Brain Slices

Brain Slices

Edited by RAYMOND DINGLEDINE University of North Carolina Chapel HilI, North Carolina

Plenum Press • New York and London

Library of Congress Cataloging in Publication Data

Main entry under title:

Brain slices.

Bibliography: p. Includes index. 1. Brain. 2. Electrophysiology-Technique. 3. Brain chemistry-Technique. 4.

Microtomy. l. Dingledine, Raymond, 1948- [DNLM: 1. Brain-Physiology. WL 300 B814j QP376.B734 1983 599'.01'88 83·22957

ISBN 978-1-4684-4585-5 ISBN 978-1-4684-4583-1 (eBook) DOI 10.1007/978-1-4684-4583-1

©1984 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1984 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

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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, or otherwise, without written permission from the Publisher

Con tribu tors

BRADLEY E. ALGER • Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland

PER ANDERSEN • Institute of Neurophysiology, University of Oslo, Oslo, Norway

MICHEL BAUDRY • Department of Psychobiology, University of California, Irvine, California

THOMAS H. BROWN • Department of Cellular Neurophysiology, Division of Neurosciences, City of Hope Research Institute, Duarte, California

BARRY W. CONNORS • Department of Neurology, Stanford Uni­versity School of Medicine, Stanford, California

S. S. DHANJAL • Sobell Department of Neurophysiology, Institute of Neurology, The National Hospital, University of London, Lon­don, England

RAYMOND DINGLEDINE • Department of Pharmacology, Univer­sity of North Carolina, Chapel Hill, North Carolina

JOHN GARTHWAITE • Department of Veterinary Physiology and Pharmacology, The University of Liverpool, Liverpool, England

A. GRINV ALD • The Weizmann Institute of Science, Rehovot, Israel MICHAEL J. GUTNICK • Unit of Physiology, Faculty of Health Sci­

ences, Ben Gurion University of the Negev, Beer-Sheva, Israel GLENN I. HATTON • Department of Psychology, and the Neuro­

science Program, Michigan State University, East Lansing, Michi­gan

v

vi Contributors

GRAEME HENDERSON • Department of Pharmacology, University of Cambridge, Cambridge, England

J0RN HOUNSGAARD • Department of Neurophysiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark

DANIEL JOHNSTON • Program in Neuroscience, Section of Neu­rophysiology, Department of Neurology, Baylor College of Medi­cine, Houston, Texas

MARKUS KESSLER • Department of Psychobiology, University of California, Irvine, California

GREGORY L. KING • Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina

H. KITA • Department of Anatomy, College of Medicine, The Uni­versity of Tennessee, Center for the Health Sciences, Memphis, Tennessee

S. T. KITAI • Department of Anatomy, College of Medicine, The Uni­versity of Tennessee, Center for the Health Sciences, Memphis, Tennessee

PETER LIPTON • Department of Physiology, University of Wiscon­sin, Madison, Wisconsin

RODOLFO R. LLiNAs • Department of Physiology and Biophysics, New York University Medical Center, New York, New York

GARY LYNCH • Department of Psychobiology, University of Cali­fornia, Irvine, California

HENRY McILWAIN • Department of Biochemistry, St. Thomas's Hospital Medical School, London, England

CHARLES NICHOLSON • Department of Physiology and Biophys­ics, New York University Medical Center, New York, New York

ALAN NORTH • Neuropharmacology Laboratory, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

PHILIP A. SCHWARTZKROIN • Department of Neurological Sur­gery, University of Washington, Seattle, Washington

T. A. SEARS • Sobell Department of Neurophysiology, Institute of Neurology, The National Hospital, University of London, London, England

M. SEGAL • The Weizmann Institute of Science, Rehovot, Israel DENNIS A. TURNER • Department of Neurosurgery, VA Medical

Center, Minneapolis, Minnesota TIM S. WHITTINGHAM • Laboratory of Neurochemistry, National

Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland

JOHN WILLIAMS • Neuropharmacology Laboratory, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

Preface

In little less than a decade brain slices have gained prominence among neurobiologists as appropriate tools to study cellular electrophysiolog­ical aspects of mammalian brain function. The purpose of this volume is to present in some detail several inquiries in the brain sciences that have benefited greatly by the use of brain slices. The book is directed primarily toward advanced students and researchers wishing to evaluate the impact these in vitro preparations of the mammalian brain are having on neurobiology.

The term brain slice has come to refer to thin (100-700 j.Lm) sections of a brain region prepared from adult mammals and maintained for many hours in vitro, for either electrophysiological or biochemical stud­ies. In addition to good accessibility, slices feature relatively intact syn­aptic connections that allow a variety of experiments not feasible with standard in vivo or tissue culture preparations. Certain electrophysiol­ogical studies once practical only with invertebrate models are becoming routine with mammalian brain slices. The ability to perform both bio­chemical and electro physiological experiments on the same piece of CNS tissue provides additional bright prospects for future research. Although most of the electrophysiological studies have dealt with hippocampal slices, it should be evident from this book that slice methodology is not limited to the hippocampus.

The Appendix, "Brain Slice Methods," is a multiauthored treatment of the technical aspects of brain slice work, collected into one document. The procedures developed in many laboratories for the preparation of

vii

viii Preface

slices are described and compared. The comparison of slice with in vivo data from the viewpoints of electrophysiology, metabolic function, and histology receives a good deal of discussion. This has represented an opportunity for the authors to comment on their experiences, and those of others, in developing a range of techniques. The appendix is thus a broad-based working handbook that should be valuable to the pro­spective slicer as well as current practitioners.

This book will have served its purpose if it conveys some of the excitement generated by these preparations, and if it helps to establish the special promise slices have for advancing our understanding of the brain.

Raymond Dingledine Chapel Hill, North Carolina

Contents

INTRODUCTION: CEREBRAL SUBSYSTEMS AS BIOLOGICAL ENTITIES......................................... 1

HENRY McILWAIN

1. COMPARATIVE ELECTROBIOLOGY OF MAMMALIAN CENTRAL NEURONS

RODOLFO R. LUNAs

1. Introduction ............................................................. 7 2. The Generalized Neuron ............................................ 8

2.1. The Axon .......................................................... 8 3. Electrophysiology of the Neuronal Somata .................... 9

3.1. The Na Conductances ......................................... 9 3.2. Ca Conductances ............................................... 11 3.3. The K Conductances .......................................... 13

4. Dendritic Electrophysiology ........................................ 16 5. Discussion ................................................................ 18 6. References ................................................................ 20

ix

x

2. PASSIVE ELECTROTONIC STRUCTURE AND DENDRITIC PROPERTIES OF HIPPOCAMPAL NEURONS

Contents

DENNIS A. TURNER and PHILIP A. SCHW ARTZKROIN

1. Introduction ............................................................. 25 1.1. Why Develop Models of Neurons? ....................... 25 1.2. Hippocampal Slices as a Substrate for Theoretical

Models ............................................................. 28 2. Electrotonic and Circuitry Models-An Overview of

Methods .................................................................. 29 2.1. Network Model................................................. 29 2.2. Continuous Cable Models ................................... 29 2.3. Compartmental Models ....................................... 31 2.4. Neuronal Circuit Models ..................................... 32

3. Models Constructed with Data from Work on Hippocampal Slices ....................................... '" ...... ... 33 3.1. Continuous Cable Models ................................... 33 3.2. The Compartmental Model.................................. 41 3.3. Neuronal Circuitry Model................................... 42 3.4. Overview of Hippocampal Slice Data .................... 42

4. Discussion ................................................................ 43 4.1. Insights from the Models of Electrotonic

Structure ........................................................... 43 4.2. Models of Cell Circuitry ...................................... 45 4.3. Contributions of the Slice Preparation to Model

Studies ............................................................. 45 4.4. Experimental Directions and Testable Predictions ... 46

5. References ................................................................ 47

3. BIOPHYSICS AND MICROPHYSIOLOGY OF SYNAPTIC TRANSMISSION IN HIPPOCAMPUS DANIEL JOHNSTON and THOMAS H. BROWN

1. Motivation for Studying the Biophysics and Microphysiology of Cortical Synapses .......................... 51

2. Criteria for Selecting a Suitable Cortical Synaptic Preparation ...... ....... .............................. ................... 52 2.1. Identifiable Neurons and Synapses ....................... 52 2.2. Stable Intracellular Recordings ............................. 53

Contents xi

2.3. Minimal Diffusional Barriers ................................ 53 2.4. Monosynaptic Connection from Stimulus Site ....... . 53 2.5. Minimal Electrotonic Distance between Sub synaptic

Membrane and Recording Site ............................. 53 2.6. Measurement of Single Quantal Events ................. 54 2.7. Application of Voltage-Clamp Techniques .............. 54 2.8. A Synapse That Satisfies These Criteria in the

Hippocampal Slice Preparation ............................. 54 3. Development of Voltage-Clamp Techniques for

Application to Hippocampal Synapses .......................... 54 3.1. Techniques for Voltage Clamping Small Cortical

Neurons ........................................................... 55 3.2. The Problem of Space Clamp ............................... 58

4. Current- and Voltage-Clamp Studies of Evoked Synaptic Events in Hippocampal Neurons ................................. 63 4.1. Analysis of Mossy-Fiber Evoked Synaptic

Potentials ....................................................... 63 4.2. Analysis of Mossy-Fiber Evoked Synaptic

Currents . . . . . . . . . . . .... .. . . . . ... . . . . . . . . . . .... . . . .... ... . . . . . . ... . 64 5. Current- and Voltage-Clamp Studies of Spontaneous

Miniature Synaptic Events in Hippocampal Neurons ...... 67 5.1. The Quantum Hypotheses .................................. 67 5.2. Discovery of Spontaneous Miniature Synaptic

Potentials in Hippocampal Neurons ...................... 69 5.3. Current- and Voltage-Clamp Studies of Single

Quantal Events .................................................. 70 6. Implications for Performing a Quantal Analysis of

Evoked Release ......................................................... 74 6.1. The Problem of Mixed Synaptic Responses ............ 75 6.2. Some Advantages of Voltage-Clamp Analysis ......... 75 6.3. The Importance of a Suitable Signal-to-Noise

Ratio ................................................................ 76 6.4. A Caveat Concerning Release and Quantal Size

Statistics ........................................................... 76 7. Significance for Selected Problems in Cortical

Physiology .... ......................................................... 76 7.1. Mechanism of Long-Term Synaptic Potentiation ..... 77 7.2. Role of Dendrites and Their Spines in Synaptic

Information Transfer and Integration .................... 78 7.3. Currents Underlying Epileptiform Discharges ........ 79 7.4. Summary and Conclusions .................................. 81

8. References ................................................................ 81

xii Contents

4. HIPPOCAMPUS: SYNAPTIC PHARMACOLOGY RAYMOND DINGLEDINE

1. Introduction ............................................................. 87 2. Localization of Transmitters and Endogenous

Neuroactive Agents in the Hippocampal Formation ........ 88 2.1. Transmitter Candidates ....................................... 88 2.2. Neuroactive Pep tides .......................................... 91

3. Cellular Actions of Neuroactive Drugs in Hippocampal Slices ....................................................................... 92 3.1. GABA .............................................................. 92 3.2. Acetylcholine..................................................... 95 3.3. Opioid Peptides ................................................. 100 3.4 .. Excitatory Amino Acids ....................................... 105

4. Conclusions and Future Directions .............................. 107 5. References ................................................................ 108

5. ENERGY METABOLISM AND BRAIN SLICE FUNCTION PETER LIPTON and TIM S. WHITTINGHAM

1. Introduction ............................................................. 113 2. Integrity of the Slice Preparation ................................. 114

2.1. Comparison of Energy-Related Parameters in Slices and in Situ ........................................................ 114

2.2. Possible Bases for Compromised Function in the Brain Slice ......................................................... 121

3. Mechanism of Anoxic Damage .................................... 129 3.1. Metabolic Changes during Anoxia ........................ 129 3.2. Effects of Metabolic Changes Occurring during

Anoxia on Neurotransmission .............................. 130 3.3. Experimental Evidence Concerning the Mechanism

of Synaptic Transmission Failure during Compromised Oxidative Phosphorylation .............. 135

4. References ................................................................ 147

6. HIPPOCAMPUS: ELECTROPHYSIOLOGICAL STUDIES OF EPILEPTIFORM ACTIVITY IN VITRO BRADLEY E. ALGER

1. Introduction ............................................................. 155 1.1. Experimental Questions ...................................... 156 1.2. Terminology...................................................... 157

Contents xiii

2. What Enables Some Cells to Fire Bursts Readily? ........... 158 2.1. Paroxysmal Depolarization Shift (PDS) .................. 158 2.2. Regenerative Components of the Burst.................. 163

3. Why Do Cells That Can Fire Burst Potentials Not Do So All the Time? ............................................................ 164 3.1. Inhibitory Postsynaptic Potentials (IPSPs) Prevent

Bursts ................ ;.............................................. 164 3.2. Feedforward Dendritic Inhibition .......................... 166

4. How Does Synchronization of Firing within a Population of Cells Occur? ......................................................... 169 4.1. Imposed Synchrony............................................ 169 4.2. "Spontaneous" Synchrony .................................. 172 4.3. Electrotonic Interactions ...................................... 174

5. What Triggers the Switch from Interictal Spiking to Seizures? .................................................................. 177 5.1. Burst Afterhyperpolarizations (AHP) ..................... 177 5.2. The Late Hyperpolarizing Potential (LHP) is a Slow

IPSP ................................................................. 179 5.3. Seizure Development .......................................... 183

6. How Can a Seizure Spread from Epileptic Tissue across Normal Tissue? ......................................................... 184 6.1. Endogenous Opiates ....... . ........... . . . . . .... .... . . ...... . . 184 6.2. Acetylcholine..................................................... 185 6.3. Use-Dependent IPSP Depression .......................... 186 6.4. Ammonia.............................. .......... .................. 190

7. Conclusions .............................................................. 192 8. References ................................................................ 193

7. CORRELATED ELECTROPHYSIOLOGICAL AND BIOCHEMICAL STUDIES OF HIPPOCAMPAL SLICES GARY LYNCH, MARKUS KESSLER, and MICHEL BAUDRY

1. Introduction ............................................................. 201 2. Modification of Stimulation Procedures and Slice

Techniques for Biochemical Experiments ....................... 202 3. Hippocampal Long-Term Potentiation .......................... 207 4. Influences of High-Frequency Stimulation on 3H_

Glutamate Binding to Synaptic Membranes ................... 209 4.1. Characteristics of 3H-Glutamate Binding to

Hippocampal Synaptic Membranes ....................... 209

xiv Contents

4.2. Effect of High-Frequency Stimulation on 3H_ Glutamate Binding ............................................. 210

4.3. Calcium Regulation of 3H-Glutamate Binding to Hippocampal Membranes .................................... 211

5. High-Frequency Stimulation and Protein Phosphorylation ........................................................ 214

6. Summary ................................................................. 220 7. References ................................................................ 222

8. OPTICAL MONITORING OF ELECTRICAL ACTIVITY: DETECTION OF SPATIOTEMPORAL PATTERNS OF ACTIVITY IN HIPPOCAMPAL SLICES BY VOLTAGE-SENSITIVE PROBES A. GRINV ALD and M. SEGAL

1. Introduction ............................................................. 227 1.1. Preview ............................................................ 227 1.2. The Limitations of Current Intracellular Electrical

Recording Techniques ........................................ . 1.3. The Principle of the Optical Recording Technique .. .

2. Optical Monitoring of Changes in Membrane Potential .. . 2.1. The Apparatus .................................................. . 2.2. Design and Synthesis of Improved Optical

Probes ........................................................... . 2.3. Example of Application to the Study of Single Cells

and the Study of Invertebrate CNS ...................... . 3. Optical Recording from Brain Slices ............................ .

3.1. Preparation of the Slices for Optical Recordings ..... . 3.2. The Correction Procedure for Light-Scattering

Signals ............................................................ . 3.3. Optical Signals from a Stained Slice ..................... . 3.4. Light-Scattering Signals from Unstained Slices ....... . 3.5. Ca2 + Dependency of the Fast and Slow

Responses ..................................................... . 3.6. Effects of Tetrodotoxin on the Fast Responses ....... . 3.7. Optical"Artifacts" near the Stimulating Electrode

3.8. Properties of the Unmyelinated Axons in the

228 228 229 229

231

232 236 237

240 241 243

244 245

245

Hippocampus Slice ............................................. 246 3.9. Postsynaptic Responses ....................................... 248

Contents xv

3.10. The Cellular Discharges ...................................... 249 3.11. Examples of Pharmacological Studies: The Effect of

Picrotoxin ......................................................... 250 3.12. Visualization of the Spread of Activity in Slices ...... 250 3.13. Comparison between Field Potentials and Optical

Recordings ........................................................ 253 3.14. Limitations and Advantages ................................ 254 3.15. Future Prospects ................................................ 257

4. References ................................................................ 258

9. PROBING THE EXTRACELLULAR SPACE OF BRAIN SLICES WITH ION-SELECTIVE MICROELECTRODES J0RN HOUNSGAARD and CHARLES NICHOLSON

1. Introduction ............................................................. 263 2. The Brain Cell Microenvironment ................................ 263 3. Extracellular Ion Changes Produced by Simultaneous

Activity of Ensembles of Neurons ................................ 268 4. Extracellular Ion Changes Evoked by Individual Cells ..... 269

4.1. Spontaneous Purkinje Cell Activity ....................... 271 4.2. Changes in [K +]0 during Simultaneous Intracellular

Recording and Current Passage ............................ 273 4.3. Changes in [K+]o Recorded outside Glia Cells

during Intracellular Current Passage ..................... 276 5. Prospects and Problems ............................................. 278 6. References ................................................................ 282

10. ELECTROPHYSIOLOGICAL STUDY OF THE NEOSTRIATUM IN BRAIN SLICE PREPARATION S. T. KITAI and H. KITA

1. Introduction ............................................................. 285 2. Methods .................................................................. 286

2.1. Electrode . . . . . . . . . . . . . . .. . .... . . . .... ...... . . . . . . . .. . ... . . .. ... . . . . 286 2.2. Slice Chamber ................................................... 286 2.3. Slice Preparation . ............................................... 288

3. Results and Discussion ............................................... 289 3.1. Field Potentials .................................................. 289

xvi Contents

3.2. Intracellular Recording ........................................ 290 3.3. Discussion and Summary .................................... 293

4. References ................................................................ 296

11. LOCUS COERULEUS NEURONS JOHN WILLIAMS, GRAEME HENDERSON, and ALAN NORTH

1. Introduction ............................................................. 297 2. Methods .................................................................. 298 3. Results .................................................................... 299

3.1. Guinea Pig Locus Coeruleus ................................ 299 3.2. Guinea Pig Mesencephalic Nucleus of the

Trigeminal Nerve ............................................... 300 3.3. Rat Locus Coeruleus ........................................... 300 3.4. Pharmacological Studies ...................................... 301

4. Discussion ................................................................ 307 5. References ................................................................ 311

12. NEOCORTEX: CELLULAR PROPERTIES AND INTRINSIC CIRCUITRY BARRY W. CONNORS and MICHAEL J. GUTNICK

1. Introduction ......................................................... .... 313 2. Early Use of Neocortical Slices .................................... 315 3. Notes on Neocortical Slice Methodology................. ...... 316 4. Properties of Neocortical Slices ........ ...... ...................... 318

4.1. Electrophysiology of Single Cells .......................... 318 4.2. Synaptic Behavior of Neocortical Neurons ............. 323 4.3. Intrinsic Connectivity of Neocortical Slices . ............ 329

5. Conclusions .............................................................. 334 6. References ................................................................ 335

13. HYPOTHALAMIC NEUROBIOLOGY GLENN I. HATTON

1. Introduction ................ ... .......................................... 341 2. The Development of the Hypothalamic Slice

Preparation ................................................ .............. 341

Contents xvii

2.1. Motivating Factors .............................................. 341 2.2. Early Problems Encountered ................................ 344

3. Hypothalamic Slice Preparations and Their Uses ............ 344 3.1. Magnocellular Areas ........................................... 345 3.2. Parvocellular Areas ............................................ 363

4. Summary and Conclusions ......................................... 368 5. References ................................................................ 370

14. BRAIN SLICE WORK: SOME PROSPECTS PER ANDERSEN

1. Introduction ............................................................. 375 2. Optimal Conditions ................................................... 375 3. Slices from New Regions ............................................ 377 4. New Uses of Slices .................................................... 378 5. New Approaches in Slice Experiments ......................... 378 6. The Need for Correlation ........................................... 379 7. References ................................................................ 379

APPENDIX: BRAIN SLICE METHODS BRADLEY E. ALGER, S. S. DHANJAL, RAYMOND DINGLEDINE, JOHN GARTHWAITE, GRAEME HENDERSON, GREGORY L. KING, PETER LIPTON, ALAN NORTH, PHILIP A. SCHWARTZKROIN, T. A. SEARS, M. SEGAL, TIM S. WHITTINGHAM, and JOHN WILLIAMS

1. Introduction ............................................................. 381 2. Preparation of Slices .................................................. 382

2.1. Slicing the Brain . . . . . . ...... . ........... . . . . ...... .... ...... .. . . 382 2.2. Slice Chambers .................................................. 391 2.3. Bathing Medium Composition ............................. 397 2.4. Slice Thickness .................................................. 404

3. Evaluation of Slice Data .............................................. 405 3.1. General Remarks ................................................ 405 3.2. Electrophysiology............................................... 406 3.3. Histology.......................................................... 413 3.4. Metabolism ....................................................... 422 3.5. Spreading Depression ......................................... 425

xviii Contents

4. Methods of Drug Application ...................................... 425 4.1. Superfusion....................................................... 426 4.2. The Nanodrop ........ ........................................... 427 4.3. Iontophoresis .................................................... 429 4.4. The Pressure Pipette ........................................... 430 4.5. Summary ................................... :................. ..... 431

5. Concluding Statement ................................................ 431 6. References ................................................................ 432

INDEX ................................................................ 439