Table Of ContentContributors
CHRISTOPHER D. ANDREWS KENNETH E. MOORE
BENITO ANTON KATHYRNE J. MUELLER
ALEJANDRO BAYON JANN A. NIELSEN
CLAES-HENRIC BERTHOLD KAZUO NUNOKI
IRENE CASTANO BRITTA NYSTROM
ANDRE CHERAMY MARIA TRINIDAD PACHECO
JOSE M. R. DELGADO ATHINEOS PHILIPPU
DAVID DIAZ-PONTONES QUENTIN J. PITTMAN
RENE DRUCKER-COLIN VICTOR D. RAMIREZ
FRANCISCO ESTRADA-VILLANUEVA PETER REDGRAVE
JACQUES GLOWINSKI RAFAEL MARTIN DEL RIO
ANDERS HAMBERGER RANULFO ROMO
DEAN R. HAUBRICH WILLIAM D. RUWE
TOSHIHIKO IIJIMA MATS SANDBERG
INGEMAR JACOBSON EUGENE P. SCHOENER
BIRGITTA KARLSSON SARA SOLANO
NORMAN W. KASTING JOSE M. SOLIS
PETER J. KNOTT H. S. SWARTZWELDER
JAKOB KORF JAN A. M. VAN DER HEYDEN
K. LEDERIS WARREN L. VEALE
ANDERS LEHMANN FRANCISCO VELASCO
JUAN LERMA MARCOS VELASCO
GREGORY E. MARTIN KOR VENEMA
IN VIVO
PERFUSION
AND RELEASE
OF NEUROACTIVE
SUBSTANCES
Methods and Strategies
Edited by
ALEJANDRO BAYON
Departamento de Biologia Molecular
Instituto de Investigaciones Biomedicas
Universidad Nacional Autonoma de Mexico
Mexico City, Mexico
RENE DRUCKER-COLIN
Centro de Investigaciones en Fisiologfa Celular
Universidad Nacional Autonoma de Mexico
Mexico City, Mexico
1985
ACADEMIC PRESS, INC.
(Harcourt Brace Jovanovich, Publishers)
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COPYRIGHT © 1985, BY ACADEMIC PRESS, INC.
ALL RIGHTS RESERVED.
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ACADEMIC PRESS, INC.
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United Kingdom Edition published by
ACADEMIC PRESS INC. (LONDON) LTD.
24-28 Oval Road, London NW1 7DX
Library of Congress Cataloging in Publication Data
Main entry under title:
In vivo perfusion and release of neuroactive substances.
Includes index.
1. Neurotransmitters. 2, Brain chemistry. 3. Per
fusion (Physiology). I. Bayon, Alejandro. II. Drucker-
Colin, Rene Raul. [DNLM: 1. Neurochemistry. 2. Brain
Chemistry. 3. Neuroregulators-physiology. 4. Per
fusion. QV 126 135]
QP364.7.I5 1985 599.01*88 84-21541
ISBN 0-12-083350-6 (alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA
85 86 87 88 9 8 7 6 5 4 3 2 1
Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Christopher D. Andrews (141), Department of Pharmacology, Marshall University
School of Medicine, Huntington, West Virginia 25704
Benito Anton (69), Departamento de Biologfa Molecular, Instituto de Investigaciones
Biomedicas, Universidad Nacional Autonoma de Mexico, 04510 Mexico City,
Mexico
Alejandro Bayon (1, 69), Departamento de Biologfa Molecular, Instituto de Inves
tigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, 04510 Mex
ico City, Mexico
Claes-Henric Berthold (119), Department of Anatomy, University of Goteborg,
S-400 33 Goteborg, Sweden
Irene Castano (69), Departamento de Biologfa Molecular, Instituto de Investiga
ciones Biomedicas, Universidad Nacional Autonoma de Mexico, 04510 Mexico
City, Mexico
Andre Cheramy (161), Chaire de Neuropharmacologie, Groupe NB, College de
France, 75231 Paris 05, France
lose M. R. Delgado (95), Departamento de Investigacion, Centro Ramon y Cajal,
Madrid 28034, Spain
David Diaz-Pontones (69), Departamento de Biologfa Molecular, Instituto de Inves
tigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, 04510 Mex
ico City, Mexico
Rene Drucker-Colin (1), Centro de Investigaciones en Fisiologfa Celular, Univer
sidad Nacional Autonoma de Mexico, 20 Mexico City, Mexico
Francisco Estrada-Villanueva (271), Division of Neurophysiology, Scientific Re
search Department, National Medical Center, Instituto Mexicano del Seguro So
cial, 03020 Mexico City, Mexico
Jacques Glowinski (161), Chaire de Neuropharmacologie, Groupe NB, College de
France, 75231 Paris 05, France
Anders Hamberger (119), Institute of Neurobiology, University of Goteborg, S-400
33 Goteborg, Sweden
xi
xii Contributors
Dean R. Haubrich* (293), Department of Pharmacology, Merck Sharp & Dohme
Research Laboratories, West Point, Pennsylvania 19486
Toshihiko lijima (41), Department of Pharmacology, Tohoku University School of
Medicine, Sendai 980, Japan
Ingemar jacobson (119), Institute of Neurobiology, University of Goteborg, S-400 33
Goteborg, Sweden
Birgitta Karlsson (119), Institute of Neurobiology, University of Goteborg, S-400 33
Goteborg, Sweden
Norman W. Kasting (233), Department of Physiology, University of British Colum
f
bia, Vancouver, British Columbia, Canada
Peter j. Knott (141), Department of Pharmacology, Marshall University School of
Medicine, Huntington, West Virginia 25704
Jakob Korf (51), Department of Biological Psychiatry, University Psychiatric Clinic,
9713 EZ Groningen, The Netherlands
K. Lederis (233), Department of Medical Physiology, Faculty of Medicine, University
of Calgary, Calgary, Alberta T2N 4N1, Canada
Anders Lehmann (119), Institute of Neurobiology, University of Goteborg, S-400 33
Goteborg, Sweden
Juan Lerma (95), Departamento de Investigacion, Centro Ramon y Cajal, Madrid
28034, Spain
Gregory E. Martin* (293), Department of Pharmacology, Merck Sharp & Dohme
Research Laboratories, West Point, Pennsylvania 19486
Kenneth E. Moore (177), Department of Pharmacology and Toxicology, Michigan
State University, East Lansing, Michigan 48824
Kathyrne], Mueller (141), Department of Pharmacology, Marshall University School
§
of Medicine, Huntington, West Virginia 25704
Jann A. Nielsen (177), Department of Pharmacology and Toxicology, Michigan
State University, East Lansing, Michigan 48824
Kazuo Nunoki (41), Department of Pharmacology, Tohoku University School of
Medicine, Sendai 980, Japan
Britta Nystrom (119), Institute of Neurobiology, University of Goteborg, S-400 33
Goteborg, Sweden
Maria Trinidad Pacheco (271), Division of Neurophysiology, Scientific Research
Department, National Medical Center, Instituto Mexicano del Seguro Social,
03020 Mexico City, Mexico
Athineos Philippu (221), Department of Pharmacodynamics and Toxicology, Uni
versity of Innsbruck, A-6020 Innsbruck, Austria
f
•Present address: Sterling-Winthrop Research Institute, Rensselaer, New York 12144.
Present address: Departments of Psychiatry and Pharmacology, The Mount Sinai Medical
Center, One Gustave L. Levy Place, New York, New York 10029.
^Present address: McNeil Pharmaceutical, Department of Biological Research, Spring
House, Pennsylvania 19477.
§Present address: Program of Pharmacology, Northeastern Ohio University, College of
Medicine, Rootstown, Ohio 44272.
Contributors xiii
Quentin I. Pittman (233), Department of Medical Physiology, Faculty of Medicine,
University of Calgary, Calgary, Alberta T2N 4N1, Canada
Victor D. Ramirez (249), Department of Physiology and Biophysics, University of
Illinois, Urbana, Illinois 61801
Peter Redgrave (11), Department of Psychology, University of Sheffield, Sheffield
S10 2TN, England
Rafael Martin del Rio, (95), Departamento de Investigacion, Centro Ramon y Cajal,
Madrid 28034, Spain
Ranulfo Romo (161), Chaire de Neuropharmacologie, Groupe NB, College de
France, 75231 Paris 05, France
William D. Ruwe (233), Department of Medical Physiology, Faculty of Medicine,
University of Calgary, Calgary, Alberta T2N 4N1, Canada
Mats Sandberg (119), Institute of Neurobiology, University of Goteborg, S-400 33
Goteborg, Sweden
Eugene P. Schoener (25), Department of Pharmacology, Wayne State University,
School of Medicine, Detroit, Michigan 48201
Sara Solano (69), Departamento de Biologfa Molecular, Instituto de Investigaciones
Biomedicas, Universidad Nacional Autonoma de Mexico, 04510 Mexico City,
Mexico
lose M. Solis (95), Departamento de Investigacion, Centro Ramon y Cajal, Madrid
28034, Spain
H. 5. Swartzwelder (201), Department of Medicine, Division of Neurology, Duke
University Medical Center, Durham, North Carolina 27705
Ian A. M. van der Heyden (51), Department of Pharmacology, Duphar B. V., 1380
AA Weesp, The Netherlands
Warren L. Veale (233), Department of Medical Physiology, Faculty of Medicine,
University of Calgary, Calgary, Alberta T2N 4N1, Canada
Francisco Velasco (271), Division of Neurophysiology, Scientific Research Depart
ment, National Medical Center, Instituto Mexicano del Seguro Social, 03020
Mexico City, Mexico
Marcos Velasco (271), Division of Neurophysiology, Scientific Research Depart
ment, National Medical Center, Instituto Mexicano del Seguro Social, 03020
Mexico City, Mexico
Kor Venema (51), Department of Biological Psychiatry, University Psychiatric Clinic,
9713 EZ Groningen, The Netherlands
Preface
The birth of neuroscience as an interdisciplinary subject largely originated
from the necessity of unifying the conceptual maps produced within the
different traditional disciplines studying the nervous system. The in vivo
techniques that allow the concerted study of neurochemical, phar
macological, electrophysiological, and behavioral aspects of brain function
have played a paramount role in this endeavor. In spite of the fact that these
techniques—the subject matter of this book—have for two decades been
our main window for the direct observation of these correlations, their use
within the scientific community has been limited by misinformation, mis
conceptions, and/or inexperience. Only recently has this panorama begun
to change. As a result of improved procedures, probes, and detectors, and
due to the availability of more specific and sensitive neurochemical assays,
these techniques have provided new strategies for the study of brain func
tion, and their success has resulted in the recognition of their unique value.
The feeling that these developments are opening a new epoch for the in
vivo study of the brain has stimulated several scientific events on this topic,
including a symposium in the Annual Meeting of the Society for Neuro
science (1981), a workshop at the European Society for Neuroscience
(1981), and recently a symposium entitled "/n Vivo Release of Neuroactive
Substances in the CNS," held in 1983 in Mexico City. From these meetings
it was clear that in this changing scientific atmosphere it is necessary not just
to analyze the emerging possibilities and alternatives, but also to evaluate
the state of the art and the depth of its accomplishments.
Our main concern in editing this book is to provide the reader with sound
criteria to engage with, use, and improve on the methods and strategies
utilized to study in vivo neurochemistry, in order to correlate it with elec
trophysiological and behavioral events. We do not intend to give the best of
recipes for in vivo perfusion and release techniques. This is possible only by
careful consideration of current knowledge and personal experience, along
xv
xvi Preface
with practical tests of the suitability of the methods for one's own neces
sities. Nonetheless, in the Introduction we present an overview of meth
odological alternatives and experimental strategies that we think are critical
for the outcome of this type of research. The chapters that follow collect
personal experiences on the study of in vivo neurochemistry and of its
correlation with other neural events, contributed by representative scientists
whose efforts have made possible the optimization of current techniques
and the development of new ones. The chapters have been written and
edited to facilitate the evaluation and comparison of the different techniques
and experimental strategies used by the authors. Chapters in Part I concen
trate on detailed descriptions of the probes and procedures used in this
research and also provide a critical evaluation of the potentialities and
limitations of the methodologies employed. Chapters in Part II exemplify the
variety of experimental strategies in which these techniques have been used,
gathering understanding on the chemical, bioelectrical, and behavioral as
pects of brain function. They also illustrate interdisciplinary techniques for
approaching problems of neuronal connectivity, neuroendocrinology,
motor behavior, neuropathology, etc.
We think this is a timely review, not just for the specialist, who surely will
find this a useful, updated reference text, but for neuroscientists and students
who have not had contact with this field and who may now find that these
developments could open new possibilities for their own research interests.
The editors are grateful to the Direccion General de Asuntos del Personal
Academico (DGAPA) and to the Coordinacion de la Investigacion Cientffica
(CIC) of the Universidad Nacional Autonoma de Mexico, without whose
support this book could not have been written. We also thank Mrs. Martha
Sarmiento for her patient clerical work.
Alejandro Bayon
Rene Drucker-Colin
1
Introduction
ALEJANDRO BAYON
Departamento de Biologia Molecular
Instituto de Investigaciones Biomedicas
Universidad Nacional Autonoma de Mexico
Mexico City, Mexico
RENE DRUCKER-COLIN
Centro de Investigaciones en Fisiologia Celular
Universidad Nacional Autonoma de Mexico
Mexico City, Mexico
Methodological Alternatives and Experimental Strategies for Studying
the in Vivo Perfusion and Release of Neuroactive Substances in the
Central Nervous System 1
References 7
METHODOLOGICAL ALTERNATIVES AND EXPERIMENTAL
STRATEGIES FOR STUDYING THE IN VIVO PERFUSION AND
RELEASE OF NEUROACTIVE SUBSTANCES IN THE CENTRAL
NERVOUS SYSTEM
Twenty-seven years ago Fox and Hilton (/) designed a system of parallel
needles to perfuse subcutaneous tissue, which was modified by J. H. Gad-
dum (2) into two concentric tubes—push-pull cannula—making it possible
to perform in vivo experiments "to localize the site of liberation of sub
stances in such tissues as the central nervous system, more precisely than by
other means." In order to better understand the importance and conse
quences of this development, which revolutionized in vivo neurochemistry,
one must compare it with alternate superfusion techniques. Perfusion meth
ods, push-pull cannulae systems included, are based on the same working
principle: controlled infusion and simultaneous withdrawal of an artificial
physiological fluid through the nervous tissue. This procedure creates a
stream that interfaces with tissular elements, allowing bidirectional diffusion
1
IN VIVO PERFUSNI OAND RELEAES Copyright © 1985 by Academic Pres,s Inc.
OF NEUROACTE IVSUBSTANSC E All rights of reproduction in any form reserved.
ISBN 0-12-083350-6
2 Alejandro Bayon and Rene Drucker-Colin
between the tissue and the exogenous fluid. The possibility of adding neu
roactive chemicals to the infusion fluid, and the fractionation, collection,
and analysis of the outcoming liquid have made perfusion techniques a
milestone in neurobiology. However, both goals can be achieved in vivo by
technical means other than Gaddum's cannulae, namely, surface cups and
ventricular needles. Surface cups (mainly cortical cups) are chambers
chronically adhered to the surface of the brain which contain inlet and
outlet ports to allow infusion and withdrawal of perfusing medium. Ven
tricular needles or cannulae are probes implanted in separate regions of the
brain ventricular system which act as inlet and outlet ports to perfuse the
cerebrospinal fluid (CSF)-filled cavity between them. Cups and ventricular
needles have been, and still are, valuable tools for in vivo neurochemistry;
both were in use at the time Gaddum's cannula was developed and their
power and limitations have been critically compared to those of concentric
cannulae (3). Unfortunately, their use is restricted to the brain surfaces and
cavities and do not allow the study of most deep-brain nuclei. The Gaddum
cannula has found a much wider use: since the inlet and outlet ports (push-
pull) are concentric (see Fig. 1 in the chapters that follow), the cannula can
be implanted just as an electrode is, using stereotaxic techniques in order to
reach discrete neural regions. Thus, the neuroanatomical selectivity and the
versatility of its use are responsible for its success. Because of these advan
tages, and since the basic working principles of surface cups and ventricular
needles are close to those of concentric cannulae, the latter will serve as a
model for discussion of in vivo perfusion methodology. Some designs such
as dialytrodes are also suitable for site-specific perfusions. Dialytrodes are
probes based on the same working principles as other perfusion devices, but
the diffusional exchange between the tissue and the perfusing medium is
mediated by a dialysis membrane. The discussion of the advantages and limita
tions in brain neurochemistry of dialytrodes—and of electrochemical probes—
deserve and need a separate treatment, given in Chapters 7, 8, and 9.
For the neuroscientist using push-pull perfusion methods it is not uncom
mon to meet colleagues who some time in their careers started using this
type of method, but were discouraged very early because of technical diffi
culties and poor, unreliable results. This is the case, particularly, when no
experienced advice backs up the setting of the experimental tools and pro
cedures. In practice, the central and usually unexpected problem with these
techniques is the difficulty in maintaining a steady flow of the perfusion
medium at the interface with the tissue. Since flow obstructions and tissue
damage are closely linked, we will first discuss the factors involving both
problems, then present those related to damage in the absence of flow
impairments.
