Table Of ContentIntracellular Staining
I• n
Neurobiology
Intracellular Staining
•
In
Neurobiology
Edited by
Stanley B. Kater
and
Charles Nicholson
•
•.•. ••
Springer-Verlag Berlin· Heidelberg· New York
1973
Library of Congress Cataloging in Publication Data
Kater, Stanley B
Intracellular staining in neurobiology.
1. Stains and staining (Microscopy) 2. Neurobiology-Technique. 3. Re
active dyes. I. Nicholson, Charles, Joint author. II. Title. [DNLM: 1.
Histological techniques. 2. Stains and staining. QH237 K19i 1973]
QH237.K33 578'.64 73-77837
All rights reserved.
No part of this book may be translated or reproduced in
any form without written permission from Springer-Verlag.
© 1973 by Springer-Verlag New York Inc.
Softcover reprint of the hardcover I st edition 1973
ISBN-13: 978-3-642-87125-2 e-ISBN-13: 978-3-642-87123-8
DOl: 10.1007/978-3-642-87123-8
To the memory of
Donald Maynard
Preface
The purpose of this book is to provide basic reference material to facilitate the
further development and application of the intracellular staining techniques which
originated with the introduction of Procion dye as a neurocellular stain. We had sev
eral specific objectives in compiling these chapters. First, we recognized that both the
techniques and results of intracellular staining have been derived from a variety of
preparations and published in diverse journals. Consequently, we tried to make this
information more readily available by incorporating several reviews by original authors.
This should provide the reader with a knowledge of the kinds of neurobiological prob
lems for which intracellular staining has already been employed. A second objective
was to facilitate extension of these methods to a wider variety of problems. To this
end there are chapters dealing with Procion dye as a marker for ultrastructural investi
gations, the use of cobalt as a staining compound and the implementation of computer
techniques for quantitative studies of neuronal relationships. As background for further
extension of these methods this volume contains material on the history of the discovery
of Procion dyes as intracellular stains, the chemistry of these dyes, and fundamentals of
fluorescence microscopy. Our final specific objective was to present details of technical
procedures that were not readily available in the literature. In addition to asking con·
tributors to stress this aspect and providing a final technical chapter, a two-day sym
posium was held at the University of Iowa in October of 1972. This meeting acted
as a forum for discussion of technical details; much of this, often animated, discussion
has been included in the book. It is hoped that, by virtue of the breadth of expertise
represented by those in attendance, this volume gives a true, "state of the art", picture
of intracellular staining technology.
The organization of this volume has proceeded smoothly, due, in large part, to the
cooperation of our contributors, each of whom met stringent deadlines and wrote
chapters which we believe make this book a relatively cohesive entity rather than simply
a collection of papers. Our publishers, Springer-Verlag, New York, cooperated fully
in realizing our desires for high quality and rapid publication. We also wish to par
ticularly acknowledge Dr. Konrad Springer who took a personal interest in this endeavor.
The whole venture was, of course, dependent upon generous financial support from sev
eral sources. We are indebted to the Alfred P. Sloan Foundation, which not only
financed much of the symposium and editing expenses but also paid for the production
of the color plates of this volume. Another major financial contributor was the University
of Iowa Interdisciplinary Committee for Neurobiology (NSF-GU 2591). Other valuable
support came from the University of Iowa Neurobehavioral Sciences Program (5 T01
MH 10641-07), the University of Iowa Graduate College Learned Publications Fund,
and the Department of Physiology and Biophysics and the Department of Zoology.
vii
viii Preface
On an individual basis, we wish to thank Dr. Rodolfo Llinas who provided a lot
of the initiative for this undertaking. Dr. Jerry Kollros likewise fully supported our
efforts and provided much timely advice. Both Dr. Llinas and Dr. Kollros assisted with
preliminary readings of this volume. Dy Mistick bore the brunt of the copy editing with
professional patience and unfailing good humor. We OWe special thanks to Chris Kaneko
who master-minded the practical details of the symposium. Joleen Brue provided a
most human interface with the computer used to store this manuscript. To these and
the many other individuals who facilitated this effort, we offer our thanks.
Iowa City, Iowa Stanley B. Kater
August 15, 1973 Charles Nicholson
List of Contributors
John N. Barrett, Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts.
M. V. L. Bennett, Department of Anatomy, Kennedy Center for Research, Albert Ein
stein School of Medicine, Bronx, New York.
D. f. Bihary, Department of Computer Science, Carnegie-Mellon University, Pittsburgh,
Pennsylvania.
Melvin f. Cohen, Department of Biology, Yale University, New Haven, Connecticut.
William f. Davis, Division Natural Sciences I, University of California, Santa Cruz,
California.
Yoko Hashimoto, Tokyo Women's Medical College, 10 Kaeada-cho, Shinjuku-Ku,
Tokyo 106, Japan.
E. Jankowska, Department of Physiology, University of G6teborg, G6teborg, Sweden.
Akimichi Kaneko, Department of Physiology, School of Medicine, Keio University,
Shinaneomachi, Shinjuku-Ku, Tokyo, Japan.
C. R. S. Kaneko, Department of Zoology, University of Iowa, Iowa City, Iowa.
Stanley B. Kater, Department of Zoology, University of Iowa, Iowa City, Iowa.
James Kelly, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts.
Donald Kennedy, Department of Biology, Stanford University, Palo Alto, California.
E. A. Kravitz, Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts.
S. Lindstrom, Department of Physiology, University of G6teborg, G6teborg, Sweden.
Rodolfo Liinas, Division of Neurobiology, Department of Physiology and Biophysics,
University of Iowa, Iowa City, Iowa.
u. f. McMahan, Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts.
William H. Miller, Department of Ophthalmology and Visual Sciences, School of Medi
cine, Yale University, New Haven, Connecticut.
IX
x List of Contributors
Brian Mulloney, Department of Biology, University of California San Diego, La Jolla,
California.
R. K. Mttrphey, Department of Zoology, University of Iowa, Iowa City, Iowa.
Charles Nicholson, Division of Neurobiology, Department of Physiology and Biophysics,
University of Iowa, Iowa City, Iowa.
R. B. Ohlander, Department of Computer Science, Carnegie-Mellon University, Pitts
burgh, Pennsylvania.
Robert N. Pitman, Department of Physiology and Biochemistry, University of Southamp
ton, Southampton, England.
D. Purves, University College London, London, England.
D. R. Reddy, Department of Computer Science, Carnegie-Mellon University, Pittsburgh,
Pennsylvania.
T akehiko Saito, Department of Ophthalmology and Visual Sciences, School of Medicine,
Yale University, New Haven, Connecticut.
Allen I. Selverston, Department of Biology, University of California San Diego, La Jolla,
California.
C. V. Stead, Research Department, Organics Division, Imperial Chemical Industries
Limited, Blackley, Manchester, England.
A. O. W. Stretton, Department of Zoology, University of Wisconsin, Madison, Wisconsin.
Tsuneo Tomita,. Department of Ophthalmology and Visual Sciences, School of Medicine,
Yale University, New Haven, Connecticut.
Charles D. Tweedle, Department of Zoology, Michigan State University, East Lansing,
Michigan.
David Van Essen, Department of Neurobiology, Harvard Medical School, Boston,
Massachusetts.
L. S. Van Orden III, Department of Pharmacology, University of Iowa, Iowa City, Iowa.
Table of Contents
Preface vii
List of Contributors lX
CHAPTER 1 The Development of Intracellular Staining 1
Charles Nicholson and Stanley B. Kater
Technology of Intracellular Staining
CHAPTER 2 Intracellular Dye Injection: The Selection of Procion Yellow and
its Application in Preliminary Studies of Neuronal Geometry in
the Lobster Nervous System 21
A. O. W. Stretton and E. A. Kravitz
CHAPTER 3 The Chemistry of Reactive Dyes and its Relevance to Intracellular
Staining Techniques 41
C. V. Stead
CHAPTER 4 Principles of Fluorescence Microscopy and Photomicrography with
Applications to Procion Yellow 61
L. S. Van Orden III
CHAPTER 5 Procion Yellow as a Marker for Electron Microscopic Examination
of Functionally Identified Nerve Cells 71
D. Purves and U. J. McMahan
CHAPTER 6 The Form of Nerve Cells: Determination by Cobalt Impregnation 83
Robert M. Pitman, Charles D. Tweedle, and Melvin J. Co hell
Recent Applications of Intracellular Staining
CHAPTER 7 Microelectrode Injection, Axonal Iontophoresis, and the Structure
of Neurons 99
Brian Mtilloney
CHAPTER 8 Permeability and Structure of Electrotonic Junctions and Intercellu-
lar Movements of Tracers 115
M. V. L. Bennett
CHAPTER 9 Characterization of an Insect Neuron which cannot be Visualized
in situ 135
R. K. Murphey
xi
Xl! Table of Contents
CHAPTER 10 Intracellular Staining Techniques in Gastropod Molluscs 151
C. R. S. Kaneko and Stanley B. Kater
CHAPTER 11 Morphological Identification of Single Cells in the Fish Retina by
Intracellular Dye Injection 157
Akimichi Kaneko
CHAPTER 12 Morphological and Physiological Identification of Retinal Cells in
the Turtle 181
Y oko Hashimoto, T akehiko Saito, William H. Miller, and Tsuneo
Tomita
CHAPTER 13 Morphological Identification of Simple, Complex and Hyper-
complex Cells in the Visual Cortex of the Cat 189
David Van Essen and James Kelly
CHAPTER 14 Procion Yellow Staining of Functionally Identified Interneurons
in the Spinal Cord of the Cat 199
E. Jankowska and S. Lindstrom
CHAPTER 15 Procion Yellow and Cobalt as Tools for the Study of Structure-
Function Relationships in Vertebrate Central Nervous Systems 211
Rodolfo Lli17as
Quantitative Analyses
CHAPTER 16 Computer AnalysIs ot Neuronal Structure 227
D. R. Reddy, W. J. Davis, R. B. Ohlander, and D. J. Bihary
CHAPTER 17 The Use of Intracellular Dye Injections in the Study of Small
Neural Networks 255
Allen I. Selverston
CHAPTER 18 Deterinination of Neuronal Membrane Properties Using Intracellu-
lar Staining Techniques 281
John N. Barrett
Practical Considerations
CHAPTER 19 Current Practice and Prospects for the Intracellular Stains 301
Donald Kennedy
CHAPTER 20 A Guide to Intracellular Staining Techniques 307
Stanley B. Kater, Charles Nicholson, and William J. D,wis
Subject Index 327
