Methods in Neurobiology

607 Pages·1981·24.48 MB·English

by  Philip Rosenberg (auth.), Robert Lahue (eds.)

Methods in
Neurobiology
VOLUME 1
Methods i n
Neurobiology
VOLUME 1
Edited by
Robert Lahue
University of Waterloo
Waterloo, Ontario, Canada
PLENUM PRESS • NEW YORK AND LONDON
Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in neurobiology.
Includes index.
1. Neurophysiology-Technique. 2. Neurobiology-Technique. I. Lahue, Robert.
QP357.M47 591.1'88'028 80-15623
ISBN 978-1-4684-3808-6 ISBN 978-1-4684-3806-2 (eBook)
DOl 1O.l007/978-1-4684-3806-2
© 1981 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1981
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N. Y. 10013
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, or otherwise, without written permission from the Publisher
Preface
Rapid advances in knowledge have led to an increasing interest in neuro
biology over the last several years. These advances have been made possible,
at least in part, by the use of increasingly sophisticated methodology.
Furthermore, research in the most rapidly advancing areas is essentially
multidisciplinary and is characterized by contributions from many investi
gators employing a variety of techniques. While a grasp of fundamental
neurobiological concepts is an obvious prerequisite for those who wish to
follow or participate in this field, critical awareness and evaluation of
neurobiological research also requires an understanding of sophisticated
methodologies.
The objective of Methods in Neurobiology is the development of such
critical abilities. The reader is exposed to the basic concepts, principles, and
instrumentation of key methodologies, and the application of each meth
odology is placed in the special context of neurobiological research. The
reader will gain familiarity with the terminology and procedures of each
method and the ability to evaluate results in light of the particular features
of neurobiological preparations and applications.
Robert Lahue
Waterloo
v
Contributors
P. Kontro, Department of Biomedical Sciences, University of Tampere,
Tampere, Finland
Robert Lahue, Department of Psychology, Renison College, University of
Waterloo, Waterloo, Ontario, Canada
E. Marani, Laboratory of Anatomy and Embryology, University of Leiden,
Leiden, The Netherlands
S. S. Oja, Department of Biomedical Sciences, University of Tampere, Tamp
ere, Finland
Neville N. Osborne, Nuffield Laboratory of Ophthalmology, University of
Oxford, Oxford OX2 6AW, England
Karl H. Pfenninger, Department of Anatomy, Columbia University, New
York, New York 10032
Kedar N. Prasad, Department of Radiology, University of Colorado Medical
Center, Denver, Colorado 80220
Philip Rosenberg, Section of Pharmacology and Toxicology, University of
Connecticut School of Pharmacy, Storrs, Connecticut 06268
Werner T. Schlapfer, Western Research and Development Office, Veterans
Administration Medical Center, Livermore, California 94550
R.J. Thompson, Department of Clinical Biochemistry, University of Cam
bridge, Cambridge, England
J. S. Woodhead, Department of Medical Biochemistry, Welsh National School
of Medicine, Cardiff CF4 4XN, Wales, and Department of Clinical
vi
Contents
Chapter 1
The Squid Giant Axon: Methods and Applications
Philip Rosenberg
1. Introduction 1
1.1. Uniqueness of the Squid Giant Axon. 2
1.2. A Brief History of Squid and Squid Users. 5
2. Methods 7
2.1. Obtaining and Maintaining Squid 7
2.2. Dissection of the Giant Axon. 9
2.3. Recording of Electrical Activity. 16
2.4. Extrusion of Axoplasm and Measurement of Penetration . 31
2.5. Perfusion of the Axon. 33
3. Applications 41
3.1. The Giant Axon: A Model for Nonmyelinated Axons . 41
3.2. The Electrical Spike . 58
3.3. The Optical Spike . 69
3.4. Biochemical Studies 77
3.5. Pharmacological Studies 92
4. Concluding Comments 109
4.1. Evaluation of Data 109
4.2. Generalization of Results 110
4.3. Some Final Thoughts 113
References 114
Chapter 2
Single-Cell Isolation and Analysis
Neville N. Osborne
1. Introduction. . . . . . . . . . .. 135
2. Single-Cell Isolation and Removal of Discrete Tissue Regions 136
vii
viii Contents
2.1. Choice and Validity of Isolated Neurons. 136
2.2. Isolation of Neurons. . . . . . 139
3. Microbiochemical Procedures. . . . . . . 146
3.1. Microanalysis of Transmitter Substances . 146
3.2. Microanalysis of Other Substances. 168
References . . . . . . . . . . . . 176
Chapter 3
Tissue and Organ Culture
Werner T. Schlapfer
1. Introduction. 183
1.1. Advantages and Disadvantages of Tissue Culture 184
1.2. Historical Developments 186
2. Development of Modern Methods of Nerve Tissue and Organ
Culture 186
3. Techniques for Long-Term Organotypic Cultures of Mammalian
and Avian Nervous Tissue . 188
3.1. General Considerations . 189
3.2. Slide Cultures 192
3.3. Long-Term Cultures in Petri Dishes 198
3.4. Tube Cultures . 201
3.5. Summary. 206
4. Short-Term Tissue Cultures of Mammalian and Avian Nervous
Tissue 207
4.1. Plasma-Clot Cultures . 207
4.2. Maximow-Chamber Cultures 207
4.3. Petri-Dish Cultures 208
5. Organ Cultures of Mammalian and Avian Nervous Tissue 209
5.1. Raft Cultures 210
5.2. Transfilter Cultures 212
5.3. Agar Technique . 213
5.4. Suspension Cultures 213
5.5. Petri-Dish Cultures 213
5.6. Flask Cultures 213
6. Tissue and Organ Cultures of Nervous Tissue of Cold-Blooded
Vertebrates 214
7. Tissue and Organ Cultures of Invertebrate Nervous Tissue. 215
7.1. Insects. 216
7.2. Molluscs 217
7.3. Other Invertebrates 218
8. Special Methodologies Associated with Mammalian and Avian Tissue
Culture 219
8.1. Histological Techniques 219
Contents ix
8.2. Special Biochemical Techniques. 223
8.3. Electron Microscopy. . . . 224
8.4. Autoradiography. .... 225
8.5. Electrophysiological Techniques. 225
9. Conclusion 229
References 229
Chapter 4
Cell Culture
Kedar N. Prasad
1. Introduction 245
2. Outline of Cell Culture Techniques 246
2.1. Explant Culture . 246
2.2. Aggregate Culture . 249
2.3. Dissociated Cell Culture 251
2.4. Monolayer Cell Culture. 256
2.5. Suspension Cell Culture 260
3. Concluding Remarks. 261
References 261
Chapter 5
Enzyme Kinetics
P. Kontro and S. S. Oja
1. Introduction . 265
2. Basic Kinetic Concepts 266
2.1. Kinetics of U ncatalyzed Reactions 266
2.2. Determination of Order of Reaction 269
2.3. Determination of First-Order Rate Constants 271
2.4. Determination of Second-Order Rate Constants 273
2.5. Effect of Temperature on Rate Constants 274
3. Kinetics of Unireactant Enzymes 275
3.1. Basic Theory of Enzyme Kinetics . 276
3.2. Time Course of Enzyme Reactions. 281
3.3. Determination of Kinetic Parameters 282
4. Inhibition Studies . 289
4.1. Basic Nomenclature 289
4.2. Inhibition Mechanisms 290
4.3. Analysis of Inhibition 291
4.4. Substrate Inhibition 299
4.5. Product Inhibition 301
x Contents
5. Cooperative Effects . 301
5.1. Sigmoid Kinetics 301
5.2. Allostery and Cooperativity 302
5.3. Hill Equation . . . . . 304
5.4. Graphic Presentation of Cooperative Effects 305
5.5. Models of Cooperativity . . 306
6. Kinetics of Two-Substrate Reactions. . 307
6.1. Reaction Mechanisms . . . . . 307
6.2. Determination of the Kinetic Parameters: Initial-Velocity
Measurements . 311
6.3. Substrate-Inhibition Studies 316
6.4. Product-Inhibition Studies. 316
6.5. Isotope-Exchange Studies . 317
7. Effects of pH and Temperature on Enzyme Kinetics. 318
7.1. Effects of pH . 318
7.2. Effects of Temperature. . . . . 322
8. Certain Neurobiological Applications. . 326
8.1. Transport of Organic Solutes across Nervous Membranes 326
8.2. Binding of Ligands to Receptors 330
9. Symbols and Their Definitions 333
References . . . . . . . . . . . 335
Chapter 6
Spectrophotometry and Fluorometry
Robert Lahue
1. Introduction. . .. ..... 339
1.1. Spectrum of Electromagnetic Radiation 340
2. Energy States of Matter. 344
2.1. Rotational Energy. . . . . 345
2.2. Vibrational Energy 348
2.3. Combination of Rotational and Vibrational Energies 350
2.4. Electronic Energy. . . . . . . 352
3. Quantum Absorption of Radiant Energy. 360
3.1. After Absorption . 362
3.2. Selectively Indexed References. . 366
3.3. Absorption Spectra . . . . . . 367
4. Quantitative Absorption Spectrophotometry 373
4.1. Laws of Absorption. . 373
4.2. Instrumentation 375
4.3. Spectrophotometer Components 377
4.4. Quantitative-Absorption Procedures 401
4.5. Fluorescence Spectra. . . 410