THE "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