Table Of ContentResearCh Melhods
in NeurochemlSlry
VOluma3
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Research Methods
in Neurochemistry
Edited by
Nevilla MarkS
New York State Research I nstitute for
Neurochemistry and Drug Addiction
Ward's Island, New York, New York
and
Richard ROdnight
Department of Biochemistry
I nstitute of Psych iatry
University of London
London, Great Britain
VOluma3
PLENUM PRESS. NEW YORK-LONDON
The Library of Congress cataloged the first volume of this title as follows:
Research methods in neurochemistry. v.1-
New York, Plenum Press, 1912-
v. Illus. 24 em.
1. Neurochemistry.
QP356.3.R46 612'.8'042 72-222263
Lib"ary of Congress
Library of Congress Catalog Card Number 72-222263
ISBN-13:978-1-4613-4460-5 e-ISBN-13:978-1-4613-4458-2
001: 10.1007/978-1-4613-4458-2
©1975 Plenum Press. New York
Softcover reprint of the hardcover 1st edition 1975
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 Floorl, 8 Scrubs Lane, Harlesden, London, NW10 6SE, England
All rights reserved
No part of this book may be reproduced, stored in a retrieval
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mechanical, photocopying, microfilming, recording, or otherwise,
without written permission from the Publisher.
Contributors
AKIRA ARIMURA Department of Medicine, Tulane
University School of Medicine, and
Veterans Administration Hospital, New
Orleans, Louisiana
HAYDEE E. P. BAZAN Instituto de Investigaciones Bioquimicas,
Universidad Nacional del Sur, Bahia,
Blanca, Argentina
NICOLAS G. BAZAN Instituto de Investigaciones Bioquimicas,
Universidad National del Sur, Bahia,
Blanca, Argentina
A. LORRIS BETZ Department of Physiology, University of
Wisconsin, Madison, Wisconsin
STEPHEN C. BONDY Department of Neurology, University of
Colorado Medical Center, Denver, Colo
rado
DAVID H. COY Veterans Administration Hospital and the
Tulane University School of Medicine,
New Orleans, Louisiana
ESTHER J. COY Veterans Administration Hospital and the
Tulane University School of Medicine,
New Orleans, Louisiana
LESTER R. DREWES Department of Physiology, University of
Wisconsin, Madison, Wisconsin
YADIN DUDAI Department of Biophysics, The Weizmann
Institute of Science, Rehovot, Israel
F. FONNUM Norwegian Defense Research Establish
ment, Division of Toxicology, Kjeller,
Norway
vi Contributors
DAVID D. GILBOE Department of Neurosurgery, University
of Wisconsin, Madison, Wisconsin
MARIA GORDON Department of Biochemistry, Institute of
Psychiatry, de Crespigny Park, London,
England
GRAHAM A. R. JOHNSTON Department of Pharmacology, John
Curtin School of Medical Research, Aus
tralian National University, Canberra,
Australia
ELLING KVAMME Neurochemical and Clinical Laboratory.
The Oslo University Psychiatric Clinic,
Vinderen, Oslo, Norway
B. E. LEONARD Neurochemistry Group, Max Planck In
stitute for Experimental Medicine, Got
tingen, G.F.R. Present Address;
Pharmacology Department, University
College, Galway, Republic of Ireland
S. S. OJA Institute of Biomedical Sciences,
University of Tampere, Tampere, Finland
N. N. OSBORNE Neurochemistry Group, Max Planck In
stitute for Experimental Medicine, Got
tingen, G.F.R.
MARTIN REDDINGTON Department of Biochemistry, Institute of
Psychiatry, de Crespigny Park, London,
England
RICHARD RODNIGHT Department of Biochemistry, Institute of
Psychiatry, de Crespigny Park, London,
England
ANDREW V. SCHALLY Department of Medicine, Tulane
University School of Medicine and
Veterans Administration Hospital, New
Orleans, Louisiana
NIKOLAUS SEILER Max Planck Institute for Brain Research,
Unit for Neurochemistry, Frankfurt/
Main, G.F.R.
A. SHELTAWY Department of Biochemistry, University
of Leeds, Leeds, England
ISRAEL SILMAN Department of Biophysics, The Weizmann
Institute of Science, Rehovot, Israel
Contributors vii
GERD SVENNEBY Neurochemical and Clinical Laboratory,
The Oslo University Psychiatric Clinic,
Vinderen, Oslo, Norway
MARIE LOUISE UHR Department of Pharmacology, John
Curtin School of Medical Research, Aus
tralian National University, Canberra,
Australia
M.-L. V AHVELAINEN Institute of Physiology, University of
Helsinki, Helsinki, Finland
MOUSSA B. H. YOUDIM MRC Unit and Department of Clinical
Pharmacology, University of Oxford,
Radcliffe Infirmary, Oxford, England
Preface
With the continued rapid expansion of neurochemical research, there
has been no shortage of new developments in methodology for this third
volume of Research Methods in Neurochemistry. As in previous volumes we
have again tried to provide some balance in the subjects represented. The
wisdom of this policy may be questioned since it can lead to delay in publica
tion, but there are many approaches to the chemical study of the nervous
system and a methods book needs to stand on its own as well as be part of a
series. In one respect, however, the present volume departs from this policy,
in that we have included two chapters on micromethods for analyzing amines
and amino acids, both giving special emphasis to dansylation techniques.
These chapters are complementary and we feel justified in publishing them in
one volume in view of the importance of such micromethods for the study of
neural systems. At the other end of the scale, particular attention may be
drawn to the chapter by D. D. Gilboe and colleagues describing their
remarkable procedures for studying metabolism in the isolated canine brain.
We were fortunate also in persuading S. S. Oja to extend the general prin
ciples of transport systems he described in Volume 2 to amino acids in brain
slices. In addition, there are the usual chapters on components of neural
tissues, which once again we have found convenient to divide into enzymes,
macromolecules, and other constituents.
Thanks are once again due to colleagues and friends for their valuable
suggestions and comments; we continue to welcome both criticisms of
published volumes and suggestions for the future. Finally, we would like to
thank our contributors for their efforts and particularly in the case of this
volume for their patience. The delay has been in no way the fault of Plenum
Press, whose co-operation it is again a pleasure to acknowledge.
Richard Rodnight, London
Neville Marks, New York
September 1975
ix
Contents
Section I
PROPERTIES OF INTACT NEURAL TISSUES
Chapter J
Use of the Isolated Canine Brain in Studies of Cerebral Metabolism,
Metabolite Transport, and Cerebrovascular Physiology ....... 3
David D. Gilboe, A. Lorris Betz, and Lester R. Drewes
I. Introduction........................................... 3
II. Methods.............................................. 5
A. Procedure for the Isolation of the Canine Brain ......... 5
B. Anesthesia.......................... . . . . . . . . . . . . .. 13
C. Perfusion Fluid .................................... 14
D. Blood Gases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19
E. The Perfusion System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20
F. Criteria of Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27
III. Experimental Procedures ................................ 29
A. Net Metabolite Flux ................................ 29
B. Unidirectional Metabolite Influx. . . . . . . . . . . . . . . . . . . . .. 30
C. Intermediary Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . .. 35
D. Vascular Physiology ................................ 38
IV. Initial Equipment Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 40
References .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41
Chapter 2
Axoplasmic Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 43
Stephen C. Bondy
I. Introduction............................. . . . . . . . . . . . . .. 43
II. Criteria............................................... 44
III. Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 45
A. The Optic Pathway ................................. 45
B. Nonvisual eNS Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 52
xi
xii Contents
IV. Peripheral Nervous System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 55
A. Radioisotopic Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 55
B. Histochemical and Enzymic Methods. . . . . . . . . . . . . . . . .. 56
C. Bidirectional and Retrograde Axoplasmic Flow ......... 58
V. Invertebrate Preparations. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. 59
VI. Radioautography....................................... 60
VII. Conclusions ........................................... 62
A. Isolated Systems ................................... 62
B. In Vivo Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 62
References 63
Chapter 3
Transport of Amino Acids in Brain Slices 67
S. S. Oja and M.-L. Vahvelainen
I. Introduction........................................... 67
II. Current Transport Mechanism Concepts ................... 69
A. Diffusion.......................................... 69
B. Carrier Transport .................................. 73
C. Simultaneous Transport Mechanisms. . . . . . . . . . . . . . . . .. 75
D. Solute Interactions in Carrier Transport ............... 79
III. The Study of Influx. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 87
A. General Technical Procedures ........................ 87
B. Determination of the Compound Transported. . . . . . . . . .. 94
C. Swelling or Shrinking of Slices During Incubation . . . . . .. 95
D. Intracellular and Extracellular Spaces ................. 97
E. Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 97
F. Evaluation of Influx Constants ....................... 100
G. A Method for Studying the Influx of Phenylalanine . . . . .. 115
IV. Studies on Efflux ....................................... 117
A. Data Analysis ...................................... 120
B. A Method of Studying the Efflux of Tryptophan ........ 125
V. Equilibrium Experiments ................................ 127
A. Incubation Conditions ............................... 129
B. Data Analysis ...................................... 130
VI. Conclusions ............................................ 133
References 134
Chapter 4
Glycine Enzymes and Uptake Systems 139
Marie Louise Uhr and Graham A. R. Johnston
I. Introduction ........................................... 139
