Modern Techniques in Neuroscience Research PDF
Preview Modern Techniques in Neuroscience Research
Uwe Windhorst • Hwn Johansson (Eds.) Modern Techniques in Neuroscience Research Springer-Verlag Berlin Heidelberg GmbH Uwe Windhorst· Hc1kan Johansson (Eds.) Modern Techniques in Neuroscience Research With 471 Figures, 43 Color Figures, 33 Tables and CD-ROM , Springer PROFESSOR DR. UwE WINDHORST PROFESSOR DR. HÄKAN }OHANSSON Center of Musculoskeletat Research Center of Musculoskeletat Research National Institute for Working Life National Institute for Working Life Petrus Laestadius väg Petrus Laestadius väg 90713 Umeä, Sweden 90713 Umeä, Sweden Department of Clinical Neuroseiences and Department of Physiology and Biophysics Faculty of Medicine The University of Calgary 333 Hospital Drive N.W. Calgary,Alberta T2N 4N1, Canada Zentrum Physiologie und Pathophysiologie Universität Göttingen Humboldtallee 23 37073 Göttingen, Germany Library of Congress Cataolging-in-Publication Data Modern techniques in neuroscience research I U. Windhorst, H. Johannson (Eds.) p.cm. Includes bibliographical referneces and index. ISBN 978-3-642-63643-1 ISBN 978-3-642-58552-4 (eBook) DOI 10.1007/978-3-642-58552-4 1. Neuroseiences Labaratory manuals. I. Windhorst, Uwe, 1946- . II. Johannson, H. (Hakän) , 1947- . RC337.M63 1999 573'.8'072--dc21 99-32065 CIP This work is subject to copyright. All rights are reserved, whether the whole or part of the ma terial is concerned, specifically the rights of translation, reprinting, reuse of illustrations, rec itations, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1999 Originally published by Springer-Verlag Berlin Heidelberg New York in 1999 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free general use. Typesetting: medio, Berlin Cover design: design & production GmbH, Heidelberg SPIN: 10574645 27/3136 - 5 4 3 2 1 o -Printed on acid free paper Preface Nothing tends so much to the advancement of knowledge as the application of a new instrument. - Sir Humphry Davy, 1778-1829 Neuroscience has become a rapidly expanding endeavor that relies on a number of other sciences, such as mathematics, physics, chemistry, engineering, computer sci ence, general biology and medicine, genetics etc. In fact, many of its recent success es result from the application of ideas and methods borrowed from these fields. In sofar, it is a true interdisciplinary undertaking. This convergence of influences ac counts for part of its enormous attractiveness and fascination to students and re searchers from diverse walks of life, or science, for that matter. It is probably fair to say that a great number of neuroscience's most creative and productive proponents have been lured into this field not only by the excitement about the possibility to un mask the secrets of the human mind, but also by the appeal of a vast unknown land needing cultivation and tools to cultivate it. Danger may arise for any science if it is dominated by methods and techniques of investigation rather than by problems to be solved and concepts to be developed. This might concentrate efforts onto the technically feasible and doable, rather than On the real issues. But, On the other hand, especially the young and growing sciences are heavily dependent on the development and application of methods, often even before a problem relying on these methods may become apparent. In fact, under fa vorable circumstances, these methods may reveal problems that have not been thought of before. Several examples could be evoked to make this point. On the oth er hand, the stream of influence is no one-way road. Techniques and findings in neuroscience may have an impact in other sciences and practical applications. All this provides for a fascinating tangle of relationships. To try and display the variety of techniques used in modern neuroscience re search is a tremendous task, just for the sheer vastness and complexity of neuro science itself and the diversity of methods used. This is well illustrated by other lab manuals in this series, where the description of a single technique, its descendants and ramifications, advantages and limitations, applications and pitfalls may occupy a sizeable volume. Quite the same completeness and richness of detail cannot be achieved in a book attempting at presenting a somewhat comprehensive overview, and quite the same lab-manual protocol style cannot be consistently maintained throughout. However, such an overview may still be worthwhile for two main rea sons. First, it may supply just that, an overview for the technically minded ever on the search for applications, that is for those who are appealed by new lands to be ex plored by tools and the challenge to apply known and to develop new tools. Second, if done well, such a book may present an introduction into neuroscience, from the angle of methods available to tackle questions. Because neuroscience is an inter-disciplinary endeavor, it is inter-methodologi cal. By this we mean that certain questions can be answered by combining methods borrowed and adapted from different fields. This important idea underlies the or ganization of this book. The organizing principle is the investigation of levels of the nervous system, from molecular and cellular to whole organisms. We have chosen this principle for two related reasons. First, we would like to show how certain or- VI Preface ganizationallevels can be studied and potentially understood by the application of an array of diverse techniques, thus demonstrating what a particular method can contribute to the understanding of a specific object. Second, we thereby hope to avoid succumbing to the danger of pure technicality, which might arise from order ing the description of techniques according to their inherent principles of coher ence (e.g. staining vs. electrophysiological techniques). This might lead to methods living their own life, detached from what they are used for. As a matter of fact, this approach can only be taken cum grano salis, for there are techniques that can be em ployed at various levels (e.g. systems analysis and synergetic approaches). Thanks are due to a vast number of people. First, we are very grateful to our au thors who have taken the time off their busy schedules to contribute one or more chapters to this book. Dr. Rolf Lange at Springer-Verlag has been the enthusiastic initiator of this project, and without his continuing encouragement one or the other editor would probably have given up along the way. Mrs. Anne Clauss at Springer Verlag has been the ever-reliable manager in Heidelberg, and particularly valuable has been her ability to nudge authors to -please -finally submit their manuscripts when the editors were on the verge of despair. Many thanks are due to supportive staff in the background, particularly the copy editors who, as always, have done an outstanding job in polishing the chapters. Last but not least we would like to thank our wives, Eva and Siggi, for their patience and indulgence in their husbands' ob session with this book. Uwe Windhorst Hakan Johansson Umea, Juni 1999 Contents Chapter 1 Cytological Staining Methods ROBERT W. BANKS Introduction. . . . . . . . . . . . . . . . . . 1 Subprotocoll: Fixation, Sectioning and Embedding 2 Subprotocol 2: Ultrastructure . . . 9 Subprotocol 3: The Golgi Method . 13 Subprotocol4: Single-Cell Methods. 17 References . . . . . . . . . . . 23 Chapter 2 Application of Differential Display and Serial Analysis of Gene Expression in the Nervous System ERNO VREUGDENHIL, JEANNETTE DE JONG and NICOLE DATSON Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27 Subprotocoll: Differential Display: Practical Approach. . . . . . . . .. 30 Subprotocol2: Serial Analysis of Gene Expression (SAGE): Practical Approach 39 Subprotocol 3: Digestion of cDNA with Anchoring Enzyme . 43 Subprotocol4: Binding to Magnetic Beads. . . . . . . . . . 44 Subprotocol 5: Addition ofLinkers. . . . . . . . . . . . . 45 Subprotocol 6: Tag Release by Digestion with Tagging Enzyme. 46 Subprotocol 7: Blunting Tags . . . . . . . 47 Subprotocol8: Ligation to Ditags. . . . . . 47 Subprotocol 9: PCR Amplification of Ditags . 48 Subprotocol 10: Ditag Isolation . . . 49 Subprotocol 11: Concatemerisation. . 51 Subprotocol12: Cloning Concatemers 52 Subprotocol13: Sequencing . 53 References . . . . . . . . . . . . 55 Chapter 3 Methods Towards Detection of Protein Synthesis in Dendrites and Axons JAN VAN MINNEN and R.E. VAN KESTEREN Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Subprotocoll: In Situ Hybridization of Cultured Neurons. . . . . . . 58 Subprotocol 2: In Situ Hybridization at the Electron Microscopic Level . 65 Subprotocol 3: Single-Cell Differential mRNA Display . . . . . . . . 75 Subprotocol4: Functional Implications of mRNAs in Dendrites and Axons: Metabolic Labeling ofIsolated Neurites. . . 81 Subprotocol5: Intracellular Injection of mRNA 84 References . . . . . . . . . . . . . . . . 87 VIII Contents Chapter 4 Optical Recording from Individual Neurons in Culture ANDREW BULLEN AND PETER SAGGAU Introduction 89 Outline .. 102 Materials. 102 Procedure. 103 Results .. 115 Troubleshooting . 117 Comments 122 References 125 ChapterS Electrical Activity ofIndividual Neurons In Situ: Extra-and Intracellular Recording PETER M. LALLEY, ADONIS K. MOSCHOVAKIS and UWE WINDHORST Introduction . . . . . . . . . . . . . . . . . . . . 127 Subprotocoll: General Arrangement and Preparation for Electrophysiological Recording and Data Acquisition . 128 Subprotocol 2: Extracellular Recording. . . . . . . . . 134 Subprotocol 3: Intracellular Recording with Sharp Electrodes . 146 Subprotocol4: Intracellular Recording and Tracer Injection. 158 Summary and Conclusions 165 Supplier List. 166 References 168 Chapter 6 Electrical Activity ofIndividual Neurons: Patch-Clamp Techniques BORIS V. SAFRONOV and WERNER VOGEL Introduction 173 Materials . 176 Procedure. 180 Results. . 187 Comments 188 Applications. 189 References 191 Chapter 7 Microiontophoresis and Pressure Ejection PETER M. LALLEY Introduction . . . . . . . . . . . 193 Subprotocoll: Microiontophoresis. . 194 Subprotocol2: Micropressure Ejection 207 Comments 209 Suppliers . 209 References 209 ChapterS An Introduction to the Principles of Neuronal Modelling KENNETH A. LINDSAY, J.M. ODGEN, DAVID M. HALLIDAY, JAY R. ROSENBERG Introduction . . . . . . 213 A Philosophy of Modelling . . . . . . . . . . . . . . . . . . . . .. 214 Contents IX Formulation of Dendritic Model. . . 216 The Discrete Tree Equations. . . . . 226 Formal Solution of Matrix Equations . 237 Solution of the Discretised Cable Equations . 242 Generating Independent and Correlated Stochastic Spike Trains . 245 Equivalent Cable Construction .... 254 Generalized Compartmental Methods . . . . . . 268 The Spectral Methodology . . . . . . . . . . . 277 Spectral and Exact Solution of an Unbranched Tree. 284 Spectral and Exact Solution of a Branched Tree 289 References . . . . . . . 299 Notations and Definitions. 302 Appendix ....... . 304 Chapter 9 In Vitro Preparations KLAUS BALLANYI Introduction. . . . 307 In Vitro Models . . 307 En bloc Preparations . 310 Brain Slices . . . . . 311 Determinants of Ex Vivo Brain Function 318 Conclusions . 324 References 325 Chapter 10 Culturing CNS Neurons: A Practical Approach to Cultured Embryonic Chick Neurons AKE SELLSTROM and STIG JACOBSSON Introduction. 327 Outline .. 329 Materials. 329 Procedure. 330 Results .. 333 Troubleshooting . 334 Comments . 334 Applications. 337 Suppliers .. 337 References . 337 Abbreviations . 338 Chapter 11 Neural Stem Cell Isolation, Characterization and Transplantation JASODHARA RAY and FRED H. GAGE Introduction. 339 Outline .. 340 Materials. 341 Procedure. 343 Results .. 351 Troubleshooting . 352 Comments ... 354 X Contents Applications. 357 References . 357 Suppliers . . 359 Abbreviations . 360 Glossary . . . 360 Chapter12 In Vitro Reconstruction of Neuronal Circuits: A Simple Model System Approach NAWEED I. SYED, HASSAN ZAIDI and PETER LOVELL Introduction 361 Outline. . 362 Materials . 364 Procedure. 368 Results . . 371 References 376 Chapter 13 Grafting of Peripheral Nerves and Schwann Cells into the CNS to Support Axon Regeneration THOMAS J. ZWIMPFER and JAMES D. GUEST Introduction . . . . . . . . . . . . . . . . . . . . . . . . 379 Subprotocoll: Harvest and Implantation ofPN Grafts into the CNS 383 Subprotocol2: Schwann Cell Guidance Channels. 392 References . . . . . . . . . . . . . . . . . . . . . . . . . 406 Chapter 14 Cell and Tissue Transplantation in the Rodent CNS KLAS WICTORIN, MARTIN OLSSON, KENNETH CAMPBELL and ROSEMARY FRICKER Introduction . . . . . . . . . . . . . . . . . . . . 411 Outline. . . . . . . . . . . . . . . . . . . . . . . 412 Subprotocoll: Dissection of Embryonic/Fetal CNS Tissue. 413 Subprotocol 2: Preparation of Tissue/Cells . . 420 Subprotocol 3: Transplantation into Adults . . 422 Subprotocol 4: Transplantation into Neonates . 426 Subprotocol 5: Transplantation into Embryos 428 References . . . . . . . . . . . . . . . . 432 Chapter lS Histological Staining Methods ROBERT W. BANKS Introduction . . . . . . . 437 Subprotocoll: Architectonics. 437 Subprotocol2: Hodology. . . 442 Subprotocol 3: Histochemical Methods: Neurochemistry and Functional Ne urohistology, Including the Molecular Biology of Neurons . 447 Subprotocol4: Silver-Impregnation Methods in the Peripheral Nervous System. 452 References . . . . . . . . . . . . . . . . . . 455
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