Research Methods in Neurochemistry: Volume 1

375 Pages·1995·11.644 MB·English

by  Betty I. Roots, Patricia V. Johnston (auth.), Neville Marks, Richard Rodnight (eds.)

Rasaarch Methods
in NeurOChemistry
VOlumal
Research Methods
In Neurochemistry
EdiladbJ
Navilla Marks
New York State Research Institute for Neurochemistry and Drug Addiction
Ward's Island, New York, New York
and
Richard ROdDiahl
Department of Biochemistry
Institute of Psychiatry
University of London
London, Great Britain
VOlumal
~PLENUM
PRESS. NEW YORK-LONDON. 1972
Library of Congress Catalog Card Number 76·183563
ISBN-13: 978-1-4615-7750-8 e-ISBN-13: 978-1-4615-7748-5
DOl: 10.1007/978-1-4615-7748-5
© 1972 Plenum Press, New York
Softcover reprint of the hardcover 1s t edition 1972
A Division of Plenum Publishing Corporation
227 West 17th Street, New York, N. Y. 10011
United Kingdom edition published by Plenum Press, London
A Division of Plenum Publishing Company, Ltd.
Davis House (4th Floor), 8 Scrubs Lane, Harlesden, London, NW10 6SE, England
All rights reserved
No part of this publication may be reproduced in any form without
written permission from the publisher
Contributors to This Volume
ALAN A. BOULTON Psychiatric Research Unit, University Hos
pital, Saskatoon, Saskatchewan, Canada
STEPHEN R. COHEN New York State Research Institute for
Neurochemistry and Drug Addiction,
Ward's Island, New York
CARL W. COTMAN Department of Psychobiology, University
of California at Irvine
ALAN N. DAVISON M.R.C. Membrane Biology Group, De
partment of Biochemistry, Charing Cross
Hospital Medical School, London
JOSEPH D. FENSTERMACHER Office of the Associate Scientific Director
for Experimental Therapeutics, National
Institutes of Health, National Cancer In
stitute, Bethesda, Maryland
M. GOLDSTEIN Department of Psychiatry, Neurochemistry
Laboratories, New York University Medical
Center, New York
LLOYD A. HORROCKS Department of Physiological Chemistry,
The Ohio State University, Columbus,
Ohio
PATRICIA V. JOHNSTON Children's Research Center and the Burn
sides Research Laboratory, University of
Illinois at Urbana-Champaign
JOHN R. MAJER Department of Chemistry, University of
Birmingham, Edgbaston. Birmingham, Eng
land
RENEE K. MARGOLIS Department of Pharmacology, State Uni
versity of New York, Downstate Medical
Center, Brooklyn
vi Contributors
RICHARD U. MARGOLIS Department of Pharmacology, New York
University School of Medicine, New York
BRUCE S. McEwEN The Rockefeller University, New York
WILLIAM T. NORTON The Saul R. Korey Department of Neurol
ogy, Albert Einstein College of Medicine.
New York
SHIRLEY E. PODUSLO The Saul R. Korey Department of Neurol
ogy, Albert Einstein College of Medicine,
New York
SIDNEY ROBERTS Department of Biological Chemistry, School
of Medicine, and the Brain Research In
stitute, University of California Center for
the Health Sciences, Los Angeles
BETTY I. ROOTS Department of Zoology, University of
Toronto
SOLOMON H. SNYDER Department of Pharmacology and Ex
perimental Therapeutics and Department of
Psychiatry, The Johns Hopkins University
School of Medicine, Baltimore, Maryland
MARTHA SPOHN M.R.C. Membrane Biology Group, De
partment of Biochemistry, Charing Cross
Hospital Medical School, London
GRACE Y. SUN Laboratory of Neurochemistry, Cleveland
Psychiatric Institute, Cleveland, Ohio
KENNETH M. TAYLOR Department of Pharmacology and Ex
perimental Therapeutics and Department
of Psychiatry, The Johns Hopkins Uni
versity School of Medicine, Baltimore.
Maryland
L. S. WOLFE Department of Neurology and Neurosur
gery, McGill University, and Donner Labo
ratory of Experimental Neurochemistry,
Montreal Neurogical Institute, Montreal,
Canada
RICHARD E. ZIGMOND The Rockefeller University, New York
CLAIRE E. ZOMZELy-NEURATH Roche Institute of Molecular Biology,
Nutley, New Jersey
Introduction
On picking up this first volume of a new series of books the reader may ask
the two questions: (a) why research methods? and (b) why in neurochem
istry? The answers to these questions are easy - they more than justify
the volumes to come and show the strong need for their existence.
It is customary to think of methods as a necessary but unexciting means
to an end - to relegate advances in methodology to a minor role in the
creative, original portion of advances in science. This is not the case; the
pace-setting function of methodology is well illustrated in most areas of
neurobiology. To formulate our questions to Nature (which is the essence
of experimental design), methodology is needed; to get answers to our ques
tions we have to devise yet new methods. The chapters of the present volume
fully illustrate how the development of a new method can cut a new path
how it can open new fields, just as the microscope founded histology. Heter
ogeneity of structures presents a formidable challenge for methodology in
the nervous system, yet methods for separating the structures are essential if
we ever want to decipher the enigma of functional contribution of the ele
ments to the whole. The problem is not only physical separation-clearly
methods are essential to study complex structures in situ. Once we separate
a structure, more new methods are needed to analyze and to identify its
elements. Much as we have tried over many years (and are still engaged
in trying), with Dr. Marks, to devise methods for measuring protein turn
over and for measuring protein breakdown in the living brain, a good
method that would open this important area to study still eludes us (no
wonder therefore that Dr. Marks became one of the concerned editors).
No method is the final one, nor can anyone be best for all purposes; in
addition to developing new, more specific, sensitive, and rapid procedures,
we often have to dust off old-established ones from our shelves and modify
them to a particular new purpose. The need for these adaptations as well
as the development of new methods makes us look forward to a number of
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viii Introduction
volumes in addition to this very well executed initial one, and to welcome
them - in spite of the ever-increasing burden of the information explosion.
I should close with the second question: why neurochemistry? Surely,
few readers of this book have to be convinced of the importance of the
nervous system. Some say that just because the brain is at the top end of the
human anatomy it does not prove that that's the important end. They might
be right; undoubtedly many functions of the nervous system-memory,
emotion, learning - generate a great deal of trouble for us. But among its
possible properties is creativity, which needs to function optimally if we want
to create ... new research methods in neurochemistry.
ABEL LAJTHA
New York, June 1972
Preface
The present volume is the first of a series which aims to provide investigators
in the neurosciences with detailed descriptions of experimental procedures
for the biochemical study of the nervous system. We hope that the tremen
dous growth in neurochemistry as a branch of biochemistry with its own spe
cialized methodology is sufficient justification for its aim. Sound methods
form the basis of advances in any experimental subject and this is especially
true in this relatively new and expanding .f ield. Neurochemistry embraces
many specialities, with the consequences that descriptions of techniques are
often minimal and scattered among a wide variety of literature sources.
The arrangement and selection of chapters for the present volume has
been dictated largely by the tradition of subdivision already established in
biochemistry. Thus chapters on subcellular fractionation and specific molec
ular constituents have been grouped separately from those concerned with
studies on intact tissues or animals. A separate section has been accorded
to biogenic amines because of their central importance and the unique roles
tbat they play in nerve function. In so complex an organ as the brain special
importance is assigned to ultrastructural studies, which are grouped together
with the section on subcellular components. A major objective of the present
series is to present within each chapter sufficient detail for ongoing experi
ments without the need for the investigator to have extensive recourse to the
literature. As such it is aimed primarily at the skilled research worker, al
though we hope it will also facilitate teaching programs and assist research
students embarking on their chosen careers. Authors have been urged to
stress methods that can be employed with available equipment rather than
describe procedures requiring expensive instruments outside the range of
most laboratories. We anticipate that one advantage of a continuing series
will be to add refinements to established methods, or provide new approaches
to old problems when deemed appropriate or justified by new developments.
However, the series is not intended to replace older texts where well proven
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x Preface
methods still have their rightful place and have yet to be superseded by newer
procedures. It is our sincere hope that this collection of modern methods
may make a lasting and valuable contribution to the advancement of neuro
chemical knowledge and ultimately to the amelioration of diseases affiicting
the nervous system.
Finally we wish to acknowledge the assistance and encouragement of
Plenum Press and above all the many contributors who have made this vol
ume possible. Suggestions for areas to be covered or improvements for future
volumes are most welcome.
RICHARD RODNIGHT, London
NEVILLE MARKS, New York
May 1972
Contents
Section I. ULTRASTRUCTURE AND FRAGMENTATION
OF NEURAL TISSUES
Chapter I
Nervous System Cell Preparations: Microdissection and
Micromanipulation
Betty I. Roots and Patricia V. Johnston
I. Introduction: A Brief History .............................. 3
II. Preparation by Microdissection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
A. Freeze-dried Tissue .................................... 5
B. Fresh Tissue .......................................... 7
III. Preparation of Neuronal Perikarya by Micromanipulation
Using Cell Suspensions .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
I V. Handling Neuronal and Glial Perikarya for Electron
Microscopy and Adjunct Techniques ........................ 14
V. Preparation of Instruments Used in the Techniques ........... 15
Appendix: Sources of Materials ................................. 16
Acknowledgments ............................................. 16
References .................................................... 17
Chapter 2
The Bulk Separation of Neuroglia and Neuron Perikarya
Shirley E. Poduslo and William T. Norton
I. Introduction ............................................. 19
II. The Isolation of Neuronal Perikarya and Astrocytes
from Rat Brain .......................................... 22
1lI. The Isolation of Oligodendroglia from Bovine White
Matter.... . . .... ........................ ................ 24
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