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National Institute on Alcohol Abuse and Alcoholism

RESEARCH MONOGRAPH - 34

Review of NIAAA's

Neuroscience and Behavioral

Research Portfolio

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

n ■■-•?

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

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

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.

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

angstrom(s)

adenylyl cyclase

ACh

acetylcholine

ACTH

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

adrenocorticotropin (Chapter 11)

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

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

computed tomography

day

dopamine

DHEA

dehydroepiandrosterone

DHT

dihydrotestosterone

deciliter(s)

DNA

deoxyribonucleic acid

DSM-IV

Diagnostic and Statistical Manual of Mental Disorders, 4th edition

DTs

delirium tremens

dizygotic

EC50

median effective concentration

ECF

executive cognitive function

ED50

median effective dose

EDE

ethanol delayed effect

EEG

electroencephalographic

EPR

electron paramagnetic resonance

EPSP

excitatory postsynaptic potential

ERP

event-related potential

embryonic stem [cells]

FAS

fetal alcohol syndrome

FDG

fluorodeoxyglucose (Chapter 6) or 18fluorine- 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

grams(s)

GABA

gamma- aminobutyric acid

GABAA

gamma-aminobutyric acid type A

glucocorticoids

GHB

y-hydroxybutyrate

GnRH

gonadotropin-releasing hormone

GRF

growth hormone-releasing factor

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 )

hertz

Xll

ICSS

ntracranial self- stimulation

icv

ntracerebroventricularly

IEG

mmediate early gene

IGF-1

msulin-like growth factor- 1

IL-lp

nterleukin-ip

123jMp

odoamphetamine 123

ntraperitoneal

IPSP

nhibitory postsynaptic potential

ntelligence quotient

ISI

nterstimulus interval

tilodalton(s)

tilogram(s)

Korsakoff s syndrome

LD50

median lethal dose

uteinizing hormone

ong sleep [mice]

LTD

ong-term depression

LTP

ong-term potentiation

MAP

mitogen-activated protein [kinase]

mCPP

w-chlorophenylpiperazine

MDMA

3,4-methylenedioxymethamphetamine

milligram(s)

mg%

milligrams percent

milliliter(s)

millimeter(s)

millimolar

MRI

magnetic resonance imaging

mRNA

messenger ribonucleic acid

MRS

magnetic resonance spectroscopy

MRSI

magnetic resonance spectroscopic imag

magnetization transfer

microgram(s)

microliters )

[xm

micrometer(s)

monozygotic

Nac

N- acetyl compounds

nACh

nicotinic acetylcholine

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

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

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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

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

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.

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

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

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

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

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

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., Sr2+-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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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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aspartate responses: Ethanol affects a
subtype of the ifenprodil-sensitive N-

methyl-D-aspartate receptors. J Pharmacol
Exp Ther 278(1): 114-124, 1996.

Yim, HJ.; Schallert, T.; Randall, P.K.;
Bungay, P.M.; and Gonzales, RA. Effect
of ethanol on extracellular dopamine in
rat striatum by direct perfusion with mi-
crodialysis. J Neurochem 68(4):1527-
1533, 1997.

Yu, D.; Zhang, L.; Eisele, J.L.; Bertrand,
D.; Changeux, J. P.; and Weight, F.F.
Ethanol inhibition of nicotinic acetyl-
choline type alpha 7 receptors involves the
amino -terminal domain of the receptor.
Mol Pharmacol 50(4):1010-1016, 1996.

Zhou, Q.; Verdoorn, T.A.; Lovinger,
D.M. Alcohols potentiate the function of
5-HT3 receptor-channels on NCB-20
neuroblastoma cells by favouring and
stabilizing the open channel state. /
Physiol (Lond) 507(Part 2):335-352, 1998.

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.

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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.

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, Ca2+, 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-[?vr-
(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

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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,

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

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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

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

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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 P0, 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 P0. Alterations in P0 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 P0, 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

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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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

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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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.

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.

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Chu, B.; Dopico, A.M.; and Treistman,
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NIAAA's Neuroscience and Behavioral Research Portfolio

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Dopico, A.M.; Anantharam, V.; and
Treistman, S.N. Ethanol increases the
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284:258-268, 1998.

Ellingson, J.S.; Taraschi, T.F.; Wu, A.;
Zimmerman, R.; and Rubin, E. Cardio-
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Frankenhauser, B., and Hodgkin, A.L. The
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1996. pp. 13-27.

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.

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

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.

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

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

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 -

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

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

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.

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

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,

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- ... .... . , , .

Tr7 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.

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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

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

NIAAA's Neuroscience and Behavioral Research Portfolio

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

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

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

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

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

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 GABAA 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 GABAA
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

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 Mg2+ binding within
the ion channel. Mg2+ 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 Zn2+ 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

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 [3H]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 [3H]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 [3H]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 [3H]MK-801 and
NMDA- specific [3H] glutamate binding
were significantly increased in hip-
pocampus by chronic ethanol treatment,
but there were no changes in binding
of [3H]glycine or [3H]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

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

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
Ca2+ (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

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

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.

GABAA 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 GABAA
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 GABAA recep-
tors in brain (table 1). Chronic ethanol
exposure is associated with a decrease
in the sensitivity of GABAA 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 GABAA receptor
function, data from radioligand binding
studies do not provide an explanation

for these effects. No consistent alter-
ations in GABAA receptor recognition
sites have been observed (see table 1).
Therefore, chronic ethanol administra-
tion induces functional tolerance of
GABAA 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 GABAA
receptor subunits, suggesting that alter-
ations in GABAA receptor expression
may account for alterations in GABAA
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
GABAA 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 GABAA 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 GABAA
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 GABAA 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 [3H]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

GABAA Receptor Function, Recognition

Receptor Property

Alteration

GAB A- mediated CI" channel function3

Decreased

GABA-mediated CI" channel function0

No change

Phenobarbital- mediated CI" flux3

Decreased

Ethanol-enhanced CI" flux0

Abolished

Benzodiazepine -enhanced CI" fluxc

Decreased

Inverse agonist modulation0

Increased

Neuroactive steroid modulation3

Increased

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

No change

Low-affinity [3H]muscimol bindingd

Decreased

[3H]flunitrazepam (flu) binding0

No change

[35S]TBPS binding0

No change or increased

GABA enhancement of [3H]flu binding01

Decreased

[3H]zolpidem binding0

Increased or no change

[3H]Ro 15-4513 binding0

Increased

al Subunit mRNA and peptides0

Decreased

a2 Subunit mRNA and peptides3

Decreased

a3 Subunit mRNA and peptides3

No change or decreased

a4 Subunit mRNA and peptides3

Increased

a5 Subunit mRNAs3

No change

a6 Subunit mRNA and peptidesb

Increased

61 Subunit mRNA and peptides3

No change or increased

62 Subunit mRNA and peptides0

No change or increased

63 Subunit mRNA and peptides3

No change or increased

yl Subunit mRNA and peptides3

Increased

y2S Subunit mRNA3

Increased

y2L Subunit mRNA3

No change

y2 Subunit peptides3

No change

y3 Subunit mRNA3

No change

8 Subunit mRNA3

No change

Note: TBPS = tert-butyl-bicyclophosphorothionate.

"Cerebral cortex.

bCerebellum.

cCerebral 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. 1997

Devaud et al. 19956

103

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,
GABAA receptor subunit mRNA levels
appear to be in a state of flux, whereas
GABAA receptor subunit peptide levels
exhibit more stable changes. GABAA
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, GABAA 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 GABAA 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 GABAA
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 GABAA
receptor a and (3 subunits in vitro
(Russek et al. 1997; S.J. Russek and
D.H. Farb, personal communication,
June 1998). Human GABAA 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 GABAA receptor al
subunit promoter activity and increases
GABAA receptor (3 subunit promoter
activity (Russek et al. 1997). This work
may identify mechanisms of regulation
of GABAA receptor genes by ethanol.

Alterations in GABAA receptor func-
tion and gene expression are regionally
as well as temporally dependent. Chronic
ethanol exposure differentially alters
GABAA 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),

104

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, GABAA receptor (32/3 subunit
peptide expression is not altered in the
hippocampus following either period
of ethanol consumption, although
GABAA 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
GABAA receptor gene expression is
differentially regulated by ethanol in the
hippocampus compared with the cerebral
cortex. Therefore, ethanol regulation
of GABAA receptor gene expression
varies across brain regions.

Alterations in native GABAA 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 GABAA receptors
(Morrow 1995). There is ample evidence
for alterations in GABAA 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
GABAA 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
GABAA receptor gene expression
(Klein et al. 1995&). Another possible
mechanism mediating neuroadaptations
of GABAA 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
GABAA 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 GABAA receptors. How-
ever, the stoichiometry of native GABAA
receptors is undetermined. Likewise, a
dissociation of subunits could account
for decrements in GABAA 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,

105

NIAAA's Neuroscience and Behavioral Research Portfolio

recent data narrowing the site of ethanol
action to the second transmembrane
domain of GABAA receptors (Mihic et
al. 1997) suggest that ethanol may affect
the conformation of GABAA 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 GABAA receptors may play a
role in the development of ethanol
tolerance and dependence. Most
major known kinases can phosphorylate
GABAA receptor subunits (most often
the (31) in vitro (Browning et al. 1990;
Porter et al. 1990; Moss et al. 1995).
Furthermore, phosphorylation of GABAA
receptors has been shown to modulate
receptor function. PKC and PKA phos-
phorylation of GABAA 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 GABAA
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 GABAA receptor function
following ethanol administration. As
discussed previously, acute ethanol
administration induces changes in
GABAA 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 GABAA receptors
expressed in vivo has precluded defini-
tively answering this question. Expres-
sion systems with stably expressed,
functional GABAA receptors under
the control of an inducible promoter
are promising model systems. When
chronically treated with ethanol, Ltk-
cells show changes in GABAA 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
GABAA 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 GABAA receptors are
found only in desensitized receptors
(Leidenheimer et al. 1992), suggesting
that the involvement of phosphorylation
in ethanol modulation of GABAA
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 GABAA

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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 GABAA 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 GABAA
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
GABAA receptor subunit assembly fol-
lowing chronic ethanol exposure. The
exact mechanisms that account for
alterations in GABAA 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 GABAA receptor regulation. ER = endoplasmic reticulum;
EtOH = ethanol; P04 = phosphate. Adapted from Grobin, A.C.; Matthews, O.B.; Devaud,
L.L.; and Morrow, A.L. The role of GABAA 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
GABAA receptors in ethanol tolerance
and dependence. Animals selected for
differences in ethanol sensitivity or
withdrawal show differences in GABAA
receptor function. Mice selected for
differential sensitivity to the hypnotic
effects of ethanol display corresponding
differences in the ataxic effects of GABAA
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 GABAA receptor subunit mRNAs
(Keir and Morrow 1994) and diver-
gent changes in GABAA 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 GABAA 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 GABAA 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 GABAA 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-
tryptamine3 (5-HT3), 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-HT3 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-HT3 receptors
in vivo. However, there is increasing
evidence that 5-HT3 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-
HT3 receptors have not been studied,
but it is likely that studies of this area
would provide insight into the role of
5-HT3 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 86Rb+ 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 Mg2+).
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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NIAAA's Neuroscience and Behavioral Research Portfolio

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.

110

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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NIAAA's Neuroscience and Behavioral Research Portfolio

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 P2x 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., P2y and P2u receptors) and
ligand-gated ion channel receptors
(e.g., P2X) (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 (EC50 = 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).

112

Neuroadaptation to Ethanol at the Molecular and Cellular Levels

Additional studies are needed to
determine if ethanol alters mammalian
P2x 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-HT1A and the 5-
HT3 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-
HT1C or 5-HT1A 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-HTlc
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-HT3
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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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 GABAB 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 GABAB receptors
(Wan et al. 1996; Siggins et al. 1999),
suggesting that the ethanol and GABAB
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 GABAB
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 GABAA 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, Mn2+) 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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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 Mn2+
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 GABAA 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
GABAA 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
(GABAA 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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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
Vx 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 Vl 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-HT2 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

132

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
GABAAergic 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 GABAB receptor antagonist
to unmask ethanol interactions with
GABAA-IPSPs (see Wan et al. 1996),
a significantly greater ethanol enhance-
ment of GABAA-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 GABAA 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 GABAA 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 GABAA 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 GABAAergic 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

GABAB antagonist in blocking paired-
pulse inhibition (a presynaptic site of
action) of IPSPs, suggesting that the
drug might act on GABAB rather than
GABAA receptors (Berton et al. 1998).
This finding also seems consistent
with the metabotropic hypothesis of
ethanol sensitivity (Siggins et al.
1999) and suggests that GABAB
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
GABAB 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
GABAB antagonist CGP 35348, sug-
gesting that the clinical efficacy of GHB
may be due to its action on GABAB
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 GABAA 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

137

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/GABAA 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
GABAA 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

140

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 GABAA receptor function
in brain (Purdy et al. 1990, 1991;
Paul and Purdy 1992). Functional
changes in GABAA 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 GABAA receptor and NMDA
receptor subunit gene expression
(Devaud and Morrow 1998; Devaud
et al. 1998). Cerebral cortical levels of
GABAA 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
GABAA 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

142

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 GABAA receptors to ben-
zodiazepines in animal studies (see
table 1). In addition, the decrease in
the density of low- affinity [3H]musci-
mol sites and the decline in GABAA
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.
[3H]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). GABAA
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 GABAA 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 GABAA 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 GABAA
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

GABAA-NMDA receptor interactions
have been documented. Grayson and
colleagues (Memo et al. 1991; Zhu et
al. 1995) have demonstrated NMDA-
mediated alterations in GABAA recep-
tor function and gene expression in
cultured neurons. Since many neurons
contain both GABAA and NMDA
receptors and ethanol acts directly on
subtypes of both GABAA 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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Neuroadaptation to Ethanol at the Molecular and Cellular Levels

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 GABAA 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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NIAAA's Neuroscience and Behavioral Research Portfolio

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.

189

NIAAA's Neuroscience and Behavioral Research Portfolio

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

190

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 [3H] 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[3H]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 GABAA 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 [3H]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
[3H]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
[3H]MK-801- and NMDA-specific
[3H]glutamate binding slightly
increased in hippocampus during
chronic ethanol treatment, but there
were no changes in [3H]glycine or
[3H]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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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.

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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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Arango, V.; Underwood, M.D.; Pauler,
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Arendt, T. The cholinergic differentiation
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Arendt, T.; Bruckner, M.K.; Magliusi,
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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

210

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 GABAA 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

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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 GABAA 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, GABAA inverse
agonists have been tested in combina-
tion with ethanol to block ethanoPs
anxiolytic responses. However, GABAA
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 GABAA 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 pA2
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.

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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 GABAA 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 GABAA 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, GABAA 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 GABAA receptors, positive
modulators at the neurosteroid site on
the GABAA 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 GABAA
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 GABAA 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 GABAA 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
GABAA receptors that contain a binding
site for Ro 15-4513 on a6 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
a6 subunits correlate with ethanol sen-
sitivity of chronic effects are not com-
pelling (Grobin et al. 1998).
Alternative to a GABAA 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
GABAA 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.

GABAA 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, GABAA 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 GABAA
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 GABAA 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 GABAA
positive modulator or an NMDA
antagonist alone. It appears reasonable
to suggest that the interaction of
ethanol's simultaneous effects at GABAA
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 GABAA 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/GABAA 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-HT1B
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-HT1B 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-HT1B receptors are important
in the control of striatal dopaminergic
release and that 5-HT1B agonists
increase striatal dopamine levels. These
effects are consistent with the ability of
5-HT1B agonists to increase locomotor
activity, disrupt prepulse inhibition,
and substitute for, or enhance, the
discriminative stimulus effects of
cocaine. The interaction between 5-
HT1B receptors and dopaminergic
release in the striatum is not clear, but
one hypothesis is that 5-HT1B receptors
function as heteroreceptors on inter-
neurons. Data gathered with drug dis-
crimination suggest a functional link
between the actions of ethanol at
GABAA receptors and 5-HT1B media-
tion of ethanol-like activity (Green
and Grant 1998).

There are only a few investigations
of 5-HT2 receptor ligands and the dis-
criminative stimulus effects of ethanol.
These studies have shown that the 5-
HT2a 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-
HT2A agonist 5-MeODMT (5-
methoxy-N,Ar-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-HT2A antagonist
MDL 100,907 (Maurel et al. 1997).
These results suggest that the 5-HT2A
receptor mediates ethanol-like dis-
criminative stimulus effects. However,

225

NIAAA's Neuroscience and Behavioral Research Portfolio

a recent study found the 5-HT2a/2c
agonist DOI did not engender or
potentiate the discriminative stimulus
effects of ethanol (Szeliga and Grant
1998). Likewise, the 5 -HT2A antago-
nist ketanserin failed to attenuate the
discriminative stimulus effects of
ethanol (Szeliga and Grant 1998).
The data collectively suggest that the
5-HT2 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 ED50 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
GABAA 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

227

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,

228

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 GABAA
and 5-HT receptor systems. The sci-
entific literature is replete with evidence
showing the involvement of GABAA
receptor systems in regulating anxiety.

As a class of compounds, benzodi-
azepines that act as positive modulators
of GABAA 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 GABAA receptor complex. Similar
to the benzodiazepines, neuroactive
steroids that positively modulate
GABAA 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 GABAA
ligands to the convulsant site and
GABAA 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

229

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 GABAA 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-
HT1A 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-HT1A receptor
agonists have been suggested to be
related to activation of 5-HT1A autore-
ceptors, which results in a reduced
serotonin neuronal function (Dourish
et al. 1986). Specifically, 5-HT1A 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-HT1A
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-HT1A 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-HT1A agonists
possibly reflects anxiogenic activity at
postsynaptic 5-HT1A 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 GABAA 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

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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
GABAA 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 D2 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 GABAA, 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-HT3
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
GABAA 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-HT3 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-HT1B knockout

mouse has been assessed for develop-
ment of ethanol place and taste condi-
tioning (Risinger et al. 1996) because
these 5-HT1B 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,
GABAA, and dopamine involvements.
Specifically, the 5-HT2 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 GABAA 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

237

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

238

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 D3 (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

241

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,

242

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 GABAA 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-HT1B 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-
HT1B receptor. As already mentioned,
there is a 5-HT1B 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
GABAA receptor systems (Blanchard
et al. 1993; Miczek et al. 1993; Weerts
and Miczek 1996). The 5-HT1B
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-HT1B receptor activation is
a component of the discriminative
stimulus effects of low doses of ethanol
(Grant et al. 1997). 5-HT1B 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-HT1B receptor system is predomi-
nantly expressed in rodents, studies in
primates will need to focus on the 5-
HT1D 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 GABAA 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-

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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

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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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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

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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

269

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-HT1A receptor substitute for
the discriminative stimulus properties
of ethanol (Signs and Schechter 1988;
Grant and Colombo 1993 b\ Krystal et
al. 1994), whereas 5-HT3 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

271

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-HTlc and
5-HT2 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-HTlc receptors in
particular, since chronic ethanol-treated
rats show enhanced susceptibility to the
anxiogenic-like effects of a selective 5-
HT1C agonist (Rezazadeh et al. 1993).
Adaptive changes in the function of 5-
HT1C receptors may, therefore, play a
significant role in ethanol withdrawal
anxiety. In addition, changes in the
sensitivity of 5-HT1A 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-HT1A receptor agonist
8-OH-DPAT (Kleven et al. 1995) and
that 5-HT1A 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

272

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.

273

NIAAA's Neuroscience and Behavioral Research Portfolio

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 GABAA 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-HT3 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-HT3 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

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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-HT3
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 GABAA 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

277

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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

279

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 GABAA-
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 D2-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

282

Neuroadaptive Changes in Neurotransmitter Systems

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

283

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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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NIAAA's Neuroscience and Behavioral Research Portfolio

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 Ca2+ 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-
HT1A 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

287

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,

288

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,

289

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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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.

315

NIAAA's Neuroscience and Behavioral Research Portfolio

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 D2/D3 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 a2 norepinephrine
autoreceptors (Choi and Kellogg 1992;
Choietal. 1997).

Dopaminergic systems undergo
substantial reorganization during ado-
lescence. Over one-third to one-half of
the Dl and D2 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 Dx and D2 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 D3
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

319

NIAAA's Neuroscience and Behavioral Research Portfolio

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 LD50
(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

320

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.

322

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

324

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

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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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13:527-532, 1989.

333

STUDIES OF ACUTE AND
CHRONIC EFFECTS OF
ALCOHOL IN HUMANS

Chapter 9

Acute Effects of Alcohol on Cognition
and Impulsive-Disinhibited Behavior

Peter R. Finn, Ph.D.

KEY WORDS: cognition; disinhibition; CNS (central nervous system) information pro-
cessing; acute AODE (effects ofAOD [alcohol or other drug] use, abuse, and dependence);
self control; memory; learning; attention; AOD impairment; biological inhibition; psy-
chomotor impairment; risk-taking behavior; etiology; psychoactive substances; aggressive
behavior; literature review

The majority of studies of the acute
effects of alcohol on cognition and
behavior have been conducted with
the general purpose of understanding
the short-term adverse consequences
of alcohol on aspects of information
processing or on factors that influ-
ence the expression of aggressive or
risky behavior. Such studies address
larger societal concerns, such as how
alcohol impairs driving, work perfor-
mance, and cognitive functioning; the
long-term and potentially permanent
effects of alcohol on cognitive function
(e.g., Korsakoff's syndrome); the asso-
ciation between alcohol consumption
and increased physical and sexual aggres-
sion; and, more recently, the associa-

tion between alcohol consumption
and engaging in behaviors that elevate
risk for contracting sexually transmit-
ted diseases, especially AIDS.

Very few studies have directly
investigated the relevance of the acute
cognitive and behavioral effects of
alcohol for the etiology of alcoholism.
However, many studies have investi-
gated the potential relevance for the
etiology of alcoholism of the acute
effects of alcohol on mood, subjective
intoxication, and autonomic or elec-
troencephalographic activity (e.g.,
Finn et al. 1992; Cohen et al. 1993;
Schuckit 1995). Lacking are studies
of the acute impairing effect of alco-
hol on factors that affect the self-reg-

P.R. Finn, Ph.D., is director of the Clinical Science Training Program and an associate professor in
the Department of Psychology, Indiana University, 1101 East 10th St., Bloomington, IN 47405-7007.

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NIAAA's Neuroscience and Behavioral Research Portfolio

ulation of drinking and studies of the
acute effect of alcohol on impulsivity,
which in turn could contribute to
an increased long-term risk of devel-
oping alcohol problems. In addition,
there is a general scarcity of studies
that attempt to control for or assess
the effect of individual differences
in response to alcohol. As is the
case for studies of long-term effects
of alcohol on cognition (cf. Parsons
and Nixon 1998), only a few studies
have assessed the influence of impor-
tant individual differences, such as a
positive family history of alcoholism
(e.g., Schuckit et al. 1988; Lex et al.
1994), personality traits (e.g.,
Nagoshi et al. 1991), level and
history of alcohol consumption (e.g.,
Salame 1991; Schandler et al.
1988«), and limb (rising or falling)
of the blood alcohol curve (e.g.,
Nicholson et al. 1992; Wang et
al. 1992).

In this chapter I review the key
findings of studies of the acute effects
of alcohol on cognition and on
factors associated with impulsive-
disinhibited behavior, identify impor-
tant gaps in knowledge, and suggest
some potentially valuable avenues for
future research. The studies reviewed
here can be divided into two areas:
( 1 ) studies of the effects of alcohol
on basic aspects of information pro-
cessing and (2) studies of the effects
of alcohol on cognitive function asso-
ciated with poor impulse control,
such as risky decision making,
response bias, and cognitive inhibi-
tion assessed with the Stroop test.
The review is restricted to studies
that include some form of alcohol

administration, either experimental or
self- administered.

ACUTE EFFECTS
OF ALCOHOL ON
INFORMATION
PROCESSING

Studies documenting the presence of
serious cognitive deficits in chronic
alcoholics (e.g., Fitzhugh et al. 1965;
Kleinknecht and Goldstein 1972; Parsons
1977) and studies suggesting that
impaired information processing is an
important factor underlying alcohol's
effect on driving performance (e.g.,
Brewer and Sandow 1980; Linnoila et al.
1986) have provided the basic impetus
for an extensive literature documenting
the acute effects of alcohol on aspects
of information processing, including
speed, capacity, attention, memory, visual-
spatial ability, problem solving, and learn-
ing. The great majority of these studies
have assessed alcohol's effect on active,
controlled (effortful) processing, as
opposed to passive, or more automatic
(preconscious), processing of informa-
tion (such as implicit memory).

Most of the studies of the acute
effects of alcohol on information pro-
cessing have focused on the effects of
alcohol on aspects of processing capac-
ity and resources, such as processing
speed, explicit memory, and attention.
Overall, the literature indicates that
moderate to high doses of alcohol
slow the speed of information process-
ing and impair attention and memory,
and that such impairments are exacer-
bated at higher alcohol doses and with
more complex tasks (Rohrbaugh et al.
1987, 1988; Maylor and Rabbitt 1993).

338

Acute Alcohol Effects on Cognition and Impulsive-Disinhibited Behavior

The general interpretation offered for
these effects is that alcohol reduces
the available processing capacity and
resources, contributing to a general
slowing of information processing
(Rohrbaugh et al. 1987; Maylor and
Rabbitt 1993; Post et al. 1996) with
its concomitant impairment in atten-
tion and explicit memory.

Processing Speed
and Capacity

Alcohol's slowing effect on information
processing is apparent from studies of
reaction time (RT) (Maylor and Rabbitt
1993), backward masking (Moskowitz
and Murray 1976), self-paced contin-
uous performance tasks (CPTs) (e.g.,
Hasenfratz et al. 1993), and the latency
of event-related potential (ERP)
components such as the P300 (e.g.,
Rohrbaugh et al. 1987). With few
exceptions, studies consistently report
that alcohol increases RT on a range
of choice RT-type tasks (e.g., Maylor
et al. 1987, 1992; Hasenfratz et al.
1993; Post et al. 1996). In addition,
impaired RT is greater on the rising
compared with the falling limb of the
blood alcohol curve (Beirness and Vogel-
Sprott 1984; Nicholson et al. 1992;
Wang et al. 1992). Most of those
studies reporting no effect of alcohol
on RT have used between-subjects
designs (Sommer et al. 1993) or simple
tasks (Pfefferbaum et al. 1980) that
may not be sensitive enough to detect
alcohol-related impairments.

In their selective meta-analysis,
Maylor and Rabbitt (1993) presented
convincing evidence of a linear effect
of alcohol on RT (RT^^^ 1.12RTno
alcohol ~ 17.85) accounting for 99 per-

cent of the variance. Their analysis
revealed that the absolute effect of
alcohol increased linearly with task
duration and complexity, or as a func-
tion of the requirements for informa-
tion-processing resources. Other
studies have also reported that the
effects of alcohol are more pro-
nounced in tasks requiring more pro-
cessing resources, such as
divided-attention tasks, tasks using
larger arrays or displays of stimuli
(e.g., Moskowitz and DePry 1968;
Maylor et al. 1990; Post et al. 1996),
or vigilance tasks with extended duration
(Rorhbaugh et al. 1988). An analysis
of Maylor and colleagues' studies of
recognition memory (see Maylor and
Rabbitt 1993) also suggests that alco-
hol's effect on recognition memory
follows a linear function (log propor-
tion correct aiCohoi= 1-31 log propor-
tion correct no alcohol - 0.004, ex-
plaining 96 percent of the variance),
with greater impairment on tasks
requiring more processing resources.

Tasks that involve backward mask-
ing also indirectly demonstrate the
cognitive slowing effect of alcohol.
Backward masking involves the pre-
sentation of a masking stimulus soon
after a target stimulus, where relatively
short interstimulus intervals (ISIs)
between target and masking stimulus
impede the accurate detection of the
target. Alcohol's cognitive slowing effect
is inferred from the relatively longer
ISIs required for accurately identifying
the target stimulus (Moskowitz and
Murray 1976; Moskowitz et al. 1985).
Cognitive slowing after alcohol is also
apparent on tasks that directly assess
speed of information processing, such

339

NIAAA's Neuroscience and Behavioral Research Portfolio

as the self-paced rapid information
processing (RIP) task (Battig and
Buzzi 1986). The RIP task is a com-
plex CPT involving the consecutive
presentation of individual numbers on
a screen and requiring a response after
the detection of three consecutive
even or odd digits (see Fillmore et al.
1998). Correct responses increase the
rate of presentation of stimuli. False
alarms or misses result in a slowing of
the stimulus presentation rate. Alco-
hol results in an overall slower rate of
stimulus presentation due to poorer
performance at higher rates (Michel
and Battig 1989; Hasenfratz et al.
1993; Fillmore et al. 1998). Interest-
ingly, the slowing effect of alcohol on
the RIP task has been shown to be
partly attenuated by cigarette smoking
(Michel and Battig 1989), caffeine
(Hasenfratz et al. 1993), and the
expectation that alcohol would mini-
mally impair performance (Fillmore et
al. 1998).

Finally, results of studies of the effect
of alcohol on the ERP provide some
indirect evidence that alcohol slows the
speed of information processing and
reduces overall central processing capacity
(Oscar-Berman 1987; Jaaskelainen et
al. 1996). The majority of studies report-
ing on the effects of alcohol on the
latency of components of the ERP show
that alcohol increases latency. A number
of studies report that alcohol increases
the latency of the P3 ERP component
(Teo and Ferguson 1986; Campbell
and Lowick 1987; Krein et al. 1987;
Rohrbaugh et al. 1987; Schuckit et al.
1988; Lukas et al. 1990). Alcohol has
also been found to increase the latencies
of the N2 (Teo and Ferguson 1986;

Rohrbaugh et al. 1987; Jaaskelainen et
al. 1995) and P2 ERP components (Teo
and Ferguson 1986). Reductions in P3
amplitudes after alcohol, which are
thought to reflect decreases in available
processing resources, have also frequently
been reported (Teo and Ferguson 1986;
Campbell and Lowick 1987; Krein et
al. 1987; Rohrbaugh et al. 1987).
Although alcohol-induced increases in
ERP latencies and reductions in P3
amplitudes frequently do not parallel
behavioral measures of alcohol effects
(Oscar-Berman 1987), increased latencies
and reduced P3s after alcohol have been
associated with slower RTs and poorer
signal detection performance (decreased
sensitivity) with more demanding tasks
(Campbell and Lowick 1987; Rohrbaugh
et al. 1987). The lack of a parallel
between alcohol-induced changes in ERP
and performance measures could be
because, though alcohol may reduce the
relative availability of processing
resources, some tasks do not supersede
the actual available processing resources
(e.g., working memory space).

What is generally lacking in this liter-
ature are studies designed to identify the
neurophysiological basis (both struc-
turally and at the receptor level) for the
acute effects of alcohol on processing
capacity. Such studies could use a variety
(combination) of brain imaging tech-
niques such as functional magnetic res-
onance imaging (fMRI) and positron
emission tomography (PET) along with
specific cognitive tasks (CPT, recognition
memory, choice RT, or word categori-
zation) under alcohol and no alcohol (and
placebo) conditions. Some candidate sys-
tems suggested by Jaaskelainen and
colleagues (1996) in their review of

340

Acute Alcohol Effects on Cognition and Impulsive-Disinhibited Behavior

alcohol-ERP studies are the gamma-
aminobutyric acid (GAB A) system (alco-
hol augmentation) and the N-methyl-
D-aspartate (NMDA) system (alcohol
attenuation). Such studies may reveal
how the acute effects of alcohol on
such systems may translate into long-
term deficits in cognitive functioning.

Attention

Studies have consistently indicated that
alcohol impairs attention (Koelega
1995) and that alcohol's disrupting
effect on attention is most apparent in
tasks involving divided or sustained
attention or high processing demands
(Rohrbaugh et al. 1987, 1988; Maylor
et al. 1990; Lex et al. 1994; Koelega
1995; Post et al. 1996). Alcohol appears
to impair attention on divided-attention
tasks even at very low (0.02-0.03 mg%)
dose levels (Koelega 1995). Rorhbaugh
and colleagues (1987, 1988) also found
that decrements in sustained attention
increased with higher doses, with a rapid
decrement in performance at high
doses. They suggested that alcohol-
induced performance deficits on signal
detection-type tasks are due to reduced
central processing capacity and reduced
availability of processing resources over
time, an interpretation shared by oth-
ers (Maylor and Rabbitt 1993;
Koelega 1995; Post et al. 1996).
A report of alcohol's suppression of
mismatch negativity of the ERP sug-
gests that alcohol may also disrupt
preconscious detection of stimulus
events (Jaaskelainen et al. 1995).

Memory

Moderate to high doses of alcohol
have consistently been found to

impair both recognition and recall
memory (e.g., Birnbaum and Parker
1977; Peterson et al. 1990; Maylor
and Rabbitt 1993). Explanations for
the acute impairing effects of alcohol
on memory include impaired consolida-
tion and storage/encoding (Jones and
Jones 1977), a slowing of the transfer
process from sensory storage to short-
term memory (Moskowitz and Murray
1977), a curtailment in deep (elabo-
rated) processing (Craik 1977) or
rehearsal (Landauer 1977), increased
forgetting (Maylor and Rabbitt 1987),
and a general reduction in processing
capacity (cognitive slowing) (Maylor
and Rabbitt 1993). Although the
debate continues, strong arguments can
be made that the overall capacity for
processing information is reduced by
alcohol, leading to deficits in memory
processes — especially those processes
with large demands, such as encoding
large stimulus arrays or retaining items
in short-term memory sequentially over
time (Maylor et al. 1987; Peterson et
al. 1990; Maylor and Rabbitt 1993).
Although not directly assessed, these
data suggest that alcohol impairs work-
ing memory at higher processing loads.
Although it is clear that alcohol
impairs explicit memory processes,
alcohol does not appear to impair
implicit memory processes (Lister et
al. 1991). The differential effect of
acute intoxication on explicit versus
implicit memory parallels the selective
deficits in explicit, but not implicit,
memory observed in Korsakoff's
syndrome (Lister et al. 1991). Not
only do these data provide credence
to the distinction between these two
types of memory, but they also sug-

341

NIAAA's Neuroscience and Behavioral Research Portfolio

gest a common neurophysiological
basis (perhaps involving cortical
circuitry) for acute and long-term
alcohol-induced impairments in
memory. Comparing the acute effects
of alcohol on explicit and implicit
learning skills (e.g., memory and
motor) in combination with brain
imaging (fMRI and PET) could be a
very fruitful line of work and might
serve clinical (chronic alcohol effects)
and basic (cognitive-neuroscience)
research objectives.

In addition to more work on the
assessment of the effects of alcohol
on implicit memory and learning
processes, systematic research is
needed on the effect of alcohol on
working memory. As noted above,
the fact that alcohol has a greater
memory impairment effect with
higher processing demands suggests
that alcohol impairs working mem-
ory. This is directly corroborated in
animal research designed to directly
assess the effects of alcohol on work-
ing memory. Studies clearly indicate
that alcohol impairs spatial and non-
spatial working memory in mice and
rats (Melchior et al. 1993; Givens and
McMahon 1997; White et al. 1997).
Since working memory is critical for
complex problem solving and the
self- regulation of behavior (Barkley
1997), both of which are thought to
be associated with risk for alcoholism
(Peterson and Pihl 1990; Finn et al.
1994), it is quite important that the
effects of alcohol on working memory
be systematically studied in humans.
Surprisingly, a literature search turned
up only one study since 1983 that
specifically assessed the acute effects of

a moderate dose of alcohol on work-
ing memory (Stokes et. al. 1991), and
this study reported no significant
effects of alcohol on working memory.
This finding seems unusual given the
results of memory studies reported
above. In addition, divided attention,
which is significantly disrupted by
even low doses of alcohol, is thought
to be a component of working memory
(Greene et al. 1995). Interestingly,
while divided-attention tasks might
tap working memory, some human
and animal work suggests that sus-
tained attention is separate from
working memory processes (Elliott et
al. 1997; Givens and McMahon 1997).
Studies of the acute effects of alcohol
on working memory and performance
in sustained and divided-attention
tasks may help facilitate the identifica-
tion of the neurophysiological basis
for the effect of alcohol on information
processing. Such studies may also reveal
valuable information about the acute
disruptive effects of alcohol on the self-
regulation of behavior, especially the self-
regulation of alcohol consumption.

Other Aspects of
Information Processing

Given that the disruptive effects of
acute alcohol intoxication are espe-
cially apparent on more complex tasks
that demand more processing
resources, it is not surprising that
alcohol disrupts basic learning and
problem-solving ability (Carpenter et
al. 1961; Peterson et al. 1990; Salame
1991). Peterson and colleagues
(1990) reported that a high dose of
alcohol impaired performance on a
paired-associates learning task, a maze

342

Acute Alcohol Effects on Cognition and Impulsive -Disinhibited Behavior

task, and measures of word fluency. A
high dose of alcohol has also been
associated with deficits in lexical
access (Maylor et al. 1988), word cat-
egorization even when combined
with semantic priming (Maylor et al.
1987), and visual-spatial ability (Lex
et al. 1988; Maylor and Rabbin
1988; Wang et al. 1992; Pearson and
Timney 1998). Interestingly, acute
intoxication has been reported to
ameliorate visual-spatial learning
deficits in alcoholics (Schandler et al.
1988&). Schandler and colleagues
(1988*, 1988&) found that the ame-
lioration of these deficits paralleled
alcohol-induced increases in right-
hemisphere ERP components (P3,
P2, Nl) and autonomic activation
after alcohol, suggesting a cognitive
compensatory effect for continued
drinking in alcoholics with alcohol-
induced chronic cognitive deficits.

Summary

The literature on the acute effects of
alcohol on information processing is
extensive and quite strong, with only
a relatively few gaps. Studies of the
acute effects of alcohol indicate rather
widespread disruption of a range of
facets of controlled information pro-
cessing. A compelling argument can
be offered that alcohol's effect on
most, if not all, aspects of controlled
processing is due to a decrement
in available processing resources
(Rohrbaugh et al. 1987; Maylor and
Rabbitt 1993). There are a number
of gaps in this literature that leave
questions about (1) the influence of
individual differences and develop-
mental processes on acute responses

to alcohol, (2) the specific effects of
alcohol on working memory, (3) the
basic neurophysiological mechanisms
responsible for the acute effects of
alcohol on information processing,
and (4) the mechanisms by which
the acute effects of alcohol are trans-
lated to the long-term, more chronic
effects of high levels of alcohol con-
sumption. Needed are studies that
combine a variety of central nervous
system imaging techniques (e.g.,
fJVlRI and PET) with electrophysio-
logical measures and behavioral and
psychological measures of cognitive
function, using creative paradigms
such as implicit and explicit memory
and learning tasks and working mem-
ory tasks. Also needed are studies
that include subject groups with low,
moderate, and high levels of alcohol
consumption with varying durations
of overall alcohol exposure and
studies that examine the influence of
the limb of the blood alcohol curve
on cognitive performance.

In general, studies of the acute effects
of alcohol have failed to adequately mea-
sure and control for important sources
in individual differences in response to
alcohol, such as drinking level, history,
mood, a family history of alcoholism,
or the presence of other traits such as
behavior problems and impulsivity (cf.
Parsons and Nixon 1998). Also, we
know absolutely nothing about the acute
effects of alcohol on cognition in young
persons (preadolescent and adolescent
samples). Ethical considerations preclude
the administration of alcohol to such
populations; however, it is possible to
use a longitudinal study design to track
younger persons and estimate the

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NIAAA's Neuroscience and Behavioral Research Portfolio

effects of alcohol exposure on various
aspects of cognition and behavior.

ACUTE EFFECTS OF
ALCOHOL ON IMPULSIVE-
DISINHIBITED BEHAVIOR

Although studies consistently indicate
that impulsive -disinhibited behavior is
associated with increased drinking and
a greater risk of alcoholism (e.g., Stewart
and Wilcox 1985; Nagoshi et al. 1991;
Sher et al. 1991; Finn et al. 1994), apart
from the extensive literature on alcohol -
related aggression there have been rela-
tively few studies of the acute effects of
alcohol on other types of disinhibited
behavior or factors that might influence
disinhibited behavior.

Disinhibited behavior includes a
number of specific, but highly correlated,
behaviors labeled as impulsive, aggressive,
risk-taking, undercontrolled, external-
izing, conduct-disordered, sociopathic,
hyperactive, and antisocial (Gorenstein
and Newman 1980; Finn et al. 1994).
The basic construct of disinhibition is
defined as a failure to inhibit a behavior
that is likely to be punished (i.e., a
behavioral and contingency focus);
however, disinhibited behavior is multi-
faceted and multidetermined, involving
the interaction of different traits (e.g.,
impulsivity and sensation seeking), motor
processes, and a range of cognitive pro-
cesses, conditioning processes, and situa-
tional determinants (Barratt and Patton
1983; Finn et al. 1994; Lykken 1995).
For instance, impulsivity (a trait com-
ponent of disinhibition) is considered to
have both cognitive and behavioral
dimensions (Barratt and Patton 1983;
White et al. 1994). Cognitive dimensions

include fast and unreflective (risky)
decision making, a lack of attention paid
to future events (poor planning), poor
time perception, and poor cognitive inhi-
bition assessed using Stroop conflict
errors. Behavioral dimensions include
poor inhibitory motor control (Logan
et al. 1997), acting without thinking
(Barratt and Patton 1983), and motor
restlessness and impatience (White et al.
1994). Individual differences in impul-
sivity, sensation seeking, and neuro-
psychological function also strongly
influence the likelihood for disinhibited
behavior (e.g., Earleywine and Finn
1991; Lau et al. 1995; Lykken 1995).
Therefore, to fully understand the
acute effects of alcohol on disinhibited
behavior it is essential that research sys-
tematically address the multidimen-
sional nature of disinhibited behavior
and carefully assess the role of individ-
ual differences.

The vast majority of studies of the
acute effects of alcohol on disinhibited-
type behavior have examined the alcohol-
aggression relationship. A handful of
studies have addressed the effects of
alcohol on other facets of impulsive -
disinhibited behavior, such as cognitive
processes (decision making and bias,
cognitive inhibition on the Stroop),
attitudes, and motor inhibition. This
section of the chapter is divided into
two parts: (1) a review of studies of
the acute effects of alcohol on aggres-
sion and (2) a review of studies of the
acute effects of alcohol on other facets
of disinhibited behavior.

Aggression

The association between alcohol
intoxication and aggression is well

344

Acute Alcohol Effects on Cognition and Impulsive-Disinhibited Behavior

documented (Pernanen 1976; Brain
1986; Bushman and Cooper 1990),
extensively researched (cf. reviews:
Pihl 1983; Bushman and Cooper
1990; Gustafson 1993), and appears
to be common knowledge in Western
society. Aggressive behavior in child-
hood is a strong predictor of adolescent
and adult alcohol problems (Farring-
ton 1991), and alcohol intoxication
appears to increase the likelihood for
aggressive behavior (Gustafson 1993).
The alcohol- aggression relationship has
been studied from various perspectives,
including epidemiologic (e.g., Perna-
nen 1976; Roizen 1997), longitudinal
(Farrington 1991; White et al. 1993),
biological (Virkkunen and Linnoila
1990), social information processing
(Sayette et al. 1993), psychological or
individual differences (Gustafson
1993; Lau et al. 1995), and interper-
sonal (Leonard and Senchak 1993).

There is an extensive literature on
laboratory studies of the acute effects
of alcohol on aggressive behavior (cf.
Gustafson 1993). A considerable amount
is known about the factors that might
mediate, or moderate, the relationship
between alcohol intoxication and aggres-
sion. For instance, the level of provoca-
tion appears to be critical in determining
whether intoxicated subjects will respond
aggressively in laboratory settings.
Without provocation, alcohol does
not appear to increase aggression
(Gustafson 1993). Other factors that
appear to increase the likelihood that
alcohol intoxication will lead to
aggression in laboratory and field set-
tings are the type of beverage (spirits
but not beer or wine) (Gustafson
1988; Murdoch and Pihl 1988;

Gustafson 1990), alcohol dose
(Sayette et al. 1993), frustration level
(Gustafson 1993; Sayette et al. 1993),
schedule of provocation (Kelly and
Cerek 1993), availability of nonag-
gressive alternatives (Gustafson 1993),
and the saliency of threat or frustra-
tion cues (Gustafson 1986; Gantner
and Taylor 1992).

Although most of the factors that
influence the likelihood of intoxication
leading to violence in laboratory studies
are contextual in nature, support for
the compelling argument that only
some individuals become violent when
drunk is offered by a longitudinal
study. White and colleagues (1993)
studied a large sample (N = 1,380) of
adolescents at 3 -year intervals (12, 15,
and 18 years of age) and modeled the
influence of parental education, alcohol
use at the three time periods, and
alcohol-related aggression at ages 15
and 18 years. Their analysis revealed
that early aggression led to later alcohol
use, but early alcohol use did not predict
later aggression (when controlling for
initial levels of aggression). Further-
more, the only predictors of alcohol-
related aggressive behavior were both
early and contemporary levels of aggres-
sive behavior (non-alcohol related). In
other words, aggressive people were
the ones who were aggressive when
drinking (White et al. 1993). Interest-
ingly, these researchers observed that
lower levels of parental education
were also directly predictive of alco-
hol-related aggression at age 15, sug-
gesting a role for familial intellectual
functioning in alcohol-related aggres-
sion. Their data are consistent with the
finding by Lau and colleagues (1995)

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NIAAA's Neuroscience and Behavioral Research Portfolio

that subjects with evidence of poor
executive cognitive function (frontal
lobe function) showed the highest
level of provocation-induced aggressive
behavior when intoxicated. Research
and theory suggest that deficits in
frontal lobe function are associated
with disinhibited, aggressive personal-
ity traits (Giancola and Zeichner
1994; Lau et al. 1995) and may form,
in part, the neuropsychological basis
for early-onset aggressive behavior.

The findings of White and colleagues
(1993) and Lau and colleagues (1995)
are also consistent with research associat-
ing deficits in central serotonergic
function with alcohol-related violence
(Virkkunen and Linnoila 1990, 1993).
Reduced brain serotonin function and
frontal lobe deficits both have been
associated with disinhibited, impulsive
behavior (Gorenstein and Newman 1980;
Miller 1987; Virkkunen and Linnoila
1993). Research on the interrelationships
between alcohol-related aggression,
impulsive or aggressive traits, serotonin
function, and executive cognitive function
is a promising avenue for research on the
biobehavioral basis for the acute effects
of alcohol on aggression. However, much
remains to be understood about ( 1 ) the
origins (biological and environmental)
of reduced brain serotonin function, poor
executive cognitive function, and early-
onset aggression and (2) the mechanisms
by which reduced serotonin function
and poor executive function lead to
aggressive behavior, alcohol problems,
and alcohol-related aggression.

The literature on the acute effects of
alcohol on aggression is extensive and
strong. The primary strength of the liter-
ature is the delineation of the important

role that a range of contextual factors
might play in influencing the likelihood of
alcohol-related aggression. Less exten-
sively researched are the biobehavioral
bases (origins and mechanisms) of the
individual differences that influence the
alcohol- aggression relationship. Research
combining brain imaging (fMRI, PET,
dipole ERP source), behavioral and
cognitive paradigms, and careful assess-
ments of environmental context
(parental influence, stress, peer factors)
using longitudinal designs (to assess the
developmental origins and processes and
the effects of early exposure to alcohol)
and cross-sectional alcohol challenge
designs (with high-risk [impulsive or
aggressive] and low- risk subjects) should
substantially increase our knowledge
of the biobehavioral bases of important
individual differences affecting alcohol -
related aggression.

Other Facets

of Disinhibited Behavior

Alcohol might increase the likelihood
for disinhibited behavior by increasing
risky- type decision making (decreasing
caution); affecting attitudes about engag-
ing in risky behaviors (e.g., attitudes
about condom use or drinking and dri-
ving); affecting skills and self-efficacy
for engaging in safe behaviors; compro-
mising planning or future-oriented think-
ing; decreasing attention to, or salience of,
stimuli signaling potential punishment;
and decreasing general impulse control
(poor inhibitory motor control).

Attitudes and Decisions About
Risky Behaviors

There is some evidence that alcohol
intoxication affects specific attitudes

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Acute Alcohol Effects on Cognition and Impulsive-Disinhibited Behavior

that, in turn, may increase the likeli-
hood of choosing to engage in risky
behaviors. In a field study, MacDon-
ald and colleagues (1995) found that
intoxicated subjects appeared to be
more willing to consider driving a
short distance after drinking than
nonintoxicated subjects, suggesting
that alcohol may result in somewhat
more risky decision making. In two
studies of the effects of a moderate
dose of alcohol on attitudes about
condom use and self- efficacy toward
negotiating condom use, acute intoxi-
cation was associated with more nega-
tive attitudes about condom use
(Gordon and Carey 1996; Gordon et
al. 1997), lower perceived self-efficacy
to initiate or negotiate condom use
(Gordon and Carey 1996), and
poorer actual behavioral skills (role
play) to negotiate condom use (Gor-
don et al. 1997), when compared
with control subjects.

This literature is still in its infancy
and is weak. The dependent measures
and experimental paradigms are more
primitive and not abreast with current
paradigms for attitude measurement
and perceptual organization used in
social and cognitive psychology (for
discussions of paradigms, see Smith et
al. 1996; McFall et al. 1998). Apart
from a role play in the study by
Gordon and colleagues (1997), the
dependent measures used in these
studies were paper-and-pencil self-
report scales of attitudes or ratings of
self-efficacy. These measures do not
employ the more sophisticated and
objective approaches to attitude
measurement that have been routinely
employed in social psychological

research, such as categorization tasks
involving similarity judgments (Smith
et al. 1996; Fazio and Dunton 1997).
Such tasks are firmly grounded in well-
researched theories and methodologies
in cognitive science (Nosofsky and
Palmeri 1996, 1997) and provide a
more objective assessment of attitude
strength (Smith et al. 1996; Fazio and
Dunton 1997). Finally, the studies by
MacDonald and colleagues (1995)
and Gordon and colleagues (1997)
also purport to have some relevance
for decision making; however, the
effects of alcohol on decision-making
processes are not directly assessed.

In a more direct assessment of the
effects of alcohol on risky decisions,
Meier and colleagues (1996) studied
the acute effect of alcohol using a risky
decision-type task situation where the
magnitude and probability to punish-
ments (failure to win money) were
varied. These investigators found, to
their surprise, that alcohol did not
suppress the effects of punishment
(loss of money). Although this study's
design represents a step in the right
direction for the literature (its design
is systematic and the dependent mea-
sures are objective), it is the only study
directly assessing the effects of alcohol
on decision-making processes. Although
it appears surprising that alcohol failed
to suppress the effects of punishment
on risky decisions, the lack of punish-
ment suppression by alcohol may have
been due to the type of punishment
(loss of money), whose salience may
have remained high for this popula-
tion (college students) in spite of
being intoxicated. Models of behav-
ioral disinhibition focus on a failure to

347

NIAAA's Neuroscience and Behavioral Research Portfolio

inhibit behavior in contexts of
impending aversive stimulation, rather
than loss of reward (Gray 1975; Finn
et al. 1994). For instance, other
research suggests that disinhibited
individuals (psychopaths) learn to
inhibit behavior when that behavior
leads to loss of money, but fail to
learn to inhibit when the behavior
leads to an aversive outcome, such as
electric shock (Schmauk 1970).

There is some evidence suggesting
that alcohol results in an overall bias
to respond (decreases the beta
[caution] parameter) and decreases
caution in perceptual decision making
on CPT tasks (Rohrbaugh et al.
1987; Mongrain and Standing 1989).
However, in CPT tasks assessing
recognition memory, alcohol has been
reported to increase the beta para-
meter (Maylor and Rabbitt 1987).
The beta parameter may be a useful
dependent measure for assessing cau-
tious versus risky shifts in decision
bias after alcohol; however, it is prob-
ably more useful in signal detection
tasks where a risk parameter is more
directly manipulated.

Research on the effects of alcohol
on decision making would be
greatly enhanced by looking to theory
and research on dynamic, stochastic
decision-making processes within the
cognitive science research literature
(Busemeyer and Townsend 1993;
Diederich 1997). In dynamic decision-
making experimental paradigms,
the influence of time constraints,
uncertainty, risk, and preferences
(as they may change over time)
on sequential decision-making pro-
cesses can be assessed over time and

mathematically predicted (Busemeyer
and Townsend 1993).

Motor Inhibition

Although the data are scant, there is
evidence that alcohol may decrease
inhibitory motor control. A recent
study using Logan and Cowan's (1984)
stop -signal task indicates that alcohol
can impair inhibitory motor control
(Mulvihill et al. 1997). The stop-signal
task is a two-choice CPT, where the
subject responds on each trial using
one of two keys depending on the
stimulus (X or O = go signal). On 25
percent of trials an acoustic stop-signal
(a tone) is presented at some latency
after the go signal. The subject is
required to inhibit his or her response
after detecting the stop-signal (see
Logan et al. 1997 for a description).
Impulsive traits in adults and child-
hood behavioral problems have been
associated with relatively poor inhibition
on this task (Logan et al. 1997).
Compared with placebo and no alco-
hol conditions, alcohol was associated
with a sevenfold decrease in inhibitory
control (Mulvihill et al. 1997). This
effect has been replicated by the same
laboratory (Fillmore and Vogel-Sprott
unpublished data).

The stop -signal task has been used
extensively in the study of inhibitory
control in patient populations (see, e.g.,
Schachar and Logan 1990; Schachar
et al. 1995), and it is associated with a
well-tested and quantifiable information-
processing theoretical framework (Logan
and Cowan 1984; Logan et al. 1997).
The stop-signal paradigm is a very
promising paradigm to systematically
apply to the study of the acute effects

348

Acute Alcohol Effects on Cognition and Impulsive -Disinhibited Behavior

of alcohol; however, researchers also
need to study other paradigms assessing
other facets of disinhibited behavior
and the influence of preexisting disin-
hibited traits, such as impulsive, aggres-
sive, and sensation- seeking traits.

Other evidence that acute intoxica-
tion may compromise inhibitory motor
control comes from Peterson and col-
leagues (1990), who found that alcohol
had a detrimental effect on Porteus
maze performance. Although the Por-
teus maze does not directly assess motor
inhibition, poor performance on the
task is associated with disinhibited
behavior after prefrontal lobotomy
(Crown 1952). Another promising
paradigm for investigating the effects
of alcohol on behavioral inhibition is
the GO/NO GO learning task, where
passive avoidance in response to differ-
ent types of punishment contingencies
can be assessed (Newman et al. 1985;
Iaboni et al. 1995). Performance on
GO/NO GO tasks can be used to
assess relative roles of the behavioral
inhibition and activation systems (Gray
1975), which have been associated
with activity in central serotonin and
dopamine systems (Gray 1975).

Cognitive Inhibition

Interference on the Stroop task is
thought to tap the ability to inhibit an
overlearned response (reading a word)
in favor of a competing novel response
(White et al. 1994). This measure of
cognitive inhibition has been modestly
associated with other measures of
impulsivity (White et al. 1994).
Gustafson and Kallmen (1990#,
1990&) reported that alcohol had no
effect on Stroop performance (i.e., no

apparent interference effect). A poten-
tial problem with these two studies
was the lack of a specific and sensitive
measure of interference.

Summary

There is a general dearth of studies of
the effect of alcohol on the cognitive
and behavioral elements of impulsivity
and disinhibition. Many of the studies
that have been done are not well
informed by research in other areas,
such as cognitive science. This is an
extremely important area of research
because of its relevance for alcohol's
effect on impulsive decision making,
motor control, and response persevera-
tion on the self- regulation of drinking.
For instance, research in this area could
shed some light on the mechanisms
by which some individuals might
impulsively extend social drinking into
binge drinking. In addition, more
research in this area would increase
knowledge about how alcohol might
influence decisions to engage in risky
and/or illegal behaviors (drinking and
driving, participating in dangerous
activities while intoxicated, engaging
in unprotected sex) and how early
exposure to alcohol might affect the
development of impulsive traits in
younger individuals, which, in turn,
would contribute to their overall risk
for alcoholism.

Clearly, a substantial amount of
research is needed in this area. Two
major avenues of research should be
undertaken: (1) longitudinal studies
of the effects of early exposure to
alcohol (preadolescent and adolescent)
on the development of disinhibited
traits and (2) cross-sectional studies of

349

NIAAA's Neuroscience and Behavioral Research Portfolio

the effects of alcohol challenge on dis-
inhibited/impulsive behavior (and fac-
tors associated with such behavior),
using multimethod measurements to
capture the multidimensional nature
of behavioral disinhibition and control-
ling for important sources of individ-
ual differences, such as preexisting
disinhibited traits, a family history of
alcoholism (and antisocial behavior),
drinking history and level, and limb of
the blood alcohol curve. Longitudinal
research is needed where the effects of
early exposure to alcohol on behavioral
disinhibition are assessed, because it is
likely that early exposure to alcohol itself
may contribute to increased impulsivity
in childhood, leading to increased risk
for serious and long-term problems
with alcohol and other drugs.

Finally, literature in the areas of
stochastic decision- making dynamics
(Busemeyer and Townsend 1993),
the measurement of impulsivity
(Barratt and Patton 1983; White et
al. 1994; Logan et al. 1997), condi-
tioning and disinhibition (Newman et
al. 1985; Finn et al. 1994; Iaboni et
al. 1995), and the cognitive correlates
of disinhibited traits (Kosson and
Newman 1986; Howland et al. 1993)
presents potentially valuable theoreti-
cal models, experimental paradigms,
and measurement methods that can
be used in the study of the effects of
alcohol on cognitive aspects of poor
impulse control.

ACKNOWLEDGMENTS

Support for this work was provided
by National Institutes of Health/
National Institute on Alcohol Abuse

and Alcoholism grants R01-AA10120
andP50-AA07611.

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Pfefferbaum, A.; Horvath, T.B.; Roth,
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356

Chapter 10

Clinical Neuroscience Studies of

Behaviors Associated With Alcohol

Consumption in Alcoholism

John H. Krystal, M.D., Ismene L. Petrakis, M.D.,
Louis Trevisan, M.D., and Neill Epperson, M.D.

KEY WORDS: AOD (alcohol or other drug) use behavior; AODE (effects ofAOD
use, abuse, and dependence); psycho-pharmacological therapy; serotonin receptors;
agonists; serotonin uptake inhibitors; antagonists; GABA receptors; NMDA re-
ceptors; glutamate; opiates; dopaminergic receptors; catecholamines; calcium
acetylhomotaurinate; steroid hormones; tryptophan; ketamine; literature review

The past decade has been an era of and craving. In most cases, preclinical
important advances in clinical neuro- research has influenced the design and
science and psychopharmacological interpretation of subsequent clinical
studies of alcoholism. These studies have research. However, clinical research
developed along parallel tracks, with data have often provided novel insights
groups studying the contributions of that reshape the expectations based on
many neurotransmitter systems to data collected in other species,
clinical phenomena associated with The purpose of this chapter is to pro-
alcoholism, intoxication, consumption, vide an overview of clinical neuroscience

J.H. Krystal, M.D., is a professor and director of the Division of Cognitive and Clinical Neuroscience,
Department of Psychiatry, Tale University School of Medicine, New Haven, CT 06510; deputy
director of the Abraham Ribicoff Research Facilities, Connecticut Mental Health Center, 34 Park St.,
New Haven, CT 06519; and director of the VA-Tale Alcoholism Research Center, Psychiatry Service
(116-A), VA Connecticut Healthcare System, 950 Campbell Ave., West Haven, CT 06516. I.L.
Petrakis, M.D., is an associate professor in the Department of Psychiatry, Tale University School of
Medicine, and director of substance abuse treatment at the Psychiatry Service of the VA Connecticut
Healthcare System and the VA-Tale Alcoholism Research Center, Psychiatry Service. L. Trevisan,
M.D., is an assistant clinical professor in the Department of Psychiatry, Tale University School of
Medicine; staff psychiatrist at the Psychiatry Service of the VA Connecticut Healthcare System; and an
investigator in the VA-Tale Alcoholism Research Center. N. Epperson, M.D., is an assistant professor
in the Department of Psychiatry, Tale University School of Medicine, and director of the Behavioral
Gynecology Program, Abraham Ribicoff Research Facilities.

357

NIAAA's Neuroscience and Behavioral Research Portfolio

approaches to understanding behaviors
associated with alcoholism. We will focus
on several neurotransmitter systems that
have been implicated in alcoholism
and its treatment: serotonin (5-HT),
glutamate, gamma-aminobutyric acid
(GABA), endogenous opiate, and
dopamine. However, there are a number
of limitations of this review and the field
of research that it considers. These neu-
rotransmitter systems have a compli-
cated matrix of interactions at multiple
sites in the brain. The diversity of mech-
anisms to be considered interacts with
the clinical complexity of alcoholism.
Because of space limitations, the inter-
face of neurotransmitter systems in
contributing to alcoholism-related
behaviors will receive relatively superficial
attention in this chapter.

SEROTONERGIC SYSTEMS

Serotonergic systems have received the
greatest extent of psychopharmaco-
logical study in humans. As a result,
this section is organized by type of
agent studied rather than by the
behavior of interest.

Serotonin Agonists

Serotonergic systems appear to contri-
bute to the discriminative properties of
ethanol in humans. Ethanol appears to
facilitate that activity of a number of 5-
HT receptors, including the 5-HT1B,
5-HT2c, and 5-HT3 receptors, and to
share discriminative stimulus properties
with drugs acting at these sites (Lovinger
1991; Grant et al. 1997b). Although
these studies have guided clinical
research, they may not be directly
applicable to humans because the

human 5-HT1B receptor, previously
called the 5-HT1D receptor, has very dif-
ferent properties from the rodent 5-
HT1B receptor (Pauwels et al. 1996).
At first glance, the clinical findings
appear to be quite similar to the pre-
clinical data. However, the human
data may differ from the preclinical
data in the receptor sites mediating
the actions of agents that have been
studied in both animals and humans.
In particular, 5-HT2 receptor subtypes
may contribute to the ethanol-like
effects of m-chlorophenylpiperazine
(mCPP) in humans (Krystal et al.
1994). In contrast, 5-HT2 receptors
do not appear to figure prominently in
the ethanol-like effects of piperazine
5-HT agonists, including mCPP, in
animals (Grant and Barrett 1991).

The administration of piperazine 5-
HT partial agonists produces a euphoric
effect that is perceived as ethanol-like in
early-onset alcoholic patients (Buydens-
Branchey et al. 1989; Benkelfat et al.
1991; Lee and Meltzer 1991; Krystal
et al. 1994; Buydens-Branchey et al.
19976; George et al. 1997). How-
ever, the effects of the partial 5-HT
agonist mCPP were not specifically
similar to ethanol; that is, the effects
were similar to several substances of
abuse (figure 1).

The two mCPP studies that adminis-
tered mCPP intravenously also reported
the induction of craving (Krystal et al.
1994; George et al. 1997), whereas
the study administering this drug
orally found the opposite (Buydens-
Branchey et al. 1997 b). The induction
of craving by intravenous mCPP sug-
gests that the similarity between the
subjective effects of mCPP and

358

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

30 SO » 120 150 180 210 240

Time (min)

fc ?4,

V 21 •

C 18 J

O 15 -

.**

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i
/

e 9

S 6-

1 1

/i\ "\

;">\ \

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90 120 150 180 210 240

Time (min)

Figure 1. Similarity of m-chlorophenylpiperazine (mCPP) and yohimbine effects to ethanol
(left; n = 21), marijuana (middle; n = 18), and cocaine (right; n = 14). Placebo (open circles),
mCPP (filled circles), and yohimbine (open squares) effects are presented. Each value repre-
sents a mean ± SEM. Increases from baseline were evaluated using the Dunnett t test, * p <
0.05, ** p < 0.01. The drug x drug of abuse x time interaction was not significant. However,
the drug x time interactions were significant for ethanol (p = 0.01) and marijuana (p = 0.04).
The drug x time interaction for similarity to cocaine was not significant (p = 0.1). Adjusting
for placebo day responses, all significant increases from baseline retained their level of signifi-
cance except one value, #, p < 0.05 on the yohimbine day, nonsignificant after adjusting for
placebo response. Reprinted with permission from Krystal, J.H.; Webb, E.; Cooney, N.;
Kranzler, H.R.; and Charney, D.S. Specificity of ethanollike effects elicited by serotonergic and
noradrenergic mechanisms. Archives of General Psychiatry 51:898-911, November 1994.
Copyright 1994, American Medical Association.

359

NIAAA's Neuroscience and Behavioral Research Portfolio

ethanol is significant clinically. In addi-
tion to the elicitation of craving, mCPP
also produced anxiety and irritability
(Krystal et al. 1994; Handelsman et al.
1996). Thus, the induction of dysphoria
by this drug may have contributed to
the elicitation of craving.

The response to mCPP and other
direct and indirect 5 -HT agonists dis-
tinguishes early-onset alcoholic patients
(type II) from late-onset alcoholic
patients and healthy control subjects.
Several studies report that the Cortisol or
prolactin response to mCPP, MK-
212, 5 -hydroxy try tophan, or fenflu-
ramine is reduced in early-onset
alcoholic patients (Lee and Meltzer
1991; Balldin et al. 1992; Coccaro et
al. 1994; Farren et al. 1995 £; Han-
delsman et al. 1996; Krystal et al.
1996; Buydens-Branchey et al. 1997b;
George et al. 1997). Furthermore, the
cerebral metabolic response to mCPP,
assessed using the positron emission
tomography (PET) flurodeoxy glucose
technique, was reduced in early-onset
alcoholics (Hommer et al. 1997). In
contrast, the euphoric responses to
mCPP appeared to be particularly
enhanced in early-onset alcoholic
patients (George et al. 1997).

The capacity to distinguish subtypes
of alcoholic patients by their response
to 5-HT agonists may have important
clinical implications. First, mCPP may
provide a tool for studying the constel-
lation of problems associated with type
II alcoholism, including familial addic-
tion, early onset, severe course, and
sociopathy (Cloninger 1987; Babor et
al. 1992). Early-onset alcoholic
patients appear to have other seroton-
ergic abnormalities, particularly lower

central 5-HT turnover, that may contri-
bute to their expression of sociopathic
or impulsive behaviors (Virkkunen et
al. 1995; Virkkunen and Linnoila
1997). If related to reduced central 5-
HT turnover in these patients, phar-
macological challenge with mCPP may
provide a phenotypic marker of trypto-
phan hydroxylase alleles associated with
risk for impulsive behavior (Nielsen et
al. 1994). Second, worsening of crav-
ing in response to mCPP may predict
poor response to particular antidipso-
tropic medications, such as fluoxetine,
that may stimulate 5-HT receptors
(Kranzler et al. 1996). A corollary of
this hypothesis is that mCPP stimulation
of craving may help to predict beneficial
therapeutic effects of drugs that block
mCPP effects. Third, the actions of
mCPP may be particularly relevant to
early-onset poly drug abuse. Supporting
this view, mCPP effects are similar to
several drugs of abuse (Krystal et al.
1994) and mCPP effects in patients
with an early onset of cocaine addic-
tion parallel the findings in early-onset
alcoholism (Buydens-Branchey et al.
19970, 1997c).

However, the site of action of the
ethanol-like effects of mCPP is not
clear. The piperazine serotonin agonists
are notoriously nonselective for 5-HT
receptor subtypes. The most potent
actions of mCPP are partial agonism
of the 5-HT2c site and antagonism of
the 5-HT3 site (Conn and Sanders-
Bush 1987; Robertson et al. 1992). Of
these effects, the 5-HT2c agonist action
appears to figure most prominently in
its general discriminative stimulus effects
(Callahan and Cunningham 1994).
mCPP also has activity at the 5-HT7

360

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

receptor (To et al. 1995). Preliminary
data suggested that ritanserin, a drug
that blocks several serotonin receptors
(5-HT2A, 5-HT2C, 5-HT6, 5-HT7, 5-
HT1D) and the dopamine-2 (D2) recep-
tor, reduced some effects of mCPP in
healthy humans (Conn and Sanders-
Bush 1987; Seibyl et al. 1991; Shi et
al. 1995; To et al. 1995; Pauwels et
al. 1996; Boess et al. 1997). The lack
of specificity regarding the site of action
of both mCPP and ritanserin limits
the interpretation of the mechanisms
underlying the ethanol-like and craving
effects of mCPP.

The lack of specificity of both 5-HT
agonists and antagonists available for
clinical research has had important
consequences. Despite preliminary
promise (Meert 1994), subsequent
larger trials failed to find that ritanserin
was efficacious for alcoholism or heavy
social drinking (Naranjo et al. 1995;
Johnson et al. 1996). Similarly, ritan-
serin did not substantially modulate
ethanol intoxication in the laboratory
(Estevez et al. 1995). Given the limi-
tations in the specificity of ritanserin,
it is difficult to know whether this lack
of efficacy arises from shortcomings in
the selection of early-onset alcoholism
patients, the dose of ritanserin employed,
the selection of ritanserin as a 5-HT2c
antagonist, or the expectation that 5-
HT2C antagonists would be effective.

Other drugs that stimulate receptor
subtypes from the 5-HTi receptor
family have been studied in alcoholic
patients. One study reported a reduc-
tion in the human growth hormone
response to sumatriptan, a human 5-
HT1B agonist, in recently detoxified
alcoholic patients (Vescovi and Coiro

1997). Buspirone is a drug with 5-HT1A
agonist and D2 antagonist activity. Its
major metabolite, l-(2-pyrimidinyl)-
piperazine (1-PP), is also a potent a2
adrenoceptor antagonist. In animals,
5-HT1A agonists have had mixed
effects on ethanol self- administration
(Tomkins et al. 1994). Acutely, bus-
pirone appears to have no ethanol-like
effects (Griffith et al. 1986). With
chronic administration, it appears to
produce modest beneficial effects on
anxiety, alcohol craving, or alcohol
consumption in anxious alcoholic
patients (Bruno 1989; Malcolm et al.
1992; Tollefson et al. 1992; Kranzler
et al. 1994; Make et al. 1996).

5-HT Transporter
Antagonists

Insights into alcoholism have been pro-
vided by 5-HT transporter antagonists
(serotonin reuptake blockers). One
line of interest has built on evidence
that deficits in central 5-HT turnover
may contribute to ethanol consump-
tion in animals and humans. This line
of research has hypothesized that the
serotonergic antidepressants would
increase synaptic availability of 5-HT
and result in reduced ethanol con-
sumption. This view builds on the
parallels between reductions in cere-
brospinal fluid (CSF) 5-hydroxyin-
doleacetic acid (5-HIAA) in depression
and early-onset alcoholism. Molecular
genetic studies further increased interest
in genetic variation associated with the
function of the 5-HT transporter. Al-
though the findings have not been repli-
cated (Nielsen et al. 1996; Gelernter
et al. 1997), two groups have associated
5-HT transporter alleles with reduced

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NIAAA's Neuroscience and Behavioral Research Portfolio

central 5-HT function or poor impulse
control in alcoholic individuals
(Nielsen et al. 1994; Sander et al.
1995). However, one study failed to
find that alleles of the 5-HT trans-
porter were associated with alcoholism
(Gelernter et al. 1997).

One hypothesis guiding these studies
was that reduction in the efficacy or
availability of synaptic 5-HT resulting
from enhanced density or function of
the 5-HT transporter would contribute
to the constellation of behaviors asso-
ciated with early-onset alcoholism
(Daoust et al. 1991; Rausch et al.
1991; Mellerup et al. 1992; Faraj et
al. 1997). Alternatively, reduced den-
sity of 5-HT transporter binding in the
brain might reflect reductions in the
density of 5-HT terminals that might
contribute to reduced central 5-HT
function (Chen et al. 1991; Tiihonen
et al. 1997). Reductions in 5-HT
nerve terminal density might also par-
allel immunohistochemical evidence
of reductions in the number of pontine
serotonergic cell bodies (Halliday et
al. 1993).

5-HT transporter antagonists have
shown utility in the treatment of
alcoholics with comorbid psychiatric
disorders. Acute administration of
clomipramine did not have ethanol-
like effects in alcoholic patients with
comorbid panic disorder, although
this drug made these patients acutely
anxious (George et al. 1995). Other
studies have suggested that 5-HT
transporter antagonists have limited
efficacy for alcoholism and may make
early-onset patients worse (Naranjo
and Bremner 1993; Naranjo et al.
1994; Kranzler et al. 1996; Pettinati

1996). However, subgroups of patients
with comorbid depression may benefit
from treatment with 5-HT transporter
antagonists (Griffith et al. 1986;
Bruno 1989; Tollefson et al. 1992).

5-HT3 Antagonists

Preclinical research suggests that ethanol
stimulates 5-HT3 receptors and that 5-
HT3 antagonists may attenuate the
discriminative stimulus properties of
ethanol and ethanol self- administration
in animals (Grant and Barrett 1991;
Tomkins et al. 1995). The capacity of
5-HT3 antagonists to reduce the stim-
ulation of limbic dopamine systems by
drugs of abuse, including ethanol, may
contribute to this effect (Grant 1995).
In humans, the 5-HT3 antagonist
ondansetron did not robustly attenuate
the discriminative stimulus effects of
ethanol (Swift et al. 1996). A clinical
trial employing ondansetron found
some evidence of potential usefulness
of this drug (Sellers et al. 1994).

Modulation of 5-HT
Precursor Availability

There is a long history of interest in
the impact of 5-HT precursor availabil-
ity on clinical phenomena related to
alcoholism. This interest arises from the
fact that a 5-HT precursor, tryptophan,
is an essential amino acid and that the
hydroxylation of tryptophan is the rate-
limiting step in 5-HT synthesis (Fern-
strom 1983; Fernstrom and Fernstrom
1995). As a result there is a direct rela-
tionship between profound acute
changes in tryptophan availability and
reductions in brain 5-HT synthesis.
Under chronic conditions, this relation-
ship is altered as protein catabolism and

362

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

other adaptive changes can compensate
to varying degrees for reductions in
plasma free tryptophan.

There are conflicting data regarding
tryptophan levels in alcoholism. Reports
suggest that central and peripheral levels
of tryptophan are increased in recently
detoxified alcoholics (Beck et al. 1980,
1983; Farren and Dinan 1996). Other
studies suggest that plasma tryptophan,
its ratio to other neutral amino acids that
compete with it for transport into the
brain, or other indices of its transport
into the central nervous system are
reduced in recently detoxified alco-
holics (Branchey et al. 1981; Branchey
et al. 1985; Friedman et al. 1988;
Buydens-Branchey et al. 1989; Roy et
al. 1990). Although a component of
acute tryptophan reductions may be
related to malnutrition, the primary
effect thought to account for reductions
in plasma tryptophan in recovering
alcoholics is enhancement of hepatic
tryptophan pyrrolase (Badawy 1996).

The clinical significance of changes
in plasma tryptophan is not clear. Two
studies suggest that reductions in tryp-
tophan availability to the brain might
be related to memory impairments or
clinical characteristics associated with
early-onset alcoholism (Branchey et
al. 1985; Buydens-Branchey et al.
1989). However, it is possible that
this relationship is mediated by the
association of greater ethanol consump-
tion with both memory impairments
and early- onset alcoholism.

Clinically significant reductions in
plasma tryptophan may be achieved
using a tryptophan-free amino acid drink.
This technique was developed after it
became evident that substantial

dietary tryptophan restrictions had no
discernible behavioral effects in humans
(Delgado et al. 1989). The amino
acid drink stimulates the use of plasma
free tryptophan for protein synthesis
and can reduce levels by over 80 percent
(Young et al. 1985, 1988; Delgado et
al. 1989, 1990). The amino acid drink
reduces CSF levels of tryptophan and
5-HIAA in healthy individuals
(Carpenter et al. 1998). In contrast to
dietary tryptophan restriction, this level
of tryptophan depletion appears to
modulate mood — most notably, tran-
siently reversing the antidepressant
effects of serotonergic antidepressant
treatment in depressed patients. Tryp-
tophan depletion also reduced cue-
induced cocaine craving (Satel et al.
1995). However, it is not yet clear
whether tryptophan depletion produces
a clinically significant reduction in
alcohol cue-induced craving in sober
alcoholic patients (Petrakiset al. 1995).
Previous studies also failed to show
effects of modulation of plasma trypto-
phan levels on ethanol intoxication or
ethanol self- administration in popula-
tions other than early- onset alcoholics
(Pihl et al. 1987; Zacchia et al. 1987).
Tryptophan depletion has also been
reported to disinhibit or even promote
aggression in normal males (Pihl et al.
1995). However, this effect has not been
replicated in patients with aggression-
related problems (Young et al. 1988;
Salomon et al. 1994). Together, these
data suggest that profound alterations
in the availability may have some
influence on mood and behaviors
associated with alcoholism. However,
these studies question the clinical signif-
icance of the relatively modest changes

363

NIAAA's Neuroscience and Behavioral Research Portfolio

in the levels of plasma free tryptophan
in recovering alcoholic patients.

5-HT Overview

A number of clinical research strategies
have been used to study the contribu-
tions of 5-HT systems to behaviors
associated with alcoholism. As has
been suggested previously (Virkkunen
and Linnoila 1997), clinical neuro-
science research studies link alterations
in 5-HT function to problems with
impulse control that are most closely
associated with the early-onset subtype
of alcoholism. Regarding addictive
behaviors, the strongest finding is that
stimulation of population(s) of 5-HT
receptors, as of yet not conclusively
identified, by mCPP results in ethanol-
like interceptive cues that may contribute
to craving. The genetic underpinnings
of mCPP response may provide insights
into early-onset alcoholism. Further-
more, drugs that effectively antagonize
the ethanol-like components of mCPP in
humans should be evaluated as potential
pharmacotherapies for alcoholism.

GABA SYSTEMS

Contribution of GABAa
Receptors to the
Discriminative Stimulus
Effects of Ethanol

GABA systems, particularly in limbic
and cortical structures, have been
implicated in the rewarding effects of
ethanol administration (Koob et al.
1994). Ethanol facilitates GABAA
receptor function (Suzdak et al. 1988b).
Consistent with this view, the discrim-
inative stimulus effects of ethanol in

animals are shared among drugs that
facilitate GABAA receptor function,
particularly the barbiturates and benzo-
diazepines (Ticku et al. 1992; Grant
and Colombo 1993b). In particular,
these GABAA receptor-facilitating
drugs appear to substitute particularly
well for the discriminative stimulus
effects of low to moderate amounts of
ethanol consumption (Grant and
Colombo 1993a, 1993b).

The five GABAA receptor subunits
may combine in multiple configura-
tions. Particular attention has focused
on GABAA receptors bearing the al5
a6, and y2 subunits. The ax subunit is
distinctive in that it is selective for
sites above the spinal cord (Luddens
and Korpi 1995). As a result, drugs
that are selective for GABAA receptors
bearing the aj subunit, such as the
hypnotic Zolpidem, do not possess the
full range of motor, muscle-relaxing,
and perhaps autonomic effects associated
with drugs that nonselectively facilitate
GABAA receptor function. Limitations
in the ability of Zolpidem to substitute
for ethanol in drug discrimination para-
digms suggest that ethanol actions at
the GABAA receptors that do not bear
the a: subunit contribute to the dis-
criminative stimulus effects of ethanol
(Bienkowski et al. 1997). However, it
is not yet clear whether the non-o^
actions contribute significantly to
ethanol self- administration in animals
with inherited propensities for ethanol
consumption. The a6 subunit is
expressed in the cerebellum, where it
may contribute to the ataxic effects of
ethanol (Luddens and Korpi 1995).
This site received significant attention
when it was found that a (3-carboline

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Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

drug with benzodiazepine inverse ago-
nist properties, Ro 15-4513, reduced
some ethanol effects and ethanol self-
administration via this site (Suzdak et
al. 1988a; June et al. 1994).

GABA Systems

and Alcoholism Vulnerability

In humans, GABAA receptors have
been implicated in the vulnerability to
alcohol consumption. In particular,
reduced sensitivity to the facilitatory
effects of ethanol on GABAA receptor
function, particularly in neural systems
mediating adverse cognitive or motor
effects, may predispose vulnerable
individuals to consume more alcohol.
Individuals at risk for alcoholism may
not receive the expected dysphoric
interoceptive feedback signals to stop
drinking until they achieve higher
blood ethanol levels than do individuals
at lower risk for alcoholism (Schuckit
and Smith 1997).

To date, no specific gene has been
clearly implicated in a GABA-related
vulnerability for alcoholism. A haplotype
relative-risk study did not find evidence
associating either the a: or a3 subunit
genes with alcoholism, although sug-
gestive data supporting further study
were obtained for the latter gene in an
association study (Parsian and Cloninger
1997). The strongest tie between
GABAA receptor involvement and the
vulnerability to alcoholism has come
from studies evaluating ethanol and
benzodiazepine effects in populations
at high risk for developing alcoholism.
Most (Cowley et al. 1994; Schuckit
1994; Schuckit and Smith 1996;
Schuckit et al. 1996, 1997), but not
all (de Wit and McCracken 1990), of

these studies found that male offspring
of alcoholics have reduced sensitivity to
the cognitive, behavioral, and motor
effects of both benzodiazepines and
ethanol. Similarly, individuals at risk
for alcoholism exhibited blunted cere-
bellar metabolic inhibition, but not
cortical metabolic inhibition, follow-
ing a dose of lorazepam (Volkow et al.
1995). However, there may be greater
sensitivity to or preference for the
euphoric effects of benzodiazepines in
this population (Cowley et al. 1992,
1994), although these effects are not
uniformly replicated (de Wit 1991).
The rewarding effects of ethanol and
benzodiazepines in humans may be
increased in anxious individuals, who
tend to experience more pronounced
anxiolytic effects of these drugs
(Chutuape and de Wit 1995).

Clinical Evidence of GABA
Dysregulation in Alcoholism

Several clinical studies suggest that the
regulation of brain GABA systems
may differ in alcoholic and nonalcoholic
populations. To date there has been
very little direct study of the potential
contributions of dysregulation of cor-
tical GABA systems to behaviors asso-
ciated with alcohol consumption in
alcoholic patients. Thus, a relatively
large group of hypotheses remain to
be tested from a clinical perspective:

( 1 ) that reductions in GABAA receptor
density (related to changes in the
expression of particular GABAA receptor
subunits) or function convey a vulner-
ability to greater alcohol consumption,

(2) that reductions in GABA release
combined with reductions in GABAA
receptor density convey a vulnerability

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NIAAA's Neuroscience and Behavioral Research Portfolio

to acute and protracted ethanol with-
drawal symptoms that might contribute
to alcohol craving and relapse, and (3)
that normalization (i.e., treatment) of
abnormalities in GABA function in
alcoholic patients would reduce the
drive to consume alcohol. By implica-
tion, the genes that might modulate
each component of these pathophysi-
ological hypotheses would be impli-
cated in the vulnerability to alcoholism
and its treatment. The current research
database provides some support for these
hypotheses. However, it is quite limited
in its capacity to determine the extent to
which current findings are explained
by genetic factors, specific pharmacolog-
ical effects of ethanol on brain GABA
systems, and other effects of ethanol
on the brain, including neurotoxicity.
There are no studies that directly
evaluate abnormalities in brain GABA
turnover in alcoholism or in vulnerable
populations. Indirect evidence of
GABAergic abnormalities comes from
measurements of GABA levels in the
plasma, CSF, and brain. In offspring
of alcoholics there are conflicting
reports that suggest that plasma GABA
may be low (Moss et al. 1990), elevated
(Garbutt et al. 1995), or unchanged
(Cowley et al. 1996). Ethanol has been
reported to elevate plasma GABA levels
in high-risk populations with low
baseline levels (Moss et al. 1990). In
contrast, in a group of high-risk offspring
who did not show baseline reductions,
benzodiazepines had no effects (Cowley
et al. 1996). Plasma GABA in this high-
risk population may correlate with behav-
ior traits, such as novelty seeking, that
are also associated with vulnerability
to alcoholism (Cowley et al. 1996).

GABA levels appear to be reduced
in plasma, CSF, and brain in recently
detoxified alcoholics. In patients, plasma
and CSF studies found reductions in
GABA levels in alcoholics over the
first month of detoxification (Coffman
and Petty 1985; Petty et al. 1993;
Adinoff et al. 1995). In these patients,
low plasma GABA levels predicted
relapse (Petty et al. 1997), perhaps
because low GABA levels were associ-
ated with protracted abstinence symp-
toms. Although there are some
questions regarding the origin and
implications of plasma GABA, CSF
and brain GABA levels show some
similar patterns of regulation (Petty et
al. 1987). The decline in CSF GABA
levels may not drop significantly
below control levels (G.D. Goldman
et al. 1981; Hawley et al. 1981; Petty
et al. 1987; Roy et al. 1990). CSF
GABA levels in alcoholics are not
influenced by levels of comorbid
depression (Roy et al. 1991). How-
ever, CSF studies are prone to error
because of a gradient in GABA levels
across CSF samples and because CSF
GABA levels are influenced by age
(Grove et al. 1982, 1983). Also, brain
and CSF levels of GABA may corre-
late (Bohlen et al. 1979). However, it
is possible that significant, perhaps
regionally localized, differences in
brain GABA levels between patients
and control subjects may not be
reflected in CSF studies. To date, a
single report suggests that occipital
cortex GABA levels are reduced in
recently detoxified alcoholic patients
assessed in vivo using [:H] magnetic
resonance spectroscopy (MRS) (Behar
etal. 1999).

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Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

Preclinical studies provide insights
into mechanisms that may underlie
alterations in brain GAB A levels. With
long-term administration, GABA lev-
els decline and drop below baseline
(Sytinsky et al. 1975; Kulonen 1983).
Reduced GABA release and stimula-
tion of glutamic acid dehydrogenase
have been implicated in the rise of
brain GABA levels produced by acute
ethanol administration (Hunt 1983;
Sherif et al. 1997). In contrast, enhan-
ced activity of the catabolic enzyme
GABA transaminase (GABA-T) has
been implicated in subcortical but not
cortical reductions in GABA levels
(Sherif etal. 1994, 1997).

To date, insights into GABAA recep-
tor changes in alcoholism come pri-
marily from receptor binding studies.
This reflects both the development of
in vivo neuroreceptor imaging strate-
gies and the dearth of postmortem
studies of GABA-related gene expres-
sion in brain tissue from alcoholic
patients or high-risk populations
(Buck 1996). Preclinical research
describing alterations in GABAA
receptor subunit gene expression,
binding, and function has produced a
complicated picture. It appears that the
expression of some subunits, such as the
ax, a2, and a3 subunits, are decreased,
while others are unchanged or increased
(Devaud et al. 1995b). Similarly,
there is evidence of both reductions
and increases in binding to GABAA
and benzodiazepine sites (Ticku and
Burch 1980; Hemmingsen et al. 1982;
Ticku et al. 1983; Tamborska and
Marangos 1986; Mhatre and Ticku
1989; Buck et al. 1991; Montpied et
al. 1991; Montpied and Paul 1992;

Ticku and Mhatre 1992; Mhatre and
Ticku 1993). Some postmortem stud-
ies have found reductions in GABAa/
benzodiazepine binding, but several
studies had conflicting results (Tran et
al. 1981; Freund and Ballinger 1988;
Kril et al. 1988; Freund and Ballinger
1991; Korpi 1994; Lewohl et al. 1996,
1997; Tanda et al. 1997). Some of
the variability between studies may
reflect a compensatory up-regulation
in GABAA/benzodiazepine binding
that may follow alcoholism-related
neurotoxicity (Dodd et al. 1996).

Three studies have now reported ben-
zodiazepine binding data using PET
and single photon emission computed
tomography (SPECT). One small study
(five patients, five control subjects)
failed to find differences in [nC]flum-
azenil binding (Litton et al. 1993).
Two larger studies (Gilman et al.
1996; Abi-Dargham et al. 1998)
reported reductions in frontal and
anterior cingulate gyrus benzodi-
azepine binding using [nC]flumazenil/
PET and [123I]iomazenil/SPECT,
respectively. There is evidence of
reduced metabolic sensitivity of these
regions in alcoholics in response to
lorazepam (Volkow et al. 1993). This
blunting did not rapidly recover with
sobriety (Volkow et al. 1997), raising
the possibility that this deficit
reflected genetic vulnerability or irre-
versible toxicity.

A number of technical strategies are
emerging to facilitate the evaluation
of the role of neurotoxicity in GABA-
related functional and biochemical
neuroimaging. First, across imaging
modality, assessments should adjust
for atrophic changes in cortical gray

367

NIAAA's Neuroscience and Behavioral Research Portfolio

matter based on structural magnetic
resonance imaging techniques for sep-
arating gray matter, white matter, and
CSF (Hetherington et al. 1996). Sec-
ond, proton MRS may play a role in
providing insights into neuronal viabil-
ity, through assessments of metabolite
levels in gray matter N-acetylaspartate
and creatine (Tsai et al. 1995; Jagan-
nathan et al. 1996; Behar et al. 1999).
Phosphorus MRS may also provide
insights to phospholipid synthesis and
catabolism through the measurement
of phosphomonoesters and phospho-
diesters (Meyerhoffet al. 1995).

Psychopharmacological studies have
provided some insights into the involve-
ment of GABA systems in withdrawal
and relapse in alcoholic patients. Clearly,
drugs facilitating GABAA receptor
function, including benzodiazepines,
barbiturates, and anticonvulsants, are
effective clinically in suppressing acute
ethanol withdrawal symptoms (Nutt
et al. 1989; Stuppaeck et al. 1996).
Preclinical studies suggest that the
benzodiazepine antagonist flumazenil
may reduce the ataxic effects of ethanol
and reduce the vulnerability to with-
drawal seizures in dependent animals
(Buck et al. 1991). In humans, flumaze-
nil also has been reported to reduce
intoxication associated with ethanol at
doses greater than what is required to
antagonize benzodiazepine effects
(Lheureux and Askenasi 1991). How-
ever, this drug does not produce ethanol
withdrawal symptoms in alcohol-
dependent individuals (Potokar et al.
1997). Ethanol dependence contrasts
with benzodiazepine dependence, where
flumazenil response is variable but more
likely to precipitate withdrawal in animals

and humans (Griffiths et al. 1993;
Gerra et al. 1996; Saxon et al. 1997;
Wala et al. 1997). These findings may
be consistent with preclinical studies
that suggest that adaptations to chronic
ethanol administration include changes
in the expression of GABAA receptor
subunits that do not possess the ben-
zodiazepine binding site (Bosio et al.
1982; De Bias 1996). Lastly, the
homotaurine derivative acamprosate is
structurally related to GABA, and its
efficacy in reducing ethanol consump-
tion in patients may be related to its
modulatory effects on brain GABA
function (Chick 1995).

Neurosteroids
and Alcoholism

There is growing interest in the role of
neuroactive intermediates in sex
steroid synthesis and metabolism in
alcoholism. Among these compounds,
allopregnanolone has received the
greatest amount of study. However,
other compounds, such as pregenolone
sulfate and dihydroepiandrosterone, also
are of interest (Melchior and Allen
1992; Melchior and Ritzmann 1992).
Allopregnanolone compound binds
to the steroid anesthetic site of the
GABAA receptor, where it acts as a co-
agonist (Luddens and Korpi 1995).
Subsequent studies indicate that allo-
pregnanolone shares discriminative
stimulus properties with ethanol and
suppresses the ethanol abstinence syn-
drome in animals (Ator et al. 1993;
Devaud et al. 1995&; K. Grant et al.
1996#). Allopregnanolone also has
anxiolytic and sedative properties, and
circulating levels may potentiate the
effects of ethanol (Brot et al. 1995,

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Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

1997). Allopregnanolone levels, like
those of its precursor, progesterone,
show marked variation during the
menstrual cycle. In late luteal phase
allopregnanolone levels drop, and
these reductions have been related to
mood disturbances during this phase
of the menstrual cycle (Rapkin et al.
1997). Similarly, there has been signif-
icant interest in the possibility that
reductions in neurosteroid levels in
the late luteal phase might contribute
to increased intensity of the discrimi-
native stimulus properties of ethanol
(Grant et al. 1997a) and increased
alcohol consumption in women dur-
ing this phase of the menstrual cycle
(Charette et al. 1990; Allen 1996).
Supporting this view, higher allopreg-
nanolone levels in the late luteal phase
are associated with reduced triazolam
self- administration in women (de Wit
and Rukstalis 1997). Thus, cyclical
variation in neurosteroid levels or
their consequences could be an
important focus for pharmacotherapy
development for female alcoholics.

GABA Overview

GABAA receptors are clearly an impor-
tant target for the rewarding effects of
ethanol in the brain. There is growing
evidence that reduced sensitivity to
ethanol effects on GABA systems may
contribute to a familial vulnerability
to alcoholism. This vulnerability is
hypothesized to be mediated, in part,
by reduced sensitivity to the dysphoric
consequences of ethanol actions at
GABAA receptors and preservation of
its rewarding effects. It is not yet clear
whether, as with 5-HT systems,
reduced sensitivity to the GABAergic

component of ethanol response is lim-
ited to a subtype of familial alcoholism.
The involvement of GABA systems in
the rewarding effects of ethanol sug-
gests that GABAergic systems may be
involved in the component of ethanol
craving involving the pairing of envi-
ronmental cues with ethanol effects in
the brain. However, this has not
received sufficient clinical study to
draw direct inferences.

Chronic ethanol administration
produces adaptations within GABA
systems that contribute to tolerance
and withdrawal. GABA agonists
clearly are effective in the treatment of
acute withdrawal symptoms. Because
a component of craving may be driven
by acute or protracted withdrawal
symptoms, disturbances in GABA sys-
tems may also contribute to craving
and relapse. Furthermore, neurosteroids
may provide a novel and important
target for medication development for
female alcoholics.

GLUTAMATE SYSTEMS

Contribution of the NMDA
Glutamate Receptor
Antagonist Properties
of Ethanol to the
Discriminative Stimulus
Effects of Ethanol

Glutamate is the most prevalent exci-
tatory neurotransmitter in the brain,
and it mediates most cortico-cortical
communication. Glutamate receptors
are divided into three classes of recep-
tors that bear ion channels and one
family of metabotropic receptors cou-
pled by G proteins to intracellular sec-

369

NIAAA's Neuroscience and Behavioral Research Portfolio

ond messenger systems. Through the
range of doses associated with human
intoxication, ethanol preferentially
and dose-dependently blocks the N-
methyl-D-aspartate (NMDA) subclass
of glutamate receptors (Lovinger
1997). This action is not competitive
with glutamate and may be mediated
by ethanol binding to a hydrophobic
pocket on the receptor (Peoples and
Weight 1995) and inhibitory effects
on the strychnine-insensitive glycine
co-agonist site of these receptors
(Michaelis et al. 1996).

A growing body of research sug-
gests that NMDA receptor antago-
nism contributes to the discriminative
stimulus effects of ethanol in animals
and humans. In drug discrimination
paradigms, animals identify NMDA
antagonists as being similar to ethanol
(Grant and Colombo 1993«; K. Grant
et al. 1996^). The highest degree of

similarity between NMDA antagonist
effects and ethanol effects is observed
when animals are trained to recognize
ethanol doses comparable to several
drinks of alcohol in humans.

The NMDA antagonist ketamine
produced dose-related ethanol-like
effects in recently detoxified alcoholic
patients (Krystal et al. 1998&).
Ethanol-like effects, measured by the
Sensation Scale, a visual analog scale of
euphoria, and the Biphasic Alcohol
Effects Scale were more prominent at
0.5 mg/kg than at 0.1 mg/kg. The
response to ketamine was rated signif-
icantly more similar to the effects of
ethanol than it was to the effects of
marijuana or cocaine (figure 2).

At 0.1 mg/kg, ketamine effects
were rated as similar to one to two
standard ethanol drinks (-15 mL
absolute ethanol); at 0.5 mg/kg, its
effects were rated similar to eight to

Base 10 20 30 40 50 60 70 80

Time (min)

i 10 20 30 40 50 60 70 80

Time (min)

i 10 20 30 40 50 60 70 80

Time (min)

Figure 2. Similarity of the effects of placebo (open circles), ketamine 0.1 mg/kg (filled circles),
and ketamine 0.5 mg/kg (filled squares) to ethanol (left), marijuana (middle), and cocaine (right)
in alcoholic patients (n = 20). Values are expressed as mean ± SEM. Ketamine effects were signifi-
cantly more similar to ethanol than both marijuana and cocaine by post hoc contrast (Fl = 6.7,
p = 0.02). Reprinted with permission from Krystal, J.H.; Petrakis, I.L.; Webb, E.; Cooney, N.L.;
Karper, L.P.; Namanworth, S.; Trevisan, L.A.; and Charney, D.S. Dose-related ethanol-like effects
of the NMDA antagonist, ketamine, in recently detoxified alcoholics. Archives of General Psychiatry
55:354-360, April 1998. Copyright 1998, American Medical Association.

370

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

nine standard ethanol drinks. Keta-
mine effects were rated as more simi-
lar to the sedative-descending limb
than the stimulant-ascending limb
effects of ethanol. Ketamine did not
stimulate ethanol craving in patients.
Perhaps contributing to this effect,
patients anecdotally reported that the
low dose of ketamine was not suffi-
ciently ethanol-like and that the high
dose of ketamine sated their desire for
ethanol. Recent data from our labora-
tory also suggest that a high dose of
the partial agonist of the strychnine -
insensitive glycine co-agonist site of
the NMDA receptor, D-cycloserine
(1,000 mg), produces ethanol-like
subjective effects in healthy subjects
and recently detoxified male early-
onset alcoholics; glycine (0.3 g/kg), a
full agonist of this site, does not pro-
duce these effects (Krystal et al.
1998a). Like ketamine, D-cycloserine
did not produce alcohol craving in
patients. These data further implicate
NMDA receptors in ethanol effects.

Modulation of Human
Ethanol Intoxication by
Glutamatergic Agents

D-cycloserine modulates ethanol
intoxication in healthy human subjects
(Trevisan et al. 1995). In animals,
ethanol potentiates the action of NMDA
antagonists (Toropainen et al. 1997).
Also, ethanol effects in neurochemical
and behavioral paradigms are potenti-
ated by high-dose glycine partial agonists
or antagonists and attenuated by
glycine agonists and low- dose glycine
partial agonists (Khanna et al. 1995;
Moraes Ferreira and Morato 1997).
Complementing these studies, prelim-

inary data suggest that D-cycloserine,
500 mg, potentiated ethanol intoxica-
tion (Trevisan et al. 1995). Also, there
was evidence that cycloserine and
ethanol had additive amnestic effects.
However, D-cycloserine reduced ethanol
intoxication beginning approximately
6 hours after D-cycloserine administra-
tion, when its levels declined. D-
cycloserine did not alter ethanol levels
at any time point. These data are con-
sistent with the view that D-cycloser-
ine has glycine/NMDA antagonist-like
effects at oral doses of 500 mg or
greater. At lower doses, D-cycloserine
primarily has agonist-like effects
(D'Souza et al. 1995). These data
suggest a potential role for NMDA
antagonists in reducing drinking in
recovering alcoholics by potentiating
the adverse effects of ethanol intoxica-
tion. They may also suggest that
glycine agonists have therapeutic
potential arising from their capacity to
attenuate ethanol intoxication.

Association of Human
Ethanol Dependence
With Protracted Up-
Regulation of NMDA
Receptor Function
and Altered Responses
to NMDA Antagonists

In humans, as in animals, acute
ethanol withdrawal activates glutamate
systems. Human ethanol withdrawal is
associated with increases in CSF gluta-
mate levels (Tsai et al. 1995).
Increases in extracellular glutamate
levels may be consistent with regional
reductions in binding to the glutamate
transporter (Cummins et al. 1990) or
increased release (Keller et al. 1983).

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NIAAA's Neuroscience and Behavioral Research Portfolio

Increased glutamate levels might be
expected to down-regulate NMDA
receptors. However, the predominant
effect appears to be NMDA receptor
up-regulation in animal studies (Gulya
et al. 1991). A postmortem study
found that the Bmax of NMDA, but
not a-amino-3-hydroxy-5-methylisox-
azole propionic acid (AMPA), receptors
was increased in the frontal cortex of
alcoholics (Freund and Anderson 1996).
In the hippocampus, NMDA receptor
affinity, but not density, was increased
(Michaelis et al. 1990). The persistent
alterations in NMDA receptor function
may potentiate the neurotoxic and
proconvulsant effects of increased glu-
tamate release during withdrawal. How-
ever, the etiology of persistent NMDA
receptor up-regulation in alcoholics is
not clear and may also reflect a
genetic predisposition to alcoholism
or alcoholism-related neurotoxicity.
Regardless of etiology, long-lasting
facilitation of NMDA receptor func-
tion in alcoholics may have ongoing
neurological implications, including
increased seizure risk (Brown et al.
1988; Lechtenberg and Worner
1991) and startle hyperreflexia (Krys-
tal et al. 1997).

Alterations in NMDA receptor func-
tion in recovering alcohol-dependent
patients may be associated with a shift
in the reward valence of the NMDA
antagonist component of ethanol
response. Recently detoxified early-
onset male alcoholic patients show
marked reductions in their sensitivity to
the perceptual and psychotogenic effects
of ketamine (Krystal et al. 1995). In
contrast, these data suggest that these
patients exhibited a trend for increased

euphoric responses to this drug. Data
from another study suggest that these
patients also showed blunted responses
to the effects of high-dose (1,000 mg)
D-cycloserine (Krystal et al. 1998&).
Together, these data suggest that
recently detoxified alcoholics are less
sensitive to the dysphoric effects of
NMDA antagonists, while the euphoric
effects of these drugs are preserved.
Shifts in the reward valence of other
NMDA antagonists in early-onset
alcoholic patients may also apply to
the NMDA antagonist component of
ethanol response. If so, then the blunted
cognitive and behavioral effects of
ethanol in alcoholic and high-risk
populations described earlier in the con-
text of GABA systems may, in part, also
reflect a glutamatergic disturbance.

However, the current clinical data-
base does not yet allow one to choose
from among the potential pathophysi-
ological processes that might result in
altered NMDA antagonist response in
alcoholic patients. One possibility is
that reduced NMDA receptor sensi-
tivity reflects cross -tolerance between
ethanol and other NMDA antagonists.
Although this cross-tolerance has
been described in preclinical research
(Fitzgerald and Nestler 1995; Grant
and Lovinger 1995), it is not yet clear
whether this concept is applicable to
the clinical studies. A second possibility
is that reduced ketamine sensitivity
reflects neurotoxic changes associated
with alcoholism. A third explanation
could be that alterations in glutamatergic
regulation reflect a pathophysiological
process predisposing individuals to
alcoholism. Additional research will be
needed to untangle these possibilities.

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Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

Acamprosate: A
Pharmacotherapy for
Alcoholism With Both
NMDA Antagonist
and Agonist Properties

Acamprosate is a homotaurine deriva-
tive without ethanol-like behavioral
effects that reduces ethanol consump-
tion in animals (Spanagel et al.
1996/j, 1996^). Several European
studies suggest that this drug is a
highly promising pharmacotherapeu-
tic support for reducing alcohol con-
sumption in alcoholics (Paille et al.
1995; Sass et al. 1996; Whitworth et
al. 1996; Pelc et al. 1997).

Acamprosate has been reported to
have NMDA antagonist activity and to
suppress ethanol withdrawal (Zeise et al.
1993; Putzke et al. 1996). Based on
these findings, some researchers have
hypothesized that acamprosate may
reduce alcohol consumption, in part,
by reducing alcohol withdrawal symp-
toms that contribute to craving (Lit-
tleton 1995). In contrast, acamprosate
may also reduce alcohol consumption
by potentiating the dysphoric effects
of ethanol. If reduced ketamine sensi-
tivity in alcoholics reflects a reduction
in the sensitivity to the dysphoric
effects of the NMDA antagonist com-
ponent of ethanol, then maintenance
treatment with acamprosate could
potentiate those very effects of
ethanol that limit alcohol consump-
tion in nonalcoholic populations.
However, acamprosate also appears to
have NMDA agonist effects in pre-
clinical research paradigms (Madamba
et al. 1996). If so, then acamprosate
may reduce the behavioral effects of
ethanol via this mechanism.

Glutamate Overview

The clinical literature now appears to
support the basic literature in suggest-
ing that NMDA antagonism is relevant
to the discriminative and perhaps the
rewarding effects of ethanol. Although
drugs that selectively block NMDA
receptors are unlikely to have therapeu-
tic potential in the treatment of alcohol-
ism due to their potential to produce
psychosis, other glutamatergic agents
may be acceptable for this purpose.
One potential target for these drugs is
the strychnine-insensitive glycine site
of the NMDA receptor complex.
Acamprosate, a drug with GAB A ago-
nist, NMDA antagonist, and NMDA
agonist effects, does not have ethanol-
like discriminative effects. Its mecha-
nism of action is currently unclear.

OPIATES

Preclinical studies suggest that the
behavioral effects of ethanol are mod-
ulated by opiate antagonists (Herz
1997). The opiate -facilitating actions
of ethanol may contribute to its
inhibitory effects on nucleus accum-
bens glutamatergic function (Nie et
al. 1994). Opiate antagonists reduced
ethanol self- administration in rodents
and primates (Myers et al. 1986;
Volpicelli et al. 1986; Honkanen et al.
1996). Similarly, most studies suggest
that naltrexone appears to reduce the
rewarding effects of ethanol and alco-
hol consumption in social drinkers
(Swift et al. 1994; Davidson et al.
1996; Doty et al. 1997). Also, naltrex-
one maintenance appears to reduce
the pleasurable aspects of alcohol con-
sumed during treatment for alco-

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NIAAA's Neuroscience and Behavioral Research Portfolio

holism (Volpicelli et al. 1995; O'Malley
et al. 1996). This property appears to
contribute to the capacity of opiate
antagonists to reduce alcohol con-
sumption in alcoholic patients
(O'Malley et al. 1992; Volpicelli et al.
1992; Mason et al. 1994).

The contributions of endogenous
opiate systems to the rewarding effects
of ethanol are further supported by
evidence of abnormalities in these sys-
tems in alcoholic patients. However,
the current data do not yield a clear
picture of these abnormalities. Post-
mortem studies have described both
increased u. receptor density (Ritchie
and Noble 1996) and decreased [i
receptor affinity (Tabakoff et al.
1985). CSF and plasma studies have
also suggested the existence of reduc-
tions in (3-endorphin levels and
ethanol -stimulated increases in plasma
(3-endorphin levels in some (Borg et
al. 1982; Genazzani et al. 1982;
Gianoulakis et al. 1996), but not all,
studies (Petrakis et al. 1997).
Naltrexone reductions in ethanol
effects appear to be particularly evi-
dent in individuals at high risk for
developing alcoholism (King et al.
1997). These data suggest a genetic
component underlying the efficacy of
naltrexone treatment for alcoholism.
To date, there have been negative
results from studies evaluating the
association of the proenkephalin
A gene and [i opiate receptor and
alcoholism (Chan et al. 1994; Bergen
et al. 1997).

Opiate Overview

Unlike the other systems reviewed here,
it remains controversial as to whether

opiate receptors are a direct target for
ethanol in the brain. However, opiate
receptor systems modulate most other
systems implicated in the rewarding
effects of ethanol. Perhaps through
these mechanisms, opiate antagonists
are effective in reducing the rewarding
effects of ethanol and reducing con-
sumption. A growing number of studies
suggest that a genetic factor associated
with the vulnerability for alcoholism
may particularly distinguish those
individuals for whom naltrexone
reduces the rewarding effects of
ethanol. However, the gene underly-
ing this interactive effect has not yet
been identified.

CATECHOLAMINES

To date, catecholamine systems have
received relatively little human study in
relation to behaviors associated with
alcoholism, despite extensive preclinical
data implicating dopamine systems, in
particular, in the rewarding effects of
ethanol (Koob 1998). Both D1 and
D2 receptors are implicated in the
rewarding effects of ethanol and in
animal models for drug craving. Fur-
thermore, ethanol withdrawal is associ-
ated with reductions in limbic dopamine
function (Hunt and Majchrowicz
1983) in animal studies and reduced
CSF homovanillic acid in some, but
not all, clinical studies (Major et al.
1977). Noradrenergic systems are less
clearly implicated in the rewarding
effects of ethanol or craving. How-
ever, a2 adrenergic antagonists have
been reported to reduce some effects
of ethanol in rats (Lister et al. 1989;
Durcanetal. 1991).

374

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

There have been relatively few
human studies evaluating the contribu-
tions of catecholamine systems to
ethanol intoxication, craving, or self-
administration. Some studies used
strategies that did not distinguish among
catecholamines. For example, one
study found that catecholamine syn-
thesis inhibition, produced by a-
methyl-para-tyrosine, modestly reduced
ethanol intoxication in healthy
humans (Ahlenius et al. 1973). Simi-
larly, dexamphetamine and metham-
phetamine pretreatment either had no
effect or modestly potentiated ethanol
intoxication in humans (Perez- Reyes
et al. 1992; Mendelson et al. 1995).
Building on preclinical findings, clinical
studies suggest that the a2 adrenergic
antagonist yohimbine potentiated the
intoxicating effects of ethanol but did
not substantially alter the subjective
sense of euphoria associated with
intoxication (McDougle et al. 1995).
In recently detoxified early-onset alco-
hol-dependent patients, yohimbine
effects have a low degree of similarity
to ethanol effects (Krystal et al. 1994).
In this study, yohimbine also reduced
craving relative to placebo and mCPP.
Conversely, clonidine does not appear
to be effective in suppressing ethanol
cue-induced craving (Petrakis and
Krystal unpublished data).

Several studies document reduced
sensitivity of both dopamine and nora-
drenergic receptors in recently detoxified
patients. Reduced sensitivity of dopa-
mine receptors is suggested by blunted
growth hormone responses to apomor-
phine or bromocriptine (Balldin et al.
1985; Balldin et al. 1992; Farren et al.
1995«; Heinz et al. 1995). These

data are consistent with neuroimaging
data suggesting that the density of
dopamine transporter binding is pre-
served but striatal D2 receptor density
is decreased in alcoholic patients
(Volkow et al. 1996; Tiihonen, et al.
1997). Down-regulation of postsy-
naptic cc2 adrenergic receptors is sug-
gested by blunted growth hormone
responses to clonidine and increased
Cortisol responses to yohimbine (Glue
et al. 1989; Krystal et al. 1996). In
contrast, presynaptic noradrenergic
activity appears to normalize rapidly
following withdrawal (Borg et al.
1983). Similarly, the presynaptic com-
ponent of the noradrenergic response
to yohimbine is normal in patients
sober for approximately 1 month
(Krystal et al. 1996).

Genetic studies relating catechol-
amine receptor alleles to the vulnera-
bility to alcoholism have been a
promising but controversial research
strategy that has not yet born fruit.
Initial studies suggested that alleles of
the D2 receptor were associated with
alcoholism, particularly severe alco-
holism (Blum et al. 1990, 1991;
Parsian et al. 1991; Higuchi et al.
1994). However, subsequent studies
using more definitive techniques were
negative (Bolos et al. 1990; Gelernter
et al. 1991; Geijer et al. 1994; Gejman
et al. 1994; D. Goldman et al. 1997).
Reanalysis of the published studies also
suggests that the positive finding may
have arisen due to ethnic differences
between the patient and control pop-
ulations (Gelernter et al. 1993). A
subsequent study also suggested that
alleles of the D2 receptor might predict
an anticraving response to bromocrip-

375

NIAAA's Neuroscience and Behavioral Research Portfolio

tine (Lawford et al. 1995). However,
studies using dopamine agonists to
treat alcoholism have so far had limited
promise (Penick et al. 1996; Naranjo et
al. 1997). There are also interesting
potential associations between versions
of the D4 receptor and novelty-seeking
(Sander et al. 1997), with some negative
reports (Sullivan et al. 1998). However,
so far there is little evidence linking the
Dl5 D3, or D4 receptor genes to alco-
holism (Adamson et al. 1995; Gorwood
et al. 1995; Sander et al. 1995; Malhotra
et al. 1996; Muramatsu et al. 1996;
Chang et al. 1997; Parsian et al. 1997).

Catecholamine Overview

Of all systems in the brain, dopamine
systems are perhaps most strongly
linked to the rewarding properties of
drugs of abuse. However, dopaminer-
gic agents have not yet shown signifi-
cant promise in modulating the
behavioral effects of ethanol, reducing
ethanol craving, or preventing
ethanol consumption. One factor that
may contribute to this problem is the
absence of clinical studies focused on
the T)1 receptor. However, overall
dopaminergic contributions have
received relatively little attention.
Noradrenergic systems have been
studied in the clinical laboratory, but
a tentative finding suggesting that a2
adrenergic antagonists might potenti-
ate ethanol effects in healthy subjects
and reduce craving in patients has not
yet been followed by a clinical trial.

DISCUSSION

There is good news and bad news
regarding achievements in the study

of behaviors related to alcohol abuse.
The good news is that human laboratory
studies support the efficacy of naltrex-
one. Although these studies did not lead
to the development of naltrexone, it is
at least reassuring that these paradigms
may have the sensitivity to uncover
other agents that might be of therapeu-
tic use in the treatment of alcoholism.
Mostly, however, the clinical studies
have produced unanswered questions.
These questions, as a group, pertain to
the importance of mechanisms under-
lying the subjective effects of ethanol
to the treatment of alcoholism. In this
regard, the therapeutic potential of
ethanol effects on brain 5-HT,
GABA, NMDA, and catecholamine
systems does not appear to be fully
realized. Although the field of alco-
holism research seemingly should be
interested in drugs that blocked the
euphoric effects of mCPP, ketamine,
benzodiazepines, and ethanol, this
line of research (with the possible
exception of attempts to block ethanol
effects) has received vanishingly little
clinical study. A major obstacle to this
line of research is the limited availability
of selective agonists and antagonists.
These agents are often in Phase II or
III of testing, but are unavailable to
clinical researchers. It would be useful
for the National Institute on Alcohol
Abuse and Alcoholism (NIAAA) to
consider establishing a program that
parallels programs in the National
Institute of Mental Health and the
National Institute on Drug Abuse
designed to target particular agents of
high interest and facilitate their avail-
ability to clinical investigators to pursue
aims consistent with those of NIAAA.

376

Clinical Neuroscience Studies of Behaviors Associated With Alcoholism

A second line of research that is
beginning to be tapped is the use of
the laboratory to study the neurobiology
of triggers for relapse to alcohol use,
particularly alcohol-related cues, the
priming effects of alcohol consumption
on drinking, and the interactive effects
of stress (negative mood induction)
and alcohol cues. If, as for naltrexone,
clinical research can successfully move
from preclinical research to full-scale
clinical trials, then deficits in the clinical
research portfolio may not be an
immovable obstacle to medications
development for alcoholism. However,
if the differences between animal and
human pharmacology are important,
as illustrated by the difference between
the animal and human 5-HT1B, then
the deficits in the clinical research
arena may be reflected in the fact that
there are relatively few effective phar-
macotherapies for alcoholism.

Alcoholism research has also not
fully integrated functional neuroimaging
techniques to facilitate the study of the
neural circuitry underlying craving or
factors related to alcohol consumption.
Some progress has been made in study-
ing the circuitry of ethanol effects on
the human brain (de Wit et al. 1990;
Volkow et al. 1990). However, alco-
holism studies lag behind studies
attempting to identify the circuitry of
cocaine craving (Grant et al. 1996) A
critical step in alcoholism research will
be the integration of evolving studies
of the neural circuitry of behavior and
psychopharmacology.

The field of alcoholism research,
however, may be leading psychiatric
research in its sensitivity to subtypes
in bridging clinical molecular genetics

and other domains of clinical neuro-
science. From the descriptive perspec-
tive, there is great interest in the
relationship between the vulnerability
to early- onset alcoholism and altered
function within several systems in the
brain. The diversity of systems impli-
cated in the vulnerability to alcoholism
could very likely reflect the worst of all
possible worlds with respect to a quick
answer to the etiology and treatment of
alcoholism: multiple genes modulating
multiple neurotransmitter systems.
Regardless of the intrinsic complexity,
the bridging of pharmacology, behav-
ioral science, neuroimaging, and mole-
cular genetics remains the brightest
hope for a "home run" in the treat-
ment of alcoholism.

ACKNOWLEDGMENTS

This work was supported by research
funds from the Department of Veter-
ans Affairs and NIAAA (ROl
AA10121-01).

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396

Chapter 11

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

Gary Wand, M.D.

KEY WORDS: hypothalamus-pituitary axis; pituitary-adrenal axis; AODE (effects
ofAOD [alcohol or other drug] use, abuse, and dependence); psychological AODC
(causes of AOD use, abuse, and dependence); physiological stress; corticotropin
RH; adrenocorticotropic hormone; genetic marker of potential AODD (AOD use
disorder); Cortisol; endocrine system; AOD use susceptibility; dopamine; opioids;
naloxone; literature review

Stress threatens homeostasis and is
counteracted by a series of physiolog-
ical and behavioral responses that
improve the chances for survival
(Chrousos and Gold 1992). A success-
ful response to stress plays an important
role in maintaining good health and
well-being. The unique characteristics
of an individual's stress response are
the product of genetic and environ-
mental determinants. A hyperactive
stress response as well as an attenu-
ated response to stress is maladaptive.
Mounting evidence suggests that
abnormalities in the stress response
heighten an individual's vulnerability to
certain endocrine, metabolic, psychiatric,

and immunological disorders. There is
also growing evidence that nonalco-
holic persons at increased risk for the
future development of alcoholism
have abnormalities in the hypothalamic -
pituitary- adrenal (HPA) component
of the stress response. Moreover, the
performance of the HPA axis becomes
impaired as a result of chronic, heavy
ethanol ingestion. Animal studies
have demonstrated that stress alters
mesolimbic dopamine accumulation,
resulting in aberrant responses to
ethanol and certain other drugs of
abuse. For these reasons it is important
to more fully understand the nature
of the HPA axis in alcohol-dependent

G. Wand, M.D., is professor of medicine and psychiatry, Departments of Medicine and Psychiatry,
Division of Endocrinology, Ross 863, The Johns Hopkins University School of Medicine, 720 Rutland
Ave., Baltimore, MD 21205.

397

NIAAA's Neuroscience and Behavioral Research Portfolio

persons and in their nonalcoholic
family members.

PHYSIOLOGY
OF THE HPA AXIS

The response to stress is generated
within two well-defined brain regions,
the hypothalamus and the brainstem.
The hypothalamic center includes cor-
ticotropin-releasing hormone (CRH)
and arginine vasopressin (AVP) neu-
rons in the paraventricular nucleus.
The brainstem center includes CRH
neurons of the paragigantocellular
and parabranchial nuclei of the
medulla, as well as the locus coeruleus
and other catecholaminergic neurons
of the medulla and pons (Tsigos and
Chrousos 1995). The HPA axis, the
efferent sympathetic/adrenomedullary
system, and components of the para-
sympathetic system constitute the
peripheral branches of the stress system
(Tsigos and Chrousos 1995).

Following perception of stress the
hypothalamus releases CRH, AVP,
and other adrenocorticotropin (ACTH)
secretagogues into the pituitary portal
circulation, resulting in release of ACTH
from the pituitary gland. In turn,
ACTH initiates the release of the adrenal
androgens and glucocorticoids. Glu-
cocorticoids prepare the organism for
stress by modulating fuel metabolism
and thereby increasing glucose and
fatty acid availability for the central
nervous system (CNS). Another
important function of the HPA axis is
its bidirectional interactions with the
immune system (Rivier 1993).
Inflammatory cytokines (e.g., tumor
necrosis factor alpha, interleukin-1,

and interleukin-6) and other humoral
mediators of inflammation stimulate
the HPA axis. In response, glucocorti-
coids inhibit cytokine production and
virtually all components of the immune
reaction, thus preventing an over-
whelming and potentially harmful
immune response to stressors. Finally,
glucocorticoids participate in their
own production through binding to
glucocorticoid receptors in the pitu-
itary, hypothalamus, and hippocam-
pus, inhibiting the synthesis and
release of CRH and ACTH.

PATHOPHYSIOLOGY
OF THE HPA AXIS

A healthy HPA axis response to stress
is characterized by rapid onset and lim-
ited duration. The time-limited nature
of the event ensures that the accom-
panying behavioral, metabolic, and
immunosuppressive effects induced by
CRH and Cortisol are beneficial and
have no adverse sequelae (Tsigos and
Chrousos 1995). Excessive HPA axis
responses to stress are maladaptive.
Sustained hypercortisolism initiates a
spiral-like cycle of HPA hyperactivity
(e.g., hypercortisolism begets hyper-
cortisolism). This is because pro-
tracted exposure to Cortisol is toxic
and injures the glucocorticoid nega-
tive feedback set-point, resulting in
worsening hyperactivity over time.

A well-studied example of inappro-
priate hypercortisolism is melancholic
depression characterized by dysphoria,
sustained activation of the HPA axis,
immunosuppression, and osteoporosis
(Gold et al. 1988^, 1988b). Cortisol
production is elevated, CRH levels in

398

The HPA Axis: Changes and Risk for Alcoholism

cerebrospinal fluid are increased, and
there are more CRH neurons found in
depressed patients on autopsy, all con-
sistent with overactivity of CRH neu-
rons (Raadsheer et al. 1994). Other
disorders associated with sustained
activation of the HPA axis include
anorexia nervosa (Chrousos and Gold
1992), obsessive -compulsive disorder
(Insel et al. 1982), panic and anxiety
disorders (Gold et al. 1988c), excessive
exercising (Luger et al. 1987), visceral
obesity (Pasquali et al. 1993), the pre-
menstrual tension syndrome (Robin et
al. 1990), and chronic active alco-
holism (Wand and Dobs 1991).
Moreover, elevated Cortisol levels have
been found in preschool children with
poor inhibitory control and in associa-
tion with boredom, impatience, irrita-
tion, fear, and anxiety (Stansbury and
Gunnar 1994).

Why should abnormal states of CRH
and/or Cortisol secretion compromise
health? First, sustained exposure to
elevated levels of glucocorticoids can
have detrimental effects on bone and
muscle mass as well as on growth,
reproductive status, and immune func-
tion. There is evidence indicating that
hypercortisolism seen in Cushing's
syndrome and depression decreases
immune function and bone mass
(Chrousos and Gold 1992). Neuro-
toxicity is another important conse-
quence of increased glucocorticoid
production. Elevated levels of gluco-
corticoids impair CNS energy metab-
olism, making the brain vulnerable to
injury. Hippocampal damage is
observed in conditions linked with
hypercortisolism, an event that may
alter learning, memory, and regulation

of the HPA axis (Sapolsky 1996). In
fact, studies have shown a negative cor-
relation between hippocampal volume
and ( 1 ) plasma Cortisol levels in Cush-
ing's syndrome, (2) duration of depres-
sion in patients with affective illness,
and (3) months of combat exposure
in patients with posttraumatic stress
disorder (Sapolsky 1996). Since CRH
modulates behavioral, neuroendocrino-
logical, and immunological responses,
altered production of CRH could also
explain certain manifestations in the
syndromes noted above.

Pathological consequences resulting
from altered HPA axis dynamics are
not limited to states of hyperfunction.
An attenuated HPA axis response to
stress can also be problematic. For
example, patients with atypical, seasonal
depression and chronic fatigue syn-
drome have decreased activity of the
HPA axis (Chrousos and Gold 1992).
Patients diagnosed with fibromyalgia
and fatigue have decreased urinary free
Cortisol values (Griep et al. 1993). Ani-
mal models have shown that a weak
HPA axis response to stress results in
excessive cytokine activation and a
predisposition for certain autoimmune
disorders (Sternberg et al. 1992).

ETHANOL

AND THE HPA AXIS

Ethanol has several contrasting effects
on the HPA axis. In social drinkers,
acute ethanol ingestion activates the
HPA axis, especially when intoxication
is accompanied by gastrointestinal
symptoms. Chronic alcohol abuse is
associated with activation of the HPA
axis, most profoundly observed during

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periods of acute withdrawal. However,
during early and even sustained absti-
nence, the HPA axis is dampened. In
the alcohol-dependent person, cyclical
episodes of intoxication and withdrawal
provoke cycles of hyper- and hypoac-
tivity, contributing to tissue injury,
craving, and relapse.

Acute Effects of Ethanol
on the HPA Axis
in Social Drinkers

In 1966, Mendelson and Stein demon-
strated that ingestion of ethanol
increased plasma Cortisol concentration.
Subsequently, investigators more fully
characterized the biological mecha-
nisms of ethanol-induced HPA axis
activation in rodents (Rivier et al.
1984; Thiagarajan et al. 1989). In the
rat, ethanol activates the HPA axis by
inducing release of CRH from the
hypothalamus (Rivier et al. 1984).
Although never directly proved in
humans, ethanol is thought to induce
ACTH and Cortisol release through
similar mechanisms.

Oral administration of ethanol to
humans stimulates the HPA axis as it
does in rodents. However, the response
is not clearly dose dependent, and is
most likely observed when blood levels
are greater than 100 mg%. More recent
studies have shown that intoxicating
doses of ethanol per se do not result in
activation of the HPA axis. However,
subjects experiencing nausea or vomit-
ing as a result of the ethanol exposure
are much more likely to develop sig-
nificant rises in ACTH and Cortisol
(Waltman et al. 1993; Aguirre et al.
1995; Inder et al. 1995/;). Interestingly,
mild levels of intoxication (blood

ethanol level 75 mg%) result in blunted
ACTH and Cortisol responses to
exogenously administered CRH, sug-
gesting that even acute ethanol expo-
sure impairs the ability of the HPA
axis to respond to physiological stressors
(Waltman et al. 1993). We have pro-
posed that the impairment results
from ethanol-induced inhibition in
AVP, a secretagogue that potentiates
the action of CRH on ACTH release
(Waltman et al. 1993).

The HPA Axis in Alcohol-
Dependent Persons

Ethanol alters the integrity of the
HPA axis in alcohol-dependent per-
sons. Paradoxically, chronic ethanol
exposure can result in episodes of
increased as well as decreased HPA
axis activity. The direct effects of
chronic ethanol ingestion on the
HPA axis have been difficult to ascer-
tain because of comorbid states such
as depression, liver disease, malnutri-
tion, and other stressors, as well as
the underlying fluctuations of intoxi-
cation and withdrawal. Depression, in
particular, is common in alcoholics
seeking treatment (Weissman et al.
1977). Although hypercortisolemia is
frequent in depression, the alterations
of the HPA axis seen in alcoholics
can be attributed, at least in part,
directly to the development of alco-
hol dependence.

A dramatic alteration in the HPA
axis observed in alcohol-dependent
subjects is pseudo-Cushing's
syndrome, which was described by
Smals and colleagues in 1976 and
later also reported by others (Paton
1976; Frajria and Angeli 1977; Rees

400

The HPA Axis: Changes and Risk for Alcoholism

et at. 1977). The syndrome is charac-
terized by an elevated plasma or urine
Cortisol level, which is not suppressed
following dexamethasone, accompa-
nied by the clinical stigmata of hyper-
cortisolism, which resolve with
abstinence from ethanol. Although
chronic ethanol exposure stimulates
Cortisol secretion, few actively drink-
ing alcoholics develop frank pseudo-
Cushing's syndrome. The mechanism
of tolerance that prevents the devel-
opment of symptomatic hypercorti-
solemia in alcoholics is not known.
However, based on our animal mod-
els, we have proposed that ethanol-
induced dampening in adenylyl
cyclase signal transduction is a factor
(Wand 1989, 1990; Wand and
Levine 1991).

Although most alcoholics do not
develop pseudo-Cushing's syndrome,
almost all have abnormalities in HPA
axis function. Most studies have
examined the HPA axis during acute
withdrawal; few studies have exam-
ined the HPA axis during periods of
active drinking. A study performed in
our laboratory evaluated the HPA
axis in a group of actively drinking,
nondepressed, alcohol-dependent
men (Wand and Dobs 1991). Subjects
were outpatients who continued their
normal drinking routine throughout
the study. Alcoholic subjects had
twofold higher 24-hour urinary free
Cortisol values compared with matched
control subjects. Paradoxically, despite
elevations in integrated Cortisol mea-
surements, the alcoholic subjects
demonstrated blunted ACTH and
Cortisol responses to CRH as well as a
blunted adrenocortical response to

the ACTH analog cosyntropin
(Cortrosyn). In fact, 25 percent of
alcoholic subjects met criteria for
secondary adrenal insufficiency as
assessed by metyrapone blockade. It
is clear that actively drinking, alcohol-
dependent persons have a deranged
HPA axis.

The HPA Axis During Acute
Withdrawal

The abnormalities in HPA axis function
during acute withdrawal are similar to
the abnormalities found during active
drinking. There is clearly an increase
in HPA axis activation during ethanol
withdrawal (Willenbring et al. 1984).
Cortisol concentrations are elevated as
a result of increases in Cortisol burst
amplitude and Cortisol mass secreted
per burst (Iranmanesh et al. 1989).
There is also disruption of the circadian
pattern of Cortisol release (Iranmanesh
et al. 1989). Moreover, there is nonsup-
pressibility of the HPA axis to low
doses of dexamethasone in alcoholics
during acute withdrawal (Swartz and
Dunner 1982; LaFuente et al. 1983;
Del Porto et al. 1985; Burrov et al.
1986; Kirkman and Nelson 1988;
Iranmanesh et al. 1989). Longitudinal
Cortisol studies on alcoholics admitted
to inpatient services for acute with-
drawal have shown that the hypercor-
tisolism subsides over time. Cortisol
levels normalize following 7-10 days
of abstinence (Adinoff et al. 1991).

The HPA Axis During
Abstinence

As the alcohol-dependent person pro-
gresses from acute withdrawal to early
abstinence, another impairment in

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HPA axis function become manifest,
namely, hyporesponsivity. A series of
HPA axis challenge studies with
CPvH, opioid receptor antagonists,
insulin-induced hypoglycemia, and
ACTH has documented dampened
hormonal responsiveness at each level
of the axis (Costa et al. 1996). Hypo-
responsiveness persists as far out as 6
months into abstinence.

Summary of Abnormalities
in the HPA Axis

These findings demonstrate that alco-
hol-dependent persons have altered
HPA axis dynamics. The nature of
the injury is twofold. First, alcohol-
dependent subjects who are actively
drinking or in acute withdrawal gen-
erate more Cortisol per 24-hour time
period relative to non-alcohol-depen-
dent subjects. Second, despite this
elevation in Cortisol production, the
HPA axis is hyporesponsive to most
forms of stress. The following sce-
nario may explain this paradox: Daily
cycles of intoxication and withdrawal
are stressful and result in a net eleva-
tion in Cortisol production; however,
ethanol-induced injury to the axis
results in dampening responsiveness
to stressors. Therefore, alcohol-
dependent subjects respond to stress
in a suboptimal manner. We speculate
that non-alcohol-dependent subjects
put under a comparable level of stress
would demonstrate much higher lev-
els of ACTH and Cortisol. Abstinent
alcohol-dependent subjects who have
fully completed the withdrawal
process only manifest HPA axis
hyporesponsivity and no longer have
elevated Cortisol production. The

period of hyporesponsiveness can last
at least 6 months and perhaps longer.

Possible Consequences
of HPA Axis Disturbances
in Alcohol Dependence

As alcohol-dependent persons cycle
through episodes of intoxication and
withdrawal alternating with periods of
abstinence, the HPA axis cycles through
periods of hyper- and hypoactivity.
This cyclical pattern of CRH and Cor-
tisol secretion may induce various
pathological states associated with
alcoholism. For example, periods of
sustained hypercortisolism may exac-
erbate osteoporosis, diabetes mellitus,
and hypertension and may alter
growth, reproductive status, and
immune function. Moreover, it has
been proposed that withdrawal-induced
hypercortisolism increases accumula-
tion of excitatory amino acids within
the CNS, resulting in neurotoxicity
(Adinoff 1994). Several studies have
suggested that excess CRH and Corti-
sol production triggered by with-
drawal actually enhance the magnitude
of withdrawal symptoms, including
seizure activity (Menzagh et al. 1994;
Schuikin et al. 1994). Rodent studies
demonstrate that glucocorticoids have
reinforcing properties (Deroche et al.
1995) and will increase mesolimbic
dopamine, a process that may accelerate
addiction to drugs of abuse. Other
studies have shown that stress-induced
increases in corticosterone result in
increased ethanol intake, whereas
medical or surgical adrenalectomy
reduces ethanol consumption (Samson
1995). Furthermore, CRH antago-
nists have been shown to block the

402

The HPA Axis: Changes and Risk for Alcoholism

stress-induced relapse to drug-seeking
behavior, indicating a significant role of
CRH in the consumption of drugs
of abuse, including alcohol (Erb et
al. 1998).

Whereas HPA axis hyperactivity
induced by chronic alcohol dependence
may increase the risk for alcohol-
induced injury and for altered neuro-
chemistry within the mesolimbic system,
hyporesponsivity of the HPA axis
observed during abstinence may gener-
ate another set of consequences. Low
levels of CRH and Cortisol have been
correlated with atypical depression,
impulsivity, lack of behavioral inhibi-
tions, and low motivation (Stansbury
and Gunnar 1994). Extrapolating from
these observations, it has been proposed
that suppressed HPA axis responsiveness
experienced during early abstinence
may provoke dysphoria, craving, and
ultimately the high rate of relapse
associated with acute abstinence (Sher
and Trull 1994). It is provocative to
speculate that part of the mechanism
through which opioid antagonists
reduce relapse is their ability to alter
responsivity of the HPA axis.

There are additional serious implica-
tions for HPA axis hyporesponsiveness
in alcohol-dependent persons. For
example, a subset of alcohol -dependent
patients have some degree of adrenal
insufficiency. Alcohol-dependent persons
are at increased risk for severe infections
and have a compromised immune
system. Once ill, alcohol-dependent
persons have increased morbidity and
mortality compared with non-alcohol -
dependent persons. It is plausible that
altered HPA axis dynamics coupled with
an injured immune function in this

patient population are the reasons for
increased susceptibility to illness and
for poor treatment outcomes. Since
ethanol can injure the HPA axis and
immune function (Rivier 1993), the
increased risk of infection may be the
result of chronic alcoholism.

THE HPA AXIS AND THE
RISK FOR ALCOHOLISM

There is strong evidence that certain
genetic determinants increase the risk
for alcoholism (Kaig 1960; Goodwin
et al. 1973; Cotton 1979; Hrubec
and Omenn 1981; Cloninger 1988;
Merikangas 1990; Pickens et al. 1991;
Kendler et al. 1992). Such findings
have led to investigations of the non-
alcoholic offspring of alcohol-dependent
persons for the purpose of uncovering
markers that identify those at increased
risk for alcohol dependence. The family
history of alcoholism research strategy
compares nonalcoholic offspring from
families with a high density of alcoholism
(referred to as family history positive
[FHP]) with offspring from families
with no history of alcoholism (family
history negative [FHN]). Using this
research design, ethanol challenge stud-
ies have compared FHPs and FHNs
on their reaction to ethanol (Begleiter
et al. 1984; Hill et al. 1988; Schuckit
et al. 1987a, 1987&; Finn et al. 1990;
Pfefferbaum et al. 1991; Gianoulakis
and De Waele 1994; Wand et al.
1994; Gianoulakis et al. 1996). The San
Diego group has reported the largest
series employing this design. In this
longitudinal investigation, over 400
men were originally studied at about
age 20 (Schuckit and Smith 1996).

403

NIAAA's Neuroscience and Behavioral Research Portfolio

Subjects were evaluated for responses
to ethanol based on changes in sub-
jective feelings, hormonal responses,
motor performance, and physiological
measures. Interestingly, a low-level
Cortisol response to ethanol character-
ized those individuals who were most
likely to develop alcohol abuse or
dependence almost a decade later.

Other studies have also shown
differences in HPA axis dynamics as a
function of family history. Women
with alcoholic fathers had higher
Cortisol levels after ingesting ethanol,
compared with women whose fathers
were not alcoholics (Lex et al. 1988).
Gianoulakis and colleagues (1996)
investigated plasma ^-endorphin
responses in a group of nonalcoholic
men and women with a three-genera-
tion family history of alcoholism.
Mild intoxication significantly
increased opioid release in FHP com-
pared with FHN subjects. It has been
proposed that increased responsive-
ness of the opioid system to ethanol
may create vulnerability for alcohol
dependence (Gianoulakis et al.
1996). The mechanisms by which
high- and low-risk persons have dif-
ferential plasma ACTH, (3-endorphin,
and Cortisol responses to ethanol
challenges have not been elucidated.

Ethanol challenges have only a
modest stimulatory effect on the
HPA axis. And, as noted earlier in
this chapter, nausea is often a requi-
site condition for ethanol-induced
activation of the HPA axis. However,
important information concerning
family history differences in HPA axis
dynamics can also be obtained by
stimulating the HPA axis with

other agents. As discussed later in this
chapter, we have demonstrated that
HPA axis responses to CRH and
naloxone differ as a function of family
history of alcoholism (Waltman et al.
1994; Wand et al. 1998).

Ethanol- Seeking Behavior
and the HPA Axis

In the preceding section I reviewed
evidence that the HPA axis may serve as
a marker for individuals at increased
risk for alcoholism. It appears that
Cortisol responses to ethanol have
predictive ability. It is also possible
that the unique HPA hormone profile
in high-risk individuals has a causal
relationship to vulnerability for alco-
holism. To understand why this might
be true, it is important to understand
the anatomical and functional relation-
ships between the HPA axis and the
mesolimbic reward center.

The mesolimbic dopaminergic sys-
tem mediates the reinforcing effects
of many drugs of abuse, including
ethanol. This system consists mainly
of A10 dopaminergic neurons whose
cell bodies are located in the ventral
tegmental area and project terminals
to the nucleus accumbens, frontal cor-
tex, amygdala, and septal area (Koob
et al. 1988). Experimental evidence
suggests that dopamine released within
the nucleus accumbens is responsible, at
least in part, for mediating the reward-
ing effects of many drugs of abuse.
For example, ethanol administration
increases synaptic dopamine accumula-
tion within this important brain region
and is thought to contribute to its
rewarding properties. The ethanol-
induced release of nucleus accumbens

404

The HPA Axis: Changes and Risk for Alcoholism

dopamine is blocked by opioid antag-
onists, implicating opioidergic activity
as an intermediary between ethanol
exposure and the release of dopamine
(Benjamin et al. 1992). Interestingly,
the HPA axis is also modulated by the
same opioid systems, which regulate
dopamine-induced reward within the
mesolimbic system. Following per-
ception of stress, efferent fibers from
several brain regions innervate hypo-
thalamic CRH neurons in the para-
ventricular nucleus. This input
regulates the glucocorticoid compo-
nent of the stress response. The CRH
neurons receive this signal through
four major neurotransmitter systems
(Tsigos and Chrousos 1995): stimu-
latory input from serotonergic and
adrenergic neurons and inhibitory
input from GABAergic and opioider-
gic neurons. These four neurotrans-
mitters, which are responsible for
modulating the activity of CRH neu-
rons, are also "candidate" neurotrans-
mitters implicated in conveying
biological vulnerability for alcoholism
(Chick et al. 1996).

Once CRH is released into the
hypophyseal-portal circulation, it, in
turn, stimulates ACTH secretion from
the pituitary. ACTH stimulates Corti-
sol release from the adrenal gland.
Because of the powerful inhibitory
effect of opioid neurons ((3-endorphin
and enkephalin) on CRH activity, an
acquired or inborn abnormality in
opioid activity in this region would
alter inhibitory tone on CRH neu-
rons. A subset of these opioidergic
neurons responsible for providing the
inhibitory effect on the CRH neurons
simultaneously provides stimulatory

input to presynaptic dopamine termi-
nals within the nucleus accumbens. If
the opioid system is a biological sub-
strate that modulates craving, then this
group of opioidergic neurons is posi-
tioned to play an important role in
mesolimbic contributions to craving
and ethanol-seeking behaviors. There-
fore, ethanol ingestion, through its
effects on opioidergic neurons, can
activate mesolimbic dopamine genera-
tion and the HPA axis simultaneously.
In turn, glucocorticoids, released fol-
lowing ethanol ingestion, modulate
the activities of the opioidergic, CRH,
and mesolimbic dopamine systems;
chronic glucocorticoid exposure
decreases CRH and opioid expression,
whereas it enhances mesolimbic
dopamine production. In this manner,
a primary brain reward pathway — the
mesolimbic dopamine system — and a
primary stress pathway — the HPA
axis — modulate one another.

Studying the release of CRH pro-
vides a window on CNS function and
can uncover differences in neuro-
transmitter systems as a function of
both alcoholism and family history of
alcoholism. It is plausible that if a
neurotransmitter system is altered in
persons at increased risk for alcoholism,
then a neuroendocrine system (e.g.,
HPA axis) regulated by the deranged
neurotransmitter system will also be
altered. Thus, we have hypothesized
that differences in hypothalamic opi-
oid activity between high-risk and
low-risk non-alcohol-dependent sub-
jects may be the basis for differences
in the dynamics of the HPA axis in
the two groups (Wand et al. 1998).
The next section describes how we

405

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designed a research strategy to test
this hypothesis.

Hypothalamic Opioid
Activity and HPA Dynamics
in Persons at Increased Risk
for Alcoholism

Several lines of evidence suggest that
the brain opioid system is part of a
neurocircuitry involved in alcohol rein-
forcement and heavy alcohol drinking
(Gianoulakis and De Waele 1994;
Chick et al. 1996). Initially, pharma-
cological studies indicated that opioid
receptor antagonists, such as naloxone
and naltrexone, decreased ethanol
self- administration in animal models
(Altshuler et al. 1980; Marfaing-Jallat
et al. 1983; Samson and Doyle 1985;
Froehlich et al. 1990; Weiss et al.
1990; Hubbell et al. 1991, 1996). Sub-
sequent clinical trials demonstrated
that the opioid antagonist naltrexone
reduced alcohol drinking, alcohol
craving, and relapse rates in detoxified,
outpatient alcoholics (O'Malley et al.
1992; Volpicelli et al. 1992). In fact,
naltrexone was approved by the Food
and Drug Administration as a pharma-
cotherapeutic agent for the treatment
of alcohol dependence. Several studies
have shown that a genetic predisposi-
tion toward alcohol drinking is associ-
ated with increased responsiveness of
the opioid system to ethanol (De
Waele et al. 1992; De Waele and
Gianoulakis 1994; Gianoulakis et al.
1996), and several "opioid hypotheses"
have been proposed to explain the
role of the endogenous opioid system
in vulnerability to and maintenance of
alcoholism (Reid and Hunter 1984;
Reid 1990; Reid et al. 1991; Gianoulakis

and De Waele 1994). To determine
the validity of an opioid model for
alcoholism, it would be necessary to
generate accurate measurements of
endogenous opioid tone in human
subjects. One technique that can mea-
sure innate differences in endogenous
opioid activity is the induction of opi-
oid receptor blockade with an opioid
receptor antagonist.

Naloxone, a nonselective opioid
receptor antagonist, induces a rise in
ACTH and Cortisol by blocking the
opioid component of the inhibitory
activity directed at the CRH neurons.
As a result of opioidergic modulation
of CRH neurons, the naloxone chal-
lenge test can identify inborn and
acquired alterations in endogenous
opioid activity (Torpy et al. 1993; Del
Campo et al. 1994; Delitala et al.
1994; Facchinetti et al. 1994; Inder et
al. 1995#; Kassimos et al. 1996). For
example, persons with less opioid
activity (less inhibitory tone directed
at CRH neurons) would be maximally
blocked by a lower dose of naloxone
compared with persons with greater
opioid activity (more inhibitory tone),
who require higher doses of naloxone
to induce blockade. In other words,
persons with less opioid activity would
be more sensitive to opioid receptor
blockade than persons with greater
opioid activity. Thus, opioid receptor
blockade by naloxone provides a non-
invasive, functional assessment of
hypothalamic opioid activity.

To explore the hypothesis that off-
spring from families with a high density
of alcohol-dependent persons have
altered hypothalamic opioid activity
compared with offspring from families

406

The HPA Axis: Changes and Risk for Alcoholism

with no history of alcoholism, we
employed opioid receptor blockade
with naloxone (Wand et al. 1998).
The impact of opioid blockade by
naloxone was evaluated by measuring
Cortisol response curves over 2 hours
following administration of different
naloxone doses. To date, we have
conducted this analysis in over 70
nonalcoholic subjects ages 18-25.
Half of the subjects were offspring
from families with a high density of
alcohol dependence and were desig-
nated as FHP subjects; the other half
of the sample population were the off-
spring of non-alcohol -dependent par-
ents and were designated as FHN
subjects. Subjects received graded
amounts of naloxone in double- blind,
randomized order, and serum Cortisol
was monitored. FHN subjects demon-
strated a graded Cortisol response to
each dose of naloxone. In contrast,
FHP subjects were maximally blocked
by the lowest dose of naloxone. These
results show that FHP subjects are
more sensitive to naloxone relative to
FHN subjects.

Although there are several potential
neurochemical explanations for this
finding (Wand et al. 1998), we specu-
late that FHP subjects have dimin-
ished hypothalamic opioid activity
compared with FHN subjects. Fur-
thermore, we speculate that chronic
ethanol ingestion may exacerbate this
underlying opioid defect. In our model,
offspring of alcoholics are born with,
or acquire, a deficit in opioid activity,
compared with offspring of nonalco-
holics ("trait"). Moreover, alcohol-
dependent persons, regardless of family
history, will evidence an opioid deficit

as a consequence of chronic alcohol
abuse ("state"). Lastly, FHP alcohol-
dependent persons will have even less
opioid activity then FHN alcohol-
dependent persons due to additive effects
of trait and state factors. These trait and
state differences in opioid activity may
simultaneously account for abnormal
mesolimbic dopamine generation and
for abnormal HPA axis dynamics in
alcoholics and in persons at increased
risk for alcoholism. We think that this
alteration in endogenous opioid activ-
ity modulates the vulnerability to and
the maintenance of alcoholism for at
least three reasons:

1. Abnormally low amounts of
endogenous opioid activity in the
offspring of alcoholics may create a
neurochemical milieu, which pre-
disposes them to drug-seeking
behaviors (trait).

2. Abnormal reductions of opioid activ-
ity in the alcoholic, as a result of
chronic alcohol exposure, may inten-
sify craving for alcohol and may alter
mood and affect in a manner that
makes relapse more likely (state).

3. Low opioid activity contributes to
a labile HPA axis; the ensuing hyper-
cortisolism may sensitize the meso-
limbic system to drugs of abuse,
including alcohol.

AREAS FOR FUTURE
INVESTIGATION

Studies of the HPA axis can inform us
about genetic and neurobiological risk

407

NIAAA's Neuroscience and Behavioral Research Portfolio

for alcohol dependence. This is not
surprising when one considers that the
CRH neuron, which is responsible for
initiating the HPA axis response, is
modulated by many of the candidate
neurotransmitter systems implicated in
genetic vulnerability for alcoholism.
CRH neurons are under inhibitory
control by opioidergic and GABAergic
neurons and under stimulatory control
by serotonergic and catecholaminergic
systems. Studying the release of CRH
provides a window on CNS function
and can uncover potential group differ-
ences in neurotransmitter systems as a
function of alcoholism and family his-
tory of alcoholism. It is difficult in liv-
ing human subjects to make direct
measurements of these important neuro-
transmitter systems in order to compare
them as a function of family history of
alcoholism and then estimate their rel-
evance for vulnerability for alcoholism.
However, neuroendocrine strategies
allow noninvasive assessment of these
crucial pathways in living human subjects.
To this end, neuropharmacological
studies designed to activate or block 5-
hydroxytryptamine (5-HT), GABA,
and opioidergic input on CRH neurons
may provide crucial information. It is
important that such studies generate
dose-response curves to provide mean-
ingful interpretations. In this manner,
important statements about opioid, 5-
HT, GABA, and catecholaminergic
tone directed at the CRH neuron can
be established. It is also important that
nonhormonal measures be included,
such as physiological and behavioral
responses to the neuropharmacological
challenges. Such studies could be per-
formed during various phases of alcohol

dependence: active drinking, acute with-
drawal, early and late abstinence. How-
ever, because chronic ethanol exposure
can injure the HPA axis, studies
would be more powerful using non-
alcohol-dependent subjects in a family
history design. Moreover, the use of
positron emission tomography or
other imaging measurements of 5-HT,
GABA, and opioids in combination
with neuroendocrine challenges may
provide powerful supplemental confir-
mation of alterations in neurotrans-
mitter systems as a function of alcoholism
and family history of alcoholism.

The San Diego group has provided
important evidence that ACTH and
Cortisol responses following an ethanol
challenge have predictive value vis-a-vis
the future development of alcoholism.
It is now important to establish the
mechanism through which high- and
low-risk persons show a differential
HPA axis response to ethanol. Is it
possible that such differences are not
only predictive but have causal impli-
cations as well? Moreover, as the num-
ber of markers for the risk of alcohol
dependence grows, it would be benefi-
cial to know if altered HPA axis
dynamics identified in high-risk nonal-
coholics co-segregates with other mark-
ers of risk (e.g., reduced P300 amplitude
or low adenylyl cyclase activity).

Differences in the dynamics of the
HPA axis may distinguish animals
with ethanol-seeking behavior from
animals that will not voluntarily drink
ethanol. As noted earlier in this chap-
ter, there are genetic as well as envi-
ronmental factors that determine the
dynamics of the HPA axis. In otherwise
healthy people, there is a continuum

408

The HPA Axis: Changes and Risk for Alcoholism

of HPA axis activity (e.g., from low to
high responders). On either end of
the HPA axis spectrum, low or high
producers of CRH, ACTH, and Corti-
sol may be at risk for certain behav-
ioral disturbances. Drug-reinforcing
properties may be related to the sensi-
tivity level of the mesolimbic system
(Samson 1995). The sensitivity of the
mesolimbic system to drugs of abuse
may be modulated by glucocorticoids.
In this manner, the onset and magni-
tude of sensitization to drug actions
may be modulated by stress. Address-
ing this issue, Samson hypothesized
that "stress, via its ability to result in
sensitization and altered mesolimbic
dopamine system function, results in an
increase in the reinforcing efficacy of
alcohoF (pp. 284-285).

Since the degree of HPA axis lability
may influence alcohol-seeking behav-
iors, it behooves us to systematically
analyze and compare HPA axis dynam-
ics as a function of family history of
alcoholism. To this end, there should
be a head-to-head comparison of the
entire HPA axis in alcohol-dependent
and non-alcohol- dependent people as
a function of family history of alco-
holism. A series of studies could compare
integrated measurements of 24-hour
ACTH and Cortisol production in
stressed and nonstressed persons.
Studies could also measure hypothala-
mic CRH/AVP responses to nalox-
one or hypoglycemia, pituitary ACTH
responses to CRH, and adrenocortical
responses to ACTH. Additionally,
much may be gained by measuring
the glucocorticoid negative feedback
set-point as a function of family history
of alcoholism. Moreover, the behavioral

(e.g., reinforcing) effects of CRH,
ACTH, and glucocorticoids should be
studied in a family history design in
both alcoholics and nonalcoholics.

One potential confounding variable
in family history designs is that the
subjects, although not alcohol abusers,
have been exposed to alcohol. This
means a certain degree of tolerance to
ethanol may already be established. In
addition, concerns for multiple blood
drawing and administering pharmaco-
logical agents in children have pre-
vented serious hormonal assessments
in preteenage, ethanol-naive offspring
of alcohol-dependent persons. The use
of salivary Cortisol measurements in
preteenagers may help in this regard.
Multiple salivary Cortisol determina-
tions in a 24-hour period can be per-
formed with little or no distress for
the subject.

Studies using humans and rodents
have indicated that anxiety and stress
are positively correlated with high
alcohol consumption as well as relapse
to heavy drinking by abstinent alco-
holics (Hore 1971; Kushner et al.
1990). The reason for this relationship
between stress and alcohol drinking is
not well understood. It has been sug-
gested that alcohol consumption
relieves anxiety and as a result helps
the individual to cope with the stress-
ful situation (Pohorecky 1991). The
possible relationship between stress
and alcohol consumption has led
some investigators to propose a "tension
reduction hypothesis" to explain a
subset of alcoholism and the ability of
alcohol to significantly dampen this
response (Pohorecky 1991). Self-
medication with alcohol for anxiolytic

409

NIAAA's Neuroscience and Behavioral Research Portfolio

purposes may be one route through
which high-risk persons begin to
become alcohol dependent. The
effects of stress dampening by ethanol
should be studied in a family history
design using the HPA axis as the end
point measures.

Although understanding the interac-
tions of the HPA axis and alcoholism
may help in developing better methods
of prevention and treatment and under-
standing neuromechanisms that con-
tribute to alcohol dependence, it is also
important to understand why alcohol-
dependent persons are at increased risk
for severe infections. Since the HPA axis
and immune system modulate one
another, the increased risk of infection
may be the result of both direct and indi-
rect sequelae on both systems. Future
studies should more clearly define the
relationship between ethanol -induced
injury to the HPA axis and immune sys-
tem. It is also important to determine if
alcohol-dependent persons would ben-
efit from stress glucocorticoid cover-
age during acute illness.

ACKNOWLEDGMENT

Preparation of this chapter was sup-
ported by grant ROl 10158 from the
National Institute on Alcohol Abuse
and Alcoholism.

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415

Chapter 12
Alcohol and Sleep

Cindy L. Ehlers, Ph.D.

KEY WORDS: Sleep disorder; respiratory disorder; acute AODE (effect ofAOD
[alcohol or other drug effects] use, abuse, and dependence); respiratory system
function; electroencephalography; REM sleep; circadian rhythm; polypeptide hor-
mones; risk factors; relapse prevention; muscle function; literature review

EFFECTS OF ALCOHOL ON
ELECTROENCEPHALO-
GRAPHIC (EEG) SLEEP
AND BREATHING
DURING SLEEP

Acute doses of alcohol (ethanol) sig-
nificantly alter the sleep of healthy
subjects, producing changes in the
electroencephalogram, eye movements
during sleep, muscle activity, and
breathing patterns. Changes in sleep
patterns produced by acute alcohol in
normal subjects include increases in
delta (slow wave, stages 3 and 4) sleep
(Yules et al. 1967; MacLean and
Cairns 1982) and delta EEG power
(Landolt et al. 1996), reductions in
rapid eye movement (REM) sleep

(Gresham et al. 1963; Knowles et al.
1968; Rundell et al. 1972; Landolt et
al. 1996; Lobo and Tufik 1997), and
increases in stage 2 sleep (Yules et al.
1966). Subjects given alcohol in exper-
imental procedures reported sleeping
more superficially and experiencing
lighter sleep, especially during the sec-
ond half of the night (Landolt et al.
1996). Periodic limb movements were
also increased in some subjects and
may contribute to sleep disturbance
following moderate alcohol consump-
tion (Aldrich and Shipley 1993).

Alcohol consumption also influences
breathing during sleep. Moderate doses
of alcohol (0.7-1.05 mL/kg) produce
respiratory depression (Johnstone and
Reier 1973), vasodilation and swelling

C.L. Ehlers, Ph.D., is an associate professor in the Department of Neuropharmacology, The Scripps
Research Institute, CVN-14, 10550 North Torrey Pines Rd., Lajolla, CA 92037.

417

NIAAA's Neuroscience and Behavioral Research Portfolio

of the respiratory mucosa (Rains et al.
1991). Alcohol increases inspiratory
resistance during stage 2 sleep in both
snoring and nonsnoring men (Dawson
et al. 1993, 1997). The mechanism
whereby alcohol increases inspiratory
resistance appears to be through a
selective reduction in respiratory motor
activity of the hypoglossal and laryngeal
nerves. (Bonora et al. 1984; Krol et al.
1984). This reduced muscle tone pre-
sumably makes the upper airway more
likely to collapse. Thus, it has been sug-
gested that alcohol consumption can
lead to an aggravation of obstructive
sleep apnea (Taasan et al. 1981; Issa
and Sullivan 1982; Scrima et al. 1982;
Block et al. 1986; Mitler et al. 1988;
Scrima et al. 1989), although the data
are still somewhat controversial
(Teschler et al. 1996).

Sleep Apnea in Alcoholics

Sleep apnea and sleep-related breathing
and movement disturbances are not
uncommon in abstinent alcoholics
(Tan et al. 1985; Vitiello et al. 1987;
Mamdani et al. 1989; Vitiello et al.
1990«, 1990&; Aldrich et al. 1993; Le
Bon et al. 1997). Although there is
evidence that sleep-related breathing
disorders are increased in alcoholics,
there are several mediating factors (see
Aldrich et al. 1993). For instance,
Vitiello and colleagues (1987) found
increased nocturnal hypoxemia in
abstinent male alcoholics, but more
than half of those alcoholics (vs. none
of the control subjects) were smokers.
It has been suggested that the increase
in sleep apnea seen in some alcoholics
is secondary to either chronic lung
disease or cardiovascular morbidity

associated with smoking combined with
alcohol use. However, an alternative
hypothesis has been put forth by Tan
and colleagues (1985), who found that
central apneas and hypopneas were
associated with the presence of nervous
system damage in alcoholics. It appears
that apneas in alcoholics may be of
mixed origin and may include both
central nervous system effects and car-
diopulmonary factors. Of interest is
the fact that sleep-disordered breath-
ing is apparently not seen in alcoholic
women (Aldrich et al. 1993). This
suggests that women may be protected
from the effects of alcohol on the mech-
anisms that generate apnea.

EEG Sleep in Alcoholics

Few studies have used polysomno-
graphic measures to evaluate the sleep-
ing electroencephalogram in alcoholics
(see Zarcone 1978). This stands in sharp
contrast to the frequency of com-
plaints of sleep disturbance in alco-
hol-dependent individuals observed
in general medical practice. Various
forms of hypersomnia, parasomnia,
disrupted circadian and ultradian
rhythms, and insomnia have all been
described in alcoholic patients (Wagman
and Allen 1975; Gross and Hastey
1976; Wagman et al. 1977; Zarcone
1979; Snyder et al. 1981; Williams
and Rundell 1981; Snyder and Karacan
1985; Ishibashi et al. 1987; Gillin et
al. 1990). In 1992, a meta-analysis
evaluated the published polygraphic
sleep studies of psychiatric and substance
abuse disorders (Benca et al. 1992).
In that meta-analysis only eight stud-
ies on alcoholism were available that
met criteria for inclusion and only

418

Alcohol and Sleep

four of those were controlled with
normal volunteers, for a total of only
123 subjects. In the smaller comparison
with matched normal control sub-
jects, sleep latency was significantly
prolonged, stages 3 and 4 (delta)
sleep percentage of total sleep time
(Delta%) was reduced, and REM%
was increased. In the larger compari-
son with normal subjects (20-50
years old), alcoholics had significantiy
reduced total sleep time, total non-
REM sleep, delta sleep, and Delta%
compared with normal control sub-
jects; sleep latency, REM latency (the
elapsed time from sleep onset to the
first REM period), and REM times
were within normal limits.

In comparison with studies of
affective disorder patients, alcoholics
were found to have significantly
shorter sleep latency and longer REM
latency but did not differ statistically
on total sleep time, delta sleep, non-
REM sleep, or REM sleep time (table
1). The sleep of alcoholics can also be
differentiated from that of abstinent
stimulant abusers (Thompson et al.
1995). Thus, the sleep disturbance in

alcoholism appears to be somewhat
distinct from the syndrome described
in affective disorder (Moeller et al.
1993), or other psychiatric disorders
and may arise from disruption of dif-
ferent brain mechanisms.

This typology must be regarded as
tentative until it is replicated in addi-
tional prospective studies. Further-
more, formal comparison of the
alcoholic groups with matched normal
control subjects would be highly
desirable to establish the normal limits
for each of the measures.

SLEEP DISTURBANCE
DURING ALCOHOL
WITHDRAWAL,
ABSTINENCE,
AND RELAPSE

Insomnia is perhaps the most common
complaint in the alcoholic patient during
acute and subacute withdrawal and
may be a key variable in predicting
relapse. Sleep is often markedly disturbed
during the first days of abstinence (Mello
and Mendelson 1970; Allen et al.
1971; Kotorii et al. 1980), even in the

Table 1. Sleep Disturbance in Alcoholics and Depressed Patients Compared With
Normal Subjects, as formulated by Gillin et al. 1990.

Diagnosis

REM

Delta

TST

Major depressive disorder
Alcohol dependence

Good prognosis

Poor prognosis
Alcohol dependence with
alcohol-induced mood disorder

Good prognosis

Poor prognosis

Note: RL = REM latency; REM = rapid eye movement sleep; RD = REM density; Delta = delta sleep; TST = total sleep time.

419

NIAAA's Neuroscience and Behavioral Research Portfolio

absence of delirium tremens (DTs), con-
vulsions, or other major withdrawal
syndromes, but may improve about
the 3d or 4th week of abstinence (Gillin
et al. 1990). Nevertheless, some recov-
ered alcoholic patients show reduced
total sleep time, loss of stages 3 and 4
(delta) sleep, and fragmented sleep
despite prolonged abstinence (Johnson
et al. 1970; Allen et al. 1971; Williams
and Rundell 1981; Snyder and Karacan
1985; Ishibashi et al. 1987). Subjec-
tively unrefreshing sleep or specific
sleep abnormalities (such as reduced
delta sleep) during chronic abstinence
have been associated with protracted tol-
erance or increased risk of renewed drink-
ing (Allen and Wagman 1975; Wagman
etal. 1978; Allen et al. 1980).

A well-controlled study by Gillin and
colleagues (1994) sheds more light on
the mechanisms underlying disturbed
sleep in withdrawal and abstinence and
its relation to relapse. Poly graphic sleep
recordings were obtained at the time of
admission to an inpatient alcohol treat-
ment program and evaluated for their
predictive value in determining absti-
nence and relapse 3 months following
hospital discharge. Abstinence and
relapse were not consistently related to
any clinical measures at the time of
hospital admission, including age,
duration and severity of alcoholism,
marital status, employment, hepatic
enzyme levels, cognitive performance,
or depression ratings. However, short
REM latency, increased REM%, and,
possibly, increased REM density at
the time of admission were successful
in predicting relapse in nondepressed
alcohol-dependent men at 3 months
following hospital discharge (Gillin et

al. 1994). Further investigation of these
patients demonstrated that some facets
of their sleep remained abnormal even
after 27 months of abstinence and that
insomnia and sleep fragmentation after
approximately 5 months of abstinence
may be related to relapse by 14 months
(Drummond et al 1998). In a recent
study of 10 abstinent and 11 relapsing
depressed alcoholics, increased REM
density at admission also predicted
relapse at 3 months (Clark et al. 1998).
Other studies have confirmed that sleep
disturbance is an important predictor
of relapse. In a study of 74 alcoholic
patients, Brower and colleagues (1998)
found that relapsed patients did not
differ from abstinent patients at baseline
in demographics or psychiatric comor-
bidity but had more difficulty falling
asleep, more complaints of abnormal
sleep, and longer sleep latencies and
less stage 4 sleep percentage than
abstinent patients.

POTENTIAL
NEUROBIOLOGICAL
MECHANISMS
UNDERLYING SLEEP
DISTURBANCE
IN ALCOHOLICS

The neurobiological mechanisms under-
lying disturbed sleep in alcoholics
remain largely unknown. Based on
concepts derived from the neurobiology
of sleep and clinical studies (Hobson
et al. 1986), considerable evidence
implicates neurochemical factors, such
as a relative increase in cholinergic
neurotransmission (Gillin et al. 1979),
which may underlie short REM
latency and reduced stages 3 and 4

420

Alcohol and Sleep

sleep seen in some alcoholics. With
respect to short REM latency, increased
REM%, and increased REM density
found in alcoholic patients, these find-
ings are consistent with current findings
suggesting that serotonergic neurons
within the dorsal raphe suppress REM
sleep by inhibiting cholinergic neurons
in the lateral dorsal tegmentum and
pedunculopontine tegmental nucleus
(Luebke et al. 1992). Data from normal
volunteers also support this hypothesis,
where using a tryptophan-free amino
acid drink challenge, which depletes the
brain of serotonin, has been found to
significantly reduce REM latency and
increase REM% and REM density
(Bhattietal. 1995).

Sleep disturbances in abstinent alco-
holics may also have a neurohormonal
basis. It has been clearly demonstrated
in animal models that hypothalamic
peptides can profoundly affect sleep
quality. Growth hormone-releasing fac-
tor (GRF) has been shown to increase
slow wave sleep in several species
(Ehlers et al. 1986; Wehrenberg and
Ehlers 1986; Obal et al. 1988), whereas
corticotropin-releasing factor (CRF)
produces hyperarousal, reductions in
slow wave sleep, insomnia, and delays
in sleep onset (Ehlers et al. 1986,
1997). There are some data to suggest
similar effects of these peptides on
sleep in human subjects (Steiger et al.
1992, 1994; Friess et al. 1995).
Whether disruption in these peptides
leads to sleep disturbance in alcoholics
is not clear.

There are also data to suggest that the
hypothalamic -pituitary- adrenal system
(HPA) is altered in some alcoholics.
Chronic alcohol ingestion has been

demonstrated to produce an activating
effect on the HPA system in both clinical
and animal studies (see Wand 1993 for
a review; see also chapter 11 in this
monograph). In certain chronic alco-
holics, hypercortisolemia can be severe
enough so that several features of
"pseudo-Cushing's syndrome" are
present, although this is not typical
(Smals et al. 1976; Rees et al. 1977).
During early withdrawal, nonsuppression
of plasma Cortisol to dexamethasone
has been reported (Swartz and Dunner
1982; Kroll et al. 1983; Newsom and
Murray 1983; Porto et al. 1985), as
well as blunted plasma adrenocorti-
cotropic hormone (ACTH) response to
metyrapone blockade (Wand and Dobs
1991) and CRF challenge (Inder et al.
1995). Reduced concentrations of
CRF in cerebrospinal fluid have also
been reported in some alcoholic patients
(Geracioti et al. 1994). Cortisol levels
generally return to normal over pro-
longed abstinence, but stress responses
may not (Errico et al. 1993; George
et al. 1994).

Although the locus of disturbed
HPA functioning in alcoholics is not
clear, studies by Rivier and colleagues
(1984, 1990) suggest that regulation
of CRF activity may be important. For
instance, in animal studies chronic
exposure to ethanol vapors decreased
the hypothalamic content and increased
the synthesis of CRF. Immunoneu-
tralization of endogenous CRF has
also been demonstrated to abolish
ethanol-induced release of ACTH in
rodents (Rivier et al. 1984; Zgombick
and Erwin 1987). Whether the
changes in the HPA system seen in
alcoholics are associated with specific

421

NIAAA's Neuroscience and Behavioral Research Portfolio

sleep difficulties is unclear, because
evaluation of the relationship between
the functioning of the HPA system
and polysomnographically recorded
sleep in alcoholic patients has received
little attention.

The hypothalamic-pituitary-soma-
totropic system (HPS) may also be
important in understanding disturbed
sleep in alcoholism. It has been suggested
that alteration in growth hormone secre-
tion may be one of the major endocrine
characteristics of alcoholism (see Badger
et al. 1993 for a review). Growth hor-
mone is normally secreted in associa-
tion with delta sleep in normal control
subjects, at least up until middle to
old age (Carlson et al. 1972; Holl et
al. 1991; Van Cauter et al. 1992). Few
sleep studies have examined nocturnal
growth hormone secretion during
poly graphically recorded sleep in alco-
holics. Othmer and colleagues (1982)
found that growth hormone secretion
was generally low throughout the
night and was dissociated from both
sleep onset and delta sleep in chronic
sober alcoholics (n - 8) compared
with normal control subjects (n = 3).
Interestingly, in those studies, acute
alcoholic intoxication increased delta
sleep after sleep onset but did not
alter the pattern of growth hormone
secretion in alcoholic patients.

Prinz and colleagues (1980) previously
reported that alcohol reduced nocturnal
growth hormone secretion in normal
volunteers. GRF secretion has also been
examined in early abstinent alcoholic
patients (De Marinis et al. 1993). Blunted
growth hormone responses to clonidine
but not GRF were found, suggesting
that the pituitary response to GRF is

intact in abstinent alcoholics; however,
hypothalamic regulation of the HPS
system may be disturbed. In another
study, growth hormone response to GRF
was intact in alcoholics; growth hormone
response to sumatriptan (5-HT1D sero-
tonergic receptor agonist), however,
was blunted in alcoholics (Coiro and
Vescove 1995). Blunted growth hor-
mone responses to apomorphine have
also been associated with early relapse
(Heinz et al. 1995).

Taken together, these studies suggest
that dysregulation of both the HPA and
HPS systems at the level of the hypothal-
amus may occur in alcoholics during
abstinence. Whether this dysregulation
contributes to the sleep disturbance that
can commonly occur at that time is still
unknown, because of the paucity of stud-
ies in both human and animal models. It
is possible that sleep and hormonal dis-
turbances in alcoholism are linked and
that a common mechanism (serotoner-
gic) may be responsible for dysregulation
in both. Alternatively, sleep disturbance
and hormonal disturbance may be causally
related to each other in either direction.
Clearly, further studies are necessary to
describe the phenomenology of sleep and
endocrine secretion within individuals
in order to begin to form hypotheses
regarding causal relationships.

RELATIONSHIP BETWEEN
CIRCADIAN RHYTHMS
AND SLEEP DISTURBANCE
IN ALCOHOL DEPENDENCE
AND ABSTINENCE

Sleep research and circadian rhythm
research are inextricably linked, because
any phenomenon that disrupts circadian

422

Alcohol and Sleep

functioning (e.g., jet lag) will disturb
sleep. Several studies have demon-
strated that alcohol can inhibit or
alter melatonin secretion (Ekman et
al. 1993; Steindl et al. 1995), a
marker of the circadian systems in
humans. Low levels of melatonin
have also been reported in alcohol
withdrawal (Schmitz et al. 1996) and
abstinence (Wetterberg et al. 1992).
If alcohol disturbs sleep through a
circadian rhythm disturbance, this
suggests that specific brain mecha-
nisms may be responsible. Some evi-
dence that this may be true is
provided in a study by Madeira and
colleagues (1997). They reported
that rats experiencing long-term
ethanol treatment (6 and 12 months)
followed by long-term abstinence (6
months) had significant, irreversible
depression of peptide immunoreactivity
and mRNA levels in the suprachias-
matic nucleus. Since the suprachias-
matic nucleus is considered the site of
the biological clock in rodents, these
data suggest that disturbance of the
biological clock is a potential mecha-
nism for disturbed sleep in long-term
abstinent alcoholics.

GAPS IN KNOWLEDGE
AND PROMISING
RESEARCH AREAS

Sleep and alcohol is clearly an under-
studied area. Although progress was
made in the 1970s defining the field,
in every decade since fewer investiga-
tors remain in the field. Clearly, an
effort needs to be made to rekindle
this area, especially considering that
sleep complaints may be a core issue

in alcohol relapse. Can newer method-
ologies shed further light on the
hypothesis that chronic alcohol intake
produces specific and persistent
changes in brain function that lead to
prolonged sleep disturbance and
increased risk for relapse?

Although previous clinical sleep
studies have been invaluable in defining
the sleep disturbance in alcoholism, they
are somewhat limited. Aside from the
small number of subjects enrolled in the
studies, all of these studies relied on
evaluating the results of manual scor-
ing of EEG sleep records. None of
these studies used any quantitative
measures of the electroencephalogram,
such as spectral analysis or period/
amplitude analysis. Additional studies
using quantitative techniques in a
larger sample should be able to better
characterize the features of the sleep
electroencephalogram that distinctly
characterize alcohol dependence and
potentially link that disturbance to
underlying neural activity.

Some clinical studies support the
notion that chronic alcohol exposure
causes direct toxic or neuroadaptive
changes in the brain areas that are
involved in the regulation of sleep.
For instance, cerebral atrophy and
slow wave sleep have been correlated
in abstinent chronic alcoholics
(Ishibashi et al. 1987), as have REM
sleep time and digit span impairment
(Benson et al. 1978). Further studies
using magnetic resonance imaging or
single photon emission computed
tomography imaging techniques in
abstinent alcoholics in combination
with better quantification of waking
and sleeping electroencephalograms

423

NIAAA's Neuroscience and Behavioral Research Portfolio

are likely to allow for a better testing
of this hypothesis.

Brain Mechanisms Underlying
Alcohol- Induced
Sleep Disturbance

There have been very few animal studies
that have attempted to link brain
mechanisms important in the regulation
of sleep and the long-term effects of
alcohol. Studies need to be conducted
in animals that are exposed to alcohol in
clinically relevant amounts over long
periods of time and then studied in
acute and chronic withdrawal, as well
as following long-term abstinence. The
specific target neurochemical systems
could include but are not limited to
serotonin, acetylcholine, and specific
neuropeptides such as GRF, CRT, and
neuropeptide Y. Molecular techniques
using such markers as COX (cyclo-
oxygenase) and c-Fos may also shed
light on target brain areas.

Risk Factors for Sleep
Disturbance and Alcohol Use

Evidence from family, twin, and adop-
tion studies suggest that alcoholism is a
genetically influenced disorder. Poten-
tially important neurophysiological
markers of alcoholism, as well as risk for
alcohol dependence, are event-related
potentials and EEG measures. Many,
although not all (Emmerson et al.
1987), studies in alcoholics have found
that their resting, waking electroen-
cephalogram was deficient in alpha
activity, was of lower voltage, and
tended to contain theta and excessive
fast activity (Varga and Nagy 1960;
Begleiter and Platz 1972; Naitoh 1973;
Jones and Holmes 1976; Coger et al.

1978; Propping et al. 1981; Kaplan et
al. 1985; Spehr and Stemmler 1985;
Krauss and Niedermeyer 1991; Pollock
et al. 1992). More recently it has been
suggested that EEG fast frequency
activity may also be related to risk of
relapse in abstinent alcoholics, with
abstinence -prone patients not differing
on this variable from control subjects
(Bauer 1994).

Low-voltage fast EEG patterns are
also known to be genetically influenced
and appear to be transmitted by an
autosomal-dominant mode of inheri-
tance. Low- voltage EEG variants have
also been linked to risk for the devel-
opment of alcoholism (see Enoch et al.
1995; Ehlers et al. 1999). However, no
studies have determined whether certain
EEG sleep patterns (e.g., sleep pheno-
types) actually pre-date the development
of alcohol dependence and thus rep-
resent preexisting risk factors rather
than the consequence of chronic alco-
hol exposure. It is also not known
whether waking EEG phenotypes are
reflected in the sleep electroencepha-
logram in alcoholics, and specifically
whether waking EEG patterns may be
predictive of sleep disturbance. If so,
this would suggest that a common set
of brain mechanisms may influence
the patterns of both the waking and
sleeping electroencephalograms in
alcohol-dependent patients and those
at risk for alcohol dependence.

To answer this set of questions,
studies need to be conducted in subjects
at differing genetic risk for alcoholism.
Sleep studies should be carried out in
children of alcoholics and appropriate
control subjects. In addition, prospective
studies should be conducted addressing

424

Alcohol and Sleep

the question of whether sleep disturbance
per se is a risk factor for developing
alcoholism. In this light, a recent study
(Roehrs et al. 1999) showed that in the
controlled conditions of the sleep labor-
atory insomniacs were more likely to
select a disguised alcohol drink before
bedtime than control subjects. Adminis-
tration of alcohol to both control sub-
jects and insomniacs was found to
decrease REM sleep in both groups but
to increase delta sleep selectively in
insomniacs. These data suggest that
alcohol may be more reinforcing for
insomniacs and thus may be potentially
more addicting.

To What Extent Is Alcohol-
Induced Sleep Disturbance
Gender Related?

There are virtually no studies that have
specifically addressed gender issues in
sleep and alcohol. The studies of Aldrich
and colleagues (1993) are provocative in
demonstrating that alcohol-related sleep
apnea may be exclusively a male phenom-
enon. Recent studies in large, healthy
subject populations have demonstrated
that the sleep of young men (30-40
years old) deteriorates at a much faster
rate than that of age-matched women
(see Ehlers and Kupfer 1997). These
data are intriguing in view of the later age
of onset of alcoholism in many women.
Studies evaluating gender issues in
sleep and alcohol need to be accom-
plished in both humans and animals.

Should New Therapies Target
Sleep as a Core Symptom
in Preventing Relapse?

If the neuroadaptive changes associated
with the protracted abstinence syndrome

include sleep and circadian rhythm dis-
turbance, then some novel treatment
modalities may be useful in preventing
relapse. In an animal study, stimulation
of GABAA receptors by muscimol or
homotaurine administration during
alcohol withdrawal was found to sig-
nificantly improve the disturbances in
the sleep-wake states in the alcohol-
dependent rats, in a time-related manner
(Rouhani et al. 1998). Thus, GAJBAergic
drugs might be specifically targeted to
treat sleep disturbance during acute
withdrawal. One study investigated
sleep in abstinent alcoholics during
ritanserin treatment (Monti et al. 1993).
In that study mood and sleep were
improved in the treated group; however,
no information was given on long-term
abstinence, drug response, and sleep
quality. In another study the efficacy
of L-tryptophan was evaluated and
found to be useful in improving sleep
and mood in alcoholics (Asheychik et
al. 1989).

In addition to pharmacotherapy,
interventions aimed at regulating cir-
cadian functioning may also be useful
in treating sleep disturbance during
abstinence. In one study, bright light
exposure was evaluated in patients dur-
ing alcohol withdrawal. Subjective mea-
sures of sleep quality, sleep maintenance,
and sleep architecture were all found
to be improved after bright light therapy
(Schmitz et al. 1997), but the study was
not placebo controlled. Dawn simula-
tion (early morning light treatment
simulating dawn) has also been used
to treat abstinent alcoholics with winter
depression, and has been found useful
(Avery et al. 1998). Further therapeutic
trials that include sleep measurements,

425

NIAAA's Neuroscience and Behavioral Research Portfolio

relapse rates, and psychiatric states are
clearly needed.

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STUDIES OF

COGNITIVE/BEHAVIORAL/

STRUCTURAL DEFICITS

IN HUMANS

Chapter 13

Neuropsychological Vulnerabilities
in Chronic Alcoholism

Marlene Oscar-Berman, Ph.D.

KEY WORDS: brain damage; neuropsychological assessment; AODR (alcohol or
other drug-related) disorder; chronic AODE (effects ofAOD use, abuse, and de-
pendence); aging; gender differences; nutrition; elderly; malnutrition; thiamine
deficiency; Korsakoff s syndrome; cerebral cortex; cerebral hemisphere; neurobio-
logical theory; emotion; memory; risk factors; literature review

Alcohol-related brain damage has been
associated with a variety of neuropsy-
chological changes, among which are
deficits in cognitive, emotional, and
behavioral functioning. This chapter
reviews accomplishments in neuropsy-
chological investigations of the residual
consequences of long-term chronic alco-
holism. (There is minimal coverage of
imaging and electrophysiological studies
in this chapter because these topics are
discussed in chapters 14 and 15, respec-
tively.) Residual effects — in contrast to
acute effects — can be studied only in
people who have abused alcohol for a
lengthy period of time but who have
been sober for at least 4 weeks before
testing. In addition, they must be free of
comorbid medical complications that
can affect brain functioning. Comorbid

conditions (e.g., head injury, history of
abuse of other drugs, concurrent psy-
chiatric diagnoses, liver disease, HIV
infection) usually are exclusionary criteria
in the study of alcohol-related brain
damage because comorbidities may
contribute to artifactual results that
complicate interpretations about the
neurobehavioral sequelae of alcoholism
(Oscar-Berman et al. 1997; Neiman
1998). The exception is when researchers
are specifically interested in understand-
ing the combined consequences of a
comorbid condition with alcoholism
(see Galanter 1998 and chapter 14 in
this monograph).

The locus and extent of alcoholism-
related brain damage, as well as the
type and degree of impairment, differ
across individuals. These differences

M. Oscar-Berman, Ph.D., is professor of neurology and professor of psychiatry, Division of Psychiatry,
M-902, Boston University School of Medicine, 715 Albany St., Boston, MA 02118; she is also a research
scientist at the Department of Veterans Affairs New England Healthcare Systems, Boston,
Massachusetts Division.

437

NIAAA's Neuroscience and Behavioral Research Portfolio

suggest vulnerability of certain sub-
groups of alcoholics (e.g., the elderly
and women), and susceptibility of par-
ticular areas of the brain to alcoholism-
related damage (e.g., cerebral cortex
and limbic and diencephalic structures).
To account for the divergent findings,
researchers have proposed different
models (or hypotheses). For context,
table 1 lists common current theoretical
frameworks proposed to explain various
residual neuropsychological effects of
alcoholism. Although these models
tend to focus on vulnerable subject
groups, brain regions, or mental pro-
cesses, they are not mutually exclusive;
some are interrelated. Supporting
data for these models come from
neurobehavioral studies, brain scans, elec-
trophysiological studies, and postmor-
tem neuropathology.

In this chapter I discuss three
significant subject characteristics that
contribute to increased vulnerability
to the effects of alcoholism: aging,
nutrition, and gender. Another focus
of the discussion is on vulnerable
cortical brain systems that are pur-
ported to mediate specific functional
deficits: the frontal lobes and the
right hemisphere. A primary goal of
this review is to help establish future
priorities in research on alcohol abuse
and alcoholism.

VULNERABILITY
RELATED TO SUBJECT
CHARACTERISTICS

Not all alcoholics are equally at risk
for brain changes and neuropsycho-
logical deficits, nor are all alcoholics

Table 1. Models Proposed To Explain the Neuropsychological Consequences of Alcohol-
Related Brain Damage.

Models Emphasizing Vulnerability in Terms of Subject Variables

• Premature aging: Alcoholism accelerates aging. Brains of alcoholics resemble brains of
chronologically older nonalcoholics.

• Gender: Alcoholism affects women more than men.

• Vitamin deficiency: Thiamine deficiency contributes to brain lesions, especially in
Wernicke - Korsakoff syndrome .

Models Emphasizing Vulnerability of Brain Regions/Systems or Functional Processes

• Diffuse cortical atrophy: Cerebral atrophy occurs throughout the brain.

• Right hemisphere functions are more vulnerable to the effects of alcoholism than are left
hemisphere functions. (Similar observations have been made with normal chronological
aging, hence support for the premature aging hypothesis.)

• Frontal lobe systems are more vulnerable to the effects of alcoholism than other cortical
systems.

• Limbic and diencephalic brain regions are vulnerable to the effects of alcoholism in
Korsakoff's syndrome.

• Neurotransmitter systems: Emphasis is placed on disruption of neurotransmitter systems,
e.g., acetylcholine, dopamine, gamma-aminobutyric acid, glutamate, and serotonin
systems.

• Reduction in efficiency of mental operations: Emphasis is placed on impairments of
underlying cognitive processes.

438

Neuropsychological Vulnerabilities in Chronic Alcoholism

affected similarly. Therefore, when
trying to understand the effects
of alcoholism on brain and behavior,
researchers must be aware of the
influence of a wide range of interre-
lated variables (figure 1). These
variables include the following indi-
vidual differences:

• Age: The very young and the
elderly are especially vulnerable
(Dufour 1996; Oscar-Berman and
Schendan in press).

• Comorbid medical, drug abuse,
and psychiatric conditions (Sher
and Trull 1994; Lieber 1995;
Galanter 1998; Bates and Convit
1999; see also chapters 14 and 15
in this monograph).

• Nutrition (Cook et al. 1998;
Shimomura et al. 1998).

• Education, intelligence, and socio-
economic background (Lezak
1995; Rourke and Loberg 1996).

• Ethnicity: In addition to the
contribution of gene variants,
ethnicity influences environmental
factors such as drinking habits
and nutrition (Keefe and New-
comb 1996).

• Gender: Deleterious effects of alco-
holism may differ for men and
women (Glenn 1993; Nixon 1993).

• Genetics and family history:
Alcoholism is genetically heteroge-
neous. Its expression is influenced
by gene variants, which also con-
tribute to ethnicity (Wall et al.
1996) and to personality traits
(e.g., antisocial personality disor-
der and attention -deficit/hyperac -
tivity disorder [Schuckit 1997]).
In addition, nonalcoholic offspring

of alcoholics show abnormal
neuropsychological signs (Pihl and
Bruce 1995; Porjesz and Begleiter
1995), and patients with alcoholic
dementia may be genotypically dis-
tinct from patients with alcoholic
Korsakoff's syndrome (Muramatsu
et al. 1997).
• Other factors: Cranial circumference
(see chapter 15); experimental
methodologies (e.g., specific tests
and procedures); size and homogene-
ity of samples; comparability of
human and nonhuman animal
models (Lancaster 1995; Rourke
and Loberg 1996; Gallagher and
Rapp 1997; Oscar-Berman and
Bardenhagen 1998).

Although the contributions of
many of these variables are not spe-
cific to the effects of alcoholism, some
have direct relevance. Among the
variables that have been assessed
recently, and that have potentially
informative consequences for eluci-
dating the neuropsychological seque-
lae of alcoholism, are the subject
characteristics of aging, nutrition, and
gender. However, it is important to
note that comorbid medical, neuro-
logical, and psychiatric conditions
occur in a large number of alcoholics.
In an attempt to clarify the nature of
residual effects of alcoholism, many
alcohol researchers cautiously exclude
alcoholic subjects with common con-
ditions that may contribute to arti-
facts in interpreting the results. Such
a strategy ignores a large population
of alcoholics who have co-occurring
risk factors. For progress to be
made on this front, future research

439

NLAAA's Neuroscience and Behavioral Research Portfolio

strategies must include samples of alco-
holics who have comorbid conditions.
For consistency in research protocols
across laboratories, study groups can
be homogeneous with respect to many
subject characteristics (e.g., age, gender,
family history), and each co-occurring
risk factor can be studied systematically.
Some laboratories are already taking
this approach (e.g., Nixon et al. 1998;
see reviews by Rourke and Loberg 1996;

Galanter 1998; Bates and Convit
1999; see also chapters 14 and 15 in
this monograph).

Aging

Background

Neuropathological analyses provided
some of the earliest insights into the
relationship between alcoholism and
aging. In postmortem specimens of

Neuropsychiatric Risk Factors

Pre-abuse: FAS/FAE

Systemic illnesses and general health

Head injury

Psychiatric comorbidity

Use of other drugs

Age

Genetics

Temperament

FH+

Alcohol Abuse

Amount per occasion
Duration of abusive drinkin
Pattern over lifetime
Recent amount/duration
Length of abstinence

Test

Characteristics

& Sample

Education
&SES

Brain
Structure

&
Function

Motivation
Expectancies

Neuropsychological
Performance

Figure 1. Variables to consider in models of alcohol -associated neuropsychological abnormali-
ties. FAS = fetal alcohol syndrome; FAE = fetal alcohol effects; FH = family history; SES =
socioeconomic status. Reprinted with permission from Rourke, S.B., and Loberg, T. The
neurobehavioral correlates of alcoholism. In: Grant, I., and Adams, K.M., eds.
Neuropsychological Assessment of Neuropsychiatric Disorders. 2d ed. New York: Oxford
University Press, 1996. p. 443.

440

Neuropsychological Vulnerabilities in Chronic Alcoholism

brains of alcoholics, cerebral atrophy
was noted to resemble the brain shrink-
age that occurs with normal chronologi-
cal aging (Courville 1966). The atrophy
was most prominent in the frontal
lobes, and it extended backward to the
parietal lobes. This finding was replicated
by others, who reported abnormal
ventricular enlargement and widening
of the cerebral sulci of alcoholics in
relation to increasing age (Pfefferbaum
et al. 1993). From the observed simi-
larities in the brains of alcoholic and
aging individuals came a search for
parallels in functional decline associated
with alcoholism and aging.

The premature aging hypothesis has
been put forth in two versions (reviewed
by Ellis and Oscar-Berman 1989).
According to the first version, the
"accelerated aging" (or "cumulative
effects") model, alcoholism is accom-
panied by the precocious onset of
neuroanatomical and behavioral changes
typically associated with advancing
age. Cognitively, or neuropsychologi-
cally, alcoholics become old before
their time. This version proposes that
alcoholics at all ages are impaired
compared with age-matched nonalco-
holic control subjects. The second ver-
sion places the timing of the changes
somewhat differently. In this view,
which has been labeled the "increased
vulnerability" interpretation, the aging
brain is more vulnerable to the delete-
rious influences of toxic substances,
including ethanol, than is the brain of
a younger person. Therefore, the cog-
nitive decline associated with normal
chronological aging (beginning at
around age 50) receives added momen-
tum when combined with alcoholism.

This version proposes that older
alcoholics are impaired compared with
age-matched nonalcoholics; however,
this would not be the case for
younger alcoholics.

Research Review

Taken together, most of the evidence
from neuropathological and neurora-
diological investigations supports the
view of a link between alcoholism and
premature aging. Furthermore, studies
favoring the increased vulnerability
model are more common than those
supporting the accelerated aging
model, although results of a study by
Belzunegui and colleagues (1995)
favor the latter. These investigators
examined neuronal nuclear size and
neuronal population of the mammillary
bodies and anterior thalamic complex
in alcoholics and control subjects ages
30-75. These subcortical structures are
of particular interest because lesions to
them are thought to be critical for
establishing the memory impairment
in alcoholic Korsakoff's syndrome (for
reviews, see Kopelman 1995; Oscar-
Berman and Evert 1997). Belzunegui
and colleagues found significant
reductions in neuronal size and number
in the alcoholics, but the youngest alco-
holics showed the greatest differences.
Most data, however, support the
idea that older alcoholics are especially
vulnerable to the effects of alcoholism.
Elderly alcoholics have an increased
risk of accidents, side effects, and toxi-
city resulting from alcohol intake. In
part this is because older people have
a decreased ability to metabolize alco-
hol and may have concomitant med-
ical problems (National Institute on

441

NIAAA's Neuroscience and Behavioral Research Portfolio

Alcohol Abuse and Alcoholism [NIAAA]
1997). As predicted by the increased vul-
nerability model, certain brain structures
show greater reduction in size in older
alcoholics than in younger alcoholics.
These are the cerebral cortex (Nicolas
et al. 1997; Pfefferbaum et al. 1997),
the corpus callosum (Pfefferbaum et
al. 1996), the hippocampus (Sullivan
et al. 1995#; but see Harding et al.
1997), and the cerebellum (Sullivan et
al. 1995^, 1996; Harris et al. 1999; see
also chapter 14 in this monograph).
Alcoholics also have shown a stronger
association between age and ventricular
dilation compared with nonalcoholic
control subjects (Di Sclafani et al.
1995; Pfefferbaum et al. 1997).

Results of neurobehavioral investi-
gations tend to support the view that
aging increases one's vulnerability to
alcohol-related decline, but controversy
prevails because the association between
alcoholism and aging is less reliable
with behavioral measures than with
imaging techniques (see reviews by
Evert and Oscar- Berman [1995] and by
Oscar- Berman and Schendan [in press]).
More important, when neuroradio-
logical and behavioral changes are
examined together in the same study,
concomitant brain damage with per-
formance impairments is not always
found. For example, Sullivan and col-
leagues (1995#) reported clear evidence
of brain abnormalities but no differ-
ences on explicit memory tests in alco-
holics relative to nonalcoholic control
subjects ages 21-70 years. Explicit
memory refers to the ability to con-
sciously remember facts and events, is
assessed by recognition and recall tests,
and is impaired in alcoholic Korsakoff

and other amnesic patients (for reviews,
see Kopelman 1995; Mayes 1995). The
hippocampus and adjacent cortical
areas have been strongly implicated in
explicit memory by a host of neuropsy-
chological, brain imaging, and nonhu-
man animal studies (Eichenbaum 1997).
Sullivan and colleagues (1995#) eval-
uated whether there were correlations
between magnetic resonance imaging
(MRI) measures of hippocampal volume
and behavioral measures of verbal and
nonverbal delayed recall, as defined by
the Wechsler Memory Scale (Wechsler
and Stone 1945). Anterior portions of
the hippocampus were found to be
smaller in the alcoholics than in the
control subjects, and this difference was
even greater in older than in younger
alcoholics. Although the MRI findings
supported the increased vulnerability
model, the behavioral significance of
the neuroanatomical reduction (i.e.,
increased hippocampal vulnerability in
aging and alcoholism) was unclear
because the explicit memory scores did
not correlate with hippocampal volumes.
Indeed, the alcoholics were not impaired
on the memory tests. Furthermore,
although Sullivan and colleagues
observed visuospatial impairments on
an IQ subtest with a memory compo-
nent (Digit Symbol), there were no
significant correlations with hippo-
campal volume.

In contrast to the findings of Sullivan
and colleagues (1995#) were those
from another study, which found no
evidence of cortical atrophy but did
show aging-related cognitive deficits
(Di Sclafani et al. 1995). In that study,
older alcoholics and control subjects
were given MRI scans and numerous

442

Neuropsychological Vulnerabilities in Chronic Alcoholism

age-normed neuropsychological tests
to compare brain measures and cognitive
abilities. The alcoholics displayed clear
impairments on memory and visuospa-
tial tasks, but there were no group dif-
ferences in global cerebral atrophy
(although two alcoholics had significant
atrophy). One reason for cognitive
dysfunction in the absence of changes
in gross brain morphology is that there
can be synaptic neuronal alterations
that affect processing but are unde-
tectable at a macroscopic anatomical
scale (see Harper 1998 for review).
However, Di Sclafani and colleagues
did find a stronger association between
age and ventricular dilation in the
alcoholics than in the control subjects.

In a neurobehavioral study, Ellis
(1990) found that alcoholics ages 48-74
were impaired compared with nonalco-
holic peers and younger alcoholics (ages
25-47) on Performance IQ subtests
of the Wechsler Adult Intelligence
Scale— Revised (WAIS-R) (Wechsler
1981). Although that finding was sup-
portive of the increased vulnerability
model, another observation in the same
study was not supportive. The older
alcoholics did not show deficits out of
proportion to their age on dichotic
listening tasks of right hemisphere
functioning (a string of piano notes).

In another study assessing the cog-
nitive domain of selective attention
(Evert and Oscar-Berman unpublished
manuscript), male and female alcoholics
and nonalcoholic control subjects ages
29-76 were given a cued detection task
in which the pre-cue provided infor-
mation about the most probable visual
field (VF) location of a subsequent
target letter. The nonalcoholic controls

showed a VE asymmetry, reflected as a
specific disruption when the cue was
presented in the Right VF and the target
was presented in the Left VF. Unlike
the younger control subjects, the alco-
holics in both age groups (and the
older nonalcoholic control subjects)
showed no evidence of a right hemi-
sphere (Left VF) advantage on our task;
they were influenced by the validity of
the cue regardless of the VF in which
it was presented. Gender differences
were absent. In sum, within the cog-
nitive domain of selective attentional
processing, the results from this
experiment provided support for the
accelerated aging model of premature
aging (and also for the right hemi-
sphere hypothesis discussed later in
this chapter).

Results of numerous additional stud-
ies examining right hemisphere func-
tional decline in relation to alcoholism
and aging have not been sufficienriy
consistent to resolve the premature aging
issue (for reviews, see Oscar-Berman
1992; Oscar-Berman and Schendan in
press; see also Coffey et al. 1998).
Consequentiy, the concept of aging as
a model for the study of alcoholism
remains unresolved.

It should be noted that patients with
alcoholic Korsakoff's syndrome (see the
next section) typically display deficits
that are more severe than those of
age-equivalent non- Korsakoff alcoholics
(Evert and Oscar-Berman 1995; Oscar-
Berman and Pulaski 1997). Interestingly,
Wilkinson and Carlen (1982) indi-
cated that the cortical brain morphology
scores of patients with Korsakoff's
syndrome were not age related, as they
were in alcoholics without Korsakoff's

443

NIAAA's Neuroscience and Behavioral Research Portfolio

syndrome. This may be because patients
with KorsakofPs syndrome already
have suffered maximal alcohol-related
brain damage, such that age-related
cortical cell loss becomes irrelevant. In
any case, it is important in considering
alcoholic populations (with and without
KorsakofPs syndrome) to be able to
differentiate, describe, and quantify
the separate contributions of aging and
alcoholism to cognitive decline.

Nutrition

Background

Malnutrition is not a common conse-
quence in alcoholics who have adequate
nourishment, but it is in those who
eat poorly or who have medical condi-
tions that affect the utilization of
nutrients. Groups at risk include the
poor, elderly people living alone, people
with liver disease, and schizophrenics
(Butterworth 1995; Casanova 1996;
NIAAA 1997; Nussbaum 1997).
Dietary factors such as ingestion of
vitamins, protein, and saturated fats
may modulate the severity of alcohol-
related organ damage, and secondary
malnutrition occurs from malabsorption
due to gastrointestinal complications
(e.g., pancreatic insufficiency, gastrec-
tomy, and changes in hepatic metabo-
lism) (Lieber 1995; Shimomura et al.
1998). Thus, malnutrition can influ-
ence brain structure and function in
many ways (Witt 1985; Marsano
1993; Tarter et al. 1993; Lieber 1995).
One area of growing interest is
damage to the cerebellum, which may
be induced by thiamine deficiency
(Welch et al. 1997). Cerebellar damage
produces problems with movement

(mainly gait, balance, and coordina-
tion). However, cerebellar atrophy can
be present in alcoholics without sig-
nificant movement abnormalities (Sul-
livan et al. 1995&). The role of the
cerebellum in cognition is controversial
and, therefore, so is the connection of
cerebellar damage to cognitive changes
in alcoholism (McGlinchy-Berroth et
al. 1995; Sullivan et al. 1995 b\ Shear et
al. 1996; Schmahmann 1997; Ivry
1998; Woodruff-Pak 1998; Harris et
al. 1999; see also chapter 14 in this
monograph). Some investigators have
suggested that alcoholic cerebellar
degeneration represents the same dis-
ease as Wernicke's encephalopathy, an
acute, transient stage of alcohol-
related neurological problems which
include confusion and abnormalities
in oculomotor and gross muscle con-
trol (Reuler et al 1985; Victor 1992;
Cooketal. 1998).

Thiamine deficiency also has long
been associated with KorsakofPs syn-
drome (see reviews by Bowden 1990;
Kopelman 1995; Langlais 1995;
Oscar-Berman and Evert 1997; Cook
et al. 1998). The syndrome is character-
ized most notably by a severe antero-
grade memory loss, which coexists
with an IQ within normal limits, but
other cognitive, emotional, and moti-
vational deficits exist (Oscar-Berman
and Evert 1997). Memory is better
for semantic, procedural, implicit, and
incidental information than for explicit
factual knowledge (Beauregard et al.
1997; Oscar-Berman and Evert 1997;
Seger et al. 1997; Oscar-Berman and
Bardenhagen 1998). Alcoholic
KorsakofPs syndrome is usually pre-
ceded by Wernicke's encephalopathy;

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Neuropsychological Vulnerabilities in Chronic Alcoholism

this acute phase typically disappears
with abstinence and good nutrition.
In the following section, I discuss
research findings relevant to purported
relationships among nutritional
considerations, brain damage, and
neurobehavioral impairments in
Wernicke -Korsakoff patients.

Research Review

The etiology of Wernicke-Korsakoff
syndrome is controversial (Butterworth
1995; Kopelman 1995; Ma and
Truswell 1995; Cook et al. 1998;
Lishman 1998). Some investigators
favor the view that thiamine deficiency
is the primary cause (Bowden 1990;
Joyce 1994; Shimomura et al. 1998);
others believe that the direct neuro-
toxic effects of ethanol are causal
(Laas and Hagel 1994); still others
have suggested that acute Wernicke's
encephalopathy is attributable to the
effects of thiamine deficiency, whereas
chronic Korsakoff s syndrome is due to
the interaction of thiamine deficiency and
ethanol neurotoxicity (Lishman 1998).
Joyce (1994) has stated that while
ethanol neurotoxicity can damage cor-
tical neurons, thiamine malnutrition
affecting the diencephalon can account
for neuropsychological deficits in all
brain-damaged alcoholics. Cullen and
Halliday's evidence (1995) placed the
locus of brain damage from thiamine
deficiency in the cholinergic nucleus
basalis of alcoholics, but in a later study
(Cullen et al. 1997) the investigators
provided evidence that cell loss in that
region was unrelated to memory loss in
alcoholic amnesia. In contrast, Lishman
(1998) suggested distinct subgroups or
clinical forms of alcoholics according

to the brain's vulnerability to alcoholism.
The vulnerability derives from two
distinct pathological influences, which
may operate independently in some
people and may interact in others. The
first, characterized as shrinkage of the
cerebral cortex, as well as possible atro-
phy of basal forebrain regions, is thought
to result from the direct neurotoxic
effects of ethanol or its metabolites. The
second, characterized by damage to
the diencephalon, is attributed to thi-
amine deficiency. According to Lishman,
alcoholics who are susceptible to ethanol
toxicity alone may develop permanent
or transient cognitive deficits associated
with cortical shrinkage. Those who are
susceptible to thiamine deficiency
alone will develop a mild or transient
Korsakoff state, with anterograde
amnesia as a salient feature. Individuals
with dual vulnerability, suffering from
a combination of ethanol neurotoxicity
and thiamine deficiency, will have wide-
spread damage to large regions of the
cerebral cortex, as well as to deep brain
structures. These people will exhibit
severe anterograde amnesia as well as
other cognitive impairments.

Evidence to support the various
models is contradictory (Langlais
1995). In Australia, an investigation
was launched of the cost- effectiveness
of thiamine supplementation alterna-
tives, on the assumption that thiamine
deficiency causes Wernicke -Korsakoff
syndrome (Connolly and Price 1996),
and a policy of thiamine enrichment of
bread flour was introduced in 1991. Ma
and Truswell (1995) conducted a ret-
rospective survey of Australian hospital
records indicating diagnoses of
Wernicke's encephalopathy or

445

NIAAA's Neuroscience and Behavioral Research Portfolio

Korsakoff's syndrome over a 16-year
period (1978-1993). The goal of the
study was to determine whether the
introduction of the thiamine enrichment
program was associated with a decline
in incidence of the disorders. The num-
bers of acute cases began to fall before
1991, but the numbers for years 1992
and 1993 were significantiy lower than
in all previous years. Ma and Truswell
concluded that although their results are
consistent with a preventive effect of
mandatory thiamine enrichment of
bread, the evidence is not conclusive. It
may be that response to treatment is a
function of age of onset of the symp-
toms and the rapidity with which treat-
ment is instituted (Tallaksen et al. 1993).
Other important factors include the
synergistic effects of thiamine deficiency,
ethanol toxicity, and liver disease
(Butterworth 1995; KrU 1995).

Not all alcoholics develop Korsakoff s
syndrome. Therefore, some investiga-
tors have suggested a possible genetic
component, or inborn predisposition
for its occurrence (reviewed by Oscar -
Berman and Evert 1997). In addition,
patients with alcoholic Korsakoff's
syndrome have been described as being
genotypically distinct from patients
with alcoholic dementia (Muramatsu et
al. 1997). Evidence in favor of a genetic
predisposition for Korsakoff's syn-
drome revolves around the demonstra-
tion of deficient transketolase activity
in Korsakoff patients, thought to be
inherited, such that thiamine metabo-
lism is compromised (Blass and Gibson
1977; Butterworth et al. 1993; Wang
et al. 1997). Indeed, Butterworth and
colleagues (1993) found significant
reductions of thiamine-dependent

enzymes in autopsied cerebellar samples
from alcoholic patients with a diagnosis
of Wernicke - Korsakoff syndrome . How-
ever, whereas the enzyme activities in
brain samples from non-Korsakoff alco-
holics were within normal limits in
one study (Butterworth et al. 1993), in
another study (Lavoie and Butterworth
1995) thiamine deficiency and reduced
enzyme activity were observed. Also,
McCool and colleagues (1993) pro-
vided evidence against genetic predis-
position as a viable mechanism, based
on their own work with genetic sensi-
tivity to thiamine deficiency and on
other studies of the development of
alcoholic Korsakoff s syndrome.

Gender

Background

Until recently, gender differences in the
neurobiological effects of alcoholism
have focused mainly on the reproductive
system and hepatic injury. Evidence
suggests that female alcoholics have
increased menstrual disturbances, spon-
taneous abortions, and miscarriages, and
women are more susceptible to alcoholic
liver disease than are men (NIAAA
1997). Only in the last decade have
women (and female nonhuman animals)
been the focus of research on alcohol-
related brain damage (Lancaster 1994,
1995). Still, sample sizes often have
been small, and insufficient attention
has been paid to gender differences in
body size and drinking habits. Because
humans and rodents have opposite
gender differences in drinking patterns
and responses to alcohol (Lancaster
1995), some animal models may have
limited generality with respect to people.

446

Neuropsychological Vulnerabilities in Chronic Alcoholism

In any event, controversy remains
about whether and to what extent
alcoholism affects the brain and neuro-
psychological vulnerability differently
in females and males (reviewed by
Glenn 1993 and Nixon 1993).

Research Review

Parsons (1994) reported that although
male and female alcoholic subjects
showed impaired performance on neu-
ropsychological tests relative to same-sex
control subjects, only the male alcohol-
ics differed from their control subjects
on measures of the brain's electrical
activity. However, other investigators
found that female alcoholics displayed
electrophysiological abnormalities
similar to those reported in male alco-
holics (Hill and Steinhauer 1993). In
still other studies, using brain scans to
measure gender differences in brain size,
evidence is contradictory. For example,
Lishman and colleagues ( 1987) reported
that female alcoholics had larger ven-
tricles compared with female control
subjects (as measured by computed
tomography [CT] scans) than did male
alcoholics compared with male control
subjects. However, Kroft and colleagues
(1991) found that the average ventric-
ular volume in female alcoholics was
within the normal range of ventricular
volumes found in MRI studies of
nonalcoholic females of similar ages.
In another study using CT brain scans
to measure atrophy (Mann et al. 1992),
there was evidence of a similar degree
of brain shrinkage (and reexpansion
after 6 weeks of abstinence) in men
and women, despite significantly shorter
drinking histories in the women. The
findings were obtained after controlling

for moderating variables such as age,
daily alcohol consumption (based on
body weight), and liver dysfunction.

Using MRIs, Hommer and col-
leagues (1996) measured the size of the
corpus callosum in hospitalized male
and female alcoholics, and they reported
that alcoholic women had smaller cal-
losal areas than alcoholic men and
nonalcoholic control subjects. The
reduction in size of the corpus callosum
was equally distributed along its length,
as revealed by comparisons among four
equal segments of the callosal images.
Alcoholic men did not differ from
nonalcoholic male control subjects in
the size of the corpus callosum. Inter-
estingly, callosal area is notably
reduced with age in alcoholics (Pfef-
ferbaum et al. 1996).

The corpus callosum enables com-
munication between the cerebral hemi-
spheres. In both genders, the left and
right sides of the brain subsume asym-
metrical abilities in linguistic (e.g.,
words or phrases) and nonverbal (e.g.,
visuospatial or musical) domains. Specif-
ically, the left hemisphere usually is more
efficient than the right for verbal signals,
and the right hemisphere is more efficient
than the left for nonverbal signals. The
two hemispheres also are complementary
in their processing modes, depending
on context and task demands. The left
hemisphere plays a special role in pro-
cessing piecemeal information analyti-
cally and sequentially, and the right
hemisphere plays a special role in inte-
grating information holistically.

Abnormalities in the structure of the
corpus callosum can occur as a conse-
quence of diffuse cortical damage and
subsequent degeneration of cortical

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NIAAA's Neuroscience and Behavioral Research Portfolio

axons. Diffuse cortical damage, in
turn, would be interpreted in conven-
tional neuropsychological testing as a
selective right hemisphere functional
deficit either because right hemisphere
functions have less cortical representa-
tion than left hemisphere functions or
because nonverbal tasks tend to be
more difficult or less familiar than ver-
bal tasks (Ellis and Oscar-Berman
1989). Alternatively, diffuse cortical
and/or callosal atrophy could interfere
with cross-callosal transfer of informa-
tion, causing some of the cognitive
deficits observed in alcoholics and
aging people (Hutner and Oscar-
Berman 1996; Rourke and Loberg
1996). For example, if the left hemi-
sphere's contribution to nonverbal
tasks normally is greater than the right
hemisphere's contribution to verbal
tasks, an interhemispheric transfer dys-
function would affect visuospatial
functions more than verbal functions.
Because the characteristics of hemi-
spheric dominance are specific to each
functional brain area, the altered pat-
terns of hemispheric dominance from
alcoholism (and aging) may be idio-
syncratic to those brain areas and
behavioral functions. It is, therefore,
important to appreciate which brain
regions are affected by chronic alco-
holism (and aging).

Results of neuropsychological stud-
ies, like the evidence from imaging
and electrophysiological studies, are
inconclusive regarding the connection
between gender and susceptibility to
alcohol-related brain damage. For
example, Parsons (1994) and Lishman
and colleagues (1987) reported no gen-
der differences in neuropsychological

functioning, despite the presence of
gender differences in alcoholics' brain
electrical activity and ventricular size.
However, women often have shorter
drinking histories, consume less alcohol,
and have been abstinent for a longer
period of time than men. Because other
individual differences in brain structure
and function tend to obscure gender
differences (Lezak 1995), one fruitful
way of studying possible gender- related
neuropsychological effects of alco-
holism is to capitalize on known sexually
dimorphic brain and behavioral char-
acteristics. This can be achieved in
conjunction with neuropsychological
tests having special sensitivity to the
functions of particular brain regions
or systems, especially in view of differ-
ences between men and women in the
organization and functioning of the
two cerebral hemispheres. Results of
meta-analytic studies of functional
brain asymmetries have shown that
men tend to be more lateralized than
women (Hiscock et al. 1994, 1995);
women also have a larger corpus callo-
sum (Voyer 1996). Such differences
suggest that bilateral representation of
verbal and nonverbal functions, along
with greater interhemispheric commu-
nication, is more prevalent in women
than men. Furthermore, gender dif-
ferences in brain size have been
reported as a function of aging: cere-
brospinal fluid (CSF) volume overall
was found to be greater in elderly men
than in elderly women, as were CSF
volumes in the sylvian fissure and parieto-
occipital regions (Coffey et al. 1998).
However, aging-related structural
cerebral asymmetries were indepen-
dent of gender. In any case, Coffey et

448

Neuropsychological Vulnerabilities in Chronic Alcoholism

al.'s normative measures of brain
aging (as well as results of a meta-analy-
sis by Meinz and Salthouse [1998])
appear to show greater decline in men
than in women, in distinct contrast to
the gender effects of alcoholism.

These differences suggest an impor-
tant area for future research, because
studies of alcoholics have consistently
revealed a pattern of poor performance
on visuospatial tasks. Visuospatial tasks
are also difficult for patients with dam-
age to the right hemisphere. The simi-
larity in performance between alcoholics
and right hemisphere patients led
researchers to hypothesize that right
brain functions are more vulnerable to
the effects of alcoholism than left
brain functions (see the section on right
hemisphere vulnerability later in this
chapter). Of interest in this regard is a
study by Errico and colleagues (1992)
that examined hormone levels and
cognitive performance in male alco-
holics. (Alcoholic women were not
included in the study.) The investiga-
tors measured testosterone levels in
alcoholics and nonalcoholic control
subjects and related the hormone levels
to performance on visuospatial and
verbal tasks. Errico and colleagues
replicated, in the control group, the
findings of Christiansen and Knussmann
(1987), who observed a positive rela-
tionship between testosterone levels
and performance on visuospatial tasks
in healthy nonalcoholic men. However,
in the alcoholics, Errico and colleagues
found that the expected relationship
between testosterone levels and perfor-
mance on visuospatial tasks was absent
compared with nonalcoholic peer con-
trol subjects. Instead, the alcoholics'

testosterone levels were significantly
correlated with performance on verbal
tests. As an explanation, Parsons sug-
gested that the alcoholics' impaired
visuospatial abilities might have led
them to rely on their intact verbal
functioning, perhaps indicating a shift
in cerebral dominance (OA. Parsons,
personal communication, May 1998).
Drake and colleagues (1990) mea-
sured gender differences in hemi-
spheric asymmetries using dichotic
listening procedures sensitive to left
and right hemisphere functioning. They
presented a series of two different
words or two different melodies to male
and female alcoholics and nonalcoholic
control subjects to measure their abili-
ties at identifying the competing verbal
or nonverbal signals. Male alcoholics
showed atypical laterality patterns.
Compared with control subjects, the
male alcoholics showed a larger left
hemisphere advantage for identifying
words and a smaller right hemisphere
advantage for identifying melodies. In
contrast, female alcoholics' laterality
patterns did not differ from those of
control subjects on either of the dichotic
listening tasks. The authors concluded
that male alcoholics showed evidence of
right hemisphere dysfunction, but female
alcoholics did not. Unfortunately, in
the alcoholism literature, other studies
comparing the separate functions of
the left and right cerebral hemispheres
(based on visual, tactual, and auditory
signals) have relied mainly on male
research participants. Results of these
studies have been inconsistent in
showing atypical asymmetries, and no
consistent pattern of abnormalities has
emerged. However, gender differences

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typically have not been examined (see
review by Oscar-Berman 1992). In
addition, the question of aging-related
gender difference in hemispheric
asymmetries has not been addressed in
most studies of alcoholics (but Evert
and Oscar-Berman [unpublished man-
uscript] found no gender differences).
This issue is ripe for future research.

Research Opportunities

As was indicated early on, numerous
individual differences contribute to
the lack of consensus about the effects
of alcoholism on brain and behavior.
However, interesting trends have
emerged when subject characteristics
have been studied systematically
(aging, nutrition, and gender). For
example, the role of malnutrition
(especially thiamine deficiency) seems
to contribute to severe cognitive
deficits (e.g., as in Wernicke -Korsakoff
syndrome), but the critical lesions and
the underlying mechanisms remain
unknown. New research is needed to
clarify the separate and combined roles
of malnutrition and ethanol neurotox-
icity to brain damage and functional
impairments. In addition, older alco-
holics and women alcoholics are espe-
cially vulnerable to the deleterious effects
of alcoholism, as evidenced by a conver-
gence of findings on these subgroups.
Typically, older alcoholic women have
not been the focus of investigation.
Future studies can capitalize on normal
gender differences in brain organization
to address questions such as whether
and how alcoholism affects perceptual
asymmetries and other neurobehavioral
measures, and how the effects are bound

to aging (Hiscock et al. 1994, 1995;
Voyer 1996; Seeman 1997).

Future research should concentrate
on the promising trends, and investi-
gations should be intensified of the
less-studied subject characteristics.
Expermiental methods should be
improved by encouraging consistency
in research protocols across laborato-
ries, employing study groups that are
homogeneous with respect to specific
individual differences (e.g., age, gen-
der, family history), and systematically
evaluating each co-occurring risk factor
(including the use of statistical covari-
ance procedures for confounding factors,
when necessary). Because there may be
differing degrees of liability that each
relevant subject characteristic contributes
to brain- behavior impairment, longi-
tudinal or retrospective methods could
be used to evaluate subgroup vulnera-
bility. In addition, topics related to
vulnerability to cognitive deficits may
be approached by using nonhuman
animal models (Gallagher and Rapp
1997; Oscar-Berman and Bardenhagen
1998). It is important to assess the
comparability of human and nonhuman
models in alcohol-related brain damage;
similarities and differences among study
species need to be clearly established
in future work on aging, nutrition, and
gender. Table 2 outlines the ideal meth-
odological strategies for future research.

VULNERABILITY OF
SPECIFIC BRAIN REGIONS

The Cerebral Cortex

Cortical changes have been reported
throughout the brain, but there is

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Neuropsychological Vulnerabilities in Chronic Alcoholism

evidence that some cortical regions, (Jernigan et al. 1991&; Pfefferbaum

especially the frontal and parietal and Rosenbloom 1993; Wang et al.

lobes, are more consistendy vulnerable 1993; Davila et al. 1994; Sullivan et

to the effects of chronic alcoholism (as al. 1995#; Shear et al. 1996; Estruch

well as aging) than other regions et al. 1997; Kril et al. 1997; Adams et

Table 2. Ideal Methodological Strategies for Future Research.

Make Subject Sample Characteristics Clear and Explicit

Age, gender, race, ethnicity

Education; IQ

Drinking history, family history of alcoholism

Medical and psychiatric comorbidity

Develop Uniform Definitions of Subgroups of Alcoholics With Brain Impairments

Mild cognitive deficits
Wernicke's encephalopathy
Korsakoff's syndrome
Alcohol-induced dementia
Marchiafava-Bignami disease
Cerebellar degeneration

Develop Operational Definitions of Functions To Be Measured

Perceptual processing/information processing
Attention (e.g., sustained attention, divided attention)
Memory (e.g., working memory, explicit/implicit memory)
Disinhibition/response perseveration
Executive dysfunction

Use Multiple Methodologies for Convergence of Evidence

Neuropsychology

Standardized psychiatric and neuropsychological assessment procedures (e.g., IQ, memory tests)

Paradigms from behavioral neuroscience (e.g., perceptual processing, attention, working

memory, disinhibition/response perseveration)
Neuroimaging

Magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging, positron

emission tomography, single photon emission computed tomography, structural and

functional magnetic resonance imaging, diffuse tensor imaging, magnetoencephalography
Electrophysiology

Electroencephalography
Event- related potentials
Neuropathology/Neurochemistry

Gross structural morphology

Cellular structure

Receptor level (neurotransmitters)

Establish Comparability Between Human and Nonhuman Animal Models

451

NIAAA's Neuroscience and Behavioral Research Portfolio

al. 1998). Frontal, cingulate, parietal,
parietooccipital, and mesial temporal
cortices in alcoholics show reduced
metabolic activity with positron emis-
sion tomography (PET) and single
photon emission computed tomography
(SPECT) (e.g., Adams et al. 1993;
Volkow et al. 1995; Gilman et al.
1996; Volkow et al. 1997; Gansler et
al. 2000) and significantly smaller gray
and/or white matter volume with
structural MRI (e.g., Jernigan et al.
1991**; Sullivan et al. 1998).

Kril and colleagues (1997) described
selective neuronal loss at a microscopic
level in the frontal lobes of alcoholics.
Pfefferbaum and colleagues (1997)
conducted regional MRI analyses of
cortical integrity and found evidence that
the frontal lobes were especially vul-
nerable to chronic alcoholism across a
wide age range, but the effects were
exacerbated in elderly people. Of inter-
est, Pfefferbaum and colleagues also
noted that temporal-parietal loss
occurred mainly in older alcoholics.
Thus, their findings favor the
increased vulnerability model of pre-
mature aging in alcoholism. Another
recent study by Sullivan and colleagues
(1998) showed fewer regional abnor-
malities than those of Pfefferbaum and
colleagues, but both studies reported
a relative sparing of gray matter (but
loss of white matter) in the posterior
superior temporal region.

In a study that compared MRI
measures of the brains of Korsakoff
and non-Korsakoff alcoholics, Jernigan
and colleagues (1991^) observed that
Korsakoff patients had greater ventric-
ular size, and smaller gray matter vol-
umes in the septal nuclei, anterior

hypothalamus, mesial temporal cortex,
and orbitofrontal cortex. However, in
a later study by Emsley and colleagues
(1996), comparing MRI measures of
cortical and subcortical regions in alco-
holics with and without Korsakoff's
syndrome, the extent of cortical gray
matter was similar in Korsakoff patients
and control subjects, even though sub-
cortical findings were similar to those of
Jernigan and colleagues. Hence, there
is controversy about the locus and
extent of brain involvement among
alcoholic subtypes.

Neurobehavioral deficits suggestive
of cortical atrophy in alcoholics have
been reported. Various studies have
disclosed difficulty with tests of problem
solving and conceptualization (Beatty
et al. 1993; Sullivan et al. 1993; Pollux
et al. 1995; Brunfaut and d'Ydewalle
1996; Sullivan et al. 1997), selective
attention (Evert and Oscar-Berman
unpublished manuscript), spatial memory
(Joyce and Robbins 1991; Verfaellie
et al. 1992; Bowden and McCarter
1993), working memory (Oscar-Berman
and Hutner 1993; Sullivan et al.
1997), visual association learning and
recognition memory (Bowden et al.
1992; Oscar-Berman and Pulaski 1997),
and tactual learning (Oscar-Berman et
al. 1990). In most studies, neurobehav-
ioral deficits have been assessed indepen-
dently of brain changes. The importance
of using both approaches (neurobehav-
ioral and structural/neuroradiological)
is to evaluate ideas related to the con-
nection between the locus of damage
and the nature of the decline in func-
tioning. In studies where both types of
measures have been obtained, results
have not revealed consistent convergence

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Neuropsychological Vulnerabilities in Chronic Alcoholism

between brain changes and presumed
functional loss.

For example, in a study by Jernigan
and colleagues (1991«), although several
significant correlations were found
between MRI measures and perfor-
mance on cognitive tests, there was little
evidence of a relationship between gray
matter measures and cognitive test
scores. In another study, Wang and col-
leagues (1993) used MRI and func-
tional imaging techniques in conjunction
with behavioral measures for comparing
long-term alcoholic patients with non-
alcoholic control subjects. The inves-
tigators noted that the degree of
cortical atrophy on MRI was associ-
ated witii decreased brain metabolism,
perhaps indicative of loss of brain tissue.
Although they did report a relationship
between certain neuropsychological
test scores and measures of frontal
brain metabolism in the alcoholics,
neuropsychological performance did
not correlate with MRI structural
changes. The lack of correlation was
interpreted as reflecting either the
preservation of cognitive abilities with
mild brain structural changes or insen-
sitivity of the tests employed for
detecting mild brain structural changes.
In a different study, CT scans and
cerebral blood flow measures were
obtained in a sample of 40 chronic
alcoholic patients, and the findings
were related to results of neuropsycho-
logical testing (Nicolas et al. 1993).
The researchers found evidence of sig-
nificant brain hypoperfusion in 65
percent of the alcoholics (26 out of
the 40), but only about 25 percent of
them (11 of the 40) had CT evidence
of cerebral atrophy (mainly in the

frontal lobes). In this study, the alco-
holics exhibited significant impairments
on tests of frontal lobe functioning
and visuospatial skills. Frontal lobe
test performance was independently
related both to frontal atrophy and to
frontal hypoperfusion.

Frontal Brain Systems

Results of neuropsychological, neuro-
physiological, and functional neuroimag-
ing studies generally are compatible
with deficits in brain systems involving
the frontal lobes (Erbas et al. 1992;
Nicolas et al. 1993; Wang et al. 1993;
Cohen et al. 1997; Pfefferbaum et al.
1997; Zhang et al. 1997; Adams et al.
1998; Hoaken et al. 1998; Gansler et
al. 2000). Melgaard and colleagues
(1990) measured regional cerebral blood
flow in alcoholic and nonalcoholic men
and found that the alcoholics showed
significant flow reduction in the antero-
mesial frontal region, in a small area
of the left parietal region, and in the
mesial part of the right occipital lobe.
Severity of alcoholism was related to
greater flow reduction in frontal cortex
and in periventricular regions. When
alcoholics were compared on the basis
of severity of intellectual impairment,
the more impaired group showed a
greater flow reduction in frontal cortical
and periventricular regions as well as in
cortical regions of the temporal lobes.
Thus, greater flow reduction was asso-
ciated with poorer test performance.
Adams and colleagues (1993) obtained
similar results: Alcoholics showed
hypometabolism in the medial frontal
region. Significant correlations were
found ( 1 ) between frontal lobe metab-
olism and errors on a test sensitive to

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NIAAA's Neuroscience and Behavioral Research Portfolio

frontal lobe damage and (2) between
cerebral metabolism and atrophy of
medial frontal cortex on CT or MRI
scans. Other studies have confirmed the
reports of a correlation between impaired
neuropsychological performance on tests
of frontal functioning and decreased
frontal lobe perfusion or metabolism in
alcoholics (Nicolas et al. 1993; Wang
et al. 1993; Adams et al. 1995, 1998).
Thus, investigators have observed
expected relationships between reduced
frontal brain activity and abnormalities
in abilities such as executive control
skills. The findings collectively support
the view that alcoholism results in
impaired metabolic and neurobehavioral
functions of the frontal cortex (Hoaken
et al. 1998). In addition, frontal lobe
hypometabolism may reflect loss of tis-
sue. In a study by Erbas and colleagues
(1992), decreased blood flow (mostly in
frontal regions) was found in 85 percent
of the alcoholics measured, but only
60 percent of the alcoholics showed
structural changes on CT scans.

Of interest also are studies demon-
strating recovery of brain functioning
with abstinence. During die first 1-2
months of abstinence from alcohol, a
partial recovery of metabolic or perfu-
sion deficits has been observed, with the
greatest improvement in the frontal
lobes (Nicolas et al. 1993; Volkow et
al. 1994). In a pilot SPECT study of
10 alcoholics with long and short
periods of abstinence, Gansler and
colleagues (2000) observed a positive
relationship between perfusion levels
in the left inferior frontal brain region
and years of sobriety. Alcoholics with
less than 4 years of sobriety had signif-
icantly reduced left inferior frontal

perfusion compared with nonalcoholic
control subjects and alcoholics having
longer periods of sobriety. However,
despite a significant correlation between
left inferior frontal perfusion and
memory scores, the short- and long-
term sobriety groups did not differ on
measures of IQ and memory. It is pos-
sible that SPECT measures are indepen-
dent of overt neurobehavioral measures
(e.g., see Dupont et al. 1996). In any
case, the effects of long-term absti-
nence on frontal lobe perfusion and
corresponding cognitive deficits have
been insufficiently investigated.

Hunter and colleagues (1989) mea-
sured cerebral blood flow in Korsakoff
patients. Compared with nonalcoholic
control subjects, Korsakoff patients
showed a trend toward reduced blood
flow in frontal areas. The Korsakoff
patients showed several significant
correlations between the degree of flow
reduction in frontal areas and the degree
of impairment on memory and orienta-
tion tests (decreased flow corresponded
to increased impairments). Hunter
(1990) noted that frontal metabolic
deficits could mean that a normal tissue
mass has reduced neuronal activity, or
that a reduced tissue mass has normal
activity levels — or some of both. Hunter
further noted that since some CT and
neuropathological studies point to
structural loss of gray and white matter
in the frontal lobes of Korsakoff patients,
the metabolic impairment in this region
probably at least in part reflects reduced
tissue mass. Paller and colleagues (1997)
used PET to measure regional cerebral
metabolism in Korsakoff patients per-
forming a continuous recognition test.
The investigators found that Korsakoff

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Neuropsychological Vulnerabilities in Chronic Alcoholism

patients demonstrated a severe memory
impairment in delayed recognition and
concomitant widespread decline in
glucose metabolism in frontal, parietal,
and cingulate regions (but not in the
hippocampus), suggesting that cortical
and neuropsychological abnormalities
are related. Neurobehavioral studies
have shown that Korsakoff patients
exhibit clinical signs associated with
damage to the frontal cortex, for exam-
ple, emotional apathy, disinhibition,
and abnormal response perseveration
(Kopelman 1995; Oscar- Berman and
Evert 1997). Research adapting tests
highly sensitive to frontal lobe damage
in monkeys also supports the view of
frontal system dysfunction in alcoholic
Korsakoff's syndrome (for a review, see
Oscar-Berman and Bardenhagen 1998).
It is important to point out that new
discoveries concerning the functioning
of healthy and diseased frontal brain
systems are appearing in neuroscience
literature unrelated to alcoholism (see
Wickelgren 1997). These discoveries
reveal an exquisite cooperation among
various brain areas, partly under control
of the frontal cortex; they provide excit-
ing insights that can be applied to the
design of studies aimed at under-
standing abnormal frontal system
functioning in alcoholism (e.g., Tracy
et al. 1995; Sullivan et al. 1997;
Freedman et al. 1998).

Right Hemisphere

Clinical reports and experimental
studies have provided evidence that
each hemisphere of the human brain
is important for mediating different
functions. The left hemisphere has a
dominant role in communication and

in understanding the spoken and written
word, and the right hemisphere plays
a dominant role in coordinating inter-
actions with the three-dimensional
world around us (e.g., spatial cognition).
Processing modes of the two hemi-
spheres complement each other.
Depending on context and task demands
(Banich and Heller 1998), the left
hemisphere processes information
analytically and sequentially, whereas
the right hemisphere integrates infor-
mation holistically. Differences between
the two cerebral hemispheres can be
seen easily in cases of unilateral brain
damage, and standard clinical neuro-
psychological tests are helpful for educ-
ing the dichotomy (e.g., see Lezak
1995). Patients with left hemisphere
damage often have problems with lan-
guage, and patients with right hemi-
sphere damage often have difficulty
with maps, designs, music, and other
nonlinguistic materials. Of interest to
this discussion is the fact that alcoholics
(and elderly people) have difficulty on
tasks that resemble those on which
patients with damage to the right
hemisphere also encounter problems.
In particular, patients with right hemi-
sphere lesions, as well as alcoholic and
elderly individuals, are disproportion-
ately impaired on nonverbal visuospa-
tial tasks, as assessed by Performance IQ
subtests (compared with Verbal IQ
subtests) of the WAIS-R These obser-
vations led, independently, to the right
hemisphere hypothesis in both the alco-
holism literature and the aging literature
(Oscar-Berman and Schendan in press).
The right hemisphere hypothesis
states simply that the right half of the
brain is more vulnerable to the effects

455

NIAAA's Neuroscience and Behavioral Research Portfolio

of alcoholism (or to the effects of aging)
than the left half of the brain. In other
words, a disproportionate decline in
nonverbal, visuospatial functions is
attributed to a greater sensitivity of
the right hemisphere to the neurobio-
logical consequences of alcoholism or
aging. Studies of right hemisphere
contributions to cognitive functions in
alcoholism and aging have been reviewed
extensively (e.g., Ellis and Oscar-
Berman 1989; Oscar-Berman 1992;
Parsons and Nixon 1993; Gerhardstein
et al. 1998; Oscar-Berman and Schendan
in press) and will not be summarized
here. In general, the results of most of
the research in this area — as was seen
earlier in reviewing gender differences
and the right hemisphere — have
provided only equivocal support for
the hypothesis that alcoholism and
aging (alone or together) differentially
affect the functioning of the two cere-
bral hemispheres.

The topic of right hemisphere
integrity is important not just from the
standpoint of contributions to cognitive
functions (such as visuospatial skills), but
also because of the role of the right
hemisphere in emotion. Emotional
abnormalities such as those known to
accompany alcoholism (Oscar-Berman
et al. 1990; Oscar-Berman 1992;
McGue et al. 1997) result from lesions
in multiple brain systems (Borod 1993;
Gainotti et al. 1993; Heller et al. 1998).
Modular models of emotion combine
concepts from lateral dominance with
ideas about cortical-subcortical inter-
actions (Borod 1993). Although many
brain centers act to take in emotional
cues, evaluate them, and execute appro-
priate reactions, the right hemisphere's

role in emotional functions generally is
more relevant than the left hemisphere's
role, and the right hemisphere is more
relevant for processing negative emo-
tions than for positive emotions (Borod
1993; Gainotti et al. 1993; Heller et
al. 1998). Therefore, emotional percep-
tion tasks (especially those that include
stimuli with a negative valence) can be
used to tap right hemisphere abilities.
If alcoholics have deficient right hemi-
sphere function, they should have more
difficulty perceiving emotional materials
than nonemotional materials, and they
should have more difficulty perceiving
negative than positive emotional stimuli.
Likewise, if alcoholism interacts with
aging, then deficits should be most
pronounced in older alcoholics (com-
pared with their nonalcoholic peers).

In a recent study (Covall and Oscar-
Berman unpublished data), we used
variations of the Stroop color-word
interference task to assess emotional
abilities in alcoholics and nonalcoholic
control subjects across a wide age
span (29-83 years). We presented
positive and negative emotional words,
and neutral words, in a color-naming
paradigm — that is, the participants
were asked to report only the colors
of the words, which were written in
different colored inks. We hypothesized
that if there are changes in emotional
perception as a function of alcoholism
and/or aging, then alcoholics (especially
those over age 50) would show abnor-
mal interference effects for emotional
as compared with neutral words, and
negative words might be less disruptive
than positive words. Results showed
expected emotional interference effects
for all subjects, and differences in

456

Neuropsychological Vulnerabilities in Chronic Alcoholism

emotional valence processing were
found among older subgroups: older
alcoholics were more susceptible than
older nonalcoholic control subjects to
the interference effects, and positive
emotional words produced a greater
interference effect than negative and
neutral words. Thus, the findings pro-
vided some support for the right
hemisphere hypothesis (as well as for
the premature aging hypothesis).

In another study, Hutner and
Oscar-Berman (1996) used a visual
backward -masking perceptual laterality
paradigm to evaluate emotional process-
ing abilities in detoxified alcoholics
compared with nonalcoholic control
subjects ages 30-69. Emotional and
nonemotional words were presented
tachistoscopically to the Left or Right
VFs, followed by a visual masking
stimulus. The research participants
were asked to judge the emotional
valence of each word (positive, negative,
or neutral), and to respond verbally or
manually (button presses). The depen-
dent measure was the critical inter-
stimulus interval needed to escape the
backward-masking effect. The alco-
holics showed a significant Right VF
advantage in both response mode
conditions, whereas the control subjects
did not. In addition, older alcoholics
showed a selective impairment in pro-
cessing negative words. Therefore, once
again the findings were in support of
suggestions that alcoholics (especially
older alcoholics) may have deficient
right hemisphere functions when
emotional materials are used.

Since distinct affective/cognitive
domains, as well as multiple brain areas,
can underlie different emotional tasks,

broadly defined neurobehavioral alter-
ations can occur from injury to only a
subset of functions. Cognitive neuro-
scientific research has made notable
progress in recent years with the advent
of new functional brain imaging tech-
niques and improvements to tradi-
tional approaches toward defining the
functional contribution of specific brain
systems to human behavior. These find-
ings with populations of neurologkally
intact and impaired nonalcoholics,
coupled with the neuroanatomical,
neuropsychological, and cognitive
neuroscientific studies of alcoholics,
have provided additional means for
more precisely testing the right hemi-
sphere hypothesis (and the premature
aging hypothesis). With the new tech-
niques available, we anticipate a suc-
cessful future in understanding the
ways the cerebral hemispheres act to
integrate and complement their func-
tions, and a similar leap in progress in
pinpointing precisely the neurobehav-
ioral consequences of long-term
chronic alcoholism, as well as the syn-
ergism of alcoholism and aging.

Other Brain Systems

Memory loss similar to the amnesia in
Korsakoff patients has long been associ-
ated with surgical lesions of the temporal
lobes, which include the hippocampus
and the amygdala (Zola-Morgan and
Squire 1993; Petri and Mishkin 1994).
Memory impairment in Korsakoff
patients has also been associated with
lesions in basal forebrain and diencephalic
areas (including the mammillary bodies
of the hypothalamus, the dorsomedial
thalamic nucleus, and the fibers connect-
ing these two structures) (Victor 1992).

457

NIAAA's Neuroscience and Behavioral Research Portfolio

In addition to memory impairments,
anomalies most probably related to
limbic system damage that have been
studied in alcoholic Korsakoff patients
include emotional changes (Douglas
and Wilkinson 1993; Hutner and Oscar-
Berman 1996; Oscar-Berman and Evert
1997) and a reduced ability to integrate
information coming in from more than
one sense modality (e.g., cross-modal
functions [Oscar-Berman et al. 1990;
Shaw et al. 1990]). Emotional changes
and multimodal deficits in Korsakoff
patients have been inadequately
researched, however, and they remain
poorly understood.

In studies by Jernigan and colleagues
(reviewed by Butters and Jernigan 1995),
MRI scans from Korsakoff and non-
Korsakoff alcoholics showed that
Korsakoff patients had — in addition to
widespread reductions in cortical gray
matter volumes and increases in CSF —
significant reductions in diencephalic
structures. Volume losses in anterior
portions of the diencephalon, medial
temporal lobe structures, and the orbito-
frontal cortex were found to differentiate
best between the Korsakoff and control
groups. These findings were used to
support the view that damage to dien-
cephalic structures is involved in antero-
grade amnesia and that other regions,
such as the hippocampus, may also con-
tribute to Korsakoff patients' amnesic
symptoms. However, in a different study
comparing MRI measures of dien-
cephalic damage in alcoholics with and
without the amnesia of Korsakoff s syn-
drome, Blansjaar and colleagues (1992)
found that diencephalic atrophy was of
similar frequency in both groups.
Based on these results, the investigators

suggested that diencephalic lesions
develop regardless of whether patients
acquire the amnesia of Korsakoff s syn-
drome, and are not so much typical of
Korsakoff s syndrome as they are of
chronic alcoholism and malnutrition.

In regard to the nature and location
of brain damage in Korsakoff s syn-
drome, it is important to note that
Korsakoff described neuropathology
in the cerebral cortex, but he did not
specify a subcortical locus of brain
damage for the syndrome bearing his
name. Rather, he described a set of
neuropsychological characteristics in
association with various etiologies
(Victor and Yakovlev 1955). The brain
regions that have been implicated in
the syndrome were described much
later by researchers using inconsistent
neuropsychological criteria to classify
the patients whose brains were subse-
quently examined at autopsy (Victor
1992; Kopelman 1995; Oscar-Berman
and Evert 1997). Controversy still exists
with regard to the critical lesion site(s).
Therefore, the idea that alcoholic
Korsakoff s syndrome can be "neuro-
pathologically confirmed" (e.g., Martin
et al. 1993; Butterworth 1995), without
knowledge and standardization of ante-
mortem neuropsychological symptoma-
tology, may be misleading. The best
approach will be one in which multiple
methodologies are used, namely, neuro-
behavioral, neuroimaging, and neu-
ropathological approaches.

Research Opportunities

Convergent results indicate that much of
the brain is vulnerable to alcohol-related
damage, but cortical changes have
been reported most consistently across

458

Neuropsychological Vulnerabilities in Chronic Alcoholism

subgroups of alcoholics. Of the corti-
cal systems that have been implicated,
frontal and right hemisphere systems
have received considerable research
attention. Nonetheless, many topics
remain poorly explored and are in
need of future research. Some of these
topics are (1) the manner in which
disruption of specific subsystems of
frontal brain regions contributes to
impairments in attention, perception,
emotional functioning (and regulation
of drinking), and executive functioning;
(2) the ways in which atrophy of the
corpus callosum interferes with inter-
hemispheric communication and/or
normal cerebral asymmetries; (3) char-
acterization of deficits in memory and
emotional functioning resulting from
damage to limbic and diencephalic
structures (and how they differ from
frontal-induced deficits); (4) the dif-
ferential contributions of cerebellar
damage to motor versus cognitive
impairments; and (5) the nature of
abnormalities in specific neurotrans-
mitter systems.

To understand the etiologies and
mechanisms of brain damage across
subgroups of brain-impaired alcoholics,
future studies should use multiple
methodologies such as structural and
functional imaging techniques, neuro-
pathological studies, electrophysiolog-
ical studies, and neurobehavioral
studies (Rabbitt 1997; Wickelgren
1997; Liu 1998; Oscar-Berman and
Bardenhagen 1998). In many instances,
diagnostic procedures for establishing
alcoholic functional impairments vary
across studies. Alcohol-induced demen-
tia, for example, is a controversial dis-
order because investigators differ with

respect to the deficits that should be
included when defining its clinical pre-
sentation (Smith and Atkinson 1995).
Likewise, there is lack of consensus
regarding criteria for classifying
patients with Wernicke -Korsakoff syn-
drome. At the outset, therefore, it is
important to establish uniform positive
diagnostic criteria for subtypes of
brain-impaired alcoholics; this can be
approached with the aid of sophisti-
cated neurological and neurobehav-
ioral techniques.

SUMMARY AND
CONCLUSIONS

Neuropsychological impairments result
from prolonged ingestion of alcohol
in vulnerable populations. I reviewed
research findings on three factors that
make certain alcoholics susceptible to
behavioral and brain changes: chrono-
logical aging, gender, and nutritional
deficiencies. A convergence of the evi-
dence supports the view that older
alcoholics are especially at risk for the
deleterious effects of alcohol on the
brain, but women are not consistentiy
found to be vulnerable. However,
older alcoholic women have not been
studied sufficiently. Future research can
capitalize on normal gender- dimorphic
differences in brain organization (e.g.,
gender differences in hemispheric asym-
metries) to address questions such as
whether and how alcoholism affects
perception, attention, emotion, and
other neurobehavioral functions, as
well as gender effects in association
with aging. Malnutrition — especially
thiamine deficiency — seems to con-
tribute to limbic and basal forebrain

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lesions leading to severe cognitive deficits unknown. Similarly, it is important to

in Wernicke -Korsakoff syndrome, but clarify the etiology and consequences

the critical lesions and the underlying of cerebellar degeneration. Research is

neuropathological mechanisms remain needed to explicate the separate and

Table 3. Important Research Questions and Topics.

How do individual differences (subject characteristics) contribute to alcohol's effects on
neuropsychological functioning?

Age

Investigate adolescents

Investigate elderly (including women)
Gender

Use sexually dimorphic human characteristics (e.g., hemispheric differences)

Compare human and nonhuman animal models
Nutrition vs. toxicity

Clarify roles in Wernicke's encephalopathy, KorsakofFs syndrome, cerebellar degeneration,

and Marchiafava-Bignami disease

Evaluate nutritional therapies (e.g., thiamine fortification)
Drinking history

Study amount and type of alcoholic beverages consumed, and duration of alcoholism

Consider duration of abstinence
Family history

Investigate genetic factors

Investigate drinking habits
Comorbidity: Evaluate the combined effects of alcoholism and

Polysubstance abuse

Psychiatric comorbidities

Medical problems (e.g., HIV and liver disease)

Head injury

Which individual differences (subject characteristics) influence recovery of structural
and/or functional integrity?

Age (when tested and at onset of drinking)

Gender

Nutrition

Drinking history

Family history

Comorbidity

What are the similarities and differences between the residual and the acute effects of
alcoholism?

How do they parallel each other (e.g., brain dysfunction and brain damage)?
How do they contribute to abusive drinking (e.g., risky behaviors, disinhibitions)?

Are animal models sensitive to problems associated with human alcoholism?

Are the behavioral paradigms comparable across species for measuring alcohol's effects?
Are "lesion effects" comparable across species?
Are gender effects comparable across species?

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combined roles of malnutrition and
ethanol neurotoxicity to brain damage
and functional impairments in these
devastating disorders, as well as in
alcoholic dementia. A prerequisite to
research on severely impaired alcoholic
patients, however, is the adoption of
uniform terminology for defining the
disorders and syndromes under study.
Methodological standardization also
is critical.

Although alcoholics have diffuse
cortical damage that affects functioning
of the two cerebral hemispheres, no def-
inite relationships have been established
between alcoholism-related damage to
specific cortical areas and concurrent
cognitive impairments. However, find-
ings from neuroimaging and neuro-
pathological studies point to increased
susceptibility of frontal brain systems.
In addition, while alcoholics' deficits
in visuospatial functions persistently
have implicated right hemisphere vul-
nerability, neuropsychological and
neuroanatomies evidence for unilateral
damage is not convincing. Research
on cognitive capabilities and differences
between the hemispheres in alcoholics
has provided only limited support for
the premature aging hypothesis, and
even less consistent support for the
right hemisphere hypothesis.

Future research would benefit greatly
from cooperation among disciplines.
Behavioral neuroscience offers excel-
lent techniques for sensitively accessing
distinct cognitive and emotional func-
tions. Electrophysiological and neu-
roimaging techniques provide a
window on the active brain, as well as
a glimpse at regions with structural
damage. Likewise, followup post-

mortem examination of brains of well -
studied alcoholic individuals should
be encouraged for clues about neuro-
transmitter abnormalities, as well as
analyses of injury at the cellular level.
Specific questions and topics in need
of research attention are outlined in
table 3. They include the following:
neurobehavioral and brain func-
tional/structural recovery with absti-
nence, the extent of multimodal
sensory and perceptual deficits, the
nature of the loss of emotional and
motivational functioning among sub-
groups of alcoholics, the synergistic
influence of common comorbid con-
ditions and alcoholism, and the direct
contribution of frontal system dys-
function to alcohol consumption and
alcoholic symptomatology.

ACKNOWLEDGMENTS

The writing of this report was sup-
ported by NIAAA grants R37-
AA07112 and K05-AA00219, and by
funds from the Medical Research
Service of the U.S. Department of
Veterans Affairs.

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471

Chapter 14

Human Brain Vulnerability to Alcoholism:
Evidence From Neuroimaging Studies

Edith V. Sullivan, Ph.D.

KEY WORDS: AOD (alcohol or other drug) dependence; neuroimaging; alcoholic
brain syndrome; brain damage; chronic AODE (effects of AOD use, abuse, and
dependence); risk factors; nutritional disorder; computed x-ray tomography; magnetic
resonance imaging; single photon emission computed tomography; magnetic resonance
spectroscopy; Korsakoff's syndrome; gender differences; biological repair; ethanol
metabolite; glucose metabolism; comorbidity; gender differences; aging; literature review

It has been suspected for centuries and
known for decades that excessive, chronic
alcohol consumption damages brain struc-
ture and impairs brain function. Before
the introduction of in vivo neuroimaging
techniques, the establishment of brain
structural compromise relied on post-
mortem data, which by definition, are
cross-sectional, biased to cases that come
to autopsy (in years gone by, samples
may have been typically indigent and
malnourished [cf. Sullivan 1899]), and
susceptible to fixation artifacts. In vivo
imaging methods have the advantage of
capturing the dynamic course of alcohol-
ism, and some methods are amenable
to experimental challenges and exami-
nation of neurochemistry, thus providing
opportunities to go beyond description

of abnormality and to identify mecha-
nisms of neuronal disruption and
vulnerability. Collection of three-
dimensional data sets of neuroimages
provides snapshots of an individual's
brain at a specific time point, can be
repeated to follow change, and, unlike
the autopsied brain, can be resliced on
later occasions to address new ques-
tions. Despite their unparalleled worth,
in vivo studies provide relatively coarse
data and are unlikely to fully replace
postmortem studies, which at least cur-
rently are essential for verifying actual
pathology and for providing microscopic
analyses of cytoarchi tectonic and cellu-
lar structure.

The initial optimism accompanying
the emergence of in vivo imaging

E.V. Sullivan, Ph.D., is an associate professor in the Department of Psychiatry and Behavioral Sciences
and the Neurosciences Program, Stanford University School of Medicine, 401 Quarry Rd., Stanford,
CA 94305-5717.

473

NIAAA's Neuroscience and Behavioral Research Portfolio

methods was somewhat damped when
the predicted brain "pathology" was
not always clearly visible and when the
predicted relationships between brain
integrity and cognition or alcohol
consumption variables were not readily
forthcoming. Brain-behavior discoveries
based on patient groups with gross,
focal brain lesions may have been pos-
sible and the application of the double
dissociation model may have been
successful because the "signal" from
the lesion was powerful enough to
override the "noise" from other factors,
such as age or comorbid disease, which
may have had an influence in cases where
the lesion was less prominent, as holds
for alcoholism. Additional factors that
may attenuate obvious relationships, such
as dose-response relationships (greater
alcohol intake and greater brain dam-
age), include age, gender, nutrition,
genetics, and comorbidities, each of
which may exert its own contribution
to brain dysmorphology and may dif-
ferentially dispose the alcoholic indi-
vidual to relatively greater or less insult.
Thus there is a multiplicity of factors
that may influence the brain and its
functions in alcoholism, and each
must be controlled and will be consid-
ered in this review.

A growing number of in vivo brain
imaging technologies are available.
Methods already applied to study
alcoholism include those for structural
imaging (computed tomography [CT],
magnetic resonance imaging [MRI],
and magnetization transfer [MT]) and
functional imaging (single photon emis-
sion computed tomography [SPECT],
positron emission tomography [PET],
and magnetic resonance spectroscopy

[MRS] and spectroscopic imaging
[MRSI]). Findings from a sample of
these studies will be summarized here
(see also reviews by Pfefferbaum and
Rosenbloom 1993; Jernigan and Cermak
1994; Fein et al. 1995; Volkow et al.
1995); this review will reflect the fact
that most imaging studies in alcohol-
ism have examined brain structure.

PATTERNS OF STRUCTURAL
BRAIN ABNORMALITIES

Early imaging studies in chronic alcohol-
ics were based on CT and revealed a
significantly dilated ventricular system
and enlarged cortical sulci throughout
the cortex (Haubek and Lee 1979;
Ron et al. 1982; Wilkinson 1985;
Pfefferbaum et al. 1988; Lishman
1990). The resolution of CT, how-
ever, did not permit reliable separation
of tissue types. With the appropriate
image acquisition sequences, MRI can
distinguish gray matter and white mat-
ter (Wehrli et al. 1984). Application
of such MRI sequences in studies of
detoxified alcoholics has provided
convincing evidence that the increases
in cortical cerebrospinal fluid (CSF)-
filled spaces, previously observed with
CT (Pfefferbaum et al. 1988, 1993),
occur at the expense of both gray mat-
ter and white matter (Jernigan et al.
1991 a; Pfefferbaum et al. 1992). This
pattern of gray and white matter tissue
involvement differs from that seen in
other neuropsychiatric conditions such
as schizophrenia (Zipursky et al.
1992; Sullivan et al. 1998&) and
Alzheimer's disease (Jernigan et al.
1991 b; Fama et al. 1997; Tanabe et
al. 1997; Pfefferbaum et al. 1999&),

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Brain Vulnerability to Alcoholism: Neuroimaging Studies

both of which have cortical gray but
little, if any, white matter volume loss.
The tissue shrinkage visually detectable
on MRI can be as extensive in "uncom-
plicated" alcoholism (figure 1) as it is
in Alzheimer's disease, yet the behav-
ioral consequences, such as memory
impairment, are not so clinically appar-
ent in alcoholism.

Cortical volume deficits are present
throughout the cortex and subjacent
white matter (Jernigan et al. 199 la;
Pfefferbaum et al. 1992, 1995; but
see Di Sclafani et al. 1995). A regional
profile of volume abnormalities, how-
ever, was not apparent in alcoholics
until age was considered. Identifying
any regional pattern of abnormality
first requires standardizing structures
of widely different fundamental sizes
(e.g., lateral and third ventricles, frontal
lobes and temporal lobes) to a common
metric. If such a metric also takes the
effects of normal aging and variations
associated with head size difference into

account, regional patterns of volume
abnormalities can be seen.

Age

The brain morphology of older alco-
holics (i.e., from about age 45 years
onward) is especially vulnerable to the
adverse effects of alcohol consumption
(Wilkinson and Carlen 1980; Ron et al.
1982; Pfefferbaum et al. 1988, 1992;
Sullivan et al. 1995«; Pfefferbaum et al.
1996#). To legitimately reach such a con-
clusion, however, the effects of normal
aging, as well as normal variation in head
size, on the brain must be measured and
statistically taken into account; this
can be done with regression analysis
(Pfefferbaum et al. 1986, 1992;
Mathalon et al. 1993). The use of age -
corrected standardized brain volumes
permits a cross-sectional approach for
estimating the dual effect of age and
alcohol on different regions of the brain.
Our studies based on this method have
consistently revealed an age-alcohol

Figure 1. Surface-rendered brains of a 57-year-old healthy man (left), who consumed about
60 kg of ethanol in his lifetime, and a 57-year-old alcoholic man (right), who consumed about
1,866 kg of ethanol in his lifetime. Note the thinner gyri and wider sulci and interhemispheric
fissure in the alcoholic man's brain compared with the healthy man's brain.

475

NIAAA's Neuroscience and Behavioral Research Portfolio

interaction, where older alcoholics
have greater tissue volume loss and
CSF volume expansion for their ages
(and for a given head size) than do
younger alcoholics.

The age-alcohol interaction holds
for a number of brain regions. Specifi-
cally, although gray matter and white
matter volume loss in alcoholics is wide-
spread throughout the cortex, the vol-
ume loss of both tissue types in the
frontal lobes is relatively greater than that
detected in the more posterior reaches
of the cortex in older (> 45 years of age)
than younger alcoholics (Pfefferbaum
et al. 1997). When regional volumes
were compared, the older groups exhib-
ited substantial deficits in prefrontal
cortical volume of gray matter and in
prefrontal and frontal white matter vol-
ume. This observation supports post-
mortem studies, which identified the
frontal lobes as being particularly
damaged in alcoholics (Courville 1955;
Harper and Kril 1990). Similar age-
related declines occur in cerebellar
hemisphere gray matter volumes in
alcoholic men (Sullivan et al. in press).

Other selective brain structures are
also deleteriously affected by chronic,
excessive alcohol consumption. Mea-
surement of these structures entails
careful tracing of the anatomical bor-
ders across multiple, contiguous MRI
slices of the target brain structure.
These analyses have shown that non-
amnesic alcoholics have significant,
age-related bilateral volume deficits in
the anterior but not posterior hippocam-
pus (Sullivan et al. 1995#). The volume
deficits were even more severe (by 1
standard deviation) in alcoholics with
global amnesia and likely Korsakoff's

syndrome (KS) (Sullivan et al. 1996;
see also Jernigan et al. 1991c).

Prenatal exposure to alcohol (PEA)
disrupts the development and integrity
of the corpus callosum (human [Peiffer
et al. 1979; Clarren 1986; Mattson et
al. 1994]; rat [Zimmerberg and Scalzi
1989; Zimmerberg and Mickus 1990;
Coulter et al. 1993]). The adult aging
corpus callosum is also affected by exces-
sive alcohol consumption. Hommer and
colleagues (1996) reported smaller cal-
losal areas in alcoholic women but not
men who ranged in age from 30 to 50
years. In a sample of alcoholic men
ages 28-67, we observed that the corpus
callosum is thinner throughout its
extent, from rostrum and genu to
splenium (Pfefferbaum et al. 1996&)
and that smaller callosal areas signifi-
cantly correlated with older age (r =
-0.56, p< 0.01).

Alcohol abuse and dependence are
prevalent in adolescents as well as older
individuals, yet alcohol's adverse effects
on the adolescent brain is unknown.
Alcohol's teratological effects in humans
are under study in children and adults
with fetal alcohol syndrome (FAS) or
PEA. To date, quantitative MRI studies
of FAS and PEA patients have revealed
abnormally small volumes of the cranial
vault, basal ganglia (in particular, the
caudate nucleus), and diencephalon (a
brain region including thalamic and
septal nuclei) (Mattson et al. 1996).
Additional sites of dysmorphology
included the anterior superior vermis
(Sowell et al. 1996) and corpus callo-
sum (Mattson et al. 1992; Riley et al.
1995), which are consistent with post-
mortem studies and animal models of
FAS (Clarren 1986; West et al. 1990;

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Brain Vulnerability to Alcoholism: Neuroimaging Studies

Goodlett and Lundahl 1996). These
teratological studies suggest that even
in adolescents free of PEA the still-
developing brain could be particularly
vulnerable to alcohol's neurotoxicity,
which could result in aberrant neural
connectivity and arrested neuronal
growth, and consequently irreversible
brain insult.

Nutrition and Other
Factors

Nutritional status is known to contri-
bute to alcohol-related brain dysfunction
and dysmorphology. Korsakoff's syn-
drome, whether or not accompanied by
alcoholism, is associated with lesions of
the diencephalon and thalamic nuclei
resulting from thiamine deficiency
(Witt and Goldman-Rakic 1983; Victor
et al. 1989; Langlais and Zhang 1997).
Thiamine replacement therapy is reg-
ularly used clinically and prophylactically
to reduce the chances of developing
KS in alcoholics who undergo with-
drawal. The dramatic syndrome of
Marchiafava-Bignami disease, often
associated with alcoholism and possi-
bly caused by deficiency in dietary
nicotinic acid or its precursor, trypto-
phan (Victor et al. 1989), pathogno-
monically involves demyelination of
the corpus callosum and the discon-
nection syndrome. Early studies of
severe alcoholism with Wernicke's
encephalopathy reported no cases of
corpus callosum degeneration (Victor et
al. 1989) and suggested that the two
conditions have different etiologies. It
is not known whether adequate nutri-
tional supplements improve the struc-
tural condition of the brain as they do
cognition and motor abilities.

Subjects in our neuroimaging stud-
ies who were scanned after a 2 8 -day
inpatient rehabilitation program were
well nourished at the time, yet they
still manifested relationships between
nutritionally dependent variables, such
as indices of macrocytic anemia and
brain volumes (Pfefferbaum et al. 1988,
1992; Davila et al. 1994). Specifically,
body mass index, hemoglobin, and
mean corpuscular volume were corre-
lated with CT measures of ventricular
volume (Pfefferbaum et al. 1988). In a
different sample, also abstinent about 1
month, mean corpuscular volume,
hemoglobin, and hematocrit were cor-
related with MRI measures of ventricu-
lar volumes (Pfefferbaum et al. 1992).

The landmark neuropathology stud-
ies of Victor, Adams, and Collins (Victor
et al. 1971, 1989) suggested that severe
cell damage in the anterior superior
vermis, dorsomedial nucleus of the thal-
amus, and in many cases the mammillary
bodies is pathognomonic for alcoholic
KS and is probably caused by thiamine
deficiency (Butterworth et al. 1993;
Langlais and Savage 1995). The KS-
specific cerebral damage to periven-
tricular and thalamic nuclei is thought
to cause the prominent global amne-
sia defining KS (Butters 1985; Butters
and Stuss 1989; Victor et al. 1989).
Some pathology studies conclude that
autopsied cases with significant atrophy
in these regions together with the vermis
have KS even in the absence of the
diagnostic constellation of aberrant
behavior in life (Torvik 1991).

With in vivo imaging, we observed
that the cerebellar vermis can be sub-
stantially affected in alcoholics who do
not show signs of KS, and the region

477

NIAAA's Neuroscience and Behavioral Research Portfolio

of greatest loss is the anterior superior
lobules (Sullivan et al. 1995 b), which is
consistent with postmortem studies of
alcoholic KS (Victor et al. 1989). The
mammillary bodies are also abnormally
small in volume in nonamnesic alcohol-
ics (Sullivan et al. 1999). Mammillary
body dysmorphology was evident even
in a series of studies based on a 3 -point
rating system of MRI films in older
alcoholics (younger alcoholics were
comparable to controls [Charness
1993]). In those studies, we observed a
significant incidence of mammillary
body shrinkage in alcoholics with or
without symptoms of KS (Davila et al.
1994), and instances of normal-sized
mammillary bodies in alcoholics with KS
amnesia (Shear et al. 1996). Mammil-
lary body volume deficits, however, were
not predictive of performance on tests
of explicit memory, such as delayed
recognition or recall.

Using anatomically determined MRI
measurement of brain structures, Squire
and colleagues (1990) observed signif-
icantly smaller area measures of the
mammillary nuclei but not of the hip-
pocampal formation in four alcoholic
KS patients relative to four non-KS
amnesic patients, whereas the non-KS
amnesics showed the opposite pattern
of regional sparing and loss. These
authors further concluded that the
amnesia in the KS patients is more likely
associated with the shrinkage observed
in the mammillary nuclei, possibly
combined with thalamic abnormalities
observed with CT in these same
patients by Shimamura and colleagues
(1988). An MRI study by Jernigan
and colleagues (1991c), using geomet-
rically determined volume measurements,

observed significant cortical sulcal
enlargement, which occurred at the
expense of cortical gray matter loss, in
nonamnesic alcoholics; these volume
abnormalities were even greater in a KS
group. Consistent with this observation,
another MRI study (Sullivan et al.
1996&) reported that relative to an
age-range-matched control group, KS
patients and nonamnesic alcoholics
had significantly and comparably smaller
gray matter volumes throughout the
cortex; in addition, an observed white
matter volume deficit was confined to
the prefrontal region. Although both
alcoholic groups had significantly smaller
anterior hippocampi than the control
group, these structures were smaller by
1 standard deviation in the KS relative
to the nonamnesic alcoholics. Together
with the observation that amnesic and
nonamnesic alcoholics share a similar
degree of clinically detectable mammil-
lary body shrinkage, these results suggest
that extensive hippocampal volume
deficits probably contribute to alcohol-
related amnesia of KS.

The extent and nature of brain dys-
morphology found in chronic alcoholics
may be exacerbated by conditions fre-
quently associated with chronic alco-
holism: liver disease (Patek 1979;
Acker et al. 1982; Rao and Butterworth
1995), use of hepatotoxic drugs (Miitzell
1992), withdrawal seizures (Sullivan
et al. 1996#), or withdrawal delirium
tremens (Daryanani et al. 1994). Signif-
icant liver disease is a standard criterion
for exclusion from brain imaging stud-
ies of alcoholism. Withdrawal-related
neurological symptoms may explain
differential vulnerability; this is particularly
relevant given reports that withdrawal

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Brain Vulnerability to Alcoholism: Neuroimaging Studies

symptoms are more severe in older
than in younger alcoholics (Brower et
al. 1994).

As many as 2 to 15 percent of chronic
alcoholics experience seizures, typi-
cally grand mal in type (Victor and
Brausch 1967), when they withdraw
from drinking alcohol (Chan 1985;
Booth and Blow 1993; Anton and
Becker 1995). Alcohol-related seizures
constitute about 50 percent of hospital-
izations for seizure in the United States
(Anton and Becker 1995). Several
attempts made to identify brain correlates
of withdrawal seizures have been largely
unsuccessful (Meyer- Wahl and Braun
1982; Daryanani et al. 1994; see also
Fox et al. 1976). Given the low seizure
threshold of the hippocampus, we asked
specifically whether alcoholics with a
positive history of withdrawal seizures,
established by retrospective chart
review, would have greater hippocampal
volume deficits than those without
such a history. Alcoholics with and
without withdrawal seizure history
demonstrated comparable tissue volume
deficits in hippocampus, as well as
frontoparietal gray and white matter
and temporal lobe gray matter. Tempo-
ral lobe white matter, however, showed
greater volume deficits in the patients
with histories of withdrawal seizures
than in those without (Sullivan et al.
1996#). Although a history of seizures
puts alcoholics at a particularly high
risk for future seizures (e.g., Victor
and Brausch 1967; Ballenger and Post
1978; Brown et al. 1988; Lechtenberg
and Worner 1990; Lechtenberg and
Worner 1991; Booth and Blow 1993),
we do not know whether the seizure -
prone alcoholics already had less white

matter, or whether repeated seizures
induced the structural change. Nonethe-
less, it appears that reduced white
matter volume in the temporal lobes
is either a risk factor for or sequela of
alcohol withdrawal seizures.

Gender Differences

There is a paucity of data on the effects
of alcoholism on the brains of women.
Anecdotal reports suggest that women
with alcoholism are more difficult to
recruit for such study than are their male
counterparts and that those women
who do volunteer for study are more
likely than men to carry comorbidities,
such as depression or eating disorders,
that would typically exclude them
from study. Another problem is that
female alcoholic subjects often consume
less alcohol than male alcoholic subjects.
Consequently, the samples of women
and men currently studied may not be
comparable. It is predicted, however,
that, as was observed with cardiovascular
disease and smoking, alcohol consump-
tion rates in women may approach those
of men as more women enter the work-
force (Parker and Harford 1992; Roman
and Blum 1992). Indeed, gender dif-
ferences in hormones and neuro-
steroids and the presence of estrous
and menstrual cycles in women pro-
vide additional factors for interaction
with alcohol that may either be pro-
tective to alcohol-consuming women
or render them more vulnerable (Lan-
caster et al. 1984; Harper et al. 1990;
Lancaster 1994^, 1994b). Further
complicating direct gender comparisons
are fundamental gender differences in
the normal population in intracranial
volume (Dorst 1971; Pfefferbaum et

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NIAAA's Neuroscience and Behavioral Research Portfolio

al. 1994), size of different brain struc-
tures, and patterns of aging (Gur et al.
1991; Blatter et al. 1995; Raz et al.
1997; Coffey et al. 1998).

Studies of alcoholic or problem drink-
ing in women have suggested that
women can be more vulnerable not only
to adverse medical consequences (Ashley
et al. 1977) but also to structural brain
deficits (Jacobson 1986; Mann et al.
\992a). The higher average percentage
of body fat in women than men means
that equivalent doses of ingested alco-
hol per pound of body weight are less
diluted in total body water in women
than men. In addition, women exhibit
lower levels of first-pass metabolism
than men (Frezza et al. 1990), result-
ing in higher levels of acetaldehyde to
affect the liver and other organs. Con-
versely, hormonal differences may
contribute to higher metabolic rates
for ethanol in women than men
(Thomasson 1995). However, an analy-
sis of cross-national epidemiologic data
showed that after controlling for drink-
ing levels (quantity and frequency), male
and female alcoholics were equally at
risk for self-reported health problems
(Fillmore et al. 1995). Epidemiologic
studies of the population as a whole
show that compared with men, women
start to drink later in life, drink less per
occasion, are more likely to be abstinent
(York and Welte 1994), and have lower
rates of alcoholism (Kessler et al. 1994).
Some studies report that drinking levels
in alcoholic women decline with old age
(Wilsnack et al. 1984; Hilton 1991).
Although epidemiologic data suggest
that alcoholism is less likely to affect
women than men, for those afflicted
the consequences pose a major public

health problem. All told, however, the
biological consequences of alcoholism
in women have been studied less thor-
oughly than in men.

There are few investigations of
morphological brain changes in alcoholic
women. To date, none has been opti-
mally designed to determine whether
the female brain is more vulnerable than
the male brain to the chronic effects of
alcohol. A neuropathological study
(Harper et al. 1990) showed similar
changes in alcoholic women compared
with control women as were found
comparing alcoholic and control men. A
CT study comparing recently detoxified
alcoholic men and women, long-sober
(Alcoholics Anonymous) men and
women, and control men and women
found that alcoholic women, despite
shorter histories of alcoholic drinking,
had CT indices of atrophy similar to
those of the men (Jacobson 1986), sug-
gesting greater vulnerability of the female
brain. Like Jacobson, Mann and col-
leagues (\992a) did not detect differ-
ences between alcoholic men and women
on a number of indices of brain shrink-
age on CT. Although the women were
most recently drinking at a comparable
level to the men, they had shorter drink-
ing histories. With MRI, Kroft and
colleagues (1991) found no structural
abnormalities in alcoholic women relative
to controls, whereas Hommer and col-
leagues (1996) found corpus callosum
reduction in alcoholic women but not
in men.

We compared MRI data from 36
alcoholic women (ages 28-63) and 48
healthy community control women
(ages 20-85). As with men, control
women had predicted age effects for

480

Brain Vulnerability to Alcoholism: Neuroimaging Studies

head size-corrected volumes of cortical
gray matter, cortical sulci, lateral and
third ventricles, and no age effect on
white matter. Alcoholic women also
showed age-related declines in gray
but not white matter volumes and
increases in volumes of CSF-filled
spaces. These alcoholic women, how-
ever, showed less structural brain deficit
relative to age and gender norms than
alcoholic men (Pfefferbaum et al.
1996^). Although the results do not
directly support earlier reports ( Jacobson
1986; Mann et al. 1992&) that brains
of alcoholic women are more vulnerable
to the effects of alcohol than those of
men, it must be noted that the recent
alcoholic drinking levels in our sample
of women were lower than those
reported for women in other studies.
Further to this point, the alcoholic
women in the sample who were scanned
following at least 2 months of sobriety
had smaller third ventricles and
tended to have more cortical white
matter than women scanned at shorter
intervals of sobriety.

Reversibility

A CT study by Carlen and colleagues
(1978) was the first neuroradiological
report to provide evidence of at least
partial reversal of alcohol-related cortical
sulcal and ventricular dilatation in a small
sample of alcoholics who remained
abstinent for about 1 year. Since that
time, a number of CT (Artmann et al.
1981; Ron et al. 1982; Carlen et al.
1984; Schroth et al. 1985; Muuronen
et al. 1989) and MRI (Schroth et al.
1988; Zipursky et al. 1989; Shear et
al. 1994; Pfefferbaum et al. 1995, 1998)
studies have reported that alcoholics

who remain abstinent can show signif-
icant reversal of cortical sulcal and ven-
tricular enlargement. Of considerable
interest is the time course of the changes,
the nature and locus of the presumed
brain tissue increases that are reflected
in sulcal and ventricular size reduc-
tions, and possible mechanisms for
this recovery.

The first study to employ a healthy
cohort to control for test-retest mea-
surement changes (Zipursky et al.
1989) used MRI and reported that a
small but significant decrease in ventric-
ular volume occurs early (10-14 days)
in the course of abstinence. Another
MRI study (Shear et al. 1994) examined
alcoholics 1 month after sobriety and
again 3.5 months later, when some
remained sober and others had relapsed.
White matter volume increased and
ventricular volume decreased in the
abstainers, whereas the relapsers showed
a trend toward further white matter
volume loss and ventricular dilatation.
A reexamination of this sample about
1 year after the first MRI revealed that
relapsers exhibited little change in CSF
volume during the interscan interval,
whereas the abstainers showed a signif-
icant decrease in cortical and subcorti-
cal CSF volumes and a trend toward
an increase in white matter volume
(Drake et al. 1994).

Differences in head placement typi-
cally occur when the same individual
is rescanned, even when great care is
taken to standardize placement. To
address this methodological confound,
we developed a rescan correction fac-
tor to statistically remove the interscan
difference in intracranial volume from the
interscan difference in regional volumes.

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NIAAA's Neuroscience and Behavioral Research Portfolio

Applying this method in a longitudinal
study (Pfefferbaum et al. 1995), we
observed that enlargement in cortical
gray matter and reduction in sulcal
and lateral ventricular volumes occur
early in the course of abstinence (about
1 month), and that reduction in third
ventricular volume appears later with
continued abstinence (about 3 to 12
months). Resumption of drinking after
a short period of abstinence arrests
third ventricular volume improvement
and produces white matter volume
loss. The shrinkage of the third ventricle
with further abstinence is consistent
with the 5 -year longitudinal CT study
of Muuronen and colleagues (1989),
who reported that third ventricular
shrinkage was related to improvement in
performance on cognitive tests, thus
providing evidence for functional rele-
vance of the brain structural change.

In a 5 -year longitudinal MRI study
of control and alcoholic men, cortical
gray matter diminished over time in
the control subjects, most prominently
in the prefrontal cortex, whereas lat-
eral and third ventricles enlarged
(Pfefferbaum et al. 1998). The alco-
holics showed similar age-related
changes with a greater rate of cortical
gray matter volume loss than the control
subjects in the anterior superior tempo-
ral lobe. Amount of alcohol consumed
during followup predicted rate of corti-
cal gray matter volume loss, particularly
in the frontal regions, as well as corti-
cal sulcal expansion.

Until recently, alcohol history factors,
such as total lifetime consumption of
alcohol, have generally proved to be dis-
appointing predictors of alcohol -related
conditions, including brain volume

abnormalities (for a review, see Parsons
19876). Estruch and colleagues (1993)
reported a dose-response relationship
with medical and neurological compli-
cations of alcoholism, such as cirrhosis
and peripheral neuropathy. Likewise,
Pfefferbaum and colleagues (1995)
observed that within abstainers, lifetime
alcohol consumption, shorter disease
duration, and less cortical white matter
volume after 1 month of sobriety were
significant and independent predictors
of cortical white matter volume increase
with prolonged abstinence. Within the
relapsers, greater lifetime consumption
of alcohol was associated with greater
decrease in anterior white matter and
increase in third ventricular volume.
This dose relationship persisted even
after accounting for age, disease dura-
tion, and brain volume at the start of
the comparison interval. In addition,
amount of alcohol intake between MRIs
in the relapsed alcoholics was related to
greater enlargement of the lateral ventri-
cles. These relationships provide support
for the reasonable speculation of dose-
related, adverse influences of alcohol on
brain structure.

The mechanisms underlying morpho-
logical recovery are still unknown. A
well-controlled study (Mann et al. 1994)
tested the hypothesis that reduction in
ventricular size with abstinence reflects
rehydration of brain tissue and demon-
strated that tissue and CSF volume
changes during withdrawal occur in
the absence of significant changes in
magnetic resonance relaxation times
(Tl and T2), measures derived from
magnetic resonance images that provide
information about the amount and dis-
tribution of free versus bound water

482

Brain Vulnerability to Alcoholism: Neuroimaging Studies

in brain tissue. CT studies also refuted
the rehydration hypothesis of recovery
by showing an increase in CT density
measures with abstinence, suggesting
a decrease in the water content of brain
tissue (reviewed in the study by Mann
et al. 1992 b). Neuropathological reports
also support the presence of increased
tissue density with abstinence (Harper
and Corbett 1990).

PATTERNS OF FUNCTIONAL
BRAIN ABNORMALITIES

MRS and MRSI Studies

MRSI permits in vivo observation of
several metabolites, some of which have
distinct profiles in different brain tissue
types. Studied in the context of struc-
tural anatomy, the concentration of
different metabolites should help to
delineate metabolite changes underlying
age- and disease-related deterioration
in tissue integrity. In vivo metabolite
imaging may help to define disease
progress, assess efficacy of treatments,
and track alcohol-related changes across
the lifespan. Most MRS alcohol studies

to date have measured metabolites on
the proton spectrum, which provides
information about four major brain
chemicals: the N-acetyl compounds
(NAc), creatine (Cr), choline (Cho)
and wzyo- inositol (figure 2). Further-
more, MRS or MRSI can be used to
determine the in vivo concentration
and distribution of ethanol per se in
the brain.

Magnetic resonance-visible NAc is
primarily, but not uniquely, N- acetyl -
aspartate, which is found almost
exclusively in neurons (Urenjak et al.
1992, 1993) and thus may serve as an
in vivo measure of neuronal integrity.
The Cr signal, generated by creatine
and phosphocreatine, is an indicator
of high-energy phosphate metabolism
(Tedeschi et al. 1995). The in vivo
visible Cho peak is generated primar-
ily by water-soluble Cho-containing
compounds — free choline, phospho-
choline, and glycerophosphocholine
(Barker et al. 1994) — and is associated
with cell membrane synthesis and
turnover. Myo-\nosito\ is present in glial
but not neuronal cell cultures (Brand
et al. 1993) and is a precursor of the

Figure 2. Axial brain images of a 30-year-old healthy man: (a) fast spin echo magnetic reso-
nance structural image segmented into compartments of cerebrospinal fluid (dark gray), gray
matter (medium gray), and white matter (light gray); (b) low-pass tissue image to match the
spatial frequency characteristics of the metabolite images; (c) ^-acetyl image; (d) creatine im-
age; (e) choline image.

483

NIAAA's Neuroscience and Behavioral Research Portfolio

inositol-triphosphate second messen-
ger pathway, where it functions to
maintain cell volume (Lien et al.
1990; Ernst et al. 1997). Also spectro-
scopically visible but more challenging
to measure are gamma- aminobutyric
acid (GABA), the primary inhibitory
neurotransmitter in the brain, and glu-
tamate, a principal excitatory neuro-
transmitter (Paul 1995).

Metabolite Studies

An MRSI study of older alcoholic men,
abstinent for 3-24 months, revealed
lower NAc/Cr ratios in frontal lobes
and somewhat higher ratios in parietal
lobes than in control subjects. The rel-
atively lower frontal than parietal meta-
bolite ratio was interpreted as reflecting
neuronal loss and glial hyperplasia in
the alcoholics (Fein et al. 1994). As
noted by the authors, use of ratios does
not permit firm conclusions to be drawn
regarding the independent rise or fall
of either or both metabolites expressing
the ratio.

Martin and colleagues (1995) used
proton MRS to examine vermian
integrity and potential changes in
recently detoxified alcoholics scanned
twice: 0-41 days abstinent and then
10-55 days abstinent. Control subjects
were scanned at comparable intervals.
NAc/Cho substantially increased with
longer abstinence, suggesting an
improvement due to either increases
in NAc levels or decreases in Cho levels,
or both. In a naturalistic experiment,
the NAc/Cho ratio increased with absti-
nence and decreased with relapse in
one alcoholic who was scanned on three
occasions. Another study of vermian
integrity observed lower NAc/Cr and

Cho/Cr ratios in the alcoholics than
in the control subjects and no group
differences in glutamate/glutamine or
wyo-inositol (Seitz et al. 1999). If the
lower ratios involving NAc are due to
the NAc rather than the Cr component,
then this study provides some evidence
for alcohol-related reduction in neuronal
size, number, or dendritic arbors in
the absence of glial proliferation in the
cerebellar vermis. Consistent with this
finding, Jagannathan and colleagues
(1996) detected lower NAc/Cr and
NAc/Cho ratios in the thalamus, frontal
lobe, and cerebellum of alcoholics
abstinent for at least 1 month relative
to control subjects.

The only study considering the
potential compounded effects of alcohol
comorbidity used 3 1 -phosphorus MRS
in alcoholics infected with the human
immunodeficiency virus (HIV+), alco-
holics HIV-, low- drinking HIV+, and
low- drinking HIV- (Meyerhoff et al.
1995). The results showed a cumulative
rather than an interactive effect of the
two diseases, where the alcoholic HIV+
group had the lowest concentrations
of white matter and subcortical gray
matter phosphodiester and of white
matter phosphocreatine. This pattern is
reflective of cell membrane degeneration,
which was greatest in the comorbid
group with greater alcohol use.

In an animal model (rat), injections
of the antagonist pyrithiamine resulted
in reduced Cho/NAc ratios. Thiamine
treatment resulted in increased Cho/
NAc ratios and no change in Cr/NAc
ratios (Lee et al. 1995). These results
suggest that the brain may be resilient
enough to reverse biochemical evidence
of KS-like pathology and, furthermore,

484

Brain Vulnerability to Alcoholism: Neuroimaging Studies

if treated early enough may escape
structural damage.

A major criticism of MRS studies
of the chronic effects of alcohol as
well as other diseases has been the
expression of one metabolite as a ratio
of the other to generate standardized
measures. Furthermore, few studies
have distinguished spectroscopic signals
arising from gray matter and white
matter, even though metabolites have
different concentrations in the two tis-
sue types. Recent studies from our
laboratory, using absolute measures,
showed disease -related decrease in NAc
concentration in gray and white matter,
age-related increase in Cr concentration
in gray matter, and age- and disease -
related increase in Cho concentration
in gray matter (Pfefferbaum et al. 1999 a,
19996), pointing to the limitation of
ratio measures.

The recently developed techniques
for estimating in vivo absolute measures
of NAc, Cho, and Cr separately for
white and gray matter, and confirmed
in vitro studies indicating different
concentrations of these metabolites in
different tissue types, complement stud-
ies by Petroff and colleagues (1995)
demonstrating that spectroscopically
visible metabolites differ in concentra-
tion between neurons and glia. In
vitro studies show that neurosomes
are enriched in glutamate, GAB A, and
NAc, whereas gliasomes are enriched
in lactate, glutamine, and myoinositol.
This opens the possibility of designing
studies in which MRSI techniques are
used to identify the contribution of neu-
ronal replacement by glia to document
mechanisms of volume loss or shrinkage
that occur through withdrawal, longer

term abstinence, and relapse. Separate
analyses of white and gray matter can
also document the relative involvement
of white and gray matter in these
processes. Such functional studies may
be more likely to yield predictable
brain -behavior relationships than have
structural studies.

Ethanol Detection Studies

MRS provides a powerful method for
tracking presence and distribution of
ethanol in the brain and the rate of its
metabolism. Ethanol is acutely detectable
with MRS and has a characteristic triplet
profile on the proton spectrum. The
ethanol peak is clearly visible when blood
alcohol levels are 0.10 percent (100
mg/dL), the legal level of intoxication
in many States (Hanstock et al. 1990).
One study reported a dose-response
effect, where regional brain alcohol levels
correlated with ascending blood alcohol
levels and also with subjective estimates
of euphoria and dysphoria (Mendelson
et al. 1990). Another study showed that
acute ethanol has a heterogeneous distri-
bution across tissue types (figure 3). When
blood alcohol levels were between 0.08
and 0.12 percent, about 1 hour after
ingesting alcohol, relative ethanol signal
intensities were highest in CSF, 30 percent
less in gray matter, and 60 percent less
in white matter (Spielman et al. 1993).
MRS of acute ethanol concentration
and distribution may also provide an
in vivo method for studying tolerance,
behavioral indices of which are used to
define alcohol dependence (American
Psychiatric Association 1994). One study
reported higher MRS-measured brain
ethanol concentrations in heavy (non-
alcoholic) drinkers than in occasional

485

NIAAA's Neuroscience and Behavioral Research Portfolio

drinkers (Chiu et al. 1994) following
alcohol consumption yielding blood
alcohol levels of 0.08 percent. These
results were interpreted as evidence of
tolerance in the heavy drinkers resulting
in membrane lipid rigidity and decreased
membrane binding of ethanol, leading
to more spectroscopically visible ethanol.
To examine the extent that tolerance
may be genetically determined, it would
be enlightening to compare estimates
of nonabsorbed ethanol in nonalcoholic,
low-alcohol-consuming persons who
have a positive family history for alco-
holism versus those with a negative
family history.

PET Studies

Functional imaging with PET requires
the use of positron-emitting radioactive
isotopes (ligands), which are injected

into study subjects and detected by
the scanner. Depending on the isotope
used, PET can track regional cerebral
metabolism in resting or behaviorally
activated states or can identify brain
regions and quantify the binding sites of
a specific neurotransmitter ligand (e.g.,
the dopa receptor with spiperone).

Studies of brain metabolism in
alcoholism have primarily relied on
three different positron emitters.
18Fluorine (half-life of 110 minutes )-
labeled deoxyglucose (FDG) is used
to image the regional distribution of
glucose in the brain and provides an
index of metabolism rate. nCarbon
(half-life of 20 minutes) can also be used
to label deoxyglucose. 15Oxygen, with
its short half-life (2 minutes), can be
injected serially while subjects perform
cognitive, sensory, and motor tasks

\ before EtOH ingestion

residual water

NAc

creatine
choline \

EtOH

60 min post-EtOH ingestion

i i i r

5 0

Chemical Shift (ppm)

Figure 3. Proton spectra acquired before alcohol (EtOH) ingestion and 60 minutes after
EtOH ingestion. The notable difference in the spectra is the appearance of the ethanol triplet fol-
lowing alcohol consumption. NAc = N-acetyl compounds; ppm = parts per million. Adapted
from Spielman, D.M.; Glover, G.H.; Macovski, A.; and Pfefferbaum, A. Magnetic resonance
spectroscopic imaging of ethanol in the human brain: A feasibility study. Alcohol Clin Exp Res
17:1072-1077, 1993.

486

Brain Vulnerability to Alcoholism: Neuroimaging Studies

under different conditions; differences
in regional brain activity from task to
task can serve to map neural networks
of regions invoked during specific,
controlled tasks. In addition, 150 is
capable of tracking temporal and
regional blood flow effects of acute
alcohol uptake. Brain glucose metabo-
lism detectable with PET occurs pri-
marily in gray matter, that is, in cell
bodies. Thus it is a critical control in
PET studies to express metabolism in
terms of the amount of gray matter
present in a target region as deter-
mined by structural imaging. In the
case of alcoholism, low FDG signal
may occur because of abnormally low
cerebral metabolism or because alco-
holics as a group have smaller gray
matter volumes, and thus appear to
have lower metabolism rates, than
their nonalcoholic cohorts.

PET studies have consistently revealed
lower whole brain glucose metabolism
in alcoholics than in control subjects
(Volkow et al. 1992; Wang et al.
1993). Wang and colleagues (1993)
showed reduced PET brain metabolic
activity in alcoholics in the absence of
structural volume deficits. Regionally, the
pattern of differences is greatest in frontal,
cingulate, parietal, and cerebellar cortices
(Wik et al. 1988; Gilman et al. 1990;
Martin et al. 1992; Volkow et al. 1992,
1995). Presence of alcoholic cerebellar
degeneration determined from signs, such
as gait ataxia, was associated with abnor-
mally low metabolic rates in the anterior
superior vermis of cerebellum compared
with that observed in healthy control
subjects and alcoholics free of cerebellar
signs; however, alcoholics, regardless of
cerebellar signs, had abnormally low

glucose metabolism in the frontal lobes
(Gilman et al. 1990, 19966). Medial
frontal hypometabolism was associated
with poor performance on the Wisconsin
Card Sorting Test, which is sensitive to
dysfunction or lesions of the dorsolateral
prefrontal cortex (Milner 1963; Sullivan
et al. 1993). KS alcoholics have especially
low glucose utilization in the cingulate
and precuneal regions (Joyce et al. 1994).
Older alcoholics may show a further
age-related decrease in cerebral metab-
olism (Sachs et al. 1987), but rigorous
studies have yet to be conducted that
control for the effect of normal aging
on cortical gray matter volume as well
as potentially normal age-related
decline in cerebral metabolism that may
occur even after age-related volume
declines are considered.

Family history of alcoholism may not
be predictive of glucose metabolism
differences in the cerebrum (Adams et
al. 1998) but may be predictive of such
differences in the cerebellum (Volkow
et al. 1995). Volkow and colleagues
used lorazepam in a PET FDG study
to seek mechanisms underlying the
often-reported blunted response of
alcoholics to alcohol. Brain regions
consistently showing decreased glucose
metabolism are those with substantial
density of GABAA and benzodiazepine
receptors (Gilman et al 1996a). Lora-
zepam is a compound with behavioral
and pharmacological effects that, like
other benzodiazepines, mimic alcohol,
including sleepiness and decreased
motor and cognitive functions. The par-
ticipants in the Volkow et al. (1995)
study were nonalcoholics, but one group
had a family history of alcoholism
whereas the other group did not. Those

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NIAAA's Neuroscience and Behavioral Research Portfolio

with a positive family history showed a
blunted effect following acute alcohol
administration, especially in cerebellar
metabolism. This response suggests
that the GAB A- benzodiazepine receptor
complex may have different sensitivi-
ties depending on genetic disposition
to alcoholism.

Regarding metabolic changes with
alcohol abstinence, the increase in
cortical gray matter volume with alco-
hol abstinence observed with MRI
(Pfefferbaum et al. 1995) is consistent
with the findings of a PET FDG study
of recovery of brain glucose metabolism
in detoxified alcoholics. An increase in
cerebral metabolism, especially in the
frontal region, occurred predominantly
16-30 days after alcohol withdrawal,
compared with PET studies done in
alcoholics 8-5 days and 31-60 days
after withdrawal (Volkow et al. 1994).

lsO studies have focused on KS
because identification of the neural sites
underlying its characteristic global amne-
sia for declarative events remains an
enigma. One study reported low cerebral
blood flow prominent in the frontal-
temporal regions bilaterally and the left
thalamus, brain regions known to con-
tribute to memory impairments (Matsuda
et al. 1997). Paller and colleagues (1997)
used high-resolution PET and observed
an anterior to posterior gradient in
cerebral metabolic rates for glucose uti-
lization that were especially marked in
frontal, anterior, and posterior cingulate
regions, less so in parietal regions and
even less so in occipital and temporal
regions. Using a cognitive activation
paradigm, these authors noted widespread
declines in glucose metabolism in frontal,
parietal, and cingulate areas in KS relative

to nonamnesic alcoholics in a delayed
recognition task. These two alcoholic
groups did not differ, however, in glucose
utilization in hippocampus, a structure
in which lesions can result in profound
amnesia (Squire and Zola 1996).

SPECT Studies

SPECT studies with ligands that detect
regional cerebral blood flow provide
evidence of hypoperfusion in frontal
lobes of detoxified alcoholics. The extent
of reduced perfusion correlated with
performance on tests of frontal lobe
function, such as the Wisconsin Card
Sorting Test and the Trail Making Test
(Nicolas et al. 1993). Frontal hypoper-
fusion was also noted to be especially
prominent in alcoholics with antisocial
personality compared with alcoholics with
dependent personality disorder (Kuruoglu
et al. 1996). Cerebral blood flow showed
different patterns of hypoperfusion in two
different diseases: frontal regions were
notably affected in KS, whereas posterior
temporal and parietal regions were most
affected in Alzheimer's disease (Hunter
et al. 1989). In a study (Dupont et al.
1996) of recovery after abstinence from
alcohol, alcoholics, whether recently
detoxified or long-term abstinent, showed
persistently low cerebral blood flow as
measured by uptake of the blood flow
tracer iodoamphetamine (123IMP). A
neuropsychological test of nonverbal
reasoning, however, did distinguish the
two alcohol groups: the long-term absti-
nent group performed better than the
short-term abstinent group on the Raven's
Progressive Matrices (Dupont et al 1996).
Interestingly, smokers had the lowest
123IMP uptake in any group, thus identi-
fying smoking as a significant confounding

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Brain Vulnerability to Alcoholism: Neuroimaging Studies

factor in functional studies of alco-
holics, who typically are smokers.

New Imaging Directions

Functional MRI

The functional significance of brain
volume loss in terms of the commonly
observed cognitive behavioral deficits
has been difficult to demonstrate (Parsons
et al. 1987). Obstacles to observation
of selective structure-function relationships
may include imprecise measures of com-
plex, multicomponent processes, poor
understanding of the neural networks
underlying them, and a limited under-
standing of the role of compensatory
mechanisms. Functional MRI (fMRI)
techniques have the potential to identify
areas throughout the brain that are acti-
vated during performance of specific
components of cognitive operations.
These activated regions can then be
mapped onto structural images to locate
the regions precisely and determine
their structural integrity. The consistent
deficits reported in executive functions
in alcoholics together with the relatively
selective cortical gray matter volume deficit
observed in older alcoholics provide a
strong rationale for using functional imag-
ing techniques to examine, in real time,
potential behavioral deficits associated
with circumscribed brain regions.

One of the few fMRI studies to date
involving alcoholics examined repetition
priming, a form of implicit memory
thought to be relatively preserved in
global amnesia. Amnesic alcoholics,
nonamnesic alcoholics, and normal
(nonalcoholic) control subjects each
showed the predicted reduction in
BOLD (blood oxygen level dependent)

signal of the left inferior prefrontal cor-
tex in conditions comparing repeated
stimuli with novel stimuli relative to
conditions comparing only novel stim-
uli (Gabrieli et al. 1996). These data
support the contention that repetition
priming is preserved even in patients
with amnesia for declarative, or explicit,
events. The apparently normal pattern
of BOLD response to primed and
unprimed stimuli in this experiment
also suggests that despite the potential
structural damage in frontal cortex of
alcoholics, at least some cognitive func-
tion remains intact. This again high-
lights the importance of incorporating
indices of brain function as well as struc-
ture in studies investigating brain-
behavior relationships in alcoholism.

Diffusion Tensor Imaging

The neuropathological literature, espe-
cially that describing alcoholics in
Australia, consistently reports volumet-
ric loss of white matter far in excess of
that seen in gray matter (Harper and
Kril 1988, 1990, 1991). In vivo neu-
roimaging studies consistently observe
volumetric decrements in both white
and gray matter volumes, and both
tissue types show some, albeit modest,
recovery with abstinence. There are
also the dramatic subsyndromes of
alcoholism, such as Marchiafava-Bignami
disease and central pontine myelinosis,
which are characterized by specific white
matter pathology. Diffusion tensor
imaging is a newly developed MRI
technique that provides a measure of
the anisotropy of water diffusion in
every voxel imaged (Belzunegui et al.
1995). Diffusion of water molecules is
essentially isotropic; that is, the diffusion

489

NIAAA's Neuroscience and Behavioral Research Portfolio

of water molecules exhibits brownian
motion with diffusion in all directions
equally as occurs in CSF. In white
matter, where there are closely packed
bundles of parallel axons, diffusion of
water molecules occurs along the axons
and is thus "anisotropic"; that is, the
diffusion proceeds in a specific direction.
Diffusion of molecules in gray matter
falls between that of CSF and white mat-
ter, as can be seen in figure 4. Diffusion
tensor imaging provides a number of
measures of diffusion anisotropy, includ-
ing its direction and magnitude, and
thus offers a potential metric for
assessing the integrity of white matter
throughout the course of alcoholism
(Pfefferbaum et al. 1999c).

DIFFERENCES IN
VULNERABILITY:
SPECIAL POPULATIONS

Psychiatric Comorbidities

Alcohol use disorders are commonly
present in major psychiatric diseases
(Regier et al. 1990; Kendler et al.

1996; Kessler et al. 1996) (figure 5)
and thus dispose patients comorbid for
both diagnoses to be susceptible to brain
volume abnormalities from both sources.
Because most studies have sought to
include only "clean" patients meeting
strict criteria for a particular disease,
patients comorbid for a psychiatric
disease plus alcoholism have been
carefully excluded. Only recently have
these combined effects been investi-
gated rigorously.

We compared regional cortical and
cerebellar volumes in samples of schiz-
ophrenics, alcoholics, patients comor-
bid for both schizophrenia and
alcoholism, and control subjects. The
first analysis (Mathalon et al. 1995)
quantified gray matter volumes across
six regions, from prefrontal to occipi-
tal cortex. The resulting profile of vol-
ume deficits was similar for the
schizophrenic and comorbid groups,
with the greatest deficits present in
the prefrontal and anterior temporal
cortex, and different from that of the
alcoholic group, which showed a flatter
deficit profile. The prefrontal cortex was

Figure 4. (a) Early (TE = 20) and (b) late (TE = 80) fast spin echo axial magnetic resonance
images at the level of the lateral ventricles; (c) fractional anisotropy image at the same anatomi-
cal location (bright signal = greater anisotropy primarily seen in the white matter).

490

Brain Vulnerability to Alcoholism: Neuroimaging Studies

particularly vulnerable, showing greater
deficits in the comorbid group than
either single -diagnosis group. Further-
more, when the comorbid patients were
divided according to which disease
developed first, the "schizophrenia-first"
group had a regional volume profile
more like the schizophrenics, and the
"alcoholism-first" group was more
like the alcoholics. Thus, alcohol-
schizophrenia comorbidity may represent
a biologically heterogeneous group of
clinical entities. For the cerebellar hemi-
spheres, the alcoholic and comorbid
groups had volume deficits in gray
matter. The alcoholics and comorbid
patients, but not schizophrenics, had
deficits in the anterior superior vermis.
Conversely, the schizophrenic and the

Figure 5. Lifetime prevalence of comor-
bidity of psychiatric and addictive disorders,
including alcoholism, in the United States.
Adapted from Regier, D.A.; Farmer, M.E.;
Rae, D.S.; Locke, B.Z.; Keith, S.J.; Judd,
L.L.; and Goodwin, F.K. Comorbidity of
mental disorders with alcohol and other
drug abuse — results from the Epidemiologic
Catchment Area (ECA) Study. JAMA
264:2511-2518,1990.

comorbid groups but not the alcoholic
group both had severely enlarged fourth
ventricles relative to control subjects
(Sullivan et al. in press). Thus, fourth
ventricular neuropathology is common
to schizophrenia and anterior superior
vermian neuropathology is common
to alcoholism. The vermian volume
deficits may underlie the residual
ataxia of detoxified alcoholics and eye-
tracking impairment of schizophrenics.
The presence of both sites of vermian
dysmorphology in the comorbid group
puts them at risk for both types of
motor compromise.

Other diseases with a high incidence
of alcohol abuse and dependence are
bipolar disorder (Regier et al. 1990) and
HIV infection (Schleifer et al. 1996).
Anonymous HIV testing at an inner-
city outpatient alcohol treatment center
revealed that 16.7 percent of patients
exclusively abusing alcohol were HIV
seropositive. HIV infection can result in
progressive ventricular volume enlarge-
ment, reduced volume of white matter,
and caudate atrophy as the disease pro-
gresses (Stout et al. 1998). Some (Lim
et al. 1999) but not all (Schlaepfer et
al. 1994; Zipursky et al. 1997) MRI
studies have detected cortical gray mat-
ter volume deficits in patients with bipo-
lar disorder without alcohol dependence.
Although both HIV infection and
bipolar disease may have at least some
degree of associated brain pathology,
few studies have rigorously examined
the added effect of alcoholism. Those
that have typically report a combined
effect of HIV and excessive alcohol
consumption. These studies are usually
based on infected individuals who are
still drinking heavily and thus reflect

491

NIAAA's Neuroscience and Behavioral Research Portfolio

the combined acute and chronic
effects of alcohol consumption.

Polydrug Abuse

Studies of structural brain abnormalities
in polydrug abusers tend to have neg-
ative results (Liu et al. 1995), unless
alcohol is a significant ingredient in
the mix of drugs abused (Cascella et
al. 1991; Aasly et al. 1993). In one
study, statistical analysis of the role of
severity of use of different drugs
abused found that, after taking age into
account, only alcohol contributed addi-
tionally to ventricular enlargement
(Cascella et al. 1991). In another study,
the authors attributed findings of
reduced cerebellar vermis and increased
incidence of clinically defined white
matter abnormalities in polydrug abusers
to alcohol consumption in their sam-
ple (Aasly et al. 1993). Thus, abuse of
other drugs provides no protection
against the known effects of alcohol
on brain structure. Whether the use
of drugs such as cocaine, marijuana,
or barbiturates potentiates the brain's
vulnerability to alcohol has not been
adequately studied.

HOW DO STRUCTURAL
AND FUNCTIONAL BRAIN
ABNORMALITIES
PREDISPOSE ALCOHOLICS
TO BEHAVIORAL AND
NEUROPSYCHOLOGICAL
PROBLEMS?

Neuronal Shrinkage
Versus Loss

Gray matter volume reduction could
be indicative of shrinkage of neuronal

cell bodies or dendrites or actual neu-
ronal loss. When postmortem studies
have documented neuronal loss, it
usually affects large cells, such as
pyramidal cells found in the frontal
lobes and in cerebellum (Courville
1955; Harper and Kril 1990, 1993;
Pentney 1993; but see Badsberg-
Jensen and Pakkenberg 1993). In
animal models (mouse), long-term
ethanol consumption produces loss of
dendritic spines on hippocampal pyra-
midal cells and dentate granule cells
(Riley and Walker 1978; Davies and
Smith 1981) and significant loss of
pyramidal and dentate gyral cells even
in the absence of malnutrition (Walker
et al. 1980). Conversely, one study
showed that abstinence following a
period of ethanol intake was accompa-
nied by increases in dendritic arboriza-
tion of hippocampal pyramidal cells
(McMullen et al. 1984). In humans,
decreased basal dendritic arborization
of layer III pyramidal neurons was
observed in alcoholic compared with
normal, nonalcoholic cases (Harper
and Corbett 1990). The critical dif-
ference between these mechanisms of
volume reduction is that cell loss is
presumably permanent, whereas cell
size reduction may well be transient
and amenable to reversal with absti-
nence from alcohol or with adequate
nutrition or other treatment.

Postmortem studies have shown
white matter volume reduction and
could reflect demyelination, axonal
shrinkage, or loss of fibers due to cell
degeneration (Harper and Kril 1990).
Longitudinal in vivo studies following
the course of alcoholism show
that white matter volume may be

492

Brain Vulnerability to Alcoholism: Neuroimaging Studies

recoverable, at least in part, with alco-
hol abstinence (Shear et al. 1994;
Pfefferbaum et al. 1995).

Regrowth of neuronal processes
does not ensure normal "rewiring"
and restitution of function (see, e.g.,
Hill and Mikhael 1979). Thus, neuro-
psychological studies are essential for
testing whether, with abstinence,
alcoholics whose brains show recov-
ery of tissue volume are the individu-
als who also show improvement in
their cognitive and motor abilities
(see, e.g., Eckardt et al. 1979; Brandt
et al. 1983; Parsons 1987^). A recent
naturalistic, longitudinal study, which
used quantitative neuroimaging and
neuropsychological methods, exam-
ined alcoholics who returned to the
community following a 2 8 -day reha-
bilitation program. Alcoholics who
maintained sobriety sustained greater
improvement than relapsers on tests
of nonverbal memory, visuospatial
functions, attention, gait, and balance
(Sullivan et al. in press). In general,
improvement in cognitive and motor
functions was related to improvement
in the condition of the brain; in partic-
ular, increases in scores on short-term
memory tests correlated with shrink-
age of the third ventricle. Neuronal
recovery could also be inferred from
proton MRSI studies if increases in
tissue volumes were accompanied by
further increases in gray matter or
white matter NAc concentrations,
which would occur with sobriety.
Such recovery would suggest that the
remaining cell bodies, although once
shrunken, were resilient and viable (cf.
Badsberg-Jensen and Pakkenberg
1993). Whether such plasticity is

curtailed by the aging process (Ron et
al. 1982; Carlen et al. 1984) also
remains to be established.

Discontinuous Syndromes
or Continuous Brain
Abnormalities

In vivo neuroimaging has enabled us
to identify brain structural abnormalities
in chronic alcoholics without clinical
symptoms of severe liver disease, amne-
sia, or alcoholic dementia that previ-
ously had been considered hallmarks
of specific subsyndromes, such as
Wernicke -Korsakoff syndrome (mam-
millary body and possibly hippocampal
shrinkage), Marchiafava-Bignami disease
(thinning of corpus callosum), and
alcoholic cerebellar degeneration with
ataxia (figure 6). These observations
were made possible through survey,

Figure 6. Alcoholism and its nutritional
and genetic attendant and disposing factors
appear central to specific neuropsychologi-
cal and neuropathological syndromes.
However, many of the same brain abnor-
malities characterizing these syndromes are
also found with in vivo neuroimaging in
"uncomplicated" alcoholism.

493

NIAAA's Neuroscience and Behavioral Research Portfolio

controlled quantification, and com-
parison of large regions and multiple
brain structures that yielded patterns
of normality and abnormality. Rather
than there being classical alcoholic
subsyndromes, the findings suggest a
continuum of graded brain dysmor-
phology, whereby lesions in specific
brain regions usually leading to syn-
dromes of selective cognitive or motor
deficits occur in clinically unsymptomatic
alcoholics with far greater frequency
than previously thought.

What needs to be established is
whether these brain volume deficits
are accompanied by predictable neuro-
psychological symptoms. Furthermore,
although alcoholic dementia has been
seldom studied with imaging tech-
niques, one hypothesis would be that
alcoholic dementia results from the
cumulative effect of volume deficits in
multiple brain structures, each of which
traditionally define only selective syn-
dromes. The question of gene-envi-
ronment interaction with respect to
apolipoprotein genotype (as seen in
Alzheimer's disease [Strittmatter and
Roses 1996]) and alcohol on the devel-
opment of dementia and accelerated
brain volume loss in alcoholics has yet
to be addressed.

Functional Outcome of
Frontal- Cerebellar Circuit
Disruption

Converging evidence from every
imaging modality, neuropsychological
examination, and neuropathological
study identifies the frontal lobes and
the cerebellum as principal targets of
alcohol-related corruption of brain
structure and function. The extent to

which these regions recover in structure
or function remains unknown despite
their importance to problem solving,
contextual memory, and execution
and learning of procedures. A recent
brain- behavior analysis revealed that
lower scores on test composites assessing
executive function and motor function,
gait, and balance showed independent
relationships with white matter volumes
of the anterior superior vermis of the
cerebellum (Sullivan et al. 1998#).
Thus, vermian volume deficits in alco-
holics appear to have functional rami-
fications for cognitive executive function
as well as motoric functions, raising
the possibility of disruption of frontal -
cerebellar circuitry in alcoholism. The
presence of cerebellar structural abnor-
malities might also set limits on the type
of procedural retraining and learning
of new habits required to maintain
sobriety in alcoholics (Finn et al.
1990) and could contribute to problem
solving and working memory deficits
known to persistent in long-detoxified
alcoholics (Butters and Cermak 1980;
Becker et al. 1983; Brandt et al. 1983;
Nixon etal. 1992).

Models of Aging

Despite the usefulness of the cross-
sectional age-regression method for esti-
mating the effects of age, it remains essen-
tial to verify cross-sectional observations
with those from controlled, longitudinal
study. Although longitudinal neuroimag-
ing studies of alcoholics have been
reported, they typically focus on issues of
relapse and abstinence. Still needed are
controlled studies of alcoholics who
remain abstinent for years in comparison
to those who drink for years. It would

494

Brain Vulnerability to Alcoholism: Neuroimaging Studies

be of great importance to know, for
example, whether treatment of and
recovery from early-onset alcoholism at
a young age can adequately protect the
individual from the usual alcohol-related
accelerated brain aging, or whether the
early bout with alcoholism reduces the
margin of safety thought to be available
to pathology- free individuals. Other
conditions, such as polio (Cosgrove et al.
1987) and head injury (Corkin et al.
1984, 1989), do increase the risk of
exacerbated functional declines with age.
Early alcoholism may be an added factor
of vulnerability and may interact with
genetic underpinnings to accelerate aging.
For instance, in a study of monozygotic
(MZ) and dizygotic (DZ) elderly normal
(nonalcoholic) twin pairs, intracranial
volume and age-corrected cortical sulcal
volume, but not ventricular volume, were
highly heritable, with evidence for
additive genetic variance. However,
highly significant MZ but not DZ intra-
class correlation for ventricular volumes
suggested a smaller role for additive
genetic effects and a larger role for gene-
gene interaction and/or shared environ-
mental effects on this morphometric
measure (Pfefferbaum et al in press).
To date, no quantitative in vivo studies
have been conducted to examine, even
cross-sectionally, potential alcohol-gene
interactions on brain morphology.
These studies are essential to deter-
mine the pattern and extent of poten-
tially preexisting differences in
individuals genetically predisposed to
alcohol addiction compared with those
not so predisposed.

The severity of the brain dysmorphol-
ogy observed in chronic alcoholics, and
possibly the degree of recovery with

abstinence, may also be related to
demographic and drinking factors,
including age, duration of alcoholism,
and lifetime alcohol consumption.
Although far more longitudinal studies
using structural than functional imag-
ing techniques have been conducted,
cohorts studied to date have not been
large and diverse enough to adequately
address these possibilities.

CONCLUSION

Human in vivo neuroimaging studies
have characterized gross dysmorphol-
ogy associated with alcoholism and in
excess of what is expected in normal
aging. Even in "uncomplicated alco-
holism," significant brain tissue volume
deficits are present in brain structures
previously thought to be affected only in
certain alcohol-induced subsyndromes.
Regional brain metabolism and selec-
tive neuroreceptor systems are also
affected in alcoholism. These struc-
tural and functional abnormalities
wax and wane with abstinence and
relapse, but exactly how they pro-
mote cognitive, sensory, and motor
dysfunction and whether the degree
of recovery has diminishing returns
with advancing age and recurrent
bouts of alcoholism remain unknown.
Furthering knowledge of these neural
functions and mechanisms may lead
to rational approaches for behavioral
and pharmacological treatment of
alcoholism and for its prevention.
Combining structural and functional
imaging modalities provides the
opportunity to identify both mecha-
nisms of vulnerability and points
of damage, some of which may be

495

NIAAA's Neuroscience and Behavioral Research Portfolio

uniquely human and present only Mental Disorders. 4th ed. Washington,
in life. DC: the Association, 1994.

ACKNOWLEDGMENTS

This work was supported by National
Institute on Alcohol Abuse and Alcohol-
ism grants AA10723 and AA05965. I
would like to thank Adolf Pfefferbaum,
M.D., my collaborator and mentor in
imaging and alcoholism, for the mag-
netic resonance images and other incal-
culable contributions to this chapter. I
also thank Daniel Spielman, Ph.D.,
for the proton spectra and Margaret J.
Rosenbloom, M.A., for helpful com-
ments on the manuscript.

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508

Chapter 15

Human Brain Dysfunction Secondary

to Alcohol Abuse: Suggestions for

New Research Initiatives

George Fein, Ph.D., Daniel Fletcher, Ph.D.,
and Victoria Di Sclafani, M.P.H.

KEY WORDS: AOD (alcohol or other drug) abuse; brain damage; electroen-
cephalography; neuroimaging; behavior; chronic AODE (effects of AOD use,
abuse, and dependence); risk factors; dysfunction; gender differences; aging; ado-
lescent; morbidity; evoked potential; literature review

Electrophysiological , neuropsycholog-
ical, and structural and functional
neuroimaging methods hold great
promise for obtaining new insights
into brain dysfunction secondary to
chronic alcohol abuse. These comple-
mentary approaches have the potential
to advance the currently limited state
of knowledge regarding ( 1 ) structural
damage to the brain pursuant to
chronic alcohol exposure, (2) func-
tional brain changes associated with
or independent of structural damage
(or both), and (3) the specific behav-
ioral effects resulting from these struc-
tural and functional alterations.

WHAT ARE THE MOST
IMPORTANT QUESTIONS
REGARDING BRAIN
DYSFUNCTION
RELATED TO CHRONIC
ALCOHOL ABUSE?

Which Brain Systems Are
Most Vulnerable to the
Morbid Effects of Chronic
Alcohol Abuse?

It is important to improve the evi-
dence linking structural damage,
functional alterations, and the specific
behavioral effects of chronic alcohol

G. Fein, Ph.D., is a senior scientist at Neurobehavioral Research Inc., 45 Gable Court, San Rafael,
CA 94903. D. Fletcher, Ph.D., is a student in the School of Veterinary Medicine at the University of
California, Davis. V. Di Sclafani, M.P.H., is a research scientist at Neurobehavioral Research Inc.

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NIAAA's Neuroscience and Behavioral Research Portfolio

abuse. Performance on most behavioral
tasks involves a complex interplay of a
number of brain systems. Further work
is needed to define the specific brain
processes and systems affected by
chronic alcohol abuse. Such work could
begin with a review of the behavioral
literature to determine tasks that con-
sistently reveal robust performance
decrements in long-term alcoholics.
This should be followed up with care-
ful behavioral, electrophysiological, and
functional imaging investigations of
performance on such tasks in non-
alcohol-abusing subjects to elucidate
the constituent operations and brain
systems involved in performing these
tasks. This approach would provide a
normative database useful in defining
abnormalities in alcoholic patients.

It is very possible that alcohol-
related changes will be subtle, reflect-
ing "bias" effects within the neural
systems that contribute to task
performance. In other words, the sys-
tems and associated sites of brain
activation may be unchanged in
chronic alcoholic patients, but the
relative contribution and timing
of activation of the constituent
brain systems may be altered.
Electrophysiological methods can
make a unique contribution to such
investigations as a result of their very
fine temporal resolution.

What Alcohol Use-Related
Factors Influence Brain
Morbidity Secondary to
Alcohol Abuse?

1 . What is the relative role of nutritional
(primarily thiamine) deficiency and

impaired liver function in the
development of brain dysfunction
secondary to alcohol abuse? Is
there a diagnostic entity of
alcoholic dementia separate from
that of dementia secondary
to alcohol abuse-related nutri-
tional deficiency or impaired
liver function (Diamond and
Messing 1994; Emsley et al.
1996)? How would one differenti-
ate these conditions from each
other and from brain changes asso-
ciated with coexisting Alzheimer's
disease (AD) or primary cere-
brovascular disease?

2. Is brain dysfunction primarily related
to the effects of alcohol consump-
tion or to the occurrence of episodes
of withdrawal? A fundamental
question is whether brain dys-
function is caused by the direct
toxic effects of heavy alcohol use or
whether it is caused by the episodes
of alcohol withdrawal (which result
in kindling in animals and
humans). This is a core issue with
very little data.

3. Is the development of tolerance to
the acute effects of alcohol associated
(either positively or negatively)
with the degree of brain dysfunc-
tion secondary to alcohol abuse?

4. What is the role of the pattern of
drinking behavior on the develop-
ment of brain dysfunction? This
issue relates to the relative impor-
tance of total alcohol consumption
versus the maximum alcohol dose
(i.e., the brain dysfunction atten-

510

Brain Dysfunction Secondary to Alcohol Abuse

dant to chronic regular drinking
versus binge drinking).

What Other Factors
Influence Brain Morbidity
Secondary to Alcohol Abuse?

Other factors that may influence brain
morbidity secondary to alcohol abuse
include genetics, gender, age, brain
functional reserve, and comorbidity.
These factors are discussed in the fol-
lowing paragraphs.

Are there genetically transmitted
vulnerabilities to alcohol abuse-related
brain morbidity? There is extensive
compelling evidence for a genetic pre-
disposition to develop alcoholism (e.g.,
Pfefferbaum et al. 1991; Cadoret et
al. 1995; Crabbe et al. 1998). It is pos-
sible that there may also be a genetic
predisposition to develop brain damage
secondary to the toxic effects of chronic
alcohol abuse. Moreover, such a
genetic predisposition may be quite
independent of the genetic predisposi-
tion to develop alcoholism.

How does gender affect the severity
and persistence of brain morbidity sec-
ondary to alcohol abuse? Most research
on cognitive function in recovering
alcoholics has used samples of male
veterans in inpatient and outpatient
treatment programs. The relative vulner-
ability of women to cognitive deficits
associated with chronic alcohol abuse
has received only limited study.
Although most studies report the
same pattern and degree of deficits in
alcoholic women as in alcoholic men
(Sparadeo et al. 1993), gender-related
differences have been reported (Leber
et al. 1981). Some of these studies have
determined that the pattern of deficits

is the same in female alcoholics as in
male alcoholics, but is more severe
among women (Acker 1986; Bergman
1987), or that deficits comparable to
those suffered by male alcoholics are
present in women after shorter drink-
ing histories and lower average daily
consumptions (Acker 1986). Some
studies have found additional deficits
in female alcoholics, such as in verbal
abstraction ability (Hatcher et al.
1977). Other studies that compare cog-
nitive functioning of alcoholic women
and men show better performance
among the women in visuospatial abil-
ities (Fabian et al. 1984) and in both
verbal and visual short-term memory
(Sparadeo et al. 1993). It is clear that
additional research on the central ner-
vous system (CNS) effects of chronic
alcohol abuse in women is needed; a
particularly important question is
whether the modulating effects of age
on the CNS morbidity of chronic alco-
hol abuse differ in men and women.

Why is brain morbidity greater in
the older alcoholic? The effect of aging
on brain dysfunction secondary to
alcohol abuse has been a long-debated
topic. There is an extensive literature
demonstrating that brain morbidity
due to alcohol abuse is greater in the
older compared with the young or
middle-aged alcoholic, even after
adjustments are made for duration
and amount of alcohol consumption
(e.g., Goldman et al. 1983; Grant et al.
1984; Fein et al. 1990; Pfefferbaum et
al. 1992). The generally accepted
"age-related vulnerability" model postu-
lates that abusive drinking during old
age has greater effects on the brain
than does comparable drinking during

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NIAAA's Neuroscience and Behavioral Research Portfolio

young adulthood or middle age. In
this model, an older alcoholic who
became abstinent in old age would have
more brain damage than an alcoholic
of the same age who became abstinent
in middle age, assuming comparable
duration and amount of drinking.

An alternative model, the "cumula-
tive effects" model, postulates the aging
process and the duration and amount
of abusive drinking as additive effects
underlying the greater brain morbidity
in the older alcoholic. In this model,
brain structure and function of two
older abstinent alcoholics would be
comparable regardless of the age at
which abstinence was attained (assum-
ing comparable durations and total
amounts of alcohol consumed). This
cumulative effects model maintains
that the young or middle-aged brain
is able to compensate for the damage
done by alcohol abuse. However,
even in such individuals, the brain
damage caused by abusive drinking in
earlier life will become unmasked as
compensatory brain processes are dimin-
ished due to the normal aging process.
There are no data in the literature to
test the differential predictions of these
two models (e.g., data on the brain
status of older individuals who were
alcoholic into middle age, but who have
been abstinent for 10 years or more)
because research to date has focused
only on recently abstinent individuals
(usually those in treatment through
the first year or so of abstinence).

The cumulative effects model and the
age-related vulnerability model have
very different implications. The age-
related vulnerability model suggests
that the long-term consequences of

alcohol abuse are attenuated if a person
stops abusing alcohol before old age
(when the brain is much more vulner-
able to lasting damage secondary to
alcohol abuse). The cumulative effects
model suggests that functional dam-
age will be revealed as a person ages
(when the brain can no longer com-
pensate for the underlying damage)
no matter when he or she stopped
abusing alcohol.

Are there subject factors that denote
a brain functional reserve mitigating
the effects of chronic alcohol abuse7.
Might genetic factors and/ or parental
alcohol abuse result in a decreased func-
tional reserve, amplifying the morbid
effects of chronic alcohol use7. The concept
of brain functional reserve was devel-
oped in the AD literature to describe
normally "unused" or "extra" brain
capacity. In the AD literature, functional
reserve has been estimated using a mea-
sure of premorbid brain volume, pos-
tulating a greater number of neurons
and synapses available in the larger
brain. Head circumference or intracra-
nial volume quantified on magnetic
resonance images or computed
tomography (CT) images is usually
used to estimate premorbid brain vol-
ume (since mature skull size is driven
by brain growth, a process mostly
complete by the end of the second
year of life). AD patients with larger
head circumference or intracranial vol-
ume on magnetic resonance or CT
images had a later onset of disease, or
a lessened severity of cognitive impair-
ment, or both (Schofield et al. 1995;
Graves et al. 1996; Mori et al. 1997).

In the first study (that we know of)
to investigate the concept of functional

512

Brain Dysfunction Secondary to Alcohol Abuse

reserve outside the AD field, Di Sclafani
and colleagues (1998), in our laboratory,
used MRI to quantitate the intracranial
volume of cocaine -dependent individ-
uals and dually addicted cocaine- and
alcohol-dependent persons abstinent
approximately 3 months. Premorbid
brain size accounted for more than 20
percent of the variance in global cog-
nitive impairment in both groups.
The investigators suggested that func-
tional reserve may be a general pro-
tective mechanism of the brain that
has consequences for the severity of
expression of cerebral disease or insult
throughout life.

In addition to the very large effect
of functional reserve on neuropsycho-
logical performance in abstinent alcohol-
and/or other drug- dependent (AOD-
dependent) subjects, Di Sclafani and
colleagues (1998) also observed a trend
for AOD-abusing subjects to have a
smaller intracranial volume than control
subjects. It is possible that the inter-
section of negative prenatal and early
childhood factors on brain growth and
the genetic predisposition to alcoholism
and addiction may result in a synergism.
The parents of AOD-dependent sub-
jects may have been AOD-dependent
themselves; therefore both prenatal and
early childhood environments for such
individuals may not be conducive to
maximal brain growth. Furthermore,
children of AOD-dependent parents
have a greater probability of becom-
ing AOD-dependent themselves. In
that case, in the context of possible
early developmental deficits, the mor-
bid effects of AOD dependence may
be a "second strike" situation for the
brain. Moreover, such individuals may

be even more vulnerable to a "third
strike" later in life secondary to cerebral
diseases such as AD, or even the declines
associated with normal aging.

Are there developing brain systems in
adolescents that are particularly vul-
nerable to the morbid effects of chronic
alcohol abuse? There are a plethora of
studies underscoring the importance
of late brain maturational processes
during adolescence (Benes et al.
1994; Pfefferbaum et al. 1994; Giedd
et al. 1996a, 1996£, 1996c)- Since
many people begin to drink heavily
during adolescence, it is important to
determine if and how alcohol exposure
affects brain maturation during this
period. Neuropsychological, electro-
physiological, and in vivo imaging
techniques can be used to monitor the
processes of brain maturation in adoles-
cence. In this regard, it has been demon-
strated that both age-related structural
changes and changing patterns of
brain activation can be observed during
late childhood and adolescence. How-
ever, the most informative studies would
require analysis of changing patterns
of brain activation associated with per-
formance of specific tasks, so that
alcohol-related alterations in the devel-
opmental trajectories could be investi-
gated in young alcohol abusers, and
so that such changes could be linked
to specific behavioral effects and out-
comes. Work by Rakic and others
(Huttenlocher 1979; Rakic et al.
1986; Huttenlocher and de Courten
1987; Zecevic and Rakic 1991) sug-
gests that peripubertal changes in
synaptic density are dramatic, and
these changes probably have important
and long-lasting functional implications.

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NIAAA's Neuroscience and Behavioral Research Portfolio

There is also evidence that gonadal
steroid hormones may play a large role
in these final "sculpting" processes
within the brain (e.g., Forget and
Cohen 1994; Panzica et al. 1996;
Rubinow and Schmidt 1996). Alcohol
exposure, either directly or through
interactions with hormonal effects,
may result in particularly resilient
alterations within neural circuits.
Carefully designed electrophysiological
and functional imaging studies of these
phenomena would be invaluable.

How does chronic alcohol abuse-asso-
ciated brain dysfunction manifest in
persons with comorbid disorders who
have independent brain morbidities?
This is a crucially important issue given
the high comorbidity of alcoholism
with psychiatric disorders and with abuse
of other substances. The effects of
alcoholism on brain dysfunction in per-
sons with comorbidity reflect at least two
processes. First, there are the additive
(and possibly synergistic) direct effects
of alcohol abuse and the comorbid
disorder (e.g., schizophrenia, HIV
infection, cocaine or other drug abuse)
on brain function. Second, alcoholism
is associated with denial, reduced care
seeking, and reduced compliance with
treatment. This would result in less
efficacious treatment of the comorbid
disorder, with corresponding greater
morbidity secondary to the comorbid
disorder. Most research studies focus-
ing on these comorbid disorders
exclude alcohol abusers. The National
Institute on Alcohol Abuse and Alco-
holism (NIAAA) should form alliances
with the institutes that have direct
responsibility for research on these
comorbid disorders to expand their

research to include subjects who
chronically abuse alcohol.

What Are the Mechanisms
Involved in Brain Recovery
From the Effects of Chronic
Alcohol Abuse?

Studies of the process of brain recov-
ery from dysfunction associated with
chronic alcohol abuse may need to
wait for progress in the areas mentioned
above. The mechanisms of recovery
from brain dysfunction secondary to
chronic alcohol abuse may also differ
across brain systems and with comor-
bid and modulating factors.

HOW CAN

ELECTROPHYSIOLOGICAL
METHODS UNIQUELY
CONTRIBUTE TO
INVESTIGATIONS OF
ALCOHOL ABUSE-
RELATED BRAIN
DYSFUNCTION?

Electrophysiological methods have
advantages over other neuroimaging
techniques in terms of temporal resolu-
tion, ease, and cost-effectiveness of data
acquisition, and the ability to collect
data during the performance of complex
cognitive tasks. The ability to collect
electrophysiological data during com-
plex cognitive tasks is particularly useful
in studies of the effects of chronic and
acute alcohol exposure. Correlating the
electrophysiological responses of the
brain with performance on specific
cognitive tasks can yield insight into
alcohol effects unobtainable with
other functional imaging methods where
the type of stimuli and tasks are more

514

Brain Dysfunction Secondary to Alcohol Abuse

severely constrained by the imaging
equipment and procedures. In addition,
the injured brain is more likely to dis-
play measurable deficiencies when
stimulated rapidly, and the increased
temporal resolution possible in elec-
trophysiological studies allows studies
using such paradigms. Finally, electro-
physiological methods have particular
promise in studies of the genetic factors
in alcoholism (and other disorders)
because of the high heritability of event-
related potentials (ERPs) and electroen-
cephalographic (EEG) characteristics
(e.g., van Beijsterveldt and Boomsma
1994; Sorbel et al. 1996; van Beijster-
veldt et al. 1996). Positron emission
tomography (PET) yields functional
information on a scale measured in
seconds to tens of seconds depending on
the tracer used, and functional magnetic
resonance imaging (fMRI) resolves
brain activity on a scale measured in
hundreds of milliseconds to seconds. In
contrast, electrophysiological recordings
can resolve activity on a millisecond time
scale. Once the electrodes are attached,
electrophysiological data acquisition
can easily be done while a subject per-
forms tasks or responds to sensory stim-
uli in any modality. Sensory inputs
and information-processing demands
can be manipulated and the effects of
these manipulations observed in the
neural responses obtained.

There are two major categories of
brain electrophysiological data: spon-
taneous EEG signals and ERPs. ERPs
reflect processes time-locked to sen-
sory stimuli or to specific information-
processing operations (e.g., orienting
to a stimulus or preparing for a motor
response). They are deterministic and

repeatable, and are usually averaged
over several occasions to increase the
signal-to-noise ratio. This reduces the
effect of spontaneous brain activity,
which is nondeterministic and repre-
sents the "background" activity of the
brain. Because spontaneous EEG sig-
nals are nondeterministic and ERPs
are deterministic signals, they must be
interpreted using different analytical
and statistical techniques.

Spontaneous EEG recordings can
be used to examine alertness, patterns
of regional brain activation, and the
integrity and efficiency of connectivity
between brain regions as these measures
are affected by experimental manipu-
lations, disease states, or acute or
chronic AOD administration. Similarly,
ERPs can be used to study brain sensory
or cognitive processes and systems as
they are affected by experimental manip-
ulations, disease states, or acute or
chronic AOD administration.

Although electrophysiological meth-
ods offer insights into alcohol abuse-
associated brain dysfunction not obtain-
able with other techniques, their use-
fulness can be improved through further
technological developments. Most
studies to date have relied on charac-
terization of the ERPs at a single elec-
trode location. However, this single -
channel waveform analysis suffers from
a number of limitations. Because of
individual variation in head shape and
brain structure, the same neural
source does not yield a maximal ERP
at the same scalp location in all
subjects. Measures of the amplitude
and latency of the component at a
single scalp location vary across sub-
jects in how well they represent the

515

NIAAA's Neuroscience and Behavioral Research Portfolio

amplitude and latency of the underly-
ing neural source.

The difficulties presented by individ-
ual variation in head shape and brain
structure are compounded by the fact
that scalp responses are rarely the result
of activation of a single region of the
brain. Usually, ERPs reflect the activity
of many spatially separated neural
sources, which are activated asynchro-
nously and simultaneously. Indeed,
even a single peak in an ERP can be
the result of several distinct neural
generators. This is hypothesized to be
the case with the P300 response,
which appears to have at least two
components, P3A and P3B. This situ-
ation makes interpretation of group
differences in ERPs extremely com-
plex, because a change in a given peak
may reflect a change in a single neural
generator or in multiple generators.
Several methods have been developed
that attempt to individually character-
ize the contributions of specific neural
generators and to yield measures that
can be compared across subjects. Fur-
ther development of these methods
would strengthen the usefulness of
ERP measures in studies of alcohol
abuse-related brain dysfunction.

EEG coherence and phase are mea-
sures of the integrity and speed of
communication between brain regions
that can be used to investigate alcohol
effects on white matter. A major
methodological problem has cast the
results of much of the previous coher-
ence literature into doubt. We have
shown (Fein et al. 1988) that common-
reference EEG recordings (which
make up over 90 percent of the data in
all published studies to date) can yield

inaccurate coherence estimates, which
depend both on the actual coherence
between the two regions of interest
and the power and phase of the activity
at the reference electrode. Reference -
free methods have been developed
which do not have these problems. An
investment in coherence studies using
this newer methodology is warranted.
The full promise of electrophysio-
logical methods can be achieved through
their use in conjunction with neu-
roimaging studies. PET imaging or
fMRI can be used to determine locality
of brain sources, and EEG data can
examine the activity of those sources
at a much higher temporal resolution.
MRI or CT imaging can be used to
construct extremely accurate three-
dimensional representations of the
shape and physical characteristics of
the brain, and EEG data collected
from co-registered spatially distributed
electrodes can be used to examine the
activity of cortical sources. That activ-
ity can then be correlated with specific
structures identified from the three-
dimensional representation of the
brain. Although a layering of methods
will provide information that each
method alone cannot (indeed, convolved
methods often produce a synergy of
information), electrophysiological
methods will remain a unique tool in the
alcohol research armamentarium because
of their unparalleled temporal resolution.

CONCLUSION

It is vitally important that NIAAA
provide a framework for research into
alcohol abuse-related brain dysfunction
in the next decades by defining the

516

Brain Dysfunction Secondary to Alcohol Abuse

essential questions to be addressed. A
mandate is needed for the establishment
of a normative database (including
behavioral, structural, and functional
indices) of brain systems affected by
alcohol abuse. An investment in
improved methods for combining
behavioral, structural, and functional
approaches is warranted, and investi-
gations using a multifaceted approach
should be encouraged.

ACKNOWLEDGMENTS

Preparation of this chapter was supported
by NIAAA grant ROl AA11311 and
National Institute on Drug Abuse
grant ROl DA09463.

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519

SUBCOMMITTEE REPORT

Chapter 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

KEY WORDS: AODD (AOD [alcohol and other drug] use disorder); research;
neurology (field); nervous system; brain; AODE (effects of AOD use, abuse, and
dependence); behavior; theory ofAODU (AOD use, abuse, and dependence);
AOD sensitivity; neuroimaging; endocrine system; biological adaptation; adoles-
cence; sleep; cognition; research funding; report

EXECUTIVE SUMMARY

The National Institute on Alcohol Abuse
and Alcoholism (NIAAA) Subcommittee
of the National Advisory Council on
Alcohol Abuse and Alcoholism on the
Review of the Extramural Research
Portfolio for Neuroscience and Behavior
met May 11-13, 1998. The charge to
the subcommittee was to examine the
appropriateness of the breadth, coverage,
and balance of the neuroscience and
behavior research portfolio, identifying
research areas that are well covered and
others that are either underinvestigated
or otherwise warrant significantly
increased attention. The subcommit-
tee was asked also to provide specific
advice and guidance on the scope and

direction of NIAAA's extramural
research activities in the neuroscience
and behavior area.

The subcommittee consisted of
two NIAAA Advisory Council co-chairs
and an advisory group of 17 individuals.
Sixteen of these individuals have demon-
strated expertise in alcohol-related areas,
and three individuals have demon-
strated expertise in non-alcohol-
related areas (see Appendix A).

The review process was initiated by
having experts (see Appendix B) in
neuroscience and behavior prepare
written assessments of the state of
knowledge, gaps in knowledge, and
research opportunities. NIAAA program
staff (see Appendix C) presented the
current extramural portfolio, categorized

523

NIAAA's Neuroscience and Behavioral Research Portfolio

into the areas of basic neuroscience
research, molecular adaptive responses,
neuroendocrine system, studies of the
acute and chronic effects of alcohol in

behavioral/structural deficits in
humans. All information was shared
with experts, selected NIAAA staff,
and the co- chairs and advisory group

animals and in humans, and cognitive/ before the meeting.

Table 1. NIAAA FY97 Awards in Neuroscience and Behavior.

Dollar Amount

Type of Award

Number

(thousands)

Research project grants

121

21,677

Research centers

6,744

Research careers

1,218

Research training

2,012

Total

171

31,651

No.

% of Total
Amount

22
29

57
25b

17
30
21
35
20b

aOf the 14 NIAAA centers, 4 are exclusively neuroscience and behavior. Seven of the 14 centers have neuroscience and
behavior components; 5 of these estimate that a 50 percent of the research budget of the center is invested in
neuroscience and behavior.
"Percentage of all NIAAA awards in FY97.

Table 2. NIAAA FY97 Research Project Grants and Research Careers Awards in Neuroscience and

Behavior.

Number of

%of

Category

Awards

FY97 Awards*

Basic neuroscience

Research project grants

Research careers

Molecular adaptive responses

Research project grants

Research careers

Neuroendocrine system

Research project grants

Research careers

Acute and chronic behavioral effects of alcohol

Animals

Research project grants

Research careers

Humans

Research project grants

Research careers

Cognitive/behavioral/structural deficits in

humans

Research project grants

Research careers

'Percentage of FY97 awards in neuroscience and behavior.

524

Subcommittee Report

At the meeting in May 1998,
experts and NIAAA program staff
made abbreviated presentations of their
material followed by discussion among
all of the participants, including rep-
resentatives from other Institutes of
the National Institutes of Health
(NIH) and guests (see Appendix D).
After completing this process, the
co-chairs and advisory group, with
input from the experts, delineated the
following list of research priorities, in
order of importance:

1. Increase emphasis on mechanisms
associated with various models of neu-
roadaptation (sensitization, tolerance,
dependence, withdrawal, recovery),
at multiple levels of analysis — that is,
molecular, cellular, and whole ani-
mal, including human and nonhu-
man primates.

2. Emphasize new genetic techniques
that have great potential for under-
standing the effects of alcohol on
brain and behavior. Techniques
include transgenics, knockouts,
knockins, antisense oligonucleotides,
tissue-specific expression, and viral-
mediated gene transfer. Enhanced
development and availability of
these genetically altered animals may
have to be subsidized by NIAAA.

3. Increase emphasis on delineating the
cellular and molecular mechanisms
of neuropathophysiology and neuro-
pathology resulting from exposure
to alcohol. This should be accom-
plished in areas of the brain previ-
ously demonstrated to be highly
sensitive to the effects of alcohol, such

as hippocampus, frontal cortex, cere-
bellum, accumbens, hypothalamus,
and ventral tegmental area.

4. Some of the research questions in neu-
roscience and behavior require col-
laborations by experts in a number
of different disciplines. Moreover,
involving the best and most appro-
priate scientists often requires
interinstitution collaboration. Conse-
quently, NIAAA is encouraged to
use appropriate funding mecha-
nisms that will facilitate such col-
laborative studies.

Other areas mentioned, in order of
importance, included:

5. Support multisite, prospective longi-
tudinal studies designed to examine
vulnerability and protective effects of
alcohol on brain and behavior. Spec-
ified individuals should be examined
longitudinally with the techniques
of imaging, electrophysiology, and
neuropsychology. Alcohol, other
drug, and neuroendocrine challenges
should be conducted when ethi-
cally appropriate.

6. Support studies of the effects of alco-
hol on intracellular signaling sys-
tems. This area is underrepresented
in the portfolio, especially given the
recent explosion of information on
intracellular signaling.

7. Increase the number of animal neu-
robehavioral phenotypes studied, such
as animal analogs of impulsive or
disinhibited behaviors and their
interactions with alcohol.

525

NIAAA's Neuroscience and Behavioral Research Portfolio

8. Develop and enhance availability of
new imaging techniques, with consid-
eration of a dedicated imaging center.

These 8 areas of importance were
derived from a list of 22. The remain-
ing 14 are listed by topic without
regard to priority.

General Considerations

• There should be an increased empha-
sis on training in the neurosciences,
especially mentored training.

• Alcohol-related ligands and reagents
should be developed and their
availability, at reduced cost, should
be increased.

• NIAAA should assist investigators
in gaining access to alcohol -related
investigational new drugs.

• An informatics type of database for
alcohol research should be devel-
oped, including information on
molecular, cellular, genetic, behav-
ioral, and pathologic effects of
alcohol. The database would be
designed to provide information in
a format that would be useful to
investigators addressing specific
and complex questions.

• Support for alcohol-related neuro-
endocrine research at all levels of
analysis should be enhanced. Repre-
sentative topics include responses to
stress and alcohol-neuroendocrine-
immune interactions.

• Dose-response relationships are par-
ticularly informative. The effects of
moderate doses of alcohol on the
central nervous system are underin-
vestigated, particularly in animal
models and humans.

Molecular

and Cellular Studies

• Effects of alcohol on lipid-protein
interactions should be emphasized;
effects of alcohol on lipids alone are
of more limited value. Particular
emphasis should be placed on the
identification of proteins with rea-
sonable sensitivity to alcohol that
can readily be studied in the presence
of different lipids.

• A program for the structural analyses
of alcohol-protein interactions,
including molecular modeling as
well as physical measurements,
should be developed. This could
be accomplished by establishing
dedicated groups with equipment
and facilities or by supplementing
existing grants to allow incorpora-
tion of structural initiatives.

Animal Models

• The use of nonhuman primate
models should be increased, includ-
ing studies of the effects of alcohol
on adolescent and aged animals.

• Support for neurobiological studies
of acute and chronic alcohol expo-
sure in adolescent animals should
be increased.

Human Studies

• An alcoholic brain bank should be
established, perhaps by supple-
menting an existing bank. Accurate
information on diagnoses, current
drug use, and concomitant medical
conditions is essential. Antemortem

526

Subcommittee Report

brain structure-function studies
would be valuable.

• Studies of alcohol-related nosology
and concomitant comorbidity
with other psychiatric and brain dis-
orders, including dementia, would
be informative.

• Emphasis on alcohol and sleep
research should be increased.

• Studies of neurobiological factors
associated with relapse are important.

Additional gaps in knowledge and
research opportunities were deter-
mined by experts in each of the areas
covered and are listed in the relevant
sections of this chapter.

OVERVIEW OF THE
NEUROSCIENCE
AND BEHAVIORAL
RESEARCH PROGRAM

Walter A. Hunt, Ph.D.

This overview provides information on the
overall programmatic balance of the port-
folio, with all analyses based on awards
made in fiscal year 1997 (FY97). Spe-
cific aspects of the portfolio will be pre-
sented in other sections of this chapter.

The overall portfolio consists of 121
grants at a total cost of $21,677,142
(see table 1). The balance of neurosci-
ence grants to behavior grants is almost
equal. Most of the research funded is
basic research, with very little of the port-
folio considered applied research.
Those few grants that are applied
research relate to the development of
medications at the preclinical or early
clinical level.

About 80 percent of the grants,
representing 77 percent of the dollar

amount of the awards, used animals;
about 20 percent, representing 23
percent of the dollar amount of the
awards, used humans. About 70 percent
of the grants were in vivo studies, repre-
senting 68 percent of the dollar amount
of the awards. Animal and human stud-
ies represent 62 percent of the number
of grants awarded, with molecular and
cellular studies reflecting the remainder
in approximately equal amounts.

The following list of priorities rep-
resents common themes among vari-
ous initiatives:

• Identify relevant targets of alcohol
in the brain.

• Identify neural circuits underlying
the behavioral effects of alcohol.

• Determine what neurobiological
actions of alcohol are responsible
for its acute and chronic behavioral
effects.

• Determine how the effects of alcohol
on the brain and behavior contribute
to the development of alcoholism.

• Determine neurobiological and behav-
ioral factors that render adolescents
more vulnerable than adults to abuse
alcohol and become alcoholic.

• Develop prototypic compounds for
potential therapeutic development.

The last recommendation evolves
from the need to translate basic
research findings into clinical applica-
tions. To that end, a Medications
Development Working Group was
recently formed to determine if
research findings from basic preclinical
work could provide a basis for devel-
oping potential agents for clinical trials
to treat alcoholism. A short-term goal

527

NIAAA's Neuroscience and Behavioral Research Portfolio

is a workshop to bring together individ-
uals from academia, industry, and gov-
ernment to build bridges among the
groups, review the process by which
compounds are developed into drugs for
clinical trials, and solicit ideas about how
NIAAA can help facilitate this process.

TRAINING AND CAREER
DEVELOPMENT IN
NEUROSCIENCE AND
BEHAVIORAL RESEARCH

Walter A. Hunt, Ph.D.

Training of new investigators is sup-
ported through several mechanisms,
depending on the educational level and
experience of the applicant. Much of
the program is based on individual fel-
lowships and training grants. The
remainder of the program supports
scientist development awards at differ-
ent levels of experience.

Overall Training Program

Forty-six competitive and noncom-
petitive awards were funded in FY97.
Of those, 19 were individual fellow-
ships, 10 were training grants, and
17 were scientist development
awards. Thirty awards supported neu-
roscience research and 16 supported
behavioral research.

Individual Fellowships

The balance between predoctoral and
postdoctoral awards is fairly even, with
a slightly greater number for predoc-
toral awards. Based on NIH policy,
the commitment to individual fellow-
ships relative to training grants should
be at least 15 percent of the available

funds. In FY97, the figure for NIAAA
was 19 percent.

Only one of the awards is for a
potential clinical researcher. Four
involve neurotransmitter receptors,
three use genetic approaches, two
study ontogenetic differences in the
effects of alcohol, three examine the
motivational effects of alcohol, four
are on the central control of alcohol
reinforcement, one is on stress and
neurosteroids, and two are minority
predoctoral fellowships.

Institutional Training Grants

The 10 training grants support
57 slots for 21 predoctoral and 34
postdoctoral trainees. The relative
balance between neuroscience train-
ing slots and behavioral training
slots is almost equal. Three grants
educate trainees in using genetic
approaches to discover the mecha-
nisms underlying actions of alcohol
on the brain, 4 deal with basic
neurochemical mechanisms, 2 with
neurotoxic actions of alcohol, 1 with
brain imaging, and 3 with behavioral
studies. Two training grants primarily
use human subjects.

Scientist Development Awards

Of the 17 awards, most were men-
tored awards, and almost half were to
junior, mentored investigators. All of
the mentored awards support investi-
gators in neuroscience research. Only
two awards support investigators in
clinical research. Most of the investi-
gators study basic actions of alcohol
on neurotransmitter receptors or sig-
nal transduction systems. Two grants
have behavioral components.

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Subcommittee Report

Recruitment

and Future Directions

Recruitment efforts involve staff
contacts with potential applicants
at scientific meetings. In addition, site
visits of NIAAA- funded training grants
were undertaken to introduce trainees
to the grant process and inform
them about the benefits of individual
fellowships and mentored scientist
development awards. These visits
resulted in increased applications but
have been discontinued because of lim-
ited travel funds.

The training program is fairly mature,
with most mechanisms of training ade-
quately represented. However, increased
training of clinical investigators could
provide an additional pool of needed
scientists to pursue alcohol research
with human subjects.

MOLECULAR AND
CELLULAR EFFECTS
OF ALCOHOL

Acute Actions of Alcohol
on Specific Neural Targets

Neurotransmission, Receptors,
Transporters, Modulators,
Ion Channels and Signal
Transduction: Gaps in Knowledge
and Research Opportunities

David M. Lovinger, Ph.D.

• A greater emphasis should be placed
on describing alcohol-induced
alterations in the molecular struc-
ture and dynamics of neural pro-
teins that are sensitive to the
effects of alcohol.

• Analysis of relationships between alco-
hol sensitivity and protein domain
structure should be encouraged in
order to characterize the molecular
sites of alcohol actions.

• Changes in protein function asso-
ciated with exposure to alcohol are
important to delineate.

• It is important to determine alco-
hol effects on synaptic transmission
using techniques that infer a pre- or
post-synaptic locus of effect. Specific
synaptic proteins should be identified.

• A greater emphasis should be placed
on discerning the effects of alcohol
on the neurophysiology of the
ventral tegmental area, amygdala,
hypothalamus, cerebellum, and
prefrontal cortex.

Lipid Involvement in the Acute
Actions of Alcohol in the Nervous
System: Gaps in Knowledge and
Research Opportunities

Steven N. Treistman, Ph.D.

• Increased emphasis on protein
function as the measure of lipid
perturbations will be informative.

• The function of a particular protein
should be well characterized before
attempts are made to assess pertur-
bations of function by alcohol. Ideally,
this should include information in
native membranes as well as in arti-
ficial bilayers.

• Reasonable (physiological) concen-
trations of alcohol should be used
in determining its effects.

• Studies should be integrated across
multiple levels of analysis: (a) a target
protein of known behavioral or physio-
logical relevance to alcohol action

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NIAAA's Neuroscience and Behavioral Research Portfolio

should be selected; (b) effects of lipid
perturbation and possibly modulation
of lipid composition on the protein in
its native environments should be
determined; (c) the target protein
should be capable of being reconsti-
tuted into a simplified lipid environ-
ment for examining alcohol effects;
and (d) a cloned protein should be
selected to permit expression studies
in a variety of native membranes and
to enable mutagenesis studies.
• The study of the significant role of
lipids in the compensatory responses
of cells exposed to alcohol should
be continued.

NIAAA Basic Neuroscience
Research Portfolio

Yuan Liu, Ph.D.

In FY97, the NIAAA Basic Neuroscience
Research Program funded 35 extramural
grants for a total of $5.5 million. Of
these, 30 were basic research project
grants, and 5 were research careers
awards (see table 2).

This program supports a wide variety
of investigations that explore different
effects of alcohol on the brain at multi-
ple levels of analysis and use a number
of different neuroscience techniques.
Several different molecular targets are

being examined, as are different mech-
anisms hypothesized to underlie the
actions of alcohol in the brain. Tables 3,
4, and 5 show the distribution of grants
by level of analysis, molecular target, and
brain region, respectively. In table 3,
the categories are not mutually exclu-
sive: some investigators are studying the
effects of alcohol at multiple levels. Table
4 shows that identifying the molecular
targets and elucidating the mechanisms
underlying the interactions between alco-
hol and these targets has been the research
focus of more than half of the basic neu-
roscience research portfolio. As shown in
table 5, identifying the cellular targets of
alcohol and their distribution in the brain
is another major focus of research.

More than half of the total awards in
the portfolio examine the acute effects
of alcohol on the brain, about one -third
study the chronic effects, and the remain-
der investigate both short- and long-term
effects. Only a few research projects study
directly the mechanisms underlying
various interactions phases of alcohol-
related problems, including tolerance and
withdrawal; the majority of projects study
the direct action between alcohol and
candidate targets. About 80 percent of
investigators use multidisciplinary
approaches, primarily a combination of

Table 3. NIAAA Basic

Neuroscience Grant Distribution

Level

of Analysis.

Level of Analysis

Number

% of Total

Molecular

Cellular

Neural circuitry

Miscellaneous

Total

100

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Subcommittee Report

electrophysiological and molecular bio-
logical techniques. Nearly 80 percent of
the research teams use in vitro prepa-
rations. Among the projects studying
molecular targets of alcohol, about 80
percent of the experiments use recom-
binant receptor-channel proteins.

Research Areas

Molecular Level (18 grants)

Alcohol, unlike most other highly
abused substances, does not act on a
single specific target in the brain. Instead,
it interacts with many targets, such as
neurotransmitter receptors and voltage -
gated ion channels on nerve cells. A
major challenge confronting molecular
studies is the lack of uniform effects of
alcohol on various cell types in different

regions in the brain. Several complemen-
tary working hypotheses address the
cellular and regional specificity of alco-
hol. The first postulates that alcohol
directly interacts with specific amino
acid residues or domains of the target
proteins. The second proposes that
expression of particular receptor sub-
unit combinations contribute to the
different degrees of alcohol sensitivity
in different neurons. A more recent
hypothesis suggests that posttransla-
tional modifications, such as the phos-
phorylation status of a target protein,
can determine sensitivity to alcohol.

Direct Molecular Action Sites (8
grants). The majority of research pro-
jects within this category are attempting
to identify the sites of alcohol action
on target molecules, including voltage-

Table 4. NIAAA Basic Neuroscience Grant Distribution

Molecular Targets

of Alcohol.

Molecular Target

Number

% of Total

Acetylcholine receptors

22.2

GABAA receptors

22.2

NMDA receptors

27.8

Glycine receptor

5.6

5-HT3 receptor

5.6

CA++ channels

11.1

K+ channels

5.6

Total

100.0

Table 5. NIAAA Basic Neuroscience Grant Distribution by

Studies of Brain

Regions.

Brain Region

Number

% of Total

Amygdala

2.8

Cortex

5.7

Cerebellum

5.7

Hippocampus

34.3

Mesolimbic system

22.8

> 4 Brain regions

20.0

N/A

37.1

Total

128.5

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NIAAA's Neuroscience and Behavioral Research Portfolio

gated Ca++ and K+ channels and
acetylcholine, gamma-aminobutyric
acid type A (GABAA), N-methyl-D-
aspartate (NMDA), and glycine recep-
tors. One working hypothesis is that
alcohol alters channel kinetics, such as
channel open time, channel closing time,
and desensitization status of molecular
targets. Other investigators study protein
targets in their native environments. The
majority use a combination of electro-
physiological and molecular biological
approaches to test alcohol effects on
recombinant proteins expressed in
Xenopus oocytes or transfected mam-
malian cell lines. Two research groups
introduced a state-of-the-art approach —
the chimeric receptor technique — to
alcohol research. Chimeric receptors
consist of complementary parts of two
target proteins that give opposite
responses to alcohol. By using a well-
designed set of chimeras, the process
of locating the sites of action of alcohol
can be significantly accelerated.

There has been a long debate over
the question of whether lipid, protein,
or the interface between them is the site
of action of alcohol. Although increasing
evidence suggests that membrane pro-
teins are the direct targets, the role of
the lipid environment in mediating
effects of alcohol is likely to be impor-
tant. Two projects are attempting to
further clarify this challenging and
confusing issue. Both projects use
preparations that incorporate well-
characterized protein candidates into
a lipid bilayer. In this environment,
each lipid component is known and
can be individually manipulated.

Subunit Composition (8 grants).
Both voltage-gated and ligand-gated

channel proteins are composed of
multiple subunits. The putative mole-
cular structure of the superfamily of
ligand-gated channels/neurotransmit-
ter receptors is a pentamer consisting
of five identical or distinct subunits. It
is evident that the number of possible
combinations of subunits is quite
large. In addition, an individual neu-
ron can express multiple types of a
given receptor, based on the combina-
tion of different subunits. When con-
sidering the many different regions in
the brain, the patterns of expression of
those subunit compositions can be
exceedingly complex.

Currently, eight research groups
supported by this program are testing
the "subunit composition" hypothesis.
The two major approaches that are
investigating the structure-function
relationship use either (a) recombinant
proteins produced in various expres-
sion systems or (b) natural proteins
expressed in intact neurons. Several
research groups are taking advantage of
some of the newest pharmacological
tools. The binding selectivity to subunits
of these agonists or antagonists appears
to correlate with alcohol specificity of
certain subunits. Other investigators
are using different approaches, such as
specific antibodies against the candidate
subunits. A novel technique — the single
cell reverse transcription-polymerase
chain reaction (RT-PCR) method —
has recently been introduced to alcohol
research. This sophisticated method,
when properly used, can identify the
profile of the subunits at the messenger
RNA level in an identified cell.

Protein Modifications (2 grants).
Previous observations suggest that

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Subcommittee Report

phosphorylation/dephosphorylation
status of some target proteins, such as
GABAA, NMDA, and 5-hydroxytrypt-
amine type 3 (5-HT3) receptors,
might be important for understanding
the interactions between alcohol and
these targets. Two research projects are
exploring this hypothesis by applying
a combination of electrophysiological,
biochemical, and molecular biological
techniques on recombinant receptors.

Cellular Level (7 grants)

Two projects are examining the
effects of alcohol at the single cell
level. One project is using the brain
slice preparation and electrophysio-
logical recording techniques to study
the acute effects of alcohol. A second
study is using both brain slice and in
vivo preparations to explore the
chronic effects of alcohol on the spon-
taneous activity of dopaminergic cells
in the ventral tegmental area.

Most projects are focused at the
synaptic level. For example, one inves-
tigator is using an in vitro NMDA
synapse model to identify cellular
mechanisms underlying the develop-
ment of acute tolerance to alcohol.
Another investigator is using both tra-
ditional and organotypic slice prepara-
tions to explore the alteration of
voltage-gated Ca++ channels following
acute and chronic alcohol exposure.

Several projects are investigating
the effects of alcohol on synaptic plas-
ticity. One project is testing the
hypothesis that subcortical inputs to
the dentate gyrus mediate the acute
actions of alcohol on both short- and
long-term plasticity in hippocampus.
Another project is exploring a possi-

ble role for the amygdala in mediating
the acute effects of alcohol on synap-
tic plasticity. One study uses hip-
pocampal activity-related spatial
memory as a model system to test the
effects of alcohol on different age
groups of animals.

Neural Circuitry Level (3 grants)

Research at the neural circuitry level
has not yet been intensively pursued.
Investigators supported by this pro-
gram have recently developed several
novel methods that are further exten-
sions of the traditional extracellular
single-unit recording techniques. One
uses a multielectrode, single-unit
recording apparatus that can perform
simultaneous recordings of neuronal
activities from different areas of a
defined neural circuit during a specific
behavioral paradigm. This allows the
analysis of spatial/temporal pattern
changes of neuronal firing related to
alcohol-induced behaviors. Another
new method combines in vivo micro-
dialysis with electrophysiological and
behavioral techniques. This provides a
means of locally delivering alcohol
and other pharmacological agents to
individual neurons, simultaneously
recording neuronal activity, and collect-
ing neurotransmitters released from
the same neuron in real time, during
ongoing behaviors.

Recommendations

Molecular Level

Direct Molecular Action Sites

• Emphasize less-studied molecular
targets (e.g., serotonin receptors,

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NIAAA's Neuroscience and Behavioral Research Portfolio

peptide neurotransmitters, neuro-
transmitter transporters).

• Identify action sites of alcohol at
the level of single amino acids.

• Examine channel kinetics of target
proteins at the single-channel level.

• Study well-characterized protein
targets in a known lipid environment.

Subunit Composition

• Emphasize studies of natural receptors
in intact neurons, rather than focusing
specifically on recombinant receptors.

• Identify subunit composition pro-
files by single-cell RT-PCR and by
subunit-specific antibodies.

• Characterize the role of subunit
composition as a mediator of alco-
hol's effects using antisense tech-
niques and inducible knockout,
tissue-specific knockout techniques.

Protein Modifications

• Examine a larger variety of protein
kinase pathways.

• Investigate the role of protein
phosphorylation and alcohol inter-
actions, using phosphorylation,
state -specific antibodies, and genet-
ically engineered animals.

• Explore mechanisms other than
phosphorylation modifications.

Cellular Level

• Investigate mechanisms of alcohol's
actions on neuronal firing rate at
molecular and cellular levels.

• Determine the effects of alcohol
on synaptic transmission (e.g.,
neurotransmitter release stat-
istics and quantal efficiency, presy-
naptic modification, postsynaptic
regulation).

• Characterize the effects of alcohol
at integrated levels (e.g., interac-
tions between excitatory and
inhibitory receptors at single-cell
and pathway levels).

• Study the effects of alcohol on synap-
tic plasticity, using methods such as
minimal stimulation, stimulation-
induced miniature potential, and
quantal analysis.

• Identify effects of alcohol on
synaptic plasticity at locations
other than hippocampal NMDA-
dependent long-term potentiation
(LTP) (e.g., non-NMDA-depen-
dent hippocampal LTP, cerebellar
long-term depression [LTD]
and LTP, and synaptic plasticity
in other areas of the brain, includ-
ing the mesolimbic system,
basal ganglia, thalamic relays,
and neocortex).

Neural Circuitry Level

• Use single- or multiple single-unit
recordings in behaving animals to
associate effects of alcohol directly
to behavior.

• Use combinations of neurochemical
(e.g., iontophoresis, microdialysis, and
voltammetry) and electrophysiological
recordings in behaving animals to
associate neurochemical and neuro-
physiological events with alcohol-
induced behaviors.

• Encourage the use of sophisticated
optical recording techniques
and voltage-sensitive and CA++
sensitive dyes in brain slice prepara-
tions to study the activity of large
neural networks under the influence
of alcohol, as well as studies of intrin-
sic optical activity using the newest

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Subcommittee Report

computer algorithms coupled with
modern photorecording techniques.
Use computational modeling meth-
ods in combination with physiolog-
ical and behavioral empirical
studies to explore how neural cir-
cuitry synchronizes functions dur-
ing alcohol-induced behaviors.

Effects of Alcohol on the
Neuroendocrine System: Gaps
in Knowledge and Research
Opportunities

Catherine Rivier, Ph.D.

Despite the involvement of
corticotropin- releasing factor (CRF) in
many alcohol-related disorders, and
the fact that this peptide appears to be
a reinforcer of drug abuse, the num-
ber of studies supported by NIAAA
that investigate its synthesis, release,
and effects on endocrine function in
general, and the hypothalamic-pitu-
itary-adrenal (HPA) axis in particular,
is quite low.

• A greater emphasis should be placed
on studies investigating the func-
tional interactions between CRF
and neurotransmitters involved in
drug- seeking behavior, reinforce-
ment, and relapse.

• Exposure of adult mice with a con-
ditional null mutation for the CRF
or CRF receptor gene to alcohol
would provide valuable informa-
tion.

• Exposure of pregnant dams lacking
the CRF or CRF-R1 gene to
alcohol could provide useful infor-
mation on the endocrine, behav-

ioral, autonomic, and immune
pathologies observed in fetal
alcohol syndrome.
Studies of the functional interac-
tions between alcohol and nitric
oxide/carbon monoxide, and
between alcohol and nuclear
regulatory factor-KB (NF-kB),
are important.

A good model of isolated cells (either
primary culture or immortalized
cells) that produce CRF is needed.
The development of potent CRF
antagonists that are long-lasting
and receptor specific should be
encouraged.

NIH-distributed reagents for the
measurement of plasma adrenocor-
ticotropic hormone (ACTH) and
corticosterone levels in rodents are
urgently needed.

It would be useful to have access to
different strains of alcohol -prefer-
ring rats and mice.

Alcohol and the
Neuroendocrine Portfolio

Samir Zakhari, Ph.D.

In FY97, the neuroendocrine port-
folio consisted of 14 grants totaling
$2.1 million. Except for 1 human
study, all of the grants study alcohol
effects on the endocrine system using
experimental animals. Five of the 14
grants focus on the HPA axis, 5 on
the hypothalamic-pituitary-gonadal
(HPG) axis, 2 on the endocrine
regulation of immune function, 1 on
hormonal regulation of alcohol
metabolism, and 1 on enkephalin
gene expression.

535

NIAAA's Neuroscience and Behavioral Research Portfolio

Twelve of the 14 grants are catego-
rized as basic research; the other 2 are
considered applied research. Two grants
focus on response to stress, and all 14
grants study alcohol at the cellular and
molecular level.

Research Areas

HPA Axis (5 grants)

Alcohol and Stress: Interactive
Effects. Prenatal alcohol exposure
elicited HPA axis hyperresponsiveness
and compromised immune integrity in
adult male and female rats.

AlcoholTnterleukin Interactions on
the HPA Axis. Prenatal exposure to
alcohol perturbed HPA responsiveness to
interleukin-l(3 (IL-1(3) by blunting the
ACTH response in immature (3 week)
male and female rats, but conversely pro-
duced a potentiating effect when these
same animals reached adulthood.

Alcohol Effects on Opiomelano-
cortinergic Regulation. Under non-
stressful conditions, moderately high
blood alcohol levels temporarily activated
the HPA axis, with concomitant activa-
tion of the forebrain opiomelanocortin-
ergic neuronal system.

HPA Axis and Alcoholism. The
HPA dynamics was different in nonal-
coholic people with a family history of
alcoholism (FHP) than in nonalcoholic
subjects without a family history of
alcoholism (FHN).

Alcohol and Neuroendocrine Func-
tion: Oxytocin Expression. Alcohol
inhibited the secretion of oxytocin, which
may play a role in the development of
tolerance to alcohol. Oxytocin secretion
was reduced during acute intoxication,
but not during alcohol withdrawal.

Chronic alcohol administration inhib-
ited oxytocin secretion in male rats
but not in female rats.

HPG Axis (5 grants)

Pubertal Alcohol and Male Repro-
duction. Acute alcohol administration
to 35-day-old (prepubertal), 45-day-
old (midpubertal), or 55-day-old (late
pubertal) male rats caused depression
of testosterone and luteinizing hormone
(LH) levels in the two older groups;
coadministered naltrexone reversed the
testosterone but not the LH depres-
sion. Chronic alcohol administration
to male rats (45 and 55 days old) that
were subcutaneously implanted with a
pellet of naltrexone 2 days before
being offered a liquid diet containing
alcohol (36 percent of total calories)
for 14 days showed similar effects.

Alcohol Testicular Effects. Alcohol
decreased testosterone secretion and
testicular interstitial fluid formation in
rats. This effect was not mediated by
endogenous opioids or nitric oxide.

Alcohol and Female Rodent Repro-
duction. A single dose of alcohol
given to female rats nearly obliterated
serum proestrum LH levels, and
serum estradiol and progesterone lev-
els fell to half of the control values.

Neuroendocrine Effects of Alcohol
on Puberty. In vivo and in vitro inves-
tigations have demonstrated that insulin-
like growth factor- 1 (IGF-1) is crucial to
the onset of puberty in females, and
that alcohol impedes the physiological
responses of the brain and ovary to IGF-
1. Alcohol-induced depression of LH
release arises from a decrease in pro-
staglandin E2 formation. Leptin also
induces the prepubertal release of LH;

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Subcommittee Report

peripherally administered leptin reverses
the depressed LH secretion by alcohol.
Alcohol also perturbs the nitric oxide/
nitric oxide synthase system at specific
phases of the reproductive cycle dur-
ing puberty, consistent with a role in
ovulation and luteal formation.

Alcohol and Hyperprolactinemia.
The human disorder gynecomastia,
observed in some alcoholic men, which
arises from elevated plasma levels of
the pituitary prolactin hormone (PRL),
is being studied in a rat model. Stud-
ies focus on elucidating the cellular
mechanisms (presumptively involving
inhibition of transforming growth fac-
tor beta 1 [TGF-(31] in the pituitary)
of hyperprolactinemia.

Endocrine Regulation of Immune
Function (Igrants)

Immunosuppression in a Binge-Drink-
ing Model. A single high dose of alcohol
in the mouse produced peak corticos-
terone levels reaching 10 times basal
levels. Humoral immune function was
suppressed, expression of IL-1(3, inter-
leukin-2, and interleukin-4 were com-
promised in the spleen, and resident
B-cell population was reduced. RU 486
(a glucocorticoid antagonist) reversed
the suppressed antibody and cytokine
responses. Exogenous administration
of corticosterone mimicked some, but
not all, of the effects produced by a high
dose of alcohol, suggesting that addi-
tional components are contributing to
the immunosuppression produced by
high-dose alcohol administration.

Natural Killer (NK) Cells and Binge
Drinking. Alcohol suppresses basal and
induced NK cell activity. The sup-
pressed NK activity arises pardy via a

glucocorticoid- based mechanism and
partly via the perturbation of the bal-
ance between Thl and Th2 cells.

Endocrine Regulation of Alcohol
Metabolism (1 grant)

Dihydrotestosterone (DHT) suppresses
alcohol dehydrogenase (ADH) transcrip-
tion in hepatocytes, possibly explaining
the higher alcohol elimination rates in
women. The suppression of rat liver
ADH activity by DHT was associated
with a decrease in ADH protein.

Effects of Alcohol on Enkephalin Gene
Expression (1 grant)

The ultimate goal of this grant is to study
the role of enkephalins in alcohol-seeking
behavior; the immediate focus is on
the mechanism by which members of
the steroid-retinoid receptor super-
family modulate expression of the pre-
proenkephalin gene.

Recommendations

More research is encouraged in the fol-
lowing areas: (1) alcohol and neuroim-
munomodulation, (2) role of peptides
in alcohol intake, (3) alcohol and the
hypothalamic-pituitary-thyroid axis, and
(4) alcohol and growth hormone.

Molecular and Cellular
Adaptive Responses to
Chronic Alcohol Exposure

Neuroadaptation: Gaps in Knowledge
and Research Opportunities

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

• It is important to establish the rela-
tionship between observed changes

537

NIAAA's Neuroscience and Behavioral Research Portfolio

and neurochemical and molecular
changes after chronic alcohol treatment
and the occurrence of neuroadaptive
events such as tolerance and depen-
dence. Studies are needed that are
interdisciplinary and translational —
studies at the molecular and cellular,
brain slice, and whole animal levels,
as well as human studies. Examples
include (1) studies of synaptic neuro-
physiology and neural circuitry after
chronic alcohol exposure, using tech-
niques to separate pre- and postsynap-
tic changes; use of models such as
slices and explants, with intact neuro-
physiology, and use of animal models
to observe neuronal ensemble activity;
(2) studies of molecular mechanisms
of changes in receptors/ion channels
after chronic alcohol treatment, includ-
ing subunit composition, posttrans-
lational modification, and receptor
localization; (3) studies of functional
importance of changes in gene expres-
sion for neuroadaptation, with a focus
on gene expression in neural circuits
that affect pharmacological responses
to alcohol; and (4) studies of the inter-
connections among various signal
transduction systems that influence cell
survival, differentiation, and responses
to stress and other external stimuli,
and the importance of these systems
for neuroadaptation to alcohol.

• It is necessary to determine the rela-
tionship between tolerance or sensi-
tization to the reinforcing/aversive
effects of alcohol and alcohol intake.
A corollary is whether tolerance or
sensitization to the reinforcing effects
of alcohol does, in fact, develop.

• Increased emphasis should be
placed on the application of simple

(e.g., invertebrate) models that
have been used to study learning
and memory, to the study of alcohol-
induced neuroadaptation (toler-
ance and dependence).

• Neuroadaptation after moderate drink-
ing (events taking place in the brain
during the transition from moderate to
abusive drinking) should be explored.
Biochemical and behavioral studies of
the effects of the full range of alco-
hol doses and concentrations should
be carried out, to determine thresh-
olds for neuroadaptive effects of
alcohol, and J- or U- shaped dose-
response curves.

• Tools are needed to study neu-
roadaptation, including genetic
models such as transgenics, knock-
outs, selected lines, recombinant
inbreds, and congenics, in order
to test hypotheses of mechanisms
of neuroadaptation and identify
genes involved in these processes.
Animal models should also
include nonhuman primates for
behavioral studies.

• Emphasis should be placed on
application of discoveries regarding
molecular and neurochemical
mechanisms of neuroadaptation to
alcohol to the treatment and inter-
vention arena.

Neurotoxicity: Gaps in Knowledge
and Research Opportunities

Fulton T. Crews, Ph.D.

• It is important to determine the
region-specific neurotoxic effects
accompanying extended alcohol
exposure as well as those accompa-
nying withdrawal.

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Subcommittee Report

Although the loss of white matter
has been documented to accom-
pany chronic alcohol exposure,
there has been relatively little
research into mechanisms.
It is recommended that a human
alcoholic brain bank be established
in the United States.
Although women represent approx-
imately 25 percent of alcoholics
and may suffer greater pathology,
they have been understudied.
Moreover, data regarding the role
of gender might provide important
fundamental insights into mecha-
nisms of brain damage.
A significant gap in knowledge
is the relationship between
acute excessive stimulation of neu-
rons by glutamate and delayed
neuronal death.

Why specific brain regions are
particularly sensitive to chronic
alcohol exposure is an important
question that needs to be
answered.

Oxidative stress is increased
in brain by alcohol and is often
postulated to contribute to brain
damage. There are, however,
relatively few data for or against
this hypothesis.

An important area of research is
to determine the role alcoholic
brain damage plays in the progres-
sion to alcoholism, recovery from
alcoholism, and other behaviors
associated with alcoholism.
Understanding the relationship
of neuropathology to behavioral
pathology is essential and funda-
mental to improving prevention
and treatment.

Molecular Neuropharmacology
Portfolio

Robert W. Karp, Ph.D.

To identify the primary effects of
chronic alcohol exposure, investiga-
tors studying neuroadaptation and
neurotoxicity have examined alcohol-
induced molecular changes in cellular
components. Some of these studies can
be performed in intact animals, thereby
permitting correlation with alcohol-
induced changes in systemic neural
function and behavior. Other studies can
be performed only in cultured cells.
Although studies in cultured cells pro-
vide information about some molecu-
lar processes that cannot be assayed in
intact animals, it is often difficult to
evaluate their behavioral significance.
Moreover, for some measures that can
be made in both cultured cells and
whole animals, different changes are
observed in these two experimental sys-
tems. For this reason, many investigators
try to confirm cellular observations in
intact animals whenever possible.

Research Areas

Table 6 shows the distribution of
molecular neuropharmacology awards
by research area.

Withdrawal

NIAAA supports a number of studies,
most of them in intact animals, of
molecular changes associated with
withdrawal from chronic alcohol
exposure. The largest group of these
studies is directed at GABAA receptors,
focusing on changes in brain -regional
distribution and pharmacological
properties (including interactions with

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NIAAA's Neuroscience and Behavioral Research Portfolio

neurosteroids), and the underlying
causes of such changes. The second
largest group of studies focuses on
similar issues related to NMD A recep-
tors. Other studies examine 5-HT
receptors and their downstream sig-
naling (especially their relationship to
anxiety during withdrawal), non-
NMDA glutamate receptors, Fos-like
immunoreactivity, mechanism of up-
regulation of voltage -gated Ca++ chan-
nels, and free radical accumulation
due to oxidative stress. Of the 11 pro-
jects in this category, 4 include studies
on cultured cells.

Tolerance

NIAAA supports studies of a variety
of molecular changes induced by
chronic alcohol exposure in various
types of neuronal cell lines. Although
the precise relationships of these changes
to alcohol-induced physiological changes
in animals are not yet understood,
they could potentially be related to
tolerance or dependence. These studies
include heterologous desensitization
of signaling from purine receptors,
intracellular translocation of protein

kinase A, global changes in gene
expression, and the normal physiolog-
ical role of a phosducin-like protein,
whose levels are raised in response to
chronic alcohol exposure.

Neurotoxicity

Most of the studies of molecular
changes associated with neurotoxicity
involve a combination of experiments
on both intact animals and culture
cells. The largest group of such studies
focuses on alcohol- induced changes in
brain-regional distribution of neuro-
trophic factors and their receptors, the
role of neurotrophic factors in protec-
tion from alcohol-induced disruption
of calcium homeostasis, and the mech-
anism of enhancement of neuro-
trophin-induced neurite outgrowth by
alcohol. Another group of studies is
directed at NMDA receptors, focusing
on changes in brain-regional distribu-
tion and pharmacological properties,
underlying causes of such changes,
and changes in downstream signaling
(especially induction of nitric oxide syn-
thase). Two studies are concerned with
alcohol's enhancement of pro-oxidant-

Table 6. NIAAA FY97 Awards

in Molecular Neuropharmacology.

Study Area

Number

Dollar Amount

Withdrawal

1,694,000

GABA

871,000

NMDA

283,000

Other

540,000

Tolerance

1,351,000

Neurotoxicity

1,761,000

Neurotrophins

706,000

NMDA

532,000

Oxidative Stress

356,000

Other

167,000

Total

4,806,000

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Subcommittee Report

induced membrane lipid peroxidation
and changes in gene expression. Finally,
individual projects study, variously, the
mechanisms of alcohol-induced changes
in the levels and/or activities of the
Na-Ca exchanger and metabotropic
glutamate receptors.

Recommendations

The projects described above study
molecular changes induced by alcohol
in intact animals and/or cultured
cells. Alcohol-induced molecular
changes can be correlated with behav-
ioral and physiological changes in ani-
mals, whereas molecular changes in
cultured cells cannot be directly corre-
lated. Investigators typically hypothe-
size that the molecular changes they
observe actually mediate particular
behavioral and physiological changes.
Since chronic alcohol exposure induces
a multitude of molecular, physiological,
and behavioral changes, correlative
evidence is insufficient to prove that a
particular molecular change mediates
a particular physiological or behavioral
change. Emphasis should be placed on
experiments in which an investigator
specifically blocks either the alcohol-
induced molecular change under
study, or the function of the molecule
whose disposition is changed by alco-
hol, and then observes whether this
intervention also blocks the behavioral
change under study. Alternatively, the
investigator can induce the molecular
change by some means other than alco-
hol treatment and observe whether
the behavioral or physiological change
still occurs. Only about one-fifth of
the projects described above attempt
to perform such an intervention.

ADDICTION AND OTHER
BEHAVIORS IN ANIMAL
MODELS

Basic Behavioral Effects and
Underlying Neurocircuitries
of Alcohol: Gaps in Knowledge
and Research Opportunities

Kathleen A. Grant, Ph.D.

• The initiation of alcohol seeking
needs additional exploration with
new paradigms using animal models.
There is also a lack of studies inves-
tigating the possibility of protecting
the individual from the develop-
ment of alcohol-seeking behavior.

• Additional studies are needed to
characterize the interrelationships
among oral self-administration of
alcohol and preference condition-
ing studies.

• Studies are needed to understand
the apparent differences between
rats and mice in sensitivity to alco-
hol's rewarding effects in the place
conditioning paradigm.

• Sophisticated neuroscientific
procedures should be combined
with sophisticated behavioral
procedures.

• As candidate genes become identified
and gene products known, there will
be a need to have ligand development
to target potential sites of action in
rats, monkeys, and humans.

• The use of monkey models for non-
invasive imaging procedures related
to alcohol abuse should be increased.
Monkeys are important models
because of their compliance in drink-
ing excessive quantities of alcohol,

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NIAAA's Neuroscience and Behavioral Research Portfolio

neuroanatomy, brain size, complex
behavior, neuroendocrine system,
and longevity.

• Cloning techniques of nuclear trans-
plantation from adult monkey cells
should at least be given thoughtful
consideration. Such approaches
could be used to address the
genetic basis of complex behavioral
responses associated with the
development of alcohol abuse and
alcoholism.

• Gender differences in the behav-
ioral neuroscience of alcohol using
animal models are understudied.

• Understanding the role of stress in
alcohol's behavioral effects requires
more sophisticated approaches.

• Depression is a risk factor that ani-
mal models in alcohol abuse have
not addressed extensively.

• The age at which individuals start
regular, heavy use of alcohol has
recently been reported to predict
the occurrence of alcohol depen-
dence. The macaque monkey has at
least a 12-month adolescent phase,
which allows a window of opportu-
nity to design appropriate experi-
mental manipulations.

• The concurrent use of alcohol and
other drugs of abuse has received
limited attention.

Neuroadaptive Changes in
Neurotransmitter Systems
Mediating Alcohol-Induced
Behaviors: Gaps in Knowledge
and Research Opportunities

Friedbert Weiss, Ph.D.

• A systematic research effort at the
behavioral, neurochemical, cellular,

and molecular levels will be needed
to identify and characterize neu-
roadaptive changes and homeosta-
tic disturbances during protracted
withdrawal, and to determine their
motivational significance in appro-
priate models of alcohol-seeking
behavior and relapse. .

• It is essential to develop new, or
modify existing, animal models of
self- administration suitable for the
longitudinal monitoring of neu-
roadaptive or homeostatic changes
during the development of chronic
alcohol drinking and over the
course of withdrawal and interven-
ing periods of abstinence.

• Effective procedures or models are
needed that permit investigation of
behavioral plasticity such as the
development of associations
between alcohol's subjective
rewarding effects and relevant envi-
ronmental stimuli that may trigger
relapse during abstinence.

• It is important to examine the role of
stressful stimuli on the reinstatement
of alcohol-seeking behavior at differ-
ent stages of the protracted abstinence
phase and to determine whether
vulnerability to relapse becomes
exacerbated with repeated with-
drawal and abstinence episodes.

• Research strategies need to be devel-
oped to identify neural or molecu-
lar mechanisms mediating the
switch or transition from nonde-
pendent social drinking to a state
of dependence.

• There is a need to better model
various aspects of alcoholism in
laboratory animals. This includes,
in particular, voluntary drinking

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Subcommittee Report

models that promote spontaneous
and persistent intake of high
alcohol concentrations or volumes
without prior need to induce
dependence. Such models will
represent an important step toward
the need for studying critical
issues such as the mechanisms
underlying the switch from nonde-
pendence to dependence, and
the reinforcement contingencies
that maintain alcohol consumption
in dependent individuals.
There is a need to study neuro-
transmitter circuitries and interac-
tions mediating alcohol reward
in the dependent and postdepen-
dent state. Although there is
increasing evidence that the acute
reinforcing actions of alcohol
depend on multiple neurochemical
systems and their interactions, lit-
tle if anything is known about
these mechanisms in dependent
individuals. In this context, it will
also be beneficial to incorporate
multiple systems approaches in
medication development efforts
and to examine the therapeutic
efficacy of combinations of rele-
vant pharmacological agents.
It is important to clarify the role of
dopamine in alcohol reinforcement.
This need involves both a better
understanding of mechanistic ques-
tions such as, for example, how alco-
hol activates mesolimbic dopamine
transmission and a better under-
standing of the precise role of
dopamine in various aspects of
alcohol-seeking behavior.
An important emerging issue is the
role of sensitization in alcohol

reinforcement, genetic preference,
and dependence. In particular, the
following questions will require
clarification: (1) does alcohol sen-
sitization augment the reinforcing
efficacy or potency of alcohol; (2)
does alcohol sensitization promote
a heightened motivational state
with increased alcohol-seeking
behavior ("craving") without nec-
essarily altering the reinforcing effi-
cacy of alcohol; (3) is alcohol
sensitization a correlate of aversive
or side effects of repeated alcohol
intoxication; and (4) if so, is alco-
hol sensitization negatively linked
with alcohol preference or vulnera-
bility to abuse?

It is important to better under-
stand the mechanisms of kindling
or sensitization of withdrawal
seizures at the molecular, cellular,
and biochemical levels. This
includes efforts to define sensitiza-
tion of psychological components
of withdrawal (e.g., anxiety, affec-
tive changes), to characterize pos-
sible changes in the subjective
perception of alcohol's intoxicating
actions (i.e., alcohol's discrimina-
tive stimulus effects), to determine
whether multiple alcohol with-
drawal experiences alter the rein-
forcing properties of alcohol, and
to examine potential changes in
susceptibility to alcohol neurotoxi-
city and associated cognitive
impairments. Finally, it is impor-
tant to examine whether condition-
ing factors contribute to the
kindling phenomenon.
The field would benefit from the
development of genetic models of

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NIAAA's Neuroscience and Behavioral Research Portfolio

alcohol-seeking behavior selected
on the basis of motivational
measures such as operant respond-
ing or even progressive-ratio per-
formance during dependence.

Adolescent Period: Gaps in
Knowledge and Research
Opportunities

Linda P. Spear, Ph.D.

• Further research is needed to exam-
ine how sensitivity to various effects
of alcohol differs in adolescents from
other aged organisms, and to explore
the neural mechanisms underlying
these ontogenetic effects.

• Research to examine the reinforc-
ing efficacy of alcohol and other
reinforcers during adolescence is
particularly critical, given evidence
for alterations during adolescence
in forebrain regions modulating
the reward efficacy of reinforcing
drugs.

• Stress effects on alcohol self- admin-
istration during adolescence should
be examined, along with funda-
mental research examining the rela-
tionships among hormonal,
behavioral, and neural responses to
stressors throughout ontogeny.

• In addition to further research
examining normal brain function in
adolescence, research is needed to
determine factors that trigger onto-
genetic changes in brain function,
alcohol sensitivity, and responsivity
to stressors during the adolescent
period.

• It is critical to determine whether
early exposure to alcohol increases
the likelihood for later alcohol
problems, and if so, why such early
exposure should be so predictive.

Portfolio of Studies of the
Addiction Process and Other
Behaviors in Animal Models

Ellen D. Witt, Ph.D.

Animal studies, representing about $6
million, are focused largely on neural
mechanisms of sensitization, tolerance,
dependence, withdrawal, relapse, and
more recently the reinforcing and
hedonic effects of alcohol that can lead
to alcohol-seeking behavior and exces-
sive drinking. Animal models have
also been constructed to study innate
neural and behavioral traits as well as
the acute and chronic effects of alcohol
on behaviors such as learning, memory,
and aggression.

Table 7. NIAAA FY97 Animal Portfolio.

Category

Number

Dollar Amount

Basic behavioral models/nondependent

3,850,412

Neuroadaptive models/dependence

630,790

Basic behavioral and dependence models

798,483

Adolescent models

520,924

Methodology development

182,256

Total

5,982,865

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Subcommittee Report

The animal portfolio has been
divided conceptually into five broad
scientific categories, as shown in table
7. Each broad category is further sub-
divided to provide a more detailed
picture of the number and types of
animal models being studied. This
breakdown is based on the specific
aims of each grant. Since a single
grant may be investigating more than
one paradigm or neurotransmitter sys-
tem, a grant could be counted more
than one time in this analysis. There-
fore, the number of grants (not dol-
lars) is indicated for each area of study
described in the text below.

Basic Behavioral Effects and
Underlying Neural Circuitry With
Limited Alcohol Exposure (Low to
Moderate/Nondependent )

Research Areas

Alcohol-Seeking Behavior/Hedonic
Effects, Neural Circuitry,
Neurotransmitters

Self- Administration: Operant Rein-
forcement, Two-Bottle Choice (7
grants). These grants are investigating
the neural circuitry and neurotrans-
mitter pathways underlying excessive
alcohol drinking using either operant
self- administration paradigms or two-
bottle choice. All seven grants use
microinjection and/or microdialysis
techniques to look at the regulation of
oral self-administration by various
neurotransmitter systems in specific
regions of the cortico-mesolimbic
reward pathway.

Drug Discrimination (4 grants).
These grants are investigating multi-

ple receptor mechanisms that mediate
the discriminative stimulus effects of
alcohol — that is, the internal subjective
effects that are reinforcing and main-
tain drinking.

Brain Stimulation Reward (2
grants). Reductions in brain stimulation
reward thresholds are used to identify
brain areas that mediate reinforcement
after drug administration, using selected
lines of alcohol-preferring rats.

Plus Maze (2 grants). The plus maze
is used to measure alcohol's anxiolytic
effects in selected lines of alcohol-
preferring and -nonpreferring rats.

Place Preference, Taste Aversion (3
grants). These grants are studying the
neural circuits and/or neurotransmit-
ter systems mediating the hedonic or
aversive properties of initial alcohol
exposure using place and taste condi-
tioning paradigms.

Tolerance/Acute Withdrawal

Place Preference /Aversion, Operant
Self- Administration, Drug Discrimina-
tion (2 grants). One project is investi-
gating whether alcohol's aversive
effects are reduced and its hedonic
effects are enhanced as tolerance
develops to alcohol's hypothermic effects.
A second project is investigating acute
withdrawal 12-30 hours after a single
high dose of alcohol, referred to as
"ethanol delayed effect" (EDE) or
"hangover," and determining whether
physiological and subjective effects of
EDE are similar to phase shift effects
associated with jet lag or work shifts.

Environmental Factors, Taste, Gender

Operant Self-Administration (6
grants). Many social and environmental

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NIAAA's Neuroscience and Behavioral Research Portfolio

factors such as stress (social status),
schedules of reinforcement, taste factors,
and temperature influence the estab-
lishment of alcohol-seeking behavior.
Four studies are investigating environ-
mental factors in an operant reinforce-
ment paradigm to evaluate reinforcing
efficacy of alcohol alone or multiple
reinforcers that differ in taste (alcohol
vs. sucrose or alcohol vs. alcohol/
sucrose). Two grants are examining
gender differences, hormonal regulation,
and effects of social status on operant
self- administration of alcohol. One
study is investigating the role of ambient
temperature in altering alcohol's hedonic
effects as measured by taste and place
conditioning and self- administration.

Conditioned Stimuli, Incentive
Motivation (3 grants). Previously neutral
environmental stimuli can acquire moti-
vational properties of a primary reinforcer
(alcohol) that contribute to alcohol seek-
ing. These grants are examining the role
of conditioned stimuli (previously neutral
stimuli paired with alcohol) in rein-
stating or maintaining alcohol self-
administration following extinction.

Acute/Chronic Brain and Behavioral
Effects (4<0rants)

These grants are investigating the
acute and chronic (nondependent)
effects of alcohol on cognition and
other behaviors, and their underlying
neuroanatomies, neurophysiological,
and/or neurochemical substrates.

Innate Behavioral Characteristics
(Disinhibition, Challenge) (3 grants)

These grants use selected rat lines (pre-
ferring/nonpreferring) to study innate
patterns of behavior (disinhibition and

electrophysiological parameters) that may
predispose to alcoholism and their under-
lying neurobiological mechanisms.

Recommendations

Behavioral Paradigms/Phenotypes

More studies are needed in the areas of
tolerance and acute withdrawal and devel-
opment of temperament phenotypes.

Neurotransmitter and Brain Regions

More grants are needed to study addi-
tional sites in the prefrontal cortex,
amygdala, hippocampus, and hypo-
thalamus, and the interactions of the
neurotransmitter NMD A, 5-HT, and
the cholinergic system.

Species/Gender

Only four grants are investigating gen-
der differences as part of their specific
aims. Since women are more susceptible
to the toxic effects of alcohol and the
menstrual cycle phase alters sensitivity
of the subjective effects of alcohol,
more studies are needed on the neural
mechanisms of gender differences in
the subjective and/or reinforcing
effects of alcohol.

Neuroadaptive Changes in
Neurotransmitters Mediating
Alcohol-Induced Behaviors:
Sensitization, Relapse, Dependence,
Repeated Withdrawals

Research Areas

Sensitization (2 grants)

Sensitization is the opposite response
to tolerance, that is, the effects of the

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Subcommittee Report

same dose of a drug become more pro-
nounced with repeated exposure. Two
grants are studying neuroadaptive
changes underlying sensitization.

Dependence, Abstinence/Relapse Models,
Neural Mechanisms (5 grants)

Dependence Models: Self-Adminis-
tration, Brain Stimulation Reward,
Incentive Motivation (2 grants).
These grants are examining models
of dependence produced by repeated
withdrawals from liquid diet or vapor
inhalation. Both grants use microdialy-
sis and/or microinjection techniques.

Prolonged Abstinence, Relapse,
"Deprivation Effect" (3 grants).
These grants are exploring models of
relapse in chronically exposed but not
dependent animals. It has been shown
that when abstinence is imposed after
chronic exposure to alcohol, drinking pat-
terns temporarily increase. This has been
referred to as the "deprivation effect."

Long-Term Effects on Brain
and Behavior (I grant)

This grant is investigating the neural
mechanisms of memory impairments
produced by long-term chronic expo-
sure to alcohol.

Neurobiological Mechanisms of
Protracted Withdrawal (1 grant)

This grant is exploring the role of 5-
HT in mediating the symptoms of
alcohol withdrawal.

Recommendations

Behavioral Paradigms/Phenotypes

More applications are needed to develop
models of dependence, craving, relapse,

and withdrawal, as well as long-term
neurobiological consequences.

Neurotransmitter Systems
and Brain Areas

Because the cortico-mesolimbic reward
system involves complex interconnections
among many structures in addition to
the ventral tegmental area, nucleus
accumbens, and "extended" amygdala,
more grants are needed to study all the
relevant transmitter systems and their
interactions in the cortico-mesolimbic
reward system, including the prefrontal
cortex and hippocampus.

Species/Gender

In that females have been shown to be
differentially sensitive to alcohol's effects
across the menstrual cycle, more studies
are needed on gender differences in
craving, development of dependence,
and relapse.

Adolescent Period: Biological Basis
for Vulnerability and Underlying
Neurobiological Mechanisms

Research Areas

Neurobiological and Behavioral
Mechanisms and Consequences of
Adolescent Drinking

Self-Administration and Neuro-
transmitter Systems (2 grants). These
grants are investigating whether charac-
teristics of the dopamine and serotonin
systems present in adults are also pre-
sent in young animals and contribute to
excessive drinking in the young. Both
grants use microdialysis, high-pressure
liquid chromatography, and autoradiog-
raphy procedures to determine if there

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NIAAA's Neuroscience and Behavioral Research Portfolio

are differences in regional 5-HT and
dopamine content, regional densities
of 5-HTj and 5-HT2 sites, and binding
of 5-HT1A and D2 sites in animals 15,
25, and 35 days of age. Brain regions
of interest are the cerebral cortex, hippo-
campus, striatum, hypothalamus, accum-
bens, and olfactory tubercle. Both grants
are using selected lines (P, NP, HAD,
LAD) to compare phenotypic differences
in these measures.

Development of Tolerance /Sensitivity,
Low- Dose Stimulatory Effect (3 grants).
These grants are looking at the presence
of and/or ontogeny of alcohol respon-
siveness by measures of low- dose alcohol
stimulation, acute and rapid tolerance to
alcohol's sedative effects (sleep time),
hypothermia, and suppression of startle,
as well as differences in emotionality and
cognitive functions (plus maze and
Morris Water Maze learning). Three
grants are using rodents and two are
using selected lines (P, NP, HAD, LAD).

Environmental Factors and Con-
sumption (Free Choice and Operant
Responding) (1 grant). This grant is
examining the ontogeny of drinking
after weaning (day 22 of age) and the
effects of various factors on this devel-
opment, such as housing conditions,
taste aversion conditioning, and phar-
macological interventions (fluoxetine,
methylphenidate, and buspirone).
Selected lines of rats are used (P, NP,
HAD, LAD).

Long-Term Behavioral Consequences
of Adolescent Drinking (2 grants).
These grants are studying cognitive and
emotional changes in adult animals from
selected lines (P, NP, HAD, LAD) that
were exposed to alcohol during adoles-
cence, such as orienting to a novel

environment, plus maze performance,
and Morris Water Maze performance.
Early Experience and Later Response
to Alcohol (3 grants). These grants are
investigating the effects of early expo-
sure to alcohol on later responsiveness
to alcohol. All grants use rodents, and
one grant uses selected lines.

Recommendations

Behavioral Paradigms

This is a relatively new initiative in the
portfolio. There is now evidence in
humans that early alcohol exposure
(especially during periods of early child-
hood, late childhood, and adolescence)
correlates with the development of alco-
holism in adulthood. More studies are
needed to develop paradigms of alcohol
seeking (self- administration and drug
discrimination) during the adolescent
period to study the underlying mecha-
nisms of ontogeny of alcoholism.

Neurotransmitters

Only one study is examining neurochem-
ical markers of alcoholism vulnerability
in selected lines of alcohol-preferring and
-nonpreferring rats. The neurotrans-
mitter systems targeted are dopamine
and 5-HT. Clearly, studies of other
neurotransmitter systems are needed,
particularly their role in the initiation of
alcohol-seeking behavior.

Species/Gender

All four grants are using rats, and two are
using selected lines. Although two are
studying both male and females, gender
difference is not a specific aim. More
studies are needed to examine gender
differences. Furthermore, primate

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Subcommittee Report

models are ideal because of their rela-
tively longer adolescent period.

Medications Development

The underlying goal of NIAAA's basic
neuroscience research program is to
understand the neurobiological mecha-
nisms of alcohol's effect in order to
develop treatments for alcoholism, partic-
ularly medications. However, for the grants
discussed in this section, at least one
specific aim is to test specific compounds,
via systemic injection, for their ability
to reduce drinking, prevent relapses,
or reduce withdrawal symptoms.

Research Areas

Operant Responding and Free-Choice
Consumption (4jjrants)

These studies are using self- administra-
tion paradigms (operant reinforcement
paradigms and two-bottle consumption)
to explore the effectiveness of several
receptor antagonists as potential thera-
peutic agents for reducing alcohol intake.

Protracted Withdrawal (1 grant)

This grant is studying the effectiveness
of various serotonergic compounds
(buspirone, a 5-HT1A partial agonist;
mianserin, a 5-HT2A/2C antagonist;
methysergide, a nonselective 5-HT
antagonist; and ICS 205-930, a 5-HT3
antagonist) in blocking withdrawal
symptoms as measured by drug discrim-
ination and plus maze paradigms.

Recommendations

The most frequently studied compounds
for reducing drinking are the opiate
antagonists. Clearly, more candidates
for potential therapeutic agents are

needed, drawing from all the relevant
neurotransmitter systems. Another rec-
ommendation is that more standardized
testing procedures for the potential
agents be developed.

Methodology Development (2 grants)

One grant is establishing procedures
to measure the effects of alcohol on
carbohydrate metabolism and rates and
synthesis of amino acid neurotransmit-
ter (glutamate, GAB A, and aspartate)
in the brain of fully intact, conscious
rats using 13C nuclear magnetic reso-
nance spectroscopy. Another grant is
developing an ultrasensitive mass spec-
trometric procedure for the analysis of
neurosteroids in tissue samples and
microdialysates of select brain areas of
freely moving rats after acute and
chronic alcohol administration and
during withdrawal.

Recommendations

Techniques that promote an understand-
ing of the underlying neural circuitry of
the addiction process and their relation-
ship to behavior should be encouraged.

STUDIES OF ACUTE
AND CHRONIC EFFECTS
OF ALCOHOL IN HUMANS

Studies of the Acute Effects
of Alcohol on Cognition

AND iMPULSIVITY/DlSINHrBrTORY

Behavior: Gaps in Knowledge
and Research Opportunities

Peter R. Finn, Ph.D.

• Research should be undertaken on the
effects of early exposure to alcohol

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NIAAA's Neuroscience and Behavioral Research Portfolio

(preadolescent/adolescent) and the
development of disinhibited traits
using longitudinal designs.
It is important to conduct cross-
sectional studies of the effects of
alcohol challenge on disinhibited/
impulsive behavior (and factors
associated with such behavior),
using multimethod measurements
to capture the multidimensional
nature of behavioral disinhibition
and controlling for important
sources of individual differences
such as preexisting disinhibited
traits, family history of alcoholism
(and antisocial behavior), drinking
history, and level and limb of the
blood alcohol curve.

Clinical Neuroscience
Studies of Behaviors
Associated With Alcohol
Consumption in Alcoholism:
Gaps in Knowledge and
Research Opportunities

John H. Krystal, M.D.

• More emphasis should be placed
on developing drugs that block the
euphoric effects of alcohol.

• NIAAA should assist investigators
in gaining access to alcohol-related
investigational new drugs.

• Increased emphasis should be
placed on studying the neurobiol-
ogy of triggers for relapse to alco-
hol use, particularly alcohol-related
cues; priming effects of alcohol
consumption on subsequent drink-
ing; and the interactive effects of
stress (negative mood induction)
and alcohol cues.

• Alcohol research should more fully
integrate functional neuroimaging
techniques to facilitate the study of
neural circuitry underlying craving
and factors related to alcohol con-
sumption.

HPA Axis: Changes and Risk
for Alcoholism: Gaps in
Knowledge and Research
Opportunities

Gary Wand, M.D.

• It is important to better understand
the relationships between specific
neurotransmitter input to hypothal-
amic CRH neurons as a function of
family history of alcoholism and to
estimate their relevance to vulnerabil-
ity for alcoholism. More specifically,
dose -response neuropharmacological
studies are needed that activate or
block 5-HT, GABA, and opioid
input. It is important that physio-
logical and behavioral responses be
examined in these studies.

• Relationships between HPA and
alcohol-seeking behavior should be
investigated. In rodent models, empha-
sis should be on relationships among
corticotropin-releasing hormone
(CRH)/cortisol states, mesolimbic
neurochemistry, and alcohol-seeking
behavior. In humans, there should
be comparisons of the entire HPA axis
between alcohol- and nonalcohol-
dependent individuals as a function
of family history of alcoholism.

• It would be useful to determine
whether (1) the magnitude of HPA
axis activity during acute withdrawal
predicts relapse rates; (2) supplements

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Subcommittee Report

with CRH, ACTH, and/or glucocor-
ticoids during early abstinence decrease
mood disturbances and attenuate
relapse; and (3) effectiveness of opioid
receptor antagonists in treating
alcoholism relates to ability to stim-
ulate HPA axis.

• It is important to determine if indi-
viduals at increased risk for the
future development of alcoholism
have enhanced opioidergic sensitiv-
ity to alcohol exposure.

• The effects of stress dampening by
alcohol should be studied in a family
history design using components of
the HPA axis as end-point measures.

• Future studies should more clearly de-
fine the relationships between alcohol-
associated alterations to HPA axis
function and immune system function.

Acute and Chronic Effects
of Alcohol on Human Sleep:
Gaps in Knowledge and
Research Opportunities

Cindy L. Ehlers, Ph.D.

• There is a clear need to increase fund-
ing in the area of alcohol and sleep.

• It is important to determine if chronic
alcohol intake produces prolonged

sleep disturbances and increased
risk for relapse.

It is not known what brain mecha-
nisms underlie alcohol-induced
sleep disturbances.
It would be valuable to determine
the risk factors for sleep disturbance
and excessive alcohol consumption.
It would be useful to investigate
gender-related, alcohol-associated
sleep disturbances.
It is important to determine if new
therapies that target sleep distur-
bances can be effective in prevent-
ing relapse.

Portfolio of Studies of Acute and
Chronic Effects of Alcohol in

Humans

Ellen D. Witt, Ph.D.

In the scientific content analysis of
the portfolio on the acute and
chronic effects of alcohol in humans
described below, each broad scientific
category is further subdivided to
give more detail on the number and
types of human paradigms being
studied. Table 8 shows the number
and dollar value of grants for these
human studies.

Table 8. NIAAA FY97 Grants for Studies of Acute and Chronic Effects of Alcohol in Humans.

Category

Number

Dollar Amount

Acute cognitive and behavioral effects
Mechanisms of alcoholic behavior
Sleep
Total

348,967

550,772

202,466

1,102,205

551

NIAAA's Neuroscience and Behavioral Research Portfolio

Acute Cognitive and Behavioral Studies

Research Areas

Acute Effects of Alcohol on Stress-
Dampening Response/ Aggressive
Behavior (Igrants)

These grants are studying the acute
behavioral effects of alcohol. One grant
is examining the effects of alcohol on
aggressive responding in women in a
laboratory setting. The other grant is
examining the ability of alcohol to
reduce stress/anxiety using "stress
dampening" techniques (i.e., alcohol's
effects on the magnitude of physiolog-
ical responses) and a "cognitive appraisal"
model of stress and emotion.

Innate Behaviors: Antisocial
Personality, Behavioral Activation/
Inhibition/ Aggression (2 grants)

These grants are investigating the rela-
tionship between innate characteristics,
such as antisocial personality and/or
temperament (behavioral activation
and inhibition), and alcohol-related
behaviors (i.e, consumption and/or
alcohol-induced aggression). One
grant is investigating the relationship
between antisocial personality, alco-
hol-induced aggression, and serotonin
function. The other grant is investi-
gating the biobehavioral mechanisms
(i.e., strength of the behavioral inhibi-
tion and activation systems) mediating
the relationship between impulsive
personality, disinhibited/antisocial
personality, and alcohol abuse.

Recommendations

There is a long history of research
on the acute effects of alcohol on cogni-

tive processes, but these studies have
largely focused on memory, divided
attention, reaction time, and visuomo-
tor skills. The currently funded
research is investigating how alcohol's
effects on cognitive processes may
have a role in increasing drinking or
other alcohol-related behaviors, such
as aggression. More research is need-
ed in other areas, such as alcohol's
effects on executive functioning, a cog-
nitive construct involving the cogni-
tive regulation of behavior, and how
that may increase drinking. More
research is also needed on the role of
temperament and/or personality char-
acteristics in contributing to excessive
drinking and other alcohol-related
behaviors.

Mechanisms of Alcoholic Behaviors

Research Areas

Neurotransmitter and Neurohormonal
Mechanisms: Behavioral
Psychopharmacology (3 grants)

These grants are examining the role
of neurotransmitters and neuropep-
tides in alcohol reinforcement and
craving in alcoholics. One grant is
studying the interactive contributions
of noradrenergic and serotonergic sys-
tems to alcohol craving in detoxified
alcoholics using cue reactivity para-
digms. A second grant is evaluating
the role of a glutamate antagonist
(ketamine) at the NMDA receptor
in producing subjective effects similar
to alcohol. A third grant is study-
ing opioidergic mechanisms of rein-
forcement for coexisting alcohol and
nicotine use.

552

Subcommittee Report

Neurotransmitter Mechanisms:
Imaging Studies (1 grant)

This grant is studying whether the
dopamine system is abnormal in alco-
holics, the functional consequences of
these abnormalities, and the effects of
detoxification. Using positron emission
tomography (PET) imaging and mul-
tiple tracer methods, this grant is eval-
uating the dopamine system in
alcoholics in two phases: the dopamine
system at rest and during pharmaco-
logical activation.

Recommendations

While many animal studies are investigat-
ing neural circuitry and neurochemical
mechanisms of alcohol- motivated behav-
iors, such as reinforcement, few studies
are exploring neurochemical mecha-
nisms in humans. With the advent of new
imaging technologies such as PET, and
the discovery of ligands and receptor ago-
nists and antagonists, more studies are
needed to investigate neural mecha-
nisms of alcohol-motivated behaviors
in humans.

Effects of Alcohol
on Sleep (1 grant)

This grant is investigating whether dis-
ruption of sleep continuity and/or loss
of slow wave sleep leads to greater
elevations in sympathetic nervous sys-
tem activity in African American alco-
holics, which in turn results in impaired
immune function and increased risk
for disease.

Recommendations

Sleep disturbance is a common prob-
lem during withdrawal and abstinence,

which could contribute to relapse to
drinking. More research is needed to
understand the underlying neural mech-
anisms of sleep disturbances in abstinent
alcoholics, and whether potential medica-
tions will ameliorate these sleep disorders.

COGNITIVE/BEHAVIORAL/
STRUCTURAL DEFICITS

Neuropsychological
Sequelae of Chronic
Alcoholism: Gaps in
Knowledge and Research
Opportunities

Marlene Oscar-Berman, Ph.D.

• Additional research is needed to
clarify the separate and combined
roles of malnutrition and alcohol
neurotoxicity in brain damage and
functional impairments.

• Additional studies would be useful
on the enhanced susceptibility to
alcoholism-associated deficits in
women and the elderly.

• Future research should include lon-
gitudinal or retrospective methods
to evaluate the likelihood of sub-
group vulnerability.

• Specific evaluation of nonhuman ani-
mal models of alcohol-associated cog-
nitive deficits should be encouraged.

• It is important to establish uniform
positive diagnostic criteria for sub-
types of brain-impaired alcoholics
(e.g., KorsakofPs syndrome vs.
alcoholic dementia); this can be
approached most effectively with
the aid of sophisticated neurological
and neurobehavioral techniques.

553

NIAAA's Neuroscience and Behavioral Research Portfolio

• Followup postmortem examination
of brains of well-studied alcoholics
should be encouraged for clues about
neurotransmitter abnormalities, as
well as analyses of injury at the cel-
lular level.

• Particular problems in need of future
research effort include the following:
neurobehavioral and brain functional/
structural recovery with abstinence;
extent of multimodal sensory and
perceptual deficits; nature of the
loss of emotional and motivational
functioning among subgroups of
alcoholics; and specific contribu-
tions of frontal system dysfunction
to alcoholic symptomatology.

Human Brain Dysfunction
Secondary to Alcohol
Abuse: Gaps in Knowledge
and Research Opportunities

George Fein, Ph.D.

• It is important to determine which
brain systems are most vulnerable
to the morbid effects of chronic
alcohol abuse.

• What alcohol use-related factors
influence brain morbidity secondary
to alcohol abuse, including nutritional
deficiency, number of episodes of
withdrawal, development of tolerance,
and role of pattern of drinking?

• Other factors influencing brain
morbidity secondary to alcohol
abuse should also be studied,
including genetically transmitted
vulnerabilities, gender, age, vari-
ability in brain functional reserve as
measured by intracranial volume,
adolescence, and comorbidity.

What are the mechanisms involved
in recovery of the brain from the
effects of chronic alcohol abuse?
Electrophysiological methods should
continue to be used because of the
advantages over other neuroimaging
techniques in terms of temporal
resolution, ease, and cost- effectiveness
of data acquisition, and the ability
to collect data during the performance
of complex cognitive tasks.
It is important to combine behav-
ioral, structural, and functional
measures of brain function.

Neuroimaging Studies

of Brain Vulnerability

to Alcoholism: Gaps

in Knowledge and Research

Opportunities

Edith V. Sullivan, Ph.D.

• Even though alcohol abuse and
dependence are prevalent in adoles-
cents as well as older individuals,
alcohol's adverse effects on the
adolescent brain are unknown.

• It is unknown whether adequate
nutritional supplements improve
the structural condition of the
brain of alcoholics.

• There is a paucity of data on the
effects of alcoholism on the brains
of women.

• The mechanisms underlying morpho-
logical recovery are still unknown.

• Increased emphasis should be placed
on in vivo metabolite imaging
because of its possible use in helping
to define disease progress, assess effi-
cacy of treatment, and track alcohol-
related changes across the lifespan.

554

Subcommittee Report

• To examine the extent that toler-
ance may be genetically determined,
it should be possible to use mag-
netic resonance spectroscopy (MRS)
to compare estimates of nonab-
sorbed alcohol in nonalcoholics
(low alcohol-consuming individu-
als) who have a positive family his-
tory for alcoholism to those with a
negative family history.

• Although the functional signifi-
cance of brain volume loss in terms
of the commonly observed alco-
holism-associated cognitive behav-
ioral deficits has been difficult
to demonstrate, functional mag-
netic resonance imaging (fMRI)
has the potential to identify areas
throughout the brain that are
activated during performance of
specific components of cognitive
operations.

• Diffusion anisotropy offers a poten-
tial metric for assessing the integrity
of white matter throughout the
course of alcoholism.

• It would be useful to examine
brain function and psychiatric
comorbidity in alcoholics in a sys-
tematic manner.

• It is important to determine whether,
with abstinence, alcoholics whose
brains show recovery of tissue vol-
ume are the individuals who also
show improvement in their cogni-
tive and motor abilities.

• It needs to be established whether
alcoholism-associated brain volume
deficits are accompanied by predictable
neuropsychological performance.

• The question of gene -environment
interaction with respect to apolipo-
protein genotype and alcohol in

the development of dementia and
accelerated brain volume loss in
alcoholics has yet to be addressed.

• The extent to which the frontal
lobes and cerebellum recover in
structure or function remains
unknown despite their importance
to problem solving, contextual
memory, and execution and learn-
ing of procedures.

• Controlled studies of alcoholics who
remain abstinent for years in com-
parison with those who drink for
years are needed.

• No quantitative in vivo studies have
been conducted to examine, even
cross-sectionally, potential alcohol-
gene interactions on brain morphol-
ogy. These studies are essential to
determine the pattern and extent of
potentially preexisting differences in
individuals genetically predisposed
to alcohol addiction compared with
those not so disposed.

Portfolio of Studies on Cognitive,
Behavioral, and Structural Deficits
in Humans

Ellen D. Witt, Ph.D.

Human studies investigate the conse-
quences of acute and chronic alcohol
on cognition and other behaviors, as
well as the underlying structural
changes associated with the behavioral
deficits using state-of-the-art imaging
technologies. The neural mechanisms
of alcohol-motivated behaviors, such as
craving, have also been studied in
humans, but to a lesser extent. Table 9
shows the number and dollar value of
grants for studies of cognitive, behav-
ioral, and structural deficits in humans.

555

NIAAA's Neuroscience and Behavioral Research Portfolio

Brain Damage, Cognitive/Motor
Dysfunction: Affected Brain Areas,
Contributions of Malnutrition,
Influences of Polysubstance Abuse,
Aging, Gender

Research Areas

Neurobehavioral/Cognitive/Motor
Dysfunction (8 grants)

These grants are investigating cogni-
tive and motor deficits and their un-
derlying neural substrates in chronic
alcoholics and Korsakoff patients, us-
ing neuropsychological, neurocogni-
tive, neurophysiological, and/or
magnetic resonance imaging (MRI)
techniques.

Motor Functioning (2 grants).
These grants are targeting alcohol's
effects on the cerebellum. One grant
uses Pavlovian techniques (delayed
classical conditioning tasks involving
heart rate, galvanic skin response
(GSR) conditioning, eyeblink condi-
tioning, and extinction) to dissociate
structures in the medial temporal lobe
(amygdala) and cerebellum in mediat-
ing associative learning deficits. The
other grant is quantifying regional
volumes of the cerebellar hemispheres
and vermis with MRI, using a com-
ponent-process approach to assess
motor coordination and motor skill

learning, and determining structure-
function relationships between spe-
cific motor processes and regional
cerebellar volumes.

Attention, Memory, Executive
Processes (4 grants). Two of these
grants study attention and/or mem-
ory processes using cognitive tests.
One grant is studying deficits in ex-
plicit and implicit conceptual memory
in chronic alcoholic and Korsakoff pa-
tients. The second grant is evaluating
cognitive efficiency (principally atten-
tion and memory) in subtypes of al-
coholics classified on the basis of
other drug use and abuse (marijuana,
stimulants).

Two other grants are studying
attention, memory, and executive
functioning in conjunction with
event-related potential (ERP) and/or
fMRI to understand the underlying
neural mechanisms of these deficits in
alcoholics. One of these grants uses
fMRI to study localized brain activa-
tion during performance of auditory
and visual working memory tasks.
The other grant uses ERP and elec-
troencephalographic techniques
to study attention and memory
processes in abstinent alcoholics
classified along several variables, in-
cluding type I and type II alcoholism,
family history of alcoholism, and

Table 9. NIAAA FY97 Grants for Studies of Cognitive, Behavioral, and Structural Deficits
in Humans.

Category

Number

Dollar Amount

Brain damage/cognitive/motor deficits

Methodology development

Total

3,173,813

103,482

3,277,295

556

Subcommittee Report

the relationship between antisocial
behavior and neurophysiological
characteristics.

Affective (Emotional) and
Conative (Intentional) Functions (2
grants). These grants are investigating
emotional and intentional abnormali-
ties in chronic alcoholics (with and
without Korsakoff s syndrome) and
whether these changes are mediated
by right frontal or bilateral frontal
lobe pathology.

Brain Metabolic Changes and Tolerance
and Long-Term Abstinence (2 grants)

One grant is using MRS, which al-
lows noninvasive quantification in
vivo of brain metabolites, to investi-
gate the mechanism of alcohol-
induced tolerance in humans.
Another grant is using MRS to char-
acterize the longitudinal course of
metabolic changes (i.e, the ratio of
visible choline to the neuronal marker
N-acetylaspartate) in the brains of ab-
stinent alcoholics.

Effect of Aging and/or HTVon Cognitive
Functioning in Alcoholics (5 grants)

Alcohol and Aging (4 grants). One
grant is using structural MRI, electro -
physiology, and neuropsychological
assessments to critically evaluate
two opposing models of central ner-
vous system effects of chronic alcohol
abuse as they interact with age
and gender. Three grants are con-
tinutions of ongoing research on
the interaction between alcohol and

aging. Two of these are investigating
emotional, attentional, and inten-
tional processes in alcoholics and age-
matched control subjects to evaluate
the ways in which the behavioral con-
sequences of aging and alcoholism are
parallel, divergent, and/or interactive.
Another grant is a continuation of an
ongoing longitudinal study of alco-
holic and control women using MRI,
ERP, and neuropsychological tests to
identify cross-sectional patterns of
sparing and loss, their interaction
with age, and comparability to find-
ings in alcoholic men.

Alcohol and HIV (1 grant). This
grant is examining the effect of
alcohol consumption on neuropsycho-
logical function across stages of HIV
infection.

Recommendations

More research is needed in relating
cognitive deficits produced by chronic
alcoholism to excessive drinking and
the inability to benefit from treatment.

Methodology Development (1 grant)

This grant is developing a method to
induce and maintain a steady-state
concentration of alcohol in the brain
and blood while multiple dependent
measures are assessed.

Recommendations

Technologies that improve our un-
derstanding of the neural mechanisms
of alcoholism and improve treatment
are encouraged.

557

NIAAA's Neuroscience and Behavioral Research Portfolio

APPENDIX A:
SUBCOMMITTEE FOR
REVIEW OF
NEUROSCIENCE AND
BEHAVIOR PORTFOLIO

Co -Chairs

Henri Begleiter, M.D., Ph.D.
Department of Psychiatry
Health Science Center at Brooklyn
450 Clarkson Ave., Box 1203
Brooklyn, NY 11203-2098

Catherine Rivier, Ph.D.
The Salk Institute
10010 North Torrey Pines Rd.
La Jolla, CA 92037-1099

Experts in Alcohol-
Related Areas

Ivan Diamond, M.D., Ph.D.

Department of Neurology/Gallo Center

University of California, San Francisco

San Francisco General Hospital

Bldg. 1, Room 101

1001 Potrero Ave.

San Francisco, CA 94110-3594

Adron Harris, Ph.D.

Department of Pharmacology, C-236

University of Colorado HSC

4200 East Ninth Ave.

Denver, CO 80262-0236

Harold Kalant, M.D., Ph.D.
Department of Pharmacology
University of Toronto
Medical Science Bldg.
Toronto, ON
Canada M5S 1A8

George Koob, Ph.D.

Department of Neuropharmacology

The Scripps Research Institute, CVN-7

10550 North Torrey Pines Rd.

La Jolla, CA 92037

Ting-Kai Li, M.D.

Indiana University School of Medicine

Emerson Hall 421

545 Barnhill Dr.

Indianapolis, IN 46202-5124

Elias Michaelis, M.D., Ph.D.
Department of Pharmacology and

Toxicology
Malott Hall
University of Kansas
Lawrence, KS 66045-2505

Oscar Parsons, Ph.D.

University of Oklahoma HSC

Rogers Bldg., Suite 410

800 N.E. 15th St.

Oklahoma City, OK 73104-1602

Adolf Pfefferbaum, M.D.
SRI International
333 Ravenswood Ave.
Menlo Park, CA 94025

Bernice Porjesz, Ph.D.
Department of Psychiatry
Health Science Center at Brooklyn
450 Clarkson Ave., Box 1203
Brooklyn, NY 11203-2098

Herman Samson, Ph.D.

Department of Physiology/Pharmacology

Wake Forest University School of

Medicine
Medical Center Blvd.
Winston-Salem, NC 27157-1083

Boris Tabakoff, Ph.D.
Department of Pharmacology, C-236
University of Colorado HSC
4200 East Ninth Ave.
Denver, CO 80262-0236

Maharaj Ticku, Ph.D.
Department of Pharmacology
University of Texas HSC
7703 Floyd Curl Dr.
San Antonio, TX 78284-7764

Nora Volkow, M.D.
Department of Medicine
Brookhaven National Laboratory
Upton, NY 11973

558

Subcommittee Report

Gary Wand, M.D.
Division of Endocrinology
The Johns Hopkins University
School of Medicine, Ross 863
720 Rutland Ave.
Baltimore, MD 21205

Experts in Non-Alcohol-
Related Areas

Robert Freedman, M.D.
Department of Psychiatry
University of Colorado HSC
Box C-268-71
4200 East Ninth Ave.
Denver, CO 80262

Peter Kalivas, Ph.D.
Department of VCAPP
Washington State University
Pullman, WA 99154

Steven Paul, M.D.
Lilly Research Laboratories
Lilly Corporate Center
893 Delaware Ave.
Indianapolis, IN 46285

APPENDIX B: EXPERTS
IN NEUROSCIENCE
AND BEHAVIOR

Fulton T. Crews, Ph.D.

University of North Carolina School

of Medicine
Center for Alcohol Studies
Thurston-Bowles Bldg., CB 7178
Chapel Hill, NC 27599-7178

Cindy Ehlers, Ph.D.

The Scripps Research Institute, CVN-14

10550 North Torrey Pines Rd.

La Jolla, CA 92037

George Fein, Ph.D.
Department of Psychiatry
University of California
Parnassus Ave.
San Francisco, CA 94143

Peter R. Finn, Ph.D.
Department of Psychology
Indiana University
1101 East 10th St.
Bloomington, IN 47405-7007

Kathleen A. Grant, Ph.D.
Department of Physiology and

Pharmacology
Wake Forest University School of

Medicine
Medical Center Blvd.
Winston-Salem, NC 27157-1083

Paula L. Hoffman, Ph.D.
Department of Pharmacology, C-236
University of Colorado Health Sciences

Center
4200 East 9th Ave.
Denver, CO 80262-0001

John H. Krystal, M.D.

Yale-VA Alcoholism Research Center

Psychiatry Service (116-A)

VA Connecticut Healthcare System

950 Campbell Ave.

West Haven, CT 06516

David M. Lovinger, Ph.D.

Department of Molecular Physiology and

Biophysics
Vanderbilt University School of Medicine
702 Light Hall
Nashville, TN 37232-0615

A. Leslie Morrow, Ph.D.

University of North Carolina School of

Medicine
Center for Alcohol Studies
3027 Thurston-Bowles Bldg., CB 7178
Chapel Hill, NC 27599-7178

Marlene Oscar-Berman, Ph.D.

Division of Psychiatry

Boston University School of Medicine

715 Albany St.

Boston, MA 02118

Catherine Rivier, Ph.D.
The Salk Institute
10010 North Torrey Pines Rd.
La Jolla, CA 92037-1099

559

NIAAA's Neuroscience and Behavioral Research Portfolio

George R. Siggins, Ph.D.

The Scripps Research Institute, CVN-12

10550 North Torrey Pines Rd.

La Jolla, CA 92037

Linda P. Spear, Ph.D.

Department of Psychology

Center for Developmental Psychobiology

Binghamton University

Binghamton, NY 13902-6000

Edith V. Sullivan, Ph.D.

Department of Psychiatry

Stanford University School of Medicine

410 Quarry Rd.

Stanford, CA 94305-5717

Steven N. Treistman, Ph.D.
Department of Pharmacology
University of Massachusetts Medical

Center
55 Lake Ave. North
Worcester, MA 01655

Gary Wand, M.D.
Division of Endocrinology
The Johns Hopkins University
School of Medicine, Ross 863
720 Rutland Ave.
Baltimore, MD 21205

Friedbert Weiss, Ph.D.
Department of Neuropharmacology
The Scripps Research Institute, CVN-15
10550 North Torrey Pines Rd.
La Jolla, CA 92037

APPENDIX C: NIAAA
PROGRAM STAFF

Walter A. Hunt, Ph.D.

Neurosciences and Behavioral Research

Branch
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

Robert W. Karp, Ph.D.
Neurosciences and Behavioral Research

Branch
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

Yuan Liu, Ph.D.

Neurosciences and Behavioral Research

Branch
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

Ellen D. Witt, Ph.D.

Neurosciences and Behavioral Research

Branch
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

Samir Zakhari, Ph.D.
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

APPENDIX D: NIAAA STAFF,
REPRESENTATIVES FROM
OTHER NIH INSTITUTES,
AND GUESTS

Megan Adamson, M.D.
Office of Collaborative Research, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Faye Calhoun, D.P.A.
Office of Collaborative Research, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Mary Dufour, M.D., M.P.H.
Deputy Director, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Michael J. Eckardt, Ph.D.
Office of Scientific Affairs, NIAAA
6000 Executive Blvd., Suite 409
Bethesda, MD 20892-7003

560

Subcommittee Report

Joanne Fertig, Ph.D.

Division of Clinical and Prevention

Research, NIAAA
6000 Executive Blvd., Suite 505
Bethesda, MD 20892-7003

Richard K. Fuller, M.D.

Division of Clinical and Prevention

Research, NIAAA
6000 Executive Blvd., Suite 505
Bethesda, MD 20892-7003

Klaus Gawrisch, Ph.D.

Laboratory of Membrane Biochemistry

and Biophysics, NIAAA
12420 Parklawn Dr., Room 116
Bethesda, MD 20892-8115

Thomas Gentry, Ph.D.
Office of Collaborative Research, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Enoch Gordis, M.D.
Director, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Daniel Hommer, M.D.

Laboratory of Clinical Studies, NIAAA

Bldg. 10, Room3C114

Bethesda, MD 20892-1256

Nancy Hondros

Planning and Financial Management

Branch, NIAAA
6000 Executive Blvd., Suite 412
Bethesda, MD 20892-7003

William M. Lands, Ph.D.
Office of the Director, NIAAA
6000 Executive Blvd., Suite 400
Bethesda, MD 20892-7003

Burt Litman, Ph.D.

Laboratory of Membrane Biochemistry

and Biophysics, NIAAA
12420 Parklawn Dr., Room 116
Bethesda, MD 20892-8115

Raye Litten, Ph.D.

Division of Clinical and Prevention

Research, NIAAA
6000 Executive Blvd., Suite 505
Bethesda, MD 20892-7003

Steve Long, M.A.
Office of Policy Analysis, NIAAA
6000 Executive Blvd., Suite 405
Bethesda, MD 20892-7003

Matthew McGue, Ph.D.
Department of Psychology
University of Minnesota
Elliot Hall, Room N-2 18
75 East River Rd.
Minneapolis, MN 55455

Antonio Noronha, Ph.D.
Office of Scientific Affairs, NIAAA
6000 Executive Blvd., Suite 409
Bethesda, MD 20892-7003

Carrie L. Randall, Ph.D.

Department of Psychiatry and Behavioral

Science
Medical University of South Carolina
171 Ashley Ave.
Charleston, SC 29425

Norman Salem, Jr., Ph.D.

Laboratory of Membrane Biochemistry

and Biophysics, NIAAA
12420 Parklawn Dr., Room 116
Bethesda, MD 20892-8115

Jules Selden, D.V.M., Ph.D.
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

Martin K. Trusty, M.S.

Office of Planning and Resource

Management, NIAAA
6000 Executive Blvd., Suite 412
Bethesda, MD 20892-7003

Ernestine Vanderveen, Ph.D.
Division of Basic Research, NIAAA
6000 Executive Blvd., Suite 402
Bethesda, MD 20892-7003

561

NIAAA'' s Neuroscience and Behavioral Research Portfolio

Kenneth Warren, Ph.D.
Office of Scientific Affairs, NIAAA
6000 Executive Blvd., Suite 409
Bethesda, MD 20892-7003

Forrest Weight, M.D.

Laboratory of Molecular and Cellular

Neurobiology, NIAAA
12420 Parklawn Dr., Room 118
Bethesda, MD 20892-8115

Herbert Weingartner, Ph.D.
National Institute on Drug Abuse
Bldg. 31, Room 1B58
Bethesda, MD 20892-1250

562

NOTES

I LliRARy

™ Amazing Research.
Amazing Help.

http://nihlibrary.nih.gov

10 Center Drive

Bethesda, MD 20892-1150

301-496-1080

3 1496 00823 7078

NIH Publication No. 00-4520
National Institutes of Health
Printed 2000

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

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