squid as experimental animals - springer978-1-4899-2489-6/1.pdfsquid as experimental animals edited...

SQUID AS EXPERIMENTAL

ANIMALS

From L. W. Williams, 1909, The Anatomy of the Common Squid, Loligo pealii, Lesueur, E. J. Brill, Leiden, Holland. In the illustration by Williams, the top figure of the whole squid is oriented with the physiological dorsal surface facing downwards and the animal appears to be swimming upside-down.

SQUID AS EXPERIMENTAL

ANIMALS

Edited by

Daniel L. Gilbert and William J. Adelman, Jr. National Institutes of Health

Bethesda, Maryland

and John M. Arnold

University of Hawaii Honolulu, Hawaii

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

L1brary of Congress Catalog1ng-1n-Publ1cation Data

Squid as experimental antmals 1 edited by Daniel L. Gllbert, William J. Adelman, Jr., and John M. Arnold.

p. cm. Includes bibliographical references. ISBN 978-1-4899-2491-9 ISBN 978-1-4899-2489-6 (eBook)DOI 10.1007/978-1-4899-2489-6 1. Squids as laboratory anlmals. 2. Nervous system--Mollusks.

3. Squtds--Cytology. I. Gilbert, Daniel L. II. Adelman, William J., 1928- III. Arno 1 d, John M. QL430.2.S66 1990 594 · . 58--dc20

© 1990 Springer Science+Business Media New York Originally published by Plenum Press, New York in 1990

Softcover reprint ofthe hardcover 1 st edition 1990

All rights reserved

90-6849 CIP

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

Contributors

William J, Adelman, Jr. Laboratory of Biophysics, NINDS, National Institutes of Health, Bethesda, MD 20892

Mario Alberghina Marine Biological Laboratory, Woods Hole, MA 02543 and Institute of Biochemistry, Faculty of Medicine, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy

Frederick A. Aldrich Ocean Studies Task Force and Department of Biology, Memorial University of Newfoundland, St. John's, Newfoundland A1C 5S7, Canada

John M. Arnold Pacific Biomedical Research Center, Cephalopod Biology Laboratory, 209A Snyder Hall, 2538 The Mall, University of Hawaii at Manoa, Honolulu, Hawaii 96822

Francisco Bezanilla Department of Physiology, Ahmanson Laboratory of Neurobiology and Jerry Lewis Neuromuscular Center, University of California at Los Angeles, Los Angeles, California 90024

F. J, Brinley, Jr. Neurological Disorders Program, National Institute of Neurological Disorders and Stroke, Federal Building, Room 814, National Institutes of Health, Bethesda, MD 20892

Anthony Brown Bio-architectonics Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Bernd U. Budelmann Marine Biomedical Institute and Department of Otolaryngology, University of Texas Medical Branch, Galveston, Texas 77550

Lawrence B. Cohen Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Rochelle S. Cohen Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois 60612

Harold Gainer Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD 20892

Daniel L. Gilbert Laboratory of Biophysics, NINDS, National Institutes of Health, Bethesda, MD 20892

vi Contributors

Robert M. Gould Marine Biological Laboratory, Woods Hole, MA 02543 and New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314

Roger T. Hanlon Marine Biomedical Institute and Department of Psychiatry and Behavioral Sciences, University of Texas Medical Branch, Galveston, Texas 77550-2772

Francis C. G. Hoskin Biology Department, Illinois Institute of Technology, Chicago, IL 60616

David Landowne Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101

George M. Langford Marine Biological Laboratory, Woods Hole, MA 02543 and Department of Physiology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599

Raymond J. Lasek Bio-architectonics Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Charlotte P. Mangum Dept. of Biology, College of William and Mary, Williamsburg, Virginia 23185

I. A. Meinertzhagen Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 411

Monica A. Meyer Marine Biological Laboratory, Woods Hole, MA 02543

RuthAnne Mueller Laboratory of Biophysics, NINDS, NIH, Bethesda, MD. 20892

Lorin J, Mullins Department of Biophysics, Medical School, University of Maryland, 660 W. Redwood Street, Baltimore, MD 21201

Ron O'Dor Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada B3H 411

Harish C. Pant Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD 20892

H. 0. Portner Institut ftir Zoologie IV, Universitllt Dusseldorf, Universitlitsstrasse 1 D-4000 DUsseldorf 1, F. R. Germany

Robert V. Rice Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, PA 15213

Helen R. Saibil Department of Zoology, Oxford University, Oxford OX1 3PS, England. Present address: Department of Crystallography, University of London Birkbeck College, Malet Street, London WC1E 7HX, England

Contributors vii

Brian M. Salzberg Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

R. E. Shadwick Biology Department, University of Calgary, Calgary, Alberta, Canada T2N1N4

Elis F. Stanley Laboratory of Biophysics, NINDS, National Institutes of Health, Bethesda. MD 20892

William C. Summers Huxley College of Environmental Studies and Shannon Point Marine Center, Western Washington University, Bellingham, Washington 98225

Carol Vandenberg Department of Biological Sciences, University of California at Santa Barbara, Santa Barbara, California, 93106

Dieter G. Weiss Marine Biological Laboratory, Woods Hole, MA 02543 and Institut ftir Zoologie, Technische Universtlit Miinchen, D-8046 Garching, Fed. Rep. Germany

ix

Preface

The predecessor to this book was A Guide to the Laboratory Use of the Squid Loligo pealei published by the Marine Biological Laboratory, Woods Hole, Massachusetts in 1974. The revision of this long out of date guide, with the approval of the Marine Biological Laboratory, is an attempt to introduce students and researchers to the cephalopods and particularly the squid as an object of biological research. Therefore, we have decided to expand on its original theme, which was to present important practical aspects for using the squid as experimental animals. There are twenty two chapters instead of the original eight. The material in the original eight chapters has been completely revised. Since more than one method can be used for accomplishing a given task, some duplication of methods was considered desirable in the various chapters. Thus, the methodology can be chosen which is best suited for each reader's requirements. Each subject also contains a mini-review which can serve as an introduction to the various topics. Thus, the volume is not just a laboratory manual, but can also be used as an introduction to squid biology. The book is intended for laboratory technicians, advanced undergraduate students, graduate students, researchers, and all others who want to learn the purpose, methods, and techniques of using squid as experimental animals. This is the reason why the name has been changed to its present title. Preceding the chapters is a list of many of the abbreviations, prefixes, and suffixes used in this volume.

Cephalopods possess the most advanced nervous system of all invertebrates. For this reason, there are a predominance of chapters on the components of the nervous systems. Part I deals with evolution, history, and maintenance. The frrst chapter is on evolution of intelligence. Then chapters follow on squid in its natural habitat, the discovery of Loligo, squid maintenance and rearing. Part 2 contains two chapters on squid mating and embryology. Part 3 includes the neural membranes. Chapters in this part are concerned with electrophysiology of the squid axon, internal dialysis in the squid axon, the cut-open axon, optical measurements on squid axons, and the squid giant synapse. Part 4 contains chapters on cell biology. These are on tissue culture techniques, squid optic lobe synaptosomes, the cytoskeleton of the giant axon, axoplasmic transport using video microscopy, and lipid metabolism in the nervous system. Part 5 has some chapters on the sensory systems, which are the squid eye, the development of the visual system, and the statocysts of squid. Finally, Part 6 concludes with integrated systems. Chapters in this part deal with the squid as a whole. Blood oxygen and carbon dioxide gas transport to and from the tissues is the topic of the frrst chapter in this part. This is followed by a chapter on a detoxifying enzyme unique to the squid. The final chapter presents the integration of all the squid systems as a whole for the functioning of the squid in its natural habitat. Due to lack of space, other aspects of squid biology, such as physiology of the central nervous system, digestion, and excretion, are not included.

X Preface

The frontispiece is taken from the frontispiece in Leonard Worcester Williams classic, The Anatomy of the Common Squid, Loligo pea/ii, Lesueur, published in 1909 by E. J. Brill, Lei den, Holland.

ACKNOWLEDGEMENTS: Thanks are given to Dr. Claire Gilbert for her editorial assistance and to the staff of Plenum Press, especially Mary P. Born, Senior Editor, John Matzka, Managing Editor and his assistant, Gregory Safford. We acknowledge the intramural support of the Basic Neurosciences Program of the National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.

DEDICATION: This book is dedicated to the memory of two distinguished scientists, who recently passed away: Kenneth S. Cole, who did the pioneering studies on the squid giant axon, and Gilbert L. Voss, who was a world renowned authority on squid.

The Editors Marine Biological Laboratory Woods Hole, Massachusetts 02543 September, 1989

xi

Contents

Abbreviations, Prefixes, and Suffixes Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxvii Prefixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxi Suffixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxi

PART I. EVOLUTION, HISTORY, AND MAINTENANCE

Chapter 1 Evolution and Intelligence of the Cephalopods

JOHN M. ARNOLD

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1. Intelligence and behavior . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2. Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.1. Evolution and competition . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2. Evolution of form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.3. Evolution of function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2 Natural History and Collection

WILLIAM C. SUMMERS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.1. The ecological dichotomy . . . . . . . . . . . . . . . . . . . . . . . . 11 1.2. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.2.1. Terminology of the squid, Loligo pealei . . . . . . . . . . 13 1.3. A functional taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.4. Biological strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.5. Distribution and fisheries . . . . . . . . . . . . . . . . . . . . . . . . . 18 1.6. Selection and handling . . . . . . . . . . . . . . . . . . . . . . . . . . 20 1.7. Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

xii Contents

Chapter 3 Lol-i-go and Far Away: A Consideration of the Establishment of the Species Designation Loligo pealei

FREDERICK A. ALDRICH

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2. Vernacular nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3. An afterword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Chapter 4 Maintenance, Rearing, and Culture of Teuthoid and Sepioid Squids

ROGER T. HANLON

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 3. Anatomical and behavioral traits relevant to laboratory handling . . . . . 37

3.1. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2. Locomotor habits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3. Sensory systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 3.4. Mode of feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.5. Egg size and hatchling behavior . . . . . . . . . . . . . . . . . . . . 41 3.6. Social behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4. Water quality and closed vs. open seawater systems . . . . . . . . . . . . 43 5. Capture and transport of eggs, juveniles and adults . . . . . . . . . . . . . 45

5.1. Egg care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.2. Juveniles and adults . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

6. Maintenance of wild-caught juveniles and adults . . . . . . . . . . . . . . 48 6.1. Tank configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 6.2. Behavior, feeding and growth . . . . . . . . . . . . . . . . . . . . . . 50

7. Rearing and culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 7 .1. Loligo spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 7 .2. Sepioteuthis lessoniana . . . . . . . . . . . . . . . . . . . . . . . . . . 54 7.3. Sepia officina/is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

8. Mortality and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 9. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

10. Future considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Contents xiii

PART II. MATING BEHAVIOR AND EMBRYOLOGY

Chapter 5 Squid Mating Behavior

JOHN M. ARNOLD

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 2. Reproductive anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

2.1. Male reproductive system . . . . . . . . . . . . . . . . . . . . . . . . . 66 2.2. Female reproductive system . . . . . . . . . . . . . . . . . . . . . . . 67 2.3. Mating behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 2.4. Copulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 2.5. Egg deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Chapter 6 Embryonic Development of the Squid

JOHN M. ARNOLD

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 2. Handling techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 3. Stages of normal cephalopod development . . . . . . . . . . . . . . . . . . 81

3.1. Fertilization and meiosis . . . . . . . . . . . . . . . . . . . . . . . . . 85 3.2. Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 3.3. Establishment of the germinal layers . . . . . . . . . . . . . . . . . . 86 3.4. Completion of the cellulation of the egg surface . . . . . . . . . . 86 3.5. Organogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

PART III. NEURAL MEMBRANES

Chapter 7 Electrophysiology and Biophysics of the Squid Giant Axon

WILLIAM J. ADELMAN, JR. and DANIEL L. GILBERT

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 1.1. Overall view of nerve function . . . . . . . . . . . . . . . . . . . . . 94

2. The giant axon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 3. Electrophysiology of the giant axon . . . . . . . . . . . . . . . . . . . . . . 95

3.1. Intracellular recording . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 3.2. From current to voltage clamp . . . . . . . . . . . . . . . . . . . . . 96 3.3. Microinjection, internal dialysis, and internal perfusion . . . . . . 97 3.4. Chemical blockers of specific ionic conductances . . . . . . . . . . 97

xiv Contents

3.5. Membrane noise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 3.6. Single channel currents . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.7. Ion channel gating currents . . . . . . . . . . . . . . . . . . . . . . 100 3.8. Gating currents indicate molecular conformational changes . . . 101

4. The giant axon preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 4.1. Location of the giant axon in the squid . . . . . . . . . . . . . . . 102 4.2. Preparations for dissecting the mantle nerves . . . . . . . . . . 103 4.3. Dissecting the mantle nerves . . . . . . . . . . . . . . . . . . . . . . 105 4.4. Storing the mantle nerves . . . . . . . . . . . . . . . . . . . . . . . 107 4.5. Isolation of the giant axon . . . . . . . . . . . . . . . . . . . . . . . 108 4.6. Physiological saline for the giant axon . . . . . . . . . . . . . . . Ill

5. Electrophysiological methods . . . . . . . . . . . . . . . . . . . . . . . . . . 112 5.1. Whole axon voltage clamp . . . . . . . . . . . . . . . . . . . . . . . 112

5.1.1. Axon preparation for voltage clamping . . . . . . . . . . 113 5.1.2. Voltage clamp chamber and electrodes . . . . . . . . . . 114 5.1.3. Internal perfusion and electrode placement . . . . . . . . 115

5.1.3.1. Piggy-back technique . . . . . . . . . . . . . . 115 5.1.3.2. Two internal electrode technique . . . . . . . 115

5.2. Forcing functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 5.2.1. Square waves . . . . . . . . . . . . . . . . . . . . . . . . . . 119 5.2.2. Sinusoidal forcing functions . . . . . . . . . . . . . . . . . 121 5.2.3. Other forms of forcing functions . . . . . . . . . . . . . . 122

5.3. Data acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 5.3.1. Storage of voltage clamp data . . . . . . . . . . . . . . . 124

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Chapter 8 Internal Dialysis in the Squid Giant Axon

LORIN J. MULLINS and F. J. BRINLEY, JR.

1. Historical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 2. Materials for internal dialysis . . . . . . . . . . . . . . . . . . . . . . . . . . 137

2.1. Evaluation of porosity to low molecular weight substances . . . 139 2.1.1. Porous acetate capillaries . . . . . . . . . . . . . . . . . . 139 2.1.2. Hollow cellulose fibers . . . . . . . . . . . . . . . . . . . 140

3. Solutions for internal dialysis . . . . . . . . . . . . . . . . . . . . . . . . . 140 4. Theoretical analysis of diffusion within the porous capillary and

axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 4.1. Efflux experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

4.1.1. End effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 4.2. Influx experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

5. Design of dialysis chambers . . . . . . . . . . . . . . . . . . . . . . . . . . 146 5.1. Efflux chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 5.2. Special precautions to remove ATP from axoplasm . . . . . . . 148 5.3. Influx chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

6. Hardware for control of internal dialysis . . . . . . . . . . . . . . . . . . 150 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Contents XV

Chapter 9 The Cut-Open Axon Technique

FRANCISCO BEZANILLA and CAROL VANDENBERG

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153 2. The cut-open axon technique for small population of channels . . . . . 153 3. The cut-open axon technique for single channel recording . . . . . . . 154 4. Method and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

4.1. Experimental set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 4.2. Experimental procedure . . . . . . . . . . . . . . . . . . . . . . . . . 156 4.3. Experimental results . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Chapter 10 Optical Measurements on Squid Axons

LAWRENCE B. COHEN, DAVID LANDOWNE, and BRIAN M. SALZBERG

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 2. Optical studies of structural changes in axons . . . . . . . . . . . . . . . 162

2.1. Light scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 2.2. Birefringence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 2.3. Optical activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

3. Screening for larger optical signals for monitoring activity . . . . . . . 165 4. Optical determination of the series resistance in Loligo . . . . . . . . . 166 5. Fast measurements of potentiometric probe response . . . . . . . . . . . 168

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Chapter 11 The Preparation of the Squid Giant Synapse for Electrophysiological Investigation

ELlS F. STANLEY

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 1.1. The stellate ganglion . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 1.2. The giant synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

2. The giant synapse as an experimental preparation . . . . . . . . . . . . . 173 2.1. Experimental advantages of the giant synapse preparation . . . 174

2.1.1. Large size of the pre- and postsynaptic giant axons (Young, 1939). . . . . . . . . . . . . . . . . . . . . . . . . . 174

2.1.2. Fast transmitting synapse . . . . . . . . . . . . . . . . . . 174 2.1.3. High release capacity . . . . . . . . . . . . . . . . . . . . . 174 2.1.4. Rapid exchange of external solutions . . . . . . . . . . . 174 2.1.5. Facilitation and depression . . . . . . . . . . . . . . . . . . 174 2.1.6. Analysis of ionic currents in a nerve terminal . . . . . 175 2.1.7. Single presynaptic input-output relations . . . . . . . . . 175

xvi Contents

2.1.8. Nerve terminal capacitance . . . . . . . . . . . . . . . . . 175 2.2. Experimental disadvantages of the giant synapse . . . . . . . . . 175

2.2.1. Unidentified transmitter substance . . . . . . . . . . . . . 175 2.2.2. Miniature excitatory postsynaptic potentials

(MEPPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 2.2.3. More than one presynaptic axon . . . . . . . . . . . . . . 176 2.2.4. The presynaptic giant projects multiple nerve

terminals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 2.2.5. Diffusion barrier . . . . . . . . . . . . . . . . . . . . . . . . 177 2.2.6. Large size of the pre- and postsynaptic giant axons . . 177 2.2.7. Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 2.2.8. Oxygen sensitivity . . . . . . . . . . . . . . . . . . . . . . . 177 2.2.9. Depletion of transmitter release . . . . . . . . . . . . . . 177

3. Dissection, mounting, and experimentation . . . . . . . . . . . . . . . . . 178 3.1. Squid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 3.2. The basic stellate ganglion preparation . . . . . . . . . . . . . . . 179

3.2.1. Coarse dissection . . . . . . . . . . . . . . . . . . . . . . . 179 3.2.2. Removal of the stellate ganglion . . . . . . . . . . . . . . 180 3.2.3. Fine dissection . . . . . . . . . . . . . . . . . . . . . . . . . 181

3.3. The stellate ganglion with aortic perfusion . . . . . . . . . . . . . 181 3.3.1. The blood supply of the stellate ganglion . . . . . . . . 181 3.3.2. Coarse dissection with cannulation of the artery . . . . 182 3.3.3. Fine dissection of the perfused ganglion . . . . . . . . . 183

3.4. Experimental considerations for studies on the stellate ganglion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

3.4.1. Artificial sea water (ASW) . . . . . . . . . . . . . . . . . 183 3.4.2. Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 3.4.3. Recording from the presynaptic nerve terminal . . . . . 185

3.4.3.1 Selection of synapses . . . . . . . . . . . . . . 185 3.4.3.2. Movement . . . . . . . . . . . . . . . . . . . . . 185 3.4.3.3. Micropipette impalement . . . . . . . . . . . . 186

3.4.4. Correlation of pre- and postsynaptic events . . . . . . . 186 3.4.5. Voltage clamp techniques . . . . . . . . . . . . . . . . . . 186

3.4.5.1. Equipment . . . . . . . . . . . . . . . . . . . . . 186 3.4.5.2. Current passing electrodes . . . . . . . . . . . 187 3.4.5.3. Voltage clamp limitations . . . . . . . . . . . . 187

3.4.6. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 3.4.7. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . 187 3.4.8. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 190


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PART IV. CELL BIOLOGY

Chapter 12 Tissue Culture of Squid Neurons, Glia, and Muscle Cells

ROBERT V. RICE, RUTHANNE MUELLER, and WILLIAM J. ADELMAN, JR.

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 1.1. Invertebrate tissue culture . . . . . . . . . . . . . . . . . . . . . . . . 196 1.2. Neurons, glia, and muscle cells . . . . . . . . . . . . . . . . . . . . 196

2. Culture strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 2.1. Reduced carbon sources . . . . . . . . . . . . . . . . . . . . . . . . . 196 2.2. Osmolarity of the media . . . . . . . . . . . . . . . . . . . . . . . . 197 2.3. Ionic composition of the media . . . . . . . . . . . . . . . . . . . . 197 2.4. Incubation temperatures . . . . . . . . . . . . . . . . . . . . . . . . . 197 2.5. Modified Eagle medium . . . . . . . . . . . . . . . . . . . . . . . . 197 2.6. Fetal bovine serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

3. Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 3.1. Manipulation of eggs . . . . . . . . . . . . . . . . . . . . . . . . . . 198 3.2. Preparation of embryos . . . . . . . . . . . . . . . . . . . . . . . . . 198 3.3. Embryo dissection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 3.4. Cell dispersion and plating out . . . . . . . . . . . . . . . . . . . . 199 3.5. A typical dispersion and plating out protocol: . . . . . . . . . . . 200 3.6. Cryopreservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 3.7. Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

4. Cell culture and growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 4.1. Effects of media on growth . . . . . . . . . . . . . . . . . . . . . . 201 4.2. Effects of aggregates, explants, and conditioned media on

growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 4.3. Effects of carbohydrates on growth . . . . . . . . . . . . . . . . . 202 4.4. Effects of temperature on growth . . . . . . . . . . . . . . . . . . . 202

5. Cell identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 5.1. Neuron identification by tetanus toxin binding . . . . . . . . . . . 203 5.2. Glial cell identification . . . . . . . . . . . . . . . . . . . . . . . . . 203 5.3. Cell imaging, photography, and video processing . . . . . . . . . 203

6. Characteristics of cultured cells . . . . . . . . . . . . . . . . . . . . . . . . 205 6.1. Morphology of soma . . . . . . . . . . . . . . . . . . . . . . . . . . 205 6.2. Are fibroblasts present in the cultures? . . . . . . . . . . . . . . . 205 6.3. Muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 6.4. Distinguishing neurons from glia . . . . . . . . . . . . . . . . . . . 206 6.5. Bipolar and pyramidal neurons . . . . . . . . . . . . . . . . . . . . 209 6.6. Characteristics of cultured bipolar cells . . . . . . . . . . . . . . . 209 6.7. Muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

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Chapter 13 Squid Optic Lobe Synaptosomes: Structure and Function of Isolated Synapses

ROCHELLE S. COHEN, HARISH C. PANT, and HAROLD GAINER

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 2. Subcellular fractionation of squid optic lobe synaptosomes . . . . . . . 214

2.1. Preparation of synaptosomes . . . . . . . . . . . . . . . . . . . . . . 214 2.2. Preparation of synaptosomal plasma membranes . . . . . . . . . . 217 2.3. Preparation of synaptic vesicles . . . . . . . . . . . . . . . . . . . . 219

3. Structure of squid optic lobe synaptosomes . . . . . . . . . . . . . . . . . 220 3.1. Morphological characterization . . . . . . . . . . . . . . . . . . . . 220 3.2. Preparation of tissue for electron microscopy . . . . . . . . . . . 221

4. Proteins in the squid optic lobe synaptosome and synaptosomal plasma membrane fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

4.1. Polypeptide composition . . . . . . . . . . . . . . . . . . . . . . . . 223 4.2. Protein phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . 226 4.3. Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

5. Squid optic lobe synaptosomes as model cholinergic endings . . . . . . 227 6. Localization and uptake of other putative neurotransmitters and

neuropeptides in squid optic lobe nerve terminals . . . . . . . . . . . . . 229 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Chapter 14 The Cytoskeleton of the Squid Giant Axon

ANTHONY BROWN and RAYMOND J. LASEK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 2. The squid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 3. The anatomy and development of the giant nerve fiber . . . . . . . . . 236 4. Dissection of the giant axons . . . . . . . . . . . . . . . . . . . . . . . . . . 239

4.1. Fine-cleaning the giant axons . . . . . . . . . . . . . . . . . . . . . 239 5. Handling the giant axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 6. Methods for obtaining axoplasm from the giant axons . . . . . . . . . . 240

6.1. Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 6.2. Slitting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 6.3. Cannulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

7. How pure is axoplasm isolated from the giant axon? . . . . . . . . . . . 246 7 .I. Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 7 .2. Slitting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 7 7.3. Cannulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

8. Handling axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 9. The composition of axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . 248

9 .I. Ions and small molecules . . . . . . . . . . . . . . . . . . . . . . . . 248 9 .2. Macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 9.3. Calcium and magnesium in axoplasm . . . . . . . . . . . . . . . . 250

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10. Artificial axoplasm solutions . . . . . . . . . . . . . . . . . . . . . . . . . . 252 10.1. Buffer P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 10.2. Buffer X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

11. The solubility and stability of axoplasm . . . . . . . . . . . . . . . . . . . 255 11.1. Salt and pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 11.2. Calcium-dependent proteolysis . . . . . . . . . . . . . . . . . . . . . 256

12. Preparation of axoplasm and sheath for SDS PAGE technique . . . . . 257 13. Preparation of axoplasm for electron microscopy . . . . . . . . . . . . . 257

13.1. A general fixation protocol . . . . . . . . . . . . . . . . . . . . . . . 257 13.2. Preservation of microfilaments . . . . . . . . . . . . . . . . . . . . . 258 13.3. Fixative penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 13.4. Removing the axon sheath . . . . . . . . . . . . . . . . . . . . . . . 259 13.5. Cannulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 13.6. Negative staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

14. The organization of the cytoskeleton . . . . . . . . . . . . . . . . . . . . . 261 14.1. Longitudinal organization . . . . . . . . . . . . . . . . . . . . . . . . 261 14.2. Helicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 14.3. Radial organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

15. The cortical cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 15.1. Axoplasm is attached to the plasma membrane . . . . . . . . . . 263 15.2. Internal perfusion of axons . . . . . . . . . . . . . . . . . . . . . . . 264 15.3. Protein composition of the cortical cytoskeleton . . . . . . . . . . 265 15.4. Architecture of the cortical cytoskeleton . . . . . . . . . . . . . . 266 15.5. The cortical cytoskeleton and membrane excitability . . . . . . . 267

16. The inner cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 17. Neurofilaments in axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . 269

17 .1. Purification of squid neurofilaments . . . . . . . . . . . . . . . . . 270 17 .2. Polypeptide composition of squid neurofilaments . . . . . . . . . 270 17 .3. Phosphorylation of squid neurofilaments . . . . . . . . . . . . . . 271 17 .4. Calcium-dependent proteolysis . . . . . . . . . . . . . . . . . . . . . 272 17 .5. The structure of the squid neurofilament . . . . . . . . . . . . . . 273

18. Microtubules in axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 18.1. Purification of microtubules . . . . . . . . . . . . . . . . . . . . . . 275 18.2. Video-enhanced contrast light microscopy of axoplasm . . . . . 275 18.3. Microtubule-associated proteins in axoplasm . . . . . . . . . . . . 276

19. Microfilaments in axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 19.1. Microfilaments are numerous in the inner cytoskeleton . . . . . 277 19.2. Two classes of microfilaments in the inner cytoskeleton . . . . 278 19.3. Organization of microfilaments in the inner cytoskeleton . . . . 278 19.4. Myosin from squid brain . . . . . . . . . . . . . . . . . . . . . . . . 279

20. Studying the stability of the cytoskeleton . . . . . . . . . . . . . . . . . . 279 20.1. Assembly dynamics of cytoskeletal polymers in axons . . . . . 279 20.2. The axoplasmic ghost . . . . . . . . . . . . . . . . . . . . . . . . . . 281 20.3. Monomer-polymer equilibria in axoplasm . . . . . . . . . . . . . . 283

21. Mechanical studies on axoplasm . . . . . . . . . . . . . . . . . . . . . . . . 284 21.1. Axoplasm has mechanical integrity . . . . . . . . . . . . . . . . . . 284 21.2. Axoplasm is anisotropic . . . . . . . . . . . . . . . . . . . . . . . . . 285 21.3. The macroscopic mechanical properties of axoplasm . . . . . . . 285 21.4. The stretch apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . 287 21.5. Stretch analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

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21.6. Interpretation of the stretch profile . . . . . . . . . . . . . . . . . . 291 21.7. A mechanical model . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 21.8. The structural basis for elasticity and flow . . . . . . . . . . . . . 293 21.9. Polymer sliding in axons . . . . . . . . . . . . . . . . . . . . . . . . 294

22. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Chapter 15 Studying Axoplasmic Transport by Video Microscopy and Using the Squid Giant Axon as a Model System

DIETER G. WEISS, MONICA A. MEYER, and GEORGE M. LANGFORD

1. Present status of the results obtained by the use of intact axons and membrane-free or cell-free preparations . . . . . . . . . . . . . . . . 303

1.1. Studies on intact axons . . . . . . . . . . . . . . . . . . . . . . . . . 303 1.2. Studies on native microtubules and organelles . . . . . . . . . . . 304 1.3. Studies on purified components and reconstituted

preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 2. Gross dissection of the squid giant axon . . . . . . . . . . . . . . . . . . 307 3. Fine dissection of the squid giant axon . . . . . . . . . . . . . . . . . . . 307 4. Preparation of the axon for light microscopy . . . . . . . . . . . . . . . . 308

4.1. Slides and holders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 4.2. Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 4.3. Intact axons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 4.4. Extrusion of axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . 310 4.5. Homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

5. Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 6. Preparation of the axoplasm for electron microscopy . . . . . . . . . . . 314

6.1. Negative contrast electron microscopy . . . . . . . . . . . . . . . . 314 6.2. Other forms of electron microscopy . . . . . . . . . . . . . . . . . 315


Chapter 16 Lipid Metabolism In The Squid Nervous System

ROBERT M. GOULD and MARIO ALBERGHINA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 2. Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

2.1. Lipid composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 2.2. Lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 2.3. Lipid enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

3. Tissue preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 3.1. The giant axon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 3.2. Extruding axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

3.2.1. Axoplasmic subfractions . . . . . . . . . . . . . . . . . . . 333

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3.3. Giant fiber lobe (GFL) . . . . . . . . . . . . . . . . . . . . . . . . . 335 3.4. Retinal fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

3.4.1. Retinal fiber axolemma . . . . . . . . . . . . . . . . . . . . 336 3.5. Optic lobe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 3.6. Fin nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 3.7. Photoreceptor membranes . . . . . . . . . . . . . . . . . . . . . . . . 337 3.8. Giant synapse and pallial nerve . . . . . . . . . . . . . . . . . . . . 338

4. Studying lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 4.1. The giant axon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

4.1.1. Incubation of giant axons . . . . . . . . . . . . . . . . . . 338 4.1.1.1. Procedures . . . . . . . . . . . . . . . . . . . . . 338 4.1.1.2. Experimental considerations . . . . . . . 340

4.1.2. Injection of giant axons . . . . . . . . . . . . . . . . . . . 340 4.1.2.1. Procedures . . . . . . . . . . . . . . . . . . . . . 340 4.1.2.2. Experimental considerations . . . . . . . . . . 341

4.1.3. Axonal transport of lipid metabolizing enzymes . . . . 341 4.1.3.1. Procedure . . . . . . . . . . . . . . . . . . . . . . 342 4.1.3.2. Experimental considerations . . . . . . . . . . 343

4.2. Extruded axoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 4.2.1. Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 4.2.2. Experimental considerations . . . . . . . . . . . . . . . . . 344 4.2.3. Axoplasmic subfractions . . . . . . . . . . . . . . . . . . . 345

4.2.3.1. Procedures . . . . . . . . . . . . . . . . . . . . . 345 4.2.3.2. Experimental considerations . . . . . . . . . . 345

4.3. Giant fiber lobe, retinal fibers, retinal fiber axolemma, optic lobe, optic lobe synaptosomes, fin nerve, retina and photoreceptor membranes . . . . . . . . . . . . . . . . . . . . . . . . 346

4.3.1. Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 4.3.2. Experimental considerations . . . . . . . . . . . . . . . . . 347

5. Useful lipid techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 5.1. Precursor selection and lipid product identification . . . . . . . . 347 5.2. Enzymes of lipid metabolism . . . . . . . . . . . . . . . . . . . . . 350

5.2.1. Phosphatidylinositol synthase (CDP-diacylglycerol: myo-inositol transferase) (EC 2.7.8.11) . . . . . . . . . . 351

5.2.2. 1,2-Diacylglycerol kinase (ATP:diacylglycerol phosphotransferase) (EC 2.7.1.-) . . . . . . . . . . . . . . 351

5.2.3. Phosphatidylinositol kinase (EC 2.7.1.67) . . . . . . . . 351 5.2.4. Phospholipase A2 (EC 3.1.1.4) . . . . . . . . . . . . . . . 352 5.2.5. Acyl-CoA: 1-acyl-sn-glycero-3-phosphocholine

acyltransferase (EC 2.3.1.23) . . . . . . . . . . . . . . . . 352 5.2.6. Serine base-exchange . . . . . . . . . . . . . . . . . . . . . 353 5.2.7. Phospholipid transfer proteins . . . . . . . . . . . . . . . . 353 5.2.8. Octopine dehydrogenase (EC 1.5.1.11); procedure

from Dr. Michael Dowdall, (1989) Univ. of Nottingham . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

5.3. Localizing radioactive lipids in squid neural tissues by quantitative EM autoradiography . . . . . . . . . . . . . . . . . . . 354

5.3.1. Lipid metabolism in squid axoplasm . . . . . . . . . . . 355 5.3.2. Inositol lipid metabolism by the squid giant synapse . 359

6. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

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7. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 7 .1. Solutions and media . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 7 .2. Chromatography solvents . . . . . . . . . . . . . . . . . . . . . . . . 362

7 .2.1. Solvent 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 7 .2.1.1. Rf values for solvent 1 . . . . . . . . . . . . . 362

7 .2.2. Solvent 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 7 .2.2.1. Rf values for solvent 2 . . . . . . . . . . . . . 363

7.2.3. Solvent 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 7 .2.3.1. Rf values for solvent 3 . . . . . . . . . . . . . 363

7 .2.4. Solvent 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 7.2.4.1. Rf values for solvent 4 . . . . . . . . . . . . . 363

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

PART V. SENSORY SYSTEMS

Chapter 17 Structure and Function of the Squid Eye

HELEN R. SAIBll..

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 1.1. The cephalopod camera eye . . . . . . . . . . . . . . . . . . . . . . 371

2. Visual information produced by the squid eye . . . . . . . . . . . . . . . 372 2.1. Spatial resolution and sensitivity . . . . . . . . . . . . . . . . . . . 372 2.2. Polarization sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . 372

2.2.1 Photoreceptor membrane and photopigment orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

2.2.2. Contrast enhancement . . . . . . . . . . . . . . . . . . . . . 373 3. The squid eye as an experimental preparation . . . . . . . . . . . . . . . 373

3.1. Disadvantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 3.1.1. Delicate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 3.1.2. Electrophysiology difficult . . . . . . . . . . . . . . . . . . 374 3.1.3. Spectral overlap . . . . . . . . . . . . . . . . . . . . . . . . 374

3.2. Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 3.2.1. Large numbers and large eyes . . . . . . . . . . . . . . . 374 3.2.2. Simple retinal neuroanatomy . . . . . . . . . . . . . . . . 374 3.2.3. Pure photoreceptor membrane preparation . . . . . . . . 374 3.2.4. Photostable metarhodopsin . . . . . . . . . . . . . . . . . . 375

3.3. Protocol for dissection of the eye . . . . . . . . . . . . . . . . . . 375 4. The retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

4.1. Cellular organization . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 4.1.1. Fixation methods for the visual cells . . . . . . . . . . . 376

4.1.1.1. Protocol . . . . . . . . . . . . . . . . . . . . . . . 379 4.1.1.2. Quality of fixation . . . . . . . . . . . . . . . . 379

4.1.2. Neuroanatomy . . . . . . . . . . . . . . . . . . . . . . . . . 381 4.2. Intracellular transport . . . . . . . . . . . . . . . . . . . . . . . . . . 382

4.2.1. Pigment granule migration . . . . . . . . . . . . . . . . . . 382 4.2.2. Retinoid transport and membrane turnover . . . . . . . . 382

Contents xxiii

4.3. Electrophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 4.3.1. Electroretinogram (ERG) . . . . . . . . . . . . . . . . . . . 382 4.3.2. Photoreceptor potentials and ionic mechanisms . . . . . 383 4.3.3. Optic nerve responses . . . . . . . . . . . . . . . . . . . . 383

5. Molecular organization of squid photoreceptors . . . . . . . . . . . . . . 384 5.1. Isolation of the microvillar membranes . . . . . . . . . . . . . . . 384

5.1.1. Preparation of the retinas . . . . . . . . . . . . . . . . . . 384 5.1.2. Protocol for isolation of the membranes . . . . . . . . . 384

5.1.2.1. Method of Saibil (adapted from Saibil and Hewat, 1987 and Baer and Saibil, 1988) . . . . . . . . . . . . . . . . . . . . . . . . 384

5.1.2.2. Method of Vandenberg (Vandenberg, 1982; Vandenberg and Montal, 1984c) . . . 386

5.1.3. Lipid and protein composition of the preparation . . . 387 5.1.4. Measurement of rhodopsin concentration . . . . . . . . . 387

5.2. Purification of retinal binding proteins . . . . . . . . . . . . . . . . 387 5.2.1. Rhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

5.2.1.1. Method of Nashima, Mitsudo and Kito (Nashima et al., 1979) . . . . . . . . . . . . . 388

5.2.1.2. Method of Vandenberg (Vandenberg, 1982; Vandenberg and Montal, 1984c) . . . 388

5.2.2. Retinochrome . . . . . . . . . . . . . . . . . . . . . . . . . . 389 5.2.2.1. Purification method of Hara and Hara

(Hara and Hara, 1982) . . . . . . . . . . . . . 389 5.2.3. Retinal binding protein .............. , . . . . . 390

5.3. Microvillus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 5.3.1. · X-ray diffraction . . . . . . . . . . . . . . . . . . . . . . . . 390 5.3.2. Electron microscope image analysis . . . . . . . . . . . . 391 5.3.3. Rapid freezing, freeze etching and freeze

substitution EM . . . . . . . . . . . . . . . . . . . . . . . . 391 5.4. Assays for rhodopsin-activated signalling enzymes . . . . . . . . 392

5.4.1. G1P-binding proteins . . . . . . . . . . . . . . . . . . . . . 392 5.4.2. Phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . 393 5.4.3. Cyclic guanosine monophosphate (cGMP) . . . . . . . . 393

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 6.1. Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 6.2. Membrane-cytoskeleton and membrane-membrane

interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

Chapter 18 Development of the Squid's Visual System

I. A. MEINER1ZHAGEN

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 2. Development of the eye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

2.1. Formation of an eye vesicle . . . . . . . . . . . . . . . . . . . . . . 401 2.2. Development of the lens . . . . . . . . . . . . . . . . . . . . . . . . 402

xxiv Contents

2.3. Development of the iris . . . . . . . . . . . . . . . . . . . . . . . . . 402 2.4. Development of the cornea . . . . . . . . . . . . . . . . . . . . . . . 403 2.5. Experimental embryology of the eye . . . . . . . . . . . . . . . . . 403 2.6. Retinal differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . 405

2.6.1. EM fixation for squid retina . . . . . . . . . . . . . . . . 406 2.6.1.1. Method of Cohen (1973a) . . . . . . . . . . . 406 2.6.1.2. Method of Yamamoto (1985) . . . . . . . . . 406

2.6.2. Electrophysiological differentiation . . . . . . . . . . . . . 406 2.6.2.1. ~ecor~ing ERG and optic nerve activity

1n sqwd . . . . . . . . . . . . . . . . . . . . . . . 406 2.6.3. Golgi impregnation of the retina . . . . . . . . . . . . . . 407

3. Development of the optic lobe . . . . . . . . . . . . . . . . . . . . . . . . . 408 3.1. Morphogenetic dependence of the optic lobe upon eye

development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 3.2. Developmental origins of the optic lobe . . . . . . . . . . . . . . 410

3.2.1. Culture conditions . . . . . . . . . . . . . . . . . . . . . . . 410 3.3. Fiber tracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411

3.3.1. Nauta degeneration method . . . . . . . . . . . . . . . . . 411 3.4. Intemeurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412

3.4.1. Silver impregnation and staining methods for cephalopod tissue . . . . . . . . . . . . . . . . . . . . . . . 412

3.4.1.1. Cajal's block silver method . . . . . . . . . . 412 3.4.1.2. Golgi-Kopsch method . . . . . . . . . . . . . . 412 3.4.1.3. Golgi-rapid method . . . . . . . . . . . . . . . . 413

3.4.2. Other methods to stain pathways . . . . . . . . . . . . . . 413 3.4.2.1. HRP labeling method . . . . . . . . . . . . . . 413 3.4.2.2. Cobalt backfills . . . . . . . . . . . . . . . . . . 413

3.5. Synaptic organization . . . . . . . . . . . . . . . . . . . . . . . . . . 414 4. Growth of the visual system . . . . . . . . . . . . . . . . . . . . . . . . . . 414

4.1. Nuclear counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 4.2. Eye growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 4.3. Brain growth ......................... , . . . . . 415

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416

Chapter 19 The Statocysts of Squid

BERND U. BUDELMANN

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 2. Short summary of research on cephalopod statocysts . . . . . . . . . . . 422 3. Dissection of the squid statocyst . . . . . . . . . . . . . . . . . . . . . . . . 423

3.1. Statocyst operations on living squid . . . . . . . . . . . . . . . . . 426 4. Structure and function of the squid statocyst . . . . . . . . . . . . . . . . 427

4.1. The receptor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 4.2. The gravity receptor system . . . . . . . . . . . . . . . . . . . . . . 428

4.2.1. Statolith growth rings and aging . . . . . . . . . . . . . . 429 4.3. The angular acceleration receptor system . . . . . . . . . . . . . . 429

4.3.1. Anticristae . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

Contents xxv

4.4. Ciliated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 4.5. K5lliker's canal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

5. Central projections of the statocyst sensory epithelia . . . . . . . . . . . 434 6. Compensatory eye and head movements . . . . . . . . . . . . . . . . . . . 434

6.1. Counterrolling of the eyes . . . . . . . . . . . . . . . . . . . . . . . 434 6.2. Post-rotatory nystagmus . . . . . . . . . . . . . . . . . . . . . . . . . 435 6.3. Compensatory head movements . . . . . . . . . . . . . . . . . . . . 435


PART VI. INTEGRATED SYSTEMS

Chapter 20 Gas Transport in the Blood

CHARLOTIE P. MANGUM

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 2. Respiratory properties of cephalopod bloods . . . . . . . . . . . . . . . . 444

2.1. Squid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 2.2. Other cephalopod Hcs . . . . . . . . . . . . . . . . . . . . . . . . . . 449

2.2.1. Gas transport . . . . . . . . . . . . . . . . . . . . . . . . . . 449 2.2.2. pH dependence: a special case . . . . . . . . . . . . . . . 451

3. Molecular properties of cephalopod Hcs . . . . . . . . . . . . . . . . . . . 452 3.1. Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452 3.2. Molecular structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453

4. Performance of the oxygen transport system . . . . . . . . . . . . . . . . 456 4.1. Oxygen transport in Loligo pealei . . . . . . . . . . . . . . . . . . 456 4.2. Oxygen transport in octopus . . . . . . . . . . . . . . . . . . . . . . 457 4.3. Oxygen transport in Nautilus pompilius . . . . . . . . . . . . . . . 459 4.4. Gas transport in Sepia officina/is . . . . . . . . . . . . . . . . . . . 459

5. Methods of investigating gas transport . . . . . . . . . . . . . . . . . . . . 460 5.1. Measurement of blood gases . . . . . . . . . . . . . . . . . . . . . . 460 5.2. Hc02 equilibrium measurements . . . . . . . . . . . . . . . . . . . . 461

5.2.1. Tonometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 6. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

Chapter 21 An Organophosphorus Detoxifying Enzyme Unique to Squid

FRANCIS C. G. HOSKIN

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 3. Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

xxvi Contents

Chapter 22 Squid as Elite Athletes: Locomotory, Respiratory, and Circulatory Integration

RON O'DOR, H. 0. P0R1NER, and R. E. SHADWICK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 2. Physiology in active squid . . . . . . . . . . . . . . . . . . . . . . . . . . . 481

2.1. Anesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482 2.2. Cannulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 2.3. Swim-tunnels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484 2.4. Ultrasonic transducer-transmitters . . . . . . . . . . . . . . . . . . . 487

3. Respiratory physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487 3.1. Ventilatory flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 3.2. Ventilatory regulation . . . . . . . . . . . . . . . . . . . . . . . . . . 489

4. Circulatory physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491 4.1. Cellular elements .................. , . . . . . . . . . . 491 4.2. Oxygen carrier pigment . . . . . . . . . . . . . . . . . . . . . . . . . 493 4.3. Circulatory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494 4.4. Circulatory regulation . . . . . . . . . . . . . . . . . . . . . . . . . . 497 4.5. Auxiliary systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505

XXVII

Abbreviations, Prefixes, and Suffixes

a f 2-P-ATP Llli Mog P5J~pH 1,10-<J> 5-HT 8-0HQ-5SA a AID AChE ADP AMP AMP-PNP ArgP ASW ATCh ATP ATP-y-S AVEC-DIC

BES BIS-TRIS BIS-TRIS-propane c CaC02 CaMgF CaNP Ca02 CD CDP CDTA cGMP Chol Ci CL CL CNS cs CsEGTA

CTA

absorption coefficient or solubility of C02 in aqueous media A TP labeled with J>32 in the y position enthalpy Bohr coefficient (used as a measure of the Bohr shift) 1, 10-phenanthroline 5-hydroxytryptamine 8-hydroxyquinoline-5-sulfonate refers to postbranchial analog to digital acetylcholinesterase adenosine 5'-diphosphate adenosine 5'-monophosphate 5'-adenylyl imidodiphosphate arginine phosphate artificial sea water acetylthiocholine adenosine-5'-triphosphate A TP with S in the y position Allen video-enhanced contrast differential interference contrast microscope N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid bis(hydroxethyl)imino-tris(hydroxymethyl)-methane 1 ,3-bis( tris(hydroxymethyl)methylamino )-propane pressure wave velocity postbranchial C02 concentration calcium and magnesium free calcium-dependent neutral protease (calpain) postbranchial 0 2 concentration circular dichroism cytidine 5'-diphosphate trans-1 ,2-diaminocyclohexane-N ,N ,N' ,N' -tetraacetic acid cyclic guanosine monophosphate cholesterol curie cardiolipin crista longitudinalis central nervous system common stellate Cs ethylene glycol-bis(2-amino ethyl ether)N,N,N',N'-tetraacetic acid crista transversalis anterior

XXVIII

CTP CTP cv CvC02 Cv02 d D D/A DC DEAE deoxyHc DFP DIC DMSO DNA dpm D1NB E Eb02 EDTA EGTA ElM EKG EM EMG EPSC EPSP ERG ERP ExtrC02

Extr02

FB FBS FCCP FET FFT FITC g G.A. I G.A. II G.A. III GFAP GFL GTP h H-H equations He Hc02

HEPES

crista transversalis posterior cytidine 5'-triphosphate crista verticalis


prebranchial C02 concentration prebranchial 0 2 concentration blood density Dalton digital to analog direct current diethylamino ethyl deoxygenated form of He diisopropyl phosphorofluoridate differential interference contrast dimethylsulfoxide desoxynucleic acid disintegrations per minute 5,5'-dithiobis-(2-nitrobenzoic acid) elastic modulus of the vessel wall efficiency of 0 2 uptake at the gill ethylenediamine tetraacetic acid ethylene glycol-bis(2-amino ethyl ether)N,N,N',N'-tetraacetic acid excitability inducing material electrocardiogram electron microscopy electromyogram excitatory postsynaptic current excitatory postsynaptic potential electroretinogram early receptor potential C02 extraction from blood 0 2 extraction from blood fixation buffer fetal bovine serum p-trifluoro methoxy carbonyl cyanide phenyl hydrazine field effect transistor fast Fourier transform fluorescein isothiocyanate acceleration due to gravity at sea level or 9.80 m/sec2

frrst order giant axon second order giant axon third order giant axon glial fibrillary acid protein giant fiber lobe guanosine 5'-triphosphate vessel wall thickness Hodgkin-Huxley equations hemocyanin oxygenated He N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

Abbreviations, Prefixes, and Suffixes xxix

HPLC

HRP Hz i IgG IM IR IR IU KEP LPT MAO MAP MEM MEPC mepp MEPP MES ML MMEM MNI MNS MOPS MSP n N N. A. NAD NADH NADP OPA ORD

OT oxyHc Pa Paraoxon PC PCMB PC02 PE PE PEM PEP PI PIP PIP2

PIPES

high pressure liquid chromatography (high performance liquid chromatography) horseradish peroxidase cycles/sec refers to inhalent immunoglobin G inverted microscope current times resistance infrared international units kinetic equilibration paradigm linear position transducer monoamine oxidase microtubule-associated protein minimal essential medium miniature excitatory postsynaptic current miniature end plate potential miniature excitatory postsynaptic potential sodium 2-(N-morpholino)ethane sulfonate mantle length mostly modified Eagle media macula neglecta inferior macula neglecta superior 3-(N-morpholino)propanesulfonic acid macula statica princeps Hill coefficient Newtons numerical aperature nictinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide 3'-phosphate organophosphorus acid optical rotatory dispersion (optical rotation as a function of wavelength) obligate thermophilic oxygenated form of He Pascal diethyl p-nitrophenyl phosphate phosphatidylcholine p-hydroxymercuribenzoate (p-chloromercuribenzoate) partial pressure of carbon dioxide phosphatidylethanolamine polyethylene photoelastic modulator phospho( enol)pyruvate phosphatidylinositol phosphatidylinositol phosphate phosphatidylinositol bisphosphate piperazine-N ,N'-bis(2-ethanesulfonic acid)

XXX

pK.

pK'

PL PMSF pN P02 ppt PS PSD psi Px Q,o R REO Rf rpm s Sarin SDS SDS PAGE SEM SML So man SPM SPM SPME

spp. STX Tabun TAME TC TCA TEA TES TLC TRIS Tris maleate TRIZMA TS TTX TV UTP uv v VCR VHS WORM


constant describing the equilibrium between the dissociated and undissociated forms of an acid constant describing the equilibrium between the components of the C02 system in aqueous media phospholipid phenyl-methylsulfonylfluoride pH at neutrality partial pressure of oxygen parts/thousand phosphatidylserine postsynaptic density pounds/in2 gauge pressure oxygen pressure in torr when He is x% oxygenated ratio of reaction mtes 10 oc apart internal mdius recordable emsable optical distance of migration of the solute relative to the solvent front revolution/min Siemens isopropyl methylphosphonofluoridate sodium dodecyl sulfate SDS polyacrylamide gel electrophoresis scanning electron microscopy sucrose monolaurate 2,3,3-trimethylpropyl methylphosphonofluoridate sphingomyelin synaptosomal plasma membrane 0.6 M sucrose, 0.1 M potassium phosphate, 10 mM magnesium chloride, 1 mM EGTA, pH 7.1 species saxitoxin ethyl N ,N-dimethylphosphommidocyanidate tosyl L-arginine methyl ester tissue culture trichloroacetic acid tetraethyl ammonium chloride N-tris (hydroxymethyl)-methyl-2-aminoethanesulfonic acid thin layer chromatography tris (hydroxymethyl) aminomethane mono[tris(hydroxymethyl)-aminomethane] maleate tris (hydroxymethyl) aminomethane-HCI (Tris-HCI) Tris-HCl, 0.25 M sucrose solution tetrodotoxin television uridine 5'-triphosphate ultraviolet refers to prebranchial video cassette recorder video home system write once read many

Abbreviations, Prefixes, and Suffixes xxxi

ww 375

z

Prefixes

Symbol

p n Jl m k

M G

5-[(1-y-triethylammonium sulfopropyl-4(1H)-quinolylidene)-2-butenylidene]-3-ethylrhodamine hydraulic impedance to blood flow

Name Factor* Examples

pi co IQ-12 pico Newtons (pN) nano 10-9 nano Siemens (nS) micro 10-6 micro curie (JJ.Ci) milli IQ-3 milli curie (mCi) kilo 10l kiloDalton (kD); kilo Hertz (kHz); kilo

Pascal (kPa) Mega 10' MegaHertz (MHz) giga 1()9 gigaohm

* factor by which quantity is multiplied

Suffixes

ase refers to an enzyme which breaks the substrate designated by the prefix. Examples are A TPase and GTPase.

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