a wonderful 
opportunity to obtain significant 
insights into this role. 

ACKNOWLEDGMENT 

I would like to thank the National 
Institute on Alcohol Abuse and Alco- 
holism for financial support. 

REFERENCES 

Barrantes, F.J. Structural -functional corre- 
lates of the nicotinic acetylcholine receptor 
and its lipid microenvironment. FASEBJ 
7:1460-1467, 1993. 

Barry, J.A., and Gawrisch, K. Direct 
NMR evidence for ethanol binding to 
the lipid- water interface of phospholipid 



bilayers. Biochemistry 33:8082-8088, 
1994. 

Bolotina, V.; Omelyanenko, V.; Heyes, B.; 
Ryan, U.; and Bregestovski, P. Variations 
of membrane cholesterol alter the kinetics 
of Ca 2+ -dependent K + channels and mem- 
brane fluidity in vascular smooth muscle 
cells. PflugersArch 415:262-268, 1989. 

Chang, H.M.; Reitstetter, R; Mason, RP.; 
and Gruener, R. Attenuation of channel 
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a unifying concept. J Membr Biol 143: 
51-63, 1995. 

Channareddy, S.; Jose, S.S.; Eryomin, V.A.; 
Rubin, E.; Taraschi, T.F.; and Janes, N. 
Saturable ethanol binding in rat liver 
microsomes. J Biol Chem 271:17625- 
17628, 1996. 

Chin, J.H., and Goldstein, D.B. 
Membrane-disordering action of ethanol: 
Variation with membrane cholesterol 
content and depth of the spin labeled 
probe. Mol Pharmacol 19:425^31, 1981. 

Chu, B.; Dopico, A.M.; and Treistman, 
S.N. Ethanol potentiation of calcium- 
activated potassium channels reconstituted 
into planar lipid bilayers. Mol Pharmacol 
54:397-406, 1998. 

Deitrich, R.A.; Dunwiddie, TV.; Harris, 
RA.; and Erwin, V.G. Mechanism of action 
of ethanol: Initial central nervous system 
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Devaux, P.F., and Zachowski, A. Main- 
tenance and consequences of membrane 
phospholipid asymmetry: Lipid domains 
in model and biological membranes. 
Chem Phys Lipids 73:107-120, 1994. 

Dopico, A.M.; Lemos, J.R; and Treistman, 
S.N. Ethanol increases the activity of large 
conductance, Ca 2+ -activated K + channels 



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in isolated neurohypophysial terminals. 
Mol Pharmacol 49:40-48, 1996. 

Dopico, A.M.; Anantharam, V.; and 
Treistman, S.N. Ethanol increases the 
activity of Ca ++ -dependent K + ( mslo) 
channels: Functional interaction with 
cytosolic Ca ++ . / Pharmacol Exp Ther 
284:258-268, 1998. 

Ellingson, J.S.; Taraschi, T.F.; Wu, A.; 
Zimmerman, R.; and Rubin, E. Cardio- 
lipin from ethanol-fed rats confers tolerance 
to ethanol in liver mitochondrial mem- 
branes. Proc Natl Acad Sci USA 85:3353- 
3357, 1988. 

Frankenhauser, B., and Hodgkin, A.L. The 
action of calcium on the electrical proper- 
ties of squid axons. J Physiol 137:218- 
244, 1957. 

Gennis, R.B. Lateral and transverse asym- 
metry in membranes. In: Cantor, C.R., 
ed. Springer Advanced Texts in Chemistry: 
Biomembranes. Molecular Structure and 
Function. New York: Springer- Verlag, 
1989. pp. 138-165. 

Goldstein, D.B. Pharmacology of Alcohol. 
New York: Oxford, 1983. 

Goldstein, D.B. The effects of drugs on 
membrane fluidity. Annu Rev Pharmacol 
Toxicol 24:43-64, 1984. 

Harris, RA.; Groh, G.I.; Baxter, D.M.; and 
Hitzemann, RJ. Gangliosides enhance the 
membrane actions of ethanol and pentobar- 
bital. Mol Pharmacol 2SA10-417, 1984. 

Ho, C.; Kelly, M.B.; and Stubbs, CD. 
The effects of phospholipid unsaturation 
and alcohol perturbation at the protein/ 
lipid interface probed using fluorophore 
lifetime heterogeneity. Biochim Biophys 
Acta 1193:307-315, 1994. 

Hoke, L.L., and Gawrisch, K. Determining 
ethanol distribution in phospholipid 



multilayers with MAS-NOESY. 
Biochemistry 36:4669^*674, 1997. 

Janes, N.; Hsu, J.W.; Rubin, E.; and 
Taraschi, T.F. Nature of alcohol and 
anesthetic action on cooperative mem- 
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31:9467-9472, 1992. 

Jorgensen, K.; Ipsen, J.H.; Mouritsen, 
O.G.; and Zuckermann, M.J. The effect 
of anesthetics on the dynamic heterogene- 
ity of lipid membranes. Chem Phys Lipids 
65:205-216, 1993. 

Moczydlowski, E.; Alvarez, O.; Vergara, 
C.; and Latorre, R Effect of phospholipid 
surface charge on the conductance and 
gating of a Ca 2+ -activated K + channel in 
planar lipid bilayers. J Membr Biol 
83:273-282, 1985. 

Orme, F.W.; Moronne, M.M.; and 
Macey, R.I. Modification of erythrocyte 
membrane dielectric constant by alcohols. 
J Membr Biol 104:57-68, 1988. 

Pawlosky, R.J., and Salem, N., Jr. Ethanol 
exposure causes a decrease in docosahex- 
aenoic acid and an increase in docosapen- 
taenoic acid in feline brains and retinas. 
Am J Clin Nutr 61:1284-1289, 1995. 

Rankin, S.E.; Addona, G.H.; Kloczewiak, 
M.A.; Bugge, B.; and Miller, K.W. The 
cholesterol dependence of activation and 
fast desensitization of the nicotinic acetyl- 
choline receptor. Biophys J 73:2446- 
2455, 1997. 

Rottenberg, H. Probing the interactions 
of alcohols with biological membranes 
with the fluorescent probe Prodan. 
Biochemistry 31:9473-9481, 1992. 

Rubin, H. On the nature of enduring 
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Salem, N., Jr. Alcohol, fatty acids, and 
diet. Alcohol Health Res World 
13:211-218, 1989. 

Treistman, S.N., and Wilson, A. Alkanol 
effects on early potassium currents in Aplysia 
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Acad Set USA 84:9299-9303, 1987a. 

Treistman, S.N., and Wilson, A. Effects of 
ethanol on early potassium currents in 
Aplysia: Cell specificity and influence of 
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1987b. 

Treistman, S.N.; Moynihan, M.; and 
Wolf, D.E. Influence of alcohols, temper- 



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in neuronal membrane. Biochim Biophys 
Acta 898:109-120, 1987. 

Welti, R, and Glaser, M. Lipid domains 
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Chem Phys Lipids 73:121-137 ', 1994. 

Wood, W.G.; Schroeder, F.; Rao, A.M.; 
Igbavboa, U.; and Avdulov, NA. 
Membranes and ethanol: Lipid domains 
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1996. pp. 13-27. 



59 



Chapter 3 

Effects of Alcohol on the 
Neuroendocrine System 

Catherine Rivier, Ph.D. 



KEY WORDS: AODE (effects of AOD [alcohol or other drug] use, abuse, and 
dependence); endocrine system; hypothalamus-pituitary axis; pituitary-adrenal 
axis; corticotropin RH (releasing hormone); glucocorticoids; cytokines; brain 
function; adrenocorticotropic hormone; homeostasis; neurotransmitters; biological 
regulation; gonad function; gonadotropin RH; luteinizing hormone; animal 
study; literature review 



Research on the effects of alcohol as 
they pertain to neuroscience can be 
broadly divided into three parts: (1) 
the search for the mechanisms that 
lead to excessive alcohol consumption 
and addiction and the associated syn- 
dromes of withdrawal and relapse; (2) 
the investigation of the pharmacologi- 
cal influence of alcohol on molecular, 
cellular, and system biology, which is 
a consequence of alcohol exposure/ 
consumption; and (3) the study of the 
possible contributing effect of specific 
hormones — in particular, those of the 
hypothalamic -pituitary- adrenal (HPA) 
axis — on alcohol consumption. 



Alcohol exerts a wide spectrum of 
effects, which affect virtually every cell 
in the body. It is not entirely clear 
whether alcohol exerts similar effects 
on most signaling pathways (e.g., by 
similarly altering cyclic adenosine 
monophosphate (cAMP)-dependent 
processes, or gene transcription, or 
binding of ligands to their receptors), 
or whether these effects are system 
specific. Indeed, the ability of the 
drug to indiscriminately distribute 
itself throughout the body (including 
the brain) renders studies of its specific 
influence within a particular system 
difficult. Also, because alcohol does 



C. Rivier, Ph.D., is a professor at The Salk Institute, The Clayton Foundation Laboratories for 
Peptide Biology, 10010 North Torrey Pines Rd., La folia, CA 92037-1099. 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



not have a receptor, the mechanisms 
through which it alters cellular function 
are not easy to elucidate. It is important 
to keep these points in mind when 
reviewing what is known and suggest- 
ing topics for future emphasis. 

This chapter focuses on the neuro- 
endocrine influence of alcohol, a topic 
that was the focus of a National Institute 
on Alcohol Abuse and Alcoholism 
(NIAAA) workshop and monograph 
(Zakhari 1993). I will therefore present 
only a brief overview of the alcohol - 
related literature and will list selected 
topics of interest for NIAAA that 
either represent a logical extension of 
present NIAAA-supported programs 
or are not represented in the portfolio. 

SCIENTIFIC REVIEW 

Study of the molecular and cellular 
aspects of the effects of alcohol, which is 
routinely done in isolated cell systems, 
presents the challenge of determining 
the molar concentrations of the drug 
within which results can be interpreted 
as being relevant for the whole organ- 
ism. Study of the influence of alcohol 
in the intact animal, on the other hand, 
presents the challenge of determining 
whether results obtained with forced 
exposure to alcohol are relevant for 
conditions associated with spontaneous 
alcohol consumption, or whether one 
should dissociate between the two. 
Most of the results in the published 
literature were obtained in laboratory 
animals exposed to alcohol through 
an experimenter- controlled procedure. 
This is primarily because unselected 
animals do not spontaneously drink 
alcohol or, if they are forced to do so 



(e.g., when presented with a chocolate- 
based alcohol diet as die sole source of 
nutrients), only consume it in limited 
amounts. There is no doubt that results 
obtained with this experimental approach 
have been very interesting and useful. 
Nevertheless, it is important to remain 
cognizant of the possibility that at least 
some of the biological effects of alcohol 
may be different in animals forcibly 
exposed to alcohol from those that 
self- administer the drug (Moolten and 
Kornetsky 1990). If there are differ- 
ences between the two paradigms, they 
may represent the fact that animals 
that are not selected for alcohol self- 
administration usually consume small 
amounts of alcohol and therefore only 
reach low blood alcohol levels (BALs); 
that forced exposure to alcohol may 
induce aversiveness; and whether or not 
there are rewarding efFects. It is extremely 
important to determine the origin of 
these differences in alcohol's effects 
because investigators may pursue mech- 
anisms that are not those that underlie 
the effect of alcohol during spontaneous 
consumption. Although I do not advo- 
cate abandoning models based on 
experimenter-induced alcohol exposure, 
the field would greatly benefit from 
the increased availability of and ease of 
access to animals selected for the 
spontaneous consumption of mean- 
ingful amounts of alcohol. 

HPAAxis 

Many studies have shown that alcohol 
administration to laboratory rodents 
causes a rapid and significant activation 
of the HPA axis (Rivier 1996). 
Increased levels of corticotropin- 
releasing factor (CRT) and possibly 



62 



Effects of Alcohol on the Neuroendocrine System 



vasopressin in the brain are important 
in modulating the effect of the drug 
on this axis (Rivier et al. 1984; Car- 
mona-Calero et al. 1995; Rivier and 
Lee 1996). There is some controversy 
regarding the acute effect of alcohol 
in humans; some investigators claim 
that increased HPA axis function in 
human volunteers consuming an alco- 
holic beverage is only present in the 
subjects who experience gastrointesti- 
nal discomfort (Inder et al. 1995 b). 
Nevertheless, the consensus appears 
to be that humans who consume large 
amounts of alcohol exhibit increased 
HPA axis activity (though with a great 
deal of variability [Wand 1993]), as 
indicated by the fact that enhanced 
basal Cortisol production is found in 
some alcoholics (Wand and Dobs 
1991) and can lead to a pseudo-Cush- 
ing's syndrome (Veldman and Mein- 
ders 1996). To state that the HPA 
axis of alcoholics is activated, however, 
is simplistic. Indeed, possibly as a con- 
sequence of increased corticosteroid 
feedback and/or down- regulated pitu- 
itary CRF receptors, many alcoholics 
show a blunted response to exoge- 
nous CRF injection or exposure to a 
non-alcohol-related stress (Wand and 
Dobs 1991). Furthermore, the HPA 
axis remains pathologically altered in 
short-term abstinent alcoholics, who 
also show blunted responses to CRF 
or exposure to non-alcohol stresses 
(Heuser et al. 1988; Adinoff et al. 
1990; Inder et al. 1995a; Ehrenreich 
et al. 1997). Therefore, while there 
appears to be reasonable evidence for 
alcohol-induced changes in the HPA 
axis of humans who abuse this drug, 
much remains to be investigated. 



The importance of studies of the 
effect of alcohol on the HPA axis of 
any mammalian species extends 
beyond the mechanisms that they will 
uncover. Because of its pivotal influ- 
ence as a general regulator and coor- 
dinator of the stress responses, and 
because its hormones exert such a 
wide range of effects, changes in the 
activity of this axis are likely to con- 
tribute to many of the effects of alcohol. 
Although CRF is the primary regula- 
tor of the HPA axis (Rivier and Plot- 
sky 1986), this peptide also exerts 
many other effects. Thanks to studies 
of the distribution of CRF through 
the brain, its pharmacological effects 
on a wide array of parameters, and the 
consequence of immunoneutralizing 
it or blocking its receptors, we now 
know that this peptide also controls or 
participates in the regulation of the 
hypo thalamic -pituitary -gonadal 
(HPG) axis, growth hormone release, 
gastrointestinal functions, and natural 
killer cell activity, which it inhibits 
(Rivier and Vale 1984; Irwin et al. 
1990; Tache et al. 1993; Rivest and Riv- 
ier 1995); opioids and catecholamines, 
which it stimulates (Brown and Fisher 
1985; Boyadjieva et al. 1997); depres- 
sion, which it appears to induce 
(Nemeroff 1996); and anxiety, which 
it promotes (Koob et al. 1993). Any 
alterations in CRF production and the 
activity of CRF -dependent circuitries 
(such as those seen after alcohol) will 
therefore have profound conse- 
quences for the organism, both under 
basal conditions and during attempts 
to restore homeostasis. A full and 
complete knowledge of what alcohol 
does and how is therefore crucial. 



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NlAAA's Neuroscience and Behavioral Research Portfolio 



Another important point is that in 
experimental animals, CRF has been 
shown to induce or participate in many 
responses that are very similar to those 
associated with fetal alcohol exposure 
in humans, such as hyperactivity, 
decreased attentiveness, aggressive- 
ness, increased incidence of infections, 
augmented activity of the HPA axis, 
abnormal sexual behavior, and prema- 
ture aging. Many of these pathologies 
might therefore be directly or indi- 
rectly caused by elevated CRF levels. 
However, while our understanding of 
the consequences of prenatal alcohol on 
CRF-dependent circuitries is increas- 
ing (Rivier 1996), there is a paucity of 
studies testing the hypothesis that this 
peptide participates in fetal alcohol syn- 
drome (FAS)-related disorders. Finally, 
there is the very interesting finding 
that animals displaying increased vol- 
untary alcohol consumption have ele- 
vated corticosterone levels under basal 
conditions (Prasad and Prasad 1995) — 
although this observation is not uni- 
versal (Tuominen and Korpi 1991). If 
true, this finding suggests the intrigu- 
ing possibility that increased brain 
CRF levels may be associated with 
increased drinking. 

CRF also exerts direct effects in many 
systems outside of the brain. In view 
of the ability of alcohol to up-regulate 
the CRF gene, it seems reasonable to 
propose that it would be of great 
interest to probe the hypothesis that 
cardiovascular and immune effects (for 
example) of the drug might be modu- 
lated through this peptide. Although 
it is outside the scope of this chapter 
to review this field, it may be useful to 
remember that CRF is reported to be 



manufactured by and to have receptors 
in macrophages (Webster et al. 1990) 
and other immune cells (Aird et al. 
1993; Kravchenco and Furalev 1994), 
and is also reported to be present in 
arthritic (Crofford et al. 1992) and 
inflamed tissues (Hargreaves et al. 
1989), where it is believed to partici- 
pate in the inflammatory process (Kar- 
alis et al. 1991; Theoharides et al. 
1997). Indeed the concept of a "tis- 
sue CRF" that is released in response 
to immune challenges and plays a local 
regulatory role (Hargreaves et al. 1989) 
has long been recognized. Although 
the effect of alcohol on this CRF has 
not been extensively studied, it has 
been described (Dave and Eskay 1986). 
CRF is also present in steroid-produc- 
ing cells (Audhya et al. 1989; Ulisse 
et al. 1989; Tortorella et al. 1993), 
where it is reported to play a (mostly 
inhibitory) physiological role in regu- 
lating sex steroid production (Fabbri 
et al. 1990; Eskeland et al. 1992; 
Dufau et al. 1993; Calogero et al. 
1996; Gnessi et al. 1997). Finally, 
CRF and/or its receptors are found in 
the gastrointestinal tract and in the 
heart (Chalmers et al. 1996). It is not 
known if alcohol influences CRF and 
CRF receptors in these tissues, and 
whether this might play a role in the 
gastrointestinal and cardiovascular 
effects of the drug. 

In addition to exerting effects by 
itself, CRF alters homeostasis by stim- 
ulating the release of glucocorticoids 
(GC). These steroids influence immune 
functions: if their levels are too high, 
infection can develop because the 
activity of immune cells is inhibited 
(Black 1994; Kusnecov and Rabin 



64 



Effects of Alcohol on the Neuroendocrine System 



1994; McEwen et al. 1997); if their 
levels are too low, inflammation can 
take place because of increased reac- 
tivity (Sternberg 1992; Chrousos 
1995; Sternberg 1997). Glucocorti- 
coids also play a critical role in the 
general metabolism by regulating car- 
bohydrate levels (Dallman et al. 
1993) and by influencing the tone of 
blood vessels (Munck and Guyre 
1986). Within the brain, GC maintain 
the integrity of neuronal networks 
(Meyer 1985) and chronic elevations 
of its levels can enhance susceptibility 
to neurodegeneration (Joels and de 
Kloet 1995) and premature aging 
(Sapolsky 1992; Seckl and Olsson 
1995). There, too, the potential for 
an influence exerted by alcohol 
through GC is enormous, and it 
should be investigated. 

Hormones of the HPA Axis in 
Drug- Seeking Behavior 

There is good evidence that CRF is 
involved in many aspects of drug- 
seeking behavior. First, individual vul- 
nerability appears to correlate well 
with various responses to stress, 
including GC release (Deminiere et 
al. 1989; Deroche et al. 1995). The 
role of GC is further supported by the 
findings that adrenalectomy prevents 
the development of alcohol preference 
in rats (Lamblin and De Witte 1996) 
and that corticosterone stimulates 
alcohol intake and the consumption 
of other drugs (Piazza et al. 1993; 
Fahlke et al. 1995). This may explain, 
at least in part, why stress reinstates 
drug-seeking behavior (Erb et al. 
1996). It must be pointed out, how- 
ever, that the respective role of CRF 



and GC in drug-seeking behavior 
remains unclear (Deroche et al. 1993; 
Shaham et al. 1997). In addition, 
there is a great deal of controversy as 
to whether brain levels of CRF are 
elevated (George et al. 1990) or 
decreased (Ehlers et al. 1992) in ani- 
mals with high preference for alcohol, 
and whether alcohol-sensitive mice 
exhibit a blunted (Tuominen and 
Korpi 1991) or enhanced (Ehlers et 
al. 1992) HPA axis response upon 
exposure to stimuli. 

Interactions Between the HPA 
Axis and Cytokines: An Example of 
How Alcohol Can Indirectly 
Compromise Homeostasis 

The appropriate release of CRF, 
adrenocorticotropic hormone (ACTH), 
and GC in response to threats to 
homeostasis is essential for the health of 
the organism. This is true, for exam- 
ple, during immune stimulation, and 
we know that hypo- or hypersecretion 
of CRF and GC leads to inflammation 
or infection, respectively. In dis- 
cussing the potential influence of alco- 
hol in altering the HPA axis's ability 
to restore homeostasis, a few intro- 
ductory remarks may be helpful. 

Stress consists of a stimulus input, a 
central processing system, and a 
response output (Levine and Ursin 
1991). Therefore, the systems that are 
responsible for restoring homeostasis 
in the face of stress, such as the HPA 
axis, need a set of components such as 
sensors to monitor stimuli or pertur- 
bations, afferent transducers of infor- 
mation, a brain region that can receive 
and be responsive to the afferent 
inputs, and efferent transducers to 



65 



NIAAA's Neuroscience and Behavioral Research Portfolio 



command appropriate responses to 
the effector system. The brain center 
that controls the activity of the HPA 
axis is the paraventricular nucleus 
(PVN) of the hypothalamus (Swanson 
1987), which contains CRF neurons 
(Swanson 1986), and the efferent sys- 
tem includes ACTH and GC. Follow- 
ing exposure to antigens in the 
periphery, activated immune cells pro- 
duce and release cytokines. These pro- 
teins convey to the brain in general, 
and the PVN in particular, the occur- 
rence of immune stimulation by 
releasing intermediates (prostaglandins, 
catecholamines, etc.), by inducing 
production of cytokines in the brain 
itself, by stimulating vagal afferents to 
the brain, or possibly by entering the 
brain themselves (Rivier 1995&; Bese- 
dovsky and Del Rey 1996). In 
response, the PVN up-regulates CRF 
synthesis, and ACTH secretion 
increases. The subsequent release of 
GC subserves many purposes in the 
metabolic adjustments that are neces- 
sary during the acute-phase response. 
Through increased feedback, these 
steroids also ensure that PVN activa- 
tion remains within limits that are not 
themselves threatening to homeostasis. 
In particular, they prevent overpro- 
duction of cytokines. The ability of 
the organism to restore homeostasis 
therefore depends on complex func- 
tional interactions (a "checks and bal- 
ances" process) between cytokines 
and the HPA axis, and on the appro- 
priate response of this axis. If the 
activity of the HPA axis has been pre- 
viously altered by other stimuli (e.g., 
alcohol), PVN CRF synthesis may be 
enhanced or inhibited, CRF receptors 



may be up- or down-regulated, and the 
HPA axis will respond inappropriately. 

Our laboratory has shown that in 
adult rats exposed to alcohol, subsequent 
exposure to exogenous cytokines or 
to a cytokine-releasing inflammatory 
process results in a significandy blunted 
response of the HPA axis (Lee and 
Rivier 1994^, 1994£, 1995). This may 
be at least in part due to increased 
steroid feedback, but many other 
mechanisms probably play a role. For 
example, increased nitric oxide (NO) 
production may be important (Rivier 
1995#). In view of the relevance of 
this gas in brain function (see, e.g., 
Vincent 1994), and possible functional 
relationships between alcohol and NO 
(Khanna et al. 1993; Fitzgerald et al. 
1995; Lancaster 1995; Calapai et al. 
1996; Zou et al. 1996; Naassila et al. 
1997), we need studies investigating 
the effect of NO and the other gaseous 
neurotransmitter, carbon monoxide, 
in regulating the response of CRF cir- 
cuitries to alcohol. 

Another second messenger-type 
factor that deserves attention is 
nuclear regulatory factor-KB (NF-kB) 
(O'Neill and Kaltschmidt 1997), 
which is emerging as a crucial regula- 
tor of brain function. The NF-kB 
family of transcription factors is a pri- 
mary regulatory component of the 
intracellular signal pathways in cells of 
the immune system, and endotoxin 
and interleukin-1 represent its key 
activators. An emerging concept is 
that NF-kB is an important stress sensor. 
If this is true, it may interact with 
CRF in the maintenance of homeostasis, 
for example, by disrupting functional 
relationships between CRF and cytokine - 



66 



Effects of Alcohol on the Neuroendocrine System 



dependent pathways. The observation 
that physiologically relevant concen- 
trations of alcohol may alter cytokine 
production by disrupting NF-kB sig- 
naling (see, e.g., Mandrekar et al. 
1997) lends support to this concept. 

In contrast to the inhibitory effect 
of alcohol postnatally, exposure to the 
drug during embryonic development 
up-regulates PVN CRF gene transcrip- 
tion (Lee et al. 1990), which may par- 
ticipate in the enhanced HPA axis 
response of the adult offspring (Taylor 
et al. 1984; Weinberg 1988; Lee and 
Rivier 1996) to immune challenges 
(Lee and Rivier 1996) and other 
stresses (Taylor et al. 1986; Weinberg 
1989). As a result, these offspring per- 
manently secrete too much CRF and 
GC in response to any stress, with the 
potential consequences outlined 
above. The mechanisms through 
which alcohol exerts this effect remain 
to be fully elucidated. 

These experiments exemplify the 
type of effects exerted by alcohol 
when it disrupts pathways that are 
essential for health, and the impor- 
tance of understanding the mecha- 
nisms responsible for these effects. 

Stress Hormones 

Broadly speaking, stress hormones 
include hormones of the HPA axis 
(CRF, ACTH, GC, opiates) and cate- 
cholamines. The opioid peptide (3- 
endorphin has important functions in 
the brain as a neurotransmitter and is 
believed to play a role in positive rein- 
forcement, adaptive processes, mood, 
and the development of alcoholism 
(Gianoulakis 1996; Boyadjieva et al. 
1997). Both (3-endorphin and another 



opioid peptide, pro-opiomelanocortin 
(POMC), are regulated in part by CRF, 
which may explain why their levels are 
elevated in both the pituitary and the 
hypothalamus following alcohol expo- 
sure (Angelogianni and Gianoulakis 
1993; Fickel et al. 1994). On the 
other hand, hypothalamic POMC levels 
are decreased in rats made dependent 
on alcohol (Scanlon et al. 1993), which 
could be due to decreased testosterone 
levels. Although there are differences 
between the brain opioid levels of 
rodents with different voluntary alcohol 
consumption, these differences are 
region specific (Gianoulakis et al. 
1992), and their importance in modulat- 
ing alcohol consumption remains to be 
fully understood (George et al. 1991). 

HPG Axis 

The fact that alcohol inhibits reproduc- 
tive functions in experimental animals 
as well as in humans abusing the drug 
is well known (see, e.g., Bannister and 
Lowosky 1987; Purohit 1993). How- 
ever, understanding of the mecha- 
nisms responsible for this influence 
has remained surprisingly elusive and 
controversial. A careful evaluation of 
the published data indicates significant 
alcohol-induced decreases in luteiniz- 
ing hormone (LH) and sex steroid 
release. Alcohol could act at one or 
several levels of the HPG axis: the 
gonadotropin-releasing hormone 
(GnRH) neurons, the afferent circuits 
to these neurons, pituitary responsive- 
ness (including GnRH receptors, 
GnRH signaling pathways, and LH 
synthesis), and steroidogenesis 
(including LH receptors, postreceptor 
events such as steroidogenic acute 



67 



NIAAA's Neuroscience and Behavioral Research Portfolio 



regulatory [StAR] protein-mediated 
cholesterol transport to the inner 
mitochondrial membrane, and the 
activity of steroidogenic enzymes). 

Alcohol can inhibit GnRH release 
(Ching et al. 1988; Ogilvie and Rivier 
1997), and the fact that this effect is 
usually only seen in the intact animal 
(Uddin et al. 1996) suggests that the 
drug may primarily act on afferent cir- 
cuits to GnRH neurons, rather than 
on GnRH production itself. Indeed, a 
decrease in catecholamine-induced 
prostaglandins secretion has been sug- 
gested to play a role (Hiney and Dees 
1991). Decreases in LH levels often 
take place at least 1 hour after alcohol 
treatment (Dees et al. 1985), and they 
probably reflect blunted GnRH secre- 
tion in the hypothalamus and/or 
release from nerve terminals (Canteros 
et al. 1995) rather than changes in 
pituitary responsiveness (Rivier et al. 
1992). Alcohol may also decrease LH 
mRNA stability (Emanuele et al. 
1991; Halloran et al. 1995). Overall, 
it appears that the influence of alcohol 
on gonadotropin production may be 
modest following acute exposure to the 
drug, but probably contributes to hypo- 
gonadism during chronic treatment. 

The inhibitory effect of alcohol on 
the activity of both male and female 
gonads is very strong, but it is inter- 
esting to note that decreases in sex 
steroid levels (particularly testosterone 
[T]) often precede measurable 
changes in LH release. This suggests 
that alcohol can directly inhibit 
steroidogenesis, a phenomenon that is 
now well recognized (Adams et al. 
1997). In addition, alcohol can act on 
Sertoli cells (Zhu et al. 1997). The 



gonadal influence of alcohol includes a 
decrease in the number of LH recep- 
tors (particularly after long-term drug 
treatment [Salonen and Huhtaniemi 
1990]), decreased availability of the 
metabolites that are necessary for 
mitochondrial activity (Orpana et al. 
1990), and impaired synthesis/activ- 
ity of steroidogenic enzymes (Akane 
et al. 1988). Alcohol may impair 
steroidogenesis through increased 
production of testicular opioids 
(Adams et al. 1997), but the possibil- 
ity that the drug can increase cytokine 
production in the liver, coupled with 
the known inhibitory influence of 
these proteins on gonadal function 
(Rivier and Vale 1989; Adashi 1990), 
suggests that a similar mechanism might 
also deserve attention in the gonads. 

One important aspect of the effect of 
alcohol on the gonads is its extreme 
rapidity. Indeed, in our laboratory, its 
intraperitoneal or intragastric injection 
significantly lowered basal T levels and 
impaired gonadotropin-induced T 
response in less than 15 minutes (Rivier 
1999). The mechanisms responsible 
for this rapid effect have not been 
explored, but they may be important 
for even the casual drinker. Consistent 
with results obtained in laboratory 
animals, chronic alcohol has been 
shown to impair the activity of the 
HPG axis in nonhuman primates 
(Mello et al. 1989) and in humans 
(Bell et al. 1995; Villalta et al. 1997). 
It is intriguing to note that although 
acute alcohol usually induces a rapid 
and significant drop in T levels, it not 
only increases concentrations of the 
steroid in women (Eriksson et al. 
1994) but also augments estradiol 



68 



Effects of Alcohol on the Neuroendocrine System 



production (Mendelson et al. 1989). 
Finally, alcohol interferes with the 
normal appearance of puberty (Cicero 
et al. 1990). 

FUTURE DIRECTIONS 

General 

Despite the pivotal role of CRF in so 
many alcohol-related disorders and as 
a reinforcer of drug abuse, the number 
of studies supported by NIAAA that 
investigate its synthesis, release, and 
effects on endocrine functions in gen- 
eral, and the HPA axis in particular, is 
quite low. There may be many rea- 
sons for this. Experiments dealing 
with endocrine axes are difficult, 
expensive, and technically challenging, 
and they require investigators who are 
very familiar with the biology of stress. 
In particular, extensive expertise with 
in vivo work of the quality required 
for a valid assessment of HPA axis 
activity is required, but is a fast- disap- 
pearing knowledge. Many of the tools 
necessary to carry out these studies are 
not widely available; these include 
CRF antagonists as well as standardized, 
easy-to-use, and affordable reagents to 
measure ACTH and corticosterone 
levels. Proposals describing studies 
pertaining to the HPA axis should be, 
but are not always, reviewed by inves- 
tigators who are keenly aware of both 
the potential and limitations of these 
studies, which results in a dwindling 
number of laboratories that are funded 
for this work. Along the same lines, 
the investigation of hypotheses related 
to the possible role of CRF in FAS 
pathologies may require experimental 



approaches that do not immediately 
yield hard data, that require many 
false starts, or that may even fail. 
Investigators who understand these 
difficulties are often the same ones 
who would be best suited to conduct 
these studies, but few may spend time 
and effort writing proposals under 
these circumstances. 

Animal Studies 

Functional interactions have been 
shown between CRF and the neuro- 
transmitters involved in drug-seeking 
behavior, reinforcement, and relapse. 
There are undoubtedly many neuro- 
transmitters that play a role in the 
development of alcohol abuse. For 
example, serotonin has been impli- 
cated in drug-seeking behavior, and 
animals or humans with low serotonin 
levels in their brains are described as 
more aggressive (Brown et al. 1982) 
and prone to depression (van Praag 
1996). Mice lacking serotonin or its 
receptors show less evidence of intoxi- 
cation and tolerance (Crabbe et al. 
1996), which supports possible corre- 
lations between low serotonin levels 
and the development of alcoholism 
(Roy et al. 1987; Schulz et al. 1998). 
Although it is known that serotonin 
does not act alone in the brain, its 
potential functional connection with 
CRF in the context of alcohol con- 
sumption has not been given much 
attention. 

Role of CRF in Adult Animals 

A powerful tool to investigate the role 
of a secretagogue is to study biologi- 
cal responses in mice lacking the gene 
for this secretagogue or its receptors. 



69 



NIAAA's Neuroscience and Behavioral Research Portfolio 



One must, however, be aware of the 
limitation of this approach. A devel- 
oping system that is deprived of a par- 
ticular component will often, if it is 
viable, rely on alternate components 
to replace the missing entity, or on 
redundant systems that are normally 
not active in the intact animal. Conse- 
quently, one needs to be very careful 
when interpreting results obtained 
with mutant animals. For example, 
NO is presently considered an essen- 
tial neurotransmitter, and its acute 
removal in the intact animal has pro- 
found consequences for brain func- 
tion. However, mice with null 
mutation of the gene for NO synthase 
(NOS) (the enzyme that is responsi- 
ble for NO formation) display surpris- 
ingly normal brain function, including 
long-term potentiation, an activity 
thought to be extremely dependent 
on NO. 

In the future, the availability of 
conditional mutants (i.e., animals in 
which a gene can be deleted in adult- 
hood) will probably represent a much 
better method to evaluate the role of 
a particular secretagogue. In the 
meantime, however, mutant ("knock- 
out") rodents provide valuable insight 
into both the role of a secretagogue 
and the pathways that come into play 
when it is absent. Mice with null 
mutation for the CRF or CRF-receptor 
gene are available, they are capable 
of sustaining many experimental pro- 
cedures, and they are fertile. Valuable 
information would be gained from 
studies investigating the effect 
of exposing rodents with null muta- 
tion of the CRF or CRF-receptor gene 
to alcohol. 



Like most other peptides, CRF exerts 
its effect through receptors. Two CRF 
receptor families have been identified 
(reviewed in DeSouza 1995). CRF- 
Rl in the pituitary (Pozzoli et al. 
1996; Sakai et al. 1996) mediate the 
stimulatory effect of CRF on ACTH 
release. CRF-R1 in the PVN of the 
hypothalamus (Sawchenko et al. 
1995) probably also play a role for the 
activity of the HPA axis, though this 
remains to be fully elucidated. Certainly, 
these receptors could participate in 
the effect of CRF on non-HPA axis- 
related events, and as such represent 
important mediators of the influence 
of this peptide. CRF-R1 are also 
found in extrahypothalamic areas such 
as the amygdala (Makino et al. 1995), 
where they may regulate emotions. 

CRF-R2, which come in two 
forms — 2a and 2(3 (Chalmers et al. 
1996) — are present in fewer brain 
structures than CRF-R1 (Lovenberg 
et al. 1995). The structures in which 
CRF-R2 are present include the limbic 
system (type 2a) and the choroid plexus 
(type 2|3) (Lacroix and Rivest 1996). 
In the hypothalamic ventromedial 
nucleus (Chalmers et al. 1995), CRF- 
R2 might regulate the anorexic effect 
of CRF (Choi et al. 1996). Some 
investigators have reported the pres- 
ence of the CRF-R2 gene in the PVN 
(Chalmers et al. 1995; Makino et al. 
1997), but this remains somewhat 
controversial. CRF-R2 are also found in 
the periphery, particularly in the heart 
(Lovenberg et al. 1995; Heldwein et 
al. 1997). Overall, the role of CRF- 
R2 remains poorly understood. 

Studies using CRF -Rl -deficient 
rodents will test hypotheses related to 



70 



Effects of Alcohol on the Neuroendocrine System 



those tested with animals lacking the 
gene for CRF itself. In addition, they 
will provide information regarding the 
type of CRF receptors that mediate a 
particular effect of alcohol, which is 
important for the future development 
of therapies aimed at alleviating or 
preventing unwanted consequences of 
alcohol exposure. 

Role of CRF in Adult Offspring of 
Dams Exposed to Alcohol 

If we postulate the hypothesis that an 
up-regulated CRF system in the brain 
is key for many of the adult endocrine, 
behavioral, autonomic, and immune 
pathologies observed in animals and 
humans exposed to alcohol during fetal 
development, there is a dire need for 
studies investigating the consequences 
of exposing pregnant dams lacking 
the CRF or CRF-R1 gene to alcohol. 
These studies should include not only 
alterations in HPA axis activity but 
also changes in behavior, reproductive 
capacity, immune functions, and drug- 
seeking behavior. 

Functional Interactions Between 
Alcohol and Nitric Oxide, Carbon 
Monoxide, and NF-kB 

Nitric oxide, carbon monoxide, and 
NF-kB are essential for normal brain 
activity. They are likely to be influenced 
by alcohol and probably participate in 
pathologies due to this drug. Studies 
of functional interactions between 
alcohol and these entities are essential. 

In Vitro Systems 

The study of the effect of alcohol on 
CRF systems depends on the availabil- 
ity of reliable systems in which to 



study CRF gene transcription. Such 
studies are seriously hampered by the 
lack of a good model of isolated cells 
(either primary culture or immortal- 
ized cells) that produce CRF. Fetal 
hypothalami, which can be main- 
tained in culture, produce little CRF 
in comparison with other peptides, 
and adult hypothalami are very diffi- 
cult to maintain in culture. Models in 
which to study CRF signaling path- 
ways and gene transcription are there- 
fore greatly needed. 

Reagents and Animal Models 

CRF Antagonists 

The investigation of the physiological 
role of CRF can be addressed via a 
number of avenues. One is the use of 
potent CRF antagonists. The devel- 
opment of these analogs has been 
surprisingly slow and difficult. If they 
are to be of experimental, and even- 
tually therapeutic, benefit, CRF 
antagonists (whether peptidic or non- 
peptidic) will need to be not only 
potent and long-lasting but also 
receptor specific. Stability, the ability 
to penetrate the brain following sys- 
temic administration, and cost of 
manufacture are also factors in the 
ultimate usefulness of these analogs. 
However, these factors cannot be 
determined until we have an array of 
CRF antagonists to choose from. 
Interactions between various agencies 
of the National Institutes of Health 
(NIH) — for example, the National 
Institute of Diabetes and Digestive 
and Kidney Diseases, the National 
Institute of Mental Health, the 
National Institute on Drug Abuse, 



71 



NIAAA's Neuroscience and Behavioral Research Portfolio 



and NIAAA — might be of great benefit 
in the development of these antagonists. 

ACTH and Corticosterone Assays 

The cost of measuring ACTH levels 
in unextracted plasmas with assays 
that can reliably handle hundreds of 
samples is escalating at an alarming 
rate and may soon threaten studies 
focused on the HPA axis. Similarly, 
the availability of reliable antibodies 
for corticosterone is dwindling. NIH- 
distributed reagents for the measure- 
ment of plasma ACTH and 
corticosterone levels in laboratory 
rodents are urgently needed. Here 
also, collaborative arrangements with 
other NIH agencies may be beneficial. 

Alcohol -Preferring Animals 

As mentioned earlier, results obtained 
in experimental animals forcefully 
administered alcohol and results 
obtained in those that spontaneously 
consume the drug may be different. It 
would be extremely useful to have 
access to different strains of alcohol- 
preferring rats and mice. These strains 
have been created, but a mechanism 
making them readily available to 
appropriate investigators would 
greatiy facilitate comparative studies. 

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MOLECULAR AND CELLULAR 

RESPONSES TO CHRONIC 

ETHANOL EXPOSURE 



Chapter 4 

Neuroadaptation to Ethanol at the 
Molecular and Cellular Levels 



Paula L. Hoffman, Ph.D., A. Leslie Morrow, Ph.D., 
Tamara J. Phillips, Ph.D., and George R. Siggins, Ph.D. 

KEY WORDS: AOD (alcohol or other drug) tolerance; biological adaptation; 
chronic AODE (effects of AOD use, abuse, and dependence); brain function; 
AOD dependence; homeostasis; AOD withdrawal syndrome; AOD sensitivity; 
memory; learning; central nervous system; membrane channel; neurotransmitter 
receptors; dopamine; serotonin; opioid receptors; adenylate cyclase; protein kinases; 
signal transduction; gene regulation; neuropeptides; arginine; vasopressin; anti 
alcohol craving agents; gender differences; genetics and heredity; literature review 



DEFINITIONS drinking has ceased or been reduced; 

the intake of alcohol to relieve signs 
Alcohol dependence currently has c ■ , , , , ,.rr- u- 

.. . ,, , r .. . . . ,,£„,, or withdrawal; and difficulties in con- 

well- denned diagnostic cntena (DSM- ... .... . , , . 

Tr 7 rA • t^ i • . • * ■ ■ trolling drinking, with a strong desire 

IV [American Psychiatric Association & & ' & 

1994] and ICD-10 [World Health or compulsion to drink (Tabakoff and 

Organization] ), including the presence Hoffman 1996a). All of these symptoms 

of alcohol tolerance; the presence of may be considered to arise as a result 

an alcohol withdrawal syndrome when of changes in brain function that 

P.L. Hoffman, Ph.D., is a professor in the Department of Pharmacology, University of Colorado 
Health Sciences Center, 4200 East 9th Ave., Box C-236, Denver, CO 80262-0001. A. Leslie Morrow, 
Ph.D., is associate director of the Bowles Center for Alcohol Studies and associate professor of psychiatry 
and pharmacology at the University of North Carolina School of Medicine, 3027 Thurston Bowles 
Bldg., CB 7178, Chapel Hill, NC 27599-7178. T.J. Phillips, Ph.D., is a professor in the Department 
of Behavioral Neuroscience, School of Medicine, Oregon Health Sciences University and a research 
geneticist at Veterans Affairs Medical Center, Research Division, R&D-32, 3710 SW US Veterans 
Hospital Rd., Portland, OR 97201. G.R. Siggins, Ph.D., is a professor in the Department of 
Neuropharmacology, CVN-12, The Scripps Research Institute, 10550 North Torrey Pines Rd., La 
folia, CA 92037. 



85 



NIAAA's Neuroscience and Behavioral Research Portfolio 



occur following chronic exposure to 
alcohol (ethanol). It is generally believed 
that these changes represent adaptations 
of the brain to ethanol: acutely, ethanol 
produces changes in the function of a 
number of neuronal systems, and the 
consequence of homeostatic responses 
to these changes, induced by the 
chronic presence of ethanol in the 
brain, is the production of the alcohol 
dependence syndrome (Tabakoff and 
Hoffman 1996#). These homeostatic 
responses may involve positive or neg- 
ative feedback mechanisms or may 
reflect more permanent, qualitative 
changes in synaptic connections, which 
may be either beneficial or harmful to 
the organism as a whole (Hyman and 
Nestler 1996). One of the challenges 
of alcohol and other drug research is 
to identify the changes in central ner- 
vous system (CNS) function that 
reflect adaptation to the chronic pres- 
ence of the drug, and to define the 
relationship of those cellular, bio- 
chemical, and molecular changes to 
various aspects of the dependence 
syndrome. In this review, we will out- 
line certain CNS changes produced 
by chronic ethanol treatment that are 
thought to be neuroadaptive, and we 
will attempt to determine how these 
changes lead to or reflect the behav- 
ioral aspects of neuroadaptation to 
ethanol, which are defined in the fol- 
lowing sections. 

Ethanol Tolerance 

Tolerance to ethanol is defined as 
acquired resistance to the effects of 
the drug, but it is a more complex 
phenomenon than is suggested by this 
definition (Tabakoff and Rothstein 



1983; Kalant 1998). Tolerance may be 
metabolic or dispositional, meaning that 
previous exposure to ethanol results in a 
change in the metabolism, distribution, 
or excretion of the drug such that the 
organism is exposed to lower blood or 
brain ethanol levels after ethanol inges- 
tion. Functional tolerance, which is the 
focus of this review, refers to an increase 
in cellular resistance to the effects of 
ethanol in the CNS. Tolerance can occur 
within the time that a single dose of 
ethanol is ingested (acute or within - 
session tolerance) or after repeated 
exposure to ethanol (chronic tolerance). 
A form of tolerance known as "rapid 
tolerance" has also been described, in 
which exposure of an animal to a single 
dose of ethanol generates tolerance 
when a second dose is administered 
8-24 hours after the first dose. It is 
not known whether similar or identical 
mechanisms underlie the development 
of these different forms of tolerance. 

Ethanol tolerance can also be influ- 
enced by environmental variables. For 
example, it has been shown that if a 
task is practiced under the influence of 
ethanol, tolerance to the effect of 
ethanol on the performance of that task 
develops more rapidly than if no prac- 
tice occurs ("behaviorally augmented 
tolerance"). In this case, it appears to 
be the rate of tolerance development 
that is affected, as the same maximal 
degree of tolerance develops eventu- 
ally in both situations (practice or no 
practice). There is also evidence for a 
role of classical conditioning in the 
development of ethanol tolerance (as 
for tolerance to other drugs [e.g., 
Siegel 1976]), and it has even been 
suggested that the occurrence of 



86 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



ethanol tolerance is entirely dependent 
on learned responses (Tabakoff and 
Hoffman 1992). It has been demon- 
strated that when ethanol administra- 
tion is repeatedly paired with a distinct 
environment, tolerance to various effects 
of ethanol can be demonstrated in that 
environment but is absent if the animal 
is tested in a different environment. 
Tolerance in this case results from a 
conditioned response, which is associ- 
ated with cues in the environment in 
which ethanol was administered and 
which is opposite to the initial effect of 
ethanol. For example, animals that have 
been treated with ethanol in a particular 
environment, and are then tested with 
saline in that same environment, show 
hyperthermia in response to the saline 
treatment. This hyperthermia counters 
the hypothermic effect of ethanol and 
produces tolerance in the "cued" envi- 
ronment, but it does not occur if the 
animal is tested in an environment dis- 
tinct from that in which ethanol was 
administered (Le et al. 1979; Mansfield 
and Cunningham 1980; Crowell et al. 
1981; Melchior and Tabakoff 1981, 
1985).This type of tolerance has been 
called conditional or environment- 
dependent tolerance. Its characteristics 
are different from those of environ- 
ment-independent tolerance^ which 
can be demonstrated regardless of the 
environment in which the animals are 
treated with ethanol and tested. Envi- 
ronment-dependent tolerance is 
induced by lower doses of ethanol, 
administered as repeated doses (e.g., 
by injection), and persists for a longer 
time than environment-independent 
tolerance, which can be induced by 
giving higher doses of ethanol in a 



continuous manner, such as in a liquid 
diet or by vapor inhalation (Melchior 
and Tabakoff 1981). It is not known 
whether environment-dependent and 
-independent forms of ethanol toler- 
ance result from different underlying 
mechanisms; however, studies in the 
area of learning and memory may pro- 
vide some clues. 

For considering the cellular, neuro- 
chemical, and molecular changes that 
may underlie ethanol tolerance, we have 
previously found it useful to consider 
tolerance within a framework that had 
been used to discuss the neurobiology 
of learning: that is, intrinsic and extrin- 
sic neuronal systems (Tabakoff and 
Hoffman 1992). Extrinsic systems are 
those that influence the development, 
maintenance, or expression of tolerance 
or other neuroadaptive phenomena, but 
do not encode tolerance within them- 
selves. Intrinsic systems, on the other 
hand, do encode specific information, 
such as tolerance to a specific effect of 
ethanol, presumably by changes in 
synaptic efficacy in a particular neu- 
ronal pathway. There are a number of 
behaviors and physiological functions 
that are affected by ethanol and that are 
commonly used to assess ethanol toler- 
ance. These include ethanol-induced 
incoordination, loss of righting reflex 
("sleep time"), changes in body temper- 
ature, and anxiolytic effects. Identifi- 
cation of intrinsic systems would be 
facilitated by knowledge of the neuronal 
systems that mediate behaviors or 
physiological functions that become tol- 
erant to ethanol, but in many cases (e.g., 
sleep time, body temperature changes) 
these systems are not well characterized 
or are very complex. Another means 



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of identifying possible intrinsic sys- 
tems is by analyzing the acute effects 
of ethanol. It is assumed that adapta- 
tions will occur in neurochemical 
and/or neurophysiological systems 
that are initially perturbed by ethanol. 
Whether the same or different neu- 
ronal systems mediate the occurrence 
of acute, rapid, and chronic tolerance, 
as well as the environment-dependent 
and -independent forms of tolerance, 
is a question that has only begun to 
be addressed. 

Ethanol Dependence 

As described above, the alcohol 
dependence syndrome comprises not 
only tolerance, but also physical 
dependence and the compulsion to 
use alcohol ("psychological depen- 
dence"). Physical dependence on 
alcohol is defined primarily by a char- 
acteristic set of symptoms and signs 
that appear when the chronic adminis- 
tration or consumption of relatively 
high doses of alcohol is abruptly ter- 
minated. The signs and symptoms of 
withdrawal are, in most instances, 
opposite in nature to the signs of 
acute intoxication, and follow a char- 
acteristic time course after the cessa- 
tion of alcohol intake (see Tabakoff 
and Rothstein 1983). In humans, 
alcohol withdrawal signs and symp- 
toms can be divided into early and 
late stages; the early stages (first 36 
hours) are characterized by tremors, 
convulsions, mild diaphoresis, and 
hallucinations, and the later stages 
include severe autonomic dysfunction 
and delirium. Many of the early signs 
of withdrawal, such as tremors, con- 
vulsions, and temperature aberrations, 



can also be observed in animal models 
of physical dependence on alcohol 
(Tabakoff and Rothstein 1983). 

The biological mechanisms that 
underlie the signs and symptoms of 
alcohol withdrawal are, by definition, 
the factors that are responsible for 
physical dependence. As discussed for 
tolerance, one can attempt to identify 
the neuronal systems involved in phys- 
ical dependence and withdrawal based 
on the systems that mediate the physi- 
ological functions that are disrupted 
during withdrawal. However, in most 
cases, the systems underlying the signs 
that are used to assess alcohol with- 
drawal in animals (e.g., spontaneous 
and "handling-induced" convulsions, 
temperature aberrations) are complex 
and poorly understood. Nevertheless, 
it is believed that the chronic exposure 
of the susceptible neuronal systems to 
alcohol results in an adaptation that 
generates exaggerated and maladap- 
tive responses to normal neuronal 
input after the cessation of alcohol 
intake. 

It has been postulated that the 
same adaptations that lead to toler- 
ance to certain effects of alcohol will, 
when alcohol intake or exposure 
is terminated, produce the signs of 
withdrawal (e.g., Goldstein and Gold- 
stein 1968). For example, a change 
that produced tolerance to the 
hypothermic effect of alcohol could, 
in the absence of alcohol, result 
in hyperthermia. However, differences 
in the time course of development 
of physical dependence and functional 
tolerance, as well as the demonstra- 
tion of treatments that can block the 
development of tolerance but not 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



withdrawal signs, and vice versa (e.g., 
Tabakoff and Ritzmann 1977; Snell 
et al. 1996^), suggest that different 
neuroadaptations may underlie 
physical dependence and tolerance to 
various effects of alcohol. 

The degree to which environmen- 
tal variables affect physical depen- 
dence on alcohol is not clear. 
Although it has been hypothesized 
that conditioned withdrawal signs 
may occur when abstinent individuals 
are exposed to the environment previ- 
ously associated with opiate with- 
drawal (O'Brien et al. 1986), there is 
very little evidence for conditioning 
associated with alcohol withdrawal. 
Such "conditioned withdrawal" 
would be important in that it could 
trigger increased alcohol consump- 
tion, to alleviate the perceived with- 
drawal signs or symptoms. However, 
another controversial issue is whether 
individuals will continue to consume 
alcohol in order to relieve the symp- 
toms of withdrawal associated with 
physical dependence. There have been 
several studies (Woods and Winger 
1971; Mello and Mendelson 1977; 
Roehrs and Samson 1981; Tang et al. 
1982) showing that neither humans 
nor animals will continue alcohol con- 
sumption in order to avoid with- 
drawal signs and symptoms. 

On the other hand, the depen- 
dence syndrome also includes the 
compulsion to consume alcohol. The 
concepts of craving and need have been 
described as psychological depen- 
dence, but they are rooted in neuro- 
logical processes. Many investigators 
have operationally defined craving by 
measuring drug-seeking behaviors 



(Schuster 1986). Drug- seeking behav- 
iors are responses that have previously 
been associated with the administra- 
tion of a drug and are believed to 
reflect the reinforcing efficacy of a 
drug (Schuster and Johanson 1981). 
Of particular interest for this review is 
the role that neuroadaptive processes 
may play in altering the reinforcing 
properties of alcohol, and thus the 
degree of alcohol-seeking behavior. 

In animals, alcohol is not an effica- 
cious reinforcer, and, in almost all 
studies of alcohol self- administration, 
the animal must be induced to drink 
alcohol by procedures such as food 
deprivation, adulteration of the taste 
of alcohol, or acclimatization to grad- 
ually increasing concentrations of 
alcohol (e.g., Meisch 1984; Samson 
1987). The difficulty in demonstrat- 
ing alcohol self- administration has 
been attributed to the aversive effects 
of alcohol, which can overshadow its 
reinforcing effects. Thus, one could 
speculate that if tolerance to the aver- 
sive properties of alcohol were devel- 
oped, the reinforcing properties might 
become more prominent. This possi- 
bility is supported by studies showing 
that repeated exposure of animals to 
alcohol can result in conditioned place 
preference and conditioned taste pref- 
erence for alcohol (e.g., Crawford and 
Baker 1982; Reid et al. 1985). How- 
ever, this hypothesis has yet to be 
tested directly, for example, by block- 
ing the development of tolerance to 
aversive properties of alcohol and 
measuring alcohol self- administration. 
It is also not clear whether tolerance 
to the reinforcing effects of alcohol 
can develop. The occurrence of such 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



tolerance could lead to continued 
alcohol intake (see chapter 7). Thus, 
neuroadaptive changes in the neu- 
ronal systems that mediate either the 
aversive or reinforcing properties of 
alcohol could conceivably contribute 
to alterations in the reinforcing effects 
of alcohol that would lead to craving, 
or a compulsion to drink alcohol. 
Another possible mechanism that may 
contribute to increased alcohol intake 
is the development of sensitization to 
the effects of alcohol. 

Ethanol Sensitization 

Drug-induced behavioral sensitization 
may be defined as the augmentation 
of a response to a drug with repeated 
exposures. The first demonstration of 
behavioral ethanol sensitization was 
that of Masur and Boerngen (1980), 
who treated mice for up to 60 days, 
once daily, with 1-3.5 g/kg ethanol 
and showed increases in the initial 
locomotor responses to some ethanol 
doses. Ethanol sensitization has been 
little investigated since publication of 
those results, but some studies do 
exist (Masur et al. 1986; Newlin and 
Thomson 1991; Phillips et al. 1991; 
Cunningham and Noble 1992; 
Phillips et al. 1994; Cunningham 
1995; Phillips et al. 1995). 

There is speculation as to whether 
ethanol sensitization is a determinant 
factor in addictive behavior (Wise and 
Leeb 1993). Hunt and Lands (1992) 
suggested that sensitization may 
increase the probability of the devel- 
opment of uncontrolled ethanol 
intake, and Newlin and Thomson 
(1991) asserted that sensitization to 
ethanol might reflect greater reward 



value of the drug. It is our view that 
this has yet to be conclusively demon- 
strated. Several key questions remain 
unanswered. Does sensitization 
develop to the reinforcing effects of 
ethanol? If so, does it contribute to 
increased alcohol consumption? Are 
alcoholics sensitized? These are all 
important questions that have yet to 
be answered. 

It is possible that locomotor sensi- 
tization reflects intensified reinforce- 
ment, or at least reflects alteration in a 
system that results in increased sensi- 
tivity to reinforcing stimuli (Stewart 
and Badiani 1993; Wise and Leeb 
1993). A recent study by Lessov and 
Phillips (1998) demonstrated that 
ethanol sensitization can be relatively 
long-lasting, suggesting that lasting 
neuroadaptive mechanisms may be 
engaged. The behavioral significance 
of sensitization may be that it results in 
increased efficacy of ethanol reinforce- 
ment and thus increases the likelihood 
that ethanol will be self- administered. 
In other words, molecular changes 
accompanying sensitization might be 
viewed as adaptive phenomena, per- 
mitting facilitation within a system, 
and making responses controlled by 
that system easier to elicit on future 
encounters (see Stewart and Badiani 
1993). If the sensitized system should 
happen to be one contributing to the 
reward experienced with ethanol intake, 
then it is easy to see how sensitization 
might result in increased drinking. More 
plainly, ethanol would come to more 
easily elicit its reinforcing effects. 

An example of increased drug reward 
with repeated administration is seen in 
a study by Lett (1989), who measured 



90 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



conditioned place preference to amphet- 
amine, morphine, or cocaine and 
found that prior exposure to these 
drugs enhanced conditioned place 
preference. Perhaps more direct evi- 
dence is provided by studies like that 
of Horger and colleagues (1990), 
who showed that rats sensitized to 
cocaine acquired cocaine self- adminis- 
tration at lower doses. Such studies 
have not been performed with ethanol. 
Sparse data using rats and chronic 
treatment with drugs other than 
ethanol show an increase in ethanol 
consumption or preference following 
amphetamine, nicotine, or morphine 
treatment (Potthoff et al. 1983; Levy 
and Ellison 1985; Hubbell et al. 
1988; McMillan and Snodgras 1991; 
Fahlke et al. 1994). Such results may 
be interpretable as increased sensitivity 
to ethanol reinforcement due to chronic 
drug treatment. However, develop- 
ment of behavioral sensitization due 
to the long-term drug administration 
was not measured in any of these 
studies. We know of no studies that 
have measured ethanol sensitization 
and then looked at its influence on 
ethanol drinking, or vice versa. A 
negative genetic correlation between 
ethanol sensitization and ethanol con- 
sumption was found in BXD recombi- 
nant inbred strains (Phillips et al. 
1995). Those strains more prone to 
sensitization consumed less ethanol. 
However, independent groups of ani- 
mals were tested for the two traits. 
There is a need for more research to 
establish the importance of the sensiti- 
zation phenomenon to addiction. 

One reason for the relative paucity 
of ethanol sensitization reports in the 



literature may be that there appears to 
be an important species difference in 
its occurrence: it can be demonstrated 
in the mouse but may be difficult to 
demonstrate in the rat (Masur et al. 
1986), the research animal that has 
been most commonly used in studies 
of sensitization to other drugs. How- 
ever, as already mentioned, there are 
genotype-dependent differences 
among mouse strains in propensity 
toward the development of ethanol 
sensitization. This has also proven to 
be important in some rat studies of 
acute ethanol stimulant effects (Waller 
et al. 1986; Krimmer 1991). How- 
ever, we do not know of any strain 
surveys or other genetic investigations 
of ethanol sensitization susceptibility 
in rats. 

Memory as Neuroadaptation: 
Cellular, Biochemical, and 
Molecular Models 

Although the focus of this review is 
ethanol-induced neuroadaptation, it 
should be recognized that the physio- 
logical processes of learning — the 
process by which new information 
about the environment is acquired — 
and memory — the process by which 
that knowledge is retained — also 
reflect adaptations at the cellular, bio- 
chemical, and molecular levels in the 
CNS. In other words, learning and 
memory, like tolerance and depen- 
dence, can be viewed as adaptive 
responses of the CNS to external 
stimuli. Therefore, studies of learning 
and memory have the potential to 
provide clues to the mechanisms 
underlying neuronal adaptation to 
alcohol and other drugs, as well as 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



approaches to investigate such adapta- 
tion at the molecular level. 

There is no question that learning 
and memory are complex processes. 
Memory has been classified, for exam- 
ple, as explicit or implicit (Bailey et al. 
1996). Explicit memory is conscious 
recall, while implicit memory is non- 
conscious recall of motor skills and 
other tasks. Implicit memory includes 
associative forms (e.g., classical condi- 
tioning) and nonassociative forms, 
and it is believed to involve changes in 
the same pathways that are used in the 
learning process. These aspects of 
implicit memory are reminiscent of 
the characteristics of neuroadaptation 
to alcohol and other drugs, such as 
tolerance, and the pathways that are 
altered in implicit memory are those 
previously defined as intrinsic systems 
in the Ethanol Tolerance section of 
this chapter. 

Memory is also often divided into 
short-term and long-term compo- 
nents. It is perhaps too simplistic to 
compare these processes, for example, 
to the acute and chronic forms of 
ethanol tolerance; however, it is of 
interest, in terms of the mechanisms 
of neuroadaptation to ethanol, to 
note that short-term memory is 
believed to involve covalent modifica- 
tion of existing proteins, whereas 
long-term memory is more stable and 
requires transcription, translation, and 
the growth of new synaptic connec- 
tions (Bailey et al. 1996). Similar 
processes may be involved in neuroad- 
aptations to ethanol, as discussed in 
more detail below. 

The studies of Kandel and his col- 
leagues provide a model for investigating 



the mechanisms that underlie neuro- 
adaptation. One of the examples of 
implicit memory that has been studied 
in detail by this group is sensitization 
of the gill/siphon withdrawal reflex in 
the marine snail, Aplysia. This animal 
learns to respond to a variety of previ- 
ously neutral stimuli once it has been 
exposed to a potentially threatening 
stimulus. The neural pathway for sen- 
sitization of the reflex has been deter- 
mined, and it involves activation of 
facilitatory interneurons that synapse 
on sensory neurons, to strengthen the 
connection between the sensory neu- 
rons and their central target neurons 
(Kandel 1991; Bailey et al. 1996). 
The Kandel group has been able to 
study the neuronal mechanisms of 
sensitization in this system by isolating 
a component of the reflex, a monosy- 
naptic connection between sensory 
neurons and their target cells. This 
monosynaptic connection can be studied 
in cell culture, where serotonin, which 
is released upon stimulation, can sub- 
stitute for the training stimulus. Bio- 
physical studies established that short- 
term (minutes to hours) changes in 
synaptic effectiveness in this pathway 
are attributable to enhanced release of 
neurotransmitter, due to a change in 
activity of a neuronal potassium channel 
(Bailey et al. 1996). This change also 
occurs in the longer term sensitization 
(days to weeks), but studies using 
inhibitors of transcription and transla- 
tion both in the intact animal and in 
cell culture demonstrated that long-term 
sensitization depends on new protein 
and RNA synthesis, and is also associated 
with structural changes — that is, the 
growth of new synaptic connections 



92 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



(Kandel 1991; Bailey et al. 1996). The 
idea that memory processes and neuro- 
adaptation to ethanol may have certain 
common mechanisms is not new, and 
it is interesting to note that studies 
have also showed that inhibitors of 
protein synthesis could block the 
development of chronic ethanol toler- 
ance in animals (LeBlanc et al. 1976; 
Bitran and Kalant 1993). 

Kandel and his colleagues have 
defined the molecular substrates of 
memory in the Aplysia system in some 
detail. Using a variety of biochemical 
and molecular biological techniques, 
they have provided evidence that the 
serotonin-induced activation of adenylyl 
cyclase (AC) and subsequent activation 
of protein kinase A (PKA) are critical 
factors in short- and long-term sensiti- 
zation. Initially, phosphorylation of the 
potassium channel or related proteins 
results in decreased activity of the channel, 
producing the enhanced neurotrans- 
mitter release that underlies short-term 
sensitization. In the cell nucleus, PKA- 
induced phosphorylation of a 3',5'-cyclic 
adenosine monophosphate response 
element binding (CREB)-like protein 
that binds to the 3 ',5 '-cyclic adenosine 
monophosphate response element (CRE) 
is necessary for long-term sensitization 
(Kandel 1991; Bailey et al. 1996). 
These investigators have also identified 
an immediate early gene (IEG), a 3,5'- 
cyclic adenosine monophosphate 
(cAMP)-regulated transcription factor 
that was demonstrated to be involved in 
the development of long-term sensitiza- 
tion by the use of antisense oligonucleo- 
tides (Alberini et al. 1994). In addition, 
the structural alterations associated with 
long-term sensitization in Aplysia have 



been suggested to be related to down- 
regulation of cell adhesion molecules that 
are related to nerve cell adhesion mole- 
cule (NCAM) (Mayford et al. 1992). 

These studies help to define the 
molecular and neurochemical pathways 
required for an elementary form of 
memory. It is interesting to note that the 
cAMP system has also been implicated 
in learning and memory in Drosophila, 
where mutants that produce defects in 
various portions of the cAMP signaling 
pathway are deficient in the ability to 
learn a classical conditioning task (Byers 
et al. 1981; Levin et al. 1992). Inter- 
estingly, recent studies with Drosophila 
also demonstrated the importance of 
proteins in the cAMP/PKA cascade for 
ethanol sensitivity and tolerance (Moore 
et al. 1998). Cyclic AMP is also impor- 
tant for the maintenance of long-term 
potentiation in certain areas of mammalian 
brain (e.g., Hopkins and Johnston 1988), 
and deficiencies in learning and long-term 
memory have been demonstrated in mice 
with mutations in AC (Wu et al. 1995) or 
CREB (Bourtchuladze et al. 1994). It 
could prove very informative to study 
ethanol tolerance and dependence in 
these mice, especially in view of the 
effects of ethanol on the cAMP signal- 
ing system that are described later in 
this chapter. 

This discussion is not meant to sug- 
gest, however, that the cAMP signal 
transduction system is the only system 
that should be investigated with regard 
to neuroadaptation to ethanol. In 
another invertebrate system, for exam- 
ple, protein kinase C (PKC) and cal- 
cium/calmodulin-dependent protein 
kinases, as well as various IEGs, have 
been implicated in associative learning 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



(Alkon and Nelson 1990). The advan- 
tage of using these invertebrate models is 
the well-characterized nature of the 
neuronal systems involved in the behav- 
iors being measured. The relative sim- 
plicity of the studied pathways allows 
for the construction of testable models 
of the biochemical and molecular 
changes that underlie learning and 
memory. Furthermore, there is substan- 
tial evidence that the signaling pathways 
that are implicated in adaptive processes 
in these simple models may also be applic- 
able to neuroadaptation in the vertebrate 
CNS. Consideration should therefore 
be given both to using simpler models, 
but those involving the whole organism 
(such as Aplysia), to study neuroadapta- 
tion to ethanol, and to using the studies 
of learning and memory that have been 
carried out with these models to guide 
research into the cellular, neurochemical, 
and molecular pathways that mediate 
ethanol-induced neuroadaptation. 

WHAT IS KNOWN ABOUT 
ETHANOL-INDUCED 
CHANGE AND 
NEUROADAPTATION 
AT THE CELLULAR, 
BIOCHEMICAL, AND 
MOLECULAR LEVELS 
IN THE CNS? 

Systems That Show 
Changes After Chronic 
Ethanol Exposure 

Ligand-Gated Ion Channels 

Within the past 10 years it has become 
increasingly apparent that a major site 
of action of ethanol is ion channels. 



Ion channels are multimeric struc- 
tures, comprising different subunits, 
that gate ions following subtle changes 
in tertiary structure. Ethanol is more 
hydrophobic than water and in some 
instances can replace water within mol- 
ecular structures, but it does not have 
the hydrogen- bonding capacity. Ethanol 
readily enters molecular sites within 
multimeric ion channels modifying 
intermolecular forces and bonds that are 
important for the open-close-inactivation 
kinetic properties of channels. The 
diversity of channel composition due 
to the multimeric structures results in 
subtypes of channels that are differen- 
tially distributed across brain regions. 
There are also regional differences in 
the sensitivity of ion channels to the 
action of ethanol. 

The acute intoxicating and incoor- 
dinating effects of ethanol may be 
related to inhibition of subtypes of N- 
methyl-D-aspartate (NMDA)-glutamate 
receptors and potentiation of certain 
subtypes of GABA A receptor ion 
channels. The effects of ethanol on 
glycinergic, nicotinic cholinergic, and 
serotonergic receptors, and voltage- 
gated calcium and potassium channels, 
are also considered in this chapter. A 
considerable amount of data suggests 
that alterations in NMDA and GABA A 
receptors, and voltage-gated calcium 
channels, contribute to the development 
of ethanol tolerance, dependence, and 
withdrawal. Many of the other chan- 
nels that are sensitive to ethanol at rel- 
evant concentrations have not been 
studied in this context. In the following 
sections we attempt to summarize the 
existing data and better understand the 
relationships between the effects of 



94 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



ethanol on ion channels and the 
chronic behavioral effects of ethanol. 

Glutamate Receptor Ion Channels. 
There are three classes of ionotropic 
glutamate receptors, including the 
NMD A, the kainate, and the AMPA 
(L-a-amino-3-hydroxy-5-methyl-4- 
isoxazole propionate) receptor subtypes 
(Sommer and Seeburg 1992; Sprengel 
and Seeburg 1993; Hollmann and 
Heinemann 1994; McBain and Mayer 
1994; Bettler and Mulle 1995). The 
kainate and AMPA receptors mediate 
fast excitatory neurotransmission. The 
NMDA receptor is coupled to an ion 
channel that, when activated, is per- 
meable to calcium as well as monovalent 
cations. The pharmacology of the 
NMDA receptor is well defined 
(Collingridge and Lester 1989; 
McBain and Mayer 1994). The func- 
tion of the NMDA receptor is voltage 
dependent, meaning that the response 
to NMDA is increased as the cell is 
depolarized. The voltage dependence 
is the result of Mg 2+ binding within 
the ion channel. Mg 2+ blocks the 
channel but is released upon cellular 
depolarization. The NMDA receptor- 
channel complex also contains bind- 
ing sites for several other agents that 
influence receptor activity. Glycine is a 
co- agonist at the receptor, and both 
glutamate and glycine are required for 
activation of the receptor. NMDA 
receptor activity is also affected by 
phencyclidine, which binds within the 
ion channel, as does the uncompeti- 
tive inhibitor, dizocilpine (MK-801). 
The complex also contains binding 
sites for Zn 2+ and polyamines. 

Both the non-NMDA glutamate 
receptors and the NMDA receptors 



consist of a number of subunits. 
GluRl-GluR4 are believed to form 
AMPA receptors, while GluR5-GluR7 
appear to form low-affinity kainate 
receptors. There are also proteins 
called KA-1 and KA-2 that can bind 
kainate with high affinity and can 
form functional receptors when 
expressed with members of the GluR 
family (Hollmann and Heinemann 
1994). The NMDA receptor consists 
of (a) an NR1 subunit, which has 
eight splice variants and is ubiqui- 
tously localized in the brain, and (b) a 
family of NR2 subunits (NR2A-D) 
(Monyer et al. 1992; Nakanishi 1992; 
Sugihara et al. 1992; McBain and 
Mayer 1994). Receptors composed of 
NR1 and NR2 subunits show 
responses to NMDA that are charac- 
teristic of native receptors, and the 
NR2 subunits significantly influence 
the pharmacological properties of the 
NMDA receptor (Ishii et al. 1993; 
Scheetz and Constantine-Paton 
1994). There is evidence that both 
the non-NMDA and the NMDA 
receptor subunits can be phosphory- 
lated by serine-threonine and tyrosine 
kinases, and phosphorylation may 
influence activity and/or localization 
of the receptors in the cell (Tabakoff 
and Hoffman 1996£). 

The characteristics of the NMDA 
receptor-gated channel, including its 
slow activation and permeability to 
calcium, contribute to its involvement 
in learning and memory processes 
(long-term potentiation [LTP]) and 
in neuronal development (Collingridge 
and Lester 1989). When overstimulated, 
the NMDA receptor plays a role in gen- 
erating seizure activity and excitotoxic 



95 



NIAAA's Neuroscience and Behavioral Research Portfolio 



(and possibly apoptotic) neuronal 
death (Choi 1988, 1992). These prop- 
erties of the NMDA receptor, as well 
as the fact that its function is potently 
inhibited by acute ethanol exposure, 
suggest that it might play a role in 
ethanol-induced neuroadaptation. 

Hyperexcitability of the CNS is a 
key component of ethanol withdrawal 
(Tabakoff and Rothstein 1983). Both 
a reduction in GABA-mediated inhi- 
bition and a supersensitive NMDA 
response may be involved. One of the 
earliest findings suggesting that chronic 
ethanol exposure produces up-regula- 
tion of glutamate receptors was an 
increase in [ 3 H]glutamate binding 
reported in hippocampus of human 
alcoholics (Michaelis et al. 1990). A 
more recent postmortem study of 
human alcoholics found an increase in 
NMDA-sensitive glutamate binding, 
and in binding of an NMDA receptor 
antagonist, in frontal cortex (Freund 
and Anderson 1996). Hoffman's labora- 
tory has reported increases in the den- 
sity of NMDA receptors in C57BL/6 
mice treated chronically with a 7 per- 
cent ethanol liquid diet. Seven days of 
chronic ethanol ingestion, leading to 
functional tolerance to and physical 
dependence on ethanol, led to signifi- 
cantly increased [ 3 H]MK-801 binding 
in hippocampal membranes (Grant et 
al. 1990). These animals were depen- 
dent on ethanol, as indicated by mea- 
surement of withdrawal seizures. An 
autoradiographic study using the same 
ethanol administration paradigm also 
reported increased [ 3 H]MK-801 
binding in cortex, hippocampus, and 
striatum (Gulya et al. 1991). Extensions 
of these experiments with membrane 



binding techniques found significant 
increases in MK-801 binding only in 
hippocampus, but not cerebral cortex 
(Snell et al. 1993). These studies 
found that both [ 3 H]MK-801 and 
NMDA- specific [ 3 H] glutamate binding 
were significantly increased in hip- 
pocampus by chronic ethanol treatment, 
but there were no changes in binding 
of [ 3 H]glycine or [ 3 H]CGS19755, a 
competitive NMDA receptor antago- 
nist. Sanna and colleagues (1993) also 
found a significant increase in MK- 
801 binding in hippocampal tissue of 
rats given ethanol for 6 days; the rats 
showed withdrawal signs upon cessa- 
tion of ethanol treatment. 

In contrast, there have been a few 
studies in which increases in NMDA 
receptor binding were not observed 
after chronic ethanol exposure. Carter 
and colleagues (1995) saw no changes 
in MK-801 binding in brains of mice 
bred selectively for differences in sus- 
ceptibility to ethanol withdrawal seizures 
(withdrawal seizure prone [WSP] and 
withdrawal seizure resistant [WSR] 
mice) following 24 hours of ethanol 
exposure, although handling-induced 
withdrawal seizures could be observed 
in these animals. This study contrasts 
with earlier work using WSP and WSR 
mice, in which the WSP mice were 
reported to have a higher density of 
MK-801 binding sites in hippocampus 
than WSR mice, and in which chronic 
(7 days) ingestion of ethanol in a liq- 
uid diet increased MK-801 binding in 
hippocampus of both lines of mice 
(Valverius et al. 1990). Differences 
between these studies include differ- 
ent durations and methods of ethanol 
exposure. In particular, in the study of 



96 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



Carter and colleagues, mice were 
exposed to ethanol vapor by inhalation 
and were given pyrazole to retard ethanol 
metabolism. Pyrazole interacts with 
the NMDA receptor (Pereira et al. 
1992) and may, therefore, confound 
the results. Another difference is that, 
with the method used in the study by 
Valverius and colleagues, both spon- 
taneous and handling-induced with- 
drawal seizures are observed; that is, 
withdrawal symptomatology is more 
severe in this paradigm. 

Rudolph and colleagues (1997) 
reported no changes or only very small, 
but significant, increases in MK-801 
binding or binding of other ligands to 
the NMDA receptor in brains of rats 
that were treated chronically with 
ethanol by a number of different meth- 
ods. Although some of these methods 
have previously been reported to pro- 
duce physical dependence in the rats, 
no measures of withdrawal signs or 
symptoms were included in this study. 

Alterations in ligand binding may 
reflect changes in NMDA receptor 
subunit composition, and there have 
also been several investigations of 
NMDA receptor subunit expression in 
brain following chronic ethanol expo- 
sure. Trevisan and colleagues (1994) 
found that 12 weeks of ingestion of 
an ethanol-containing liquid diet by 
rats resulted in an increase in the level 
of NR1 immunoreactivity in the hip- 
pocampus, but not in the cortex, stria- 
tum, or nucleus accumbens. Long-term 
treatment of rats with ethanol (12 
weeks) was also found to be required 
to increase NRI immunoreactivity in 
the ventral tegmental area (VTA), 
whereas 1 and 6 weeks of chronic 5 



percent ethanol liquid diet were not 
sufficient (Ortiz et al. 1995). Interest- 
ingly, studies of the levels of mRNA 
for NMDA receptor subunits have 
indicated that chronic ethanol treat- 
ment of rats (by repeated gavage for 
several days) does not change NRI 
mRNA, but increases NR2A and 
NR2B mRNA levels in hippocampus 
and cortex (Follesa and Ticku 1995). In 
contrast, Snell and colleagues (1996#) 
found that ingestion of an ethanol- 
containing liquid diet by C57BL/6 
mice for 7 days resulted in an increase 
in NRI and NR2A proteins in several 
brain areas, with no change in mRNA 
levels. They suggested that the increase 
in these two receptor subunits in hippo- 
campus was consistent with their pre- 
vious finding of an increase in MK-801 
binding in this brain region. Since 
MK-801 binding involves both an 
NRI and NR2 subunit, an increase in 
binding could be due to changes in 
the subunit expression (presumably 
receptor subunit stoichiometry), with- 
out necessarily an increase in the den- 
sity of receptors. 

A factor that may influence changes 
in NMDA receptor properties following 
chronic ethanol treatment is suggested 
by the fact that both stress and treat- 
ment with glucocorticoids have been 
shown to increase NMDA receptor 
binding in a manner similar to ethanol 
treatment (Yoneda et al. 1994; Tabakoff 
and Hoffman 1996&). Since ethanol 
increases glucocorticoids and is a stres- 
sor, it is possible that stress-induced 
glucocorticoids may play a role in 
chronic ethanol-induced increases in 
NMDA receptor binding in brain. The 
degree of stress induced by different 



97 



NIAAA's Neuroscience and Behavioral Research Portfolio 



ethanol administration paradigms 
could then influence the results. 

For the most part, the literature 
supports the view that chronic ethanol 
administration resulting in physical 
dependence and withdrawal convulsions 
is accompanied by an up-regulation of 
NMDA receptors and/or increased 
expression of NMDA receptor subunit 
proteins in various brain areas. Little 
or no work has yet been done to deter- 
mine if these changes in NMDA 
receptor properties are reflected in 
receptor function in the adult brain. 
However, a number of studies of chronic 
ethanol effects on NMDA receptor 
properties in neuronal culture have 
been performed. Chronic exposure of 
primary cultures of cerebellar granule 
neurons or cerebral cortical neurons 
to ethanol (e.g., 100 mM ethanol for 
3 days) resulted in enhanced NMDA- 
stimulated increases in intracellular 
Ca 2+ (Iorio et al. 1992; Ahern et al. 
1994), as well as increased NMDA- 
stimulated nitric oxide formation 
(Chandler et al. 1997). Dizocilpine 
binding was also increased in intact 
cerebellar granule neurons that had 
been treated chronically with ethanol, 
indicating an increase in NMDA 
receptor number after this treatment 
(Hoffman et al. 1995). Increases in 
expression of NMDA receptor subunits 
have also been reported. In cerebellar 
granule neurons, chronic ethanol treat- 
ment produced a small increase in NRI 
protein and a decrease in NR2A pro- 
tein, with no change in mRNA for 
either subunit. The same treatment 
produced larger increases in the gluta- 
mate binding protein (mRNA and 
protein levels), which has been sug- 



gested to be a component of a complex 
of proteins that has ligand binding 
sites characteristic of NMDA receptors 
(Hoffman et al. 1996). In primary cul- 
tures of cerebral cortical cells, chronic 
ethanol exposure (50 mM, 5 days) 
increased the mRNA level for the NR2B 
subunit and increased the expression 
of NRI and NR2B proteins (Follesa 
and Ticku 1996; Hu et al. 1996). In 
HEK 293 cells transfected with 
NMDA receptor subunits, chronic 
ethanol treatment (50 mM or greater 
for 24 hours) did not alter the expres- 
sion of any receptor subunit but 
changed the sensitivity of the recep- 
tors to ifenprodil, a ligand that is 
selective for receptors containing the 
NR2B subunit (Blevins et al. 1997). 

Caution must be used in extrapolat- 
ing the results seen in primary cultures 
of neurons to the situation in the 
adult brain, since the neurons in culture 
are undergoing development. The 
mechanism by which ethanol induces 
NMDA receptor changes in the cultures 
may be different from the mechanism 
in the adult brain, even though the 
receptor changes themselves may appear 
to be similar. The results obtained 
with recombinant receptors are even 
more problematic, because the receptor 
subunits are overexpressed, and both 
regulation of receptor expression and 
receptor stoichiometry are likely to be 
very different from native receptors. 

Nonetheless, the consequences of 
NMDA receptor up-regulation caused 
by chronic ethanol exposure can be 
readily measured in the cell culture 
models. Ethanol-exposed cerebellar 
granule neurons and cerebral cortical 
neurons display enhanced sensitivity 



98 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



to glutamate- induced excitotoxicity 
(Chandler et al. 1993#; Crews and 
Chandler 1993; Iorio et al. 1993; 
Ahern et al. 1994). It is likely that this 
increased sensitivity to neurotoxic dam- 
age is a consequence of ethanol with- 
drawal, because ethanol, while present 
in the cellular milieu, inhibits NMDA 
receptor function and blocks neuro- 
toxicity (Takadera et al. 1990; Chandler 
et al. 1993^). Enhanced susceptibility 
to glutamate receptor-mediated neu- 
rotoxicity has also been reported in 
rats that were exposed chronically to 
ethanol by inhalation and then 
injected intrahippocampally with 
NMDA (Davidson et al. 1993). This 
ethanol withdrawal-induced increase in 
susceptibility to glutamate excitotoxi- 
city may represent the basis for the 
observation of neuronal damage in 
alcoholics (Charness 1993). 

In the intact animal, another con- 
sequence of NMDA receptor up- 
regulation may be the generation of 
alcohol withdrawal seizures and/or 
convulsions. Competitive and non- 
competitive antagonists of the NMDA 
receptor can reduce ethanol withdrawal 
convulsions in mice and rats (Grant et 
al. 1990; Morrisett et al. 1990; Liljequist 
1991; Kotlinska and Liljequist 1996). 
Furthermore, the time course for the 
increase in hippocampal dizocilpine 
binding sites in mice treated chroni- 
cally with ethanol paralleled the time 
course for appearance of ethanol with- 
drawal convulsions (Gulya et al. 
1991). Tremwel and colleagues 
(1994&) reported no change in dizo- 
cilpine binding at 48 hours after ethanol 
withdrawal, a time when overt with- 
drawal signs have dissipated, consistent 



with the hypothesis that NMDA recep- 
tor up-regulation is a transient phe- 
nomenon that contributes to withdrawal 
seizures and convulsions. Snell and 
colleagues (1996&) reported that gan- 
glioside treatment of mice during 
chronic ethanol exposure resulted in a 
significant attenuation of withdrawal 
seizures and blocked the up-regula- 
tion of NMDA receptors (MK-801 
binding) in the hippocampus. The 
interpretation of these findings was 
that ganglioside treatment may have 
prevented the development of physi- 
cal dependence in the mice. All of 
these studies are consistent with the 
hypothesis that an increase in NMDA 
receptor number and/or function 
plays a role in the generation of ethanol 
withdrawal signs, specifically seizures 
and convulsions (i.e., ethanol with- 
drawal hyperexcitability). Electrophys- 
iological studies with hippocampal slices 
obtained from mice treated chronically 
with ethanol also indicated the pres- 
ence of an enhanced NMDA recep- 
tor-mediated component of synaptic 
excitation during ethanol withdrawal 
(Whittington et al. 1995). 

NMDA receptor up-regulation may 
also contribute to changes in dopamine 
release following chronic ethanol 
administration, which could, in turn, 
be associated with altered reinforcing 
effects of ethanol. During ethanol 
withdrawal, the firing rates and number 
of spontaneously firing dopaminergic 
neurons in the VTA are significantly 
reduced, and the release of dopamine 
in the nucleus accumbens of rats 
undergoing withdrawal is diminished 
(Diana et al. 1993). This decreased 
release of dopamine could be reversed 



99 



NIAAA's Neuroscience and Behavioral Research Portfolio 



by administration of dizocilpine (MK- 
801). That is, the up-regulation of 
NMDA receptors during ethanol with- 
drawal may increase the level of tonic 
inhibition of dopamine release by glu- 
tamate (Imperato et al. 1990; Tabakoff 
and Hoffman 1996#), leading to 
changes in dopaminergic function that 
could influence ethanol intake. 

The data reviewed suggest that 
changes in NMDA receptor function 
induced by chronic ethanol exposure 
can contribute to ethanol withdrawal 
hyperexcitability and withdrawal- 
induced neurotoxicity. These findings 
suggest the possibility of developing 
therapeutic agents that would not 
only reduce ethanol withdrawal signs 
but could also reduce the neuronal 
damage seen in chronic alcoholics. 

An investigation by Miyakawa and 
colleagues (1997) also suggests the pos- 
sibility that acute ethanol tolerance may 
be related to NMDA receptor function. 
Mice that lacked Fyn, a tyrosine kinase, 
were found to be more sensitive to the 
hypnotic effect of ethanol than wild- 
type mice. Furthermore, "acute toler- 
ance" to ethanol inhibition of NMDA 
receptor responses in hippocampal slices 
developed in control mice, but not in 
the Fyn-deficient mice. These findings 
suggest that posttranslational modifi- 
cation of the NMDA receptor could 
contribute to short-term tolerance to 
ethanol effects (i.e., the NMDA recep- 
tor might represent an "intrinsic system" 
for some aspect of acute tolerance to 
ethanol), but behavioral measures of 
acute tolerance to ethanol were not 
included in this study. 

Several studies have suggested that 
chronic ethanol exposure may also 



modify AMPA receptor properties. 
Although 12 weeks of ethanol liquid 
diet ingestion did not increase GluPvl or 
GluR2 in hippocampus (Trevisan et 
al. 1994), this treatment did increase 
GluRl immunoreactivity in the VTA 
and substantia nigra (Ortiz et al. 
1995). Chronic oral ethanol exposure 
(20 percent v/v, 28 weeks) increased 
GluR3 subunit mRNA in hippocampus 
by 15 to 30 percent, while GluRl and 
GluR2 subunit mRNAs were unaltered 
(Buckner et al. 1997). Furthermore, 
Breese and colleagues (1995) found 
significant increases in GluR2 and 
GluR3 subunit immunoreactivity in 
human postmortem hippocampal tis- 
sue of patients with alcohol abuse his- 
tories. Thus, there is accumulating 
evidence that changes in AMPA recep- 
tors may also occur following chronic 
ethanol exposure. 

GABA A Receptors. GABA is the 
most ubiquitous inhibitory neuro- 
transmitter in the brain. It interacts 
with a family of receptors containing 
recognition sites for the anxiolytic 
and sedative benzodiazepines, barbi- 
turates, and endogenous neuro- 
steroids. These binding sites are linked 
allosterically to a GABA recognition 
site, and each site is involved directly 
or indirectly in the gating properties 
of integral CI" channels. GABA 
receptor-mediated activation of CI" 
conductance results in membrane 
hyperpolarization and decreased neu- 
ronal excitability (Skolnick and Paul 
1982). Ethanol acutely alters the gating 
properties of this receptor complex; 
however, ethanol binds with little or 
no affinity to recognition sites for 
GABA, benzodiazepines, barbiturates, 



100 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



and cage convulsants on GABA A 
receptors (Davis and Ticku 1981). 

Chronic ethanol treatment, using 
paradigms that are known to produce 
tolerance and physical dependence, alters 
many of the properties of GABA A recep- 
tors in brain (table 1). Chronic ethanol 
exposure is associated with a decrease 
in the sensitivity of GABA A receptor- 
mediated responses in cerebral cortex 
(Morrow et al. 1988; Sanna et al. 1993), 
nucleus accumbens (Szmigielski et al. 
1992), spinal cord cultured neurons 
(Mehta and Ticku 1988; Ticku 1989), 
and medial septal nucleus (Criswell et 
al. 1993). In cerebral cortex, muscimol- 
or phenobarbital-stimulated CI" uptake 
is decreased following chronic ethanol 
inhalation (Morrow et al. 1988). The 
ability of ethanol to potentiate GABA 
or muscimol-stimulated Cl~ uptake is 
also lost following chronic ethanol 
administration in both cortex and 
cerebellum (Allan and Harris 1987; 
Morrow et al. 1988; Sanna et al. 
1993). Benzodiazepine enhancement 
of muscimol-stimulated chloride flux 
is reduced in the cerebral cortex of 
mouse microsacs, while the functional 
efficacy of inverse agonists is enhanced 
(Mehta and Ticku 1989; Buck and 
Harris 1990). In contrast, potentia- 
tion of Cl~ uptake by the neuroactive 
steroids 3a,5a-tetrahydroproges- 
terone (THP) and tetrahydrodeoxy- 
corticosterone (THDOC) is enhanced 
in ethanol-dependent rats (Devaud et 
al. 1996). 

While behavioral and functional data 
clearly suggest that chronic ethanol 
administration alters GABA A receptor 
function, data from radioligand binding 
studies do not provide an explanation 



for these effects. No consistent alter- 
ations in GABA A receptor recognition 
sites have been observed (see table 1). 
Therefore, chronic ethanol administra- 
tion induces functional tolerance of 
GABA A receptors without reducing 
the total number of GABA-gated 
chloride channels. This apparent paradox 
has driven researchers to identify alter- 
native mechanistic explanations for these 
phenomena. Many studies have shown 
that chronic ethanol administration alters 
the expression of various GABA A 
receptor subunits, suggesting that alter- 
ations in GABA A receptor expression 
may account for alterations in GABA A 
receptor function (Morrow et al. 1990; 
Buck et al. 1991; Montpied et al. 
1991; Mhatre and Ticku 1992; Mor- 
row et al. 1992; Mhatre et al. 1993; 
Devaud et al. 1995^, 1996, 1997). 

Chronic ethanol administration dif- 
ferentially alters the expression of distinct 
GABA A receptor subunit mRNAs in 
the cerebral cortex (Morrow et al. 1990; 
Montpied et al. 1991; Devaud et al. 
1995^) and cerebellum (Mhatre and 
Ticku 1992; Morrow et al. 1992). The 
levels of GABA A receptor al subunit 
mRNAs and peptides are reduced, 
whereas a4 subunit mRNAs and pep- 
tides are increased by approximately 
equal amounts in cerebral cortex 
(Devaud et al. 1995&, 1997). In the 
cerebellum, decreases in GABA A 
receptor al subunit mRNA and increases 
in a6 subunit mRNA levels are found 
(Mhatre and Ticku 1992; Morrow et 
al. 1992). These changes in subunit 
expression suggest that alterations in the 
assembly of GABA A receptors could 
account for the observed changes in 
receptor function and binding. For 



101 



NIAAA's Neuroscience and Behavioral Research Portfolio 



example, the increases in a4 and ct6 
subunit expression could explain the 
increases in [ 3 H]Ro 15-4513 binding 
(Mhatre et al. 1988) and inverse agonist 



sensitivity (Mhatre et al. 1988; Buck 
and Harris 1990) following chronic 
ethanol administration. The increased 
expression of ct4 subunits may underlie 



Table 1. Effects of Chronic Ethanol Administration on 


GABA A Receptor Function, Recognition 


Receptor Property 


Alteration 


GAB A- mediated CI" channel function 3 


Decreased 


GABA-mediated CI" channel function 


No change 


Phenobarbital- mediated CI" flux 3 


Decreased 


Ethanol-enhanced CI" flux 


Abolished 


Benzodiazepine -enhanced CI" flux c 


Decreased 


Inverse agonist modulation 


Increased 


Neuroactive steroid modulation 3 


Increased 


High -affinity [ 3 H] muscimol binding*-' 


No change 


Low-affinity [ 3 H]muscimol binding d 


Decreased 


[ 3 H]flunitrazepam (flu) binding 


No change 


[ 35 S]TBPS binding 


No change or increased 


GABA enhancement of [ 3 H]flu binding 01 


Decreased 


[ 3 H]zolpidem binding 


Increased or no change 


[ 3 H]Ro 15-4513 binding 


Increased 


al Subunit mRNA and peptides 


Decreased 


a2 Subunit mRNA and peptides 3 


Decreased 


a3 Subunit mRNA and peptides 3 


No change or decreased 


a4 Subunit mRNA and peptides 3 


Increased 


a5 Subunit mRNAs 3 


No change 


a6 Subunit mRNA and peptides b 


Increased 


61 Subunit mRNA and peptides 3 


No change or increased 


62 Subunit mRNA and peptides 


No change or increased 


63 Subunit mRNA and peptides 3 


No change or increased 


yl Subunit mRNA and peptides 3 


Increased 


y2S Subunit mRNA 3 


Increased 


y2L Subunit mRNA 3 


No change 


y2 Subunit peptides 3 


No change 


y3 Subunit mRNA 3 


No change 


8 Subunit mRNA 3 


No change 


Note: TBPS = tert-butyl-bicyclophosphorothionate. 




"Cerebral cortex. 




b Cerebellum. 




c Cerebral cortex and cerebellum. 




"Whole brain. 





102 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



the reduced sensitivity to GABA receptors with a4^2y2 subunits are 

(Morrow et al. 1988) and benzodi- less sensitive to GABA agonists and 

azepine agonists (Buck and Harris benzodiazepines than al(32y2 recep- 

1990), since recombinant GABA tors (Whittemore et al. 1996). 



Sites, and Subunit Expression in Cerebral Cortex and Cerebellum. 



Source 



Martz et al. 1983; Gonzalez and Czachura 1989; 

Criswell et al. 1993; Sanna et al. 1993; Devaud et al. 1996 

Frye et al. 1983; Allan and Harris 1987; Buck and Harris 1990 

Morrow et al. 1988 

Allan and Harris 1987; Morrow et al. 1988 

Buck and Harris 1990 

Mehta and Ticku 1989; Buck and Harris 1990 

Devaud et al. 1996 

Volicer 1980; Volicer and Biagioni 1982 

Ticku and Burch 1980; Unwin and Taberner 1980 

Karobath et al. 1980; Volicer and Biagioni 1982; Rastogi et al. 1986 

Thyagarajan and Ticku 1985; Rastogi et al. 1986; Sanna et al. 1993 

DeVries et al. 1987 

Devaud et al. 1995a, 19956 

Mhatre et al. 1988 

Morrow et al. 1990; Montpied et al. 1991; Mhatre and Ticku 1992; 

Morrow et al. 1992; Mhatre and Ticku 1993; Mhatre et al. 1993; 

Devaud et al. 19956, 1997 

Morrow et al. 1990; Montpied et al. 1991; Mhatre et al. 1993; 

Mhatre and Ticku 1994# 

Morrow et al. 1990; Montpied et al. 1991; Mhatre et al. 1993; 

Mhatre and Ticku 1994« 

Devaud et al. 19956, 1997 

Devaud et al. 19956 

Mhatre and Ticku 1992; Morrow et al. 1992; Mhatre and Ticku 19946 

Mhatre and Ticku 19946; Devaud et al. 19956 

Morrow et al. 1992; Devaud et al. 19956, 1997 

Devaud et al. 19956, 1997 

Mhatre and Ticku 19946; Devaud et al. 19956, 1997 

Devaud et al. 19956 

Devaud et al. 19956 

Devaud et al. 1997 

Devaud et al. 19956 

Devaud et al. 19956 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



Ethanol-dependent and -withdrawn 
rats are also sensitized to the anticon- 
vulsant effects of the neurosteroid 
3a,5a-THP (Devaud et al. 1995a, 
1996). This effect may be related to 
the increase in |3 and yl subunit mRNAs 
and peptides following chronic ethanol 
exposure (Mhatre et al. 1993; Mhatre 
and Ticku 1993; Devaud et al. 1995£, 
1996, 1997), because homologous (3 
subunit expression is sufficient for 
neurosteroid potentiation of GABA 
responses (Puia et al. 1990) and yl 
subunits enhance neurosteroid sensitiv- 
ity in recombinant expression studies 
(Puia et al. 1993). 

At the peak of ethanol withdrawal, 
GABA A receptor subunit mRNA levels 
appear to be in a state of flux, whereas 
GABA A receptor subunit peptide levels 
exhibit more stable changes. GABA A 
receptor al, a4, and yl subunit mRNAs 
return nearly to control levels, but (32 
and (33 subunit mRNA levels increase, 
compared with both control and 
dependent rats (Devaud et al. 1996). 
At this time point, 6-8 hours after 
removal of ethanol, GABA A receptor 
protein expression remains similar to 
that found in ethanol-dependent rats; 
withdrawn animals show reduced levels 
of al protein and elevated levels of a4, 
(32/3, and yl protein relative to pair-fed 
controls (Devaud et al. 1997). Thus, 
changes in GABA A receptor subunit 
mRNA expression are dynamic and 
reflect the rapidly changing state of CNS 
excitability. In contrast to the changes 
in mRNA levels, changes in GABA A 
receptor subunit peptides may reflect 
the long-term changes associated with 
ethanol dependence and addiction. 
Clearly, the measurement of peptide 



expression is important for this reason 
and should represent an important 
mechanism of adaptation to chronic 
ethanol consumption. 

Recent evidence suggests that ethanol 
modulates promoter activity for GABA A 
receptor a and (3 subunits in vitro 
(Russek et al. 1997; S.J. Russek and 
D.H. Farb, personal communication, 
June 1998). Human GABA A receptor 
promoter regions were cloned and 
transfected in cultured embryonic 
neurons that were exposed to ethanol. 
Although the data are still preliminary, 
it appears that chronic ethanol exposure 
decreases human GABA A receptor al 
subunit promoter activity and increases 
GABA A receptor (3 subunit promoter 
activity (Russek et al. 1997). This work 
may identify mechanisms of regulation 
of GABA A receptor genes by ethanol. 

Alterations in GABA A receptor func- 
tion and gene expression are regionally 
as well as temporally dependent. Chronic 
ethanol exposure differentially alters 
GABA A receptor subunit expression 
in the hippocampus compared with 
the cerebral cortex. For example, 
chronic ethanol consumption for 40 
days (Matthews et al. 1998) or 60 
bouts of chronic, intermittent exposure 
(Mahmoudi et al. 1997) increase a4 
subunit expression in hippocampus. 
However, shorter duration treatments 
with ethanol (14 days) do not alter a4 
subunit peptides. In contrast, a4 sub- 
unit peptide levels are significantly 
increased in the cerebral cortex fol- 
lowing both 40 and 14 days of ethanol 
consumption (Devaud et al. 1997). 
The level of hippocampal al subunit 
peptide is not altered following 14 
days, 28 days (Charlton et al. 1997), 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



or 40 days (Matthews et al. 1998) of 
chronic ethanol consumption. How- 
ever, a significant decrease in the al 
subunit peptide level is found in the 
cerebral cortex of the same animals 
following both 14 (Devaud et al. 

1997) and 40 days (Matthews et al. 

1998) of chronic ethanol consumption. 
Finally, GABA A receptor (32/3 subunit 
peptide expression is not altered in the 
hippocampus following either period 
of ethanol consumption, although 
GABA A receptor (32/3 subunit expres- 
sion was previously shown to increase 
in the cerebral cortex of the same ani- 
mals (Devaud et al. 1997; Matthews 
et al. 1998). These findings suggest that 
GABA A receptor gene expression is 
differentially regulated by ethanol in the 
hippocampus compared with the cerebral 
cortex. Therefore, ethanol regulation 
of GABA A receptor gene expression 
varies across brain regions. 

Alterations in native GABA A recep- 
tor subunit assembly in vivo may be 
an adaptation elicited by chronic drug 
exposure (Morrow et al. 1992; Devaud 
et al. 1995 b). An alteration in subunit 
assembly could confer alterations of 
functional properties of receptors, 
with no change in the total number of 
receptors expressed. This mechanism 
may play a role in neuroadaptations to 
chronic exposure of drugs other than 
ethanol that modulate GABA A receptors 
(Morrow 1995). There is ample evidence 
for alterations in GABA A receptor sub- 
unit composition during development 
(see Morrow 1995 for review), suggest- 
ing that changes in subunit expression 
may be an endogenous regulatory 
mechanism controlling the activity of 
GABA A receptors. Similarly, alterations 



in subunit expression of nicotinic 
cholinergic (Mishina et al. 1986), gluta- 
mate (Sheng et al. 1994), and glycine 
receptors (Malosio et al. 1991) are 
also observed during neuronal devel- 
opment. Therefore, we propose that 
ligand-gated ion channels may be sub- 
ject to alterations in subunit assembly 
that serve to modulate receptor func- 
tion in vivo. 

Other evidence suggests that chronic 
ethanol administration may result in 
the functional uncoupling of GABA 
and benzodiazepine recognition sites 
(DeVries et al. 1987; Klein et al., 
1995#), independent of alterations in 
GABA A receptor gene expression 
(Klein et al. 1995&). Another possible 
mechanism mediating neuroadaptations 
of GABA A receptors following chronic 
ethanol exposure involves internalization 
of the receptor complex. Although there 
is some evidence that GABA receptors 
can be internalized (Calkin and Barnes 
1994), this would explain the loss of 
GABA A receptor function without 
altering receptor number only if the 
receptors remain attached to the 
membrane and remain detectable in 
radioligand binding studies. It is also 
possible that ethanol affects the stoi- 
chiometry of GABA A receptors. How- 
ever, the stoichiometry of native GABA A 
receptors is undetermined. Likewise, a 
dissociation of subunits could account 
for decrements in GABA A receptor 
function while preserving receptor 
number, yet be nearly impossible to 
detect using currently available tech- 
niques. Conformational changes in 
receptor structure are another potential 
adaptation. Although this mechanism 
is more suited to explain rapid tolerance, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



recent data narrowing the site of ethanol 
action to the second transmembrane 
domain of GABA A receptors (Mihic et 
al. 1997) suggest that ethanol may affect 
the conformation of GABA A receptors 
as they reside in the membrane. 

The tertiary structure of proteins is 
profoundly affected by posttranslational 
modifications, such as phosphorylation 
and glycosylation. Several lines of con- 
vergent data support the suggestion 
that phosphorylation or dephosphory- 
lation of GABA A receptors may play a 
role in the development of ethanol 
tolerance and dependence. Most 
major known kinases can phosphorylate 
GABA A receptor subunits (most often 
the (31) in vitro (Browning et al. 1990; 
Porter et al. 1990; Moss et al. 1995). 
Furthermore, phosphorylation of GABA A 
receptors has been shown to modulate 
receptor function. PKC and PKA phos- 
phorylation of GABA A receptors reduces 
receptor activation (Kellenberger et al. 
1992; Leidenheimer et al. 1992; 
Krishek et al. 1994), whereas phos- 
phorylation by Ca ++ /calmodulin- 
dependent protein kinase II or 
tyrosine kinase enhances GABA A 
receptor function (Valenzuela et al. 
1995; Wang et al. 1995). 

Several studies have provided evi- 
dence for the suggestion that post- 
translational modifications may underlie 
changes in GABA A receptor function 
following ethanol administration. As 
discussed previously, acute ethanol 
administration induces changes in 
GABA A receptor function that may be 
dependent on phosphorylation of par- 
ticular proteins. Prolonged ethanol 
exposure might be expected to result in 
long-term changes in second messenger 



systems, including kinase activity. The 
heterogeneity of GABA A receptors 
expressed in vivo has precluded defini- 
tively answering this question. Expres- 
sion systems with stably expressed, 
functional GABA A receptors under 
the control of an inducible promoter 
are promising model systems. When 
chronically treated with ethanol, Ltk- 
cells show changes in GABA A receptor 
function that cannot be explained by 
changes in gene expression, but could 
be explained by posttranslational reg- 
ulation such as phosphorylation (Klein 
et al. 19956; Harris et al. 1997). How- 
ever, none of these studies has directly 
demonstrated that phosphorylation is 
involved in ethanol modulation of 
GABA A receptor function. 

Chronic ethanol administration alters 
PKC and PKA activity in neuronal cells 
(for a review, see Diamond and Gordon 
1997), and these alterations have been 
linked to the development of cellular 
tolerance to the effects of ethanol on 
adenosine uptake (Coe et al. 1996). 
The proposed mechanism for this 
effect involves compartmentalization 
of the kinase to specific regions of the 
cell (Dohrman et al. 1996). Interest- 
ingly, the PKC -activating effects of 
ethanol on GABA A receptors are 
found only in desensitized receptors 
(Leidenheimer et al. 1992), suggesting 
that the involvement of phosphorylation 
in ethanol modulation of GABA A 
receptors may be state dependent. Alter- 
natively, the functional consequences 
of phosphorylation may be dependent 
on the subunit composition (Krishek 
et al. 1994) and fine-tuning of func- 
tion may be achieved by selective com- 
partmentalization of either GABA A 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



receptors or kinases. Kinase activation/ 
inactivation may be involved in the 
changes in gene expression observed 
after chronic ethanol exposure. As 
mentioned, preliminary evidence sug- 
gests that ethanol modulates promoter 
activity for GABA A receptor a and (3 
subunits (Russek et al. 1997; S.J. 
Russek and D.H. Farb, personal com- 
munication, June 1998). In addition, 
altered subunit assembly of GABA A 
receptors may involve induction of chap- 
erone proteins, whose putative cellular 
role is trafficking of proteins between 
organelles, thereby controlling receptor 
assembly (Haas 1994). In support of 
this possibility, chronic ethanol expo- 
sure up -regulates expression of certain 
chaperones in neural cell cultures 
(Hsieh et al. 1996). Thus, it is con- 
ceivable that certain ethanol-sensitive 



chaperones play a role in altering 
GABA A receptor subunit assembly fol- 
lowing chronic ethanol exposure. The 
exact mechanisms that account for 
alterations in GABA A receptor func- 
tion following chronic ethanol admin- 
istration remain under avid 
investigation. These mechanisms may 
ultimately encompass several of the 
possibilities described in figure 1 . 

As a caveat to the proposed postsy- 
naptic mechanisms, it is also possible 
that ethanol tolerance and depen- 
dence involve alterations in presynap- 
tic processes. Early studies suggested 
that chronic ethanol administration 
did not affect GAB A turnover (Hunt 
and Majchrowicz 1983). However, 
more recent studies using in vivo micro- 
dialysis sampling suggest that chronic 
ethanol treatment increases basal GABA 



Altered receptor assembly 



Post-translational 
modifications 



Altered stoichiometry 



Dissociation of subunits 




Figure 1. Possible mechanisms of GABA A receptor regulation. ER = endoplasmic reticulum; 
EtOH = ethanol; P0 4 = phosphate. Adapted from Grobin, A.C.; Matthews, O.B.; Devaud, 
L.L.; and Morrow, A.L. The role of GABA A receptors in the acute and chronic effects of 
ethanol. Psycho-pharmacology (Berl) 139:2-19, 1998. 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



levels (Dahchour et al. 1996). Since 
multiple GABA transporters have now 
been cloned, the hypothesis that 
ethanol modulates GABA neurotrans- 
mission via altering GABA release or 
uptake merits reinvestigation with 
modern molecular techniques. 

Genetic models have also implicated 
GABA A receptors in ethanol tolerance 
and dependence. Animals selected for 
differences in ethanol sensitivity or 
withdrawal show differences in GABA A 
receptor function. Mice selected for 
differential sensitivity to the hypnotic 
effects of ethanol display corresponding 
differences in the ataxic effects of GABA A 
receptor agonists (Martz et al. 1983). 
Cerebral microsacs prepared from mice 
selected for high sensitivity to the hyp- 
notic effects of ethanol show ethanol 
potentiation of muscimol stimulated 
Cl~ flux, whereas mice insensitive to 
the hypnotic effects of ethanol do not 
(Allan and Harris 1986). WSP and 
WSR mice show differential expression 
of GABA A receptor subunit mRNAs 
(Keir and Morrow 1994) and diver- 
gent changes in GABA A receptor sub- 
unit mRNA levels after chronic ethanol 
treatment (Buck et al. 1991). Thus, 
genetic models of ethanol dependence 
selected for divergent behavioral respon- 
ses to ethanol support the hypothesis 
that GABA A receptors underlie the neu- 
roadaptations promulgated by ethanol 
(see Crabbe et al. 1994 for review). 

Inbred mouse strains (such as the 
BXD recombinant inbreds) have been 
used to correlate the magnitude of acute 
ethanol withdrawal severity with bar- 
biturate withdrawal, suggesting that 
common genes may influence with- 
drawal from these two GABAergic drugs 



(Finn and Crabbe 1997). Recent stud- 
ies, using a powerful two-step approach 
to quantitative trait loci (QTL) map- 
ping, have identified loci on murine 
chromosomes 1, 2, 4, and 11 that 
influence alcohol withdrawal severity 
(Buck et al. 1997). Twenty percent of 
the candidate genes put forth in this 
study are related to GABA neuro- 
transmission, including the three genes 
that encode GABA A receptor al, a6, 
and y2 subunits. QTL mapping, along 
with our rapidly expanding knowl- 
edge of mammalian genomes, will 
allow future researchers to identify and 
test candidate genes in genetic linkage 
studies of human and animal popula- 
tions susceptible to the effects of ethanol. 
Taken together, findings from genetic 
animal models add support to the 
suggestion that GABA A receptor 
modulation underlies many of the 
behavioral manifestations of ethanol 
dependence and withdrawal. 

Glycine, 5-Hydroxytryptamine3, and 
Neuronal Nicotinic Acetylcholine Ion 
Channel Receptors. At pharmacologically 
relevant concentrations, ethanol alters 
the function of glycine, 5-hydroxy- 
tryptamine 3 (5-HT 3 ), and nicotinic 
acetylcholine (nACh) receptors, 
although the physiological consequences 
of these actions are largely unknown. 
Ethanol potentiates glycine receptor- 
mediated CI" currents in spinal cord 
neurons (Celentano et al. 1988). 
Williams and colleagues (1995) showed 
that intraventricularly administered 
glycine enhances the loss of righting 
reflex produced by ethanol. A similar 
effect could be produced by microin- 
jection of the glycine precursor D-serine. 
Strychnine attenuated the effects of 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



these agents on the loss of righting, indi- 
cating that a strychnine-sensitive glycine 
site was involved. The effect of chronic 
ethanol exposure on glycine receptors 
has not been studied directly. Gonzalez 
(1993) demonstrated there was no 
change in sensitivity to strychnine- 
induced seizures during ethanol with- 
drawal, but the sensitivity of glycine 
receptors was not investigated. Further 
studies are needed to determine the 
role of glycine receptors in ethanol- 
induced hypnosis and to determine 
whether adaptations in these receptors 
are related to tolerance to the hypnotic 
effects of ethanol. 

Ethanol potentiates 5-HT 3 receptors 
in cell culture by increasing the initial 
amplitude of the ion current induced by 
stimulation with serotonin (Lovinger 
and Peoples 1991; Lovinger and White 
1991). This action is dependent on 
the open channel state of the recep- 
tor; if, for example, the channel is in a 
desensitized state, ethanol has no 
effect on ion currents (Sellers et al. 
1992). Little is known about the 
action of ethanol on 5-HT 3 receptors 
in vivo. However, there is increasing 
evidence that 5-HT 3 receptors con- 
tribute to the activating and reinforcing 
effect of ethanol (see Sellers et al. 
1992 for a review). The effects of 
chronic ethanol administration on 5- 
HT 3 receptors have not been studied, 
but it is likely that studies of this area 
would provide insight into the role of 
5-HT 3 receptors in ethanol reinforce- 
ment and perhaps craving. 

Electrophysiological studies have 
demonstrated that ethanol has effects 
on nicotine-evoked responses in brain. 
Mancillas and colleagues (1986) 



reported that ethanol enhanced the 
effect of acetylcholine in hippocampal 
pyramidal cells without affecting 
GABA-mediated inhibition. Criswell 
and colleagues showed that ethanol 
affects responses to nicotine from 
some, but not all, neurons in the sub- 
stantia nigra reticulata and blocks the 
inhibitory response to nicotine on 
some, but not all, nACh receptors in 
the medial septum. Nicotine was 
demonstrated to act on presynaptic 
nACh receptors on medial septal neu- 
rons to facilitate the release of GABA 
(Yang et al. 1996&), an interpretation 
congruous with other data indicating 
that nicotine can have a presynaptic 
action (Schwartz et al. 1984; Wonna- 
cott et al. 1989; McGehee et al. 1995; 
Sershen et al. 1995; Vizi et al. 1995). 
It is known that nicotine-induced 
dopamine release from striatal synap- 
tosomes is blocked by ethanol (Con- 
nolly et al. 1996), whereas ethanol 
does not affect 86 Rb + efflux induced 
by nicotine from thalamic synapto- 
somes (Collins 1996). In addition to 
its action on presynaptic nACh recep- 
tors, ethanol has been demonstrated 
to have an action on postsynaptic 
responses to nicotine (i.e., the nico- 
tine response not blocked by Mg 2+ ). 
In the cerebellum, ethanol blocks the 
nicotine-induced inhibitor)' response 
and enhances the excitatory response 
to nicotine (Yang et al. 1996#). 

The chronic effects of ethanol on 
nACh receptors have not been evalu- 
ated. Cross-tolerance between certain 
physiological effects of chronic nicotine 
and ethanol administration have been 
observed (Collins et al. 1987; Marks 
et al. 1987; Collins et al. 1990; Welzl 



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et al. 1990; deFiebre and Collins 1993). 
Additionally, the degree of nicotine 
tolerance was found to differ between 
long sleep (LS) and short sleep (SS) 
mice bred for differing responses to 
ethanol (Collins et al. 1993; deFiebre 
and Collins 1993). These studies sug- 
gest a change in nACh receptor func- 
tion after chronic ethanol treatment. 
However, further studies are clearly 
needed to establish these effects and 
their physiological significance. 

Voltage-Sensitive Ion Channels 

Voltage-Gated Calcium Channels. 
Voltage-gated calcium channels are 
classified into L-, N-, P-, Q-, and T- 
types based on their electrophysiological 
and pharmacological properties (for a 
review, see Stea et al. 1995). T-type 
calcium channels consist of a variety of 
low voltage-activated channels that 
activate transientiy and are very sensitive 
to changes in resting potential. The L-, 
N-, P-, and Q-types are high volt- 
age-activated channels, which overlap 
considerably in their electrophysiolog- 
ical characteristics. 

Immunoprecipitation studies indi- 
cate that L-type calcium channels are 
heteroligomeric complexes consisting 
of five distinct protein subunits, al, 
a2, (3, y, and 6 (Campbell et al. 1988; 
Catterall et al. 1988). The al subunit 
is the major voltage -sensitive and pore- 
forming subunit; the other four sub- 
units are believed to be ancillary, 
modulatory molecules (Ellinor et al. 
1993). In addition, different classes of 
al subunits have been isolated and 
cloned. Three known al subunits, alS, 
alC, and alD, form L-type channels 
(reviewed in Stea et al. 1995). The 



alC and alD subunits are expressed 
in rat brain, heart, and PC 12 cells. 

Ethanol has been shown to inhibit 
depolarization-induced calcium influx 
through voltage -gated calcium channels 
in synaptosomes and presynaptic nerve 
terminals without altering basal uptake 
(Leslie et al. 1983; Skattebol and 
Rabin 1987; Dildy-Mayfield and Harris 
1995). Ethanol also reduces voltage- 
dependent calcium influx in cultured 
neuronal and PC 12 cells (Messing et al. 
1986; Skattebol and Rabin 1987). Most 
studies have focused on L-type voltage - 
gated calcium channels. Twombly and 
colleagues (1990) found that both T- 
type and L-type voltage-gated calcium 
channels were inhibited by ethanol, 
with the L-type voltage-gated calcium 
channels showing greater inhibition at 
the same concentration of ethanol. 
Further support for the involvement 
of L-type voltage-gated calcium chan- 
nels in ethanol's response comes from 
studies showing that dihydropyridines 
potentiate the acute pharmacological 
effects of ethanol, including ethanol- 
induced hypothermia, motor incoordi- 
nation, and sedation (see Leslie et al. 
1990 for a review). Inhibition of L- 
type voltage-gated calcium channels 
has also been related to ethanol-induced 
decreases in arginine vasopressin (AVP) 
release that contribute to the diuretic 
effect of ethanol (Wang et al. 1991). 

A variety of studies suggest a role for 
voltage -sensitive calcium channel reg- 
ulation in ethanol dependence. The L- 
type calcium channel antagonists, 
nitrendipine, nimodipine, and PN200- 
110, prevent the behavioral signs of 
ethanol withdrawal (handling-induced 
or audiogenic seizures) (Little et al. 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



1986; Littleton et al. 1990; Whittington 
and Little 1991; Watson et al. 1994). 
Extracellular recordings made from 
mouse hippocampal slices prepared after 
chronic treatment with ethanol or 
ethanol and nitrendipine suggest that 
the electrophysiological changes that 
occur during ethanol withdrawal are 
also prevented by L-type calcium 
channel blockers (Whittington and 
Little 1991). Furthermore, Bay K 8644, 
an L-type calcium channel activator, 
increases withdrawal-induced hyperex- 
citability in mouse hippocampal slices 
prepared after chronic ethanol treat- 
ment (Whittington and Little 1993), 
whereas PN200-110, an antagonist, 
decreases withdrawal-induced hyper- 
excitability (Whittington and Little 
1991). Several studies have indicated 
that chronic ethanol treatment increases 
dihydropyridine binding sites in mem- 
branes prepared from whole brain and 
cerebral cortex from mouse and rats 
(Dolin et al. 1987; Dolin and Little 
1989; Whittington et al. 1991). 
Chronic ethanol administration has also 
been shown to increase the functional 
effects of Bay K 8644 — inositol lipid 
breakdown and neurotransmitter release 
(Dolin et al. 1987). These effects on 
dihydropyridine binding and function 
are blocked by calcium channel antag- 
onists (Dolin et al. 1987; Dolin and 
Little 1989; Whittington et al. 1991). 
Clearly, regulation of dihydropyridine 
binding sites is important in the devel- 
opment of ethanol dependence and the 
manifestations of ethanol withdrawal. 
Chronic ethanol exposure has been 
found to produce tolerance to the in 
vitro inhibitory effects of ethanol on 
calcium uptake (Harris and Wood 1980; 



Leslie et al. 1983), but the development 
of tolerance is pronounced in some 
brain areas (e.g., hypothalamus) and 
does not occur in other areas (e.g., 
cerebellum) (Daniell and Leslie 1986). 
Thus, the adaptive changes in voltage- 
gated calcium channels may vary with 
the brain region. 

Interestingly, striking increases in 
dihydropyridine binding are seen in 
heart tissue of ethanol-dependent rats 
(Guppy and Littleton 1994). This may 
be related to the cardiovascular effects 
of alcohol reported in humans. There 
is considerable evidence that chronic 
heavy alcohol consumption is associated 
with cardiovascular disease such as 
cardiomyopathy, hypertension, and 
arrhythmia (e.g., Lands and Zakhari 
1990). Any or all of these afflictions 
could be related to calcium channel 
deregulation caused by alcohol depen- 
dence. Since different calcium channel 
subunits may be expressed in distinct 
tissues (e.g., the heart vs. the brain), 
the differential regulation of various L- 
type channel subunits may have broader 
implications for ethanol pathology 
than just the CNS. 

Cells in culture have been used as in 
vitro models for the study of chronic 
ethanol effects on voltage-sensitive cal- 
cium channels. Increases in L-type cal- 
cium channel binding sites have been 
reported in both adrenal chromaffin 
cells and PC 12 cells (a clonal line of 
neural crest origin) after growing cells 
in 200 mM ethanol for 6 days (Messing 
et al. 1986; Harper et al. 1989; Brennan 
and Littleton 1990, 1991). It is believed 
that such changes in calcium channel 
binding following chronic ethanol expo- 
sure constitute an adaptive response to 



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ethanol, since acute alcohol decreases 
voltage -dependent calcium influx (e.g., 
Harris and Wood 1980; Leslie et al. 
1983; Messing et al. 1986). Although 
the biochemical mechanisms involved 
in this up-regulation of dihydropyridine 
binding sites are as yet unclear, several 
studies implicate the involvement of a 
PKC (Brennan and Littleton 1990; 
Messing et al. 1990, 1991). The 
increase in dihydropyridine binding sites 
can be prevented by inhibitors of pro- 
tein and mRNA synthesis, suggesting 
that transcription of some gene(s) may 
be involved in the response (Harper et 
al. 1989). It has yet to be shown, how- 
ever, that regulation of the genes encod- 
ing calcium channel subunits, per se, is 
a component of the adaptive process. 
Dihydropyridines appear to bind to 
the al subunit of the L-type calcium 
channel. However, it is unknown how 
changes in dihydropyridine binding 
relate to changes in a 1 or other subunit 
protein or mRNA. Also, since no lig- 
ands exist that specifically bind to other 
L-type channel subunits, it is unknown 
whether other subunits are changed 
following chronic ethanol exposure. 

Innate differences in susceptibility 
to dependence and withdrawal have 
been demonstrated (e.g., WSP and 
WSR mouse strains [Crabbe et al. 
1983]). Differences in the effect of 
chronic ethanol treatment on dihy- 
dropyridine binding sites in WSP and 
WSR mouse strains have been observed 
(Brennan et al. 1990), implying that 
these innate genetic differences in the 
susceptibility to dependence and 
withdrawal may be explained, in part, 
by the differential regulation of volt- 
age-sensitive calcium channels. Thus, 



voltage-gated calcium channels likely 
contribute to the acute actions of 
ethanol as well as the development of 
tolerance and dependence to ethanol. 
Innate differences in these channels 
may contribute to innate differences 
in sensitivity. Significantly more 
research will be required to under- 
stand the role of various subtypes of 
voltage-gated calcium channels in 
ethanol responses. 

~Purinergic P 2 x I° n Channel 
Receptors. Adenosine triphosphate 
(ATP) has been recognized as an 
excitatory transmitter in both the 
CNS and the peripheral nervous system, 
acting through purinergic receptors 
that include G protein-coupled recep- 
tors (e.g., P 2 y and P 2 u receptors) and 
ligand-gated ion channel receptors 
(e.g., P 2X ) (Kennedy and Leff 1995). 
Weight's laboratory has investigated 
the effects of ethanol on ATP-gated 
channels using whole cell patch-clamp 
on neurons from bullfrog dorsal root 
ganglion neurons. The amplitude of 
ATP-activated currents was decreased 
by ethanol (EC 50 = 68 mM). Unlike 
NMDA receptors where ethanol is 
noncompetitive, inhibition of ATP 
followed competitive kinetics by 
right-shifting the dose-response curve 
for ATP (Li et al. 1993). Studies of a 
series of alcohols indicated that alco- 
hol potency correlated with lipid sol- 
ubility from one to three carbons; for 
example, methanol < ethanol < pro- 
panol. For alcohols of four or more 
carbons, no effect was found. These 
studies suggest that a hydrophobic 
pocket exists within the receptor ion 
channel complex that has a specific 
molecular volume (Li et al. 1994). 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



Additional studies are needed to 
determine if ethanol alters mammalian 
P 2 x receptors and to understand the 
role of this family of ion channel 
receptors in ethanol adaptations. 

Voltage-Gated Potassium Channels. 
Potassium ion channels dominate the 
resting membrane potentials of almost 
all cell types and bring stimulated, 
depolarized cells back to their resting 
potential. Potassium ion channels are 
diverse and generally divided into two 
broad categories: delayed rectifiers 
and inward-rectifiers. Osmanovic and 
Shefner (1987, 1994) studied the 
effect of ethanol on locus coeruleus 
neuronal inward-rectifier currents and 
found that ethanol shifted the inward 
rectification in the depolarizing direc- 
tion, essentially increasing current. 
Ethanol-induced hyperpolarization of 
hippocampal neurons has also been 
suggested to be due to increased potas- 
sium conductance (Carlen et al. 1985). 
Similarly, single -channel currents stud- 
ied using the patch-clamp cell-attached 
technique found that ethanol increased 
the open probability of potassium 
channels in human T cells at concen- 
trations of 35-50 mM ethanol (Oleson 
et al. 1993). Ethanol selectively inhibits 
a voltage-dependent potassium current, 
known as the muscarinic cholinergic- 
responsive M-current, in hippocampal 
CA1 neurons (Madamba et al. 1995). 
Ethanol had little or no effect on other 
K + conductances in CA1 pyramidal 
cells (Moore et al. 1990). The regula- 
tory role of these channels makes them 
likely to be sites of adaptation involved 
in the development of tolerance to and 
dependence on ethanol. Additional stud- 
ies are needed to determine if adaptive 



changes in potassium channels occur 
during chronic ethanol treatment. 

Neurotransmitter and 
Neuromodulator Systems: 
Dopamine, Serotonin, and Opiates 

In the past two decades, a series of 
behavioral and pharmacological reports 
from several laboratories have reinforced 
the idea that the nucleus accumbens 
and particularly the VTA- accumbens 
(mesolimbic) system may play a central 
role in reinforcement for, and depen- 
dence on, an assortment of addictive 
drugs (Koob and Bloom 1988; see also 
chapter 7 in this monograph). Because 
considerable data support synaptic 
transmission as the most ethanol-sensitive 
central site, it is instructive for the 
purpose of this review to evaluate the 
types of transmitters and neuromodula- 
tors, and their interactions with ethanol, 
in these two brain regions. There is an 
abundance of opioid peptides and their 
receptors in both brain regions (see, 
e.g., Herkenham et al. 1984; Mansour 
et al. 1988), and 5-HT and associated 
receptors are also abundant in these 
regions (see, e.g., Li et al. 1989; 
Lavoie and Parent 1990; Matsuzaki et 
al. 1993; Van Bockstaele et al. 1993), 
with convergence of DA- and 5-HT- 
containing fibers in accumbens (Phe- 
lix and Broderick 1995) suggesting an 
interaction of these two transmitters. 
Of course, the major projection neu- 
rons from VTA to accumbens contain 
dopamine, and these neurons also 
receive 5-HT-containing afferent 
inputs (Van Bockstaele et al. 1994). A 
significant number of ethanol studies 
have reported changes in, or a significant 
role for, other nucleus accumbens 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



transmitters, such as GABA (Hodge 
et al. 1995) and glutamate (Nie et al. 
1993; Moghaddam and Bolinao 1994; 
Nie et al. 1994). 

The fact that the accumbens is an 
integral part of the extended amygdala 
adds more significance to the role of the 
nucleus accumbens in alcohol-seeking 
behavior and suggests the need to 
evaluate the role of various known 
transmitters in the amygdala as well. 
In the final analysis, the multifaceted 
nature of alcohol abuse and alcoholism 
will likely involve a complex interplay 
or interaction among the several neu- 
rotransmitters and neuromodulators 
found in these and other (e.g., inferior 
colHcular cortex [McCown and Breese 
1993]) brain regions thought to be 
involved in these phenomena. The 
reader is directed to the excellent review 
by Weiss in chapter 7 for a more detailed 
analysis of the changes in, and the role 
for, the dopamine, 5-HT, opioid, and 
corticotropin-releasing factor systems 
in the various aspects of alcohol prefer- 
ence, reinforcement, dependence, crav- 
ing, and sensitization. The following 
sections present cellular/biochemical/ 
molecular data obtained from some of 
these models, but also briefly highlight 
some aspects of acute ethanol effects as 
a baseline to aid in understanding the 
changes (e.g., whether tolerance devel- 
ops) occurring in the chronic or prefer- 
ence models, or as hints for what to 
examine in such models in future studies. 

Dopamine. A dopamine link is seen 
in a large percentage of literature cita- 
tions on neurochemical and behavioral 
effects of ethanol (see, e.g., Weiss et 
al. 1993). Acute systemic ethanol 
increases extracellular dopamine levels 



in accumbens (see Weiss et al. 1993 
and chapter 7 in this monograph), 
consistent with early electrophysiolog- 
ical studies showing enhanced VTA 
neuron firing (Gessa et al. 1985; Brodie 
et al. 1990) and perhaps predictive of 
a possible dopamine link in ethanol 
reinforcement. Unfortunately, charac- 
terizations of dopamine effects in 
accumbens slices have shown that the 
effects of this transmitter per se, and 
its interaction with acute ethanol, are 
rather complex (i.e., both state and 
cell dependent) (Cepeda et al. 1993; 
Surmeier and Kitai 1993, 1994; 
Surmeier et al. 1995; Levine et al. 
1996; Surmeier and Kitai 1997; Yan 
and Surmeier 1997; Yan et al. 1997). 
The cellular mechanisms of action of 
acute ethanol (let alone chronic 
ethanol) on the dopamine system 
have not been well defined. Nonethe- 
less, the Henriksen group has found 
an interesting role for dopamine in 
several actions of acute ethanol tested 
in vivo, with respect to a modulating 
role in hippocampal function (Criado 
et al. 1994). 

The importance of these acute effects 
of ethanol on the dopamine system is 
underscored by the burgeoning litera- 
ture on this system in various models 
of alcoholism. Of the alcohol-preferring 
rat genetic models, the Indiana P and 
HAD rat lines show abnormalities in 
forebrain dopamine function, and 
there is an increased sensitivity to the 
dopamine-releasing and locomotor 
effects of ethanol in the Indiana lines 
as well as in the Sardinian alcohol-pre- 
ferring (sP) lines (Fadda et al. 1980; 
Waller et al. 1986; Gongwer et al. 
1989; McBride et al. 1990; Weiss et 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



al. 1993). However, the Finnish AA/ 
ANA rats show dopamine changes 
opposite to those in the Indiana P and 
HAD lines (see, e.g., Sinclair et al. 
1989; Kiianmaa et al. 1991). In addi- 
tion, self- administration of ethanol is 
accompanied by dopamine release in 
the accumbens (Weiss et al. 1993), 
and rats will self- administer ethanol 
directly into the VTA (Gatto et al. 
1994). Other pharmacological data also 
show that ethanol preference or rein- 
forcement can be altered by dopamine - 
related drugs. Furthermore, in contrast 
to acute ethanol, ethanol withdrawal 
is accompanied by reduced firing of 
dopamine neurons (Chiodo and Berger 
1986; Diana et al. 1992, 1993) and 
reduced dopamine release in accumbens 
(Rossetti et al. 1992&; Weiss et al. 
1996). Interestingly, chronic ethanol 
treatment also decreases acute ethanol- 
evoked dopamine release, suggesting 
tolerance to the dopamine-releasing 
effect. Weiss (see chapter 7) proposes 
that these and other related data sup- 
port a role for dopamine in accumbens 
in continued ethanol abuse and depen- 
dence, in relapse after protracted absti- 
nence, and perhaps in the motivational 
aspects of these states; however, a link 
between dopaminergic systems and 
ethanol sensitization has been more 
difficult to verify. 

The mechanism of the "tolerance" 
to the dopamine-releasing ethanol effect 
seen after chronic ethanol exposure is 
still under investigation, but it may 
involve reduced presynaptic Ca ++ 
influx (Kim et al. 1994), uncoupled 
Ca ++ entry for dopamine release (Leslie 
et al. 1986), or depolarization inactiva- 
tion of the dopamine cells (Shen and 



Chiodo 1993). Despite such evidence 
for reduced dopamine release, recent 
biochemical studies showing that 
chronic ethanol increases tyrosine 
hydroxylase expression (and other 
measures of activation) in VTA suggest 
that VTA neurons are actually acti- 
vated by such treatment (Ortiz et al. 
1995). The apparent discrepancy 
between these findings and the obser- 
vation of reduced numbers of sponta- 
neously firing neurons in VTA under 
these conditions (Shen and Chiodo 
1993) has not been clarified. 

Unfortunately, other than this one 
extracellular in vivo study by Shen and 
Chiodo (1993), few studies have 
investigated the effect of chronic 
ethanol treatment or other alcoholism 
models on the cellular (electrophysio- 
logical) aspects of dopamine function, 
and to our knowledge no reports using 
intracellular or patch-clamp analyses 
of dopamine function in such models 
have appeared. Only recently has an 
intracellular study of acute ethanol 
effects on VTA neurons appeared 
(Brodie and Appel 1998), to reveal a 
few mechanisms (depolarization, 
increased "h" current, reduced spike 
afterhyperpolarization) that may cause 
the increased VTA firing previously 
reported. The lack of such studies in 
chronic ethanol models may arise 
because of the complexity of the models 
and the dopamine receptor system and 
the often multiple, covert actions of 
dopamine in electrophysiological studies 
(see, e.g., Surmeier and Kitai 1994; 
Surmeier et al. 1995; Surmeier and 
Kitai 1997; Yan and Surmeier 1997; 
Yan et al. 1997). For example, little is 
known about cellular mechanisms of 



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possible changes in dopamine autore- 
ceptor function under these conditions. 
Furthermore, it should be emphasized 
for future work that dopamine recep- 
tors, as G protein-linked receptors, 
would fall into the generic "metabotro- 
pic" category that, according to one 
hypothesis (see the Neurotransmitter 
Systems section, p. 138), could regu- 
late the ongoing sensitivity of ligand- 
and voltage-gated ion channels to 
ethanol (Siggins et al. 1999). 

5-Hydroxytryptamine. Serotonin (5- 
HT) has also been strongly implicated 
in ethanol-seeking behavior. To summa- 
rize, as with dopamine, there are clear 
differences in 5-HT levels of the Indi- 
ana P and HAD lines (see, e.g., McBride 
et al. 1990, 1995); unfortunately, no 
such differences have been seen in the 
Finnish AA alcohol -preferring rat lines 
(Sinclair et al. 1989; Kiianmaa et al. 
1991). Also, acute ethanol increases 5- 
HT release in accumbens after passive 
administration or self- administration 
(Yoshimoto et al. 1991; Yoshimoto 
and McBride 1992; Weiss et al. 
1996). Furthermore, pharmacological 
manipulations that should alter 5-HT 
levels or receptors alter ethanol-seeking 
behavior in animals (Sellers et al. 1992; 
LeMarquand et al. \99Aa) and in 
humans (see, e.g., Monti and Alterwain 
1991; Naranjo and Bremner 1993; 
LeMarquand et al. 1994&), and drugs 
related to both the 5-HT 1A and the 5- 
HT 3 receptors alter ethanol's discrimi- 
native stimulus properties (Signs and 
Schechter 1988; Grant and Barrett 1991; 
Grant and Colombo 1993; Krystal et 
al. 1994). The latter findings are consis- 
tent with clinical data showing that 5- 
HT receptor-related drugs can produce 



ethanol-like feelings (Benkelfat et al. 
1991; Lee and Meltzer 1991; Krystal 
et al. 1994) and alter alcohol craving 
and relapse rates in alcoholics (Lud- 
wig et al. 1974; Modell et al. 1993; 
Make et al. 1996; Buydens-Branchey 
etal. 1997). 

Neuroadaptation with chronic ethanol 
treatment in animals also seems to 
involve central 5-HT systems, as indi- 
cated by the following findings: 

1. Withdrawal from chronic ethanol 
suppresses 5-HT release, levels, and 
metabolism in brain (Kahn and 
Scudder 1976; Tabakoff et al. 
1977; Badawy and Evans 1983; 
Yamamura et al. 1992), including 
nucleus accumbens (Yoshimoto 
and McBride 1992; Yoshimoto et 
al. 1992). As with dopamine, this 
suppression can be reversed by 
ethanol self- administration (Weiss 
et al. 1996). 

2. Pharmacological manipulation of 
5-HT receptors (and especially 5- 
HT 1C or 5-HT 1A receptors) can 
alter the anxiogenic effects of such 
withdrawal for up to a week (Lai et 
al. 1993; Rezazadeh et al. 1993; see 
also Lai et al. 1991; Kleven et al. 
1995; and chapter 7). These findings 
have strong implications for the 
clinical treatment of alcohol depen- 
dence and withdrawal phenomena, as 
discussed later in this chapter. 

Unfortunately, despite such strong 
evidence for a role of 5-HT in ethanol- 
related phenomena, little has been done 
to reveal the molecular or cellular mech- 
anisms behind these ethanol-induced 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



changes in the 5-HT system. Acute 
ethanol has been shown to inhibit 
responses to activation of 5-HT lc 
receptors in an oocyte expression system, 
probably by interfering with (uncou- 
pling) a G protein-PKC linkage and 
involving PKC-mediated receptor phos- 
phorylation (Sanna et al. 1994). In 
another acute study, 5-HT and two 
other 5-HT receptor agonists potenti- 
ated the excitatory effects of acute 
ethanol on VTA dopamine neurons in 
three different brain slice preparations 
(Brodie et al. 1995), further supporting 
the likely interplay of dopamine and 5- 
HT systems in ethanol's central actions. 
These researchers have additionally 
explored this interaction by showing that 
a 5-HT uptake inhibitor (clomipra- 
mine, but not zimelidine), such as 
those used to treat alcoholism clinically, 
can markedly enhance the excitatory 
effect of acute ethanol on dopamine 
neurons in the VTA in vitro (Trifunovic 
and Brodie 1996). 

Despite these interesting findings 
on the interaction of acute ethanol 
with 5-HT receptors, as far as we are 
aware there are no such mechanistic 
studies on these interactions in any 
chronic ethanol or ethanol-seeking 
model. As with dopamine, this lack 
may be in part due to the complexity 
of 5-HT neuronal anatomy and 5- 
HT receptors. And again, for future 
work on the interaction between 
ethanol and 5-HT receptors, it 
should be emphasized that most 5- 
HT receptors (all except 5-HT 3 
receptors) fall into the G protein- 
linked metabotropic category that 
may regulate the sensitivity of ligand- 
gated ion channels to ethanol (see the 



Neurotransmitter Systems section, p. 
138; see also Siggins et al. 1999). 

Opiates. Opiate receptors constitute 
another group of such generic meta- 
botropic receptors. Therefore, it may 
not be surprising that the alcohol lit- 
erature is strewn with references to 
the opiate -like effects of ethanol (cf. 
Rossetti et al. 1992&), including early 
data showing cross-tolerance between 
ethanol and opiates (Mayer et al. 1980) 
and showing that opiate antagonists 
could alter ethanol self- administration 
in animals (Altshuler et al. 1980). 
Although the Koob group and others 
found that the opiate antagonist 
naloxone does block responding for 
ethanol in rats, responding for water 
was also reduced (Weiss et al. 1990). 
There is also evidence that a lack of 
transsynaptic opioid peptides may be 
linked to a genetically determined 
preference for ethanol consumption in 
mice (George et al. 1991), and a later 
study by the Koob group showed that 
naloxone could alter some aspects of 
ethanol withdrawal (Schulteis et al. 
1994). It has been suggested that 
ethanol's ability to activate the dopa- 
mine system may involve the opiate sys- 
tem as an intermediary (Badawy and 
Evans 1983; Widdowson and Holman 
1992; Acquas et al. 1993; Di Chiara 
et al. 1996; Gonzales and Weiss 
1998). Recent clinical data (see chapter 
7), particularly data showing a benefi- 
cial therapeutic effect of naltrexone in 
reducing craving and relapse in abstain- 
ing alcoholics (O'Malley et al. 1992; 
Volpicelli et al. 1992; see also the section 
on craving later in this chapter), further 
reinforce the relationship of the endoge- 
nous opioid systems to alcohol abuse 



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and alcoholism. Interestingly, animal 
studies have shown that both naltrexone 
and the 6 opiate receptor antagonist 
naltrindol can reduce ethanol-stimulated 
dopamine release in accumbens (Acquas 
et al. 1993; Benjamin et al. 1993), 
and a recent study has shown that this 
effect of naltrexone correlates directly 
with decreases in ethanol self- adminis- 
tration (Gonzales and Weiss 1998). 

Furthermore, at the functional, cellu- 
lar level, there are often striking simi- 
larities between the electrophysiological 
actions of ethanol, dopamine, and 
opiates in the nucleus accumbens. In 
fact, an early in vivo extracellular study 
found that condensation products of 
ethanol and dopamine evoked a pattern 
of effects across several brain regions 
very similar to the effects elicited by 
ethanol and opiates (Siggins et al. 
1982). In hippocampus, both ethanol 
and 6 opiate receptor agonists can excite 
pyramidal neurons by reducing the M- 
current, a voltage -sensitive K + conduc- 
tance (Moore et al. 1990, 1994; Siggins 
et al. 1995). Analysis of several studies 
in nucleus accumbens shows that 
ethanol, dopamine, and opiates all 
predominantly reduce spontaneous 
discharge in vivo (Hakan and Henrik- 
sen 1987, 1989) and evoked discharge 
in vitro (Uchimura et al. 1986; Yuan 
et al. 1992), and all three agents in 
the accumbens slice preparation can 
markedly reduce glutamatergic excita- 
tory postsynaptic potential (EPSP) 
amplitudes, with little or no effect on 
membrane potential or resistance (Yuan 
et al. 1992; Nie et al. 1993). Naloxone 
significantly reverses ethanol-induced 
reduction of glutamatergic EPSPs in 
accumbens core neurons, especially 



when these EPSPs are evoked by lower 
stimulus strengths applied to the peri- 
tubercle region (Nie et al. 1993). This 
ethanol-naloxone interaction does not 
apply to effects of exogenously applied 
NMDA or kainate (Nie et al. 1994) 
and thus is likely to be a presynaptic 
effect. These data support a role for 
endogenous opioids or opiate receptors 
in ethanol actions in reducing gluta- 
mate release at presynaptic sites. This 
mechanism could provide the physio- 
logical, cellular underpinnings (see 
Siggins et al. 1995) for the reported 
efficacy of another opiate antagonist, 
naltrexone, in reducing relapse in 
abstaining alcoholics (O'Malley et al. 
1992; Volpicelli et al. 1992). However, 
it also should be noted that in vivo 
studies using different stimulus sites 
(subiculum, amygdala) found no 
influence of systemic naloxone on 
ethanol inhibition of these inputs 
(Siggins et al. 1995; Criado et al. 
1997), showing that there are sites of 
ethanol action that clearly do not 
involve opioid systems. 

As noted above, recent evidence 
suggests that ethanol effects in certain 
mesolimbic brain regions may involve a 
more selective opiate receptor action on 
6 receptors (Acquas et al. 1993; see 
also chapter 7). It is therefore of some 
interest that chronic treatment with 
ethanol and/or naloxone up -regulates 
delta opioid receptor gene expression in 
neuroblastoma hybrid NG108-15 cells 
(Jenab and Inturrisi 1994), an effect 
that appears to be mediated by a reduc- 
tion of PKA activity (Jenab and Inturrisi 
1997). To our knowledge, similar 
molecular studies performed in vivo 
have not been reported, but such studies 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



would be very useful for comparison 
with the effects of chronic treatment 
with other abused drugs (see, e.g., 
J.Q. Wang et al. 1994; McGinty and 
Wang 1998). As yet there appear to 
be no cellular studies of the opioid 
systems in any chronic ethanol or 
preference model. 

Synaptic Transmission; 
Presynaptic Mechanisms 

The foregoing discussion implies that 
synaptic transmission would be highly 
affected by acute and chronic ethanol 
exposure, at least by virtue of the fact 
that ethanol affects most of the post- 
synaptic transmitter receptors discussed 
so far. In fact, the idea that the synapse 
might be the most sensitive substrate 
for ethanol action originated in part 
from early electrophysiological findings 
showing a greater ethanol effect in 
multisynaptic than monosynaptic path- 
ways (Berry and Pentreath 1980). 
Subsequent cellular studies on cerebel- 
lar, hippocampal, accumbens, and other 
neurons have helped confirm this idea 
(see, e.g., Carlen et al. 1982; Mancillas 
et al. 1986; Siggins et al. 1987; 
Lovinger et al. 1990; Lin et al. 1991; 
Proctor et al. 1992; Nie et al. 1993, 
1994). Because synapses are storehouses 
of messenger agents, neurochemical 
studies have been able to elucidate 
ethanol effects on transmitter release 
and metabolic pathways in brain. 
Early studies showed reproducible 
effects of ethanol on release of some 
transmitters. For example, acetylcho- 
line release evoked from brain slices is 
reduced by ethanol (Erickson and 
Grahm 1973), and the newer micro- 
dialysis and electrochemical methods 



have shown increased extracellular 
dopamine and 5-HT levels in nucleus 
accumbens with systemic ethanol 
treatment (see chapter 7); although 
more difficult to measure, release of 
corticotropin-releasing factor has also 
been found. 

Much attention has focused on the 
ionic consequences of transmitter and 
ethanol interactions — for example, 
biochemical findings that GABA- 
induced chloride fluxes are increased 
by ethanol in cultured neurons (Mehta 
and Ticku 1988) and synaptoneuro- 
somes (Suzdak et al. 1988; Allan et al. 
1991). It has also been found that 
ethanol reduces calcium influx into 
cultured cerebellar granule cells 
evoked by the NMDA glutamate 
receptors (Hoffman et al. 1989£). Still, 
electrophysiological methods have been 
particularly useful for finding trans- 
mitter effects most sensitive to ethanol, 
with the implication that the most 
sensitive systems are involved in ethanol 
intoxication and perhaps in alcohol 
dependence and abuse as well. The area 
of ethanol effects on electrophysiological 
membrane and synaptic properties and 
transmitter responses has been well 
reviewed (Deitrich et al. 1989; Shefher 
1990; Weight 1992); therefore, we 
will not provide an exhaustive review 
here. However, it should be noted that 
the finding of potent ethanol inhibition 
of NMDA receptors (Hoffman et al. 
1989/*, 1989£; Lovinger et al. 1989, 
1990; White et al. 1990) not only has 
greatly boosted interest in the study 
of ethanol and synaptic transmission 
but also has provided an underlying 
explanation for the important finding 
that ethanol reduces LTP (Morrisett 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



and Swartzwelder 1993; Tremwel and 
Hunter 1994; Swartzwelder et al. 
1995), the cellular model of learning 
and memory. 

Although electrophysiological stud- 
ies would seem suited for examining 
ethanol effects on synaptic transmission, 
the results from such studies have not 
always been consistent with the behav- 
ioral and biochemical findings, as best 
exemplified by GAB A studies. A well- 
known example of this problem is that, 
in contrast to the CI" flux studies, many 
past studies in hippocampal pyramidal 
and other neurons either in vivo 
(Mancillas et al. 1986) or in vitro 
(e.g., Siggins et al. 1987) have had 
difficulty showing an influence of 
acute ethanol on responses to exogenous 
GABA or evoked GABAergic inhibitory 
postsynaptic potentials (IPSPs) (GABA- 
IPSPs). A more recent revaluation of 
this subject found that ethanol sensi- 
tivity of CA1 hippocampal and 
accumbal IPSPs was conditional: that 
is, their augmentation by acute ethanol 
in vitro depended upon activation of 
PKC (Weiner et al. 1994, 1997) or 
inhibition of GABA B receptors (Wan 
et al. 1996; Siggins et al. 1999). How- 
ever, responses to locally applied 
exogenous GABA (in the presence of 
tetrodotoxin to minimize presynaptic 
effects) were still not altered by ethanol, 
even after block of GABA B receptors 
(Wan et al. 1996; Siggins et al. 1999), 
suggesting that the ethanol and GABA B 
receptor effects may be exerted presy- 
naptically, to enhance GABA release. 
This example illustrates the critical 
need to closely examine the synaptic 
effects of ethanol, as well as ethanol's 
effects on responses to exogenous 



transmitter, for a complete under- 
standing of mechanisms of chronic 
ethanol action relevant to behavior and 
clinical phenomena. 

This conditional effect of acute ethanol 
on synaptic transmission has also been 
evaluated in accumbens neurons, as a 
prelude to studies of chronic ethanol. 
Here, as in most other brain regions 
(see Siggins et al. 1987), ethanol (like 
opioid peptides [Yuan et al. 1992]) 
clearly reduces excitatory glutamatergic 
transmission evoked by either local or 
distal stimulation (Nie et al. 1993, 
1994). Detailed evaluation of the sensi- 
tivity of ethanol's effects in this region, 
using pharmacological isolation of 
synaptic components, has shown that 
ethanol has both pre- and postsynaptic 
inhibitory effects on the glutamatergic 
components of EPSPs, with the former 
being somewhat more potent and 
involving an opiate receptor link (Nie 
et al. 1993, 1994). Interestingly, inhi- 
bition of apparently presynaptic GABA B 
receptors blocks the depressant effect 
of ethanol on the NMDA receptor- 
mediated component of the EPSPs 
(NMDA-EPSPs) (Nie et al. 1996; 
Siggins et al. 1999), by an as yet 
unknown mechanism. Findings such as 
these have led to a metabotropic hypo- 
thesis of ethanol sensitivity for neuro- 
transmission mediated by ligand- gated 
ion channels (Siggins et al. 1999). 

Several groups have specifically 
examined the effects of chronic 
ethanol treatment on synaptic activity. 
Little and colleagues focused on the 
effects of withdrawal from chronic 
ethanol on CA1 hippocampal function, 
using extracellular field recordings in 
a slice preparation. They showed that 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



the hyperexcitability (lowered stimu- 
lus thresholds for population EPSPs, 
increased paired-pulse facilitation, 
epileptiform activity) seen in such 
slices is not mediated by changes in 
IPSP function (Whittington et al. 
1992), but rather by an up-regulation 
of Ca ++ currents and NMDA receptor 
activation (Whittington et al. 1995; 
Ripley et al. 1996). Similar electro- 
physiological findings of up-regulation 
of NMDA-mediated synaptic hyperex- 
citability after withdrawal from chronic 
ethanol treatment have been seen in 
the dentate gyrus, again in a slice prepa- 
ration. Here, in vitro ethanol exposure 
with subsequent withdrawal was asso- 
ciated with an enhancement and pro- 
longation of evoked NMDA receptor- 
dependent afterdischarges (Morrisett 
1994). Again, these data were consid- 
ered to be consistent with the involve- 
ment of NMDA receptors in ethanol 
withdrawal hyperexcitability. More 
recent studies of organotypic explant 
cultures of the hippocampal CA1 
region have shown that long-term in 
vitro ethanol exposure with subse- 
quent withdrawal causes a specific 
enhancement of NMDA receptor- 
mediated synaptic responses preceding 
the expression of frank epileptiform 
events (Thomas et al. 1998). A similar 
enhancement of NMDA receptor func- 
tion, leading to neurotoxicity and loss 
of neurons, has been seen in primary 
hippocampal cultures (Smothers et al. 
1997). It is of some relevance to alcohol 
dependence and alcohol-seeking behav- 
ior that a similar significant up-regulation 
of NMDA receptor function (assessed 
by application of exogenous NMDA) 
has been seen in nucleus accumbens 



neurons after chronic ethanol treatment 
in vivo (via the vapor chamber method) 
and subsequent withdrawal in vitro 
(Nie et al. 1995). 

A long, elegant series of studies 
highly relevant to the subject of neu- 
roadaptation has come from the Walker 
and Hunter group. This team used 
extracellular recording in vivo and in 
vitro to examine the effects of very 
long-term (20-28 weeks) exposure to 
ethanol via liquid diet, followed by 
long-term withdrawal (at least 8-28 
weeks). This protocol resulted in (1) 
neuronal loss and synaptic reorganiza- 
tion in both CA1 and dentate (Walker 
et al. 1980; Abraham and Hunter 1982; 
Abraham et al. 1982; King et al. 
1988; Orona et al. 1988); (2) reduction 
of recurrent paired-pulse inhibition 
(Rogers and Hunter 1992); (3) reduc- 
tion in muscarinic cholinergic function 
(Rothberg and Hunter 1991; Rothberg 
et al. 1993, 1996); (4) an enhance- 
ment of GABA release without change 
in GABA A receptor function (Tremwel 
et al. 1994£; Peris et al. 1997); and 
(5) a persistent reduction of LTP 
(Tremwel and Hunter 1994). The find- 
ing of item 1 may be conceived of as a 
relatively "permanent" type of neuroad- 
aptation to chronic ethanol, similar 
to the neurotoxicity of the NMDA 
receptor-mediated type described by 
Crews and colleagues (Chandler et al. 
1997). The finding of item 5 is of 
particular relevance as a synaptic locus 
of the type of neuroadaptation that 
might be involved in alcoholism and 
could provide the basis for the 
reduced memory function seen in 
alcoholics and chronically treated 
animals. It was thought that the 



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enhancement of GABA release (item 
4) could underlie the persistent reduc- 
tion in LTP (Peris et al. 1997); how- 
ever, the possible involvement of 
persistent changes in NMDA receptor 
structure or function has not been 
ruled out. 

Enhanced GABA release again sup- 
ports a role for presynaptic influences in 
chronic ethanol effects. The mechanisms 
for such presynaptic effects (e.g., 
enhancing GABA release or reducing 
glutamate release) are not completely 
known, although the several studies 
showing acute ethanol inhibition of 
voltage-activated Ca ++ currents in several 
neuron types (e.g., Camacho-Nasi and 
Treistman 1987; Twombly et al. 1990; 
Jahromi and Carlen 1991; Mullikin- 
Kilpatrick and Treistman 1994; see 
also the Voltage-Sensitive Ion Channels 
section earlier in this chapter) are sug- 
gestive of an underlying mechanism. 
Interestingly, chronic ethanol treatment 
of Aplysia neurons caused no toler- 
ance effect: that is, Ca ++ currents 
appeared normal and the inhibitory 
response to a test dose of ethanol was 
not changed (Treistman and Wilson 
1991), in contrast to undifferentiated 
PC 12 cells where chronic ethanol led 
to significantly larger voltage-gated 
Ca ++ currents (Grant et al. 1993) and 
tolerance to a standard test dose of 
ethanol (Mullikin-Kilpatrick and 
Treistman 1994). 

The cellular mechanisms behind 
the presynaptic effects of both acute 
and chroruc ethanol exposure have 
been addressed by a series of elegant 
studies on a model of transmitter 
release: presynaptic vasopressinergic 
nerve endings isolated from the rat 



neurohypophysis and studied by patch- 
clamp electrophysiological methods 
(including single -channel recording) 
in vitro (X. Wang et al. 1994; Dopico 
et al. 1996, 1998). These studies have 
determined that acute ethanol reduces 
vasopressin release in this model by 
acting on two ionic conductances: (1) 
enhancing the open-duration of voltage- 
sensitive, dihydropyridine-sensitive 
L-type Ca ++ channels, in a manner con- 
sistent with the interaction of a single 
drug molecule with a single target 
site, possibly the L-channel itself (X. 
Wang et al. 1994); and (2) enhancing 
a Ca ++ -dependent K + conductance 
(probably BK channels: mslo, alpha 
subunit) via a direct interaction of 
ethanol with the channel alpha sub- 
unit protein, resulting in a modifica- 
tion of channel gating properties to 
increased open state durations (Dopico 
et al. 1996, 1998). These two effects 
of ethanol have been correlated with 
the reduced release of vasopressin 
after ethanol ingestion in the intact 
animal. More recent studies of chronic 
ethanol effects in this model indicate 
that rats chronically exposed to 
ethanol show significantly less inhibi- 
tion of release from their terminals 
when acutely challenged with ethanol. 
The Treistman group is currently 
examining the acute effects of ethanol 
on the ion channels in terminals iso- 
lated from these chronically treated 
animals, to determine if they change 
in a manner that would explain the 
shift in sensitivity of release (S. Treist- 
man, personal communication, April 
1998). These results promise to pro- 
vide exciting new information on the 
molecular and cellular mechanisms 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



behind the apparent tolerance seen in 
this model. 

In summary, these findings have 
profound implications for the mecha- 
nisms underlying the effects of acute 
and chronic ethanol on presynaptic 
release mechanisms in central neurons 
in general. Such mechanisms in turn 
may provide important clues as, to 
critical cellular and molecular sites of 
neuroadaptation in alcoholism that 
may lead to new treatment strategies. 

Signal Transduction Systems: 
Adenylyl Cyclase.and Protein Kinases 

AC and PICA. Cyclic AMP is a ubiq- 
uitous intracellular second messenger 
formed when a hormone or neuro- 
transmitter acts at the cell surface to 
activate AC. The receptor is coupled to 
AC through the heterotrimeric guanine 
nucleotide binding proteins (G pro- 
teins), which can be either stimulatory 
(Gs) or inhibitory (Gi) to AC. To date, 
nine isoforms of AC have been identi- 
fied, and each has distinct regulatory 
characteristics and localization (Sunahara 
et al. 1996). The membrane -bound AC 
signaling system, is sensitive to acute 
perturbation by pharmacologically 
relevant concentrations of ethanol, 
and changes in this system after 
chronic exposure of cells or animals to 
ethanol have also been noted (Hoff- 
man and Tabakoff 1990; Tabakoff 
and Hoffman 1998). As discussed ear- 
lier in this chapter, the AC signaling 
system, which also involves the 
cAMP-stimulated protein kinase 
(PKA), has been implicated in learn- 
ing and memory in both invertebrate 
systems and mammalian brain, and 
thus also represents a key candidate 



for adaptations induced by alcohol and 
other drugs. 

In general, after chronic ethanol 
exposure, a decreased response of AC 
to various stimulatory agents 
is observed; this is the opposite of 
the acute effect of ethanol to potenti- 
ate agonist-stimulated AC activity 
(Hoffman and Tabakoff 1990; 
Tabakoff and Hoffman 1998). Early 
studies with brain tissue from mice 
and rats that had been chronically 
treated with ethanol revealed decreased 
neurotransmitt-er-stimulated AC activ- 
ity, compared with controls (Hoffman 
and Tabakoff 1990; Tabakoff and 
Hoffman 1998). Later investigations 
confirmed these findings, but also 
showed that stimulation of AC activity 
by agents that acted at the level of the 
G protein and/or directly on the cat- 
alytic unit of AC (e.g., forskolin, fluo- 
ride, Mn 2+ ) was reduced (e.g., Saito et 
al. 1987; Tabakoff et al. 1995). These 
results suggested that the change in 
AC activity following chronic ethanol 
exposure had the characteristics of 
heterologous desensitization. Heterol- 
ogous desensitization is defined by 
the refractoriness of AC to stimulation 
by multiple activators, acting through 
various receptors, following prolonged 
exposure of the system to a particular 
agonist. It is distinguished from 
homologous desensitization, where 
only the response to the agonist to 
which the cells were exposed is reduced 
(Clark 1986). 

Decreased agonist and guanine 
nucleotide-stimulated AC activity has 
been reported in striatal, cortical, 
and hippocampal tissue of chron- 
ically ethanol-treated mice and rats 



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(Tabakoff and Hoffman 1979; Saito 
et al. 1987; Valverius et al. 1989). In 
some, but not all, studies, a decrease 
in stimulation of cerebellar AC has 
also been reported (Valverius et al. 
1989; Wand and Levine 1991). Dif- 
ferences among studies may reflect 
differences in the duration of ethanol 
exposure, or in the development of 
tolerance and/or physical dependence 
on ethanol, which is not always mea- 
sured. The chronic effect of ethanol 
on AC activity may also vary among 
cells and brain areas, due to differen- 
tial localization of various forms of G 
proteins or isoforms of AC. In this 
regard, it is important to note that the 
various isoforms of AC show differen- 
tial sensitivity to the acute effects of 
ethanol (Yoshimura and Tabakoff 
1995), which could have an impact 
on adaptations induced by chronic 
ethanol exposure. 

"Desensitization" of AC activity after 
chronic ethanol exposure has also been 
a frequent finding when activity is 
measured in cultured neuronal (and 
nonneuronal) cells, although there are 
some exceptions (see Tabakoff and 
Hoffman 1998). Chronic in vitro expo- 
sure of N1E-115 or NG108-15 neu- 
roblastoma cells, S49 lymphoma cells, 
or primary cultures of cerebellar granule 
neurons to concentrations of ethanol 
ranging from 25 raM to 200 mM for 
several days resulted in reduced respon- 
siveness of AC to stimulation by various 
agonists. Most of these studies were 
carried out using membrane prepara- 
tions of the cells to assay AC activity, 
but in PC 12 cells the reduced response 
to stimulation was only observed in an 
assay where cAMP production in intact 



cells was measured (Rabin 1993, 
1988). With certain cell culture prepara- 
tions and brain areas, it has been 
reported that chronic ethanol exposure 
does not alter AC activity (e.g., Charness 
et al. 1988), and these exceptions may 
be important in understanding the 
mechanism of the chronic ethanol 
effect. For example, it would be of 
interest to compare the elements of 
the AC system (G proteins, isoforms of 
AC) and the modulators of this system, 
such as protein kinases or phosphatases, 
that could contribute to the differing 
responses to chronic ethanol exposure. 
The cell culture systems have, in fact, 
been utilized for investigations of the 
mechanism by which chronic ethanol 
exposure leads to desensitization of 
AC activity. In NG108-15 cells, a 30 
percent decrease in the amount of 
mRNA for Gsa, and a corresponding 
decrease in Gsa protein, was reported 
(Mochly-Rosen et al. 1988). This 
decrease was accompanied by a decreased 
response of AC to stimulation by ago- 
nist (adenosine), but not forskolin. In 
these cells, the mechanism of the 
change induced by ethanol was sug- 
gested to be a result of agonist-induced 
heterologous desensitization. Evidence 
was presented that ethanol inhibited 
adenosine transport into the NG108- 
1 5 cells by inhibiting a particular form 
of the adenosine transporter, resulting 
in accumulation of extracellular adeno- 
sine. Prolonged exposure of the cells 
to adenosine was proposed to result in 
the observed decrease in Gsa and 
agonist- stimulated AC activity (Nagy 
et al. 1989, 1990; Krauss et al. 1993). 
Later studies indicated that PKA is 
necessary for the ethanol-induced 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



"heterologous desensitization" in S49 
cells, supporting the hypothesis that 
adenosine-stimulated increases in 
cAMP led to the observed desensiti- 
zation of the AC system (Nagy et al. 
1991; Coe et al. 1995). 

Somewhat similar results were 
reported for experiments with PC 12 
cells, where chronic ethanol exposure 
resulted in a decrease in agonist-stim- 
ulated AC activity and a decrease in 
Gsa (Rabin 1993). PKA was also sug- 
gested to be involved in this desensiti- 
zation (Rabin 1993). However, in 
PC 12 cells ethanol treatment did not 
result in an accumulation of extracel- 
lular adenosine (Rabin et al. 1993). 
Furthermore, in more recent studies 
of NG108-15 cells, although chronic 
ethanol exposure was found to reduce 
the level of Gsa (as well as Gia), AC 
activity was increased, and these changes 
were reported not to be due to accu- 
mulation of extracellular adenosine 
(Williams et al. 1995). Therefore, the 
mechanism by which chronic ethanol 
treatment produces desensitization of 
the AC system is still controversial. In 
some cell culture systems, for example, 
decreases in stimulated AC activity 
have been found to be accompanied by 
increases in Gia, although this is by 
no means universal (Charness et al. 
1988; Rabin 1993; Wand et al. 
1993). In addition, the relationship 
between changes in G protein levels 
and activity of AC is not clear. Increases 
in Gia levels (for example) have been 
reported to be associated with both 
higher and lower AC activity (Reithmann 
et al. 1991). Furthermore, studies of 
the stoichiometry of proteins involved 
in regulation of AC activity suggest 



that Gsa is not the rate -limiting element 
in activation of AC activity. It has been 
reported that large reductions (90 per- 
cent or greater) in Gsa are necessary 
to lower agonist-stimulated AC activity 
(Milligan 1996), much greater than 
the changes observed with chronic 
ethanol exposure of cells. 

Nevertheless, changes in G protein 
levels have also been investigated in 
brains of animals treated chronically 
with ethanol, where desensitization of 
AC activity also occurs. A 30 percent 
decrease in Gsa was found in pituitary 
tissue of LS mice, and a two- to-fourfold 
increase in Gia was observed in cere- 
bellar tissue of LS and SS mice, along 
with decreased agonist-stimulated AC 
activity (Wand and Levine 1991; Wand 
et al. 1993). However, there was no 
significant change in the content of a 
number of G protein subunits in various 
brain areas of C57BL/6 mice treated 
chronically with ethanol using a regi- 
men known to produce alcohol tolerance 
and physical dependence, although 
stimulation of AC by various receptor 
agonists, guanine nucleotides, and 
forskolin was reduced (Tabakoff et al. 
1995). A similar lack of change in G 
protein subunits in several brain areas 
was reported for rats treated chronically 
with ethanol using a paradigm that gen- 
erates tolerance and physical depen- 
dence (Pellegrino et al. 1993). 

The overall conclusion that can be 
drawn from the literature is that changes 
in the total quantity of G proteins 
cannot well account for the desensiti- 
zation of AC activity that is produced 
by chronic ethanol exposure. It has 
been argued that the heterogeneity of 
brain tissue preparations precludes an 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



accurate determination of the relation- 
ship of G protein levels to AC activity 
in particular cells; however, even in 
more homogeneous cell culture 
preparations, the evidence linking 
changes in total G protein content to 
the decreased AC activity is not 
strong. It is still possible that changes 
in G protein levels do play a role in 
ethanol-induced AC desensitization, 
however. There is evidence, for example, 
that G proteins can interact with tubu- 
lin, and that microtubule-disrupting 
agents can increase stimulated AC activ- 
ity, suggesting enhanced accessibility 
of Gsa to AC (Popova et al. 1994; 
Yan et al. 1996). It has also been sug- 
gested that the level of Gsa-AC com- 
plexes is considerably lower than the 
total amount of Gsa in the cell (Milligan 
1996). Thus, if ethanol treatment dis- 
rupted the cellular cytoskeleton, result- 
ing in changes in accessibility of G 
protein subunits to AC and/or in the 
number of Gsa-AC complexes, alter- 
ations in AC activity might be observed 
in the absence of changes in the total 
content of G proteins. 

Another possible basis for the 
desensitization of AC activity after 
chronic ethanol treatment is a quanti- 
tative or qualitative change in the cat- 
alytic unit of AC. In some instances, 
changes in stimulation of AC by Mn 2+ 
and forskolin, both of which can 
interact directly with the AC catalytic 
unit, have been observed in brain tis- 
sue of animals treated chronically with 
ethanol (Wand et al. 1993; Tabakoff 
et al. 1995). Chronic morphine treat- 
ment of rats has been reported to alter 
the expression of a particular isoform 
of AC in the brain (Matsuoka et al. 



1994), but this possibility has not been 
addressed in animals or cells treated 
chronically with ethanol. 

Although the molecular mechanism 
of the chronic ethanol-induced change 
in AC activity and cAMP production 
is not yet clear, there is evidence for a 
role of this system in the development 
of alcohol tolerance. In mice, depletion 
of brain norepinephrine by treatment 
with 6-hydroxydopamine (6-OHDA) 
prevents the development of functional 
tolerance to ethanol (Tabakoff and 
Ritzmann 1977). However, when the 
lesioned mice were treated repeatedly 
with forskolin during chronic ethanol 
exposure, tolerance developed normally 
(Szabo et al. 1988#). These findings 
can be interpreted to indicate that 
ethanol potentiation of norepinephrine - 
mediated increases in cAMP production 
may be necessary for tolerance to 
develop. Chronic activation of AC by 
ethanol in the presence of an agonist, 
or chronic exposure to forskolin, may 
be necessary to produce the desensiti- 
zation of the AC system that is associ- 
ated with ethanol tolerance. This 
possibility is supported by a study 
showing that, while acute ethanol 
treatment of rats results in an increase 
in PKA activity and an increase in the 
phosphorylated form of the CREB 
protein in brain, these increases are no 
longer observed in animals that have 
been chronically treated with ethanol 
(Yang et al. 1996£, 1998). This 
change could result from a reduction 
of PKA activity, due to desensitization 
of the AC system, in cells exposed 
chronically to ethanol. However, 
decreased PKA activity has been sug- 
gested to occur, at least in part, from 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



chronic ethanol-induced translocation 
of the catalytic unit of PKA to the 
nucleus (Dohrman et al. 1996), which 
might be expected to enhance CREB 
phosphorylation. It is difficult to com- 
pare the studies of CREB phosphory- 
lation in animals with the investigations 
of PKA in cultured cells, and measures 
of PKA activity and translocation in 
brains of alcohol-tolerant or -dependent 
animals would be of interest. In partic- 
ular, the studies of Palmer and colleagues 
have shown that ethanol potentiation 
of GABA A receptor-mediated responses 
in cerebellar Purkinje cells is greatly 
enhanced in the presence of a |3- 
adrenergic agonist (Lin et al. 1993) 
and that this enhancement of the effect 
of ethanol involves the cAMP/PKA 
signal transduction system (Freund 
and Palmer 1997). Desensitization of 
the AC/PKA system would be expected 
to reduce the ability of ethanol to 
potentiate the effect of GABA at the 
GABA A receptor on Purkinje cells 
("tolerance" to ethanol). Since ethanol 
enhancement of GABA responses in 
Purkinje cells has been related to the 
hypnotic effect of ethanol (e.g., 
Sorensen et al. 1980), one might 
speculate that the desensitization of 
the AC system would play a role in 
tolerance to this effect of ethanol. In 
this model, the extrinsic system (nor- 
epinephrine-stimulated AC activity) 
would impinge on the intrinsic system 
(GABA A receptors) to produce toler- 
ance to a behavioral effect of ethanol. 
PKC. There is also some evidence 
for changes in PKC following chronic 
ethanol exposure in cells of cultures. 
The levels of two novel PKC isoforms, 
6 and e, are increased in PC 12 cells 



and NG108-15 cells after chronic 
ethanol treatment (Diamond and 
Gordon 1997). The change in PKC- 8 
has been found to mediate the effect 
of chronic ethanol treatment to 
increase neurite outgrowth in PC 12 
cells (Messing et al. 1991; Hundle et 
al. 1995; Roivainen et al. 1995). 

Changes in Signal Transduction 
and Neuroadaptation. These studies 
raise an important question regarding 
the applicability of investigations 
using cultured neurons to adaptation 
to ethanol in the intact animal. In a 
number of instances, "tolerance" to 
the effect of ethanol can be observed 
in cultured cells. For example, ethanol 
inhibition of the adenosine transporter 
in NG108-15 cells is reduced in cells 
that have been exposed chronically to 
ethanol, and this change has been 
cited as an example of tolerance to 
ethanol at the cellular level (Nagy et 
al. 1990; Diamond and Gordon 1997). 
However, in the absence of behavioral 
measures of tolerance in the intact 
organism, one cannot determine the 
relationship between such cellular 
resistance to the effect of ethanol on a 
biochemical system and functional tol- 
erance (or physical dependence) in the 
animal. Similarly, changes in neurite 
outgrowth can be suggested to be 
related to synaptic plasticity and remod- 
eling of synapses, but this hypothesis 
cannot be tested in the absence of mea- 
surable behavioral changes. Although 
cell culture models provide simpler 
systems to study adaptation to ethanol, 
the results of these studies must be 
integrated with behavioral or physio- 
logical responses, as has been done in 
the studies of learning and memory 



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described earlier in this chapter, in 
order to relate adaptation at die mole- 
cular level to adaptations in ethanol- 
affected behaviors. 

IEG/Transcription Factor 
Expression and Expression 
of Chaperone Proteins 

IEGs. Immediate early genes such as c- 
fosarc a class of genes whose expression 
is rapidly and transiently stimulated in 
response to a wide variety of extracellular 
factors. These genes encode transcrip- 
tion factors that regulate the expression 
of other genes ("late genes"), and 
these factors were originally thought 
to be involved in growth and differen- 
tiation. However, the induction of IEGs 
by neurotransmitters in adult brain led 
to the suggestion that these genes may 
also play a role in neuronal plasticity, 
that is, in the processes of adaptation 
such as memory (Robertson 1992). It 
has been hypothesized that the same or 
similar molecular events that regulate 
growth and development may also 
regulate long-term synaptic changes in 
the adult brain (Robertson 1992). The 
IEGs may in this sense represent third 
messengers that mediate communication 
between neurotransmitters acting at 
cell surface receptors and gene expres- 
sion leading to long-term changes in 
neuronal function. 

Most studies of changes in IEG 
expression after chronic ethanol expo- 
sure have investigated genes of the fos 
and jun families. Protein products of 
these two genes can interact to form 
heterodimeric transcription factor 
complexes. For example, the c-Fos and 
c-Jun proteins dimerize to form a 
transcription factor that binds to the 



AP-1 consensus sequence on DNA 
(Curran and Franza 1988). There are 
a large number of Fos and Jun family 
proteins, and different homo- and 
heterodimers can influence transcrip- 
tion in different ways (Robertson 
1992). It is not yet clear which genes 
are regulated by AP-1, although there 
is some evidence that this transcrip- 
tion factor can influence the expres- 
sion of proenkephalin and nerve 
growth factor (NGF) (Sonnenberg et 
al. 1989; Hengerer et al. 1990). 

The expression of c-fos was found 
to be induced in brains (hippocampus, 
cortex, and cerebellum) of C57BL/6 
mice that displayed withdrawal 
seizures following the induction of 
physical dependence on ethanol by 
ingestion of a liquid diet containing 
ethanol for 7 days (Dave et al. 1990). 
In these mice, no increase in c-fos 
mRNA was seen if the mice did not 
undergo ethanol withdrawal seizures. 
A later study of IEG expression in rat 
brain showed increases not only in 
expression of c-fos but also of c-jun 
and another IEG, zif/268 (also called 
Egr-T), during the period that overt 
withdrawal signs were evident follow- 
ing cessation of chronic exposure to 
ethanol by vapor inhalation (Mat- 
sumoto et al. 1993). As in the first 
study, changes in c-fos expression in 
the hippocampus were observed only 
in rats that demonstrated withdrawal 
seizures, although the other IEGs 
were induced even in the absence of 
such convulsions. In a third study, 
increases in c-fos mRNA were observed 
in the dentate gyrus and piriform cortex 
of rats undergoing withdrawal after 
cessation of 7 days of exposure to 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



ethanol by vapor inhalation. The 
increase in c-fos mRNA peaked at 8 
hours after withdrawal. Interestingly, 
this increase could be prevented by 
administration of an NMDA receptor 
antagonist, suggesting that the up- 
regulation of NMDA receptors observed 
during ethanol withdrawal contributes 
to the increased c-fos expression. 
However, in this study, increased c-fos 
mRNA was observed regardless of 
whether the animals displayed with- 
drawal convulsions (Morgan et al. 
1992). Knapp and colleagues (1995) 
also reported an increase in Fos-like 
immunoreactivity in several brain 
regions of ethanol-withdrawn rats, in 
the absence of withdrawal seizures. 

The functional significance of the 
changes in IEG expression was con- 
firmed by the finding of an increase in 
DNA binding activities of AP-1 and Egr 
proteins in brains of ethanol-withdrawn 
rats (Beckmann et al. 1997). This 
increased DNA binding was observed 
at 16 hours after ethanol withdrawal 
(i.e., when withdrawal signs were evi- 
dent). It was suggested that the increase 
in IEG expression and transcription 
factor binding might be related to 
long-term neuroadaptive changes 
associated with ethanol physical 
dependence and/or withdrawal. One 
specific possibility derives from the 
finding that ethanol withdrawal 
seizures become more severe upon 
repeated episodes of chronic ethanol 
exposure and withdrawal. This change 
has been likened to kindling of 
seizures (Ballenger and Post 1978; 
Becker and Hale 1993), which is also 
associated with changes in IEG 
expression (Dragunow and Robertson 



1987). However, the hypothesis that 
changes in expression of transcription 
factors play a role in neuronal plasticity, 
possibly in generating the structural 
changes in synaptic connections asso- 
ciated with long-term adaptations, is 
consistent with the IEGs playing a 
role in the broader aspects of adapta- 
tion to ethanol. 

There have also been studies of the 
chronic effects of ethanol on IEG 
expression in cultured cells. In SH- 
SY5Y neuroblastoma cells, ethanol 
exposure for 2-A days resulted in an 
increase in mRNA for c-jun and junD 
and an increase in AP- 1 binding activity 
(Ding et al. 1996). Chronic ethanol 
exposure (50 mM, 3 days) also selec- 
tively enhanced NMDA-induced AP-1 
transcription factor binding activity in 
primary cultures of rat cerebellar gran- 
ule cells (Cebers et al. 1996). The pro- 
tein composition of the AP- 1 complex 
was not altered by ethanol exposure. 
Although these findings are somewhat 
similar to those in animal brain, it is not 
clear whether these particular changes 
would be related to neuroadaptation 
to ethanol in the whole animal. 

Chaperone Protein Expression. 
Another mechanism of ethanol adap- 
tation may involve the induction of 
chaperone protein transcription (Miles 
et al. 1991, 1994; Hsieh et al. 1996). 
At pharmacologically relevant concen- 
trations, ethanol induces Hsc70 mRNA 
and protein expression in NG108-15 
neuroblastoma x glioma cells (Miles et 
al. 1991). In addition, two other mol- 
ecular chaperones, GRP94 and GRP78, 
are ethanol-responsive genes that are 
induced more than threefold by 
ethanol exposure (Miles et al. 1994). 



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These genes encode proteins that could 
serve protein -trafficking roles, produc- 
ing widespread changes in cellular 
membrane functioning. Chaperone 
proteins are required for the assembly 
of multimeric ion channels (Haas 
1994) and may therefore play a role in 
adaptations of membrane receptors that 
are sensitive to ethanol. The relation- 
ship of alterations in the expression of 
these genes to tolerance and depen- 
dence is completely unknown. 

Systems That Influence 
the Development, 
Maintenance, and Loss 
of Tolerance, Dependence, 
and Sensitization 

Tolerance 

In the studies described thus far, ani- 
mals or cells have been treated chroni- 
cally with ethanol, and changes in 
various neurochemical systems have 
been investigated. As has been 
pointed out, however, changes in 
neuronal function in one area of brain 
will have many downstream effects, 
such that it becomes very difficult to 
determine whether an observed 
change is really an underlying mecha- 
nism for neuroadaptation to alcohol 
(Kalant 1998). Another strategy to 
investigate the cellular/molecular 
basis for neuroadaptation is to alter 
the activity of a neural system in a spe- 
cific manner and then evaluate the 
effects of the alteration on neuroadap- 
tation. This approach has been partic- 
ularly effective for investigations of 
ethanol tolerance. The paradigm bor- 
rowed from studies of learning and 
memory — the definition of intrinsic 



and extrinsic systems — is useful in dis- 
cussing the systems that modify the 
development, expression, and mainte- 
nance of ethanol tolerance (extrinsic 
systems). The systems described in the 
following sections represent such 
extrinsic systems; one characteristic of 
these systems is that they affect toler- 
ance to a number of behavioral effects 
of ethanol. 

Neurotransmitters. In mice, the 
development (but not expression) of 
functional tolerance to the hypnotic 
and hypothermic effects of ethanol 
was blocked by partial destruction of 
noradrenergic neurons with 6-OHDA 
before the ingestion of ethanol by the 
animals (Tabakoff and Ritzmann 
1977). As already discussed, stimulation 
of AC activity by norepinephrine in 
the presence of ethanol seems to be 
important for tolerance development 
(Szabo et al. 1988a). The noradrenergic 
system in the rat brain, on the other 
hand, does not appear to play a primary 
role in tolerance development, since 
6-OHDA lesions did not block the 
development of tolerance to the hyp- 
notic effect of ethanol, although this 
tolerance could be blocked by treatment 
of rats with the noradrenergic toxin 
DSP-4 (Le et al. 1981a; Trzaskowska 
et al. 1986). Development of tolerance 
in the rat was also reported to be 
blocked by combined destruction of 
noradrenergic and serotonergic systems 
in brain (Le et al. 198 la). Depletion 
of serotonin alone delayed the develop- 
ment of chronic tolerance to the motor- 
impairing and hypothermic effects of 
ethanol in rats (Le et al. 1981 £), and 
also delayed the development of acute 
ethanol tolerance (Campanelli et al. 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



1988). It was shown in these studies 
that a specific lesion of the serotonergic 
pathway connecting the raphe nucleus 
and the forebrain was important in 
modulating tolerance development. 
The development of rapid tolerance in 
rats also appears to involve the sero- 
tonergic system, since rapid tolerance 
development is facilitated if mice are 
treated with the serotonin precursor, 
tryptophan (Khanna et al. 1994). In 
mice, the development of environment- 
dependent tolerance to ethanol was also 
shown to be slowed by lesions of the 
serotonin system (Melchior and 
Tabakoff 1981). These studies suggest 
that both the catecholaminergic and 
serotonergic systems, and possibly 
interactions between these systems, are 
important for the normal development 
of tolerance to several effects of ethanol. 
An important conclusion from these 
studies is that the presence of ethanol 
in the brain is a necessary, but not suf- 
ficient, condition for the development 
of tolerance; concomitant activity of 
certain neurochemical pathways, 
including postsynaptic effects on the 
AC system, is also required. 

The key role of the NMDA receptor 
in the development of LTP (Collingridge 
and Lester 1989) — that is, its putative 
role in learning and memory processes — 
led investigators to investigate whether 
NMDA receptor activity also plays a 
role in ethanol tolerance. NMDA receptor 
antagonists, including ketamine and 
dizocilpine (MK-801), were reported 
to prevent the development of rapid 
and chronic tolerance to the motor- 
impairing and hypothermic effects of 
ethanol (Khanna et al. 1992; Wu et 
al. 1993). Because, acutely, ethanol is 



a potent inhibitor of NMDA receptor 
function, it seems paradoxical that 
NMDA receptor antagonists should 
block the development of tolerance to 
alcohol. However, it is important to 
note that when the effects of dizocilpine 
on environment-dependent ethanol 
tolerance (produced by ethanol injec- 
tions) and environment-independent 
ethanol tolerance (produced by liquid 
diet ingestion) were directly compared, 
dizocilpine only blocked the development 
of environment-dependent tolerance 
to the hypothermic and ^coordinating 
effects of ethanol. The same or higher 
doses of dizocilpine did not block the 
acquisition of environment-independent 
tolerance to the hypothermic, incoor- 
dinating, or hypnotic effects of ethanol 
(Szabo et al. 1994). The explanation 
for this difference was postulated to be 
the different contributions of learning 
or conditioning to the two forms of 
tolerance. Thus, it was suggested that 
dizocilpine did not block the develop- 
ment of ethanol tolerance per se, but 
blocked learning or conditioning that 
is necessary for the development of envi- 
ronment-dependent tolerance. Simi- 
larly, dizocilpine had a much greater 
effect on the development of behav- 
iorally augmented tolerance than on 
tolerance that did not involve intoxi- 
cated practice (learning) (Khanna et 
al. 1994). 

Neuropeptides: AVP and Neuro- 
trophins. Arginine vasopressin is a 
mammalian antidiuretic hormone that 
is synthesized primarily in the hypo- 
thalamus, as well as in some extrahy- 
pothalamic brain areas. The studies of de 
Wied and colleagues (1997) demon- 
strated that AVP could influence learning 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



and memory and led to investigations 
of the effect of the peptide on tolerance. 
In both mice and rats, the administra- 
tion of AVP has been shown to main- 
tain (reduce the rate of loss of) 
functional tolerance to a number of dif- 
ferent effects of ethanol (hypnosis, 
hypothermia, incoordination) (Hoffman 
1994). The action of AVP on tolerance 
was shown to be mediated by CNS 
receptors, since analogs without periph- 
eral activity could maintain tolerance. 
Furthermore, administration of AVP 
intracerebroventricularly (icv), at doses 
with no discernible peripheral effect, 
also maintained tolerance (Hung et al. 
1984). The action of vasopressin on 
alcohol tolerance is mediated by the 
V x subtype of vasopressin receptor, as 
determined by studies with selective 
agonists and antagonists (Szabo et al. 
1988^). These studies also showed 
that a Vi receptor antagonist could 
increase the rate of loss of tolerance, 
indicating a role of the endogenous 
peptide in maintenance of tolerance. 

It is important to note that vasopressin 
does not appear to facilitate the induc- 
tion of chronic tolerance to ethanol, 
but in fact was found to retard the devel- 
opment of tolerance when given in con- 
junction with chronic ethanol treatment 
(Mannix et al. 1986). On the other 
hand, it has been reported that a single 
dose of AVP, given together with a 
low dose of ethanol, resulted in the 
production of long-lasting tolerance 
to the motor-incoordinating effect of 
ethanol, whereas the same dose of 
ethanol alone did not produce tolerance. 
These findings were taken to indicate 
a role for AVP in facilitating the 
acquisition of acute tolerance to ethanol 



(Wu et al. 1996). Such disparate findings 
could reflect differences in the mecha- 
nisms underlying acute and chronic 
tolerance or in the role of learning and 
memory in the two forms of tolerance. 

The neurochemical and molecular 
actions of AVP that may influence tol- 
erance may be pre- or postsynaptic. In mice, 
depletion of norepinephrine after toler- 
ance had developed did not block the 
expression of tolerance to the hyp- 
notic effect of ethanol, but did block 
the ability of AVP to maintain toler- 
ance (Hoffman et al. 1983). It was 
also shown that the 6-OHDA lesions 
used to deplete norepinephrine reduced 
the number of vasopressin receptors in 
the lateral septum, an area of high con- 
centration of V l receptors (Ishizawa et 
al. 1990). These data suggested that a 
portion of the vasopressin receptors 
involved in maintaining ethanol toler- 
ance may be localized to the terminals 
of catecholaminergic neurons in mouse 
brain, and that vasopressin's actions 
could be at least partially explained by 
an effect of the peptide on neurotrans- 
mitter release. In the rat, serotonergic 
systems have been reported to play a 
key role in the action of AVP on 
ethanol tolerance. The peptide could 
no longer maintain tolerance in animals 
in which the forebrain serotonin ter- 
minals had been destroyed (Speisky and 
Kalant 1985). In such animals, infusion 
of a 5-HT 2 receptor agonist restored 
the ability of AVP to maintain toler- 
ance (Wu et al. 1996). 

Kalant has postulated, and pro- 
vided evidence for, a circuit involving 
serotonin-, glutamate-, vasopressin-, and 
GABA-containing neurons in the septum 
and hippocampus, and inputs to these 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



regions, that are required for the 
development and retention of ethanol 
tolerance in the rat (Kalant 1998). 
Slightly different pathways may be 
involved in the mouse, where cate- 
cholaminergic systems play a more pri- 
mary role. In these pathways, both pre- 
and postsynaptic effects of vasopressin 
may be important for maintenance of 
tolerance. When AVP was administered 
icv to mice, it was found to increase the 
mRNA levels for the IEG c-fos in the 
lateral septum, and later studies showed 
that AVP administration also led to an 
increase in Fos protein (Giri et al. 1990; 
Andreae and Herbert 1993). The effect 
of AVP on c-fos expression was medi- 
ated by Vj receptors, and a positive 
correlation was found between the abil- 
ity of several AVP-related peptides to 
maintain tolerance and to increase c- 
fos expression in the septum (Giri et 
al. 1990; Szabo et al. 1991). Further- 
more, icv administration of an antisense 
oligonucleotide to c-fos blocked the 
ability of vasopressin to increase expres- 
sion of this IEG in the septum and 
also blocked the ability of AVP to 
maintain tolerance (Szabo et al. 1996). 
Fos can form transcription factors that 
may generate long-term changes in 
CNS function in response to neuro- 
transmitters or neuropeptides that act 
at the cell surface. In particular, Fos 
family proteins can dimerize with pro- 
teins of the Jun family to form the 
transcription factor AP-1. The genes 
whose expression is affected by AP-1 
have not been identified, although 
NGF has been suggested to be regu- 
lated by this transcription factor. 

To determine whether AVP might 
be acting by inducing the expression of 



NGF or other neurotrophins, the ability 
of these agents to maintain ethanol tol- 
erance in mice was tested. It was found 
that, whereas NGF was relatively inef- 
fective in maintaining tolerance to the 
hypnotic effect of ethanol (Szabo et al. 
1991), brain-derived neurotrophic 
factor (BDNF), as well as neurotrophins 
3 and 4/5, could maintain tolerance 
(Szabo and Hoffman 1995). It has 
been reported that a vasopressin analog 
that can maintain ethanol tolerance can 
also induce the expression of BDNF 
in rat brain (Zhou et al. 1997). Whether 
or not the effect of vasopressin on tol- 
erance is mediated through neurotro- 
phins, the ability of neurotrophins to 
maintain tolerance to ethanol indicates 
that these compounds, believed to 
play a role in growth and differentiation 
in the developing brain, can also affect 
neuroadaptation to ethanol in the adult 
brain. As previously postulated, changes 
in synaptic efficacy in the adult brain, 
including those that underlie cognitive 
function and adaptations to drugs, may 
arise from the same mechanisms that 
influence growth in the developing 
brain. In this sense, while studies of 
learning and memory may provide 
guidelines for investigation of neuro- 
adaptive processes involved in alcohol 
tolerance and dependence, studies of 
tolerance may also provide insight into 
the mechanisms of cognitive function 
in the adult brain. 

Dependence 

The foregoing discussion provides sev- 
eral clues as to the factors or systems 
that cause or modulate alcohol depen- 
dence, defined operationally here in 
part as a compensatory rebound to 



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chronic ethanol exposure following ces- 
sation of that exposure (alcohol with- 
drawal syndrome, as described earlier in 
this chapter), combined with a strong 
desire to drink (craving). It should be 
noted at the outset that, at the cellular 
level, the expected neuronal hyperex- 
citability following withdrawal from 
chronic ethanol does not occur in all 
brain regions (e.g., in accumbens core 
neurons [Nie et al. unpublished man- 
uscript]), despite satisfaction of behav- 
ioral criteria for dependence. However, 
data from other brain regions suggest 
that several candidate systems could 
be involved in, or modulate, the with- 
drawal or craving aspects following 
chronic ethanol exposure, including 
several ligand- gated transmitter systems 
(e.g., those for NMDA, GABA, 5- 
HT, acetylcholine, and ATP), Ca ++ 
channels, and the G protein-linked 
(metabotropic) transmitter or modula- 
tors. Detailed understanding of these 
factors could lead to powerful therapeu- 
tic treatments for alcohol dependence 
in the future. In this regard, at least 
two different strategies may be used 
to develop therapeutic approaches to 
dependence: (1) developing new types 
of drugs based on an understanding of 
the mechanisms of the underlying per- 
turbation^) involved and (2) developing 
new drugs or improving old drugs based 
on a newly discovered site or mecha- 
nism of action of an existing effective 
but empirically discovered drug. 

Pharmacotherapy of Withdrawal. It 
might be assumed from the previous dis- 
cussion of the effects of chronic ethanol 
on transmitters and synaptic transmis- 
sion that the best strategy for treating 
withdrawal phenomena would be to 



compensate in some way for changes in 
those channel and transmitter systems 
most affected by ethanol and/or its 
withdrawal. Therefore, likely candidates 
for such therapeutic approaches would 
include various ion channels, the GABA, 
NMDA, ATP, cholinergic, 5-HT, 
dopamine, and opioid systems, and per- 
haps their associated second messengers. 
To date, the most obvious and frequently 
discussed treatments include the ben- 
zodiazepines, for enhancement of 
GABA A ergic systems to overcome per- 
ceived hyperexcitability of central neu- 
rons; dopamine receptor-related drugs 
(perhaps useful primarily as anxiolytics); 
and, more recently, 5-HT receptor- 
related drugs, such as the 5-HT 
uptake inhibitors. 

Recent research findings on the role 
of GABAergic systems in withdrawal, 
and especially after chronic intermit- 
tent ethanol (CIE) treatment, are very 
instructive and relevant to the devel- 
opment of long-lasting dependence. 
The CIE models of the Becker and 
Olsen groups were developed largely 
to mimic the human condition of 
alcoholism more closely than the stan- 
dard continuous long-term treatment 
models, and they indeed lead to a kin- 
dling of greater susceptibility to with- 
drawal hyperexcitability and seizure 
than do standard models (Becker and 
Hale 1993; Kokka et al. 1993). The 
Olsen group has used the CIE rat model 
for study of hippocampal GABAergic 
function in withdrawal and has found 
prolonged changes in some indices of 
GABAergic inhibition, such as decreased 
muscimol-induced Cl~ flux and 
paired-pulse inhibition (Kang et al. 
1996) and increased expression of the 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



GAB A receptor a4 subunit (Mahmoudi 
et al. 1997). By contrast, more recent 
intracellular studies of the same hip- 
pocampal model after CIE (Rang et al. 
1998) showed no change in the overall 
size of GABAergic IPSPs. Nonetheless, 
after pretreatment of hippocampal 
slices with a GABA B receptor antagonist 
to unmask ethanol interactions with 
GABA A -IPSPs (see Wan et al. 1996), 
a significantly greater ethanol enhance- 
ment of GABA A -IPSP areas occurred 
in hippocampal slices from CIE animals 
than in control animals (Kang et al. 
1998). This finding shows that toler- 
ance to this ethanol effect does not 
occur in the CIE model; rather, a sensi- 
tization develops that could play some 
role in dependence (Kang et al. 1998). 
However, there is still a need for clarifi- 
cation as to whether the ethanol effect 
on IPSPs, and therefore its sensitization 
in the CIE-treated hippocampus, is 
exerted pre- or postsynaptically (Wan 
et al. 1996; Siggins et al. 1999)— that 
is, indirectly via GABA release or 
uptake or directly at the GABA A recep- 
tor. A further major finding in the 
Kang et al. study of considerable rele- 
vance to treatment strategies was a 
possible sensitization of the GABAergic 
system to benzodiazepine inverse ago- 
nists and neurosteroids in the hip- 
pocampus of CIE rats. This finding 
supports the possibility raised by Suzdak 
and colleagues (1986) that binding 
sites on the GABA A receptor for benzo- 
diazepine inverse agonists may be linked 
to changes associated with tolerance 
and dependence, and it also supports 
the possibility that neurosteroids might 
be a useful avenue for alcohol depen- 
dence therapy. 



Based on the more recent cellular and 
neurochemical findings (see, e.g., the 
Synaptic Transmission; Presynaptic 
Mechanisms section earlier in this chap- 
ter), it would seem wise also to focus on 
the newer agents acting on NMDA 
receptors and Ca ++ channels, or perhaps 
on systems downstream from these ele- 
ments (e.g., the nitric oxide or eicosanoid 
systems). Thus, agents like the NMDA 
receptor antagonists MK-801, meman- 
tine, or one of the D-CPPene com- 
pounds (NMDA receptor antagonists 
developed by Novartis Pharma that can 
be injected peripherally), Ca ++ channel 
antagonists like nifedipine, nitrendipine, 
or nimodipine, or nitric oxide antago- 
nists such as nitroglycerin should come 
under scrutiny as therapeutic agents for 
early withdrawal hyperexcitability and 
associated neurotoxicities. These com- 
pounds might be used early in the 
detoxification stage. 

Pharmacotherapy of Craving 
(Acamprosate, Naltrexone). Recently, 
distinctions have made between the 
mechanisms underlying the withdrawal 
syndrome and craving or relapse. There- 
fore, it is interesting that two of the 
newest therapeutic approaches for 
alcoholism involve treating the more 
long-term effects of alcohol dependence 
(i.e., neuroadaptation to chronic abuse, 
leading to craving). These approaches 
are based on a different rationale than 
that for treating withdrawal and thus 
are often prescribed several weeks after 
detoxification or initiation of abstinence. 
Initial positive clinical trials in Europe 
for acamprosate and in the United 
States for naltrexone have led to the use 
of these two drugs found to be effec- 
tive in reducing relapse in abstaining 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



alcoholics (Lhuintre et al. 1985; 
O'Malley et al. 1992; Volpicelli et al. 
1992). Obviously, the rationale behind 
the clinical use of naltrexone may be 
found in the lands of opiate studies in 
animals and humans described earlier 
in this chapter and in chapter 7. By con- 
trast, the original rationale for acam- 
prosate was to develop a congener of 
GABA (a homotaurinate derivative) 
that might counteract ethanol's pre- 
sumed GABA A receptor-enhancing 
effects (Lhuintre et al. 1985). Behavioral 
studies in animals have shown that 
acamprosate can reduce the ethanol 
deprivation effect (in a forced absti- 
nence paradigm) that produces 
rebound enhanced responding for 
ethanol (Heyser et al. 1996; Spanagel 
et al. 1996). 

Despite the fact that acamprosate 
was developed as a GABA receptor 
agonist, initial in vivo and in vitro 
electrophysiological studies of cortical 
neurons found that high concentrations 
of acamprosate had no discernible 
GABAergic effect but instead acted to 
reduce glutamatergic responses (Zeise 
et al. 1990, 1993). Subsequent studies 
in two different slice preparations, hip- 
pocampus and nucleus accumbens, 
showed that acamprosate again had 
no GABA A ergic action, but instead 
significantly augmented NMDA- 
EPSPs and responses to exogenous 
NMDA (Madamba et al. 1996; 
Berton et al. 1998). However, in con- 
trast to the studies of Zeise and col- 
leagues (1990, 1993), acamprosate had 
no significant effect on non-NMDA 
glutamatergic EPSPs in these brain 
regions. Interestingly, in accumbens 
neurons acamprosate also acted like a 



GABA B antagonist in blocking paired- 
pulse inhibition (a presynaptic site of 
action) of IPSPs, suggesting that the 
drug might act on GABA B rather than 
GABA A receptors (Berton et al. 1998). 
This finding also seems consistent 
with the metabotropic hypothesis of 
ethanol sensitivity (Siggins et al. 
1999) and suggests that GABA B 
receptors would be an interesting tar- 
get for alcoholism therapy. 

This idea is strengthened by pre- 
liminary electrophysiological findings 
with another drug, y-hydroxybutyrate 
(GHB), shown to have some efficacy 
against alcohol dependence in humans 
and in animal alcohol preference mod- 
els (Biggio et al. 1992; Gallimberti et 
al. 1992; Gessa and Gallimberti 1992). 
Intracellular recording of CA1 pyramidal 
neurons in a hippocampal slice prepa- 
ration has shown that GHB, like the 
GABA B agonist baclofen, hyperpolarizes 
these neurons and augments the inward- 
rectifying Q or h current (Madamba 
et al. 1997). The effects of both GHB 
and baclofen were blocked by the 
GABA B antagonist CGP 35348, sug- 
gesting that the clinical efficacy of GHB 
may be due to its action on GABA B 
receptors, and providing additional 
support for study of these metabotropic 
receptors as therapeutic targets against 
alcoholism. However, it should be 
noted that GHB is also being used in 
the detoxification stage, probably 
because it has alcohol-like effects. 

Sensitization 

Evidence for a direct role of sensitization 
in alcoholism might come from the 
demonstration that similar biological 
systems underlie both sensitization and 



136 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



vulnerability to drug self-a<iministration 
or reinforcement. Dopamine systems 
have been widely investigated with 
regard to sensitization to other drugs 
of abuse, and research results have 
supported their involvement in drug 
reward. Evidence for this involvement 
has been found, most commonly, by 
analysis of the role of dopamine systems 
in drug self- administration (Koob et 
al. 1987; Britton et al. 1991; Hubner 
and Moreton 1991; Caine and Koob 
1993; Maldonado et al. 1993; 
Richardson et al. 1993; Ng and George 
1994) or by measuring dopaminergic 
changes associated with drug self- 
administration (Goeders and Smith 
1993; Weiss et al. 1993; Laurier et al. 
1994). Enduring changes in mesoac- 
cumbens dopamine transmission have 
also been postulated to be involved in 
drug sensitization (Trulson et al. 
1987; Robinson et al. 1988; Peris et 
al. 1990; Segal and Kuczenski 1992; 
Kalivas et al. 1993; Parsons and Jus- 
tice 1993; Burger and Martin -Iverson 
1994; Self and Nestler 1995). How- 
ever, some studies support involve- 
ment of projections involving other 
pathways and neurotransmitters inter- 
acting with the dopamine system 
(Kalivas and Alesdatter 1993; Kalivas 
et al. 1993; White et al. 1995), as well 
as effects independent of the dopamine 
system (Koob and Cador 1993). This 
work has not been accomplished for 
ethanol sensitization. Nestby and col- 
leagues (1997) did show neurochemi- 
cal changes associated with repeated 
ethanol administration that paralleled 
those associated with repeated amphet- 
amine, morphine, and cocaine admin- 
istration; there was increased dopamine 



and acetylcholine release from nucleus 
accumbens slices. However, they pro- 
vided no evidence of ethanol sensiti- 
zation in that study. 

A growing body of literature sup- 
ports hypothalamic-pituitary-adrenal 
axis involvement in cocaine, ampheta- 
mine, and morphine sensitization. A 
series of studies addressed the involve- 
ment of this axis in ethanol sensitiza- 
tion (Roberts et al. 1995). The principal 
findings were as follows: (a) repeated 
exposure to restraint stress sensitized 
mice to the locomotor stimulant effects 
of ethanol, (b) stress-induced sensitiza- 
tion of ethanol's locomotor stimulant 
effects was attenuated by a glucocorti- 
coid receptor antagonist, and (c) the 
glucocorticoid receptor antagonist was 
also capable of preventing sensitization 
to ethanol produced by repeated ethanol 
injections. The only other study we 
know of that has directly assessed any 
neuropharmacological mechanism 
associated with ethanol sensitization is 
that of Broadbent and colleagues 
(1995), who reported no effect of a 
dopamine antagonist on ethanol sen- 
sitization. This mechanistic work 
should be expanded. 

Critical Gaps 

in Our Knowledge 

Ligand- Gated Ion Channels 

Mechanisms of Receptor Adaptations. 
Ethanol alters both GABA A and 
NMDA receptor function and results 
in alterations in the expression of vari- 
ous subunits that comprise these 
receptor channels. Numerous investi- 
gators have hypothesized that alterations 
in gene expression for the subunit 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



proteins could result in alterations in 
receptor assembly that could explain the 
functional alterations in these receptors. 
Although this hypothesis has clear 
heuristic value, alterations in subunit 
assembly have not been directly demon- 
strated in response to ethanol admin- 
istration. We have very little knowledge 
of the regulation of receptor assembly 
for these receptors. It is clear that chap- 
erone and other proteins are involved 
in these processes. This is an important 
area for future research. 

Most ion channel proteins are subject 
to posttranslational modifications that 
modify the functional properties of 
these receptors. Ethanol has been shown 
to activate various kinases that are 
capable of modifying ion channel recep- 
tors. Does this process underlie ethanol - 
induced alterations in ion channel 
receptor function? Is this process 
involved in ethanol regulation of ion 
channel gene expression? There is a great 
deal of indirect evidence suggesting 
that posttranslational receptor modifi- 
cations may be an important mechanism 
involved in ethanol adaptations, but 
there has never been a direct demon- 
stration that ethanol induces such an 
alteration or that such alterations can 
explain any of the physiological effects 
of chronic ethanol administration. Such 
investigations are of critical impor- 
tance and interest. 

Another possible mechanism medi- 
ating neuroadaptations of ion channel 
receptors following chronic ethanol 
exposure involves internalization of 
the receptor complex. There is substan- 
tial evidence that various receptors can 
be internalized (Calkin and Barnes 
1994), and this mechanism could 



explain alterations in receptor function. 
It is also possible that ethanol affects 
the stoichiometry of ion channel 
receptors. However, the stoichiometry 
of most native receptors that are sensitive 
to ethanol remains undetermined. 
Likewise, a dissociation of subunits could 
account for decrements in ion channel 
function while preserving receptor num- 
ber, yet be nearly impossible to detect 
using currently available techniques. 
Conformational changes in receptor 
structure are another potential adap- 
tation that may explain rapid changes 
in receptor function. Recent studies 
demonstrating ethanol action at the 
second transmembrane domain of 
glycine/GABA A receptor chimeras 
(Mihic et al. 1997) suggest that 
ethanol may affect the conformation of 
ion channel receptors. Finally, although 
we have focused on postsynaptic regu- 
lation by ethanol, physiological adap- 
tations may also involve presynaptic 
mechanisms. This possibility should 
be reexamined in the light of identifi- 
cation of multiple novel neurotrans- 
mitter transporters and more sensitive 
molecular techniques to investigate 
their function. 

Significance of Regional Differences in 
Gene Regulation. Both glutamate and 
GABA A receptor subunits are differentially 
regulated by ethanol in hippocampus 
versus cortex and other brain regions. 
Since ethanol mediates distinct physio- 
logical effects in distinct brain regions, 
this differential regulation may have 
significant physiological implications. 
Few studies have addressed the role of 
gene regulation in specific neuronal 
circuits that control specific ethanol- 
mediated physiological/behavioral 



138 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



responses. Such studies could lead to a 
better understanding of the relation- 
ship of molecular adaptations to the 
behavioral manifestations of tolerance, 
dependence, and sensitization. 

What are the signal transduction 
pathways involved in ethanol regulation 
of ion channel genes? Do regional dif- 
ferences in the regulation of a single 
gene suggest multiple mechanisms of 
gene regulation? Is ion channel gene 
regulation dependent on stimulation of 
the ion channel independent of action 
on the membrane receptors? If ethanol 
acts directly on gene promoters, what 
are the signal transduction pathways 
involved in this activity? Will this 
action differ in various brain regions? 

Ethanol-Nicotine Interactions. Con- 
verging clinical data have shown that 
alcoholism and excessive drinking are 
several times more prevalent in smok- 
ers than in nonsmokers (Deher and 
Fraser 1976; Bien and Burge 1990). 
It is also known that the ability to 
treat alcoholism is enhanced if indi- 
viduals stop smoking and vice versa 
(Griffiths et al. 1976; Johnson and 
Jennison 1992; Joseph 1993; Gulliver 
et al. 1995; Murray et al. 1995). 
Because of the interactive association 
of these abused substances, the chal- 
lenge will be to resolve the basis of 
this strong interaction between the 
actions of chronic ethanol and chronic 
nicotine exposure. Consequently, it is 
extremely important that we have a 
complete understanding of the direct 
interactions these chronically abused 
substances have on various nACh 
isoreceptors in brain, alone and when 
combined. The critical question to be 
answered is how changes in nACh 



receptor function, induced by the 
presence of chronic nicotine and 
ethanol, contribute to and perpetuate 
alcoholism and the desire to smoke. 

Neurotransmitter Systems 

With respect to the effect of chronic 
ethanol on transmitter systems, and 
given the important role the dopamine 
system is likely to play, one of the first 
questions that might be asked is: what 
happens to the membrane and synaptic 
properties of VTA neurons in any of 
the alcohol-preferring, chronic ethanol, 
ethanol -withdrawn, or relapse models? 
It would also be useful to know if there 
are changes in dopamine autoreceptor 
function in these models, given the 
importance many researchers place on 
this interesting regulatory property of 
dopaminergic neurons. It would also 
be helpful to know if there are changes 
in possible dopamine -ethanol interac- 
tions in target areas of the VTA dopa- 
mine projection. 

There are even more gaps in our 
knowledge of the 5-HT system in these 
alcohol models. First and foremost, 
we have no information on what hap- 
pens to the neuronal or molecular prop- 
erties of 5-HT-containing neurons 
(e.g., in the raphe nuclei) even with 
acute ethanol, as we have for the effects 
of opiates (see, e.g., Pan et al. 1990). 
Understanding this aspect in chronic 
models would be even more useful. In 
addition, it would be helpful to know 
whether there are ethanol-5-HT inter- 
actions at the level of the various 5 - 
HT receptors in the chronic models, 
and whether there are interactions 
between 5-HT and dopamine cells in 
these models (i.e., the studies of the 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



Brodie group need to be repeated in 
the chronic models). 

Similar questions apply to the role of 
the opioid systems in the chronic ethanol 
or preference models. Fortunately, there 
is a great background of data from mul- 
tiple brain regions on opiate effects or 
functions of opioid systems that can 
be used for comparison in the chronic 
or preference models. As for the 
dopamine and 5-HT systems, under- 
standing possible changes in the opi- 
oid-ethanol interactions in the 
chronic/preference models would also 
be very instructive. 

As all three of these transmitters act 
mostly on the G protein-linked, 7- 
transmembrane -spanning- domain 
superfamily of receptors, they fall into 
the generic category of metabotropic 
receptors capable of altering, through 
various kinases and phosphatases, the 
phosphorylation state of other recep- 
tors, ion channels, and other (intracel- 
lular) regulatory proteins. Therefore, some 
effort should be made toward under- 
standing how these three transmitter 
systems might regulate the ethanol 
sensitivity of, for example, ligand-gated 
ion channels, especially in the depen- 
dence or preference models (see Sig- 
gins et al. 1999). Such studies of 
metabotropic, posttranslational systems, 
and their related second messengers 
and kinases/phosphatases, have the 
potential to lead to new and exciting 
treatments for alcoholism or with- 
drawal phenomena. 

Since the synapse seems to be the most 
ethanol-sensitive neuronal element, 
the study of synapses either in isolation 
or under strong experimental control 
would seem to be paramount. In fact, 



at the cellular and molecular levels, 
there is a need especially for electrophys- 
iological studies of the role of all 
ethanol-sensitive transmitter systems 
in protracted abstinence, relapse, or crav- 
ing models. We encourage the use in 
these chronic models of new cutting- 
edge techniques to isolate pre- versus 
postsynaptic sites of action: for example, 
statistical analyses of spontaneous and 
miniature synaptic potentials or cur- 
rents, analysis of paired-pulse facilitation, 
and study of single-fiber synaptic units 
or synaptic pairs in slices, or autaptic 
synapses in cultures (Malinow 1991; 
Dobrunz et al. 1997; Goda and Stevens 
1998). It appears that some aspects of 
these exciting new avenues in alcohol 
research are now getting under way; 
for example, analysis of miniature 
synaptic currents for elucidation of 
presynaptic ethanol actions (R. Mor- 
risett, personal communication, July 
1998; J. Weiner, personal communi- 
cation, July 1998). 

There also is a need for additional 
cellular studies, but now in the depen- 
dence, relapse, and preference models, 
of drugs known to be efficacious in 
treating alcoholics. For example, it 
would be very interesting to know what 
membrane or synaptic effects acam- 
prosate, naltrexone, or GHB would 
have at various time points in a relapse 
or protracted abstinence model, or 
how long-term treatment with these 
drugs could alter the neuroadaptations 
seen in these models. This informa- 
tion could then be used strategically 
to develop additional new therapies. 

In terms of finding the neuroadap- 
tion(s) most important for alcoholism, 
we also need to examine the role of 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



neurotoxicity, as, for example, that 
produced by NMD A receptor actions, 
activation of excessive Ca ++ conduc- 
tances, or eicosanoids. Here we might 
benefit from review of the findings of 
the Walker/Hunter, Morrisett, Little, 
and Crews groups. The recent studies 
of the Collins group (see Corso et al. 
1998) represent a significant step in 
this direction. It would be important to 
know if the newer antagonists of NMDA 
receptors and Ca ++ currents block 
ethanol preference, relapse, or craving. 
In a related area, there is an urgent 
need for understanding possible changes 
in the anatomical distribution of trans- 
mitter receptors or their subunits in 
the dependence and preference models 
by using, for example, quantitative 
immunocytochemical confocal imaging 
of receptor subunits tagged with mon- 
oclonal antibodies, as carried out by the 
Morrison group (Siegel et al. 1995; 
Gazzaley et al. 1996; Nimchinsky et 
al. 1996). Such studies could tell us if 
neuroadaptation involves subtle trans- 
locations of receptors (e.g., from den- 
dritic spines to somata or dendritic 
shafts) or frank loss of receptors. The 
use of patch-clamp recording com- 
bined with quantitative single-cell 
reverse transcriptase-polymerase chain 
reaction (RT-PCR) in chronic ethanol, 
relapse, or preference models, similar 
to that currently being performed in 
acute ethanol paradigms by the Yeh 
group (Eberwine et al. 1992; Grigorenko 
and Yeh 1994), could tell us if neu- 
roadaptation involves changes in sub- 
unit composition (stoichiometry) in 
single cells. However, as with all such 
studies, there will be variability con- 
cerns and problems of quantification 



of measures across subjects, neuron 
types, or treatment groups. Therefore, 
there will be a great need in the future 
to develop new methods (e.g., statistical 
designs, quantification of immunocy- 
tochemical and RT-PCR data, stan- 
dardization of synaptic stimulation, 
and dose-response analyses) to deal 
with these sources of variability. 

Finally, there is a gap in our knowl- 
edge of the more long-term molecular 
changes that might occur in specific 
brain regions (e.g., the VTA, accumbens, 
or amygdala) in the chronic ethanol, 
relapse, or preference models. For this 
we need studies of the expression of 
IEGs (e.g., c-fos and c-jun), the RNA 
for important transmitter receptors 
(e.g., dopamine, 5-HT, and opiate 
receptors) and their proteins. For 
example, it would be important to 
know the time course of possible 
changes in opiate receptor, IEG, or 
CREB expression with chronic 
ethanol, relapse, or preference, as has 
been seen with chronic psychostimu- 
lant treatment (J.Q. Wang et al. 
1994; McGinty and Wang 1998). 

The Influence of Gender 
on Ethanol Dependence 
and Withdrawal 

Many neuroactive steroids are derived 
from progesterone and so occur at dif- 
ferent levels in males and females. 
Female rats show higher 3a,5a-THP 
levels in plasma and brain than male 
rats, and these levels fluctuate across 
the estrous cycle (Purdy et al. 1990; 
Paul and Purdy 1992; Corpechot et 
al. 1993). The levels of 3ct,5ct-THP 
in brain during estrus in female rats, or 
acute stress in male rats, are sufficient 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



to modulate GABA A receptor function 
in brain (Purdy et al. 1990, 1991; 
Paul and Purdy 1992). Functional 
changes in GABA A receptor stimulation 
and responses are observed during the 
estrous cycle and differ between 
female and male rats (Westerling et al. 
1991; Wilson 1992; Finn and Gee 
1993). In addition, levels of the excita- 
tory neuroactive steroid dehy- 
droepiandrosterone sulfate (DHEAS), 
are significantly higher in men than in 
women (Orentreich et al. 1984). 
Therefore, males and females operate 
with a differing hormonal regulation at 
the receptor level, in addition to gen- 
der differences in genomic regulation. 
The differences in the steroid hormone 
environment between males and 
females could have an effect on the 
influence of ethanol or other drugs in 
the brain. 

Studies have shown gender differences 
in measures of ethanol dependence and 
withdrawal and the response to 3a,5a- 
THP. Ethanol- with drawn female rats 
show greater sensitization to the anti- 
convulsant effect of 3a,5a-THP than 
ethanol- with drawn male rats (Devaud 
et al. 1995a). Female rats also exhibit 
increased sensitization to the anticonvul- 
sant effects of THDOC compared with 
male rats (Devaud et al. 1998). Further- 
more, we recently observed differences 
in the effects of ethanol dependence 
on both GABA A receptor and NMDA 
receptor subunit gene expression 
(Devaud and Morrow 1998; Devaud 
et al. 1998). Cerebral cortical levels of 
GABA A receptor al subunit peptide 
are not decreased in dependent female 
rats, although a decrease is consistently 
observed in male rats (Devaud et al. 



1997). Furthermore, chronic ethanol 
administration increases NMDA recep- 
tor NR1 subunit peptide expression in 
female cerebral cortical homogenates, 
but not in male cortex (Devaud and 
Morrow 1998). NMDA receptor NR2B 
subunit peptide expression is increased 
in both male and female cortical homo- 
genates (Devaud and Morrow 1998). 
Since both males and females exhibit 
comparable signs of ethanol depen- 
dence, including equivalent seizure 
thresholds during ethanol withdrawal 
(Devaud et al. 1995#), what is the 
significance of gender differences in 
the effects of ethanol dependence on 
GABA A and NMDA receptor subunit 
expression? How do such divergent 
alterations in these receptors produce 
similar behavioral adaptations? What 
are the modulatory mechanisms that 
control these responses? 

Studies in Human Alcoholics 

Are the alterations in receptor expression 
observed in animal models of ethanol 
dependence predictive of changes in 
human alcoholics? Like ethanol- 
dependent rodents, human alcoholics 
exhibit behavioral adaptations to pro- 
longed alcohol intake, including alco- 
hol tolerance and benzodiazepine and 
barbiturate cross-tolerance (Woo and 
Greenblatt 1979). Unfortunately, there 
is a paucity of data in human alcoholics, 
and the data that exist often are quite 
different from the effects of chronic 
ethanol exposure in animal models. 
There are examples of this dichotomy 
with respect to both GABA and NMDA 
receptor expression. 

Positron emission tomography 
(PET) studies show a decrease in 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



benzodiazepine binding in the frontal 
lobes of human alcoholics relative to 
normal control subjects (Gilman et al. 
1996). Preliminary single photon emis- 
sion computed tomography (SPECT) 
studies suggest a decrease in benzodi- 
azepine binding in the inferior medial 
frontal cortex and in the temporal and 
parietal cortices (Abi-Dargham et al. 
1995; Lingford-Hughes et al. 1998). 
PET studies of the metabolic (2- 
deoxyglucose) responses to a benzo- 
diazepine challenge show a reduction 
in the inhibitory effects of benzodi- 
azepines and further support the 
decrement of benzodiazepine recep- 
tors in specific brain regions of alco- 
holics (Volkow et al. 1997). These 
decrements in benzodiazepine receptor 
density are inconsistent with studies in 
animal models of ethanol dependence, 
where no change in benzodiazepine 
receptor binding is observed (see table 
1). However, there is a decrease in the 
sensitivity of GABA A receptors to ben- 
zodiazepines in animal studies (see 
table 1). In addition, the decrease in 
the density of low- affinity [ 3 H]musci- 
mol sites and the decline in GABA A 
receptor al subunit expression 
observed in the cerebral cortex of 
ethanol-dependent rodents are not 
observed in human studies. 

In postmortem human brain, the 
GABAergic correlates of ethanol depen- 
dence also appear to differ from rat 
models of ethanol dependence. 
[ 3 H]muscimol binding density is 
greater in alcoholic cerebral cortex 
(Tran 1981) and in the superior 
frontal gyrus of noncirrhotic alcoholics 
(Dodd et al. 1992) compared with 
normal control subjects. Significant 



increases, decreases, and no change in 
benzodiazepine binding have been 
reported (Freund and Ballinger 1988; 
Dodd et al. 1992; Dodd 1995). GABA A 
receptor al subunit mRNA levels 
were reported to increase in the frontal 
cortex of noncirrhotic alcoholics (Lewohl 
et al. 1997), although we recently 
found no change in GABA A receptor 
al or a4 subunit mRNA or peptide 
levels in frontal cortex of alcoholics 
(Mitsuyama et al. 1998). These results 
do not correlate with data from ani- 
mal models of ethanol dependence, 
which have shown reproducible 
changes in GABA A receptor gene 
expression. The most parsimonious 
explanation for these discrepancies, 
supported in part by the distinct 
regional differences in benzodiazepine 
binding observed with in vivo imaging 
techniques (Abi-Dargham et al. 
1995), is that changes in GABA A 
receptor expression are localized to 
particular cortical regions. In addition, 
differences between the longevity of 
human alcoholism and animal models 
of ethanol dependence probably con- 
tribute to these discrepancies. 

There are also discrepancies between 
human studies and animal studies with 
respect to changes in glutamate recep- 
tors. The kainate receptors GluR2 and 
GluR3 appear to be elevated in human 
postmortem hippocampus of alcohol 
abusers (Breese et al. 1995), but not in 
ethanol-dependent rats (Trevisan et al. 
1994; Buckner et al. 1997). Clearly, 
further studies of human alcoholics are 
critically needed. Human imaging 
studies will be particularly useful to our 
understanding of the adaptations 
induced by ethanol in human alcoholics. 



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The fact that ion channels represent 
a clear and prominent site of action of 
ethanol prompts speculation regarding 
the role of these channels in individual 
responses to ethanol. The findings that 
ethanol can act directly on specific ion 
channel subtypes suggests that allelic 
differences in channel subunit expression 
and/or amino acid composition could 
have dramatic effects of responses to 
ethanol. Individuals with a family history 
of alcoholism are known to have an 
innately greater tolerance (or lesser 
sensitivity) to ethanol than those with- 
out a family history (Schuckit 1994). 
Since even a minor mutation in subunit 
structure has been shown to change the 
sensitivity of an ion channel to ethanol 
(Mihic et al. 1997), it is possible that 
allelic differences in ion channel sub- 
units underlie genetic differences in 
ethanol responses. These speculations 
require additional research; however, the 
continuously accumulating evidence 
that ethanol acts on ion channels pro- 
vides the impetus to determine if these 
sites of ethanol action also contribute 
to the development of alcoholism. 

Interactions Among 
Neurotransmitter Systems 

GABA A -NMDA receptor interactions 
have been documented. Grayson and 
colleagues (Memo et al. 1991; Zhu et 
al. 1995) have demonstrated NMDA- 
mediated alterations in GABA A recep- 
tor function and gene expression in 
cultured neurons. Since many neurons 
contain both GABA A and NMDA 
receptors and ethanol acts directly on 
subtypes of both GABA A and NMDA 
receptors, this type of modulatory 
activity may play an important role in 



adaptations to ethanol. This type of 
interaction may contribute to the 
complexity of factors that regulate ion 
channel function and expression and 
may help explain regional differences in 
the effects of chronic ethanol adminis- 
tration on ion channel function and 
gene regulation. 

Integration of Ethanol's Effects 
at the Level of Signal Transduction 

The AC/cAMP system in neurons has 
been the most extensively studied signal 
transduction pathway with respect to 
chronic effects of ethanol. However, 
the mechanism of the desensitization 
of this system following chronic 
ethanol treatment is not yet clear. 
New knowledge regarding the isoforms 
of AC, their differential sensitivity to 
the acute effect of ethanol, and their 
differential modes of regulation, sug- 
gests that investigations of quantitative 
or qualitative changes induced by chronic 
ethanol exposure in these enzymes may 
prove fruitful. In addition, more 
detailed analysis of G protein quantity — 
in particular, in neurons — may provide 
a better understanding of the role of 
these proteins in AC desensitization. 
It is important to realize that G proteins 
affect certain isoforms of AC through 
their |3y subunits, as well as the a sub- 
units, and therefore G proteins other 
than Gs or Gi could be involved in 
changes in AC activity produced by 
chronic ethanol treatment. 

In addition to AC, some studies of 
the effects of chronic ethanol expo- 
sure on PKA and PKC have been 
performed. There is significant cross- 
talk between these systems; for exam- 
ple, PKC can modulate the activity of 



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certain isoforms of AC, leading to 
changes in cAMP generation and 
PKA activity. Although chronic ethanol 
treatment has been shown to increase 
neurite outgrowth mediated by PKC 
in vitro, the significance of such effects 
in vivo (e.g., new synaptic connec- 
tions) and the interaction of the PKC 
and PKA pathways to produce such 
changes have not been investigated. 

Other signal transduction system 
interactions that have not yet been 
addressed are those between the het- 
erotrimeric G proteins and the "small" 
GTPases of the Ras superfamily 
(Bokoch 1996). There is substantial 
evidence that G protein (3y subunits 
mediate activation of the mitogen- 
activated protein (MAP) kinase path- 
way by G protein-coupled receptors, 
and do so through activation of Ras. 
Because the classical MAP kinase 
pathway (i.e., ERK1 and ERK2) is 
mitogenic, changes in the activity of 
this pathway could conceivably affect 
neuronal survival by opposing apoptotic 
effects and could also be involved in 
the generation of new synaptic con- 
nections, that is, the structural changes 
associated with neuroadaptation. A study 
by Brambilla and colleagues (1997) 
demonstrated a role for the Ras signaling 
pathway in a process of memory consol- 
idation in mice, suggesting that this 
pathway may play a more general role 
in neuroadaptation. 

The myriad interconnections among 
various signal transduction systems 
(Hop kin 1997) that influence cell sur- 
vival, differentiation, and responses to 
stress and other external stimuli provide 
an important area for investigation of 
the chronic effects of ethanol that lead 



to neuroadaptation. The studies of the 
AC signaling system have already pro- 
vided evidence for a role of this system 
in ethanol neuroadaptation. Investiga- 
tions of the interactions of this system 
with other cellular signaling pathways, 
and modification of these interactions by 
ethanol, are promising areas of research. 

Integration of Ethanol's Effects at 
the Level of Neural Circuitry 

Systems (neural circuitry) analyses of 
ethanol actions and preference are among 
the most laborious and time-consum- 
ing, perhaps accounting for the relative 
paucity of such studies in the alcohol 
field. The two most popular approaches 
for such investigations are ( 1 ) the use 
of multiple recording and stimulus 
sites in anesthetized animals and (2) 
the use of single- or multi-unit record- 
ings in freely moving animals that may 
be self- administering ethanol in an oper- 
ant situation. Several recent and prelim- 
inary studies highlight the possibilities 
these approaches offer for understand- 
ing the neural circuit correlates of 
ethanol preference. 

Hints of the first type of study, using 
long-term ethanol treatment and with- 
drawal followed by electrophysiological 
studies in anesthetized animals, appeared 
in the early reports from the Walker/ 
Hunter group (cited in the Synaptic 
Transmission; Presynaptic Mechanisms 
section earlier in this chapter). How- 
ever, these were studies on relatively 
local circuits (e.g., Schaffer collaterals 
to CA1 pyramidal neurons) within the 
hippocampal CA1 and within the den- 
tate. Similar but more systems-ori- 
ented studies are now emerging: for 
example, those based on earlier findings 



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that the function of (local) recurrent 
inhibitory circuits (e.g., as expressed 
in paired-pulse inhibition) recorded in 
CA1 and dentate was markedly regu- 
lated by extrinsic sources from VTA 
and lateral septum, and that acute 
ethanoPs effect in enhancing this 
inhibitory function was exerted at 
these extrinsic sites (Criado et al. 
1994). Two weeks of chronic ethanol 
vapor treatment followed by withdrawal 
for 2-8 hours (under halothane anes- 
thesia) reduced paired-pulse inhibition 
(only in dentate) with a return to nor- 
mal within 24 hours. Interestingly, 
acute ethanol injected at this point pro- 
duced a paradoxical decrease in paired- 
pulse inhibition, in contrast to ethanol's 
effect in controls, suggesting complex 
adaptive circuit responses between 
hippocampus and VTA/lateral septum 
(Steffensen and Henriksen 1997). 
Similar studies now planned for the 
hippocampal/accumbens/amygdala 
complex could reveal critical informa- 
tion regarding the adaptation to chronic 
ethanol in the mesolimbic, extended 
amygdala reward pathways. 

The second favored approach, study- 
ing ethanol preference in awake ani- 
mals, is best exemplified by a recent 
publication from the Woodward group 
(Woodward et al. 1998; see also Givens 
et al. 1998). This work elegantly illus- 
trates what can be learned about the 
function of temporal-spatial functioning 
of distributed neural elements during 
operant responding for drugs of abuse. 
Here, simultaneous groups of many 
individual neurons in one or more brain 
regions are recorded during specific, 
controlled behavioral events. Performing 
such recordings from accumbens and 



amygdala neurons of the rat meso- 
corticolimbic circuit during operant 
responding for ethanol has revealed 
that accumbens neurons show multiple 
activity patterns within each cell just 
prior to and during ethanol responding, 
with slowing of firing predominating. 
Recording of neuronal ensembles (e.g., 
consisting of 25 neurons) has begun 
to reveal what appears to be patterns 
of response activity across multiple 
neurons that, with the use of sophisti- 
cated statistical pattern recognition 
techniques, may ultimately be used to 
reveal how networks of neurons con- 
trol behavior (e.g., via propagation of 
complex conditioned cues within the 
mesolimbic system to initiate ethanol - 
seeking behavior). 

As pointed out by these authors, 
the power of this method for under- 
standing the systems complexities of 
ethanol actions and alcohol addiction 
is great. For example, in this study 
neuronal recordings began only after 
the rats were trained to self- administer 
ethanol; it would be highly desirable 
to observe neuronal ensemble activity 
during learning of this behavior, to bet- 
ter understand the events leading up 
to the addiction process. Alternatively, 
one could apply this method to a pro- 
tracted abstinence model to follow 
changes in ensemble activity just prior 
to relapse, when craving might be at 
its highest. In addition, similar recording 
methods may be used in conjunction 
with microdialysis (Ludvig et al. 1998) 
or voltammetric (Rebec 1998) methods, 
to assess neurochemical correlates (e.g., 
transmitter release) of the electrophys- 
iological and behavioral events in these 
alcohol models. 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



Finally, the use of organotypic 
(explant) brain slice cultures is a relatively 
new approach for the future study of 
adaptation in neural circuits with 
chronic ethanol (Thomas et al. 1998). 
These cultures can be prepared from 
horizontal slices of whole forebrain to 
include, for example, a functional corti- 
costriatal pathway that can be stimulated 
selectively and recorded at multiple sites 
with extracellular field and patch-clamp 
techniques. These cultures may then 
be examined before and after chronic 
ethanol treatment (by bath application), 
followed by ethanol withdrawal. It also 
may be of interest to apply an in vitro 
sort of "protracted abstinence" protocol 
to such cultures. Major advantages of 
such a model system include the possi- 
bility of using a within-subjects design 
and allowing cutting-edge recording and 
pharmacological techniques for analysis 
of synaptic events across neural circuits 
or neuronal ensembles during various 
stages of the ethanol protocol. 

STRATEGIES 

FOR FUTURE WORK 

Paradigms for Ethanol 
Administration and Choice 
of Neural Systems for Study 

The most difficult issue to address in 
studies of cellular and molecular changes 
induced by chronic ethanol exposure is 
whether these observed changes are in 
fact related to neuroadaptation to 
ethanol. The previous sections have 
focused on neurochemical and molec- 
ular alterations induced by chronic 
ethanol ingestion or exposure, and in 
some cases the relationship of changes 



in the function of a particular neuro- 
chemical system to ethanol-induced 
neuroadaptation has been discussed. 
However, more work is needed to 
determine the importance of each of 
these "candidate systems" for ethanol- 
induced neuroadaptations. As pointed 
out earlier, any change in neuronal 
function is likely to have many down- 
stream effects. Therefore, one will find 
effects of chronic ethanol treatment on 
any number of neuronal systems, but the 
question of which is the primary effect 
that mediates behavioral aspects of 
neuroadaptation remains. Added to 
this complexity is the fact that different 
investigators use differing paradigms 
of ethanol administration, often not 
measuring blood and brain ethanol 
levels, let alone the development of 
tolerance, physical dependence, or 
sensitization. Either it is assumed that 
the ethanol treatment has produced a 
neuroadaptation, or this aspect is 
ignored, and cellular or neurochemical 
changes are simply ascribed to the 
chronic ethanol exposure. A more prof- 
itable approach is to use or develop 
ethanol administration paradigms that 
lead to the desired, measurable behaviors 
or physiological changes characteristic 
of tolerance or physical dependence, for 
example, and to correlate candidate 
cellular and molecular changes tempo- 
rally and quantitatively with the partic- 
ular behavioral aspect of neuroadaptation 
that is under investigation. 

There are a number of other 
approaches that can lead to a better 
understanding of the mechanisms of 
adaptation to ethanol. One of these 
approaches has been used with some 
success in the studies of tolerance 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



already described — alteration, prior to 
alcohol exposure, of brain systems that 
are believed to play a role in neuroad- 
aptation (extrinsic systems). Studies 
can then be performed to determine 
whether these molecular or neurochem- 
ical interventions affect the develop- 
ment, expression, or loss of tolerance, 
physical dependence, or sensitization. 
This approach includes newer methods 
such as generation of transgenic or 
knockout mice. It should be kept in 
mind that the extrinsic systems do not 
necessarily undergo changes during 
the course of chronic ethanol adminis- 
tration (although they can). However, in 
the absence of these systems, particular 
neuroadaptive processes cannot occur. 
More consideration also needs to be 
given to the adaptation that is to be 
studied. For example, the complex 
nature of tolerance means that one 
cannot simply administer ethanol to 
animals and generate a generic form of 
"tolerance." First, the form of func- 
tional tolerance that is being studied 
must be considered, including the role 
of learning and memory. The develop- 
ment of environment-dependent tol- 
erance, for example, may entail changes 
that are different from those occurring 
in association with environment- 
independent tolerance (Melchior and 
Tabakoff 1985). Second, it is impor- 
tant, when assessing changes at the 
cellular level, to consider which effect 
of ethanol is demonstrating tolerance. 
For instance, one might examine 
changes in the GABA A receptor in 
relation to the development of toler- 
ance to the anxiolytic and/or incoor- 
dinating effects of ethanol — but is this 
system as important in tolerance to 



the hypothermic effect of ethanol? 
This question bears on the notion of 
"intrinsic" systems — that is, the systems 
that encode tolerance to particular 
effects of ethanol. If we know which 
neurochemical systems are involved in 
a particular behavior, we can devise 
experiments to determine whether 
changes in those systems are important 
for tolerance to the effects of ethanol 
on that behavior. It is often assumed 
that those systems most sensitive to 
acute perturbations by ethanol will 
adapt to the chronic presence of 
ethanol. While this is undoubtedly 
true, these systems may or may not be 
important for a particular neuroadaptive 
response in the whole organism. 

Similar considerations can apply to 
studies of physical dependence; that is, 
it is important to consider the under- 
lying mechanisms of withdrawal signs 
and symptoms when investigating cel- 
lular or neurochemical changes that 
may be responsible for these signs. 
The glutamatergic and GABA systems 
may well play important roles in gen- 
erating alcohol withdrawal hyperex- 
citability and convulsions, but not in 
other withdrawal signs or symptoms 
such as autonomic disturbances. 

These considerations raise the issue of 
the relevance of studies of the chronic 
effects of ethanol on neurons in culture. 
In such systems, no behavioral correlates 
can be assessed, and "adaptation" is 
defined entirely on the basis of 
changes in response to ethanol of an 
in vitro system, or changes that occur 
after alcohol has been removed from the 
system (withdrawal). While these sys- 
tems can be extremely useful for assay- 
ing biochemical and molecular effects 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



of ethanol, a careful analysis of the 
relationship of the in vitro system to 
the in vivo situation is required. For 
example, primary neuronal cultures 
are often used to assess ethanol 
effects, but these neurons are in the 
developmental stage, and results 
obtained with these systems may not 
reflect effects of ethanol in the adult 
brain. Similarly, transformed neurons 
may have different characteristics from 
native neurons. In all in vitro systems, 
many of the interconnections among 
neurons may be lost, resulting in mis- 
leading conclusions. On the other 
hand, if the cell culture system can be 
shown to have characteristics of a 
neuronal system in the adult brain, 
the neuronal culture may provide an 
excellent model to investigate adaptive 
responses to ethanol that may be 
extrapolatable to the behaving animal. 

Application of Simple 
Models That Have Provided 
Information About Learning 
and Memory 

As discussed earlier, invertebrate systems 
have been used extensively to study learn- 
ing and memory processes, and these 
animals could also be used to advantage 
to study adaptation to alcohol and other 
drugs. The advantage of the invertebrate 
systems is their relative simplicity; that is, 
only a limited number of neurons may 
be involved in the measured behaviors. 
These systems can be investigated both in 
vivo and in vitro, but in contrast to the 
cell cultures alluded to in the previous 
section, it may be easier to demonstrate 
the similarity of the cell culture system to 
the intact animal. As illustrated by the 
work of Kandel and his colleagues, the 



relative simplicity of the Aplysia system 
has allowed for a detailed analysis of 
the cellular and molecular mechanisms 
involved in the adaptations that underlie 
learning and memory, and these mecha- 
nisms also appear to play a role in adap- 
tation in the mammalian brain. The 
introduction of ethanol into such systems 
would provide a model in which toler- 
ance and physical dependence, as well as 
sensitization, might be induced, and the 
pathways leading to these adaptive 
changes could be determined in the man- 
ner described for the studies of learning 
and memory. It is interesting to note that 
ethanol has been shown to accelerate 
the rate of decay of posttetanic potentia- 
tion at an identified synapse in a ganglion 
preparation from Aplysia, and that resis- 
tance to this effect of ethanol occurs 
rapidly after ethanol exposure (Barondes 
et al. 1979). Although high concentra- 
tions of ethanol were used in these par- 
ticular studies, this type of system has the 
potential to provide significant infor- 
mation about the pathways involved in 
ethanol-induced neuroadaptation. More 
recently, studies of ethanol sensitivity 
and tolerance in Drosophila have begun 
to illustrate the use of simpler, inverte- 
brate models in alcohol research 
(Moore et al. 1998). 

More Complex Cellular 
Models for Correlations of 
Physiology and Behavior 

Other, less simple models also offer 
advantages in the search for behaviorally 
meaningful neuroadaptations to 
ethanol. Earlier in this chapter we dis- 
cussed the effects of chronic ethanol 
on hippocampal LTP, which has been 
much discussed as a cellular correlate of 



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spatial memory and learning (see, e.g., 
Stevens 1998). In fact, this is just one 
form of synaptic plasticity that may be 
altered by chronic ethanol and could 
provide a mechanism underlying or 
related to tolerance. Other forms 
include posttetanic potentiation, short- 
term potentiation, and long-term 
depression (LTD) of synaptic transmis- 
sion. These measures may be extracellu- 
larly or intracellularly recorded, both in 
vivo as well as in vitro in a slice prepara- 
tion, and correlated with behavioral 
changes occuring in the source animal 
just prior to or during the recordings. 
The major considerations and 
advantages of the brain slice plasticity 
models are as follows: 

1. Mammalian tissue may be used for 
closer modeling of human disor- 
ders. 

2. Generally low, known concentra- 
tions of ethanol and other drugs 
may be applied. 

3. Anesthetics are not required. 

4. Relatively small numbers of neurons 
can be included. 

5. Ethanol can be chronically admin- 
istered to the animal followed by 
later slice preparation and continued 
incubation in ethanol in vitro, for 
subsequent controlled withdrawal. 

6. The several mechanisms (e.g., 
NMD A receptor and AMPA recep- 
tor activation, GABA receptors, 
Ca ++ channels, kinases) involved in 
LTP and the other forms of synaptic 



plasticity are precisely the ones most 
sensitive to acute and chronic ethanol. 

7. The genetically defined or altered 
animals described in the next section, 
as well as those subjected to the newer 
protracted abstinence protocols, can 
be subjects for these models. 

These forms of synaptic plasticity could 
serve as ideal candidates for sites or mech- 
anisms underlying some behavioral neu- 
roadaptations occuring with ethanol 
preference, alcohol-seeking behavior, or 
alcohol craving. Such multiple plasticity 
measures in transgenic mice have pro- 
vided a considerable depth of correlates 
for behavioral and neuropathologies 
changes (see, e.g., Krucker et al. 1998). 
Given the possible role of the hippo- 
campus in spatial memory, such plastic- 
ity changes could also be highly relevant 
for sites of adaptations responsible for 
spatial conditioned cues related to drug- 
seeking behavior. Interestingly, our pre- 
liminary studies have shown that the 
anticraving agent acamprosate enhances 
LTP in rat hippocampal slices (Madamba 
and Siggins manuscript in preparation). 
It also may be relevant that several forms 
of hippocampal LTP and LTD have been 
shown to involve opiate mechanisms 
(see, e.g., Francesconi et al. 1997). 

Brain slices of other brain regions (e.g., 
accumbens, VTA, amygdala) known to 
be involved in ethanol preference and 
ethanol-seeking behavior, taken from 
genetically manipulated animals or those 
previously subjected to chronic ethanol 
protocols (including forced abstinence), 
may also be excellent candidates for 
elucidation of cellular sites or molecular 
mechanisms underlying behavioral 



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Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



neuroadaptations occuring in ethanol 
dependence or craving. Ideally, data from 
these slices should be correlated with 
the behaviors expressed by the animals 
from which the slices were taken. 

Two other quite different but poten- 
tially very powerful cellular models for 
correlations to ethanol-induced behav- 
ioral neuroadaptations, discussed earlier 
in this chapter, are worth highlighting 
here: organotypic forebrain cultures 
(Thomas et al. 1998) and multineuron 
recording in freely moving animals 
(Woodward et al. 1998). In both these 
cases neural circuits could be studied 
under a variety of conditions, including 
elicitation of synaptic plasticity and the 
various treatment protocols for chronic 
ethanol, self- administration, and pro- 
tracted abstinence. In the case of organ- 
otypic cultures, tissue from transgenic and 
null-mutant mice could be investigated. 
In the case of multineuron recording from 
freely moving animals, generation of the 
various forms of synaptic plasticity could 
be obtained via stimulation electrodes, 
together with measures of precisely con- 
trolled and monitored behavioral cor- 
relates (e.g., using learning and 
memory paradigms such as the Y 
maze). Superimposition of drug treat- 
ments for the various target systems 
(e.g., the therapeutic agents mentioned 
earlier in this chapter, in the section 
Systems That Influence the Development, 
Maintenance, and Loss of Tolerance, 
Dependence, and Sensitization) could 
provide several more fruitful avenues 
of research in both of these models. 

Genetics 

There is substantial evidence that 
genetic variation is one determinant of 



individual differences in neuroadapta- 
tion to ethanol. Environmental variation 
and genotype-environment interaction 
likely also bear some responsibility for 
individual variation. Investigations of 
genetically heterogeneous populations 
are limited in their ability to separate 
genetic from environmental sources of 
variation. However, there are a number 
of methods that can be used to ascer- 
tain genetic influence on neuroadaptive 
processes. Much of the work that has 
been done in animals has relied on 
inbred strains, which provide well- 
defined genetics. Differences between 
inbred strains, when environmental 
factors have been carefully controlled, 
can be interpreted as genetically deter- 
mined differences. Differences between 
individuals within a strain must be due 
to nongenetic factors. 

Selected lines provide another pow- 
erful tool to assess genetic influences on 
ethanol-induced neuroadaptation. Work 
done with animals that display differ- 
ential sensitivity to alcohol withdrawal 
seizures (WSP and WSR mice), 
described earlier in this chapter, as well 
as work with a second selection, high 
alcohol withdrawal (HW) and low alco- 
hol withdrawal (LW) mice (Hoffman 
et al. unpublished data), has suggested 
certain mechanisms that may underlie 
alcohol withdrawal seizures. In addition, 
mice have been selectively bred for 
differences in acute functional tolerance 
to an incoordinating effect of ethanol 
(high acute functional tolerance and 
low acute functional tolerance mice), 
and these lines are currently being 
used for a variety of studies. Selected 
lines do not currently exist that have been 
bred for differences in ethanol-induced 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



sensitization, but FAST and SLOW 
mouse lines, bred for differences in 
sensitivity to the acute activating effects 
of ethanol, have been found to differ 
in susceptibility to ethanol sensitization 
as well. In theory, biochemical differ- 
ences between selected lines of animals 
are related to the trait for which they 
have been selectively bred, and contin- 
uation of studies with the selected lines, 
as well as the generation of lines selected 
for differences in other aspects of neu- 
roadaptation, should be encouraged. 

Recombinant inbred strains com- 
prise a powerful genetic animal model 
appropriate for gene mapping of alco- 
hol neuroadaptation. For example, the 
BXD/Ty recombinant inbred strains 
are a set of 26 inbred mouse strains 
produced from the F2 cross of the 
C57BL/6J and DBA/2J strains. They 
are particularly useful for examination 
of genetic correlations and for mapping 
of genes affecting the measured pheno- 
type. The observation that two traits 
differ between two inbred strains does 
not provide evidence of a genetic cor- 
relation. However, if many strains are 
tested and sensitivity for trait one is 
predictive of sensitivity for trait two, 
this is suggestive of common genetic 
mediation of the two traits. 

In large part, the traits in question 
are under polygenic control: more than 
one gene contributes to the magnitude 
of the trait. Quantitative trait loci are 
genes whose collective effects con- 
tribute to the determination of such 
traits. The goal in behavioral genetic 
studies of the constituents of alcohol 
dependence is gene identification and, 
ultimately, gene cloning, to provide a 
specific target for disease intervention. 



QTL analysis represents one step in a 
multistep approach to gene identifica- 
tion. Some QTL mapping work has 
been performed for phenotypes like 
tolerance, withdrawal, and sensitization, 
but additional work is needed to convert 
provisional markers from recombinant 
inbred studies to confirmed linkages. 

Certain sophisticated genetic prepa- 
rations are beginning to be utilized in 
alcohol research and should be applied 
to studies of neuroadaptation. Congenic 
strain production through repetitive 
backcrossing permits the creation of 
animals that are genetically segregating 
at only one genetic locus that has been 
targeted. Such congenic strains can be 
produced by direct genotyping to 
provide further verification of QTLs 
and to facilitate the identification of 
important functional genes. 

New or altered genes can be stably 
introduced into the mouse genome by 
the use of transgenic technology, 
which is now readily available. These 
genes can be expressed in all tissues or 
targeted to particular organs or tissues, 
such as brain. The extension of this 
technology to the study of behavior is 
growing, and it can certainly be used to 
study the role of specific genes, sug- 
gested by past investigations, in neuro- 
adaptation to ethanol. The production 
of knockout mice involves homologous 
recombination in embryonic stem 
cells combined with the generation of 
chimeric mice. These chimeric mice 
can then be bred to an inbred strain, 
and the offspring genotyped for the 
presence of the mutation. In some cases, 
knockout of a gene critical to develop- 
ment can mean death to the recipient. 
However, techniques have now been 



152 



Neuroadaptation to Ethanol at the Molecular and Cellular Levels 



devised to permit creation of condi- 
tional knockouts in which a gene can be 
eliminated in specific classes of cells. 
Inducible knockout procedures are also 
being developed to circumvent prob- 
lems associated with knockout of a gene 
during development. There is infinite 
opportunity to develop transgenic and 
knockout mice targeting any number 
of neurochemical processes that might 
be expected to be involved in neuroad- 
aptation to ethanol. 

Application of Findings 
at the Cellular, Biochemical, 
and Molecular Levels to the 
Development of Effective 
Intervention Strategies 

In order to achieve a level of knowledge to 
develop rational strategies for inter- 
vention, it is important to understand 
mechanisms that underlie neuroadaptive 
responses to ethanol. Once mechanisms 
are understood, potential intervention 
strategies include pharmacotherapy and 
genetic therapies. Gene therapy has 
emerged as both a novel treatment 
modality and a powerful tool for basic 
science investigations. Viral vectors 
can transfer and express foreign genes 
in a wide variety of nondividing mam- 
malian cells, and in the case of adeno- 
associated virus (AAV) vectors, studies 
have demonstrated stable, long-term, 
nontoxic gene expression in brain 
(McCown et al. 1996; Xiao et al. 1997). 
Studies are needed that will utilize 
gene transfer techniques to probe ques- 
tions central to ethanol's pathological 
actions. However, before this goal 
becomes a reality, many studies are needed 
to refine the technology of gene delivery. 
Vectors must be designed that allow 



long-term expression throughout brain 
without producing toxic side effects. 
Inducible vectors — for example, vec- 
tors that can be turned on by adminis- 
tration of exogenous agents — could 
be highly advantageous for therapeu- 
tic applications. These studies are very 
promising and well worth the invest- 
ment for the promise of future thera- 
peutic applications. 

ACKNOWLEDGMENTS 

Portions of this chapter were adapted 
from the following reviews: Tabakoff 
and Hoffman 1992, Morrow 1995, 
Tabakoff and Hoffman 1996£, 
Grobin et al. 1998, and Tabakoff and 
Hoffman 1998. 

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Chapter 5 

Neurotoxicity of Alcohol: Excitotoxicity, 
Oxidative Stress, Neurotrophic Factors, 
Apoptosis, and Cell Adhesion Molecules 

Fulton T. Crews, Ph.D. 



KEY WORDS: toxic drug effect; AODR (AOD [alcohol or other drug] related) 
disorder; neuron; chronic AODE (effects of AOD use, abuse, and dependence); 
brain damage; NMD A receptors; physiological stress; oxidation-reduction; growth 
promoting factors; cytolysis; literature review 



Studies of alcohol-induced brain dam- 
age have clearly indicated that alcohol 
is neurotoxic. Alcoholics are at 
increased risk for brain damage from a 
variety of causes, including poor 
nutrition, liver disease, and head 
trauma. Furthermore, alcoholic 
dementia is the second leading cause 
of adult dementia in the United 
States, accounting for approximately 
10 percent of the cases (Alzheimer's 
disease is the leading cause, account- 
ing for 40 to 60 percent of cases). A 
variety of studies report that 50 to 75 
percent of sober, detoxified, long- 
term alcohol-dependent individuals 
suffer from some degree of detectable 



cognitive impairment, with approxi- 
mately 10 percent suffering from serious 
dementia (Martin et al. 1986; Char- 
ness 1993; Dufour 1993). Although 
more research is required to precisely 
delineate the effects of alcohol on var- 
ious types of brain function, there 
appears to be a continuum of moderate 
deficits in the majority of long-term 
alcoholics, progressing to much more 
severe deficits of Wernicke's disease 
and Wernicke's encephalopathy with 
Korsakoff's amnestic syndrome (But- 
terworth 1995; Pfefferbaum et al. 
1996). A variety of lifestyle factors, 
including nutrition, are implicated in 
the more severe cases. However, all of 



F.T. Crews, Ph.D., is director of the Center for Alcohol Studies and professor of psychiatry and 
pharmacology at the University of North Carolina at Chapel Hill, CB#7178, Thurston Bowles Bldg., 
Chapel Hill, NC 27599-7178. 



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these cases on the continuum appear to 
be related to alcohol consumption and 
to amount of alcohol consumed. That 
is, the more severe cases are associated 
with more severe and chronic long- 
term alcoholism (Butterworth 1995; 
Pfefferbaum et al. 1996). 

Alcohol-induced changes in the 
structure of adult brain have been 
studied in both humans and rodents 
(Charness 1993). A variety of post- 
mortem histological analyses as well as 
supporting imaging analyses suggest 
that chronic alcohol changes brain 
structure. Computed tomography 
(CT) and magnetic resonance imag- 
ing (MR1) studies of human brain 
have repeatedly shown enlargement 
of the cerebral ventricles and sulci in 
most alcoholics. The enlargement of 
the ventricles and sulci essentially 
reflect a shrinking of the brain mass. 
This is consistent with studies on 
postmortem brain tissue showing that 
alcoholics have a reduction in total 
brain weight. In particularly severe 
alcoholics, global cerebral hemisphere 
and cerebellar brain weights are sig- 
nificantly reduced compared with 
control subjects and moderate 
drinkers (Harper and Kril 1993). 
Some of this loss of brain mass is 
likely due to actual loss of neurons 
and resulting loss of myelin sheath 
white matter, which normally envelops 
neuronal extensions. However, a por- 
tion of the loss in brain mass is also 
likely to be due to a reduction in the 
brain parenchyma — that is, the size of 
the cells and their processes — during 
chronic alcohol abuse. Studies have 
indicated that within 1-5 months of 
recovery from alcoholism, and with 



sustained abstinence, the size of the 
brain returns toward normal levels 
(Muuronen et al. 1989; Pfefferbaum 
and Rosenbloom 1993; Pfefferbaum 
et al. 1998). It is likely that this return 
involves an increase in neuronal cell 
size, arborization, and density of the 
neuronal processes that make up cel- 
lular brain mass, as well as increases in 
the number and size of glial cells 
(Franke et al. 1997). Although it is not 
clear exactly how alcoholism leads to a 
reduction in brain weight and volume, it 
is clear that this does occur during active 
alcohol abuse, and that some recovery 
of brain mass does occur during absti- 
nence. More studies are needed to more 
clearly understand the mechanisms 
underlying these events. 

Some studies have focused on the 
frontal lobes as being particularly sen- 
sitive to alcohol-induced changes 
(Jernigan et al. 1991). Quantitative 
morphometry suggests that the frontal 
lobes of the human brain show the 
greatest loss and account for much of 
the associated ventricular enlarge- 
ment. Specific types of brain cells 
appear to be disrupted. Both gray 
matter, which is composed largely of 
neurons, and white matter, which 
involves neuronal tracks surrounded 
by myelin sheaths, appear to be 
decreased. Harper and colleagues 
(1987) found that neuronal density in 
the superior frontal cortex was 
reduced by 22 percent in alcoholics 
compared with nonalcoholic control 
subjects, in contrast to other areas of 
the cortex, which were not different 
between the groups. Furthermore, the 
complexity of the basal dendritic 
arborization of layer III pyramidal 



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Neurotoxicity of Alcohol 



cells in both superior frontal and motor 
cortices was significantly reduced in 
alcoholics compared with control sub- 
jects. A reduction in dendritic arboriza- 
tion of Purkinje cells in the anterior 
superior vermis of the cerebellum 
was also found in alcoholics. Taken 
together, the data demonstrate a 
selective neuronal loss, dendritic sim- 
plification, and reduction of synaptic 
complexity in specific brain regions of 
alcoholics. It is uncertain how these 
cellular lesions relate to selective loss 
of white matter that appears to occur 
particularly in frontal lobes. One rea- 
son these frontal lobe changes are 
more evident is the greater proportion 
of white matter to cortical gray matter 
in the frontal regions. Frontal lobe 
shrinkage has been reported with or 
without seizures, with some studies 
suggesting that temporal lobe shrink- 
age occurs particularly in individuals 
with alcohol withdrawal seizure his- 
tory (Sullivan et al. 1996). Decreases 
in the amounts of N-acetylaspartate in 
the frontal lobe, a measure of neuron 
levels, also illustrate frontal lobe 
degeneration in alcoholics (Jagan- 
nathan et al. 1996). Alcoholics with 
more severe brain disorders, such as 
Wernicke's and/or Korsakoff's syn- 
drome, show more significant reduc- 
tion in white matter and more 
extensive brain region degeneration, 
which is consistent with the greater 
alcohol consumption associated with 
more severely damaged individuals. 

Studies have found that in addition 
to the global shrinkage of brain 
regions, certain key neuronal nuclei 
that have broad-ranging effects on 
brain activity are selectively lost with 



chronic alcohol abuse. Perhaps the 
most extensively studied are the 
cholinergic basal forebrain nuclei, 
which are also lost in Alzheimer's dis- 
ease. Animal studies and some human 
studies have suggested that this region 
is particularly damaged in alcoholic 
subjects. Arendt (1993) found a sig- 
nificant loss of neurons in this region 
in alcoholic Korsakoff's psychosis 
patients. Additional brain nuclei that 
appear to be particularly sensitive are 
the locus coeruleus and raphe nuclei. 
These two nuclei contain many of the 
noradrenergic and serotonergic neurons 
within the brain, respectively. Although 
these nuclei are small in size, they are 
particularly important because their 
neuronal processes project throughout 
the brain and modulate global aspects 
of brain activity. Chemical studies 
have shown abnormally low levels of 
serotonergic metabolites in the cerebro- 
spinal fluid of alcoholics with Wernicke - 
Korsakoff syndrome, and more recent 
morphological studies have found a 
significant reduction (e.g., 50 per- 
cent) in the number of serotonergic 
neurons from the raphe nuclei of 
severe alcoholic cases studied com- 
pared with control subjects (McEntee 
and Mair 1990; Halliday et al. 1995; 
Baker et al. 1996; Higley and Bennett 
1999). Thus the serotonergic system 
appears to be disrupted in alcoholics, 
especially in severe alcoholics. Several 
investigators have also reported signif- 
icant noradrenergic cell loss in the 
locus coeruleus (Arendt et al. 1995; 
Arango et al. 1996; Lu et al. 1997), 
although not all have found this loss 
(Harper and Kril 1993). Certain neurons 
that contain the peptide vasopressin 



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may be sensitive to chronic ethanol- 
induced neurotoxicity in both rats and 
humans (Harding et al. 1996; Madeira 
et al. 1997). Damage to hypothalamic 
vasopressin and other peptide-con- 
taining neurons could disrupt a vari- 
ety of hormone functions as well as 
daily rhythms that are important for 
healthy living. Additional studies are 
needed to determine which specific 
cell groups within the brain might be 
particularly damaged. The findings of 
specific neuronal loss in small but 
functionally significant brain areas 
could result in global changes in 
attention, mood, and personality that 
are difficult to quantify but have a 
great impact on brain function and 
overall behavior. 

Long-term ethanol intoxication is 
not necessary to cause brain damage. 
Studies have shown that as little as a 
few days of intoxication can lead to 
neuronal loss in several brain areas, 
including dentate gyrus; entorhinal, 
piriform, insular, orbital, and perirhi- 
nal cortices; and the olfactory bulb 
(Collins et al. 1996). These structures 
are involved in frontal cortical neu- 
ronal circuits, including the limbic 
and association cortex. These findings 
are consistent with human studies 
reporting damage to entorhinal cortex 
(Ibanez et al. 1995) and significant 
hippocampal shrinkage in alcoholics 
(Harding et al. 1997). Hippocampal 
damage during chronic ethanol treat- 
ment has been correlated with deficits 
in spatial learning and memory 
(Franke et al. 1997). Thus, cortical 
and hippocampal damage also occurs 
with chronic ethanol treatment, and 
relatively short durations of alcohol 



abuse may cause some form of damage. 
Additional studies are needed to under- 
stand the molecular mechanisms in- 
volved in selective neuronal death and 
the factors that regulate brain regional 
sensitivity to ethanol neurotoxicity. 

Some exciting studies have begun to 
address the effects of gender on brain 
damage. Interestingly, alcoholic women 
appear to have an increased sensitivity 
for brain damage, when compared 
with alcoholic men (Hommer et al. 
1996). This appears to be true for liver 
disease as well. Although there are 
more men diagnosed as alcoholic, the 
number of alcoholic women is increas- 
ing. The increased susceptibility of 
women to alcoholic pathology is an 
area that needs further research. 

Alcoholics who do not have Kor- 
sakoffs syndrome problems show 
decreased neuropsychological perfor- 
mance compared with peer nonalco- 
holics on tests of learning, memory, 
abstracting, problem solving, visu- 
ospatial and perceptual motor func- 
tioning, and information processing 
(Parsons 1993). Alcoholics are less 
accurate and take considerably longer 
to complete tasks. Alcoholics are dif- 
ferentially vulnerable to these deficits, 
and many of the deficits appear to 
recover to age -appropriate levels of per- 
formance over a 4- to 5 -year period of 
abstinence (Parsons 1993). Although 
global cerebral atrophy returns to nor- 
mal levels with extended abstinence, 
not all cognitive functions return. Some 
abstinent alcoholics appear to have 
permanent cognitive impairments, 
particularly in memory and visual-spatial- 
motor skills (Di Sclafani et al. 1995). 
Other studies support a loss of logical 



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Neurotoxicity of Alcohol 



memory and paired association learn- 
ing tasks in alcoholics that may be 
long-lasting (Eckardt et al. 1996). 

Cognitive deficits are not the only 
factors that suggest long-term changes 
in brain function following chronic 
ethanol treatment. Electrophysiological 
studies using brain electroencephalo- 
grams and event-related potentials 
have suggested that alcoholics have 
difficulty differentiating relevant and 
irrelevant, easy and difficult, and 
familiar and unfamiliar stimuli (Porjesz 
and Begleiter 1993). These deficits 
appear to be consistent for alcoholics 
and may be related to frontal cortical 
function. Both clinical and experimen- 
tal studies support a role of frontal 
cortical involvement in neuropsycho- 
logical dysfunction in alcoholics, par- 
ticularly those with Korsakoff's 
syndrome ( Oscar- Berman and Hutner 
1993). Areas affected include emotional 
abilities, disinhibition, perseverative 
responding, reduced problem- solving 
abilities, and poor attention. Prefrontal 
damage typically is associated with 
changes in personality and elusive 
cognitive abnormalities. In the last few 
years studies have emphasized the role 
of the prefrontal cortex in executive 
cognitive function (ECF) (Giancola 
and Moss 1998). Executive cognitive 
function is the ability to use higher 
mental functions such as attention, plan- 
ning, organization, sequencing, abstract 
reasoning, and the ability to use external 
and internal feedback to adaptively mod- 
ulate future behavior (Foster et al. 
1994). ECF is dysfunctional in alco- 
holics and in individuals with other 
diseases showing prefrontal damage 
(Boiler et al. 1995), and disruption of 



abilities has been implicated in the 
underlying aggression associated with 
substance abuse (Hoaken et al. 1998). 
Although these types of changes in 
brain function are more difficult to 
assess, they are consistent with the 
morphological changes found in 
frontal cortex and with the findings of 
studies on damage to association cortex 
in animals (Hunt and Nixon 1993; 
Giancola and Moss 1998). 

EXCITOTOXICITY 

The mechanisms of ethanol neurotox- 
icity have only recently begun to be 
elucidated. There are several reports that 
N-methyl-D-aspartate (NMDA)- 
glutamate receptors may be involved 
in tolerance to and dependence on 
ethanol as well as ethanol-induced 
brain damage. When MK-801 (dizo- 
cilpine), an antagonist to NMDA- 
glutamate receptors, was coadminis- 
tered with ethanol, the tolerance to 
ethanol was eliminated (Khanna et al. 
1992; Szabo et al. 1994). Khanna and 
colleagues (1993) found that inhibition 
of nitric oxide also inhibited the devel- 
opment of tolerance to ethanol, and 
NMDA receptors are closely coupled 
to nitric oxide formation (Chandler et 
al. 1994). Thus, NMDA receptors 
appear to be involved in the develop- 
ment of tolerance to ethanol. 

Hyperexcitability of the central 
nervous system is a key component of 
ethanol withdrawal, and a supersensi- 
tive NMDA-glutamate response 
appears to be involved, although a 
reduction in gamma-aminobutyric 
acid (GABA)-mediated inhibition 
may also contribute (Crews et al. 



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1996). One of the earliest findings 
suggesting glutamate involvement was 
that [ 3 H] glutamate binding is increased 
in human hippocampus of alcoholics 
(Michaelis et al. 1990). Although the 
subtype of glutamate receptor involved 
is not clear, this finding is consistent 
with increased glutamate receptor 
density and sensitivity. It has. been dis- 
covered that NMD A- glutamate recep- 
tors have a unique property, in that 
excessive stimulation of these recep- 
tors triggers a process in neurons that 
leads to neuronal death. This appears 
to play a key role in neurodegenera- 
tive diseases in general as well as in 
stroke, brain trauma, and other types 
of brain damage (Crews et al. 1996). 
Basic studies on alcohol have con- 
tributed significantly to the under- 
standing of this process. 

Several studies in isolated neuronal 
cells have indicated that a few days of 
chronic ethanol treatment leads to super- 
sensitive NMDA-stimulated calcium 
flux (Iorio et al. 1992; Ahern et al. 
1994), as well as NMDA-stimulated 
excitotoxicity (Crews and Chandler 
1993; Crews et al. 1993; Iorio et al. 
1993) and NMDA-stimulated nitric 
oxide formation (Chandler et al. 
1995). Nitric oxide has been impli- 
cated in neuronal toxicity because of 
the formation of highly oxidative 
metabolites (Crews and Chandler 
1993). Although NMD A supersensi- 
tivity was found in all of these 
responses, changes in[ 3 H]MK-801 
binding or the amounts of NMDAR1 
or NR2A or NR2B immunoreactivity 
were not found in all cases, suggest- 
ing that posttranslational changes in 
the NMD A receptor structure, not 



density or subunit composition, may 
be responsible for the supersensitivity. 
Administration of ethanol has been 
shown to enhance tyrosine phospho- 
rylation of the NMDA receptor, and 
this has been associated with acute 
tolerance to ethanol's inhibition of 
NMDA-mediated excitatory postsy- 
naptic potentials (Miyakawa et al. 
1997). Mice lacking a Fyn nonreceptor 
tyrosine kinase do not show acute tol- 
erance and are hypersensitive to ethanol 
(Miyakawa et al. 1997). The Fyn 
kinase appears to be involved in both 
NMDA and GABA A function and 
thus could play a role in ethanol toler- 
ance and dependence (Miyakawa et al. 
1997). Although the mechanisms are 
not totally resolved, it is clear that 
chronic ethanol can induce NMDA 
supersensitivity. Supersensitive 
NMDA responses likely contribute to 
the hyperexcitability and seizures asso- 
ciated with ethanol withdrawal, as well 
as causing neurotoxicity. 

Hoffman's laboratory has reported 
increases in the density of NMDA 
receptors in C57BL mice treated 
chronically with a 7 percent ethanol 
liquid diet. Seven days of chronic 
ethanol increased [ 3 H]MK-801 bind- 
ing in hippocampal membranes by 
approximately 16 percent (Grant et al. 
1990). These animals were dependent 
upon ethanol, as indicated by with- 
drawal seizures. An autoradiographic 
study also reported increased 
[ 3 H]MK-801 binding in cortex, hip- 
pocampus, and striatum (Gulya et al. 
1991), which returned to control val- 
ues in approximately 24 hours, a time 
course similar to the return of seizure 
scores to control levels. Extensions of 



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Neurotoxicity of Alcohol 



these experiments with membrane 
binding found only changes in hippo- 
campus, not cerebral cortex (Snell et 
al. 1993). These studies found that 
[ 3 H]MK-801- and NMDA-specific 
[ 3 H]glutamate binding slightly 
increased in hippocampus during 
chronic ethanol treatment, but there 
were no changes in [ 3 H]glycine or 
[ 3 H]CGS19755, a competitive NMDA 
antagonist, in hippocampus. No 
changes in any ligand binding were 
found in cerebral cortex (Snell et al. 
1993). Changes in some NMDA lig- 
and binding sites may be involved in 
subunit composition changes but not 
in an increased density of channels. 

Trevisan and colleagues (1994) 
found that 12 weeks of ethanol liquid 
diet to rats increased the levels of 
NMDAR1 immunore activity in the 
hippocampus, but not in cortex, stria- 
tum, or nucleus accumbens. Other 
studies of levels of NMDAR1 mRNA 
have indicated that chronic ethanol 
does not change NMDAR1 mRNA 
but increases NMDAR2A and 
NMDAR2B mRNA levels in hip- 
pocampus and cortex (Follesa and 
Ticku 1995). Binding changes were 
not reported in this study. Since MK- 
801 apparently requires both an 
NMDAR1 subunit and an NMDAR2 
subunit for binding, an increase in 
binding could be due to changes in 
channel subunits without necessarily 
an increase in the density of channels. 
Other studies have not found 
increases in MK-801 binding follow- 
ing chronic ethanol treatment of mice 
(Carter et al. 1995) or rats (Rudolph 
and Crews 1996). These differences 
could be due to different ethanol 



treatment protocols or the responses 
of different strains of animals. Long-term 
treatment of rats with ethanol (12 
weeks) was found to be required to 
increase NMDAR1 immunoreactivity 
in the ventral tegmental area, whereas 
1 and 6 weeks of chronic 5 percent 
ethanol liquid diet were not sufficient 
(Ortiz et al. 1995). Although the 
exact molecular processes require 
additional experimentation, a number 
of studies support the hypothesis that 
chronic ethanol results in supersensitive 
NMDA receptors and that this could 
be a significant factor in ethanol- 
induced brain damage. 

The mechanism of the neurode- 
generation in alcoholic Wernicke's 
syndrome also appears to involve excito- 
toxicity from glutamate in the neural 
destructive process similar to the less 
severe alcoholic brain damage (Langlais 
and Zhang 1993). In animal studies, 
extracellular concentrations of gluta- 
mate in brain increase severalfold dur- 
ing seizures in thiamine-deficient 
animals (Langlais and Zhang 1993). 
Furthermore, MK-801, an NMDA 
antagonist, reduces experimental neuro- 
biological symptoms and severity of 
neural lesioning in a thiamine-deficient 
rat model (Langlais and Mair 1990). 
It is not known whether coadministra- 
tion of ethanol and thiamine deficiency 
would enhance the degree of neuro- 
degeneration seen in experimental 
Wernicke's encephalopathy (i.e., thi- 
amine deficiency). A complicating fac- 
tor of Wernicke's encephalopathy is 
Korsakoff's amnestic syndrome (Victor 
et al. 1989), in which there is a major 
loss of memory. The memory dysfunc- 
tion correlates best with the presence 



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of lesions in the thalamus (Victor et 
al. 1989). It must be recognized that 
KorsakofPs amnestic syndrome can 
occur in the absence of ethanol, 
although it is extremely rare. How- 
ever, in susceptible individuals, 
chronic ethanol clearly facilitates the 
course of this disease. In any case, there 
is strong evidence that ethanol with- 
drawal hyperexcitability is related at 
least in part to NMDA supersensitivity 
and that this supersensitivity could 
underlie ethanol-induced brain dam- 
age. In summary, chronic ethanol 
appears to cause NMDA receptor 
supersensitivity in a variety of systems. 
NMDA supersensitivity is likely 
involved in ethanol tolerance, depen- 
dence, withdrawal, and neurotoxicity. 

OXIDATIVE STRESS 

Another likely mechanism of ethanol- 
induced brain damage involves increased 
oxidative stress of neurons. Cells use 
oxygen for energy metabolism, but they 
normally have protective mechanisms 
against oxidative damage. Studies exam- 
ining the effects of both acute and chronic 
ethanol administration upon cellular 
oxidation have primarily focused on either 
ethanol's effects on intracellular antiox- 
idant mechanisms such as a-tocopherol, 
ascorbate, glutathione, catalase, and 
superoxide dismutase activity (Ledig et 
al. 1981; Nordmann 1987; Rouach et 
al. 1987; Montoliu et al. 1994) or 
potential sources of oxidative radicals 
such as CYP2E1, an ethanol-inducible 
form of cytochrome P-450 and a 
potent generator of oxidative radicals 
(Montoliu et al. 1994, 1995). Chronic 
ethanol-induced increases in CYP2E1 



and other oxidases have been related to 
increased lipid peroxidation and reactive 
oxygen radicals in brain (Montoliu et 
al. 1994). However, levels of antioxi- 
dant enzymes such as catalase and 
superoxide dismutase appear to increase 
as a compensatory mechanism to 
ethanol-induced oxidant enzyme levels 
(Montoliu et al. 1994). The brain is 
particularly susceptible to lipid peroxi- 
dation, because it consumes a large 
amount of oxygen and is rich in poly- 
unsaturated fatty acids, which are espe- 
cially prone to reactive oxygen injury. It 
has been demonstrated that a single dose 
of ethanol results in both the elevation of 
lipid hydroperoxide levels and decreases 
in glutathione levels in rat brain homo- 
genates (Uysal et al. 1986, 1989; 
Nordmann et al. 1990, 1992). How- 
ever, it is not clear how this increased 
oxidation translates to increased brain 
damage, if it does at all. Although most 
studies have focused on the whole brain, 
a recent study of ethanol-induced 
depression of glutathione and gluta- 
mine synthetase levels, two indices in 
increased oxidative radical formation, 
found changes only in striatum, but 
not in cerebral cortex or cerebellum 
(Bondy and Guo 1995). 

Oxidative stress has been implicated 
in a variety of conditions, particularly 
aging, Alzheimer's disease, parkinson- 
ism, stroke, and other neurodegenera- 
tive diseases. Much more research is 
needed to completely understand how 
oxidation damages neurons and how 
other brain cells respond to increased 
oxidative stress. Ethanol-induced 
neurodegeneration may be related to 
an induction of oxidative enzymes, 
and alcohol research provides an 



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Neurotoxicity of Alcohol 



opportunity to clearly address this 
aspect of neurodegeneration. 

NEUROTROPHIC FACTORS 

Growth factors are specific protein ele- 
ments of brain that stimulate growth and 
extensions of neurons and are essential 
for the survival of certain neurons. Fur- 
thermore, growth factors are known to 
increase neuronal antioxidant and exci- 
totoxic protective mechanisms. Ethanol 
has been found to alter brain levels of 
growth factors (Arendt et al. 1995; 
MacLennan et al. 1995; Baek et al. 
1996; Nakano et al. 1996). Chronic 
ethanol does not lead to a loss of all 
growth factor activity, but appears to be 
somewhat selective. Chronic ethanol 
reduces brain-derived neurotrophic factor 
but does not alter nerve growth factor, 
neurotrophin 3 factor, or fibroblast 
growth factor levels (MacLennan et al. 
1995; Baek et al. 1996). Receptors for 
the growth factors remain intact after 
chronic ethanol abuse (Arendt et al. 
1995; MacLennan et al. 1995) and 
present the promising possibility that 
growth factors may be used to treat 
ethanol-induced brain damage as well as 
other neurodegenerative conditions. 
Studies of growth factor action and their 
role in ethanol-induced brain damage 
represent an exciting area of discovery 
with the tremendous potential to pro- 
vide a variety of new approaches to 
treating neurodegeneration. 

APOPTOSIS 

Apoptosis is a physiological form of 
cell death with characteristic morpho- 
logical and biochemical features that 



include nuclear pyknosis, DNA frag- 
mentation, and dependence on new 
protein synthesis. A cascade leading 
to apoptotic cell death includes 
induction of p53 protein; recruitment 
of other transcription factor proteins, 
such as bax, bcl, and bad; and activa- 
tion of a series of caspase proteases 
and tyrosine kinases. Few of these 
markers of apoptosis have been stud- 
ied in intact brain. Although some 
reports have suggested a role in exci- 
totoxic cell death, most studies have 
found excitotoxicity to be primarily 
necrotic (Cheung et al. 1998; Martin 
et al. 1998). A few studies have 
reported that adrenalectomy-induced 
loss of dentate granule cells 
(Schreiber et al. 1994) or kainate- 
induced cell death (Sakhi et al. 1994) 
is associated with induction of p53, 
suggesting that apoptosis may occur 
in intact adult brain. However, few 
other studies have extended these 
findings, and most studies have been 
done in cell culture with neonatal 
neurons. Recent studies have sug- 
gested that apoptosis is common dur- 
ing development, but that necrosis 
predominates in adult brain (Portera- 
Cailliau et al. 1997). 

Few studies of ethanol and apopto- 
sis have been done. Ethanol has been 
found to inhibit NMDA inhibition of 
apoptosis in cerebellar granule cell 
cultures (Hoffman et al. 1989). In 
astroglial cultures, ethanol-induced 
death was found to be due to 
necrotic, but not apoptotic, mecha- 
nisms (Holownia et al. 1997). Thus, 
few studies support a major role of 
apoptotic death in ethanol-induced 
brain damage in adults, although 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



important actions may occur as part of 
ethanol's teratogenic actions. 

CELL ADHESION 
MOLECULES 

Cell adhesion molecules clearly play a 
role in neuronal structure and func- 
tion. Chronic treatment with NMDA 
antagonists results in neuronal dam- 
age and increased NMDA receptor 
clustering (Rao and Craig 1997). Since 
ethanol is an NMDA antagonist, it is 
possible that this results in structural 
changes in cell adhesion proteins or 
cytoskeletal elements. Charness's lab- 
oratory found that ethanol potently 
and completely inhibits LI -mediated 
cell adhesion in transfected cells, but 
has no effect on other adhesion mole- 
cules such as NCAM 140 (Ramanathan 
et al. 1996). Studies in chick develop- 
ment have suggested that ethanol's 
teratogenic effects are due to a disrup- 
tion of cell adhesion molecule synthesis 
and function (Kentroti et al. 1995). 
This is a new and exciting area, but 
there is not enough data relating adhe- 
sion proteins to ethanol's neurotoxic 
actions to prompt a significant invest- 
ment in this field at this time. 

BRAIN DAMAGE 
AND ALCOHOLISM: 
IMPLICATIONS FOR 
THE PROGRESSION TO 
ALCOHOL DEPENDENCE 

Alcoholism is a progressive disease that 
starts with experimentation and pro- 
gresses to addiction, usually over the 
course of several years. Addiction 
involves the loss of control over the 



ability to abstain from the drug and per- 
severative preoccupation with obtaining 
and using the drug. Although earlier 
studies focused on alcohol-induced 
changes in cognition, more recent 
studies have focused on the frontal 
cortex and the role of this brain 
region in behavior. Researchers have 
begun to investigate ECF as an 
important function of the prefrontal 
cortex. A variety of evidence has 
focused attention on the prefrontal 
cortex as an area of brain that is partic- 
ularly sensitive to alcohol-induced 
brain damage. At the same time scien- 
tists have developed ways to investi- 
gate the role of ECF in behavior. 
ECF/prefrontal cortical characteristics 
are associated with decreased regulation 
of human social behavior, including 
disinhibition syndrome characterized 
by impulsivity, socially inappropriate 
behavior, and aggression (Giancola 
and Moss 1998). Neuroimaging stud- 
ies have indicated that hypofunction 
of the frontal lobes is associated with 
violence (Raine et al. 1994). Experi- 
mental subjects with poor prefrontal 
functioning appear unable to inhibit 
impulsive behavior (Lau and Pihl 
1996), particularly violence (Lau et al. 
1995). Taken together, these studies 
suggest that some of the greatest 
sociopathic problems of alcoholism, 
such as violence and loss of control 
over the drug, may be directly related to 
the neurotoxic effects of ethanol on pre- 
frontal cortical function. Animal stud- 
ies have shown that chronic exposure 
to ethanol and repeated withdrawal 
episodes increase self- administration 
of ethanol if particularly high blood 
levels are reached (Schulteis et al. 



198 



Neurotoxicity of Alcohol 



1996). More studies are needed to 
directly determine the relationship of 
prefrontal cortical function to alcohol- 
induced brain damage and addiction. 

SUMMARY 

Alcohol can be neurotoxic. Recent stud- 
ies have indicated that the prefrontal 
cortex is particularly sensitive to the 
neurotoxic actions of alcohol, as are 
specific groups of neurons that project 
throughout the brain, including the 
frontal cortex, biogenic amine, and 
peptidergic neurons. Areas that need 
increased emphasis include the specific 
types of neurons that are most sensi- 
tive to ethanol neurotoxicity and the 
mechanisms of neurotoxicity, particu- 
larly NMDA excitotoxicity, oxidative 
mechanisms of neuronal stress, and 
protein induction during chronic 
ethanol consumption. 

GAPS IN KNOWLEDGE AND 
RECOMMENDATIONS FOR 
FUTURE DIRECTIONS 

Neurotoxicity 

Neurons. Human data supports loss of 
neurons, particularly in frontal cortex. 
Animal data indicates specific limbic 
cortical neuronal damage after 4 days 
of intoxication. The role of ethanol 
withdrawal and extended intoxication, 
as well as other factors in alcoholic 
neurotoxicity, is a significant gap in 
our knowledge. 

White Matter Loss. Human studies 
indicate loss of white matter (e.g., 
myelin). This loss could be secondary 
to the loss of neurons or due to specific 



toxicity to various types of glia. This is 
a crucial question that needs to be 
addressed to clearly understand the pri- 
mary site of alcoholic insults to the brain. 

U.S. Brain Bank. Most of the 
human postmortem data comes from 
one group in Australia. Although this 
is an excellent group, there is a critical 
need to establish resources within the 
United States to provide opportunities 
for U.S. investigators. It is recom- 
mended that a consortium — through 
the Research Society on Alcoholism, 
existing brain bank facilities sponsored 
for other diseases, or a specific U.S. 
institution — be established to begin 
forming a human alcoholic brain bank 
that U.S. investigators will be able to 
use for experimentation. 

Gender. Although women represent 
approximately 25 percent of alcoholics 
(Grant et al. 1991), they may suffer 
greater pathology. Data regarding the 
role of gender could provide important 
fundamental insights into the mecha- 
nisms of brain damage as well as 
important new information. There is a 
significant gap in our knowledge in 
this area that needs to be addressed. 

Mechanisms of Brain Damage 

Mechanisms of NMDA Excitotoxicity. 
Both in vitro and in vivo data suggest 
that NMDA excitotoxicity contributes 
to neurodegeneration in a variety of 
pathologies, including alcoholic brain 
damage, Alzheimer's disease, stroke, and 
Parkinson's disease. A significant gap 
in knowledge exists with regard to the 
processes between the acute excessive 
stimulation of neurons by glutamate 
and the processes that lead to delayed 
neuronal death. Understanding these 



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events will greatly enhance our under- 
standing of the mechanisms of neu- 
ronal death. 

Selective Neuronal Death. Studies 
have indicated that frontal cortex neu- 
rons as well as specific neuronal popu- 
lations, such as cholinergic forebrain 
nuclei, raphe nuclei, locus coeruleus, 
and vasopressin neurons, may be lost 
during chronic ethanol consumption. 
Why these neuronal nuclei are specifi- 
cally sensitive is an important question 
that needs to be answered. 

Role of Oxidation. A variety of stud- 
ies have suggested that oxidative stress 
is increased in brain by alcohol. It is 
often hypothesized that this contributes 
to brain damage; however, there is a 
gap in our knowledge, with littie data 
for or against this hypothesis. 

Consequences 
of Brain Damage 

Cortical Function and Alcoholism. A 
variety of studies have indicated loss 
of specific memory tasks and other 
cognitive abilities with alcoholic brain 
damage. An important future direction 
will be to understand the role alcoholic 
brain damage plays in the progression 
to alcoholism, recovery from alcoholism, 
and other behaviors associated with 
alcoholism (e.g., violence, relapse, and 
trauma injury). There is a significant 
gap in our knowledge in this area. 
Understanding the relationship of 
neuropathology to behavioral pathology 
is essential and fundamental to improv- 
ing prevention and treatment. 

Adolescent Factors. Researchers 
need to investigate the effects of alcohol 
on the adolescent brain. Recent stud- 
ies (e.g., Grant and Dawson 1998) 



have suggested that teenagers who start 
drinking earlier are more likely to 
develop alcoholism. Adolescents 
respond differentiy to alcohol, but little 
or no biological data address this issue. 
Relationship to Other Pathology. 
Alcoholics are at greater risk of 
trauma, seizures, and stroke-induced 
brain damage. The interaction of 
these pathologies and the behavioral 
and physiological factors that con- 
tribute to these pathologies are poorly 
understood. 

ACKNOWLEDGMENT 

The preparation of this chapter was 
supported by the National Institute 
on Alcohol Abuse and Alcoholism. 



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content in rat brain. Drug Alcohol Depend 
23(3):227-230, 1989. 

Victor, M.; Adams, R.D.; and Collins, 
G.H. The Wernicke-Korsakoff Syndrome 
and Related Disorders Due to Alcoholism 
and Malnutrition. Philadelphia, FA. 
Davis, 1989. 



206 



ADDICTION 

AND OTHER BEHAVIORS 

IN ANIMAL MODELS 



Chapter 6 

Basic Behavioral Effects and Underlying 
Neurocircuitries of Alcohol 

Kathleen A. Grant, Ph.D. 



KEY WORDS: AODE (effects of AOD [alcohol or other drug] use, abuse, and 
dependence); neuron; brain function; animal behavior; locomotion; uncondi- 
tioned response; anxiety state; conditioned response; dose effect relationship; drug 
discrimination; neurotransmitter receptors; taste perception; reinforcement; 
AOD preference; animal model; self administration of drugs; learning; memory; 
cognition; aggressive behavior; risk factors; literature review 



Higher organisms interact with the 
environment primarily through condi- 
tioned behavioral processes, and the 
nervous system is where environmen- 
tal information is integrated and 
behavioral responses are generated. 
Thus, the integrity of behavior reflects 
the integrity of the nervous system 
(Weiss and Cory-Slechta 1994). Alco- 
holism is mediated through the ner- 
vous system, but its primary diagnosis 
is behavioral, not neurological. Thus, 
to understand the behavioral disorder 
of alcoholism we must integrate what 
we know about the determinants of 
behavioral responses with our knowl- 
edge of alcohol's action on the ner- 



vous system. The neurobiological 
basis of alcohol's behavioral effects 
can be characterized by co-application 
of modern approaches to measuring 
behavior and brain function. 

NEUROBIOLOGICAL 
CHARACTERIZATIONS 
OF NEURAL CIRCUITRY 
IN BEHAVIOR 

The vertebrate brain appears to 
process information through anatomical 
specialization of function. For nearly a 
century it has been clear that specific 
brain areas are involved in the process- 
ing of sensory, motor, or cognitive 



K.A. Grant, Ph.D., is a professor in the Department of Physiology and Pharmacology, Wake Forest 
University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083. 



209 



NIAAA's Neuroscience and Behavioral Research Portfolio 



information, in an arrangement com- 
monly referred to as localization of 
function (Cohen and Bookheimer 
1994). However, localization does 
not imply isolation, and it is also clear 
that there is an orchestration of infor- 
mation from all brain areas that 
results in appropriate and predictable 
behavior. There are a number of tech- 
niques currently available that have 
been used to study the integration of 
information within the brain as it 
relates to behavior. These techniques 
have differing degrees of resolution 
along several dimensions, suggesting 
that complementary and simultaneous 
use of two or more techniques will 
ultimately provide the extensive infor- 
mation necessary to understand the 
neural basis of behavior. No technique 
to date has completely identified the 
neural circuitry associated with a com- 
plex behavior such as drinking alcohol 
to intoxication. However, we do have 
a basis for beginning to speak in 
terms of neural function, brain cir- 
cuitry, and behavior. 

The major modern techniques for 
understanding neural function in a 
behaving organism can be divided into 
two categories, based upon whether the 
technique measures changes within 
only a single locus of a neural network 
or within the entire neural network. 
Those techniques that gather measures 
only from a discrete locus are single - 
unit recordings of neurons; in vivo 
voltammetry of extracellular ions and 
neurotransmitters; in vivo microdialy- 
sis of extracellular ions and neuro- 
transmitters; and site -specific injections 
of receptor ligands or neurotoxins. 
Those techniques that gather measures 



from entire neural networks or trans- 
mitter systems have the potential 
advantage in identifying the conglom- 
eration of relatively small effects that 
sum together and correlate with 
behavior. For example, relatively small 
effects noted with individual neurons 
may be overlooked when using serial 
sampling of single-unit recording, but 
simultaneous occurrence of many 
small effects may show much larger 
effects when measured by ensemble 
recording (multi-unit recording). 
Important procedures that address 
network circuitry are ensemble 
recording of multiple neurons with 
microwire arrays; site-specific injec- 
tions of multiple sites; quantitative 2- 
deoxyglucose sequestration reflecting 
energy utilization; neurotransmitter 
turnover rates reflecting activity of 
neurotransmitter systems; functional 
magnetic resonance imaging (MRI) of 
blood flow; positron emission tomog- 
raphy (PET) of glucose utilization, 
blood flow, or ligand binding; trans- 
genic knockouts, knockins, and 
knockdowns; and event-related poten- 
tials (ERPs). 

Each of these techniques has strengths 
and weaknesses in measuring neuronal 
activity associated with behavioral events. 
These strengths and weaknesses can be 
summarized by examining the resolution 
of brain activity along spatial and tem- 
poral parameters, as well as the inva- 
siveness and longevity of the preparation 
(Cohen and Bookheimer 1994). Since 
it is the spatiotemporal pattern of 
neuronal firing, within and between 
neural networks, that is believed to 
underlie the actual orchestration of a 
behavioral pattern, the resolution of 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



information along these dimensions is 
critical to understanding the neural 
control of behavior. The invasiveness 
and longevity of a preparation will 
limit either the behaviors that can be 
studied or the repeated analysis of a 
behavior. 

Along the spatial dimension, the 
best resolution is at the level of the 
neuron, which can be achieved with 
the genetic techniques of gene inser- 
tion and deletion. Electrophysiologi- 
cal recordings such as single-unit and 
ensemble recordings can discriminate 
at the level of individual neurons (10- 
50 \im). The technique of voltammetry 
has a similar resolution, within the 
30-50 ^im range. Quantitative 2- 
deoxyglucose and functional MRI 
(fMRI) have spatial resolutions in the 
50-100 n-m range and can sample 
from neuronal layers or at the subnuclei 
level. Similar resolution is achieved 
with neurotransmitter turnover stud- 
ies that use micropunches of tissue, 
depending on the transmitter being 
studied. Microdialysis requires a probe 
that is in the 100-250 |am range; how- 
ever, this technique is still able to detect 
columnar or subnucleic resolution. Site- 
specific injections of ligands or toxins 
result in a spread of material generally 
agreed to be in the 200-500 \im 
range, depending on the brain area. 
The resolution of PET depends on 
the scanner but is generally at the 
level of brain nuclei, 2-8 mm, and can 
be increased if images are co-registered 
with nuclear magnetic resonance 
(NMR). Finally, the resolution of 
ERPs is at the level of cortical lobes 
and in the range of centimeters. The 
most important point of spatial reso- 



lution is the size of the brain under 
study. For most animal models, the 
useful cutoff is below the resolution of 
PET and ERPs unless large species of 
nonhuman primates, such as macaques 
or baboons, are used as subjects. 

When choosing a technique to cor- 
relate neural function with behavior, a 
temporal dimension is another very 
important consideration. The fastest 
resolution is direct neural recording, 
with the ability to reliably separate 
spike trains in the range of 10 mil- 
liseconds. This resolution holds 
equally well for single -unit or ensem- 
ble recording. Event-related potentials 
have a resolution in the range of mil- 
liseconds to seconds from stimulus 
onset to potential recording. Voltam- 
metry also has a resolution in the 
200-500 milliseconds range, depend- 
ing on the length of the applied 
oxidative voltage and the rate of sam- 
pling (commonly 5 Hz). The tempo- 
ral resolution of fMRI is 100 
milliseconds, depending on vascular 
response mechanisms. Microdialysis 
has a relatively long resolution time of 
2-20 minutes, primarily depending on 
the sensitivity of the chromatography. 
As the sensitivity of high-pressure liq- 
uid chromatography increases, the 
quantity of dialysate necessary for 
analysis decreases, resulting in less 
time needed to gather the sample. Site- 
specific injections of ligands require 
several minutes, primarily due to 
actual handling of the animal, but also 
to the kinetics of the drug. Quantitative 
2-deoxyglucose typically requires an 
incorporation time of 45 minutes, but 
a majority of the radioactivity is taken 
up within a 20-minute period. Fluo- 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



rodeoxy glucose (FDG) incorporation 
is 30 minutes. Using a repetitive task, 
one can measure circuits because each 
unit of the circuit has been repeatedly 
assessed and cumulated over time. In 
essence, 2-deoxyglucose and FDG 
procedures provide an integrated his- 
tory of neural activation associated 
with a behavior. PET analysis of lig- 
and binding reflects receptor changes 
in number or affinity and, therefore, 
reflects neural changes that occur over 
the course of hours to days, depend- 
ing on the stimulus presentation (e.g., 
chronic ethanol exposure). 

Thus, if the interest is in neural cir- 
cuitry related to emission of a fast 
response, for example, an approach 
behavior or reaction time, the electro- 
physiological techniques are most appro- 
priate. In addition, if one is interested in 
a component analysis of neuronal activity 
in relation to aspects of the behavioral 
chain, single-unit or ensemble record- 
ing is appropriate. However, if repeti- 
tive tasks are used, microdialysis and 
turnover studies can help identify the 
actual neurotransmission pathways 
involved in a response. Likewise, glu- 
cose utilization procedures and fMRI 
reflect the integrated history of the 
circuitry involved in the response. 

Another dimension to consider is that 
of invasiveness, in terms of both tissue 
damage and restricting behavior. The 
techniques of single-unit recordings, 
ensemble recordings, voltammetry, 
microdialysis, and site-specific injections 
require placement of electrodes, probes, 
or cannulas into neural tissue, resulting 
in direct tissue damage along the probe or 
electrode track. In addition, the electrode 
leads and probe tubing need to be 



physically attached to additional equip- 
ment, often restricting movement of the 
animals, or at the very least requiring 
that a tether be attached to the animal 
during data gathering. Lesion studies 
are by definition invasive. Quantitative 
2-deoxyglucose is not invasive to brain 
tissue because the radiotracer is injected 
into die peripheral circulatory system, 
leaving the central nervous system 
(CNS) intact. However, quantitative 
2-deoxyglucose requires surgical place- 
ment of arterial catheters, which can 
restrict movement. Although the tech- 
niques of fMRI and PET are considered 
noninvasive, the measurements are very 
sensitive to head motion, and misreg- 
istrations due to movement can create 
significant artifacts. Thus, animals need 
to be anesthetized or severely restricted 
in movement during the scanning pro- 
cedure. The technique of FDG imag- 
ing with PET is somewhat immune to 
these criticisms, since the incorpora- 
tion of FDG can occur during an active 
behavioral task and the scanning can 
be done under anesthesia without 
complicating the analysis of what brain 
areas were active during the behavior. 
Finally, although the ERP technique 
is noninvasive, the procedure requires 
the animals to sit quietly. Although 
small head movements are not nearly as 
problematic as with fMRI or PET, 
extensive training of nonhuman sub- 
jects may be required. 

A final dimension to consider when 
linking a technique to behavior is the 
patency of the preparation and 
repeated measures. For example, to 
measure the incorporation of 2- 
deoxyglucose with autoradiographic 
techniques requires decapitation and 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



histology. Likewise, neurotransmitter 
turnover studies requires rapid removal 
by decapitation and freezing of neural 
tissue. Therefore, only a single experi- 
mental manipulation can be measured 
in an animal, and group designs are 
necessary for parametric examinations, 
such as dose-response determinations, 
with these techniques. The techniques 
of microdialysis and voltammetry have 
experimental half-lives in terms of 
days, and probably less in terms of 
voltammetry specificity. Site-specific 
injections are patent for 5-7 injections 
of 1-2 [iL before direct tissue damage 
can confound interpretation of the 
data. Single-unit recording is patent 
for a number of days. Ensemble 
recording is patent for an average of 2 
weeks, but clear identification of the 
same neuron across days is problematic. 
Thus many, if not all, of the techniques 
are limited in longitudinal designs. On 
the other hand, fMRI, PET, and ERP 
are techniques that can be repeatedly 
administered, with caution provided 
for the effect of repeated exposure to 
anesthetics in the use of fMRI and PET. 
From the descriptions given here, it 
is clear that there are many approaches 
to studying the neural circuitry of 
behavior. It is equally clear that each 
technique has particular strengths and 
weaknesses. The complementary use 
of techniques can increase the resolution 
in defining the neural networks and 
help investigators gain a more com- 
plete understanding of the circuitry of 
behavior. For example, single-unit 
recordings can be measured in con- 
junction with microdialysis to identify 
neural activity and the neurotransmit- 
ters released during this activity. This 



information allows the investigator to 
identify an area of the brain as well as 
a receptor system, which can then be 
used to test hypotheses addressing the 
neural basis of behavior. 

BEHAVIORAL 
CHARACTERIZATIONS 
IN ANIMAL MODELS 

To understand the behavioral mecha- 
nisms of alcohol (ethanol), we must 
first understand the variables that con- 
trol behavior and then understand 
how alcohol interacts with these vari- 
ables. Several techniques have been 
developed to model aspects of behavior 
associated with the administration of 
alcohol and other drugs. Similar to the 
techniques to study neural circuitry, the 
behavioral procedures focus on different 
aspects of an overall constellation of 
effects when alcohol is administered 
to an awake animal. Thus, any single 
behavioral procedure in isolation will 
not provide information about all the 
behavioral effects of alcohol. In addi- 
tion, each procedure has particular 
strengths and weaknesses, including 
the amount of training, the ability to 
conduct parallel studies across different 
species, the requirement for specialized 
equipment and behavioral expertise, 
and the ability to incorporate the neuro- 
biological techniques listed in the pre- 
ceding section. A description of the 
most commonly used procedures is 
provided in the following sections, 
arranged by the class of behavior 
being modeled. 

Psychoactive drugs produce a num- 
ber of effects on behavior. Those effects 
of a drug that uniquely covary with an 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



aspect of behavior are called stimuli. 
Unconditioned stimuli elicit reflex 
responses without prior co-occurrence 
of stimulus and response. The uncon- 
ditioned effects of ethanol are respon- 
sible for unconditioned behaviors 
such as loss of righting reflex and for 
unconditioned physiological responses 
such as hypothermia. Conditioning 
involves a process whereby a new asso- 
ciation is formed between the stimulus 
presentation and the occurrence of 
behavior. For example, new behaviors 
can be established in relation to the 
presentation of reinforcing stimulus 
effects, with either increases or 
decreases in behaviors that were instru- 
mental in producing the stimulus. 

The study of the neurobiological 
bases of ethanoPs behavioral effects 
necessitates using an ethanol concentra- 
tion range compatible with behavior. 
Hypotheses concerning the neu- 
ropharmacological mechanisms of 
ethanol's activity and the transference 
of these concepts to the realm of psy- 
chopharmacology have changed sig- 
nificantly over the past two decades. 
Ethanol, along with other anesthetics, 
was considered an exception to the 
receptor theory of drug action (Smith 
and Reynard 1995), in which there is 
a physical combination of a drug with 
a specific macromolecule (Goldstein 
et al. 1968). The effects of ethanol were 
viewed as nonspecific, disrupting the 
electrochemical communication between 
neurons by generally disordering all 
membrane activity through a lipid par- 
titioning effect. However, advancements 
in the application of electrophysiological, 
biochemical, and molecular techniques 
provide conclusive data to show that 



ethanol acts as a modulator at particu- 
lar receptor complexes, selectively 
altering neurochemical processes in 
discrete regions of the CNS (see chap- 
ter 1). The specificity in the mecha- 
nisms of ethanol's activity, in some 
studies localized to a handful of amino 
acids (Mihic et al. 1997), reflects 
tremendous advances in defining the 
basic neuropharmacology of ethanol. 
These advances have also provided 
rational avenues of research for identi- 
fying the receptor mechanisms 
involved in mediating ethanol's stimu- 
lus effects. 

Unconditioned Behavior 

Unconditioned behavior is often 
referred to as "innate" or "reflexive," 
and does not require learning on the 
part of the organism. Generally speak- 
ing, organisms on the lower end of the 
phylogenetic scale predominanriy dis- 
play unlearned behaviors; however, 
learning has been demonstrated in most 
multicellular organisms. Although 
these behaviors do not require associa- 
tive processes to be established, uncon- 
ditioned behaviors such as a motor 
reflex (or salivation) can be the basis 
of a conditioning paradigm. Thus, 
through learning paradigms, uncondi- 
tioned behaviors are associated with 
stimuli and produce conditioned 
behaviors, and these will be discussed 
at greater length later in this chapter. 
In alcohol research, unconditioned 
behaviors are often measured only in 
the context of single -dose exposures. 

Motor Responses 

With ethanol administration, most 
reflexive responses studied have been 



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Behavioral Effects and Underlying Neurocircui tries of Alcohol 



locomotion responses, but there are 
notable exceptions. 1 Three responses — 
open field activity, righting reflex, and 
ability to remain on a moving 
rod/track — have been heavily used in 
alcohol research addressing neurobio- 
logies correlates, and these responses 
are discussed in this section. Other 
examples of unconditioned motor 
responses include exploration in mazes, 
orientation of an inclined plane, and 
grip strength on an inverted screen. 
With the exception of elevated plus 
mazes to measure anxiety (discussed in 
the next section), these responses have 
not been used in alcohol research to any 
great extent and will not be described 
in detail here. 

Open field activity, in which hori- 
zontal and vertical motor activity is 
detected by photobeams, is a common 
measure of an unconditioned locomo- 
tor response in alcohol research. Open 
field locomotion implies motor coor- 
dination, but animals can move though 
the open field in uncoordinated activity. 
Thus, an intoxicated animal that could 
not stay on a moving belt could exhibit 
increases in open field activity. In 
addition, patterns of behavior, such as 
intense locomotion followed by 
immobility versus constant activity, are 
often not measured. Open field loco- 
motion can detect changes induced by 
ethanol in the 1.0-3.0 g/kg dose range 
and has been used to measure both 
the stimulatory and sedative effects of 
ethanol. The animals of choice for 
these procedures have been the mouse 
or the rat, and neurochemical measures 
associated with open field activity 
have focused on the dopaminergic or 
GABAergic system. A sophisticated 



neuroscience technique, the use of 
genetically selected mice, has been 
applied to this analysis. In addition, 
quantitative trait loci (QTL) analyses 
have been performed and have pro- 
vided provisional loci that may contain 
genes important for the expression of 
ethanol-induced unconditioned stim- 
ulatory locomotion. 

Another common measure is the 
loss of a righting reflex following a 
high dose of ethanol. The primary 
measures of behavior are the latency 
to lose the reflex and the duration the 
reflex is lost. Loss of righting reflex is 
associated with high doses of ethanol 
and is incompatible with most other 
alcohol-related behaviors in the non- 
tolerant animal. Mice and rats are the 
most common subjects, and the neu- 
rotransmitter system studied most 
extensively has been the GABAergic 
system, although N-methyl-D-aspar- 
tate (NMDA), serotonin, and neu- 
ropeptidergic receptor systems have 
also been explored. Most of the neu- 
rotransmitter systems have been inves- 
tigated in isolation, primarily through 
the use of receptor binding, with little 
information on circuitry. As with the 
locomotor response, the most sophis- 
ticated technique applied to this 
analysis has been the use of genetically 
selected rodents. QTL analyses using 
this measure have provided provi- 
sional loci for genes important for the 
expression of ethanol-induced uncon- 
ditioned impairment of motor 
reflexes. This measure has also been 
used extensively to study the develop- 
ment of tolerance to ethanol. 

Direct measures of motor coordi- 
nation are best represented in alcohol 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



research by rotorod or moving belt 
performance. In these procedures the 
animals are required to stay on a dowel 
or a track that is rotating at either a 
constant or an accelerating speed. 
Although the response of staying on 
the rotorod or track is unconditioned, 
animals are often given a few training 
sessions to have baseline performances 
matched in group designs. Therefore, 
there is the opportunity for condition- 
ing if several pre-ethanol trials are 
given. Latency to fall off the rotorod 
or step off the track following the 
administration of ethanol is generally 
the measure of interest. This response 
is sensitive to lower doses of ethanol 
compared with doses associated with 
the loss of righting reflex. To date, 
the response has been used most com- 
monly to assess sensitivity to ethanol 
and the development of tolerance in 
mice and rats. Neurochemical mea- 
sures associated with this response 
have also focused on the GABAergic 
system, although the serotonin system 
has been examined in the develop- 
ment of tolerance. 

Effects of ethanol on these uncon- 
ditioned behaviors have been the basis 
for selective breeding using alcohol- 
related phenotypes. Because selective 
breeding requires that the same pheno- 
type be accurately characterized each 
generation, it is easy to understand why 
these unconditioned motor responses 
were chosen to demonstrate a genetic 
basis to an ethanol-related behavior. 
However, it is clear that drugs from a 
wide variety of pharmacological classes 
can disrupt performance on these 
tasks. Thus, using these uncondi- 
tioned responses to investigate genetic 



or neural bases for ethanol-induced 
behavioral responses appears to be based 
more on replicating the response than 
on specificity or sensitivity to ethanol. 
For example, GABAergic mechanisms 
may represent a focal point of the 
effects of ethanol on the nervous sys- 
tem, or they may be secondary to the 
functional integrity of another system 
disrupted by ethanol. Motor reflexive 
behaviors are rather robust and gener- 
ally require high doses of ethanol for 
alterations in response generation. In 
turn, the high doses of ethanol are 
likely to alter the integrity of multiple 
neural circuits, and pinpointing which 
neural mechanism(s) is(are) involved 
may be impossible. To date, the use 
of specific receptor ligands, such as 
inverse agonists at GABA A receptors, 
has attenuated but not completely 
antagonized the effects of ethanol on 
these tasks. 

With the technology available today 
to selectively knock out genes, it seems 
unnecessary to continue to extrapolate 
from these unconditioned responses to 
mechanisms associated with the behav- 
ioral basis of alcohol abuse and alcohol- 
ism. Possible exceptions to this conclusion 
could be the study of unconditioned 
motor responses to ethanol in testing 
specific hypotheses such as the con- 
cordance of tolerance to the motor 
effects of ethanol and increases in 
ethanol self- administration. However, 
such tolerance to ethanol is easily gath- 
ered within self- administration or place 
preference procedures, in the context of 
appropriate dose ranges. Thus, although 
unconditioned motor behaviors are very 
useful for determining dose ranges of 
activity and can serve as initial screens for 



216 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



receptor-mediated activity, they are 
not particularly useful for understanding 
the neural circuitry involved in the 
process of alcohol addiction. 

Anxiolytic Responses 

A constellation of unconditioned 
responses that has implications for the 
abuse potential of alcohol is derived 
from unfamiliar and extreme environ- 
mental settings. Unconditioned behav- 
iors exhibited in extreme heights, in 
the presence of an unfamiliar individual, 
or in the presence of bright lighting have 
all been used as measures of anxiety in 
mice and rats (Lister 1990). The ele- 
vated plus maze measures the uncon- 
ditioned responses of rodents to stay in 
enclosed spaces and to avoid cliffs. In 
this procedure, rats or mice are placed 
in a maze with a central post that elevates 
the maze. Radiating out from the central 
post are four arms that are perpendicular 
to each other. Two of the maze arms, 
opposite to one another, have sides 
(closed arms), and the other two arms 
have no sides (open arms). When 
placed in the maze, undrugged animals 
spend most of their time in the closed 
arms of the maze; anxiolytics and alcohol 
increase the amount of time spent in the 
open arms of the maze (Lister 1990; 
but see Dawson and Tricklebank 1995). 
The social interaction test measures the 
unconditioned responses of rodents to 
engage in social investigation. The 
social interaction test capitalizes on the 
observation that social interactions 
decrease when animals are placed in 
unfamiliar or brightly illuminated envi- 
ronments. Typically, anxiolytics increase 
the social interactions of rats in these 
environments, and alcohol has also 



been shown to increase social interac- 
tions under these conditions. The light- 
dark box test measures the tendency 
of rats and mice to avoid bright light by 
placing the animal in a two-compartment 
shuttle box in which one side is brightly 
illuminated and the other is darkened. 
Anxiolytics typically increase time spent 
in the illuminated side of the chamber. 
In the mouse, ethanoPs anxiolytic 
effects are in the dose range of 1.5-2.5 
g/kg ethanol (intraperitoneal [ip]), 
corresponding to blood ethanol con- 
centrations of 150 mg/dL. Lower 
doses in the range of 0.5-1.0 g/kg 
(ip), corresponding to 100 mg/dL 
ethanol, produce anxiolytic-like effects 
in rats. The receptor system most 
extensively studied with regard to 
unconditioned anxiolytic behaviors is 
the GABA A receptor system, and this is 
the system that has been most consis- 
tently implicated in the effects of ethanol 
on these unconditioned responses. No 
specific information is available con- 
cerning the neural circuitry of ethanoPs 
anxiolytic responses using these mea- 
sures. Most often, GABA A inverse 
agonists have been tested in combina- 
tion with ethanol to block ethanoPs 
anxiolytic responses. However, GABA A 
inverse agonists are anxiogenic, and 
the attenuation of ethanoPs effects 
could be due to two separate mecha- 
nisms, canceling each other's effects. 
Flumazenil does not appear to block 
the anxiolytic effects of acute ethanol 
in conditioned conflict procedures. 
Finally, correlations have been found 
between preference for alcohol solutions 
and basal expression of these responses 
in some (P/NP and SP/SNP rats), 
but not all, genetic lines selectively 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



bred for alcohol preference (AA/ANA) 
(reviewed in Eckhardt et al. 1998). 

Vocalization is another type of 
unconditioned response that is elicited 
under circumstances associated with 
changes in extreme changes in the 
environment, including maternal or 
conspecific separation. Although 
ethanol administration will increase 
auditory vocalizations, particularly in 
nonhuman and human primates, these 
vocalizations are more affiliative and 
associated with increased social inter- 
action. In contrast, many laboratory 
species, including monkeys, have a 
range of auditory information that is 
in the ultrasonic range of human per- 
ception. These ultrasonic vocalizations 
are often associated with distress, either 
social separation or hypothermia. Low 
doses of ethanol tend to suppress 
stress-induced ultrasonic vocalizations; 
however, investigation of the neuro- 
circuitry involved in ethanol-associated 
changes in vocalizations has been rudi- 
mentary. The GABAergic and 5-ETTi 
receptor systems have been investigated 
through the use of specific ligands, 
but pathway-specific delineations have 
not been made. 

In general, the neurobiological 
investigation of unconditioned anxi- 
olytic responses to ethanol has not 
extended beyond correlational studies 
with selected or inbred lines of rodents 
or use of specific receptor ligands. The 
unconditioned responses associated with 
anxiety appear to provide relatively more 
construct validity than unconditioned 
motor responses in efforts to under- 
stand the neurobiological basis of 
ethanol related to its abuse. However, 
very few studies have actually linked 



initial anxiolytic effects of ethanol in 
these measures and subsequent ethanol 
self- administration or preference in a 
within-subjects design. An important 
caveat to remember is that the "anxi- 
olytic" effects of ethanol measured in 
these procedures involve the response 
to the first injection of ethanol in a naive 
animal. Under these circumstances, it 
has clearly been shown that ethanol 
dramatically heightens activity of the 
hypothalamic-pituitary-adrenal (HPA) 
axis, an effect that is normally correlated 
with increased stress and anxiety (Rivier 
1996). Clearly, there is a complicated 
interaction between the expression of 
the unconditioned responses associated 
with "fear" and the effects of ethanol 
(see the discussion of conditioned 
anxiolytic stimulus effects later in this 
chapter). Important factors include the 
motoric responses to ethanol, including 
both stimulatory and sedative effects, 
either of which could alter the mea- 
surement and interpretation of these 
responses. These confounds are not 
unique to ethanol, prompting leading 
investigators to conclude that "it is 
difficult to justify [use of the elevated 
plus maze] as anything other than a 
preliminary screen as a prelude to test- 
ing more robust animal models of 
anxiety" (Dawson and Tricklebank 
1995, p. 36). 

On the other hand, the use of 
unconditioned responses to demonstrate 
anxiogenic states associated with ethanol 
withdrawal may be useful in identifying 
mechanisms underlying these states. 
For example, the GABA A benzodi- 
azepine site antagonist flumazenil can 
block the anxiogenic effects of ethanol 
withdrawal in an elevated plus maze 



218 



Behavioral Effects and Underlying Neurocircui tries of Alcohol 



(Moy et al. 1997). This is an unusual 
finding, since ethanol is not believed 
to interact directly with the benzodi- 
azepine site, and flumazenil does not 
block the anxiolytic effects of ethanol. 
It has been suggested that ethanol 
withdrawal increases the level of 
diazepam binding inhibitor (DBI) 
protein (see Moy et al. 1997). Another 
study found that a corticotropin- 
releasing factor (CRF) antagonist can 
block the effects of ethanol withdrawal 
on the elevated plus maze, suggesting 
that ethanol withdrawal increases CRF 
(Baldwin et al. 1991). The promise of 
these unconditioned responses in 
studies of ethanol withdrawal lies in 
the need to characterize the short 
time course of the ethanol withdrawal 
in rodents. 

First Dose Effects 

Unconditioned responses to ethanol 
have been studied primarily in the 
context of first dose effects. Although 
easy to implement, first dose effects 
need to be evaluated in terms of the 
potential to help understand the alco- 
hol addiction process. A vast majority 
of people have encountered a "first 
dose" of alcohol, yet only 5 to 10 per- 
cent of the population establish a pat- 
tern of behavior that results in alcohol 
abuse or addiction. The neurophar- 
macologies basis of these responses 
has been difficult to discern, often 
because false-negative data can be 
extensive (Dawson and Tricklebank 
1995) or because the response can be 
produced (and attenuated) by many 
different pharmacological agents. 
More sophisticated apparent pA 2 
analyses of antagonists could help 



identify receptor heterogeneity in 
behavioral assays (Kenakin 1993), but 
these analyses have not been applied. 
Thus, responses associated with the 
first dose effects have not been, and 
may never be, useful in determining 
the underlying neuropharmacology of 
ethanol associated with abuse and 
addiction. However, these uncondi- 
tioned responses to ethanol have been 
successfully used in selectively breeding 
animals for response to ethanol. The 
selective breeding approaches have 
shown beyond a doubt that there is a 
genetic basis of these responses to 
ethanol. However, this no longer seems 
an appropriate goal for behavioral 
genetic studies, because techniques 
are now available to target specific 
genes in relation to more complex 
behaviors associated with ethanol. 
The characterizations of first dose 
effects are also necessary in following 
the development of tolerance or sensi- 
tization to ethanol. However, toler- 
ance and sensitization are processes 
that primarily use a repeated dosing 
design. Therefore, it would seem that 
studying the effects of only an initial 
single dose of alcohol is not sufficient 
to understand the neurobiological 
basis of behavior associated with alco- 
hol's use and abuse. 

Conditioned Behavior 

Behaviors that are classified as learned, 
acquired, or conditioned can be mod- 
ified by environmental events, and the 
resultant behaviors can, in turn, alter the 
immediate environment. The dynamic 
interaction between the environment 
and behavioral responses serves to pro- 
duce, refine, and eliminate conditioned 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



behaviors (Weiss and Cory-Slechta 
1994). This constant redefining of 
behavioral responses within environ- 
mental contexts provides a rich behav- 
ioral repertoire, emanating from a 
complex set of neurobiological 
processes. Alcohol can interact to dif- 
ferent degrees with any of these 
neurobiological processes and alter 
the behavioral response to the envi- 
ronment. It is incumbent upon behav- 
ioral science to direct research in 
animal models that is accessible to 
modern neurobiological techniques 
and to translate the results of this 
research into meaningful principles to 
guide research in humans. In alcohol 
research, conditioned behaviors have 
primarily focused on alcohol's stimu- 
lus effects associated with discrimina- 
tion, anxiety, reinforcement, learning 
and memory, and aggression. In addi- 
tion, the processes of tolerance and 
the roles of acute withdrawal and 
stress on these stimulus effects have 
been the subject of intensive studies 
and theoretical debates. 

Discriminative Stimulus Effects 

Discriminative stimuli specifically 
covary with the availability of rein- 
forcement. Most early investigations 
of discriminative stimuli used external 
environmental stimuli perceived 
through sensory mechanisms. However, 
it is clear that the internally produced 
(interoceptive) stimulus effects of a 
psychoactive drug exert robust stimulus 
control. In simple drug discrimination 
studies, the animal is trained, through 
differential reinforcement, to engage 
in a particular behavior in the presence 
of the internal effects of the drug, and 



to engage in a different behavior in 
the absence of the internal effects of 
the drug. Thus, the discrimination 
paradigm provides a measure of the 
association between interoceptive sen- 
sations and observable behaviors, and 
is often interpreted to be an animal 
model of the subjective effects of 
drugs (Preston and Bigelow 1991). 

Drug discrimination procedures have 
been refined to provide one of the most 
powerful avenues of research for char- 
acterizing the pharmacological aspects 
of the drug in relation to behavior. As 
with other procedures in the analysis of 
behavior, the reliable baseline of behavior 
produced by drug discrimination pro- 
cedures allows a systematic approach to 
characterizing the influence of pharma- 
cological variables. Over the past half- 
century, these procedures have been used 
extensively to characterize the pharmaco- 
logical effects of many drugs of abuse 
(Colpaert 1986). Data accumulated over 
these years have demonstrated that the 
discriminative stimulus effects of drugs 
vary along quantitative and qualitative 
dimensions and have characteristics 
indicative of receptor- mediated activity 
(Holtzman 1990). Drug discrimina- 
tion procedures have proved to be a 
reliable and valuable tool for screening 
substances for abuse potential, character- 
izing potential antagonists, identifying 
active metabolites, and establishing 
structure-activity relationships of psy- 
choactive substances. Because of their 
usefulness in these areas, drug discrim- 
ination procedures have become an 
important method for categorizing and 
adding information regarding the phar- 
macological action of drugs with simi- 
lar behavioral profiles. 



220 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



The specificity of drug discrimination 
procedures, as they apply to ethanol, 
is perhaps best illustrated by the abil- 
ity of these procedures to separate the 
effects of ligands at the various sites 
on the GABA A receptor complex. 
Specifically, muscimol and 4,5,6,7- 
tetrahydroisoxazolo( 5 ,4-c )pyridin- 3 - 
ol (THIP) do not substitute for 
midazolam, diazepam, pentobarbital, 
or ethanol (Ator and Griffiths 1989; 
Grech and Balster 1993; Shelton and 
Balster 1994). Pentobarbital and 
midazolam result in partial substitution 
for muscimol and THIP (Grech and 
Balster 1997). These data imply that 
positive modulators of GABA A recep- 
tors, including ethanol, produce stim- 
ulus effects that are fundamentally 
different than those produced by 
direct GAB A agonists. 2 In contrast to 
the GAB A- site agonists, GABA A posi- 
tive modulators consistently produce 
ethanol-like discriminative stimulus 
effects. Specifically, barbiturates that 
produce ethanol discriminative stimu- 
lus effects are pentobarbital, pheno- 
barbital, or barbital in rats and 
pentobarbital in monkeys (Grant et al. 
1996). Ligands at the benzodiazepine 
site that produce ethanol-like effects 
include chlordiazepoxide, lorazepam, 
and midazolam in rats; chlordiazepox- 
ide in gerbils; diazepam in pigeons; 
and oxazepam in mice (Sanger 1997). 
In addition to positive modulators at 
the barbiturate and benzodiazepine 
site on GABA A receptors, positive 
modulators at the neurosteroid site on 
the GABA A receptors also produce 
ethanol-like discriminative stimulus 
effects in rats (Ator et al. 1993) and 
monkeys (Grant et al. 1996). Thus, 



positive modulation of the GABA A 
receptor system appears to be a robust 
component of the ethanol cue. 

Diazepam-sensitive receptors can 
be divided into BZ1 receptors, with 
high affinity for Zolpidem, and BZ2 
receptors, with low affinity for Zolpi- 
dem. There is mounting evidence that 
drug discrimination procedures differ- 
entiate BZ1 ligands from benzodi- 
azepines that have activity at both 
BZ1 and BZ2 receptor subtypes. 
Zolpidem results in only partial sub- 
stitution in chlordiazepoxide discrimi- 
nation, and benzodiazepines result in 
partial substitutions for Zolpidem 
(Sanger et al. 1987). Similar to benzo- 
diazepines, the BZ1 selective agonists 
Zolpidem, zaleplon (CL 284,846), and 
SX 3228 produce only partial substi- 
tution in rats trained to discriminate 1.0 
g/kg ethanol (Sanger 1997). These 
data suggest that activity only at BZ1 
receptors is insufficient to produce an 
ethanol-like effect. Although these 
data fit well with ethanol potentiating 
activity at both BZ1 and BZ2 receptor 
subtypes, they are in contrast to the in 
vitro data of Criswell and colleagues 
(1995), who suggested that selective 
BZ1 activity predicts sensitivity to 
ethanol. It should be noted that the dis- 
criminative stimulus effects of ethanol 
are not antagonized by flumazenil 
(Hiltunen and Jarbe 1986). These 
data suggest that ethanol does not 
interact directly with the benzodi- 
azepine binding site. 

Functional antagonism of ethanol- 
enhanced activity at GABA A receptors 
using negative modulators of CI" flux 
active at the receptor (inverse agonists) 
has been extensively characterized 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



with the partial inverse agonist Ro 15- 
4513 (see Grant and Lovinger 1995). 
Numerous in vitro assays demonstrate 
that Ro 15-4513 blocks the effects of 
ethanol. In contrast, the benzodiazepine 
inverse agonists block some, but not 
all, of the behavioral effects of ethanol. 
In general, the behavioral effects of 
ethanol that are resistant to the effects 
of benzodiazepine inverse agonists 
often are associated with higher ethanol 
doses. The mechanisms underlying the 
specificity of Ro 15-4513 in attenuating 
some of ethanofs actions are unknown. 
Since Ro 15-4513 has intrinsic activity 
at the GABA A receptor and has anxio- 
genic and proconvulsant activity, it 
has been suggested that the ability to 
block some of the behavioral effects of 
ethanol is due to an additive interac- 
tion rather than a pharmacological 
antagonism at a single receptor mech- 
anism (Mihic and Harris 1996). 

There are mixed reports about the 
efficacy of Ro 15-4513 to block the 
discriminative stimulus effects of 
ethanol. The discriminative stimulus 
effects of 1.0 g/kg ethanol were 
antagonized in mice and rats. However, 
other studies using rats have failed to 
report any substantial antagonism of Ro 
15-4513 on the discriminative stimulus 
effects of ethanol. Gatto and Grant 
(1997) reported a wide range of sensi- 
tivities to the ethanol-attenuating effects 
of Ro 15-4513, and the potency of Ro 
15-4513 to block ethanol's discrimina- 
tive stimulus effects generally decreased 
as the substitution dose of ethanol 
increased. Overall, it appears that the 
ethanol-blocking effects of Ro 15-4513 
are surmountable with higher doses of 
ethanol, indicating a competitive antag- 



onism. Current studies using cynomolgus 
monkeys trained to discriminate ethanol 
are replicating these general findings, 
showing a blockade of the cUscriminative 
stimulus effects of 1 .0 g/kg ethanol but 
not 2.0 g/kg ethanol (Grant unpub- 
lished data). 

An intriguing hypothesis to account 
for the attenuation of ethanol's actions is 
the presence of benzodiazepine-insensitive 
GABA A receptors that contain a binding 
site for Ro 15-4513 on a 6 subunits 
(Grobin et al. 1998). However, it is 
clear that Ro 15-4513 has activity at 
benzodiazepine -sensitive receptors and 
can block the discriminative stimulus 
effects of benzodiazepines (Hiltunen 
and Jarbe 1988; Rees and Balster 
1988; Hiltunen and Jarbe 1989). In 
addition, the in vitro data showing that 
a 6 subunits correlate with ethanol sen- 
sitivity of chronic effects are not com- 
pelling (Grobin et al. 1998). 
Alternative to a GABA A hypothesis, 
overcoming the attenuating effects of 
Ro 15-4513 in an ethanol discrimina- 
tion with increased doses of ethanol 
may be due to other neural targets 
(i.e., NMDA and/or serotonin recep- 
tors) that serve as the basis of ethanol 
discrimination. Thus, by increasing the 
dose of ethanol, discriminative stimulus 
effects of ethanol not mediated by the 
GABA A receptor can serve as the basis 
for the discrimination. Since rats can 
be trained to discriminate ethanol from 
pentobarbital using a two-choice or a 
three-choice method (Bowen et al. 
1997), there is evidence that these 
other receptor systems can play an 
important role in ethanol discrimina- 
tion. One of these systems is the 
NMDA receptor system. 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



Shortly after the in vitro biochemical 
and electrophysiological findings that 
ethanol attenuated NMDA receptor- 
mediated ion flux, the NMDA channel 
blockers ketamine, phencyclidine 
(PCP), and dizocilpine, or ( + )MK- 
801, were shown to substitute for 
ethanol in mice and pigeons (Grant et 
al. 1991). This work was quickly 
extended to rats, and NMDA channel 
blockers have been shown to substitute 
for ethanol in a number of laboratories 
(Gauvin et al. 1994; Grant 1994; 
Harrison et al. 1998; Hundt et al. 
1998). A recent study found that five 
channel blockers — dizocilpine, 
memantine, PCP, ( + ) pentazocine, 
and ( + )NAMN ( TV-allynormeta- 
zocine) — substituted for a 1.0 g/kg 
(ip) training dose of ethanol in rats 
(Hundt et al. 1998). This study also 
found no substitution of the sigma- 
opioid selective antagonists rimcazole 
and FH 510, indicating that the sub- 
stitution of the NMDA channel 
blockers was due to their activity at 
the NMDA channel and not the 
sigma site, where they also bind. The 
substitution of competitive NMDA 
antagonists for ethanol is less reliable, 
with one study showing full substitution 
of the competitive antagonist CGS 
19755 and other studies showing par- 
tial substitution of the competitive 
antagonists CGS 19755, NPC 17742, 
and CPPene (cited in Hundt et al. 
1998). No study has found substantial 
substitution of glycine site antagonists 
for ethanol, including L-701,324, 
MRZ-2/504, ACEA 1021 (cited in 
Hundt et al. 1998), and (+) HA-966 
(Grant unpublished data). Finally, the 
polyamine site antagonists eliprodil, 



arcaine, and spermidine all failed to 
produce even partial ethanol-like 
responding (cited in Hundt et al. 
1998). In addition, the ct-amino-3- 
hydroxy-5-methyl-4-isoxazole propi- 
onic acid (AMPA) antagonist GYKI 
52466 did not substitute for ethanol, 
suggesting that the NMDA antagonism 
effects of ethanol are more prominent 
than the AMPA antagonist effects. 
However, these results need to be 
interpreted cautiously, because only a 
single AMPA antagonist has been 
tested in an ethanol discrimination. 

The composite picture from the 
NMDA complex ligands suggests that 
ethanoPs discriminative stimulus 
effects that are mediated by NMDA 
antagonism are generated by channel 
blockade and not interaction with the 
NMDA, glycine, or polyamine sites. 
The molecular composition of the 
NMDA channel complex is still being 
characterized, but there is now con- 
siderable evidence that the subunit 
composition confers pharmacological 
sensitivity. The competitive NMDA 
antagonists are more potent at 
NR1/NR2A heteromeric receptors, 
the glycine site antagonists are more 
potent at NR1/NR2C heteromeric 
receptors, the polyamine site antago- 
nists are more potent at NR1/NR2B 
heteromeric receptors, and the chan- 
nel blockers and ethanol are equipo- 
tent at NR1/NR2A and NR1/NR2B 
combinations (Sucher et al. 1996). 
Interestingly, detoxified alcoholics 
report that the subjective effects of 
ketamine are similar to the effects of 
high doses of ethanol (Krystal et al. 
1998). A recent study by Hodge and 
Cox (1998) found that dizocilpine, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



but not the competitive antagonist 
CPP, administered directly into the 
core of the nucleus accumbens or the 
CA1 region of the hippocampus was 
sufficient to produce ethanol-like 
effects in rats. In the same study, 
dizocilpine administration into the 
amygdala or the prelimbic cortex did 
not engender ethanol substitution. 

GABA A receptors may be present on 
a majority of CNS neurons, and there is 
considerable evidence that GABAergic 
activity can regulate glutamatergic, 
particularly NMDA-mediated, neuro- 
transmission (Lovinger 1993). Specifi- 
cally, when GABAergic transmission is 
decreased, neuronal depolarization is 
more likely to occur, releasing the 
Mg ++ blockage of the NMDA receptor 
and leading to increased functional 
activity of the NMDA channel. Con- 
versely, GABA A receptor activation 
decreases the probability of the NMDA 
receptor being activated due to the 
greater Mg ++ blockage of the NMDA 
channel at more hyperpolarized 
potentials. In addition, the inhibitory 
postsynaptic potentials mediated by 
GABAergic transmission and the excita- 
tory postsynaptic potentials have a similar 
time course (Lovinger 1993). Thus, it 
is believed that NMDA-mediated glu- 
tamatergic transmission is normally 
regulated by GABAergic transmission. 
The evidence to suggest a functional 
interaction of ethanoPs effects at GABA A 
and NMDA receptors largely comes 
from studies aimed at characterizing 
the hyperexcitability following chronic 
ethanol exposure (see Grant and 
Lovinger 1995). Nevertheless, the acute 
action of ethanol to potentiate the 
effects of GABA A activity may result in 



an enhancement of NMDA channel 
attenuation, in addition to ethanol's 
direct actions in antagonizing the 
NMDA channel. This hypothesis pre- 
dicts a greater effect of ethanol on 
inhibition of localized neuronal activity 
than the effects of either a GABA A 
positive modulator or an NMDA 
antagonist alone. It appears reasonable 
to suggest that the interaction of 
ethanol's simultaneous effects at GABA A 
and NMDA channels is an important 
aspect of ethanol's stimulus effects. 

Several laboratories have begun to 
address the simultaneous activity of 
ethanol at NMDA and GABA A receptor 
systems in producing discriminative 
stimulus effects. One demonstration 
administered combinations of dizo- 
cilpine or CPP and muscimol in specific 
brain regions. Concentrations of mus- 
cimol and dizocilpine (or CPP) that did 
not engender ethanol substitution when 
administered separately into the nucleus 
accumbens produced robust ethanol sub- 
stitution in this demonstration (Hodge 
and Cox 1998). Using a different 
strategy, a combination of diazepam 
and ketamine was used as the training 
condition, and ethanol produced sub- 
stitution for the mixture (Harrison et 
al. 1998). In ethanol-trained rats, this 
combination of ketamine and diazepam 
substituted for ethanol. This is a 
demonstration of cross -generalization 
between ethanol and an NMDA 
antagonist/GABA A positive modulator 
mixture. Given the overwhelming data 
showing asymmetrical generalizations, 
the data suggest that co-occurrence of 
GABA^NMDA activity is an impor- 
tant aspect of the receptor mediation 
of ethanol's discriminative stimulus 



224 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



effects. Finally, in humans the benzo- 
diazepine lorazepam can attenuate 
ketamine-induced emotional distress 
and perceptual alterations while exacer- 
bating the sedative, attention-impairing, 
and amnestic effects of ketamine and 
having no effect on the subjective report 
of high (Krystal et al. 1998). Notably, 
subjects described the high as ethanol- 
like ("like when I'm drinking, but 
hazier than when I'm drinking"). 

Studies have shown that 5-HT 1B 
receptors are up-regulated following 
chronic ethanol exposure and are lower 
in the CNS of rats bred to prefer 10 
percent ethanol (Grant and Lovinger 
1995). In drug discrimination studies, 
it was initially reported that the rela- 
tively nonselective 5-HT\ receptor 
agonist m-trifluoromethylphenylpiper- 
azine (TFMPP) substituted for the 
discriminative stimulus effects of ethanol 
in rats (Signs and Schechter 1988). 
Subsequent studies replicated this find- 
ing in rats trained to discriminate 1.0 
and 1.5 g/kg ethanol but not in rats 
trained to discriminate 2.0 g/kg ethanol 
(Grant and Colombo 1993; Green and 
Grant 1998). The finding that a 5-HT 
receptor agonist was similar to the dis- 
criminative effects of ethanol is intrigu- 
ing because another 5-HT agonist, 
w-chlorophenylpiperazine (mCPP), 
has been reported to produce ethanol- 
like subjective effects and alcohol crav- 
ing in recently detoxified alcoholics 
(Benkelfat et al. 1991; Krystal et al. 
1994; Buydens-Branchey et al. 1997). 

5-HT 1B receptors are negatively 
coupled to cyclic adenosine monophos- 
phate through G : proteins and act as 
presynaptic autoreceptors and hetero- 
receptors to decrease neurotransmitter 



release. There is accumulating evidence 
that 5-HT 1B receptors are important 
in the control of striatal dopaminergic 
release and that 5-HT 1B agonists 
increase striatal dopamine levels. These 
effects are consistent with the ability of 
5-HT 1B agonists to increase locomotor 
activity, disrupt prepulse inhibition, 
and substitute for, or enhance, the 
discriminative stimulus effects of 
cocaine. The interaction between 5- 
HT 1B receptors and dopaminergic 
release in the striatum is not clear, but 
one hypothesis is that 5-HT 1B receptors 
function as heteroreceptors on inter- 
neurons. Data gathered with drug dis- 
crimination suggest a functional link 
between the actions of ethanol at 
GABA A receptors and 5-HT 1B media- 
tion of ethanol-like activity (Green 
and Grant 1998). 

There are only a few investigations 
of 5-HT 2 receptor ligands and the dis- 
criminative stimulus effects of ethanol. 
These studies have shown that the 5- 
HT 2 a antagonists ketanserin in 
pigeons (Grant and Barrett 1991) and 
cinanserin in rats (Winter 1977) do not 
block the discriminative stimulus 
effects of ethanol. Likewise, the 5- 
HT 2A agonist 5-MeODMT (5- 
methoxy-N,A r -dimethyltryptamine) 
does not appreciably substitute for 
ethanol in rats (Signs and Schechter 
1988). In contrast, the 5-HT transport 
inhibitor fluoxetine produces substitu- 
tion in an ethanol discrimination, and 
this substitution can be selectively 
blocked by the 5-HT 2A antagonist 
MDL 100,907 (Maurel et al. 1997). 
These results suggest that the 5-HT 2A 
receptor mediates ethanol-like dis- 
criminative stimulus effects. However, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



a recent study found the 5-HT 2 a/2c 
agonist DOI did not engender or 
potentiate the discriminative stimulus 
effects of ethanol (Szeliga and Grant 
1998). Likewise, the 5 -HT 2A antago- 
nist ketanserin failed to attenuate the 
discriminative stimulus effects of 
ethanol (Szeliga and Grant 1998). 
The data collectively suggest that the 
5-HT 2 receptor ligands DOI and 
ketanserin interact with receptors that 
are not substantially involved in medi- 
ating the discriminative stimulus 
effects of ethanol associated with 
approximately 40-120 mg/dL blood 
ethanol concentrations. 

To date, only a limited number of 
studies have investigated the 
pharmacological basis of ethanoPs dis- 
criminative stimulus effects in selectively 
bred ethanol-preferring (P) and ethanol - 
nonpreferring (NP) rats. Cholinergic, 
serotonergic, and GABAergic receptor 
systems are preliminarily implicated in 
mediating differences in the stimulus 
effects of rats differentially bred to 
prefer ethanol. Data implicating the 
nicotinic cholinergic system is derived 
from nicotine's partial substitution for 
1.0 g/kg ethanol in P rats, with no 
substitution in NP rats (Gordon et al. 
1993). However, ascribing receptor 
mechanisms based on partial substitu- 
tion in drug discrimination procedures 
is controversial (see Colpaert 1986). 
The serotonergic system is implicated 
by the substitution of the hallucinogenic 
amphetamine MDMA (3,4-methyl- 
enedioxymethamphetamine), a com- 
pound whose discriminative stimulus 
effects are believed to be primarily 
serotonergic in nature (Schechter 
1989). In an ethanol discrimination 



using a training dose of 0.6 g/kg 
ethanol, MDMA substituted in HAD 
but not in LAD rats, suggesting a 
greater relevance of serotonergic acti- 
vation in the effects of ethanol in the 
preferred line (Meehan et al. 1995). 
Finally, GABAergic system differences 
in rat lines selected for ethanol prefer- 
ence are suggested by data showing a 
greater sensitivity to the ethanol-like 
effects of pentobarbital HAD rats 
compared with LAD rats, as measured 
by lower ED 50 value for pentobarbital 
substitution (Krimmer 1991). How- 
ever, line differences were only noted 
between HAD and LAD rats trained 
to discriminate the stimulus effects of 
0.75 g/kg ethanol 30 minutes after 
injection. There were no line differ- 
ences in rats trained to discriminate 
0.75 g/kg ethanol 2 minutes postin- 
fection (Krimmer 1992). Overall, 
there is a scarcity of data available to 
address the basis for ethanol's discrim- 
inative stimulus effects in rat lines 
selected for ethanol preference. 

There are a host of other receptor 
systems that have been only superfi- 
cially addressed in terms of discrimina- 
tive stimulus effects. Most notable are 
the opiate, dopaminergic, and voltage- 
gated calcium channel (VGCC) systems. 
In general, the opiate agonist morphine 
does not produce ethanol-like discrim- 
inative stimulus effects. However, nalox- 
one has been reported to block the 
discriminative stimulus effects of ethanol 
associated with the rising phase of the 
blood ethanol curve (Spanagel 1996). 
Only partial antagonism by naloxone (50 
percent ethanol- appropriate responding) 
was found with a 1.0 g/kg (ip) ethanol 
training dose. In the same study, delta 



226 



Behavioral Effects and Underlying Neurocircui tries of Alcohol 



and kappa opioid antagonists had no 
effect (Spanagel 1996). Dopaminergic 
agonists do not substitute and dopa- 
minergic antagonists do not block the 
discriminative stimulus effects of ethanol. 
However, apomorphine and ampheta- 
mine potentiate the discriminative 
stimulus effects of ethanol (Schechter 
1985). Similar findings have been 
reported with nicotine, where substi- 
tution is not found but potentiation 
of ethanol's stimulus effects is evident. 
Finally, VGCC antagonists have been 
reported to partially substitute (De Beun 
et al. 1996), to have no effect (Schechter 
1994), or to block (Colombo et al. 
1994) the discriminative stimulus 
effects of ethanol. Recent data have 
shown that VGCC agonists can antag- 
onize and VGCC antagonists can 
potentiate ethanol's discriminative 
stimulus effects (Green and Grant 
unpublished data). These findings 
illustrate an important strategy in drug 
discrimination: investigating the modu- 
latory effects of candidate receptor sys- 
tems on discriminative stimulus effects. 
The drug discrimination procedure 
is one technique where the multiple 
stimulus effects of ethanol have been 
extensively studied and can be readily 
compared. An important issue in 
using the discriminative stimulus 
effects of ethanol to help identify can- 
didate receptor systems to explore in 
other behavioral paradigms is the 
importance of dose. The training dose 
of a drug that acts with a high degree 
of specificity at a single receptor system 
may be analogous to stimulus intensity. 
In contrast, the evidence reviewed 
above clearly shows that ethanol has 
interactions at multiple receptors. 



Thus, the training dose of ethanol 
may determine both the intensity and 
the qualitative effects of the drug 
stimulus. That is, ethanol's functional 
activity at the various receptor systems 
may not amplify in equal proportion 
as the dose of ethanol is increased, 
resulting in ethanol having qualita- 
tively different discriminative stimulus 
effects at different doses. Ethanol has 
a well-known biphasic profile, with 
low doses resulting in activation and 
high doses resulting in sedation. Some 
of these biphasic effects may reflect 
differential sensitivity of the receptor- 
linked ionophores to a given dose of 
ethanol. The relative contribution of 
receptor systems to the qualitative 
effects of ethanol as a function of dose 
can be addressed by examining the 
discriminative stimulus effects of dif- 
ferent ethanol training doses. 

In summary, drug discrimination 
procedures can be used in alcohol 
research to provide candidate receptor 
systems and receptor mechanisms to 
target for altering the behavioral effects 
of ethanol. For example, in vitro data 
suggest that ethanol interacts with 
GABA A receptors to increase chloride 
flux. However, there are several sites 
on this receptor system that interact 
with ligands to alter chloride flux. 
Drug discrimination procedures can 
be used to differentiate these sites on 
a behavioral level. By acting as a dis- 
criminative stimulus, factual evidence is 
provided that these effects of the drug 
can be perceived and control behavior. 
Thus, the discriminative stimulus 
effects of a drug provide information on 
the realm of possible stimulus effects 
that can serve other functions, for 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



example, reinforcing stimulus effects. 
After identifying the possible receptor 
effects that can act as stimuli, addi- 
tional studies must characterize the 
role of particular receptor mechanisms 
in mediating other stimulus effects of 
ethanol. Although these additional 
stimulus effects of ethanol can be (and 
have been) studied without first being 
characterized in discrimination studies, 
it is not always possible to separate 
ethanol -specific from task- specific recep- 
tor mechanisms underlying behavior. 
Drug discrimination studies are also 
providing hypotheses of simultaneous, 
but independent, receptor activity by 
ethanol that may alter in prominence as 
the dose of ethanol changes. Thus, the 
candidate receptor systems that may 
mediate the behavioral effects associ- 
ated with specific doses of ethanol can 
be identified and then tested within 
more appropriate paradigms. 

Anxiolytic and Anxiogenic 
Stimulus Effects 

It has been hypothesized for many years 
that the ability of alcohol to reduce 
stress underlies its ability to serve as a 
reinforcer (Williams 1966; Pohorecky 
1981). Indeed, accumulating clinical 
evidence indicates a high degree of 
co-occurrence of anxiety and alcohol 
dependence (see Langenbucher and 
Nathan 1990; Crum et al 1995). Two 
mechanisms are likely responsible for 
this comorbidity, both involving condi- 
tioning. First, excessive alcohol intakes 
may be due to the anxiolytic properties 
of this drug alleviating a constant 
"basal" state of anxiety in some individ- 
uals. Second, abstinence subsequent to 
excessive and prolonged consumption 



of alcoholic beverages gives rise to 
dysphoric effects, including anxiety, 
that may be relieved by alcohol con- 
sumption. Evidence for an association 
of alcohol's anxiolytic effects with the 
use of alcohol as a basis for promoting 
future alcohol consumption is derived 
from both human and animal studies. 
When social drinkers believe that they 
have consumed alcohol, imbibing a 
nonalcoholic drink results in decreased 
levels of anxiety (Abrams and Wilson 
1979). Thus, humans can associate 
the consumption of alcohol with its 
anxiolytic effects. However, caution is 
necessary in extrapolating from the 
anxiolytic effects of alcohol and sub- 
sequent reinforcing effects of alcohol 
in humans. First, an important consid- 
eration is the generally weak anxiolytic 
effects of alcohol noted in humans. A 
possible exception may be people with 
signs of anxiety disorder who report 
anxiolytic effects of alcohol (Chutuape 
and deWit 1995). However, these 
individuals did not choose to drink 
alcohol, again suggesting a dichotomy 
between the weak anxiolytic effects 
and the reinforcing effects of alcohol. 
In animal models, anxiety has been 
associated with increases in ethanol self- 
administration and consumption. For 
example, early stressful rearing condi- 
tions and social separations are associ- 
ated with behavioral and physiological 
reactions associated with stress, and 
these conditions are sufficient to initi- 
ate and maintain ethanol consump- 
tion (Kraemer and McKinney 1985; 
Blanchard et al. 1987; Higley et al 
1991). Consistent with these findings, 
higher innate levels of anxiety have 
also been detected in selectively bred, 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



ethanol-preferring P (Stewart et al. 
1993), SP (Colombo et al. 1995), 
and HARF (Le 1996) rats, compared 
with ethaiiol-nonpreferring NP, SNP, 
and LARF rats, respectively. Thus, 
genetic models appear to have both 
increased baseline levels of anxiety and 
increased ethanol consumption, suggest- 
ing a genetic basis for a functional link 
between the anxiolytic effects of ethanol 
and increased ethanol consumption. 
This hypothetical association is similar 
to findings that alcohol attenuates stress 
reactions in young adults at risk for 
alcohol dependence (Sher and Leven- 
son 1982). 

The evidence for anxiolytic effects 
of ethanol in animal models has been 
reviewed (Pohorecky 1990). Condition- 
ing procedures frequently use conflict, 
in which responding maintained by 
food or water presentation in a deprived 
animal is occasionally punished, usually 
by the delivery of shock (Pohorecky 
1981). These procedures result in a sup- 
pression of behavior that can be rein- 
stated with typical anxiolytics such as 
benzodiazepines. These effects of 
ethanol are fairly robust, although 
ethanol is somewhat less efficacious as 
an anxiolytic compared with benzodi- 
azepines. The anxiolytic effects of 
ethanol occur at low to moderate 
dosages and are not always separable 
from other behavioral effects of 
ethanol (Koob and Britton 1996). 

The receptor mechanisms involved 
in the anxiolytic effects of ethanol 
have investigated primarily the GABA A 
and 5-HT receptor systems. The sci- 
entific literature is replete with evidence 
showing the involvement of GABA A 
receptor systems in regulating anxiety. 



As a class of compounds, benzodi- 
azepines that act as positive modulators 
of GABA A receptors produce the most 
robust anxiolytic effects in experimental 
models (e.g., Pellow et al. 1985; Jones 
et al. 1994; Rex et al. 1996) and con- 
stitute the first-line therapy for the 
treatment of anxiety in general practice 
(e.g., Ballinger 1990; Ashton 1994). 
Recent attention has been focused on 
the interactions of neurosteroids with 
the GABA A receptor complex. Similar 
to the benzodiazepines, neuroactive 
steroids that positively modulate 
GABA A receptors produce anxiolytic 
activity (Crawley et al. 1986; Britan et al. 
1991; Wieland et al. 1991; Fernandez- 
Guasti and Picazo 1995). The intrigu- 
ing aspect of the neurosteroids is that 
these are endogenous compounds, 
synthesized within the CNS as well as 
being derived from adrenal and gonadal 
steroids. Furthermore, endogenous 
levels of neurosteroids are associated 
with stressful events (see Paul and 
Purdy 1992). Anticonflict effects of 
ethanol can be blocked by GABA A 
ligands to the convulsant site and 
GABA A inverse agonists, but not by 
benzodiazepine site antagonists (Koob 
and Britton 1996). Interestingly, 
naloxone has been reported to reverse 
the anticonflict effects of ethanol and 
benzodiazepines (Koob and Britton 
1996). Corticotropin-releasing factor 
also reverses the anticonflict effect of 
ethanol, although it has proconflict 
activity so the effect may not be spe- 
cific to ethanol. CRF appears to inter- 
act through extrahypothalamic sites 
within the mesolimbic dopaminergic 
system, notably the amygdala, in 
response to stressful events, including 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



ethanol withdrawal (Pich et al. 1995). 
Only a few studies have used site-specific 
injections to investigate neural cir- 
cuitry of conflict behavior. These 
studies suggest that the basolateral 
and central nuclei of the amygdala, 
areas that are rich in GABA A recep- 
tors, are important in the mediation 
of anticonflict activity by benzodi- 
azepines and barbiturates. 

Over the last two decades, the poten- 
tial role of serotonin in the modulation 
of emotional states, including anxiety, 
has been addressed by several animal 
studies (reviewed in Barrett and 
Vanover 1993; Griebel 1995). It has 
been proposed that a reduction in 
central serotonin results in anxiolysis 
(Griebel 1995). Several studies have 
focused on the involvement of the 5- 
HT 1A subtype of the serotonin receptor 
in modulating anxiety (Lucki 1992; 
Barrett and Vanover 1993; Barrett et 
al. 1994; Griebel 1995), although 
inconsistent results have been reported 
(Dawson and Tricklebank 1995). The 
anxiolytic effects of 5-HT 1A receptor 
agonists have been suggested to be 
related to activation of 5-HT 1A autore- 
ceptors, which results in a reduced 
serotonin neuronal function (Dourish 
et al. 1986). Specifically, 5-HT 1A soma- 
todendritic autoreceptors in the raphe 
nuclei result in a reduction in 5-HT 
neuronal firing rate and subsequent 
decreases in 5-HT release in terminal 
areas of the limbic system, such as the 
hippocampus. Postsynaptic 5-HT 1A 
receptor activation in the amygdala and 
dorsal hippocampus has been reported 
to be anxiogenic, suggesting that an 
overall reduction in 5-HT neurotrans- 
mission to the dorsal hippocampus is 



anxiolytic (File et al. 1996). Direct 
administration of 5-HT 1A agonist 8- 
OH-DPAT into the raphe nucleus 
produces anxiolytic activity (see File et 
al. 1996), conditioned place preference 
(Fletcher et al. 1994), and selective 
increases in ethanol consumption 
(Tomkins et al. 1994#). Interestingly, 
when given peripherally, very low 
doses of 8-OH-DPAT (30-60 
^xg/kg) increase ethanol consumption 
(Tomkins et al. 1994&), whereas 
higher doses of 8-OH-DPAT, as well 
as buspirone and ipsapirone, signifi- 
cantly reduce voluntary ethanol intake 
in rats, mice, and monkeys (DeVry 
1995). The reduction in ethanol 
intake following peripheral injections 
of higher doses of 5-HT 1A agonists 
possibly reflects anxiogenic activity at 
postsynaptic 5-HT 1A receptors (File et 
al. 1996). In a social confrontation 
procedure using consecutive "phases" 
of increasing threat, ethanol was simi- 
lar to benzodiazepines and the 5-HTi 
agonist gepirone in decreasing signs 
of anxiety associated with future 
antagonistic encounters (e.g., tachy- 
cardia, hyperthermia, vocalizations) 
(Tornatzky and Miczek 1995). Addi- 
tionally, buspirone has been success- 
fully tested in clinical trials of anxiety 
associated with abstinence from alco- 
hol in alcoholics (Litten et al. 1996). 

Pentylenetetrazol (PTZ) binds to the 
picrotoxin site of the GABA A receptor 
complex, resulting in a reduction of 
the chloride ion flux, an increase in 
CNS excitability, and convulsions at 
high doses (Simmonds 1982). PTZ 
increases the subjective reports of anxiety 
in humans (Rodin 1958) and has been 
reported to be anxiogenic in laboratory 



230 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



animals in several experimental ap- 
proaches (e.g., Buczek et al. 1994; 
Rodgers et al. 1995). The discriminative 
stimulus effects produced by PTZ have 
been promoted as an animal model of 
anxiety (Lai and Emmett-Oglesby 
1983). Features of this discrimination 
that address the validity of an animal 
model of anxiety include the genera- 
tion of PTZ- appropriate responding 
following (a) exposure to stressful 
exteroceptive stimuli, such as a novel 
environment (Carey et al. 1990), a cage 
intruder (Carey et al. 1990), and a 
predator-prey interaction (Gauvin and 
Holloway 1991); (b) withdrawal from 
chronic benzodiazepines (Emmett- 
Oglesby et al. 1990) and ethanol (Lai 
et al. 1988); and (c) administration of 
anxiogenic drugs, such as FG 7142 
(Leidenheimer and Schechter 1990), 
Ro 5-3663, Ro 15-4513, and (3-car- 
bolines (Emmett-Oglesby et al. 
1990). Furthermore, anxiolytic com- 
pounds, mainly those acting at the 
GABA A receptor, have been reported to 
completely block the interoceptive cues 
of PTZ (Andrews et al. 1989). Ethanol 
is effective in blocking a training dose 
of 10 mg/kg PTZ, indicating that the 
anxiolytic effects of ethanol are assess- 
able in a PTZ discrimination (Emmett- 
Oglesby et al. 1990). 

In addition to serving as an animal 
model of anxiety, PTZ discriminations 
have also been used to assess anxio- 
genic aspects of ethanol withdrawal 
(Lai and Emmett-Oglesby 1983; Lai 
et al. 1988; Emmett-Oglesby et al. 
1990). It is important to note that 
acute ethanol withdrawal, several 
hours following administration of 
moderate to large (2-4 g/kg) doses 



of ethanol, resulted in PTZ generaliza- 
tion (Gauvin et al. 1989, 1992). An 
important process in the high ethanol 
intakes may be the onset of with- 
drawal- or hangover- associated anxiety 
several hours following the consumption 
of similar doses of ethanol, initiating 
further consumption of ethanol. PTZ 
discriminations also show considerable 
promise for studying the anxiety asso- 
ciated with alcohol withdrawal (Lai et 
al. 1988; Emmett-Oglesby et al. 1990). 
Following termination of chronic 
ethanol treatment rats respond on the 
PTZ-appropriate lever, indicating 
withdrawal has effects similar to the 
PTZ stimulus. This state lasts from 12 
to 48 hours, at which time the per- 
centages of rats choosing the PTZ lever 
are 80 and 30, respectively. The length 
of chronic ethanol treatment necessary 
to show this withdrawal effect is 
apparently 3 days at 12.5 g/kg/d 
(Emmett-Oglesby et al. 1990). How- 
ever, in a more recent analysis of the 
ethanol withdrawal state, acute admin- 
istration of moderate to large (2-4 
g/kg) doses of ethanol resulted in PTZ 
generalization (Gauvin et al. 1989, 
1992). Thus, PTZ discrimination 
appears to be sensitive to ethanol with- 
drawal effects following acute (i.e., 
hangover) or chronic (i.e., withdrawal) 
ethanol treatment. It may be possible to 
use this model to quantify and char- 
acterize the severity and time course 
of interoceptive stimuli associated 
with ethanol withdrawal. 

The neurocircuitry involved in con- 
ditioned anxiolytic effects of fear- 
potentiated startle responses has been 
extensively studied (Davis 1992). In this 
procedure, the startle response to an 



231 



NIAAA's Ncuroscience and Behavioral Research Portfolio 



acoustic stimulus is augmented by 
presenting the eliciting acoustic stimulus 
with a cue that has previously been 
paired with shock. The conditioned 
fear is defined by the increase in startle 
amplitude in the presence of the cue 
previously paired with shock compared 
with the startle amplitude in the 
absence of the cue. This procedure can 
be used in both humans and laboratory 
animals (Krystal et al. 1997). In 
humans, startle amplitude has been 
used to assess posttraumatic stress dis- 
order and sensory gating in schizo- 
phrenia. Ethanol attenuates startle in 
rodents and humans. Benzodiazepines 
reduce fear-potentiated startle, as do 
5-HTj agonists and morphine. On 
the other hand, drugs that increase 
reports of anxiety in humans, namely 
yohimbine and (3-carbolines, increase 
this response in rats. 

An important finding with this pro- 
cedure is the synergistic effect of 5- 
HTj agonists and D } receptor 
antagonists. The D 2 receptor antagonist 
raclopride also decreases potentiated 
startle at doses that do not alter baseline 
startle levels. It is known that dopa- 
minergic tone in the mesolimbic and 
mesocortical pathways increases follow- 
ing stressful events, and the effect of 
dopamine antagonists suggests that 
one effect of this increase in dopamine 
is the potentiation of response to fearful 
events. Dopaminergic tone in these 
pathways has also been suggested to 
potentiate response to pleasurable 
stimuli, and has led to speculation that 
ventral tegmental area (VTA) 
dopaminergic projections are not sig- 
naling pleasure but accentuating con- 
ditioned responses to significant 



stimuli (Salamone 1994; Wickelgren 
1997). In potentiated startle, informa- 
tion from the conditioned stimulus 
(light cue) and information from the 
unconditioned stimulus (shock) con- 
verge in the lateral and basolateral 
amygdala. After pairing with shock, 
the conditioned stimulus can activate 
the lateral and basal amygdala, which 
projects to the central amygdala. Acti- 
vation of the central amygdala then 
facilitates startle through the reticu- 
laris pontis caudalis, which mediates 
the startle response from acoustic 
information through the lateral lem- 
niscus to the spinal cord neurons 
(Davis 1992). 

There are several compelling hypo- 
theses that have yet to be addressed 
with ethanol. Namely, local infusion of 
NMDA antagonists into the accum- 
bens attenuates the acquisition of fear- 
potentiated startle, suggesting that 
this may also be an action of ethanol, 
an outcome that could be extrapolated 
to increases in risk-taking behaviors due 
to decreased conditioned fear. In this 
light it is interesting that the amygdala 
is one area where NMDA antagonist 
application produced ethanol-like 
stimulus effects (Hodge and Cox 
1998). Postconditioning anticonfiict 
effects in punished responding can be 
shown by direct infusion of benzodi- 
azepines, GABA, or muscimol into 
the amygdala, as discussed earlier in 
this section. Thus, it is possible that 
ethanol could act in the amygdala to 
inhibit learning about fearful stimuli 
through NMDA antagonism and also 
to inhibit response to fearful stimuli 
even after it has been learned. In addi- 
tion to NMDA and GABA A , it has 



232 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



been estimated that 25 percent of the 
neurons in the central amygdaloid 
nucleus contain CRF, somatostatin, 
and neurotensin, as well as 5-HT 3 
receptors (Davis 1992). Thus, this 
pathway and the potentiated startle 
response would appear to be an 
important area to characterize for 
alcohol responsiveness in future 
behavioral studies. 

In summary, ethanol appears to 
have attenuating effects on condi- 
tioned responses associated with anxi- 
ety. A few studies have addressed the 
correlation between anxiolytic 
responses and ethanol consumption. 
Other studies have investigated the 
neurotransmitter systems involved in 
the anxiolytic responses, primarily the 
GABA A and 5-HT systems. One area 
of study that appears promising is the 
potentiated startle response, because 
the response can be studied in both 
humans and laboratory animals and 
because the neurocircuitry has been 
established. Notably, in the startle 
procedures, NMDA antagonists 
decrease fear responding, allowing the 
incorporation of this neurotransmitter 
system into hypotheses of ethanol's 
activity on conditioned anxiolytic 
effects. Other neurotransmitter recep- 
tors, such as 5-HT 3 and neuropep- 
tides, that have previously been 
implicated in other behavioral effects 
of ethanol have yet to be extensively 
studied in mediating the acute anxi- 
olytic effects of ethanol. 

Place/Taste Conditioning Effects 

Ethanol reinforcement is a conditioned 
process that is pivotal in animal models 
of alcohol addiction. Two behaviors, 



found under different experimental 
conditions, are representative of ethanol 
reinforcement. These behaviors are 
ethanol self- administration and ethanol- 
conditioned preferences. Other responses 
may reflect the ability of ethanol to serve 
as a reinforcer. For example, effects 
such as anxiolytic, aversive, discrimi- 
native, motor, or amnestic effects each 
address important aspects of ethanol's 
ability to serve as a reinforcer. How- 
ever, these behavioral outcomes are 
not direct measures of ethanol rein- 
forcement. Likewise, the effects of 
ethanol on the threshold for intracra- 
nial self-stimulation are believed to 
reflect the reinforcing effects of 
ethanol (Wise et al. 1992); however, 
this is also an indirect measure of 
ethanol reinforcement. 

Conditioned preference refers to the 
process of a laboratory animal becom- 
ing attracted to the place associated 
with the delivery of ethanol (Mucha 
et al. 1982; Hoffman 1989). The ani- 
mal is given a specific dose of ethanol 
and placed in a distinct environment. 
Through the association of ethanol's 
effects with the specific environmental 
stimuli, these environmental stimuli 
come to serve as incentive stimuli and 
elicit approach behaviors. When given 
a choice between two environments, 
only one of which is associated with 
the effects of ethanol, a preference is 
apparent if the animal spends a greater 
proportion of time in the environment 
associated with ethanol. An aversion 
would be apparent if a smaller propor- 
tion of time was spent in the environ- 
ment associated with ethanol. These 
procedures emphasize environmental 
stimuli as generating motivational 



233 



NIAAA's Neuroscience and Behavioral Research Portfolio 



states, reflected in approach or avoid- 
ance behaviors that are indicative of 
the reinforcing effects of ethanol. 

These procedures also provide 
important alternatives to oral self- 
administration paradigms for studying 
the motivational effects of ethanol. 
The strengths of the preference proce- 
dures include the ability to detect both 
positive and negative motivational 
effects, the ability to test for motiva- 
tional impact in the absence of 
ethanol's direct sensory-motor effects, 
the ability to assess ethanol dose 
effects in the absence of confounding 
influence of taste/palatability factors, 
the ability to assess potential pharma- 
cotherapies without the need to assess 
nonspecific effects on ingestive behav- 
ior, the ability to separately assess 
manipulations that influence acquisition 
versus expression of ethanol-induced 
motivational effects, and the ability to 
implement procedures without 
surgery or lengthy periods of training. 

Both place and taste preference 
procedures serve as models for studying 
mechanisms underlying the acquisition 
and extinction of associations between 
taste or other environmental cues and 
ethanol. The understanding of these 
mechanisms is important because con- 
ditioned learning involving exterocep- 
tive stimuli is thought to contribute to 
craving and relapse to ethanol-seeking 
behavior after withdrawal and long 
periods of abstinence. For example, 
Schuster and Woods (1968) found that 
response-contingent presentation of 
stimuli previously associated with 
morphine self- administration increased 
responding under extinction condi- 
tions. Similarly, rats withdrawn from 



morphine drank more vehicle when 
placed in environments where mor- 
phine self-administration had been 
acquired than did rats placed in envi- 
ronments not associated with mor- 
phine self- administration (Thompson 
and Ostland 1965; Hinson et al. 1986). 
Finally, noncontingent amphetamine 
given to monkeys under extinction 
conditions reinstated responding pre- 
viously maintained by amphetamine 
only if a masking noise, present dur- 
ing the self-administration sessions, 
was also present (Stretch et al. 1971). 
Thus, contact with the drug may not 
be sufficient to elicit drug-seeking 
behavior outside an environment in 
which the drug was normally taken. A 
related area of research is the modu- 
lating role of tolerance or sensitization 
in the ability of conditioned stimuli to 
affect motivation for and self- adminis- 
tration of ethanol. 

An overwhelming amount of evidence 
shows that place conditioning and taste 
conditioning are sensitive to ethanol 
dose, number of trials, trial duration 
(place conditioning), the temporal rela- 
tionship between the paired stimulus 
and ethanol, and environmental condi- 
tions such as ambient temperature 
(Bormann and Cunningham 1997; 
Cunningham et al. 1997; Bormann 
and Cunningham 1998; Dickinson and 
Cunningham 1998). Thus, the condi- 
tioning aspects of the tasks reflect a 
learning process. Motor activation can 
also be measured within the context of 
place preference procedures (Cunning- 
ham and Noble 1992; Risinger et al. 
1992#, 1992&). A study investigating 
both the stimulant and rewarding 
effects of ethanol in a place preference 



234 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



procedure reported blockade of the 
motor- activating effects of ethanol with 
a dopaminergic antagonist without 
blocking the expression of place pref- 
erence (Risinger et al. 1992#). In addi- 
tion, the locomotor effects of 2.0 g/kg 
ethanol were enhanced by fluoxetine 
pretreatment within the preference 
apparatus; however, there was no effect 
on place preference (Risinger 1997). 
Thus, motor-activating effects of 
ethanol are neither necessary nor suffi- 
cient for the development of ethanol 
reinforcement. 

The neurochemical basis of these 
conditioned effects has been examined 
with genetic and pharmacological 
tools. Genetic differences in ethanol's 
rewarding and aversive effects and 
genetic correlations with other ethanol 
effects have been studied using inbred 
lines and selectively bred lines of mice 
and rats (e.g., Froehlich et al. 1988; 
Cunningham et al. 1991; Krimmer 
1991; Crabbe et al. 1992; Cunningham 
et al. 1992; Krimmer 1992; Cunning- 
ham 1995; Risinger and Cunningham 
1995; Broadbent et al. 1996; Risinger 
et al. 1996; Stewart et al. 1996; 
Chester et al. 1998). For example, 
inbred strains (C57BL/6J) or selec- 
tively bred lines (COLD, FAST) that 
drink elevated amounts of ethanol show 
reduced sensitivity to an ethanol- 
induced taste aversion (Cunningham 
et al. 1991; Risinger et al. 1994). In 
addition, place conditioning and taste 
conditioning have been used success- 
fully to identify several provisional 
QTL that may contain genes influenc- 
ing ethanol's rewarding and aversive 
motivational effects (Cunningham 
1995). Finally, the 5-HT 1B knockout 



mouse has been assessed for develop- 
ment of ethanol place and taste condi- 
tioning (Risinger et al. 1996) because 
these 5-HT 1B deficient mice drink sig- 
nificantly larger amounts of ethanol 
compared with wild-type mice (Crabbe 
et al. 1996). The knockout mice were 
more sensitive to ethanol-induced 
conditioned place preference, but 
equally sensitive to ethanol-induced 
taste aversion. These results show that 
taste aversion and place preference are 
separable and that oral ethanol con- 
sumption correlated with place prefer- 
ence but not taste aversion outcomes. 

Pharmacological investigation of 
ethanol-induced conditioned place 
preference has examined opioid, 5-HT, 
GABA A , and dopamine involvements. 
Specifically, the 5-HT 2 antagonist 
mianserin enhances (Risinger and 
Oakes 1996) but the 5-HT reuptake 
blocker fluoxetine has no effect 
(Risinger 1997) on ethanol-induced 
conditioned place preference. However, 
fluoxetine enhances ethanol-induced 
taste aversion (Risinger 1997). Inter- 
estingly, fluoxetine has been reported 
to enhance (Risinger 1997) or substi- 
tute (Maurel et al. 1997) for the dis- 
criminative stimulus effects of ethanol. 
Neither the inverse agonist Ro 15-4513 
nor the dopaminergic antagonist 
haloperidol inhibits ethanol-induced 
place preference (Cunningham et al. 
1992; Risinger et al. 1992^, 1992&). 
However, additional GABA A studies 
using a wider range of ligands at each 
receptor system are clearly needed. 

Pharmacological studies of ethanol's 
place preference using the opiate antag- 
onist naloxone have clearly differentiated 
the acquisition from the expression of 



235 



NIAAA's Neuroscience and Behavioral Research Portfolio 



ethanol-induced conditioning (e.g., 
Cunningham et al. 1995; Broadbent 
et al. 1996; Risinger and Oakes 1996; 
Risinger 1997; Cunningham et al. 
1998). Of particular interest are studies 
showing that pretreatment with the 
opiate antagonist naloxone at the time of 
testing facilitates extinction of ethanol- 
induced conditioned place preference, 
but retards extinction of conditioned 
place aversion (Cunningham et al. 
1995, 1998). These outcomes suggest 
that the opioid system maintains the 
learned association between appetitive 
events and responses, while also retard- 
ing the maintenance of learned associa- 
tion of aversive events. Thus, naloxone 
appears to have a detrimental impact 
on conditioned rewarding effects of 
ethanol (weakening approach behav- 
iors), while enhancing a conditioned 
aversive effect of ethanol (enhancing 
avoidance behaviors) (Cunningham et 
al. 1998). 

In addition to measuring preferences 
associated with ethanol administration, 
conditioned place/taste procedures can 
assess the aversive effects of ethanol. 
The dose range for the aversive effects 
of ethanol are 1.0-2.0 g/kg for rats 
(Davies and Parker 1990; Gauvin and 
Holloway 1992; Holloway et al. 
1992; Schechter 1992; Schechter and 
Krimmer 1992) and 2.0-3.0 g/kg for 
mice (Cunningham et al. 1991; 
Risinger and Cunningham 1992). 
Selectively bred NP rats, but not P 
rats, develop conditioned taste aversion 
associated with 1.0 g/kg ethanol 
(Froehlich et al. 1988). However, at 
higher doses of ethanol, P rats also 
show conditioned taste aversions 
(Froehlich et al. 1988). Using place 



preference procedures, rather than 
conditioned taste procedures, both the 
P and NP selected lines developed place 
aversions following 1.0 g/kg ethanol 
(Schechter 1992). Where conditioned 
preferences are found using rats, 
repeated exposure to ethanol is neces- 
sary (Grant et al. 1990). The necessity 
of multiple exposures to ethanol 
implies that tolerance to the aversive 
effects may expose the positive rein- 
forcing effects of ethanol. However, it 
is worth noting that at least some 
strains and lines of mice do not show 
the initial aversive effects of ethanol 
when tested in place preference para- 
digms. Both DBA/2J mice and mice 
selected for hyperthermia in response 
to ethanol develop conditioned place 
preferences associated with 2-A g/kg 
ethanol (Cunningham et al. 1991, 
1992; Risinger et al. 1992&). Future 
studies will need to continue to delin- 
eate the experimental determinants of 
ethanol-induced conditioned prefer- 
ence and conditioned aversion and 
why ethanol induces conditioned pref- 
erences in some circumstances but 
conditioned aversion in others. 

In summary, conditioned place and 
taste preference are reliable procedures 
that allow the investigation of receptor 
mechanisms mediating either the posi- 
tive or negative hedonic effects of 
ethanol. These procedures are particu- 
larly useful in studying the condi- 
tioned effects of ethanol in mice. Thus, 
they appear well suited to characterize 
the genetic bases of ethanol-related 
effects. Indeed, several provisional 
QTL have been identified for place 
preference by typing recombinant 
inbred strains. In addition, knockout 



236 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



mice have been characterized in pref- 
erence procedures and have generated 
hypotheses concerning a common 
genetic basis for ethanol's effects across 
selected lines of mice. These proce- 
dures appear to provide an important 
window into the genetic basis of 
ethanol-related effects. Combined with 
procedures such as drug discrimina- 
tion, specific receptor mechanisms can 
be identified and verified as important 
in mediating ethanol-associated 
behaviors in the mouse and provide 
potential mechanisms for study in 
other species. To date, there is no 
information available on the specific 
circuitry involved in conditioned taste 
or place preferences. 

Self- Administration 
and Reinforcing Effects 

Self- administration refers to the process 
of a laboratory animal or human engag- 
ing in a behavior that results in the 
administration of alcohol. The most 
common self- administration procedure 
involves the presentation of alcohol 
following a set number of specific 
responses in a distinct environment. 
The presentation of alcohol in self- 
administration procedures has been 
accomplished by several routes, 
including oral, intravenous, intragastric, 
and intracranial delivery (see Carroll 
et al. 1990). In short, self- administra- 
tion procedures emphasize the conse- 
quences of behavior in the role of 
alcohol seeking. 

While it is clear that alcohol has 
reinforcing effects that maintain the 
consumption of alcoholic beverages, 
ethanol is not an efficacious reinforcer 
to drug-naive laboratory animals 



(Meisch 1977; Grant et al. 1990). In 
a vast majority of the studies of oral 
ethanol self- administration in labora- 
tory animals, simply allowing access to 
ethanol is not sufficient to result in 
repeated consumption of intoxicating 
quantities. The low levels of ethanol 
intake of uninitiated animals have been 
attributed to the taste of ethanol, the 
delay between the consumption of 
ethanol and its pharmacological 
effects, the volume of ethanol needed 
for a pharmacological effect, and the 
particular pharmacological effects of 
ethanol (including positively reinforc- 
ing and aversive effects). To circumvent 
these difficulties, it is now standard to 
use an induction procedure to estab- 
lish ethanol drinking in animals that 
have not been specifically bred to drink 
large amounts of an ethanol solution. 
Induction procedures include food 
deprivation, adulterating the taste of 
ethanol, associating the consumption 
of ethanol with the presentation or 
removal of other reinforcers, acclimating 
the animal to gradually increasing con- 
centrations of ethanol, and restricting 
access to the ethanol solution (see 
Meisch 1977; Samson 1987). 

Although a wide variety of species 
have been studied in ethanol self- 
administration procedures (see Caroll 
et al. 1990), most of the extensive 
investigations of the neural basis of 
ethanol self- administration have been 
in rats. Species differences have not 
been extensively studied with self- 
administration procedures, primarily 
because the rat has been the overwhelm- 
ing animal of choice. Monkeys were 
initially investigated in the 1970s using 
oral, intravenous, and intragastric 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



procedures. However, not all primate 
species are alike in a predisposition to 
drink ethanol. Similar to rodents, female 
vervet monkeys {Cercopithecus aethiops) 
drink more ethanol than males (Juarez 
et al. 1993). In contrast, female rhesus 
monkeys {Macaco mulatto) are less 
likely to initiate and maintain ethanol 
consumption compared with males 
(Grant and Johanson 1988). 
Although monkeys can acquire ethanol 
self- administration when investigators 
provide access without an induction 
procedure (Macenski and Meisch 1992; 
Stewart et al. 1996) the average intakes 
are low (0.2-1.0 g/kg/3 h). When 
induction procedures or food depriva- 
tion is imposed, ethanol intakes increase 
to more than 1.0 g/kg/h (Carroll et 
al. 1990; Macenski and Meisch 1992; 
Williams et al. 1998). Preliminary 
results following a schedule induction 
procedure to establish ethanol self- 
administration and then 16 h/d access 
to 4 percent (w/v) ethanol and water 
show that cynomolgus monkeys drank 
1-4 g/kg ethanol per day and developed 
signs of fatty liver after 6 months of 
drinking these quantities (Grant et al. 
1998). Interestingly, the female mon- 
keys were in the lower 50th percentile 
of ethanol intake and still developed 
signs of fatty liver. These results suggest 
that increasing the access to 16 h/d 
can result in excessive intakes with 
biomedical consequences. 

Some induction procedures use 
stressful conditions. In one study, mon- 
keys were either continuously housed in 
individual cages or housed socially and 
subjected to social disruption (Krae- 
mer and McKinney 1985). Monkeys 
in the socially disrupted group were 



intermittently separated from and 
reunited with their cage mates for 1- 
week periods. Overall, the socially dis- 
rupted monkeys drank more ethanol 
than the individually caged monkeys 
and drank more ethanol when they 
were isolated than when they were 
socially housed. The authors interpreted 
these findings as evidence that the stress 
of intermittent social isolation pro- 
moted ethanol consumption (Kraemer 
and McKinney 1985). Social stress 
was also invoked as an explanation of 
induced drinking in socially subordi- 
nate male rats housed in social groups 
(Blanchard et al. 1987). Subordinate 
social status is thought to be stressful 
in mammals because subordinates of 
many species receive more aggression, 
spend more time alone, and hyper- 
secrete stress hormone relative to their 
dominant counterparts (Shively et al. 
1986, 1990). However, in the only 
available study of social status effects 
on ethanol consumption in macaques, 
dominant monkeys drank more than 
subordinates (Crowley et al. 1990). 
The authors' explanation was that only 
one source of ethanol was available to 
all social group members, and domi- 
nants exercised their status by control- 
ling access to the drinking station. 
With access to the ethanol source 
controlled by dominants, investigators 
could not detect social stress effects 
on ethanol consumption among sub- 
ordinates (Crowley et al. 1990). 

An important aspect of self- 
administration procedures is the ability 
to use schedules of reinforcement that 
maintain drug- seeking behavior in the 
absence of drug delivery. These are 
termed "complex schedules," and 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



they have been noticeably absent from 
the characterization of ethanol self- 
administration. With such schedules 
(chain schedules, second order sched- 
ules), in which responses are reinforced 
with the presentation of conditioned 
stimuli associated with the presentation 
of ethanol, but not directly with 
ethanol, the neurocircuitry involved in 
conditioned reinforcement can be 
addressed in self- administration proce- 
dures. A series of studies have shown 
that limbic innervation of the nucleus 
accumbens from the hippocampus 
and basolateral amygdala is essential 
to behavior controlled by drug-associ- 
ated stimuli (Hitchcott and Phillips 
1997; Hitchcott et al. 1997a, 1997*). 
These authors suggest that the basolat- 
eral projection to the nucleus accumbens 
determines the degree of discriminative 
control over conditioned responses, 
while hippocampal/subiculum input 
determines the efficacy of the condi- 
tioned stimulus. The input to the 
accumbens from the subiculum is glu- 
tamatergic, and from the amygdala is 
dopaminergic, possibly D 3 (Hitchcott 
et al. 1997 a). These data fit well with 
the data from Samson and Hodge 
(1996) showing that intra-accumbens 
injections of dopaminergic agonists 
increase ethanol self- administration, 
possibly through increasing the discrim- 
inative control exerted over behavior by 
drug-associated stimuli. However, these 
data also have important implications 
for the hippocampus, amygdala, and 
nucleus accumbens as target sites when 
studying the discriminative stimulus 
effects of ethanol. Enhanced discrimi- 
nation with glutamatergic, GABAergic, 
or dopaminergic compounds may affect 



discriminated responses without con- 
veying information concerning ethanol- 
specific activity. 

An emerging principle in studying 
the neurochemical basis of self- admin- 
istration is that the ability of ethanol 
to function as a positive reinforcer is 
not due to an immutable neurochem- 
istry that mediates positive affect. Ani- 
mal models involving several drug 
classes support the dissociation between 
drug presentation (and presumably 
pharmacological action in the CNS), 
positive affect, and subsequent self- 
administration. For example, several 
studies have shown that the responding 
of morphine-dependent monkeys can 
be maintained by the administration 
of naloxone (Goldberg et al. 1971; 
Woods et al. 1975; Goldberg et al. 
1978) at doses that produce avoidance 
responding in the same (Goldberg et 
al. 1971) or other (Kandel and Schuster 
1977) morphine-dependent monkeys. 
Furthermore, within-subject designs 
have shown that the same doses of 
cocaine can be either actively avoided 
or self- administered (Spealman 1979). 
Thus, any other intrinsic effect of a 
drug — for example, dopamine release 
in the nucleus accumbens — is likely 
only to influence, rather than determine, 
drug seeking. Alcohol's ability to serve 
as a reinforcer at any given time will be 
due to a combination of antecedent 
events, current environmental contin- 
gencies, and the pharmacological basis 
of alcohol's stimulus effects. Clearly, 
alcohol-seeking behavior can be viewed 
as malleable and not wholly determined 
by intrinsic pharmacological effects. 

An additional finding with self- 
administration procedures is that the 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



neurochemical effects of the drug are 
determined, in part, by the act of self- 
administration. That is, the neuro- 
chemical effects of self-administered 
drugs are different from the neuro- 
chemical effects of the same doses when 
administered irrespective of ongoing 
behavior (e.g., yoked administrations). 
The interaction of self- administration 
behavior and neurochemical conse- 
quences of drugs has parallels in the 
tolerance literature. According to toler- 
ance models, the initial effects of a drug 
or the environment in self- administration 
can serve as conditioned stimuli to 
compensate for effects of the drug. 
Indeed, conditioned drug effects and 
conditioned stimuli altering drug 
effects have now become central tenets 
in drug abuse research. Investigating 
the neurocircuitry of ethanol within 
a self-administration paradigm is 
central to our understanding of 
ethanol's effects that mediate alcohol 
abuse and alcoholism. 

The neurochemical basis of the 
reinforcing effects of ethanol within 
self-administration or consumption 
procedures has been studied for 
decades and has filled volumes of sci- 
entific literature. Practically every 
neurotransmitter, neuromodulator, or 
neuropeptide system has been investi- 
gated for effects on ethanol intakes. 
Most of these studies use receptor lig- 
ands, but there are also genetic, elec- 
trophysiological, neurochemical, and 
imaging techniques, as well as lesion 
techniques, that have been applied to 
the quest for understanding ethanol's 
reinforcing effects within the drinking 
context. However, making a list of 
these findings may not be as useful as 



examining the functional role of the 
various neurotransmitter systems and 
the circuitry in which they reside. For 
example, the mesolimbic dopamine 
system has been extensively studied, 
and we now have evidence that 
mesolimbic dopamine is released within 
a self- administration context in antici- 
pation of and response to ethanol con- 
sumption (Weiss et al. 1993). We also 
know that we can inject dopamine 
agonists and antagonists directly into 
different sites of this circuit (VTA, 
accumbens, amygdala, frontal cortex) 
and alter ethanol self- administration 
(see Samson and Hodge 1996). 
Finally, there is evidence that ethanol 
injected directly into the VTA will 
support self- administration (Gatto et 
al. 1994). However, it is clear that the 
dopamine pathway is involved in medi- 
ating reinforced responses in general 
(Salamone 1994; Hitchcott et al. 
1997#, 1997b). In short, activity in 
this complex system appears to be 
related to the more complex organiza- 
tion of appetitive-consummatory behav- 
ior, in which the actions of reinforcing 
stimuli integrate with other stimuli to 
direct and then maintain goal-ori- 
ented behaviors (Wilner and Scheel- 
Kruger 1991). Therefore, just knowing 
that this pathway is involved in ethanol 
reinforcement is insufficient to under- 
stand the mechanisms unique to 
ethanol and potential targets for 
ethanol -specific pharmacotherapies. 

An example of modifying circuitry 
involved in appetitive behaviors rather 
than ethanol-specific interactions is the 
reduction in alcohol self- administration 
in the presence of opioid antagonists. 
Activation of the endogenous opioid 



240 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



system has been postulated to mediate 
the reinforcing effects of low to mod- 
erate doses of ethanol. This hypothesis 
is supported by data in rats showing 
that mu and delta opioid antagonists 
selectively decrease ethanol intake 
when water is available (Froehlich and 
Li 1993). Data from macaque mon- 
keys show that naltrexone decreases 
oral ethanol consumption in a dose- 
dependent manner but also has effects 
on concurrently available water (Myers 
et al. 1986; Kornet et al. 1991). When 
a sweetened solution is the alternative 
reinforcer, mu and delta opioid antag- 
onists decrease both ethanol and 
sucrose consumption in rats (Samson 
and Doyle 1985; Krishnan-Sarin et al. 
1995) and monkeys (Williams et al. 
1998). Thus, the opioid system most 
likely does not selectively mediate 
ethanol's effects. In fact, endogenous 
opioid peptides mediate a variety of 
ingestive behaviors, and naloxone can 
suppress intake of water, fats, and 
sucrose in rats, monkeys, and humans 
(Brown and Holtzman 1981; Locke 
et al. 1982; Krishnan-Sarin et al. 
1995). It is important to note that 
naltrexone decreases ethanol intake 
via the intravenous route in monkeys 
(Altshuler et al. 1980; Williams et al. 
1998) and the intragastric route in rats 
(Sinden et al. 1983), suggesting that 
the effect of the opiate attenuation is 
through a centrally mediated pathway. 
There is evidence that both alcohol and 
palatable substances increase endoge- 
nous opioid peptide synthesis and release 
(Wand 1989; Gianoulakis 1990; De 
Waele et al. 1992; Froelich and Li 
1993), and preferences for alcohol and 
sweet solutions are correlated in some 



outbred rat strains (Overstreet et al. 
1993) and lines selectively bred for 
ethanol preference (Sinclair et al. 
1992; Stewart et al. 1994). However, 
it is also clear that there is not a com- 
plete correspondence between sweet 
preference and alcohol preference 
(Phillips et al. 1994). It appears that 
the opiate system mediates information 
about ingesta, including ethanol's 
effects. However, ethanol is not unique 
in its ability to activate this pathway, 
and ligands of this pathway should 
not, in turn, be expected to selectively 
attenuate the self- administration of 
alcohol. Nevertheless, naltrexone has 
some efficacy in preventing relapse, and 
recent animal studies have focused on 
the endogenous opioid system in 
mediating aversive effects of ethanol 
(Cunningham et al. 1998; Froehlich 
etal. 1998). 

Similarly, many of the neuropeptides 
alter ethanol intake when administered 
peripherally or centrally. These include 
CRF (Bell et al. 1998), cholecystokinin 
(CCK), and bombesin (Kulkosky 
1985), which decrease oral ethanol 
consumption, and neuropeptide Y, 
which increases ethanol intake 
(Kulkosky et al. 1988). However, in 
the same studies CRF, CCK, and 
bombesin also decreased food intake, 
whereas neuropeptide Y increased 
food intake. Thus, the manipulation 
of ethanol self- administration using 
these peptides appears to be through 
circuitry that regulates food intake 
and consummatory behaviors in gen- 
eral, rather than information about 
ethanol specifically. 

The overriding theme is that "con- 
summatory /regulatory" systems are 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



active when ethanol is consumed. 
Through conditioning processes, the 
unique pharmacological effects of 
ethanol interact with these regulatory 
systems to establish a pattern of con- 
sumption that is regulated by both 
ethanol's stimulus effects and the 
functioning state of the regulatory sys- 
tem. If ethanol has deleterious effects 
on consummatory systems, dysregula- 
tion will result. If, however, other events 
in the environment produce dysregu- 
lation in the consummatory system, 
elevated ethanol intake could be an 
outcome (as could overeating, gam- 
bling, and some other behaviors), which 
in turn, can further alter regulatory 
control. The challenge for behavioral 
neuroscience is to define the consum- 
matory/regulatory systems and inte- 
grate mechanisms of how ethanol 
self- administration is both an antecedent 
and a consequence to dysregulation. 

Learning, Memory, 
and Cognitive Effects 

As reviewed thus far the acute actions 
of alcohol produce a constellation of 
physiological and behavioral effects in 
humans and laboratory animals. The 
subset of these actions that affect cog- 
nitive function can be subjective in 
nature, such as how a drink of alcohol 
makes a person feel, or they can be 
objective in nature, such as the effects 
of alcohol on the recall of recently 
learned material. Since the 1940s lit- 
erally thousands of scientific studies 
have been conducted to classify and 
measure both the objective and sub- 
jective stimulus effects of alcohol that 
alter cognitive function and subse- 
quent reaction to alcohol (see NIAAA 



1995). However, these studies have 
largely been conducted in human sub- 
jects, and the application of animal 
models to study the underlying neu- 
rocircuitry has been limited. 

The classification of the cognitive 
effects of alcohol depends on the tasks 
used to measure these effects. Most of 
these tasks focus on characterizing three 
broad areas of interactive processes, usu- 
ally referred to as information processing, 
psychomotor skills, and subjective effects. 
Briefly, information processing involves 
the ability to perceive, learn, and remem- 
ber information. Psychomotor skills 
predominantly include measures of 
reaction time, proprioceptive ability (such 
as tracking), and vigilance (attending 
to a particular stimulus when there are 
distracting stimuli presented). Subjective 
reactions to alcohol are most often mea- 
sured by the perceived degree of intox- 
ication, pleasant affect, dysphoria, or 
sedation, or by other mood descriptors. 
Clearly, the cognitive processes that 
determine the outcome of these tasks are 
not mutually exclusive, and the alteration 
of one of these processes can change 
the outcome of the others. Sophisti- 
cated techniques have been developed 
in research with human subjects to sep- 
arate and quantify different aspects of 
cognitive ability, including measures of 
overall mental ability, verbal/visuospa- 
tial learning, conceptual learning, and 
perceptual/motor abilities. Human 
studies have also correlated physiolog- 
ical measures of brain function with 
the cognitive effects of alcohol (e.g., 
electroencephalographic, hormonal, 
and functional imaging measures). 

Research in humans has suggested 
areas for study in animal models, 



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Behavioral Effects and Underlying Neurocircuitries of Alcohol 



including increasing the complexity of 
the task. Performance tasks that are 
complex are very sensitive to the effects 
of alcohol and can be disrupted by 
one to two drinks, with corresponding 
blood alcohol concentrations in the 
range of 40 mg/dL. In contrast, simple 
reaction time tasks may require three 
drinks before disruption. Another 
finding from the human literature is 
the relatively high threshold dose for 
amnestic effects, which usually occurs 
at blood alcohol levels that are reported 
as blatantly intoxicating (e.g., stu- 
porous). This finding is in contrast to 
the benzodiazepines, which can dis- 
rupt memory at doses that are not 
subjectively perceived. Another vari- 
ables that is known to influence both 
performance measures and subjective 
effects of alcohol is family history of 
alcoholism. A family history may 
dampen the effects on performance 
measures and enhance the subjective 
effects of the rising phase of the blood 
alcohol curve. 

Compared with human studies, 
animal studies lag far behind. A 
notable recent addition to the literature 
is the use of eyeblink conditioning to 
study the effects of neonatal ethanol 
exposure in rats (Stanton and 
Goodlett 1998). The eyeblink condi- 
tioning procedure uses classical condi- 
tioning and is mediated through an 
identified and characterized circuit 
from the brainstem to the cerebellum 
(Harvey 1987). Cerebellar damage 
noted with neonatal ethanol exposure 
was found to have functional conse- 
quences. However, acute effects of 
ethanol in this exposure have not been 
explored. Two procedures used in 



animals, particularly nonhuman pri- 
mates, that are sensitive to impairments 
produced by GABA A positive modula- 
tors and NMDA antagonists are a 
delayed nonmatching to sample pro- 
cedure (Ogura and Aigner 1993) and 
a repeated acquisition procedure 
(Baron and Moerschbaecher 1996). 
However, ethanol has not been stud- 
ied in either procedure. One procedure 
that has been used in animals exposed 
to ethanol is a spatial working memory 
task, which is disrupted by 0.75 and 
1.0 g/kg ethanol, an effect that has 
been correlated with ethanol's impair- 
ment of hippocampal theta activity 
(Givens 1995). There have also been 
some studies involving maze perfor- 
mance and working memory in mice 
(Melchior et al. 1993). Givens (1995) 
has described a circuitry involving 
ethanol-induced disruption of medial 
septal area activity via a GABAergic 
mechanism, thereby disrupting septo- 
hippocampal input and interfering 
with information processing in the 
hippocampal circuitry. Recent work 
has used single-neuron recording 
techniques to measure cognitive 
processes affected by ethanol (Givens 
etal. 1998). 

Aggression 

Aggression has been studied in animal 
models to only a limited extent. There 
are two distinct types of aggressive 
behavior, predatory and affective. 
Predatory aggression is interspecific, 
normally related to feeding and not 
associated with increased irritability. 
This type of aggression is accompa- 
nied by minimal vocalization, stalking 
posture, and lethally directed attacks 



243 



NIAAA's Neuroscience and Behavioral Research Portfolio 



(e.g., at the back of the prey's neck). 
Affective agression is intraspecific and 
involves intense autonomic arousal, 
vocalizations, and threatening and 
defensive postures. 

By far, the receptor system most 
widely implicated in both predatory 
and affective aggression is serotonin 
(Olivier et al. 1990). Increases in preda- 
tory behavior are associated with low 
serotonin levels produced by serotonin 
depletion with neurotoxins. Stimula- 
tion of serotonin systems by electrical 
stimulation of dorsal raphe nucleus, 
serotonin precursors, or 5-HT reuptake 
blockers decreases aggressive acts. Affec- 
tive aggression has also been closely 
linked to serotonergic function, but 
the data are not as clear as with preda- 
tory aggression. Nearly all pharmaco- 
logical manipulations that either 
increase or decrease 5-HT neurotrans- 
mission can inhibit offensive aggres- 
sion (Miczek et al. 1989; Olivier et al. 
1990). These mixed results probably 
reflect differential effect on neural cir- 
cuitry, in that stimulation of hypothal- 
amic serotonergic system increases 
aggression, whereas ablation of amyg- 
dala serotonin decreases aggression 
(Fileetal. 1981). 

The serotonin subtype most impli- 
cated in animal models of affective 
aggression is the 5-HT 1B receptor. A 
class of substituted phenlypiperazine 
analogs that display remarkable anti- 
aggressive activity in animal models 
has been termed "serenics" (Olivier et 
al. 1990). Serenics specifically reduce 
offensive behavior without resulting in 
sedation, muscle relaxation, or motor 
stimulation. The two serenics with the 
greatest specificity are TFMPP and RU 



24969, phenlypiperazines with modest 
selectivity and high affinity for the 5- 
HT 1B receptor. As already mentioned, 
there is a 5-HT 1B knockout mouse, 
and this animal is highly aggressive 
(Saudou et al. 1994). 

The effect of ethanol on aggression is 
dose dependent, with low doses increas- 
ing aggressive acts and higher doses 
decreasing aggression, probably due to 
sedation (Blanchard et al. 1987; Miczek 
et al. 1993). Increased aggression with 
low doses of ethanol has been found 
with both experimenter-administered 
and self- administered ethanol studies 
(van Erp and Miczek 1997). However, 
increased aggression is not found in 
every animal tested, and a proportion of 
the population shows reduced aggres- 
sion after consuming similar doses of 
ethanol as animals showing aggression. 
There is evidence that the social context 
can determine the direction and mag- 
nitude of ethanol's effects on aggres- 
sion, particularly in monkeys (Weerts 
and Miczek 1996). The neurotrans- 
mitter systems studied in relation to 
ethanol-induced aggression in animals 
models have been the 5-HT and the 
GABA A receptor systems (Blanchard 
et al. 1993; Miczek et al. 1993; Weerts 
and Miczek 1996). The 5-HT 1B 
receptor system has not been specifi- 
cally studied in the context of ethanol- 
induced aggression or antiaggression, 
but drug discrimination data show 
that the 5-HT 1B receptor activation is 
a component of the discriminative 
stimulus effects of low doses of ethanol 
(Grant et al. 1997). 5-HT 1B receptors 
are autoreceptors that result in decreased 
5-HT release, and low 5-HT is associ- 
ated with increased levels of aggression. 



244 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



However, the neuroanatomies path- 
ways that mediate these effects are 
largely unknown. Very recent micro- 
dialysis data show decreased prefrontal 
cortex 5-HT levels in aggressive rats 
(van Erp and Miczek 1997). Since the 
5-HT 1B receptor system is predomi- 
nantly expressed in rodents, studies in 
primates will need to focus on the 5- 
HT 1D receptor system, using the 
appropriate ligands. 

The interaction of alcohol and 
aggression is a major public health con- 
cern. The genetic, neurochemical, and 
neuroanatomical bases of this interac- 
tion are largely unknown. There are 
promising data implicating both the 
5-HT and the GABA A receptor sys- 
tems, but there is a large degree of 
individual variability in response to 
ethanol that is highly dependent on 
environmental circumstances. 

FUTURE RESEARCH 
BASED ON CURRENT 
APPROACHES 

A priority for future research efforts 
should be transferring information 
between various levels of analysis: 
molecular, cellular, physiological, animal 
behavioral, human behavioral, and 
epidemiologic. The transfer from cel- 
lular data to behavioral models appears 
to be well in hand, and the use of 
conditioning procedures such as place 
preference and drug discrimination 
should be emphasized in mouse mod- 
els. The transfer from animal models 
to human studies is less apparent. 
Behavioral neuroscience can address 
the transfer from animals to humans 
by applying the same noninvasive 



technologies in animal models that are 
used in human studies. These tech- 
nologies include brain imaging tech- 
niques of PET, single-photon 
emission computed tomography, and 
functional NMR and electrophysio- 
logical techniques such as ERP. 

RISK FACTORS 

Perhaps one of the greatest challenges 
to our future research is to address the 
neuroscientific basis of risk factors 
know to affect alcohol dependence in 
humans. Risk factors can be viewed as 
an additional independent variable in 
the design of our animal models, but 
their characterization will undoubt- 
edly require that data be obtained 
using several approaches. Notable risk 
factors from human epidemiologic 
data include genetics, gender differ- 
ences, stress, depression, age of onset 
of drinking, and the concurrent use of 
other drugs. 

The most progress has been made in 
addressing the role of genetics. Alco- 
hol research clearly leads the way in 
studying the influence of genes on 
behavior. Our approaches in this area 
are strong, but the behaviors under 
study are often of questionable extrap- 
olation to the human condition and 
the animal models are currently lim- 
ited to the use of rodents. Cloning 
techniques of nuclear transplantation 
from adult monkeys cells should at 
least be given thoughtful considera- 
tion. Such approaches could be used 
to address the genetic basis of com- 
plex behavioral responses associated 
with the development of alcohol 
abuse and alcoholism. 



245 



NIAAA's Neuroscience and Behavioral Research Portfolio 



Gender differences in the behav- 
ioral neuroscience of alcohol using 
animal models are understudied. The 
hypothalamic-pituitary-gonadal axis 
hormones have been implicated in 
behavioral outcomes, primarily cognitive 
function, aggression, and stress. Drug 
discrimination studies have shown 
that sensitivity to alcohol is altered by 
menstrual cycle phase. Gender differ- 
ences in self- administration are under- 
studied, as are the anxiolytic effects of 
ethanol. That progesterone derivatives 
have been shown to produce ethanol- 
like subjective effects and alter self- 
administration is an important clue to 
follow in future studies. These find- 
ings suggest that gender differences 
must take into account the menstrual 
cycle phase. 

The role of stress in ethanol's behav- 
ioral effects requires more sophisti- 
cated approaches. It is important to 
note that ethanol has both anxiolytic 
and anxiogenic effects. This dual 
nature of ethanol can initiate a cycle 
of behavior centered around the issue 
of stress, but reflecting different 
aspects (circuitry?) of ethanol's pharma- 
cology and environmental interaction. 
Studies of the HPA axis hormones, 
particularly the central action, but also 
the extrahypothalamic action, of hor- 
mones such as CRT and glucocorti- 
coids, are beginning to yield promising 
avenues of research. 

Depression is a risk factor that ani- 
mal models of alcohol abuse have not 
addressed extensively. The basic ques- 
tions of cause and effects are still 
unanswered for depression and its 
relationship to alcohol abuse. Animal 
models are best suited to address these 



questions, since ethical and economic 
considerations limit experimental designs 
using human beings. Links between 
the 5-HT and dopamine receptor sys- 
tems with depression are encouraging 
and should be pursued. 

The age at which individuals start 
regular, heavy use of alcohol has 
recently been reported to predict the 
occurrence of alcohol dependence. In 
particular, individuals who begin to 
drink heavily as adolescents have 
increased risk to develop alcohol 
dependence. The adolescent period 
has been well documented in nonhu- 
man primates, but its occurrence in 
rodents is debatable. If solely defined 
as hypothalamic-pituitary-gonadal 
maturation, the adolescent period in 
rats would be very limited and studies 
of complex or conditioned behaviors 
might not be possible. The macaque 
monkey has at least a 12-month ado- 
lescent phase, which allows a window 
of opportunity to design appropriate 
experimental manipulations. In addi- 
tion, the social behavior of macaques, 
particularly in response to aggressive 
behavior, provides a rich data set to 
test hypotheses of predisposition (trait) 
versus reactivity (state) in antisocial 
outcomes following alcohol consump- 
tion within a social context. 

The concurrent use of alcohol and 
other drugs of abuse has also received 
limited attention. Heavy alcohol use is 
correlated with benzodiazepine, cocaine, 
opiate, marijuana, and tobacco abuse. 
Given this wide pharmacological diver- 
sity, explicit receptor interactions are 
not likely to explain the patterns of 
abuse. Behavioral patterns of drug 
abuse appear to be robust enough to 



246 



Behavioral Effects and Underlying Neurocircuitries of Alcohol 



incorporate a wide variety of psy- 
choactive substances once those 
behavioral patterns are entrenched. 
How these codependent use patterns 
are related to ethanol- specific interac- 
tion in the CNS remains unclear. 

NOTES 

1 . A measure of coordinated muscle 
movement not involved in locomotion 
or anxiolytic responses, per se, is taste 
reactivity. This measure involves the 
reflexive ingestive or expulsive oral 
movements in response to stimuli 
affecting taste sensory pathways. The 
procedure involves placing small 
amounts of a tastant, such as ethanol, 
on the caudal portion of the tongue 
and measuring coordinated tongue 
movements that result in either the 
ingestion or the expulsion of material 
from the oral cavity. Preference for 
ethanol has been assessed with this 
measure, as well as tolerance to expulsive 
response and sensitivity to ingestive 
response given repeated exposure to 
ethanol. Since the taste of ethanol 
could serve as a conditioned stimulus 
for postingestional effects of ethanol, 
changes in response to ethanol exposure 
could be an important indicator of 
reinforcement development. However, 
taste reactivity needs to be investigated 
in conjunction with reinforcement and 
not simply ethanol exposure to address 
these possibilities. Very few data are 
available on the underlying neural cir- 
cuitry involved in these unconditioned 
responses, and no data address the cir- 
cuitry in response to ethanol. 

2. An exception to this generaliza- 
tion is recent evidence suggesting that 
muscimol can produce ethanol-like 



discriminative stimulus effects if 
injected into core of the nucleus 
accumbens or amygdala in the brain 
(Hodge and Aiken 1996; Hodge and 
Cox 1998). 

ACKNOWLEDGMENT 

Financial support for the preparation of 
this chapter was provided, in part, by 
P50AA11997 from the National Insti- 
tute on Alcohol Abuse and Alcoholism. 

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260 



Chapter 7 

Neuroadaptive Changes in 

Neurotransmitter Systems Mediating 

Ethanol-Induced Behaviors 

Friedbert Weiss, Ph.D. 



KEY WORDS: AOD (alcohol or other drug) dependence; AOD withdrawal syn- 
drome; animal model; neurobiological theory; neurochemistry; AODD (AOD use 
disorder) relapse; AOD abstinence; reinforcement; chronic AODE (effects of AOD 
use, abuse, and dependence); AOD use behavior; neurotransmitters; AOD sensi- 
tivity; compulsion; literature review 



This review will highlight current under- 
standing of the neurobiologies basis of 
ethanol dependence, compulsive ethanol- 
seeking behavior associated with depen- 
dence, and persistent neuroadaptive 
changes in animals with a history of 
dependence that may motivate relapse 
and perpetuate alcohol abuse. 

MOTIVATIONAL SIGNIFI- 
CANCE OF ETHANOL 
DEPENDENCE AND 
WITHDRAWAL 

Theoretical Perspectives on 
Addiction and Compulsive 
Ethanol- Seeking Behavior 

Recent views in addiction theory 



involve the recognition of adaptations 
within the central nervous system 
(CNS). These adaptations induced by 
chronic drug use are thought to result 
in a disruption or desensitization of 
the neural mechanisms that mediate 
reward (Koob and Bloom 1988; Koob 
et al. 1993; Koob 1996; Wise 1996). 
This view is concerned not so much 
with physical withdrawal symptoms as a 
motivating factor in continued drug tak- 
ing but rather with symptoms that result 
from a compromised state of the reward 
system, which leads to affective states 
(e.g., dysphoria, depression, and anxi- 
ety) that are opposite to the initial 
mood-elevating effects of drugs. The 
significance of this emerging view of 
dependence is that it identifies a single 



F. Weiss, Ph.D., is an associate professor in the Department of Neuropharmacology, CVN-15, The 
Scripps Research Institute, 10550 North Torrey Pines Rd., Lajolla, CA 92037. 



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motivational withdrawal effect, negative 
affect, rather than a constellation of aver- 
sive, mostly physical withdrawal events 
in continued and escalating drug-seeking 
behavior, and it suggests a common 
basis for withdrawal from many, if not 
all, classes of drugs of abuse. 

Another form of neuroadaptation, 
sensitization, has received growing atten- 
tion as a possible mechanism in compul- 
sive drug use. Sensitization involves a 
dramatic augmentation of behavioral 
and neurochemical responses associated 
predominantly with meso-accumbens 
dopamine (DA) transmission that often 
develops with chronic, intermittent expo- 
sure to drugs of abuse (e.g., Robinson 
and Becker 1986; Kalivas and Stewart 
1991; Robinson and Berridge 1993). 
One of the most prominent theoretical 
positions on the significance of drug- 
induced sensitization holds that compul- 
sive drug-seeking behavior is the result of 
a progressive and persistent hypersen- 
sitivity of neural systems that mediate 
"incentive salience," resulting in a 
transformation of ordinary "wanting" 
into excessive craving (Robinson and 
Berridge 1993). According to this view, 
this process can occur independently 
of changes in neural systems that medi- 
ate the subjective pleasurable effects of 
drugs or systems that mediate with- 
drawal. Thus, this view does not neces- 
sarily rule out the possibility that other 
elements of the drug response, such as 
mood elevation or anxiolysis, undergo 
tolerance or become desensitized. 

Ethanol Dependence and 
Withdrawal 

The physical symptoms that accom- 
pany ethanol withdrawal have been 



well characterized and include auto- 
nomic hyperactivity such as hyperten- 
sion and increased heart rate, neuronal 
hyperexcitability with tremor and 
seizures, and perceptual distortions 
such as hallucinations and delirium 
(e.g., Rosenblatt et al. 1972; Cas- 
taneda and Cushman 1989; Turner et 
al. 1989; Benzer 1994; O'Brien 
1996). Animal models exist for these 
different ethanol withdrawal symp- 
toms, and physical signs have been 
used to study the neural basis of the 
alcohol withdrawal syndrome (Meert 
and Huysmans 1994). The neuro bio- 
logical basis for these physical signs of 
ethanol withdrawal involves CNS 
rebound hyperexcitability, such as a 
decrease in the function of the 
inhibitory amino acid transmitter 
gamma-aminobutyric acid (GABA) 
receptors or increases in the function 
of the excitatory transmitter glutamate 
and its receptors (Grant et al. 1990; 
Morrisett et al. 1990; Hoffman and 
Tabakoff 1994). More recently other 
neurotransmitter and neuromodula- 
tory systems, including serotonin, DA, 
norepinephrine, adenosine, ganglio- 
sides, and neurosteroids, have been 
implicated in neural hyperexcitability 
associated with ethanol withdrawal 
(Crabbe 1992; Concas et al. 1994; 
Hoffman and Tabakoff 1994; Meert 
1994; Adams et al. 1995; Finn et al. 
1995; Kotlinska and Liljequist 1996; 
Snell et al. 1996; Riihioja et al. 1997). 
In addition to these physiological 
symptoms, ethanol withdrawal has a 
variety of behavioral and affective con- 
sequences that are more "motiva- 
tional" in nature. Acute withdrawal 
from ethanol is associated with a neg- 



262 



Neuroadaptive Changes in Neurotransmitter Systems 



ative affective state consisting of dys- 
phoria, depression, irritability, and 
anxiety (e.g., Bjorkqvist 1975; Moss- 
berg et al. 1985; Turner et al. 1989; 
Bockstrom and Balldin 1992; O'Brien 
1996), which appear independent of 
physical signs of withdrawal. These 
affective withdrawal symptoms may 
motivate resumption of drinking 
(Koob and Bloom 1988; Koob et al. 
1993; Wise 1996), and by ameliorat- 
ing these symptoms ethanol could 
then serve as a negative reinforcer for 
continued alcohol use and abuse. Two 
major categories of withdrawal 
responses reflecting the motivational 
aspects of ethanol withdrawal will be 
the focus of this review: changes in 
reward function and anxiogenic con- 
sequences. 

Behavioral and Reward Functions 
in Dependence and Withdrawal 

Upon acute administration, ethanol 
has anxiolytic and mild euphorigenic 
effects, which are thought to be cen- 
tral to its reinforcing properties and 
abuse potential. These actions have 
been well documented in animal 
behavioral models. For example, like 
other drugs of abuse, ethanol stimu- 
lates locomotor activity (Waller et al. 
1986; Lewis and June 1990; Wolff- 
gramm 1991; Broadbent et al. 1995; 
Cohen et al. 1997) and can lower the 
thresholds for intracranial self-stimula- 
tion (ICSS) under relevant dosing and 
treatment conditions (Bain and Kor- 
netsky 1989; Lewis and June 1990; 
Moolten and Kornetsky 1990; Lewis 
1991). Both stimulation of locomotor 
activity and facilitation of brain stimu- 
lation reward are generally thought to 



reflect activation of the mesolimbic 
DA system and are good predictors of 
rewarding properties and abuse liabil- 
ity. Similarly, like classical anxiolytic 
drugs, ethanol exerts antianxiety 
effects in several behavioral models. 
Ethanol attenuates suppression of 
exploratory activity in the elevated 
plus maze (Pellow and File 1986; Lis- 
ter 1987) and social interaction test 
(Lister 1988; File 1980) and can 
effectively reverse behavioral suppres- 
sion in conflict tests (Koob and Brit- 
ton 1996). Although these positive 
affective responses are considered a 
critical factor in the positive reinforc- 
ing effects of ethanol, evidence is 
accumulating to suggest that, with 
chronic exposure and dependence, 
adaptive processes within the CNS 
develop that oppose the acute rein- 
forcing actions of drugs, leading to 
the emergence of affective changes in 
the absence of the drug. 

Negative Affect: Anxiogenic 
Responses and Reward Deficits During 
Withdrawal. In humans, chronic alco- 
hol use and alcohol withdrawal pro- 
duce anxiety, and these symptoms can 
persist long after physical withdrawal 
and detoxification (Bjorkqvist 1975; 
Mossberg et al. 1985; Roelofs 1985; 
Bockstrom and Balldin 1992). In ani- 
mals, anxiogenic-like consequences of 
withdrawal have been extensively docu- 
mented by suppression of exploratory 
activity on the unprotected arms of 
the elevated plus maze (e.g., Baldwin 
et al. 1991; File et al. 1991; Prather et 
al. 1991; File et al. 1993; Rassnick et 
al. 19936; Moy et al. 1997; Watson et 
al. 1997). Ethanol withdrawal anxiety 
has also been demonstrated in drug 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



discrimination studies where rats 
undergoing ethanol withdrawal 
selected a lever previously associated 
with the interoceptive effects of the 
anxiogenic compound pentylenetetra- 
zol (PTZ), and this effect was reversed 
by administration of ethanol (Lai et al. 
1988). Interestingly, the generaliza- 
tion to PTZ during ethanol with- 
drawal occurred before the onset of 
overt signs of withdrawal. This obser- 
vation is indicative of a dissociation 
between physical and affective-motiva- 
tional withdrawal effects, and suggests 
that the anxiogenic consequences of 
ethanol withdrawal may play an 
important role in the maintenance of 
ethanol abuse independent of physical 
withdrawal distress. 

Evidence is also accumulating that 
chronic use of ethanol compromises 
neural mechanisms that mediate posi- 
tive reinforcement. This is illustrated 
by the finding that in contrast to the 
acute effects of ethanol on ICSS 
thresholds, withdrawal in dependent 
animals leads to a significant impair- 
ment in the rewarding efficacy of elec- 
trical brain stimulation that lasts up to 
48 hours after termination of expo- 
sure to ethanol (Schulteis et al. 1995). 
This withdrawal-associated reward 
deficit is similar to that induced by all 
other major drugs of abuse, including 
opiates, tetrahydrocannabinol, psy- 
chostimulants, and nicotine (Markou 
and Koob 1991; Legault and Wise 
1994; Schulteis et al. 1995), and may 
reflect both adaptations within the 
mesolimbic DA system, which has 
been implicated in mediating the posi- 
tive reinforcing actions of alcohol and 
other drugs (AODs) (Leith and Bar- 



rett 1976; Markou and Koob 1991; 
Parsons et al. 1995; Pich et al. 1997), 
and the recruitment of brain stress 
systems. Like the anxiogenic effects of 
withdrawal, ICSS reward deficits are 
observed well before the onset of 
overt physical signs of ethanol with- 
drawal (Schulteis et al. 1995), con- 
firming a dissociation between 
affective -motivational and physical 
withdrawal symptoms. 

Together, these data suggest that 
while physical withdrawal symptoms 
have a role in the aversive aspects of 
ethanol withdrawal, withdrawal- 
induced anxiety and reward deficits 
may be critical in the maintenance of 
alcoholism. Since these consequences 
of withdrawal accrue well before the 
emergence of physical withdrawal 
symptoms, they may motivate contin- 
ued or increased ethanol consumption 
to avoid their occurrence and thereby 
contribute to the negative reinforcing 
properties of ethanol. 

Sleep. Ethanol withdrawal is also 
associated with marked sleep and cir- 
cadian disturbances. Alcoholics show 
less total sleep time during acute with- 
drawal, with reductions in both rapid 
eye movement (REM) and non-REM 
sleep (Gillin et al. 1990; Thompson et 
al. 1995), and these sleep disturbances 
seem coupled to a suppression of 
melatonin secretion (Schmitz et al. 
1996). Disruptions in circadian vigi- 
lance states, in particular reduced 
REM sleep, have also been reported 
during withdrawal in ethanol-depen- 
dent rats (Rouhani et al. 1990). With- 
drawal-related circadian and sleep 
abnormalities may have an important 
role in the susceptibility to relapse 



264 



Neuroadaptive Changes in Neurotransmitter Systems 



during withdrawal and the protracted 
abstinence phase. However, little is 
known about the persistence and 
long-term consequences of these dys- 
functions. This is an area of active 
research (Viglinskaya 1992; Peter et 
al. 1995; Brower et al. 1998; Clark et 
al. 1998; Drummond et al. 1998; 
Mackenzie et al. 1999) that promises 
to provide a better understanding of 
homeostatic disturbances in the main- 
tenance of ethanol abuse habits. 

ANIMAL MODELS OF 
EXCESSIVE DRINKING 
AND DEPENDENCE 

Alcoholism, by definition, involves 
compulsive and excessive use of alcohol 
(ethanol). Thus, the concepts of rein- 
forcement and motivation are crucial 
to the understanding of this syn- 
drome, and ethanol self- administra- 
tion and ethanol-seeking behavior 
have emerged as the primary behav- 
ioral measure of interest in contempo- 
rary research on alcohol abuse and 
addiction (Grant 1995). Two princi- 
pal categories of motivated behavior 
supported by ethanol can be distin- 
guished: (1) a consummatory aspect 
where drinking is reinforced and 
maintained by the rewarding conse- 
quences resulting from the consumption 
of ethanol and (2) an incentive-motiva- 
tional aspect which elicits and maintains 
behavior that brings the organism into 
contact with the reinforcing stimulus 
(i.e., ethanol). The immediate, pharma- 
cological effects of ethanol are thought 
to maintain consumption through 
positive or negative reinforcement 
of the drinking habit, where negative 



reinforcement would involve self- 
medication of an existing aversive state 
or self- medication of a drug-generated 
aversive state such as withdrawal 
(Wilder 1973). The incentive-motiva- 
tional aspects of ethanol-seeking 
behavior, on the other hand, involve 
association of previously neutral stim- 
uli with either the pleasurable subjec- 
tive effects of ethanol or relief from 
the aversive effects of withdrawal, and 
these aspects are thought to be 
involved in the initiation of alcohol- 
seeking behavior, craving, and relapse. 
Reliable self- administration models 
have been established for measuring 
the positive reinforcing effects of 
ethanol (Samson 1986, 1987) and, 
more recently, the negative reinforc- 
ing effects of ethanol self- administra- 
tion (Roberts et al. 1996; Schulteis et 
al. 1996). However, few effective 
models suited for the study of incen- 
tive-motivational effects of ethanol 
and their role in relapse are currently 
available. Efforts to develop such 
models are under way (Heyser and 
Koob 1997; Katner et al. 1999) and 
should eventually aid greatly in the 
investigation of the neurobiological 
basis of ethanol craving and relapse. 

Ethanol Self-Administration 
During Withdrawal 

As already pointed out, the consump- 
tion of ethanol, not simply for its 
euphorigenic effects but to avoid or 
reverse symptoms of withdrawal, may 
be an important factor in the perpetu- 
ation of ethanol dependence (for 
reviews, see Cappell 1981; Edwards 
1990). Indeed, withdrawal symptoms, 
in particular depression and anxiety, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



were found to provoke drinking in 83 
out of 100 male alcoholics (Hershon 
1977). However, it has traditionally 
been difficult to demonstrate that 
ethanol withdrawal motivates ethanol- 
seeking behavior in animals. Numer- 
ous studies indicate that the mere 
induction of physical dependence is 
not sufficient to promote ethanol 
intake, because ethanol-dependent 
rats and monkeys subjected to with- 
drawal often refuse to consume 
ethanol even though ethanol con- 
sumption would alleviate withdrawal 
distress (Veale and Myers 1969; Myers 
et al. 1972; Begleiter 1975; Cicero 
1980; Meisch 1984; Winger 1988; 
Samson and Harris 1992; Meisch and 
Stewart 1994). More positive results 
have, however, been obtained in rats 
with procedures that either bypass the 
aversive taste effects of ethanol or pro- 
vide for repeated opportunities to 
associate ethanol consumption with 
the alleviation of withdrawal symp- 
toms (Deutsch and Koopmans 1973; 
Hunter et al. 1974; Samson and Falk 
1974; Deutsch and Walton 1977; 
Trapold and Sullivan 1979). 

It has been more conclusively estab- 
lished that ethanol can serve as a potent 
negative reinforcer in dependent ani- 
mals undergoing withdrawal (Roberts 
et al. 1996; Schulteis et al. 1996; Weiss 
et al. 1996). Specifically, rats made 
dependent on ethanol via a liquid-diet 
procedure were shown to operantly 
respond for oral ethanol during with- 
drawal, and these animals consumed 
significantly more ethanol than non- 
dependent rats (Schulteis et al. 1996; 
Weiss et al. 1996). An even more strik- 
ing demonstration of the motivational 



effects of ethanol dependence is the 
finding that rats given access to oral 
ethanol in an operant self- administra- 
tion task immediately after removal 
from chronic ethanol vapor exposure 
responded for ethanol in a manner 
that maintained blood alcohol levels 
(BALs) in excess of 100 mg% over a 
12-hour "withdrawal" period and pre- 
vented withdrawal symptoms present 
in dependent rats not given access to 
ethanol during the withdrawal phase 
(Roberts et al. 1996). Interestingly, 
responding for ethanol became more 
stable over the course of four repeated 
withdrawal episodes, suggesting not 
only that ethanol became more firmly 
established as a negative reinforcer but 
also that rats learn to regulate ethanol 
intake in a manner that stabilizes 
BALs and minimizes or prevents with- 
drawal discomfort. 

In comparing the positive reports 
with studies that have failed to 
observe ethanol intake during with- 
drawal, it appears that procedures 
designed to overcome the aversive 
taste cues of ethanol and the opportu- 
nity to learn that ethanol consumption 
can alleviate withdrawal discomfort 
are essential for the demonstration of 
reinforcing effects of ethanol during 
withdrawal. An additional factor in 
contributing to the differences 
between these studies may be with- 
drawal severity. Severe tremors and 
seizures such as those reported in ear- 
lier work with intragastric intubation 
or forced consumption of high 
ethanol concentrations (Veale and 
Myers 1969; Myers et al. 1972; 
Begleiter 1975; Winger 1988) are 
rarely observed with the liquid-diet 



266 



Neuroadaptive Changes in Neurotransmitter Systems 



procedure or moderate ethanol vapor 
exposure in the more recent studies 
(Majchrowicz 1975; Lai et al. 1988; 
Emmett-Oglesby et al. 1990; Baldwin 
et al. 1991; Rassnick et al. 1992«; 
Merlo Pich et al. 1995; Macey et al. 
1996). It is, therefore, possible that 
milder forms of withdrawal may more 
readily support ethanol self- adminis- 
tration, whereas severe withdrawal 
distress may have general inhibitory 
effects on behavior, including self- 
administration (Winger 1988; Meisch 
and Stewart 1994), and thereby retard 
learning that consumption of ethanol 
can alleviate withdrawal. 

Ethanol Self- Administration 
After Periods of Abstinence 

Early experiments revealed that rats 
show marked increases in voluntary 
ethanol consumption after periods of 
forced abstinence (LeMagen 1960; 
Sinclair and Senter 1967, 1968; Sinclair 
1972, 1979). This so-called alcohol 
deprivation effect has since been con- 
firmed in mice (Salimov and Salimova 
1993), rats (Wolffgramm and Heyne 
1995; Spanagel et al. 1996; Heyser et 
al. 1997; Holter et al. 1997), mon- 
keys (Kornet et al. 1990, 1991), and 
human social drinkers (Burish et al. 
1981), and it is well established as a 
robust and reliable phenomenon in 
animal models of alcohol drinking. 

The alcohol deprivation effect can 
be readily demonstrated in nondepen- 
dent animals and may provide a 
potentially valuable model for under- 
standing changes in the reinforcing 
efficacy of ethanol that occur with 
abstinence (e.g., Heyser et al. 1996, 
1997; Holter et al. 1997). More 



importantly, however, under appro- 
priate conditions, this phenomenon 
appears to become resistant to manip- 
ulations of ethanol concentration, 
taste, and environmental factors 
(Wolffgramm and Heyne 1995; 
Spanagel et al. 1996) and, therefore, 
may prove useful as a model for com- 
pulsive ethanol-seeking behavior and 
loss of control that characterize sub- 
stance dependence on alcohol (per 
DSM-IV [American Psychiatric Asso- 
ciation 1994]). Studies that have char- 
acterized the alcohol deprivation 
effect in rats given long-term (8 to 24 
months) continuous free access to dif- 
ferent concentrations of ethanol and 
water, interspersed with deprivation 
periods of varying lengths, indicate 
that ethanol consumption increases 
significantly over baseline as a result of 
deprivation (Wolffgramm and Heyne 
1995; Spanagel et al. 1996), reaching 
levels of intake similar to those in rats 
selectively bred for alcohol preference 
(Li et al. 1979). The increase in 
ethanol intake associated with alcohol 
deprivation was characterized not only 
by enhanced preference for ethanol 
over water but also by preference for 
higher ethanol concentrations (> 10 
percent v/v) and resistance to change 
by altering the palatability of the 
ethanol solution (by either quinine or 
sucrose) or by manipulating environ- 
mental and social conditions (such as 
isolation housing or changing domi- 
nance hierarchies) (Wolffgramm and 
Heyne 1991, 1995; Spanaget et al. 
1996). Moreover, ethanol deprivation 
under these exposure conditions revealed 
a behavioral withdrawal syndrome, as 
measured by lowered thresholds of 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



footshock reactivity, which reached 
maximum on the 2d day of abstinence 
and persisted for up to 5 days post- 
ethanol (Heyne et al. 1991). 

Finally, the alcohol deprivation 
effect appears to outlast long abstinence 
phases (Spanagel et al. 1996). Indeed, 
it has been suggested that this effect 
is irreversible since it remained unaltered 
after 9 months of abstinence (Wolff- 
gramm 1991; Wolffgramm and Heyne 
1991, 1995). In particular, the loss of 
reversibility of this effect and the 
reduced adaptability of ethanol-seek- 
ing behavior in response to environ- 
mental or taste manipulations suggest 
that these procedures may provide an 
effective model to study mechanisms 
underlying specific aspects of ethanol- 
maintained addictive behavior and loss 
of control. As discussed later in this 
chapter, measures of the alcohol 
deprivation effect may also offer 
promise as a tool to study aspects of 
the relapse process in dependent and 
postdependent animals. 

Selective Breeding for High 
Ethanol Intake 

Several lines of rats genetically selected 
for traits of ethanol aversion or self- 
selection have been developed to 
model voluntary excessive drinking 
and alcohol abuse (Li et al. 1986; 
Kiianmaa et al. 1992; Li and McBride 
1995); these lines fulfill many of the 
established criteria as animal models 
of alcohol abuse (Lester and Freed 
1973). Lines that have been best char- 
acterized behaviorally and neuro- 
chemically include the Indiana P/NP, 
HAD/LAD, and Alko AA/ANA rats 
(Murphy et al. 1982; Li et al. 1986; 



Murphy et al. 1987; Gongwer et al. 
1989; McBride et al. 19906; Kiianmaa 
and Saito 1991; Kiianmaa et al. 1991; 
Gianoulakis et al. 1992; Kiianmaa et 
al. 1992; McBride et al. 1992). Simi- 
larities exist among these lines in 
ethanol-related behavioral and physi- 
ological characteristics, including the 
development of rapid tolerance, volun- 
tary 24-hour ethanol intake, and acqui- 
sition of ethanol-reinforced operant 
behavior. Common neurochemical 
markers of preference that have been 
identified to date include abnormali- 
ties in the function of forebrain DA 
and 5-hydroxytryptamine (serotonin 
or 5-HT) neurotransmission in the 
Indiana P and FIAD lines (Murphy et 
al. 1982, 1987; Gongwer et al. 1989; 
McBride et al. 1990#), and heightened 
sensitivity to the DA release-enhancing 
and locomotor activating effects of 
ethanol in the P and Sardinian alcohol- 
preferring (sP) lines (Fadda et al. 
1980; Waller et al. 1986; Cloninger 
1987; Engel et al. 1992; Weiss et al. 
1993). These neurochemical abnor- 
malities involve the same neural sys- 
tems that have been implicated in 
neuroadaptive changes associated with 
chronic ethanol consumption (as dis- 
cussed later in this chapter), suggest- 
ing that both environmental and 
genetic factors can converge to drive 
excessive drinking. 

In spite of these neurochemical 
commonalities and the existence of 
certain behavioral similarities among 
lines of alcohol-preferring rats, the 
neurochemical mechanisms underlying 
ethanol preference remain unclear in 
that it has been difficult to demonstrate 
common neurochemical markers of 



268 



Neuroadaptive Changes in Neurotransmitter Systems 



ethanol preference across multiple 
selected lines (e.g., Murphy et al. 1982, 
1987; Gongwer et al. 1989 vs. Sinclair 
et al. 1989; Kiianmaa et al. 1991). In 
contrast to the P and HAD lines, no 
reductions and even elevated levels of 
DA and 5-HT have been found in 
limbic and cortical forebrain regions 
of alcohol-preferring AA compared 
with nonp referring ANA rats (Sinclair 
et al. 1989; Kiianmaa et al. 1991). 
Similarly, AA rats show lower con- 
tents of (3 -endorphin in the periaque- 
ductal gray and amygdala than ANA 
rats (Gianoulakis et al. 1992), and P 
rats have increased levels of met- 
enkephalin in the hypothalamus and 
striatum relative to NP rats (Froehlich 
etal. 1987). 

Additionally, though there is good 
consistency across alcohol-preferring 
lines in 24-hour home cage ethanol 
intake, differences exist among these 
animals in other ethanol-related 
behaviors. These include differences 
in the magnitude of the alcohol depri- 
vation effect (Sinclair and Tiihonen 
1988; Sinclair and Li 1989), the 
degree to which availability of a palat- 
able alternative fluid attenuates 
ethanol intake (Lankford et al. 1991; 
Lankford and Myers 1994) and, in 
particular, ethanol-reinforced operant 
responding. Specifically, in operant 
self- administration tasks ethanol can 
serve as a reinforcer in NP rats, which 
avoid ethanol in a 24 -hour preference 
test (Files et al. 1993; Rassnick et al. 
1993#; Ritz et al. 1994). Moreover, 
when the response requirements for 
ethanol are increased, NP rats are will- 
ing to "work harder" than HAD rats, 
a line that shows high ethanol intake 



in preference tests (Ritz et al. 1994). 
A reduced "willingness" to work for 
ethanol under higher response require- 
ments has also been reported in the 
alcohol-preferring AA rats (Ritz et al. 
1989). Only in P rats was ethanol intake 
in operant tests consistent with their 
high ethanol consumption in 24-hour 
preference tests. These studies suggest 
that inherited factors that determine 
whether ethanol will come to serve as 
a reinforcer per se (i.e., in preference 
tests) differ from those that mediate 
the motivating value of ethanol, as 
inferred from the amount of work 
that rats will expend to gain access to 
the drug. Findings such as these indicate 
that it will be important to incorporate 
animals' "motivation" or degree of 
intensity and persistence in the effort 
to obtain ethanol at a particular time 
or set of circumstances in pharmaco- 
genetic models of high ethanol intake. 

MECHANISMS OF 
REINFORCEMENT 
ASSOCIATED WITH 
CHRONIC DRINKING 

Compromised Reward Systems 
and Negative Reinforcement 

As discussed earlier in this chapter, 
measures of ICSS reward thresholds 
indicate that, in contrast to the acute 
effects of ethanol, withdrawal is 
accompanied by a decrease in brain 
stimulation reward. It is becoming 
increasingly clear that at the neuro- 
chemical level, as well, the same sys- 
tems that have been implicated in the 
acute reinforcing effects of ethanol 
show adaptive changes after chronic 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



exposure, and may have an important 
role both in the affective changes 
associated with abstinence and the 
reinforcing actions of ethanol in the 
dependent state. 

Dopamine 

The role of DA neurotransmission in die 
acute reinforcing actions of ethanol is 
well established. Electrophysiological 
(Gessa et al. 1985; Brodie et al. 1990; 
Diana et al. 1992#), neurochemical 
(Imperato and DiChiara 1986; Wozniak 
et al. 1991; Yoshimoto et al. 1992«; 
Engel et al. 1992; Rossetti et al. 
1993; Kiianmaa et al. 1995), and 
behavioral (Imperato and DiChiara 
1986; Waller et al. 1986; Pecins- 
Thompson and Peris 1993) data indi- 
cate that behaviorally relevant doses of 
ethanol activate the mesolimbic DA 
reward pathway. Direct evidence of a 
role for DA in the acute reinforcing 
actions of ethanol comes from find- 
ings that operantly self-administered 
ethanol stimulates DA release in the 
nucleus accumbens (Weiss et al. 1993), 
that rats will self- administer ethanol 
directly into the ventral tegmental cell 
body region of the meso-accumbens 
DA reward pathway (Gatto et al. 
1994), and that ethanol preference or 
ethanol-maintained reinforcement is 
modified by pharmacological agents 
that interact with DA neurotransmis- 
sion (e.g., Weiss et al. 1990; Samson 
et al. 1992, 1993; George et al. 1995; 
Panockaetal. 1995). 

Although ethanol acutely activates 
mesolimbic DA neurotransmission, 
this effect shows tolerance in the depen- 
dent state. Extraneuronal DA concen- 
trations in the nucleus accumbens after 



consumption of ethanol liquid-diet in 
dependent rats are indistinguishable 
from those in ethanol-naive rats 
(Weiss et al. 1996), and stimulation of 
DA synthesis, commonly observed with 
acute ethanol, is attenuated after 
chronic ethanol (Tabakoff and Hoff- 
man 1978; Fadda et al. 1980). These 
observations suggest that long-term 
exposure to ethanol suppresses meso- 
accumbens DA activity to "balance" 
chronic stimulation by ethanol. This 
hypothesis is supported further by 
findings that ethanol withdrawal is 
associated with deficient DA release in 
the nucleus accumbens (Rossetti et al. 
1992&; Weiss et al. 1996) and a pro- 
found decrement of mesolimbic neu- 
ronal activity (Diana et al. 1992&, 
1993; Shen and Chiodo 1993). Inter- 
estingly, the suppression of DA release 
during withdrawal can be reversed by 
systemic injection of ethanol (Rossetti 
et al. 1992/7), and rats given the 
opportunity to self- administer ethanol 
during withdrawal regulate their 
ethanol intake in a manner that 
restores accumbal DA release to pre- 
withdrawal levels (Weiss et al. 1996). 
The reversal of this neurochemical 
deficit by systemic ethanol and, more 
importantly, the apparent behavioral 
"titration" of ethanol intake in self- 
administering rats to regain prewith- 
drawal conditions implicate accumbal 
DA release in ethanol-maintained neg- 
ative reinforcement and, by extension, 
in continued abuse and dependence. 

Although there is clear evidence for a 
role of neuroadaptive changes in forebrain 
DA transmission in the motivational 
effects of ethanol dependence and with- 
drawal, the mechanisms underlying 



270 



Neuroadaptive Changes in Neurotransmitter Systems 



these changes are presently not well 
understood. Acute ethanol administra- 
tion stimulates DA synthesis, but this 
effect is blunted in chronically ethanol- 
treated animals (Tabakoff and Hoff- 
man 1978; Fadda et al. 1980). Also, 
chronic ethanol exposure suppresses 
K + -stimulated DA release (Darden and 
Hunt 1977), possibly via inhibition of 
Ca ++ influx (Kim et al. 1994) or by 
uncoupling of calcium entry and DA 
release (Leslie et al. 1986). In addi- 
tion, depolarization inactivation has 
been proposed as a possible mechanism 
(Shen and Chiodo 1993). These findings 
point toward changes at the biochem- 
ical and cellular level. Future research 
will need to more precisely characterize 
these potential mechanisms, including 
molecular changes, to provide a basis 
for the pharmacotherapeutic reversal 
of ethanol-induced neuroadaptive 
alterations in DA transmission. 

5 - Hydroxytryptamine 

Ample evidence exists for an involvement 
of 5-HT in ethanol-seeking behavior as 
well. Ethanol increases 5-HT release in 
the nucleus accumbens after local, sys- 
temic, and self- administration (Yoshi- 
moto and McBride 1992; Yoshimoto et 
al. 1992&; Weiss et al. 1996; Yoshimoto et 
al. 1996). Pharmacological treatments 
that increase the synaptic availability of 
5-HT, or direct activation of 5-HT 
transmission by receptor agonists, sup- 
press voluntary ethanol intake in animals 
(for reviews, see Sellers et al. 1992; 
LeMarquand et al. 1994&) and can 
reduce alcohol consumption in humans 
(Naranjo et al. 1987, 1990; Monti and 
Alterwain 1991; Naranjo and Bremner 
1993; LeMarquand et al. 1994^; 



Naranjo et al. 1995). A serotonergic role 
in ethanol abuse is supported also by 
findings that the subjective effects of 
ethanol depend, at least partially, on 5- 
HT neurotransmission, since agonists 
of the 5-HT 1A receptor substitute for 
the discriminative stimulus properties 
of ethanol (Signs and Schechter 1988; 
Grant and Colombo 1993 b\ Krystal et 
al. 1994), whereas 5-HT 3 antagonists 
block these properties (Grant and Bar- 
rett 1991 b). 

Neuroadaptive changes similar to 
those observed with DA have been 
reported in serotonergic systems after 
chronic ethanol exposure. Ethanol 
acutely activates central 5-HT trans- 
mission (for a review, see LeMar- 
quand et al. 1994&). In contrast, 
ethanol withdrawal after induction of 
dependence leads to reductions in 5-HT 
metabolism and content of 5-HT or 
its metabolite, 5-hydroxyindoleacetic 
acid (5-HIAA), in whole brain, lim- 
bic, and striatal tissues (Kahn and 
Scudder 1976; Tabakoff et al. 1977; 
Badawy and Evans 1983; Kempf et al. 
1990; Wahlstrom et al. 1991; Yama- 
mura et al. 1992). More recently it 
was shown that whereas ethanol 
acutely enhances the release of 5-HT 
from the nucleus accumbens (Yoshi- 
moto and McBride 1992; Yoshimoto 
et al. 1992£, 1996), ethanol with- 
drawal in dependent rats is associated 
with a progressive suppression of 5- 
HT release in this brain region (Weiss 
et al. 1996). These findings of seroton- 
ergic deficiencies during withdrawal 
are consistent with clinical studies that 
have revealed deficits in 5-HT synthesis, 
turnover, or receptor function in alco- 
holics (Ballenger et al. 1979; Linnoila 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



et al. 1983; Thomson and McMillen 
1987; Lee and Meltzer 1991) and 
implicate impaired 5-HT function as 
an important neurochemical factor in 
alcohol abuse and dependence 
(LeMarquand et al. 1994#). 

Studies in ethanol-dependent rats 
implicate adaptations in 5-HT receptors 
in addition to presynaptic changes in 
the development and maintenance of 
alcoholism. Blockade of 5-HT lc and 
5-HT 2 receptors by a single large dose 
of a pharmacological antagonist pre- 
vented the anxiogenic effects of 
ethanol withdrawal in rats for up to 7 
days after treatment as measured on 
the elevated plus maze (Lai et al. 
1993). It appears that chronic ethanol 
exposure may up-regulate or enhance 
the sensitivity of 5-HT lc receptors in 
particular, since chronic ethanol-treated 
rats show enhanced susceptibility to the 
anxiogenic-like effects of a selective 5- 
HT 1C agonist (Rezazadeh et al. 1993). 
Adaptive changes in the function of 5- 
HT 1C receptors may, therefore, play a 
significant role in ethanol withdrawal 
anxiety. In addition, changes in the 
sensitivity of 5-HT 1A receptors may have 
a role in the anxiogenic consequences of 
ethanol withdrawal, as indicated by find- 
ings that chronic ethanol exposure alters 
the sensitivity to several pharmacological 
effects of the 5-HT 1A receptor agonist 
8-OH-DPAT (Kleven et al. 1995) and 
that 5-HT 1A agonists can reverse ethanol 
withdrawal-induced suppression of 
exploratory activity in the open arms of 
the elevated plus maze (Lai et al. 1991). 

Gamma- Aminobutyric Acid 

The anxiolytic effects of ethanol, in addi- 
tion to its mood-elevating actions, are 



thought to be an important mecha- 
nism promoting its abuse. Studies 
exploring the mechanisms by which 
ethanol exerts its anti-anxiety effects 
have implicated interactions with the 
GABA-benzodiazepine (BZD) receptor 
complex. In general, GABA-BZD 
antagonists and inverse agonists reverse 
the anxiolytic effects of ethanol in 
conflict tests (Liljequist and Engel 
1984; Koob et al. 1986) and the ele- 
vated plus maze (Lister 1988; Criswell 
et al. 1994; Prunell et al. 1994). In 
addition, studies examining the effects 
of ligands interacting with the GABA- 
BZD receptor complex on ethanol 
self-administration have provided evi- 
dence for a role of GABA in ethanol- 
maintained reinforcement. Partial 
inverse BZD agonists such as Ro 15- 
4513 or Ro 19-4603 dose-depen- 
dently suppress ethanol intake in both 
free-drinking and operant self-admin- 
istration models without concomitant 
reduction in the consumption of 
water or saccharin (McBride et al. 
1988; Samson et al. 1989; June et al. 
1991; Rassnick et al. 1993#; June et 
al. 1994b). Moreover, decreases in 
responding for ethanol induced by 
BZD inverse agonists are reversed by 
coadministration of the BZD antago- 
nist flumazenil, confirming that the 
effect of the partial agonist on self- 
administration is specific to the BZD 
site of the GABA-BZD receptor 
(Samson et al. 1989; Rassnick et al. 
1993#; June et al. 1994a, 1994&). 

Evidence supporting a role for neu- 
roadaptive processes in behavioral 
changes associated with chronic 
ethanol treatment and dependence is 
emerging also in the case of GABA. It 



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Neuroadaptive Changes in Neurotransmitter Systems 



is well documented that in contrast to 
acute ethanol, which potentiates 
GAB A- stimulated Cl~ flux, chronic 
ethanol administration decreases 
GAB A- dependent CI" influx (Morrow 
et al. 1988; Kuriyama et al. 1993). 
This inhibition of GABAergic func- 
tion persists for some time during 
withdrawal from ethanol (Kuriyama et 
al. 1993; Kang et al. 1996) and is 
thought to contribute to ethanol 
withdrawal symptoms such as seizure 
susceptibility, anxiety, and negative 
affect, which may motivate continued 
ethanol consumption. Consistent with 
this view, microinjections of the 
GABA agonist muscimol into the cen- 
tral nucleus of the amygdala (CeA) of 
ethanol self-administering rats 
decreased enhanced responding for 
ethanol associated with ethanol with- 
drawal, at doses that did not alter 
ethanol intake in nondependent rats 
(Roberts et al. 1996). Counteradapta- 
tions in GABAergic mechanisms after 
chronic ethanol are also suggested by 
a finding that a single dose of the 
BZD antagonist flumazenil, adminis- 
tered 14 hours before withdrawal, 
reversed behavioral manifestations 
of ethanol withdrawal in mice (Buck 
et al. 1991). This observation sug- 
gests that brief occupation of BZD 
receptors by an antagonist may per- 
haps "reset" adaptive cellular mecha- 
nisms responsible for the development 
of dependence. 

Overall, these findings are suggestive 
of the development of counteradap- 
tive responses within dopaminergic, 
serotonergic, and GABAergic systems 
that oppose the acute pharmacologi- 
cal actions of ethanol such that these 



systems exhibit functional deficiencies 
in the absence of continued stimula- 
tion by ethanol (Koob and Bloom 
1988). These changes, in conjunction 
with adaptive responses that develop 
in systems that are not involved in the 
acute reinforcing effects of ethanol 
but that when engaged counter the 
positive, mood-elevating effects of 
ethanol (see the next section), may 
provide a neurobiological basis for 
aspects of the ethanol withdrawal 
symptomatology — in particular, affec- 
tive changes opposite to those pro- 
duced by ethanol acutely. 

Disruption of Brain 
Stress Systems After 
Chronic Ethanol 

Chronic alcohol abuse has profound 
effects on the hypothalamic -pituitary- 
adrenal (HPA) axis. Alcoholics exhibit 
a blunted adrenocorticotropic hormone 
(ACTH) response to corticotropin- 
releasing factor (CRT) administration 
(Wand and Dobs 1991), suggesting 
that chronic ethanol induces HPA axis 
injury, which results in impaired 
responsiveness to non-ethanol-induced 
stress. A blunted stress response as well 
as abnormal circadian Cortisol secretion, 
perturbations of the noradrenergic 
system, and changes in CRT-norepi- 
nephrine interactions are also observed 
during ethanol withdrawal (Risher- 
Flowers et al. 1988; von Bardeleben 
et al. 1989; Adinoffet al. 1991; Hawley 
et al. 1994; Inder et al. 1995; Costa et 
al. 1996; Ehrenreich et al. 1997). These 
changes can persist beyond the acute 
withdrawal phase and thus may con- 
tribute to the physiological and behav- 
ioral complications of chronic alcoholism. 



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In animals, ethanol withdrawal is 
associated with HPA axis activation at 
both the adrenal and hypothalamic/ 
pituitary levels (Freund 1969; 
Tabakoff et al. 1978; Rivier et al. 
1984; De Soto et al. 1985; Ehlers and 
Chaplin 1987; Redei et al. 1988; 
Kleven et al. 1995; Lamblin et al. 
1996). These neuroendocrine effects 
are accompanied by increases in emo- 
tionality that resemble the effects of 
stress (Freund 1969; Tabakoff et al. 
1978; De Soto et al. 1985) and, 
therefore, may contribute to the moti- 
vational effects of ethanol withdrawal. 
A role for neuroendocrine stress sys- 
tems in excessive alcohol drinking is 
suggested also by findings that P rats 
show enhanced electrophysiological 
responses to administration of CRF, a 
finding that has been attributed to up- 
regulation of CRF receptors in these 
animals (Ehlers et al. 1992). These 
observations are consistent with find- 
ings that P rats are more "anxious" in 
behavioral tests of anxiety (Stewart et 
al. 1993) and suggest that a relation- 
ship may exist between responsivity to 
stress, the status of the HPA axis 
including the CRF system, and alco- 
hol preference. 

Much attention has been directed 
recently at understanding the role of 
the nonneuroendocrine CRF system 
in the CeA in the affective conse- 
quences of ethanol withdrawal. Grow- 
ing evidence suggests that the CRF 
neuropeptide system in the CeA has 
an essential role in the mediation of 
emotional responses to stress such as 
anxiety (Dunn and Berridge 1990; 
Heinrichs et al. 1992; Swiergiel et al. 
1993; Koob et al. 1994), symptoms 



which are also an integral part of the 
alcohol withdrawal syndrome and, 
therefore, may involve activation of 
CRF mechanisms in the CeA. In sup- 
port of this hypothesis, marked 
increases in CRF release in the CeA 
have been observed during withdrawal 
in ethanol-dependent rats (Merlo Pich 
et al. 1995), and microinjection of the 
CRF receptor antagonist a-helical 
CRF(9-41) into the CeA selectively 
reversed the anxiogenic effects of 
ethanol withdrawal as measured in the 
elevated plus maze (Baldwin et al. 
1991; Rassnick et al. 1993£). These 
results suggest that in addition to the 
classic HPA axis activation, discrete 
extrahypothalamic CRF systems may 
be affected by chronic exposure to 
ethanol, and this may be reflected in 
an overactivity of these systems during 
withdrawal. As discussed later in this 
chapter, disruptions in both HPA 
function and the CRF system in the 
CeA may be an important factor in 
protracted withdrawal and vulnerabil- 
ity to relapse. 

Neurosteroids 

A comparatively novel area of research 
concerned with the biological basis of 
alcohol addiction focuses on a group 
of endogenous steroids, termed "neu- 
roactive steroids," because these com- 
pounds are synthesized from cholesterol 
in the brain or can be formed in the 
brain as metabolites of the gonadal 
steroids and mineralocorticoids. In 
contrast to the slow, delayed intracel- 
lular effects of traditional steroids, 
neuroactive steroids have a fast action 
on neuronal membranes. Neuroactive 
steroids bind to a specific recognition 



274 



Neuroadaptive Changes in Neurotransmitter Systems 



site at the GABA-BZD receptor com- 
plex, the neurosteroid site, where they 
can modulate the activity of GABA 
(Majewska et al. 1986; Morrow et al. 
1987). Some of these compounds, 
such as allopregnanolone and 3a, Sa- 
te trahydrodeoxy cor ticoster one 
(THDOC), act as agonists at the 
GABA-BZD receptor complex pro- 
ducing neuronal inhibition and, 
behaviorally, exert hypnotic and anxi- 
olytic-like effects (Crawley et al. 1986; 
Bitran et al. 1991; Wieland et al. 
1991); others, such as pregnenolone 
sulfate and dehydroepiandrosterone 
(DHEA), act as antagonists at the 
GABA-BZD receptor, increase neu- 
ronal excitability, and can produce 
anxiogenic-like and proconvulsant 
effects (Majewska and Schwartz 1987; 
Majewska et al. 1990; Melchior and 
Ritzmann 1994&). 

Since both ethanol and neuroactive 
steroids produce many of their biolog- 
ical and behavioral effects by interacting 
with the GABA-BZD receptor, it is 
possible that neurosteroids modulate 
the actions of ethanol and play a role 
both in the acute intoxicating effects of 
ethanol and in neuroadaptive changes 
associated with chronic ethanol con- 
sumption. Indeed, allopregnanolone 
and THDOC share sedative and anxi- 
olytic discriminative stimulus properties 
with ethanol (Ator et al. 1993). Neuro- 
active steroids can also potentiate (or 
reverse [Melchior and Ritzmann 
1994^]) the anxiolytic actions of ethanol 
in the elevated plus maze (Melchior 
and Ritzmann 1994#) and can enhance 
the hypnotic effects of ethanol by 
increasing ethanol-induced sleep time 
(Melchior and Ritzmann 1992). 



These findings confirm the exis- 
tence of interactions between neuro- 
active steroids and the acute behavioral 
effects of ethanol, but there is also 
growing evidence for a modulatory 
role of neurosteroids in dependence 
and withdrawal. Plasma levels of allo- 
pregnanolone and THDOC, the most 
potent endogenous positive modula- 
tors of GABA-BZD receptors, were 
markedly reduced in alcoholic subjects 
during the early withdrawal phase, 
when anxiety and depression scores 
were elevated. Allopregnanolone and 
THDOC levels recovered during the 
late withdrawal phase at a time when 
anxiety and depression scores returned 
to normal (Romeo et al. 1996), and 
these observations suggest that chronic 
ethanol-induced decrease in neuroactive 
steroid biosynthesis may contribute to 
ethanol withdrawal symptoms. Inter- 
estingly, these deficiencies in neurosteroid 
synthesis appear to be accompanied 
by a sensitization of the GABA-BZD 
receptor to neuroactive steroids dur- 
ing withdrawal. In both rats and mice, 
administration of the anxiolytic neu- 
rosteroid 3a,5a-tetrahydroproges- 
terone abolished anxiety and seizure 
susceptibility during ethanol with- 
drawal (Devaud et al. 1995; Finn et 
al. 1995), although this effect was 
strain dependent in mice (Finn et al. 
1995). These data not only provide 
further evidence for an interaction 
between ethanol and neuroactive 
steroids at the GABA-BZD receptor 
but also suggest that genetic factors in 
neuroactive steroid sensitivity and 
biosynthesis may contribute to 
ethanol withdrawal severity, and that 
neurosteroids may be an important 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



target for the development of pharma- 
cological agents capable of antagoniz- 
ing acute or chronic effects of ethanol. 
The precise interactions of neu- 
roactive steroids with ethanol are only 
beginning to be understood. 
Nonetheless, the discovery of these 
endogenous modulators of GABA 
function and their unique binding site 
at the GABA A receptor complex pro- 
vides new perspectives and tools for 
the investigation of the mechanism by 
which ethanol exerts its behavioral 
actions, and for the development of 
novel compounds that selectively 
block the intoxicating effects of 
ethanol. 

Neurotransmitter 
Interactions in the 
Control of Ethanol- 
Seeking Behavior 

Research on the neuropharmacological 
basis of ethanol reinforcement over the 
past decade has led to the recognition 
that the acute reinforcing effects of 
ethanol depend on multiple transmit- 
ter systems and their interactions 
(e.g., Engel et al. 1992). For example, 
evidence is now accumulating to sug- 
gest that one mechanism by which 
ethanol activates the dopaminergic 
"reward circuitry" involves an action 
on endogenous opioid systems (Wid- 
dowson and Holman 1992; Acquas et 
al. 1993; Benjamin et al. 1993; Di 
Chiara et al. 1996; Gonzales and 
Weiss 1998). There is also growing 
evidence for interactions between 5- 
HT and DA in the control of ethanol- 
seeking behavior. Serotonin potentiates 
ethanol-induced excitation of meso- 
limbic DA neurons (Brodie et al. 



1995). In contrast, 5-HT 3 antagonists 
suppress ethanol-induced DA release 
in the nucleus accumbens and ventral 
tegmental area (VTA) (Yoshimoto et 
al. 1992a; Campbell and McBride 1995; 
Campbell et al. 1996), reduce ethanol 
intake (Fadda et al. 1991; Knapp and 
Pohorecky 1992; Hodge et al. 1993; 
lohnson et al. 1993), block the anxi- 
olytic effects of ethanol (Grant and 
Barrett 1991a), and attenuate the dis- 
criminative stimulus properties of 
ethanol (Grant and Barrett 1991a, 
1991/7). These findings suggest that 
5-HT 3 antagonists block these behav- 
ioral effects of ethanol by interfering 
with ethanol-induced DA release. 
Interactions relevant to the reinforc- 
ing actions of ethanol may also exist 
between DA and glutamate neuro- 
transmission (Rassnick et al. 1992&). 
The nucleus accumbens receives neu- 
ronal projections using glutamate, 
serotonin, and endogenous opioids as 
their transmitters from limbic and 
midbrain regions that play a role in 
motivational and emotional processes. 
Interactions among these transmitters 
in the nucleus accumbens can, per- 
haps, be viewed as "orchestrating" the 
rewarding effects of ethanol by orga- 
nizing the functional output from this 
structure (Engel et al. 1992). How- 
ever, the investigation of such interac- 
tions has yet to be expanded to their 
role in addiction and withdrawal. 

Isolated findings have, in fact, 
begun to suggest that neurotransmit- 
ter interactions may be important in 
the development of dependence and 
regulation of ethanol- seeking behavior 
in dependent animals. For example, 
alterations in the modulation of DA 



276 



Neuroadaptive Changes in Neurotransmitter Systems 



release in the nucleus accumbens by 
5-HT develop over the course of 
chronic ethanol exposure. These 
involve, in particular, changes in the 
regulation of DA release by 5-HT 3 
receptors, which appear to compen- 
sate for deficient serotonergic activity 
associated with chronic ethanol expo- 
sure and maintain or even enhance 
responsiveness of the dopaminergic 
system to ethanol (Yoshimoto et al. 
1996). Interactions between neuro- 
chemical systems in the development 
of dependence can perhaps also be 
inferred from the regionally specific 
effects of chronic ethanol on the 
expression of GABA A receptor sub- 
units. In particular, a finding that pro- 
longed 12-week, but not 4-week, 
exposure to ethanol decreased alpha- 1 
subunit immunoreactivity in the VTA 
and hippocampus (Charlton et al. 
1997) may suggest that interactions 
exist between GABA and other trans- 
mitter systems involved in reward and 
cognitive functions in these respective 
brain regions. As discussed later in 
this chapter, there are also putative 
interactions between endogenous opi- 
oids and mesolimbic DA transmission 
that may be relevant to mediation of 
ethanol-seeking behavior in depen- 
dent subjects. 

Sensitization 

Ethanol -Induced Sensitization: 
Determinant of Heightened Drug- 
Seeking Behavior? 

Repeated administration of drugs can 
result in an enhancement of their 
behavioral and other pharmacological 
effects, particularly if the treatment 



regimen involves intermittent, non- 
continuous administration (Robinson 
and Becker 1986; Kalivas and Stewart 
1991), and it has been suggested that 
drug-induced sensitization may play 
an important role in the development 
of compulsive drug- seeking behavior, 
craving, and perhaps relapse (Hunt 
and Lands 1992; Robinson and 
Berridge 1993). 

Ethanol-induced sensitization has 
predominantly been studied in mice, a 
species that shows augmented loco- 
motor stimulant responses to ethanol 
after repeated treatment (Phillips et al. 
1994; Broadbent et al. 1995; Phillips 
et al. 1995; Roberts et al. 1995; 
Phillips et al. 1996, 1997). This effect 
seems to be highly strain dependent in 
that it is limited largely to DBA/2J 
mice, implicating genetic factors in 
ethanol sensitization (Phillips et al. 
1994, 1995). Since the psychomotor 
stimulant effects of ethanol (Broad- 
bent et al. 1995; Cohen et al. 1997) 
and other drugs of abuse involve acti- 
vation of mesolimbic DA transmission 
and, consequently, are thought to 
reflect reinforcing properties, such 
strain differences in locomotor sensiti- 
zation may provide important clues 
with regard to genetic factors in 
ethanol preference. However, the 
interpretation of a link between sensi- 
tization and ethanol preference has 
been complicated by several issues. 

Quantitative trait loci analysis 
revealed a negative association between 
ethanol preference and both the acute 
locomotor response to ethanol and 
ethanol sensitization (Phillips et al. 
1995), a finding that supports a rela- 
tionship between ethanol preference 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



and reduced susceptibility to the sensi- 
tizing effects of ethanol but is inconsis- 
tent with the notion that sensitization 
is associated with enhanced drug- 
seeking behavior (Hunt and Lands 
1992; Robinson and Berridge 1993; 
Piazza and Le Moal 1996). The 
understanding of the significance of 
sensitization in ethanol-seeking 
behavior and genetic ethanol prefer- 
ence is further complicated by the 
question of whether the enhanced 
locomotor activity associated with 
repeated ethanol treatments reflects 
"true" sensitization or rather toler- 
ance to the sedative effects of ethanol. 
This remains an open issue. If ethanol 
sensitization is the result of sedative 
tolerance, then a positive relationship 
should exist between sensitization and 
tolerance in strains of mice exhibiting 
greatest susceptibility to sensitization. 
Using ataxia as a measure of tolerance, 
genetic correlations did not support 
the existence of such relationships in 
recombinant inbred mouse strains 
(Phillips et al. 1996). In contrast, 
complex dose-response analyses of 
ethanoPs motor effects in DBA/2J 
mice confirmed that apparent sensiti- 
zation to ethanol was accounted for 
by selective tolerance in the sedative 
limb of the dose-response function 
(Tritto and Dudek 1997). A similar 
effect has been reported in Wistar rats 
given free access to ethanol for 8 weeks. 
In these animals the sedative effects of 
higher ethanol challenge doses were 
abolished, and the animals showed 
greatly increased motor activation 
over the range of ethanol doses that 
produced sedation in naive animals 
(Wolffgramm and Heyne 1995). 



Thus, interpretation of this rather 
complex phenomenon seems to hinge 
on the particular dependent measure of 
tolerance used. Clear tolerance to motor 
activation by ethanol was also observed 
in rats after 2 weeks of 24-hour access 
to ethanol (Pecins-Thompson and 
Peris 1993), suggesting that the devel- 
opment of sensitization or tolerance 
may also be a function of the mode of 
administration (i.e., self vs. forced). 

If it is presumed that ethanol sensi- 
tization can develop independently of 
sedative tolerance, an important issue 
that arises is whether sensitization has 
a role in altered reward or heightened 
susceptibility to the addictive effects of 
ethanol. The genetic analyses dis- 
cussed above suggest that ethanol 
preference is negatively correlated with 
ethanol sensitization (Phillips et al. 
1995). On the other hand, female rats 
previously sensitized to the locomotor 
stimulant effects of amphetamine 
show enhanced ethanol intake (Fahlke 
et al. 1994), a finding that seems con- 
sistent with a role of sensitized dopa- 
minergic transmission in heightened 
ethanol-seeking behavior or prefer- 
ence. Whether such increases in ethanol 
intake reflect heightened ethanol- 
seeking behavior or tolerance to the 
psychoactive effects of ethanol 
remains a matter of debate. Moreover, 
robust changes in ethanol intake were 
not observed in amphetamine-sensi- 
tized male rats (Samson 1995), so 
gender differences may be another 
variable that must be considered when 
evaluating the relationship between 
ethanol sensitization and preference. 

It also remains unclear on the basis 
of available data whether or to what 



278 



Neuroadaptive Changes in Neurotransmitter Systems 



extent ethanol-induced behavioral 
sensitization has a dopaminergic basis. 
Rats made dependent on ethanol by a 
chronic liquid-diet procedure showed 
an enhanced propensity to develop 
sensitized locomotor responses to 
cocaine and amphetamine (Manley 
and Little 1997), suggesting that a 
history of ethanol dependence may, 
indeed, lead to a sensitization of the 
meso-accumbens DA system. In con- 
trast, repeated ethanol pretreatment in 
rats enhanced the locomotor effect of 
morphine but not amphetamine, raising 
doubt as to whether ethanol-induced 
sensitization has a dopaminergic basis 
(Nestby et al. 1997). Similarly, findings 
that the DA antagonist haloperidol 
prevented ethanol-stimulated locomotor 
activity in DBA/2J mice but failed to 
block ethanol-induced sensitization 
suggest that there is a dissociation of the 
neurobiological mechanisms that medi- 
ate the acute stimulant versus sensitiza- 
tion effects of ethanol (Broadbent et 
al. 1995) and that ethanol sensitization 
may not involve alterations in dopami- 
nergic function in mice. This view is 
further supported by the failure of 
haloperidol to reverse ethanol prefer- 
ence in ethanol -sensitized C57 mice, 
a finding that, in fact, suggests that 
the mechanisms mediating the acute 
reinforcing actions of ethanol are distinct 
from those mediating ethanol-drinking 
behavior after the development of sensi- 
tization (Ng and George 1994). Thus, 
the understanding of the neurobiological 
basis of sensitization, the question as 
to how the neurobiological changes 
observed with intermittent ethanol 
treatment relate to psychostimulant- 
induced sensitization, and elucidation 



of the significance of sensitization in 
ethanol-seeking behavior remain great 
challenges for future studies. 

Sensitization to stimulant drugs can 
also be induced by prior exposure to 
stress (Kalivas and Duffy 1989), and 
there appears to be a role for both the 
HPA axis and extrahypothalamic CRF 
in stress-induced sensitization (Koob 
and Cador 1993; see also Richter et 
al. 1995). Both stress and repeated 
administration of glucocorticoids can 
increase the behavioral effects of psycho- 
stimulants, and it has been hypothe- 
sized that circulating glucocorticoids 
may convey susceptibility to sensitiza- 
tion (and thus impart heightened sus- 
ceptibility to psychostimulant abuse) 
and may maintain the sensitized state 
once induced (Piazza and Le Moal 
1996, 1997). Several findings indicate 
that, similar to previous findings with 
psychostimulants, repeated exposure 
to stress sensitizes DBA/2J mice to the 
locomotor activating effects of ethanol. 
Moreover, both stress and ethanol- 
induced sensitization were attenuated 
by the glucocorticoid receptor antagonist 
RU 38486 (Roberts et al. 1995; Phillips 
et al. 1997). These findings implicate a 
role for the HPA axis in ethanol sensiti- 
zation and cross-sensitization with stress 
consistent with the mechanisms proposed 
to contribute to sensitization to other 
drugs of abuse. Whether a link exists 
between ethanol reward and sensitiza- 
tion involving HPA neuroendocrine 
mechanisms remains to be deter- 
mined, however, in future research. 

Repeated Withdrawal and Kindling 

Another form of sensitization that may 
contribute to excessive drinking and 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



vulnerability to relapse is the enhanced 
withdrawal response after repeated 
intoxication and withdrawal. Initial 
observations in humans indicated that 
the severity of withdrawal symptoms 
increases with increased duration of 
alcohol abuse, leading to the hypothe- 
sis that a "kindling" process similar to 
limbic kindling stimulation (Goddard 
et al. 1969) was responsible for the 
exacerbation of withdrawal severity 
(Ballenger and Post 1978). It was 
later found that the number of with- 
drawal episodes was more predictive 
of the likelihood and severity of alco- 
hol withdrawal seizures than the 
absolute amount of alcohol consumed 
over the course of the addicts' history 
of alcohol abuse (Brown et al. 1988). 
The kindling hypothesis has received 
support from numerous studies 
employing animal models of repeated 
intoxication and withdrawal (Branchey 
et al. 1971; Carrington et al. 1984; 
McCown and Breese 1990; Becker 
and Hale 1993; Kokka et al. 1993; 
Ulrichsen et al. 1995; Becker et al. 
1997^, 1997b). Mice exposed to 
chronic ethanol vapor and then sub- 
jected to repeated withdrawal episodes 
show progressive increases in the 
intensity of withdrawal seizures 
(Becker and Hale 1993; Becker et al. 
1997a, 1997b). Similarly, rats repeat- 
edly withdrawn from chronic ethanol 
treatment exhibit "kindling" effects in 
seizure activity (McCown and Breese 
1990; Ulrichsen et al. 1995). Studies 
aimed at examining the brain sites and 
mechanisms responsible for this phe- 
nomenon have implicated the inferior 
collicular cortex (McCown and Breese 
1990; see also Kang et al. 1996; 



N'Gouemo et al. 1996) rather than 
the traditional limbic foci in the kindling 
action of repeated ethanol withdrawal. 
These studies also suggest that the pro- 
gressive increase in seizure activity 
may be linked to impaired GABA A - 
mediated inhibitory input (Kang et al. 
1996), because this effect was associ- 
ated with deregulated flunitrazepam 
binding (Ulrichsen et al. 1996) and 
was blocked by diazepam (Ulrichsen et 
al. 1995). An important question for 
future research will be to examine the 
possible relationship between repeated 
withdrawal and kindling phenomena 
and changes in the reinforcing and 
motivational effects of ethanol. 

PROTRACTED 
ABSTINENCE AND 
RELAPSE 

Theoretical Considerations 
on the Relapse Process 

Alcoholism is a chronic relapsing dis- 
order, and the resumption of alcohol 
abuse after periods of abstinence is 
one of the principal characteristics of 
substance dependence on alcohol. Two 
major theoretical positions exist to 
explain the persistence of addictive 
behavior and the high risk of relapse 
long after withdrawal: conditioning 
and homeostatic hypotheses. 

Conditioning hypotheses are based on 
observations that relapse is often asso- 
ciated with exposure to ethanol-related 
environmental stimuli. This view holds 
that environmental stimuli that have 
become associated with the subjective 
actions of ethanol by means of classical 
conditioning, or act as discriminative 



280 



Neuroadaptive Changes in Neurotransmitter Systems 



stimuli signaling the availability of 
ethanol and "setting the occasion" to 
engage in drug-taking behavior, elicit 
subjective states that trigger resumption 
of drug use. Homeostatic hypotheses 
relate relapse to the persistence of resid- 
ual neuroadaptive changes and disrup- 
tion of neuroendocrine homeostasis, 
which are thought to underlie mood 
dysregulation and somatic symptoms 
such as insomnia that are often observed 
during the "protracted abstinence" 
phase. This view, therefore, implicates 
alleviation of discomfort and negative 
affect and, consequently, negative 
reinforcement as a motivational basis 
in relapse. The homeostatic and con- 
ditioning hypotheses are not mutually 
exclusive but may, in fact, be additive 
in that exposure to ethanol-associated 
environmental stimuli may augment 
vulnerability to relapse imparted by 
homeostatic disturbances alone (Koob 
and Le Moal 1997). 

Homeostatic Factors: Protracted 
Abstinence 

The persistence of affective changes and 
heightened susceptibility to relapse after 
withdrawal suggests that a history of 
alcohol dependence leads to long-last- 
ing changes in cellular or molecular 
mechanisms associated with the con- 
trol of drug- seeking behavior. Evidence 
of such persistent neurobiological 
alterations is beginning to accrue, 
although research directed at the 
identification and understanding of 
the nature of these changes is still in 
its infancy. 

Clinical studies have identified retar- 
dation in the recovery of dopaminergic 
transmission after detoxification and 



withdrawal as a possible factor in relapse 
(Heinz et al. 1995a, 1995&). In 
patients with good subsequent treat- 
ment outcome, apomorphine-stimulated 
plasma growth hormone levels (as a 
measure of DA receptor sensitivity) and 
G protein-induced inhibition of adeny- 
lyl cyclase activity in platelet mem- 
branes (as an index of DA D 2 -receptor 
coupled second messenger mecha- 
nisms) returned to normal within the 
first 24 hours of withdrawal. In con- 
trast, in patients who subsequently 
relapsed, normalization of dopaminer- 
gic function was delayed and still 
showed signs of disruptions after 8 
days of abstinence (Heinz et al. 1995 &). 
These data suggest that blunting of the 
growth hormone response to DA ago- 
nists and, by inference, an impairment 
in DA receptor function or DA- 
dependent signal transduction mecha- 
nisms are associated with the risk of 
early relapse in alcoholics (Heinz et al. 
1995 a) and poor treatment outcome 
(Heinz et al. 1995^). The possibility 
of a sustained dopaminergic dysfunction 
during protracted abstinence has also 
received some support from animal 
studies. Electrophysiological measures 
suggest that mesolimbic DA function 
in dependent rats is still reduced 72 
hours after termination of chronic 
ethanol treatment, although behav- 
ioral manifestations of the alcohol 
withdrawal syndrome recede within 
48 hours (Diana et al. 1996). 

Other possible mechanisms in pro- 
tracted abstinence symptoms are changes 
in neuroendocrine function. A history 
of alcohol dependence can change the 
responsiveness of the HPA axis to stres- 
sors during early abstinence (Muller et 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



al. 1989; Costa et al. 1996), and some 
HPA axis dysfunction was still evident 
up to 12 weeks postwithdrawal in alco- 
holics (von Bardeleben et al. 1989; 
Ehrenreich et al. 1997). In addition, 
alcoholics show abnormalities in the 
hypothalamic-pituitary- thyroid axis 
(Baumgartner et al. 1994), sleep distur- 
bances (Gillin et al. 1990; Thompson 
et al. 1995), and circadian dysregula- 
tion (Schmitz et al. 1996) during and 
after withdrawal. The persistence of 
such changes beyond the acute with- 
drawal phase may convey enhanced 
vulnerability to relapse. Indeed, mea- 
sures of HPA axis dysfunction have 
been proposed as a useful clinical 
measure of susceptibility to relapse 
during protracted abstinence (Wilkins 
etal. 1992). 

Other neuroendocrine mechanisms 
may be affected as well. Withdrawal 
from chronic ethanol consumption in 
humans causes a significant increase in 
plasma nerve growth factor (Aloe et 
al. 1996). Although the functional 
significance of this phenomenon is not 
clear, it is possible that the increased 
levels of circulating nerve growth fac- 
tor might be involved in homeostatic 
adaptive or reparative processes. 
Finally, the activation of the CRT sys- 
tem in the CeA by ethanol withdrawal 
(Rassnick et al. 1993 b\ Merlo Pich et al. 
1995) implicates changes in the func- 
tional activity of this extrahypothalamic 
CRT system as a possible mediator of 
protracted withdrawal effects. 

The long-lasting nature of protracted 
abstinence symptoms after withdrawal 
and detoxification has naturally begun to 
draw attention to the molecular mech- 
anisms and regulation of gene expression 



that may underlie homeostatic or neuro- 
adaptive changes within brain circuit- 
ries mediating AOD reinforcement. 
Although not the focus of this review, 
research on the molecular basis of pro- 
tracted abstinence has identified tran- 
scription factors including CREB (cyclic 
adenosine monophosphate response 
element binding) and novel Fos-like 
proteins (chronic FRAs or Fos-related 
antigens) as possible mediators of per- 
sistent drug effects (Hope et al. 1992; 
Hyman 1996; Widnell et al. 1996) 
that may also underlie long-term 
changes at the molecular level induced 
by chronic ethanol. 

Together, these observations indicate 
that neuroadaptive or homeostatic 
changes induced by chronic ethanol 
can outlast physical withdrawal and 
detoxification. However, little is 
known about the persistence, time 
course, and reversibility of these 
changes and the relationship of these 
parameters to vulnerability to relapse. 
Thus, a challenge for future research 
will be to better characterize the func- 
tional and behavioral consequences of 
protracted abstinence in animal mod- 
els, and to relate alterations in the 
HPA activity, regulation of specific 
transcription factors, or receptor sys- 
tems to specific aspects of drug rein- 
forcement in animals with different 
histories of ethanol exposure (e.g., 
sensitization to acute challenges vs. 
changes in set-point associated with 
protracted abstinence). 

Conditioning Factors: Ethanol - 
Associated Environmental Stimuli 

Environmental stimuli associated with 
the availability or the subjective effects 



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of ethanol can induce alcohol craving 
and reinstate alcohol -seeking behavior 
in humans (Heather and Stallard 
1989; Rohsenow et al. 1994; Stormark 
et al. 1995; Cooney et al. 1997). In 
alcoholics the sight and smell of a pre- 
ferred alcoholic beverage elicits large 
changes in measures of heart rate, 
desire to drink, and self-reported 
withdrawal symptoms (Staiger and 
White 1991). Similarly, cue reactivity 
such as salivation and the urge to 
drink is significantly enhanced in alco- 
holics after exposure to the odor of 
their favorite alcoholic beverage, but 
not after exposure to water (Monti et 
al. 1993). The motivational signifi- 
cance of ethanol-associated environ- 
mental cues is also illustrated by the 
finding that abstinent alcoholic 
patients worked harder for alcohol 
and experienced greater subjective 
and physiological responses in a "bar- 
like" environment than in a neutral 
setting (Ludwig and Stark 1974). 

Significance of Stress in the 
Relapse Process 

Stress has an established role in drug 
abuse and dependence. Stress is a major 
determinant of relapse in humans and 
has been implicated in the resumption 
of drug abuse habits for ethanol as well 
as other drugs of abuse (Marlatt 1985; 
Wallace 1989; Brown et al. 1995; 
McKay et al. 1995). The significance 
of stress as a factor in AOD-seeking 
behavior has also been amply docu- 
mented in animal studies. In both 
rodents and nonhuman primates, 
physical, social, and emotional stress 
facilitates acquisition or increases self- 
administration of ethanol (Kraemer 



and McKinney 1985; Blanchard et al. 
1987; Nash and Maickel 1988; Schenk 
et al. 1990; Higley et al. 1991; 
Hilakivi- Clarke and Lister 1992; Mol- 
lenauer et al. 1993; Roske et al. 1994) 
and other drugs of abuse (e.g., Ram- 
sey and Van Ree 1993; Shaham 1993; 
Goeders and Guerin 1994; Shaham 
and Stewart 1994; Haney et al. 
1995). In addition, studies showing 
that exposure to stress can reinstate 
cocaine, heroin, and ethanol-seeking 
behavior in drug-free animals have 
provided direct experimental evidence 
that stress has a role in relapse (e.g., 
Shaham and Stewart 1995; Ahmed 
and Koob 1997). However, with the 
exception of a single recent study in 
which footshock stress reinstated 
extinguished ethanol-seeking behavior 
(Le et al. 1998), the effects of stress 
have not yet been systematically exam- 
ined in animal models of relapse. 

Animal Models of Relapse 

In spite of the significance of stress and 
environmental stimuli in relapse to 
alcohol abuse, studies examining these 
factors and their neurobiological basis 
in the relapse process have been 
scarce, and appropriate animal models 
suited for such investigations are still 
under development. 

Ethanol-Associated 
Environmental Stimuli 

One recent study examined the effects 
of olfactory discriminative stimuli pre- 
dictive of alcohol availability (ethanol 
odor) or nonavailability (water odor) 
on ethanol-seeking behavior (Katner et 
al. 1999). Rats were given the opportu- 
nity to self-administer 10 percent 



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ethanol or water and then subjected to 
extinction procedures where lever 
presses had no scheduled consequences. 
After extinction of ethanol-seeking 
behavior, the animals were again pre- 
sented with the respective discrimina- 
tive stimuli, but without the 
availability of ethanol or water. Pre- 
sentation of the ethanol-associated 
cue but not the water- associated cue 
elicited and maintained significant 
responding despite the continued 
unavailability of ethanol, and this effect 
was selectively attenuated by the opiate 
antagonist naltrexone (Katner et al. 
1999). Similar effects were obtained 
when rats were presented with a cue 
light that had been contiguously paired 
with lever responses for ethanol during 
self-adrninistration training (Heyser and 
Koob 1997). Moreover, the ethanol- 
associated cue light increased the resis- 
tance to extinction when the cue light 
was presented in the absence of the 
ethanol reinforcer (Heyser and Koob 
1997). These results suggest that 
ethanol-associated cues can both 
maintain ongoing ethanol-seeking 
behavior and reinstate extinguished 
ethanol-seeking behavior in rats. A 
further striking demonstration of the 
control of ethanol-seeking behavior 
by ethanol-related environmental cues 
is the finding that exposure to such 
cues during a period of extinction, but 
not abstinence alone, drastically 
reduces subsequent reacquisition of 
ethanol self- administration (Krank and 
Wall 1990). 

Alcohol Deprivation Effect 

The increase in voluntary ethanol 
consumption after forced deprivation 



(alcohol deprivation effect) discussed 
earlier in this chapter is receiving 
attention as a tool to study the neuro- 
biology of alcohol craving and relapse 
(Heyser et al. 1996; Holter et al. 1996; 
Spanagel et al. 1996; Heyser et al. 
1997; Holter et al. 1997). Similarities 
exist between the alcohol deprivation 
effect in animals and certain character- 
istics of human alcohol abuse, includ- 
ing enhanced alcohol consumption after 
abstinence in social drinkers (Burish et 
al. 1981); binge drinking, in which 
alcohol consumption is followed by a 
period of abstinence (Mendelson and 
Mello 1966; Woods and Winger 
1971); and aspects of the "loss of 
control" phenomenon surrounding 
the first drink after abstinence in alco- 
holics (Ludwig and Wikler 1974; 
Ludwig et al. 1974; O'Donnell 1984). 
In view of these similarities, this pro- 
cedure appears to have appropriate 
face validity as a model for certain 
aspects of the relapse process. More- 
over, findings that pharmacological 
agents that suppress ethanol intake 
and reduce the likelihood of relapse in 
humans effectively attenuate the alco- 
hol deprivation effect in animals (see 
the section Excitatory and Inhibitory 
Amino Acids later in this chapter) 
have added support for the predictive 
validity of this procedure as a model 
of relapse. 

The alcohol deprivation effect can 
be demonstrated under both limited- 
access and unlimited-access conditions 
and with both home -cage free drink- 
ing and operant self-administration 
(e.g., Sinclair and Senter 1967; Kor- 
net et al. 1990; Wolffgramm and 
Heyne 1995; Spanagel et al. 1996; 



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Neuroadaptive Changes in Neurotransmitter Systems 



Heyser et al. 1997; Holter et al. 1997). 
Given the stability and robustness of 
the alcohol deprivation effect under a 
variety of experimental conditions, 
this phenomenon may have great utility 
for the exploration of diverse variables 
in the relapse process with a variety of 
experimental approaches, including 
motivational measures such as pro- 
gressive ratio performance and analyses 
involving behavioral economics. 

Neurochemical Basis 
of Relapse 

Available information on the possible 
neuropharmacological basis of relapse 
processes is limited. In the case of psy- 
chostimulants and opiates, pharmaco- 
logical manipulations that activate 
meso-accumbens DA transmission, as 
well as footshock stress, have been 
shown to reinstate drug self- adminis- 
tration in animals trained and then 
extinguished on intravenous drug self- 
administration (deWit and Stewart 
1981; Stewart and deWit 1987; Shaham 
1993; Shaham and Stewart 1994, 
1995; Weissenborn et al. 1995; Self et 
al. 1996). This suggests that conditions 
that activate the mesolimbic DA system 
may exert a "priming effect" and thereby 
elicit drug-seeking behavior. 

Dopamine 

There is, indeed, evidence that the 
anticipation of ethanol availability 
stimulates DA release. Rats placed in 
an environment previously associated 
with ethanol self- administration consis- 
tently show a transient rise in extracel- 
lular DA levels in the nucleus accumbens 
(Weiss et al. 1993; Gonzales and Weiss 
1998; see also Vavrousek-Jakuba et al. 



1992). There is also evidence that 
self- administration of saccharin, which 
is not normally associated with signifi- 
cant dopaminergic activation, will 
produce stimulation of DA release 
when ethanol, rather than saccharin, is 
the expected reinforcer (Katner et al. 
1996). These effects presumably are a 
consequence of the presence of incen- 
tive motivational stimuli, which are 
predictive of impending availability of 
ethanol. One may speculate, therefore, 
that ethanol-related cues may have a 
role in relapse by exerting a priming 
action (Kaplan et al. 1983, 1984, 
1985; Laberg 1986; Laberg and 
Ellertsen 1987; Heather and Stallard 
1989), because, like ethanol, these 
stimuli increase the release of DA in 
the nucleus accumbens. Consistent 
with this possibility, the DA antago- 
nist haloperidol reversed craving and 
difficulty in resisting additional alcohol 
consumption induced by a "priming 
dose" of alcohol in detoxified alco- 
holic patients (Modell et al. 1993). 
However, "anticipatory" increases in 
DA release have also been observed 
with saccharin and food reinforcers 
(Weiss et al. 1993; Wilson et al. 
1995), and expectation of a nondrug 
reinforcer has been associated with a 
discrete increase in the firing rate of 
mesolimbic DA neurons (Schultz et 
al. 1997). Stimulation of DA neuronal 
activity and release appears to be asso- 
ciated, therefore, with the anticipation 
of reinforcing stimuli in general and is 
not restricted to drug reinforcers. 

Thus, a better understanding of the 
neurochemical basis of relapse elicited by 
ethanol-related cues is urgently needed. 
Moreover, the priming hypothesis 



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remains to be confirmed by demon- 
strations that pharmacological interfer- 
ence with the dopaminergic activation 
produced by ethanol cues reduces their 
efficacy to reinstate ethanol-seeking 
behavior. Finally, DA release associated 
with the expectation of ethanol and the 
effects of ethanol-related cues on the 
reinstatement of ethanol-seeking behav- 
ior have, to date, only been demon- 
strated in nondependent animals. It 
will be important to confirm and char- 
acterize these effects in animals with a 
history of alcohol dependence in 
appropriate "reinstatement" models. 

Endogenous Opioids 

A growing number of clinical studies 
suggest that naltrexone is an effective 
pharmacological adjunct for reducing 
ethanol craving and relapse in human 
alcoholics (O'Malley et al. 1992; 
Volpicelli et al. 1992, 1995#, 1995 b, 
1995 r, O'Brien et al. 1996; Oslin et al. 
1997), and recent preliminary work 
indicates that naltrexone can prevent the 
reinstatement of extinguished respond- 
ing by ethanol-associated environmen- 
tal cues in rats (Katner et al. 1999). 
The mechanisms underlying the atten- 
uation of volitional ethanol intake by 
opiate receptor antagonists are not 
well understood but may involve an 
interaction with mesolimbic DA neu- 
rotransmission in addition to DA- 
independent effects. Both the nucleus 
accumbens and VTA are rich in opi- 
oid peptides and receptors (Wamsley 
et al. 1980; Lewis et al. 1983; Dilts 
and Kalivas 1989, 1990). Afferent 
projections to these areas as well as 
local interneurons (Khachaturian et al. 
1993; de Waele et al. 1995) provide a 



potential anatomical substrate by which 
endogenous opioids may modulate 
the dopaminergic and, ultimately, the 
rewarding effects of ethanol. In effect, 
both naltrexone and the delta-selec- 
tive opiate antagonist naltrindole have 
been shown to blunt ethanol -induced 
increases in DA release from the 
nucleus accumbens after systemic or 
local administration of ethanol 
(Acquas et al. 1993; Benjamin et al. 
1993). Thus, it is possible that interfer- 
ence with ethanol-induced stimulation 
of DA release may be a mechanism by 
which opiate antagonists suppress 
ethanol-seeking behavior. This hypoth- 
esis has received strong support by the 
recent demonstration that decreases in 
ethanol-reinforced operant respond- 
ing produced by naltrexone are 
directly coupled to naltrexone- 
induced decreases in the efficacy of 
ethanol to increase DA release in the 
nucleus accumbens (Gonzales and 
Weiss 1998). 

Excitatory and Inhibitory 
Amino Acids 

A potential role for GABAergic and 
glutamatergic mechanisms in relapse has 
emerged on the basis of both clinical and 
basic research involving the drug acam- 
prosate (calcium N^cetylhomotaurine). 
Acamprosate has been used successfully 
for relapse prevention in detoxified 
alcoholics in Europe (Lhuintre et al. 
1985, 1990; Soyka and Sass 1994; 
Paille et al. 1995; Sass et al. 1996; 
Whitworth et al. 1996; Pelc et al. 
1997; for reviews, see Chick 1995; 
Wilde and Wagstaff 1997). In behav- 
ioral studies that have used the alcohol 
deprivation procedure as a model of 



286 



Neuroadaptive Changes in Neurotransmitter Systems 



relapse in rats, acamprosate effectively 
reversed the increase in ethanol intake 
associated with forced abstinence 
(Heyser et al. 1996; Spanagel et al. 
1996; Holter et al. 1997; Spanagel 
and Zieglgansberger 1997). 

Although the neurobiological mech- 
anisms by which acamprosate acts to 
exert its putative anticraving and 
antirelapse action are not well under- 
stood, there is some evidence that this 
agent reduces ethanol withdrawal- 
induced neuronal hyperexcitability by 
interacting with glutamatergic/NMDA 
(N-methyl-D-aspartate) and, perhaps, 
GABAergic transmission (Zeise et al. 
1993; Spanagel et al. 1996; Holter et 
al. 1997). Withdrawal-associated 
hyperexcitability is predominantiy medi- 
ated by changes in GABA systems, 
voltage-gated Ca 2+ channels, and glu- 
tamate/NMDA systems, and evidence 
from functional studies confirms that 
acamprosate interacts with these sys- 
tems (Littleton 1995; Spanagel and 
Zieglgansberger 1997). In particular, 
acamprosate may have a modulatory 
action on glutamatergic mechanisms 
by enhancing glutamate transmission 
under some conditions (Madamba et 
al. 1996) but inhibiting it under other 
conditions (Zeise et al. 1993). Acam- 
prosate also suppresses elevated c-fos 
expression in rats undergoing with- 
drawal within brain reward regions 
(Putzke et al. 1996), a finding that is 
consistent with the putative antire- 
lapse properties of this agent. 

Overall, there is growing, albeit still 
tentative, evidence that acamprosate 
can suppress neuronal excitability and 
associated motivational effects during 
ethanol withdrawal, and that these 



effects involve, in particular, interactions 
with excitatory (NMD A) amino acid 
transmission. A possible role for NMDA 
receptors in ethanol-seeking behavior 
and relapse has also recently been impli- 
cated by the finding that memantine 
(l-amino-3,5-dimethyl-adamantane), 
an uncompetitive NMDA antagonist, 
suppressed ethanol deprivation- 
induced increases in ethanol consump- 
tion (Holter et al. 1996). 

5 -Hydroxytryptamine 

Clinical studies implicate a possible 
involvement of serotonergic mechanisms 
in alcohol craving and relapse. Several 
pharmacological agents that interact 
with 5-HT receptors, including the 
nonselective partial 5-HT agonist m- 
chlorophenylpiperazine (mCPP) 
(Malec et al. 1996) and the partial 5- 
HT 1A agonist buspirone (Buydens- 
Branchey et al. 1997), reduce alcohol 
craving and rates of relapse in alco- 
holic patients. In the case of bus- 
pirone, this effect has been attributed 
to the established anxiolytic efficacy of 
this drug, which is consistent with the 
hypothesis that anxiety associated with 
ethanol withdrawal and protracted 
abstinence is a major motivational fac- 
tor in relapse. The anticraving effect 
of mCPP was observed with oral 
administration (Malec et al. 1996); 
this is an interesting finding because, 
when administered intravenously, par- 
tial 5-HT receptor agonists have been 
shown to produce an ethanol-like 
feeling of "high" and craving for alco- 
hol in detoxified alcoholics (Benkelfat 
et al. 1991; Lee and Meltzer 1991; 
Krystal et al. 1994). These observa- 
tions are consistent with data from 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



animal studies that have implicated 5- 
HT in die discriminative stimulus prop- 
erties of ethanol (Grant and Barrett 
.19916; Grant and Colombo 1993^, 
19936) and suggest that, depending 
on the rate of rise in their blood and 
brain concentrations, these agents 
may either have priming effects lead- 
ing to enhanced craving (e.g., Ludwig 
et al. 1974; Jaffe et al. 1989; Modell 
et al. 1993) or substitute for aspects 
of ethanoPs psychoactive effects and, 
thereby, reduce craving. 

Stress and Neuroendocrine 
Mechanisms 

As discussed earlier, the literature iden- 
tifies stress as a major factor in relapse. 
There are currently no effective animal 
models to study the role of stress in 
relapse to ethanol-seeking behavior, 
and understanding of the neurobiol- 
ogy of stress-induced relapse in the 
case of both alcohol and other drugs 
of abuse is limited. Stressful stimuli 
are known to activate the HPA axis, 
and stress-induced increases in psy- 
chostimulant (Haney et al. 1995) and 
ethanol (Nash and Maickel 1988) 
intake have been directly linked to HPA 
activation. Stress can also increase the 
release of DA in the nucleus accum- 
bens (Imperato et al. 1992; Shaham 
and Stewart 1995; Weiss et al. 1997), 
which may, in turn, perhaps serve as a 
discriminative or "priming stimulus" 
for the initiation of AOD-seeking 
behavior. Other studies suggest that 
certain forms of stress may exacerbate 
dopaminergic deficits associated with 
psychostimulant withdrawal (Rossetti 
et al. 1992&) and, thereby, perhaps 
contribute to the increased likelihood 



of relapse associated with stress (Weiss 
et al. 1997). 

In addition to HPA activation, dis- 
turbances in the amygdaloid CRF sys- 
tem by chronic drug use may have a 
role in stress-induced drug-seeking 
behavior. Amygdaloid CRF neurons 
project to brain regions involved in 
autonomic and neuroendocrine func- 
tions such as hypothalamic nuclei, but 
also innervate midbrain monoaminer- 
gic neurons that regulate behavioral 
and reward functions. It has, therefore, 
been suggested that amygdaloid CRF 
neurons are a component of an intrin- 
sic CRF brain circuitry that activates 
other, more classical, central neuro- 
transmitter systems which, in turn, 
initiate or control components of 
behavioral and autonomic responses 
to stress (Gray 1993). Thus, by acti- 
vating these autonomic and behavioral 
centers, CRF neurons in the amygdala 
may not only contribute to withdrawal 
distress and behavioral withdrawal 
responses but also play a role in the 
initiation of AOD-seeking behavior 
and relapse. 

GAPS IN SCIENTIFIC 
KNOWLEDGE AND 
FUTURE RESEARCH 
PRIORITIES 

Although substantial advances in the 
understanding of the neurobiology of 
alcohol addiction have been made, there 
are numerous areas in which current 
knowledge is limited. The recommen- 
dations below represent those research 
needs that are considered most critical 
for the advancement of present under- 
standing of neurobiological, genetic, 



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Neuroadaptive Changes in Neurotransmitter Systems 



and environmental factors in alco- 
holism, and for the development of 
more effective pharmacotherapeutic 
tools to treat this condition. 

Protracted Abstinence 
and Relapse 

Alcoholism is a chronic relapsing dis- 
order. Yet, the precise conditions that 
lead to relapse, whether environmentally 
determined or a result of persistent 
homeostatic-neuroadaptive disruptions, 
are poorly understood. A systematic 
research effort at the behavioral, neuro- 
chemical, cellular, and molecular level 
will be needed to identify neuroadaptive 
changes and homeostatic disturbances 
during protracted withdrawal, and to 
determine their motivational significance 
in appropriate models of ethanol-seeking 
behavior and relapse. At the same time, 
it will be important to better under- 
stand the role and neurobiological basis 
of conditioning factors, external stres- 
sors, and alterations in the reinforcing 
qualities of ethanol, as well as interac- 
tions between these variables and neu- 
roadaptive changes associated with a 
history of ethanol dependence. 

Mechanisms of Reinforcement 
in Dependent Preparations 

There is a need to better model various 
aspects of alcoholism in laboratory ani- 
mals. This includes, in particular, volun- 
tary drinking models that promote 
spontaneous and persistent intake of 
high ethanol concentrations or volumes 
without prior need to induce depen- 
dence. Such models will represent an 
important step toward the need for 
studying critical issues such as the mech- 
anisms underlying the switch from 



nondependence to dependence and 
mechanisms that maintain alcohol 
consumption in dependent individuals. 

Neurocircui tries 
and Transmitter Interactions 
Mediating Ethanol Reinforcement 
in Dependent Subjects 

There is a need to study neurotrans- 
mitter circuitries and interactions 
mediating ethanol reward in the 
dependent and postdependent state. 
Although there is increasing evidence 
that the acute reinforcing actions 
of ethanol depend on multiple neuro- 
chemical systems and their interac- 
tions, little, if anything, is known 
about these mechanisms in dependent 
subjects. In this context, it will also 
be beneficial to incorporate multiple 
systems approaches in medication 
development efforts and to examine 
the therapeutic efficacy of combina- 
tions of relevant pharmacological 
agents. 

The DA Hypothesis of Ethanol 
Reward: Revisited 

It will be important to clarify the role 
of DA in ethanol reinforcement. This 
need involves both a better under- 
standing of mechanistic questions 
(e.g., how ethanol activates mesolim- 
bic DA transmission) and a better 
understanding of the precise role of 
DA in various aspects of ethanol-seek- 
ing behavior, such as craving, relapse, 
and loss of control. 

Ethanol-Induced 
Sensitization 

An important emerging issue is the role 
of sensitization in ethanol reinforcement, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



genetic preference, and dependence. In 
particular, the following questions will 
require clarification: (1) Does ethanol 
sensitization augment the reinforcing 
efficacy or potency of ethanol? (2) 
Does ethanol sensitization promote a 
heightened motivational state with 
increased ethanol-seeking behavior 
("craving"), without necessarily alter- 
ing the reinforcing efficacy of ethanol? 
(3) Is ethanol sensitization a correlate 
of aversive or side effects of repeated 
ethanol intoxication, and, if so, is 
ethanol sensitization negatively linked 
with ethanol preference or vulnerabil- 
ity to abuse? 

Significance of Repeated 
Intoxication and Withdrawal 

The kindling phenomenon associated 
with repeated intoxication and with- 
drawal may have important implications 
with regard to changes in motiva- 
tional and reinforcement processes. It 
is important to better understand the 
mechanisms of kindling or sensitiza- 
tion of withdrawal severity at the 
molecular, cellular, and biochemical 
levels. This includes efforts to define 
sensitization of psychological compo- 
nents of withdrawal (e.g., anxiety, 
affective changes), to characterize 
possible changes in the subjective per- 
ception of ethanol's intoxicating 
actions (i.e., ethanol's discriminative 
stimulus effects), to determine 
whether multiple ethanol withdrawal 
experiences alter the reinforcing prop- 
erties of ethanol, and to examine 
potential changes in susceptibility to 
ethanol neurotoxicity and associated 
cognitive impairments. Finally, it is 
important to examine whether condi- 



tioning factors contribute to the kin- 
dling phenomenon. 

Development of New 
Pharmacogenetic Models 

Motivational measures of ethanol rein- 
forcement have revealed that there can 
be overlap between aspects of ethanol- 
seeking behavior in alcohol-preferring 
animals of certain lines with the behavior 
of nonpreferring animals of other lines. 
This situation makes it difficult to con- 
duct comparative neurobiologies and 
behavioral investigations among the 
present rat models. Thus, some consen- 
sus will be required of what constitutes 
a valid animal model of ethanol drink- 
ing. Free-choice drinking preference is 
one criterion, but it may be insufficient 
by itself for defining a good animal 
model. Human alcoholics will expend 
considerable time and effort to secure 
an adequate supply of their preferred 
beverage, so motivational characteris- 
tics such as persistence of ethanol-seek- 
ing behavior and "willingness" to 
expend effort in obtaining ethanol 
would seem to be essential criteria of 
animal models of alcoholism. Such 
models, which would incorporate ani- 
mals that show robust ethanol-seeking 
behavior when subjected to motiva- 
tional tests (e.g., second-order and 
progressive-ratio schedules), are likely 
to provide an invaluable means for the 
study of genetic and neurobiologies 
factors underlying compulsive ethanol- 
seeking behavior and dependence. 

ACKNOWLEDGMENTS 

The author gratefully acknowledges 
financial support by the National 



290 



Neuroadaptive Changes in Neurotransmitter Systems 



Institute on Alcohol Abuse and 
Alcoholism Extramural Advisory 
Board, and thanks Mike Arends for 
excellent assistance in the preparation 
of the manuscript. 

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Wolffgramm, J., and Heyne, A. From 
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Woods, J.H., and Winger, G.D. A critique 
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Wozniak, K.M.; Pert, A.; Mele, A.; and 
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Yamamura, T.; Hishida, S.; Hatake, K.; 
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Yoshimoto, K.; McBride, W.J.; Lumeng, 
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313 



Chapter 8 

Adolescent Period: Biological Basis of 

Vulnerability To Develop Alcoholism 

and Other Ethanol-Mediated Behaviors 

Linda Patia Spear, Ph.D. 



KEY WORDS: AOD (alcohol or other drug) dependence; adolescence; biological 
AOD use disorder theory; psychological AODC (causes of AOD use, abuse, and 
dependence); AOD use susceptibility; AOD use behavior; growth and develop- 
ment; brain function; physiological stress; dopamine; AOD sensitivity; AOD tol- 
erance; hormones; AOD use initiation; prevalence; predictive factor; animal 
study; etiology; literature review 



THE IMPORTANCE OF 
ADOLESCENCE IN 
ALCOHOL STUDIES 

Adolescents across a variety of species 
are faced with the similar developmental 
challenge of acquiring the necessary skills 
to permit survival away from parental 
caretakers. At this critical juncture 
between childhood and adulthood, 
adolescents undergo marked hormonal 
and neural alterations. Among the brain 
regions showing prominent alterations 
during adolescence are the prefrontal 
cortex (PFC) and other forebrain dopa- 
mine (DA) projection regions, areas 
implicated in mediating the reinforcing 
effects of alcohol and other drugs of 



abuse (see Koob 1992; Goeders 1997 
for review). 

It is in this unique neurobehavioral 
state of adolescence that most humans 
begin alcohol use, yet little is known 
about alcohol in adolescence. This age 
is critical for study for three reasons. 
First, brain function in regions modu- 
lating drug reinforcement is altered 
during adolescence, and it cannot be 
assumed that factors precipitating 
alcohol use or abuse would be the 
same in adolescence as in adulthood. 
Second, rapidly changing systems are 
particularly vulnerable to disruption, so 
there may be long-term consequences 
of alcohol exposure during this time of 
rapid neural and endocrine maturation. 



L.P. Spear, Ph.D., is a Distinguished Professor in the Department of Psychology and director of the 
Center for Developmental Psychobiology at Binghamton University, Binghamton, NY 13902-6000. 



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Third, early onset of alcohol use is 
currently one of the most powerful 
predictors of later alcohol abuse. 
Before considering these issues in 
more detail, a few pertinent age -spe- 
cific characteristics of adolescence will 
be briefly summarized. 

CHARACTERISTICS OF 
ADOLESCENCE IN 
HUMANS AND OTHER 
ANIMALS 

The process of adolescence is not syn- 
onymous with puberty. Whereas ado- 
lescence subsumes the entire process of 
transition from childhood to adulthood, 
puberty is a more temporally restricted 
phase during which the physiological 
and neuroendocrine alterations associ- 
ated with sexual maturation occur. 
Puberty is but one of the numerous 
ontogenetic alterations occurring during 
adolescence, with the timing of this 
maturational event within the broader 
framework of adolescence varying 
noticeably among human adolescents 
(see, e.g., Dubas 1991). In humans, 
adolescence spans the age range from as 
early as 9 years to approximately 18 
years (see, e.g., Buchanan et al. 1992). 
In rats, commonly cited times for ado- 
lescence onset are postnatal days 28-32 
(P28-32), with offsets between P38 
and P55 (see, e.g., Ojeda and Urban- 
ski 1994), although this timing is 
somewhat disputed (Odell 1990) and 
may depend on the growth rate of the 
animals (Kennedy and Mitra 1963) and 
the maturational index used. Spear and 
Brake (1983) operationally defined 
"periadolescence" as the age period 
around the time of sexual maturation 



when age-specific behavioral and psycho- 
pharmacological discontinuities are evi- 
dent; using this criteria the age period of 
approximately P30-42 was conserva- 
tively designated as periadolescence, 
with animals of this age showing 
numerous neurobehavioral alterations 
from significantly younger (pre- or 
postweanlings) as well as more mature 
(P60 and older) animals. Adolescence 
in monkeys typically occurs in the age 
range of 2-\ years (Lewis 1997). 

Hormonal Concomitants of 
Adolescence 

Puberty represents a reactivation, after 
a prolonged period of suppression during 
the childhood/juvenile period, of pul- 
satile release of gonadotropin-releasing 
hormone (GnRH) that was evident 
perinatally. This reinstatement of pulsatile 
GnRH release induces pulsed release of 
follicle-stimulating hormone and luteiniz- 
ing hormone, which in turn stimulate 
release of gonadal hormones (e.g., testos- 
terone in males and estrogen in females) 
(see, e.g., Brooks-Gunn and Reiter 
1990). Pulsatile release of growth hor- 
mone also increases more than tenfold 
during the growth spurt of adolescence 
(Gabriel et al. 1992). It remains to be 
determined which neural and behavioral 
features of adolescence are driven by 
maturational changes in gonadal hor- 
mones, and which instead may emerge 
independently from (or in the case of 
neural alterations, might even contribute 
to) processes of sexual maturation per se. 

Behavioral Characteristics 
of Adolescence 

Periadolescents differ behaviorally from 
younger and older individuals on a 



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Adolescence: Biological Basis of Vulnerability to Alcohol Abuse 



number of dimensions consistent with 
a developmental trajectory toward the 
goal of independence. Rats in the age 
range from approximately P30 to P42 
are often hyperactive and explore more 
relative to rats of other ages (see, e.g., 
Spear et al. 1980). They also spend more 
time in social interactions with conspecifics 
(Primus and Kellogg 1989) and exhibit 
peak levels of play behavior (see, e.g., 
Fassino and Campbell 1981). Sex dif- 
ferences in behavior also begin to 
emerge in adolescence, with some of 
these differences being driven in part by 
organizational influences of pubertal 
hormones (see, e.g., Beatty and Fessler 
1977; Brand and Slob 1988). Human 
adolescents likewise exhibit increases in 
social behavior, as well as a dispropor- 
tionate amount of reckless behavior, 
sensation seeking, and risk taking rela- 
tive to individuals at other ages (Arnett 
1992). Such age-related modifications 
in behavior are consistent with the need 
of the adolescent to explore novel 
domains and establish new social rela- 
tionships during the process of achieving 
independence from their parents. 

In addition to the continuing devel- 
opment of cognitive function during 
adolescence (see, e.g., Levin et al. 1991), 
age-specific discontinuities are seen in 
some learning tasks. Adolescent rats 
sometimes exhibit enhanced perfor- 
mance on tasks in which increases in 
activity/exploration could facilitate 
performance (e.g., radial arm maze 
[Chambers et al. 1996] and active 
avoidance [Bauer 1980]). However, 
relative to younger or older rats ado- 
lescents tend toward impaired perfor- 
mance on more complex avoidance 
tasks (such as discriminated escape 



and Sidman avoidance tasks), perhaps 
as a function of increased distractibility 
and difficulties in focusing attention 
on salient cues and reward contingen- 
cies (see Spear and Brake 1983 for 
review and references). 

Adolescents also exhibit characteris- 
tic alterations in psychopharmacological 
sensitivity. For instance, adolescent rats 
are less sensitive than their younger or 
older counterparts to the stimulatory 
effects of catecholaminergic agonists 
such as amphetamine and cocaine, but 
conversely are more sensitive to the 
DA antagonist haloperidol, a psy- 
chopharmacological pattern sugges- 
tive of a temporary hyposensitivity of 
one or more DA systems during ado- 
lescence (see Spear and Brake 1983 
for references and discussion). Adult- 
typical suppressant effects of low doses 
of D 2 /D 3 DA agonists also emerge 
during early adolescence (see, e.g., 
Shalaby and Spear 1980; Arnt 1983; 
Van Hartesveldt et al. 1994), an effect 
formerly thought to be associated 
with development of DA autorecep- 
tors (Shalaby et al. 1981; Hedner and 
Lundborg 1985) but that instead may 
be mediated by maturation of a sub- 
population of postsynaptic DA recep- 
tors (see Andersen et al 1997a for 
evidence refuting an autoreceptor 
explanation; see also Stahle 1992). 

Neural Alterations During 
Adolescence 

The adolescent brain is unique and in 
a state of transition as it undergoes 
both progressive and regressive 
changes. One brain region prominently 
altered during adolescence across a 
variety of species is the PFC, an area 



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thought to subserve higher cognitive 
abilities such as the bridging of tem- 
poral delays in memory (see, e.g., Dia- 
mond 1991). For instance, absolute 
PFC volume declines in adolescence 
in humans (Jernigan et al. 1991) and 
in rats (van Eden et al. 1990). Sub- 
stantial synapse elimination occurs 
during adolescence in the PFC and 
other cortical regions in humans 
(Huttenlocher 1984) and nonhuman 
primates (Zecevic et al. 1989). At 
least a portion of this synapse elimina- 
tion in the PFC appears to be associ- 
ated with the marked developmental 
loss of presumed glutaminergic excita- 
tory input (Zecevic et al. 1989). In 
contrast, DA input to the PFC in 
nonhuman primates increases during 
adolescence to peak at levels well 
above those seen earlier or later in life 
(Rosenberg and Lewis 1994; see Lewis 
1997 for a review); increases in PFC 
DA input through adolescence are also 
evident in rats (Kalsbeek et al. 1988). 
Cholinergic innervation of the PFC 
likewise increases in adolescence to 
reach mature levels in rats (Gould et al. 
1991) and humans (Kostovic 1990). 

Maturational changes during ado- 
lescence are also evident in other brain 
regions such as the hippocampus of 
rodents (Wolfer and Lipp 1995; Dumas 
and Foster 1998) and humans (Benes 
1989). Alterations evident in the hypo- 
thalamus include qualitative differences 
in norepinephrine release in adolescents 
relative to younger or older rats, along 
with pharmacological alterations consis- 
tent with the suggested emergence in ado- 
lescence of inhibitory a 2 norepinephrine 
autoreceptors (Choi and Kellogg 1992; 
Choietal. 1997). 



Dopaminergic systems undergo 
substantial reorganization during ado- 
lescence. Over one-third to one-half of 
the D l and D 2 receptors present in 
the striatum of juveniles are lost by 
adulthood in both humans (Seeman 
et al. 1987) and rats (Gelbard et al. 
1989; Teicher et al. 1995). This peak 
in D x and D 2 binding during adoles- 
cence and subsequent decline is much 
more pronounced in striatum than in 
nucleus accumbens (Teicher et al. 
1995), and in male than in female rats 
(Andersen et al. 1997b). Not all DA 
receptors show this overproduction 
and pruning, with juveniles having 
only 40 percent of adult-typical D 3 
receptor levels in striatal and accum- 
bens regions (Stanwood et al. 1997). 
The DA transporter likewise under- 
goes a protracted period of develop- 
ment in mesolimbic and mesocortical 
brain regions, with only about 70 per- 
cent of adult uptake levels being seen 
prior to adolescence onset in rats 
(Coulter et al. 1996). 

Developmental events during adoles- 
cence may alter the relative balance of 
DA activity between the PFC and stri- 
atal or mesolimbic terminal regions. 
Basal DA synthesis increases during 
adolescence in the nucleus accumbens 
and the striatum of rats, while the rate 
of DA synthesis peaks in the PFC at 
P30 before declining to much lower 
levels by late adolescence (Andersen et 
al 1997 a). Similar data are obtained 
from estimates of DA turnover, with 
basal turnover increasing during ado- 
lescence in the nucleus accumbens and 
the striatum and decreasing in the 
PFC (Teicher et al. 1993). Interestingly, 
although the PFC is seemingly devoid 



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Adolescence: Biological Basis of Vulnerability to Alcohol Abuse 



of synthesis-modulating autoreceptors 
in adulthood (see, e.g., Galloway et 
al. 1986), convincing evidence has 
been obtained for a transient expres- 
sion of DA autoreceptor-like modula- 
tion of DA synthesis in the PFC early 
in life that disappears during adoles- 
cence (Teicher et al. 1991; Andersen 
etal. 1997 a). 

The brain of the adolescent is clearly 
in transition. Neural regions showing 
prominent alterations during adoles- 
cence include the PFC as well as other 
forebrain DA projection regions. Given 
the important role of these brain areas 
in modulating reward efficacy of rein- 
forcing drugs (Koob 1992), sensitiv- 
ity to the environment and stressors 
(see, e.g., Dunn and Kramarcy 1984), 
and the association between the two (see, 
e.g., Piazza et al. 1991; Goeders 1997), 
it is not surprising that adolescents vary 
notably from more mature animals in 
their responsivity to ethanol, stressors, 
and their interaction. 

ONTOGENY OF 
RESPONSIVITY TO 
ETHANOL 

Prevalence of Alcohol Use 
in Adolescents 

In a 1996 National Institute on Drug 
Abuse survey, 26 percent of 8th 
graders, 40 percent of 10th graders, 
and 51 percent of 12th graders 
reported that they had used alcohol in 
the past month. Ten percent of 8th 
graders, 21 percent of 10th graders, 
and 31 percent of 12th graders 
(Mathias 1997) also reported getting 
drunk on one or more occasions during 



the past month. Clearly, many adoles- 
cents use alcohol, with evidence of exces- 
sive use emerging in some individuals. 

Adolescent rats display two to three 
times higher levels of ethanol intake 
relative to their body weights than do 
more mature animals (Lancaster et al. 
1996; Bannoura et al. unpublished 
manuscript), although ethanol prefer- 
ence per se does not peak until well into 
adulthood (around 5 months of age 
[Parisella and Pritham 1964; Goodrick 
1967]). The notably different ontoge- 
netic conclusions reached when using 
grams per kilogram intake versus per- 
cent total fluid to index ethanol con- 
sumption seemingly reflect ontogenetic 
differences in total fluid consumption, 
with adolescent rats exhibiting greater 
overall fluid (and food) consumption 
than adults. Indeed, during the ado- 
lescent growth spurt, adolescent rats 
consume the greatest caloric intake 
relative to their body weight of any 
time in the lifespan (see, e.g., Nance 
1983). Adolescent humans also 
exhibit elevated metabolic activity and 
developmental hyperphagia (see, e.g., 
Post and Kemper 1993; Ganji and 
Betts 1995), with heavy alcohol use 
often being "adolescence-limited" 
(see, e.g., Bates and Labouvie 1997). 

The elevated consummatory behav- 
iors of adolescence could contribute to 
high levels of ethanol intake by these 
growing individuals relative to their 
body weight. As discussed below, 
adolescents might be able to sustain 
comparatively large ethanol intakes 
due to their relative insensitivity to the 
sedative and locomotor incoordinating 
effects of ethanol, which may be in part 
related to their greater propensity to 



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develop acute and functional tolerance 
relative to more mature organisms. 

Acute Responsivity 
to Ethanol 

Studies using a variety of measures in 
laboratory animals have observed increases 
in ethanol sensitivity from infancy, with 
further increases in sensitivity during the 
aging process (see, e.g., York and Chan 
1993). This early attenuation in ethanol 
sensitivity is evident despite slower rates 
of ethanol metabolism in younger ani- 
mals (see, e.g., Zorzano and Herrera 
1989; Silveri and Spear 2000) and is 
evident using measures such as LD 50 
(Hollstedt and Rydberg 1985), tilting- 
plane performance (Hollstedt et al. 1980), 
ethanol-induced hypnosis (Ernst et al. 
1976; Little et al. 1996; Silveri and 
Spear 1998), and hypothermia (Spiers 
and Fusco 1991; Silveri and Spear 
2000). However, this finding of early 
attenuation in ethanol sensitivity is not 
universal — see, for example, Keir and 
Deitrich(1990). 

Tolerance Development 

The resistance of young organisms to 
ethanol may be in part attributable to the 
development of pronounced acute toler- 
ance early in life, with the propensity for 
acute tolerance to ethanol gradually 
declining to reach adult levels only fol- 
lowing puberty (Silveri and Spear 1998). 
This ontogenetic decline may be specific 
to within- session tolerance, with other 
forms of tolerance such as rapid toler- 
ance showing ontogenetic increases 
(Silveri and Spear 1999). 

Grieve and Littleton (1979) reported 
that preweanling mice showed no 
evidence of functional tolerance to 



ethanol-induced sleep, whereas adoles- 
cents showed more pronounced tolerance 
development than adults. Adolescents 
also have been reported to exhibit more 
chronic tolerance to ethanol-induced 
hypothermia than adult rats (Swartz- 
welder et al. 1998). This greater propen- 
sity for adolescents to develop acute and 
chronic tolerance may contribute to their 
relative resistance to the motor-impair- 
ing and sedative effects of ethanol relative 
to their more mature counterparts. 

Adolescent Vulnerabilities 
to Ethanol Disruption 

In some respects, young rats may be un- 
usually sensitive to ethanol. Swartzwelder 
and his group found that hippocampal 
slices from preadolescent (PI 5-25) rats 
were more sensitive than adult slices to 
ethanol disruption of both NMDA- 
mediated excitation and stimulus-induced 
long-term potentiation (Swartzwelder 
et al. 1995^, 1995£). Behaviorally, P30 
adolescents were found to be more 
impaired than adult rats by ethanol in a 
spatial memory task in the Morris maze, 
while nonspatial performance was unaf- 
fected by ethanol at either age (Markwiese 
et al. 1998). Although reduced sensi- 
tivity to motor-impairing and sedative 
consequences of ethanol may permit 
adolescents to consume greater 
amounts of ethanol, this exposure might 
have more adverse effects on hip- 
pocampally-related memory processing 
than later in life. 

STRESS, ADOLESCENCE, 
AND ALCOHOL ABUSE 

Navigating the developmental transition 
toward independence may be stressful 



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Adolescence: Biological Basis of Vulnerability to Alcohol Abuse 



for adolescents. In humans, for 
instance, levels of anxiety have been 
reported to peak at around 13-15 
years of age (see Buchanan et al. 1992 
for discussion and references). This 
presumed increase in stress during 
adolescence has been postulated to 
contribute to the frequent initiation of 
alcohol and other drug use in adoles- 
cence (see, e.g., Pohorecky 1991; 
Wagner 1993), as well as to the fre- 
quent emergence in adolescence of 
schizophrenic symptomology in vul- 
nerable individuals (Walker and 
Diforio 1997). In addition to the 
actual frequency of life stressors possi- 
bly being greater in adolescence than 
at other ages, adolescents may also 
respond differently to stress than indi- 
viduals at other ages. 

Hormonal Response to 
Stressors in Adolescents 

Exposure to a stressor activates the 
hypothalamic-pituitary- adrenal (HPA) 
axis, resulting in a cascading sequence 
of hormone release from the hypo- 
thalamus (corticotropin-releasing fac- 
tor), pituitary (adrenocorticotropic 
hormone [ACTH]), and adrenals 
(corticosterone in rats; Cortisol in 
humans). Ontogenetic increases in 
stress-induced activation of the HPA 
system have been explored systemati- 
cally in animal studies. Peak ACTH 
and corticosterone responses to stress 
generally increase during ontogeny to 
reach an asymptote in rats around 
adolescence, at least in males (Ramaley 
and Olson 1974; Meaney et al. 1985&; 
Walker et al. 1986; Bailey and Kitchen 
1987; Rivier 1989). Gender differences 
in the corticosterone response to stress 



begin to emerge late in adolescence, 
with elevated levels in female rats 
compared with males and with prepu- 
bescent females (Ramaley 1972; Cir- 
ulli et al. 1996). 

Adolescent rats sometimes exhibit 
more prolonged stress-induced increases 
in corticosterone than adults (Gold- 
man et al. 1973; Sapolsky et al. 1985; 
Choi and Kellogg 1996). This delayed 
poststress recovery presumably reflects 
immature feedback regulation mediated 
in part by glucocorticoid receptors in 
hippocampus (see, e.g., Meaney et al. 
1985«, 1985&). Thus, adolescence 
may be associated with a greater over- 
all corticoid response to stress, with 
this stress-induced increase being ele- 
vated relative to younger animals and 
prolonged relative to adults. 

Behavioral and 
Physiological Stress 
Responses of Adolescents 

Although limited in number, studies 
in laboratory animals have shown that 
adolescents are sometimes more dis- 
rupted behaviorally by stressors than 
are adults. Compared with adults, 
adolescent rats show more stress- 
induced immobility during forced swim 
testing (Walker et al. 1995) or in the 
presence of intermittent footshock 
(Campbell et al. unpublished manu- 
script). Tail-pinch-induced feeding 
also has been reported to peak in 
"juvenile" rats (Heinrichs et al. 
1992), although the precise ontogeny 
of this response has apparently not 
been well characterized. Digging in 
novel or other mildly stressful situations 
is another response that appears to peak 
in adolescence in gerbils (Wiedenmayer 



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1997) and rats (S. Barron, personal 
communication, June 1998). Stone 
and Quartermain (1998) reported 
that chronic social stress (placement in 
the cage of an isolated adult male for 
5 minutes daily for 5 days) had 
a greater impact on adolescent 
(P28-32) than adult male mice, sup- 
pressing food intake, body weight 
gain, and time spent on open arms of 
a plus maze in adolescents but not 
adults. Chronic restraint stress was 
also found to suppress body weight 
gain in adolescents but not adults. 

Adult-typical environmental inhibi- 
tion of social behavior emerges in 
adolescence, with an unfamiliar envi- 
ronment decreasing social interactions 
in adult (P60) and midadolescent 
(P35), but not early adolescent (P28), 
male rats (Primus and Kellogg 1989). 
Rats at P28 were similarly reported to 
be insensitive to effects of the environ- 
ment on the benzodiazepine/gamma- 
aminobutyric acid receptor complex 
(Primus and Kellogg 1991) and to show 
less stress-induced Fos immunoreac- 
tivity than adult rats in brain regions 
such as the anterior olfactory nucleus, 
anterior cingulate cortex, and medial 
and cortical amygdaloid nuclei (Kellogg 
etal. 1998). 

Choi and Kellogg (1996) observed 
a blunted hypothalamic norepineph- 
rine response to stress in late adolescent 
rats (P42), a transition between the 
increased stress-related norepinephrine 
utilization seen in early adolescence 
(P28) and the decreased utilization 
seen in adulthood. A similar adolescent 
transitional period was seen in terms 
of autonomic reactivity to stressor 
stimuli; whereas preweanling rat pups 



exhibit heart rate bradycardia to an 
aversive stimulus, heart rate tachycardia 
emerges by adolescence, with this 
increased heart rate mediated by para- 
sympathetic withdrawal in adolescents 
but primarily by sympathetic activation 
in adults (Kurtz and Campbell 1994). 
Taken together, these data suggest 
that adolescents may differ hormonally, 
behaviorally, and neurally in the way 
they respond to stressors when com- 
pared with animals of other ages. 

Stress and Alcohol 
Consumption in Adolescents 

Corticosterone levels in rats generally 
have been positively related to rates of 
self- administration of ethanol or other 
drugs, with adrenalectomy suppress- 
ing ethanol consumption (Fahlke et 
al. 1994) and stress-induced eleva- 
tions in corticosterone increasing 
ethanol consumption (see, e.g., Bowers 
et al. 1997), although the interaction 
of stress and ethanol intake is complex 
(see Pohorecky 1990 for a review). 
Stressors may also enhance the rate of 
development of tolerance to ethanol 
(Maier and Pohorecky 1986), which 
could indirectly contribute to the 
capacity for increased consumption. 
The overall greater corticoid response 
to stress that adolescents seem to 
exhibit relative to individuals at other 
ages may increase their propensity for 
self- administration of ethanol. 

Indeed, perceived levels of stress may 
be one of a number of factors exacer- 
bating the already elevated propensity 
of human adolescents to exhibit alco- 
hol use and other drug-taking behav- 
ior (Wills 1986; Baer et al. 1987; 
Deykin et al. 1987; Tschann et al. 



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Adolescence: Biological Basis of Vulnerability to Alcohol Abuse 



1994; but see also Hansell and White 
1991). In her review of the literature 
on stress effects on alcohol consumption 
in humans, Pohorecky (1991) con- 
cluded that stress is most convincingly 
associated with alcohol consumption 
in adolescence, with more mixed findings 
evident in studies conducted in adults. 
Indeed, after peer substance use, the next 
most powerful predictor of adolescent 
alcohol and drug use was found by 
Wagner (1993) to be levels of perceived 
stress, with the appraisal of events as 
being stressful of more importance than 
the absolute number of such events. 

Much remains to be explored about 
the relationship between adolescent stress 
and alcohol use. Such investigations may 
anticipate intriguing complexities. For 
instance, lower cardiovascular stress 
responses were correlated with high-risk 
behavior in adolescent boys (Liang et al. 
1995), and low heart rate reactivity to 
alcohol was associated with familial risk 
for developing alcoholism (Peterson et 
al. 1993). King and colleagues (1990) 
reported that abstinent adult drug abusers 
exhibited lower basal levels of Cortisol than 
control subjects, with impulsivity being 
inversely correlated with basal Cortisol lev- 
els in the control group. It is unclear how 
these findings will ultimately relate to 
evidence discussed previously that 
enhanced physiological responses to stress 
may exacerbate drug-seeking behavior. 

THE EARLY EXPOSURE 
EFFECT: EARLY ALCOHOL 
USE AS A PREDICTOR 
OF LATER ABUSE 

Early onset of alcohol use has been shown 
in both prospective and retrospective 



studies to be a powerful predictor of 
later alcohol abuse and dependence 
(Rachal et al. 1982; Friedman and 
Humphrey 1985; Deykin et al. 1987; 
Fergusson et al. 1994; Grant and Dawson 
1997; Hawkins et al. 1997). In a study 
of 27,616 current and former drinkers 
interviewed for the 1992 National 
Longitudinal Alcohol Epidemiologic 
Survey, the rate of lifetime alcohol 
dependence was found to be 40 percent 
when individuals started drinking at or 
before 14 years of age, but only 10 
percent when drinking was not initiated 
until 20 years or later (Grant and Dawson 
1997). Overall, with each year of delay in 
onset of alcohol use, the odds of depen- 
dence decreased by 14 percent and the 
odds of abuse decreased by 8 percent. 
Effects of early ethanol experience were 
evident with exposures occurring as 
early as 6 years of age (Fergusson et 
al. 1994). 

This early exposure effect has been 
suggested to be one of the strongest 
predictors of subsequent alcohol abuse 
(Robins and Przybeck 1985; Barnes 
and Welte 1986; Hawkins et al. 1997) 
and is seen in relation to other drugs as 
well. Early alcohol exposure is correlated 
with increased later use and abuse of 
other drugs (Yamaguchi and Kandel 
1984; Robins and Przybeck 1985; 
Deykin et al. 1987; Robins and McEvoy 
1990), and early exposure to illicit 
drugs is associated with increased later 
abuse of alcohol (Robins and Przybeck 
1985) as well as other drugs of abuse 
(Yamaguchi and Kandel 1984; Robins 
and Przybeck 1985; Kandel and 
Davies 1992). 

There are at least two possible expla- 
nations of this powerful effect. First, 



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NIAAA's Neuroscience and Behavioral Research Portfolio 



traits associated with early alcohol use 
might independently predict later 
problem use, regardless of prior drug 
history; according to this view, early 
alcohol use serves as a marker, not a 
precursor, of a later abuse disorder. For 
instance, high novelty seeking in pre- 
teens was predictive of alcohol abuse 
at 27 years of age (Cloninger et al. 
1988); high novelty seeking is one of a 
number of traits that seem to facilitate 
initiation of alcohol and other drug 
use (Baumrind 1987). Second, early 
exposure to alcohol may alter develop- 
mental processes during adolescence, 
with long-term effects on neurobehav- 
ioral function that increase the propen- 
sity for later abuse. Indirect support 
for this possibility was obtained via 
path analysis of data from 10- to 11- 
year-old children collected prospec- 
tively for 7 years; in this study, effects 
of all significant risk factors for alcohol 
misuse were found to be mediated 
through age of alcohol initiation, other 
than a modest independent influence 
of gender (Hawkins et al. 1997). 

Animal models could help determine 
whether there is a causal relationship 
between early exposure and later alcohol- 
related problems, and could help in 
exploring the mechanisms underlying 
this association. Although chronic 
exposure of adolescent rats to ethanol 
has been reported to induce long-last- 
ing alterations in cognitive function- 
ing (Osborne and Butler 1983) and 
to disrupt puberty-associated increases 
in reproductive endocrinology in both 
males (Cicero et al. 1990) and females 
(Dees et al. 1990), no functional animal 
models of the early exposure effect per 
se have yet been developed. Although 



there are reports that pre- (Hayashi 
and Tadokoro 1985) or postweaning 
(Ho et al. 1989) exposure to ethanol 
can increase later ethanol preference, 
several groups have reported no increase 
in later consumption following periods 
of ethanol exposure that include ado- 
lescence (Kakihana and McClearn 1963; 
Parisella and Pritham 1964; Tolliver 
and Samson 1991). In the develop- 
ment of animal models of the early 
exposure effect, it may prove useful to 
consider the intriguing suggestion of 
Tolliver and Samson (1991) that 
stress may serve to unmask effects of 
early exposure on later intake. 

SUMMARY AND 
RECOMMENDATIONS 

Study of the relationship between alco- 
hol use and adolescence is still in its 
infancy, despite the frequent initiation 
of alcohol use by adolescents and the 
implications that this early use has for 
later problem use. A number of impor- 
tant goals for future research in this 
critical but underinvestigated area are 
suggested in the following paragraphs. 
Although studies in laboratory 
animals have shown that adolescents 
are relatively resistant to the motor- 
impairing and sedative effects of alco- 
hol, they conversely appear to be 
more sensitive to ethanol-induced dis- 
ruptions in hippocampally-related spa- 
tial memory. Further research is needed 
to specify the circumstances under 
which ethanol responsivity is exacer- 
bated or attenuated in adolescents, and 
to determine the neural mechanisms 
underlying these differential effects. This 
work should consider the ontogeny of 



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Adolescence: Biological Basis of Vulnerability to Alcohol Abuse 



ethanol tolerance and sensitization, 
given that reported differences in the 
ontogeny of within- and between- 
session tolerances could contribute to 
the insensitivity to ethanol often 
observed in adolescents, and could 
potentially contribute to greater levels 
of use later in life. 

We also need to know more about 
adolescence and the ontogeny of stress 
responsivity, particularly given clinical 
evidence that stress is more strongly 
associated with alcohol consumption 
in adolescents than in adults. In addi- 
tion to elucidating the relationship 
among hormonal, behavioral, and 
neural responses to stressors during 
ontogeny, stress effects on alcohol 
self-administration should also be 
considered within a developmental 
timeframe. Conclusions about the 
relationship between stress and adults' 
self-administration of alcohol and 
other drugs may bear little resem- 
blance to the relationship between 
these variables in adolescence, the age 
period when most alcohol and drug 
use is initiated. 

Additional research is needed to 
examine normal brain function in 
adolescence. The limited work to 
date has revealed various neural alter- 
ations during adolescence, but these 
rather piecemeal observations remain 
to be confirmed and more completely 
characterized and integrated. What 
are the functional implications of 
potential alterations in the balance 
among different forebrain DA regions 
during adolescence? How widespread 
are adolescent-associated changes 
among other neural systems and 
brain regions? 



Despite evidence for alterations 
during adolescence in forebrain 
regions (such as the PFC and meso- 
corticolimbic DA systems) modulat- 
ing the reward efficacy of reinforcing 
drugs, little is yet known regarding 
the reinforcing efficacy of alcohol and 
other reinforcers during adolescence. 
Such knowledge is obviously critical. 

Work is also needed to determine 
the factors that trigger these critical 
developmental changes in brain func- 
tion as well as in ethanol sensitivity 
and responsivity to stressors during 
adolescence. Do puberty-associated 
increases in gonadal hormones or 
adrenal hormones play significant acti- 
vational roles? 

It is critical that future efforts also be 
directed to the question of why early 
exposure to alcohol is seemingly so much 
more dangerous than later use, and to 
determine whether early exposure indeed 
increases the propensity for later alcohol 
problems. This issue is highly relevant 
for prevention efforts, with numerous 
clinicians suggesting that prevention 
be directed toward "just say later" efforts 
of postponing first use (see, e.g., Robins 
and McEvoy 1990). 

Multiple research approaches will 
be needed to explore the neurobehav- 
ioral and environmental antecedents 
influencing alcohol sensitivity and 
alcohol use during adolescence, as well 
as lasting consequences of this use. 
While some aspects of adolescence can 
be properly and productively modeled 
in laboratory animals, others clearly can- 
not and will require studies in human 
adolescents. Rodent studies can be 
used to rapidly and cost-effectively 
characterize neural, hormonal, and 



325 



NIAAA's Neuroscience and Behavioral Research Portfolio 



behavioral features of adolescence as 
well as the interrelationships among 
these factors, environmental stressors, 
and their association with alcohol use 
and later abuse. For some research 
questions, use of nonhuman primates 
may prove preferable. The protracted 
ontogeny generally evident in primates 
relative to other laboratory animals 
might potentially prove beneficial for 
long-term studies of the pharmacology 
of adolescent drug self- administration, 
although more needs to be understood 
regarding the time course of adolescence 
in nonhuman primates — particularly 
that of seasonal breeders where pubertal 
changes unfold in a compressed time- 
frame temporally synchronized with 
the mating season (see, e.g., Coe et al. 
1981; Plant 1996). In all of this work it 
will be critical for researchers to remem- 
ber that adolescents cannot be treated 
just as immature adults. The distinctive 
characteristics and proclivities of adoles- 
cents must be considered when devel- 
oping appropriate animal models, 
approaches, and techniques to study 
this unique developmental stage. 

ACKNOWLEDGMENT 

Preparation of this manuscript was sup- 
ported in part by National Institute on 
Alcohol Abuse and Alcoholism grant 
R01 AA10288. 



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