Table Of ContentTHE "DRUNKEN" SYNAPSE
Studies of Alcohol-Related Disorders
THE "DRUNKEN" SYNAPSE
Studies of Alcohol-Related Disorders
Edited by
Yuan Liu and
Walter A. Hunt
National Institute on Alcohol Abuse and Alcoholism
National Institutes of Health
Bethesda, Maryland
Springer Science+Business Media, LLC
Proceedings of a National Institute on Alcohol Abuse and Alcoholism Symposium on The "Drunken" Synapse:
Studies of Alcohol-Related Disorders, held in conjunction with the 27th Annual Meeting of the Society for
Neuroscience on October 25, 1997, in New Orleans, Louisiana
ISBN 978-1-4613-7148-9 ISBN 978-1-4615-4739-6 (eBook)
DOI 10.1007/978-1-4615-4739-6
© 1999 Springer Science+Business Media New York
Originally published by Kluwer Academic/Plenum PubJishers in 1999
10 9 8 7 6 5 4 3 2 1
A C.I.P. record for this book is available from the Library of Congress
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, Of otherwise, without written permission
from the Publisher
PREFACE
Over the past two years, the National Institute on Alcohol Abuse and Alcoholism
(NIAAA) has begun a series of symposia to highlight the need for more integrative re-
search to understand how ethanol alters behavior. Much of the research to date has dealt
either at the molecular level or has been whole animal studies. More studies are needed to
build our base of knowledge between these two extremes by focusing more on cellular and
network levels of organization.
To begin this focus on the intermediate steps in this scheme, the NIAAA presented a
satellite symposium entitled "Approaches for Studying Neural Circuits: Application to Al-
cohol Research" held at the Annual Meeting of the Society for Neuroscience in Washing-
ton, DC, on November 16, 1996. This symposium brought together a group of scientists
who presented their work on techniques used to study neural circuits. The proceedings of
that symposium were published (Y. Liu (Ed.) Approaches for Studying Neural Circuits:
Application to Alcohol Research. Alcohol Clin Exp Res 1998 Feb; 22: 1--{j6).
The following year the NIAAA convened a symposium on the latest research on the ac-
tions of ethanol on the synapse. Entitled "The 'Drunken' Synapse: Studies of Alcohol-Related
Disorders" and held in conjunction with the Annual Meeting of the Society for Neuroscience
on October 25, 1997, in New Orleans, LA, the symposium brought together a distinguished
cast of scientists who study synaptic function from various perspectives. This book represents
the proceedings of this symposium and will provide not only scholarly accounts of present a-
tions of the speakers, but also the discussion that ensued in response to these presentations.
The symposium was organized around three sessions: synaptic transmission, synaptic modu-
lation, and synaptic plasticity. The overview provides a synopsis of the chapters to follow.
More specific details can be found in the individual chapters. In addition, we edited all the
discussions between the audience and the speakers, grouped them under related subtitles, and
organized them into three chapters placed at the end of each section. We highly encourage the
readers to go through these chapters as well-the in-depth discussions during the symposium
provided a wealth of information, and it is reflected in these chapters.
The NIAAA presented another symposium in its series at the Annual Meeting of the
Society for Neuroscience on November 7, 1998, in Los Angeles, CA, to discuss the appli-
cation of gene knockout techniques to alcohol research. This symposium specifically ad-
dressed how these techniques can be used to explore the roles of various gene products in
biological changes induced by ethanol and its behavioral effects. Our hope is that these
symposia will generate more interest in these exciting areas of alcohol and neuroscience
research.
v
vi Preface
We would like to thank the National Institute on Alcohol Abuse and Alcoholism on
sponsoring and supporting this symposium. We would also like to thank Ms. Brenda
Hewitt for designing the cover of this book.
Yuan Liu, Ph.D.
Walter A. Hunt, Ph.D.
CONTENTS
1. Overview of the Symposium ........................................ .
Walter A. Hunt and Yuan Liu
2. A Perspective on the Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Gordon M. Shepherd
Section I. Synaptic Transmission
3. Molecular Targets Underlying Ethanol-Mediated Reduction of Hormone
Release from Neurohypophysial Nerve Terminals . . . . . . . . . . . . . . . . . . . . 27
Steven N. Treistman, Benson Chu, and Alejandro M. Dopico
4. Alcohol and General Anesthetic Modulation ofGABAA and Neuronal Nicotinic
Acetylcholine Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Toshio Narahashi, Gary L. Aistrup, Jon M. Lindstrom, William Marszalec,
Haruhiko Motomura, Keiichi Nagata, Hideharu Tatebayashi, Fan Wang,
and Jay Z. Yeh
5. Alcohol and the 5-HT3 Receptor ...................................... 51
David M. Lovinger and Qing Zhou
6. Questions and Answers of Session I: Synaptic Transmission 63
Section II. Synaptic Modulation
7. Depolarization-Induced Suppression oflnhibition (DSI) Involves a Retrograde
Signaling Process that Regulates GABAA-Mediated Synaptic Responses
in Mammalian CNS ........................................... 79
Bradley E. Alger
8. Native GABAA Receptors Get "Drunk" but Not Their Recombinant
Counterparts ................................................. 109
Hermes H. Yeh and Douglas W. Sapp
vii
viii Contents
9. Adenosine and Ethanol: Is There a Caffeine Connection in the Actions of
Ethanol? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Thomas V. Dunwiddie
10. A Metabotropic Hypothesis for Ethanol Sensitivity of GABAergic and
Glutamatergic Central Synapses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
George R. Siggins, Zhiguo Nie, and Samuel G. Madamba
II. Questions and Answers of Session II: Synaptic Modulation 145
Section III. Synaptic Plasticity
12. Alcohol, Memory, and Molecules ..................................... 159
Michael Browning, James Schummers, and Scott Bentz
13. Of Mice and Minis: Novel Forms and Analyses of Ethanol Effects on Synaptic
Plasticity .................................................... 167
Richard A. Morrisett and Mark P. Thomas
14. Ethanol Suppression of Hippocampal Plasticity: Role of Subcortical Inputs 183
Scott C. Steffensen
15. Questions and Answers of Session III: Synaptic Plasticity 205
Contributors ........................................................... 215
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 217
1
OVERVIEW OF THE SYMPOSIUM
Walter A. Hunt and Yuan Liu
Neurosciences and Behavioral Research Branch
National Institute on Alcohol Abuse and Alcoholism
Bethesda, Maryland 20892-7003
1. INTRODUCTION
Ethanol is the most abused drug in the country. According to the Ninth Special Report
to the U.S. Congress on Alcohol and Health, Americans consume 2.24 gallons of ethanol per
year. Although this is the lowest level of consumption since 1964, ethanol still underlies a
multitude of ills in this country. Alcohol contributes to many highway deaths, homicides,
suicides, and accidents, and causes numerous medical problems in long-term abusers, in-
ducing liver cirrhosis, pancreatitis, brain damage, and of course dependence. Almost 14 mil-
lion people are classified as either alcohol abusers or alcohol dependent, using standard
psychiatric instruments such as DSM-IY. Economic costs of alcohol abuse to the nation are
enormous at $ 148 billion due to lost productivity and health care expenditures.
Most of the problems caused by ethanol consumption relate to its actions on the
brain. Ethanol being a drug with many effects on neurons, researchers have been chal-
lenged to find the relevant targets on which ethanol acts. Around the tum of the century,
anesthetics including alcohols were believed to act on the membranes of neurons. The
Meyer-Overton Principle was formulated to explain their effects based on lipid solubility
by stating that the more lipid soluble the anesthetic, the more potent it was (Meyer, 1899;
Overton, 1896). Although numerous alcohols followed this principle within limits
(McCreery & Hunt, 1978), actions just on the lipids themselves were insufficient to ex-
plain the various, sometimes apparently specific, effects of ethanol. Because ethanol is a
simple molecule and is also amphiphilic, it unlikely has a specific receptor, as do most
other drugs. Thus, research over the last decade has focused on common actions of ethanol
on various functional entities in membranes responsible for neuronal excitability and
neurotransmitter release. These entities are complex proteins that traverse the neuronal
membrane and dangle in the intracellular and extracellular plasma. Collectively, these pro-
teins constitute the receptors and ion channels that regulate the excitability of neurons and
transmit impulses from one neuron to another. An important site on the neuron where
many of these important actions of ethanol take place is at the synapse.
The "Drunken" Synapse, edited by Liu and Hunt.
Kluwer Academic / Plenum Publishers, New York, 1999.
2 W. A. Hunt and Y. Liu
1.1. History of Synaptic Research
Gordon Shepherd from Yale University opened the symposium with an overview de-
scribing the history of the discovery of the synapse. He outlined how the synapse was found,
in part as a result of a feud over one hundred years ago between two famous neuroanatomists,
Camillo Golgi and Santiago Ramon y Cajal. Golgi used his new staining technique to identify
the first axon collaterals but saw these collaterals as connecting with each other into a fine
network. On the other hand, Cajal noticed "breaks" between neurons and believed that neu-
rons were not directly connected. In the 1890s, Sir Charles Sherrington coined the term "syn-
apse", from a Greek word meaning "connection" or "junction", to describe the gap that Cajal
saw. Chemical transmission between neurons had not yet been discovered and raised the
question about how impulses could jump such a gap ifnot electrically.
For the first half of the 20th century, a debate raged as to whether transmission was
electrical or chemical, a debate described by Shepherd as the "soup versus sparks" debate.
By the 1950s, with synapses visualized with the electron microscope and its electrical
properties characterized with microelectrodes, the notion that chemical mediators were re-
sponsible for transmission between neurons was increasingly accepted. Research in the
1970s consolidated the thinking that the synapse released mediators in a quanta I fashion
from stored vesicles. The vesicular membrane fused with the neuronal membrane, with the
transmitters being released by diffusion into the synaptic cleft.
Finally, Shepherd emphasized the importance of neural circuits. When neurons con-
nect with one another, they are often part of defined circuits that traverse the nervous system
from one part to another. These circuits mediate a variety of functions including motivation
and reward systems, which are relevant to the problem of alcoholism. He made a connection
to his own research with the olfactory bulb to suggest that odor could contribute to the sen-
sory input underlying eventual rewarding properties of ethanol. The olfactory system at the
molecular level involves receptors, some of which are metabotropic, coupled to G-proteins.
These are systems also affected by ethanol, as presented "in chapters of this book.
As we proceed into the rest of this book, Shepherd reminds us that the synapse not only
serves to relay impulses between neurons but can also alter the manner in which impulses are
generated. This may be a clue into the complexities of action of ethanol on neurons.
2. SYNAPTIC TRANSMISSION
A prevailing belief in alcohol research is that the synapse is the most sensitive part
of the neuron to ethanol. This belief is reflected in the pattern of research with ethanol
over the last 25 years. Traditional notions state that synaptic transmission is the predomi-
nant activity at the synapse and involves the release of a transmitter, its reuptake and stor-
age in vesicles, and its actions on pre- and postsynaptic receptors. In recent years,
mechanisms by which transmitter release could be modulated have become increasingly
important in understanding synaptic transmission. In this section, the discussion will re-
volve around research on the kinetics of neurotransmitter release and on synaptic recep-
tors, which have been postulated as molecular targets for ethanol.
2.1. Mechanisms of Transmitter Release
To lead the session on synaptic transmission, Richard Tsien from Stanford Univer-
sity presented cutting-edge research on mechanisms of transmitter release and provided
Overview of the Symposium 3
some insights on two aspects that had not yet been investigated in alcohol research-ki-
netics and regulation of presynaptic vesicle recycling and saturation of postsynaptic gluta-
matergic receptors.
Transmitter release has been considered an "all or none" event when synaptic ves-
icles release their entire store of transmitters after fusing with the presynaptic membrane.
The overall recycle time of synaptic vesicles was estimated as about 40 seconds. Through
a series of elegantly designed experiments, Tsien and colleagues provided evidence that
this "classical" view may need to be modified (Klingauf et aI., 1998). By using the fluo-
rescent dye FMI-43, which can reversibly stain vesicle membranes, Tsien's group fol-
lowed the time course of exocytosis and endocytosis closely. They first preloaded the dye
into presynaptic terminals at hippocampal synapses in culture, then stimulated the cells
with a high K+, normal Ca++ solution to induce exocytosis and consequential endocytosis.
In the classical view, if a vesicle fuses completely with the presynaptic membrane, it will
lose its entire transmitter contents, as well as the dye. When monitoring with quantitative
fluorescent microscopy, this event will be captured as a continuous decay of the fluores-
cence intensity---destaining of the vesicle. However, this was not the sole picture that
Tsien's group saw. Before bleaching completely, more than one-third of the stained termi-
nals displayed what Tsien called a "kink", a second de staining phase 20-60 seconds after
the initial destaining. Tsien's interpretation of this interesting phenomenon is that some of
the vesicles are performing a "kiss and run" show. Instead of collapsing with the terminal
membrane completely, these vesicles pinch-off early, retaining some unreleased transmit-
ters inside the vesicle and part of the dye in the vesicle membrane. In other words, a rapid
endocytosis happened after exocytosis, then followed by a second-phase release of the
freshly retrieved vesicle. To test the rate of this rapid endocytosis, they repeated the ex-
periments with another fluorescent dye, FM2-10, which has much lower affinities to the
lipid membrane than FMI-43. As predicted, terminals loaded with FM2-10 had a larger
first de staining phase and a smaller second phase. The time constant of the rapid endocy-
tosis process estimated by these experiments is about 6 seconds. Furthermore, it appears
that this process of rapid endocytosis is under some regulatory control, by both calcium
and staurosporine, a non-selective kinase inhibitor that only reduces dye de staining but
not transmitter releasing. If this phenomenon of variable rates of vesicular recycling has
physiological significance, this could be one more potential action site of ethanol in modi-
fying synaptic transmission.
During the second part of his presentation, Tsien tackled another challenging ques-
tion in synaptic research of the central nervous system-what causes the unitary synaptic
current at the central fast glutamatergic synapse to be so variable? Is it due to the variabil-
ity of presynaptic release or the saturation of the postsynaptic receptor? To answer this
question, they again used the FMI-43 dye to visualize individual presynaptic boutons. In
order to isolate individual synapses, a very small local application of the high K+, normal
Ca+ + solution to one bouton resulted in release from the same bouton without affecting
others 2 !lm away. The synaptic events in the postsynaptic cell were recorded with whole-
cell patch-clamp in the soma. Consistent fast time-course of the excitatory postsynaptic
currents (EPSCs) was seen, however, the EPSC amplitudes varied significantly among
many trials of a single bouton (Liu and Tsien, 1995). Two interpretations could explain
this phenomenon: first, variations of presynaptic glutamate release without saturating
postsynaptic receptors; and second, functional changes of saturated postsynaptic receptors.
By comparing the local high K+ -evoked synaptic events with local "puff' of glutamate-in-
duced synaFltic events, they found the answer is the former. The dose-response curves of
the two clearly indicated that postsynaptic receptors were not reaching saturation by
