Rasaarch Methods in NeurOChemistry

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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

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~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 1 st 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

STEPHEN R. COHEN

CARL W. COTMAN

ALAN N. DAVISON

JOSEPH D. FENSTERMACHER

M. GOLDSTEIN

LLOYD A. HORROCKS

PATRICIA V. JOHNSTON

JOHN R. MAJER

RENEE K. MARGOLIS

Psychiatric Research Unit, University Hos­pital, Saskatoon, Saskatchewan, Canada New York State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York Department of Psychobiology, University of California at Irvine M.R.C. Membrane Biology Group, De­partment of Biochemistry, Charing Cross Hospital Medical School, London Office of the Associate Scientific Director for Experimental Therapeutics, National Institutes of Health, National Cancer In­stitute, Bethesda, Maryland Department of Psychiatry, Neurochemistry Laboratories, New York University Medical Center, New York Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio Children's Research Center and the Burn­sides Research Laboratory, University of Illinois at Urbana-Champaign Department of Chemistry, University of Birmingham, Edgbaston. Birmingham, Eng­land Department of Pharmacology, State Uni­versity 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 Neurol­ogy, Albert Einstein College of Medicine. New York

SHIRLEY E. PODUSLO The Saul R. Korey Department of Neurol­ogy, Albert Einstein College of Medicine, New York

SIDNEY ROBERTS Department of Biological Chemistry, School of Medicine, and the Brain Research In­stitute, 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 Ex­perimental Therapeutics and Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland

MARTHA SPOHN M.R.C. Membrane Biology Group, De­partment 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 Ex­perimental Therapeutics and Department of Psychiatry, The Johns Hopkins Uni­versity School of Medicine, Baltimore. Maryland

L. S. WOLFE Department of Neurology and Neurosur­gery, McGill University, and Donner Labo­ratory 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 neurochem­istry? 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 ques­tions 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 path­how it can open new fields, just as the microscope founded histology. Heter­ogeneity 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 ele­ments 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 turn­over 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 tremen­dous growth in neurochemistry as a branch of biochemistry with its own spe­cialized 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 . field. 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 molec­ular 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 experi­ments without the need for the investigator to have extensive recourse to the literature. As such it is aimed primarily at the skilled research worker, al­though 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 neuro­chemical 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 vol­ume possible. Suggestions for areas to be covered or improvements for future volumes are most welcome.

May 1972

RICHARD RODNIGHT, London NEVILLE MARKS, New York

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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IV. Miscellaneous Applications and Modifications. . . . . . . . . . . . . . .. 25 V. General Comments ....................................... 26

VI. Cell Properties ........................................... 28 Acknowledgment .............................................. 31 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31

Chapter 3

Separation of Myelin Fragments from the Central Nervous System

Martha Spohn and Alan N. Davison

1. Introduction ............................................. 33 II. Recommended Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 34

A. Step I ................................................ 34 B. Step 2a and 2b ........................................ 35 C. Step 3 . . . . .. .. . . .... .. .. .. .. . . . . . . . .. . . . .. . . . . . . . . .. .. 35 D. Step 4 . . . . .. .. . . . . .. . . .. .. .. . . . . . . . ... .. . . . . . . . . .. . . .. 35 E. Step 5 .. . .. . .. . . . . .. . . .. . . . . . . . . . . . .. . . . .. . . . . . . . . . . .. 36 F. Steps 6 and 7 ......................................... 36

III. Alternative Methods for the Isolation of Myelin .............. 37 IV. Criteria for Purity ........................................ 38 V. Yield of Myelin .......................................... 41

References .................................................. " 42

Chapter 4

Principles for the Optimization of Centrifugation Conditions for Fractionation of Brain Tissue

Carl W. Cotman

I. Introduction ............................................. 45 II. Factors Affecting Subcellular Separations ........ . . . . . . . . . . .. 45

A. Properties of Subcellular Particles ....................... 45 B. Properties of the Centrifuge Rotor ....................... 69

III. Techniques for Preparing and Evaluating Subcellular Separations .............................................. 71 A. Operation of the Zonal Centrifuge System ................ 71 B. Evaluation of Subcellular Separations .................... 73

IV. Application of Zonal Centrifugation to Brain Separations. . . . .. 75 A. Measurements of S20,w Values for Mitochondria and

Synaptosomes ....................................... " 76 B. Use of Sedimentation Coefficients for Determining

Separation Conditions ................................. 78 V. Analytical Differential Centrifugation ....................... 82

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VI. Conclusions and Future Applications of Centrifugation to the Study of Brain .................................. 86

VII. Summary ...................................... " . . . . . . .. 89 VIII. Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 90 Acknowledgment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 91 References ...................................... . . . . . . . . . . . . .. 92

Chapter 5

Brain Ribosomes

Claire E. Zomze1y-Neurath and Sidney Roberts

I. Introduction ............................................. 95 II. General Considerations ................................... 96

III. Preparation of Microsomal Fractions ....................... 98 A. Large and Small Microsomes ........................... 98 B. Total Microsomes ..................................... 99

IV. Preparation of Ribosomal Fractions ........................ 99 A. Mixed Ribosomes ..................................... 100 B. Polyribosomes ........................................ 105 C. Stripped Ribosomes ................................... 119

V. Ribosomal Amino Acid-Incorporating Systems ............... 119 A. The pH 5 Enzyme Preparation .......................... 120 B. Ribosomal Factor Protein .............................. 121 C. Amino Acids ......................................... 121 D. Ribosomes ........................................... 122 E. Cerebral Messenger RNA .............................. 122 F. Other Additives ....................................... 123 G. Measurement of Incorporation .......................... 124

VI. Amino Acid-Incorporating Properties of Cerebral Ribosomal Systems ....................................... 125 A. Microsomal and Mixed Ribosomal Systems ............... 125 B. Polyribosomal Systems ................................. 127 C. Purified Ribosomal Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 130

VJI. Conclusions ............................................. 133 Acknowledgments ............................................. 135 References ........................................ . . . . . . . . . . .. 135

Chapter 6

Isolation of Brain Cell Nuclei

Bruce S. McEwen and Richard E. Zigmond

I. Introduction 140

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II. Equipment .............................................. 140 A. Homogenizer ......................................... 140 B. Centrifuges ........................................... 140 C. Centrifuge Rotors ..................................... 140

Ill. Solutions ................................................ 140 IV. Nuclear Isolation Procedures ............................... 141

A. Whole Brain .......................................... 141 B. Brain Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 142 C. Nuclear Isolation in Combination with Cell

Fractionation ......................................... 142 D. Nuclear Isolation without Triton ........................ 143

V. Determining Purity of the Isolated Nuclear Pellet ............. 143 A. Light Microscopy ..................................... 144 B. Electron Microscopy ............. . . . . . . . . . . . . . . . . . . . . .. 144 C. Enzymic Determinations of Purity ....................... 144

VI. Chemical Analysis of the Isolated Nuclei and Nuclear Yield Procedure .................................. 148

VII. Other Methods for Isolating Brain Cell Nuclei ............... 150 vm. Separation of Nuclear Types ............................... 152

IX. Properties of Cell Nuclei .................................. 153 A. Energy Metabolism .................................... 153 B. Protein Synthesis ...................................... 153 C. RNA Synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 154 D. DNA Synthesis ....................................... 154 E. Modification of Nuclear Macromolecules ................. 154 F. Nucleocytoplasmic Interactions. . . . . . . . . . . . . . . . . . . . . . . . .. 155

X. Uses of Isolated Brain Clel Nuclei .......................... 156 A. Steroid Hormone-Binding Macromolecules

in Brain Cell Nuclei ................................... 156 B. Study of Other Nuclear Components after in Vivo

Labeling ............................................. 156 C. Study of Nuclear Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 157 D. Study of Biosynthetic Reactions in Isolated

Brain Nuclei .......................................... 157 XL Summary of Some of the Factors Affecting Nuclear

Isolation and the Choice of Isolation Procedures ............. 158 Acknowledgments ............................................. 159 References .. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 159

Contents 1.

Section II. PROPERTIES OF INTACT NEURAL TISSUF..8

Chapter 7

Ventriculocistemal Perfusion as a Technique for Studying Transport and Metabolism within the Brain Joseph D. Fenstermacher

I. Introduction ............................................. 165 II. Method ................................................. 167

A. General Surgical Procedures ............................ 167 B. Placement of Inflow and Outflow Needles ................ 168 C. Perfusion Solution ..................................... 17] D. Procedure for Perfusion ................................ 172 E. Duration of Perfusion .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173 F. Tissue Sampling ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 173 G. Analysis of Data ...................................... 175 H. Use of Drugs ......................................... 176

III. Equipment Needs ........................................ 176 IV. Variations of the Technique ................................ 177 V. Conclusions ............................................. 177

Acknowledgments ............................................. 178 References .................................................... 178

Chapter 8

The Estimation of Extracellular Space of Brain Tissue in Vitro Stephen R. Cohen

I. Introduction ............................................. 179 II. Estimation of the Extracellular Space from the Penetration

of "Extracellular Space Markers" in Vitro. . . . . . . . . . . . . . . . . .. 180 A. General Considerations ................................ 180 B. Extracellular Spaces and Markers . . . . . . . . . . . . . . . . . . . . . . .. 183 C. Recommended Markers and Conventions ... . . . . . . . . . . . . .. 192

I II. Method for Determining Marker Space in Brain Slices ........ 195 A. Procedure ............................................ 195 B. Notes ................................................ 195 C. Computations and Bases for Expressing Marker

Spaces ................................................ 198 IV. Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 204

A. From Steady-State Efflux Kinetics of a Marker .. . . . . . . . . .. 205 B. From Marker Spaces in Vivo .... . . . . . . . . . . . . . . . . . . . . . . .. 208 C. By Electron Microscopy ................................ 211

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D. From Electrical Resistance of Tissue ..................... 212 V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 214

VI. Addendum ............................................... 216 Acknowledgments ............................................. 217 References ................................................ . . .. 218

Section III. COMPONENTS OF NEURAL TISSUES

Chapter 9

Ethanolamine Plasmalogens

Lloyd A. Horrocks and Grace Y. Sun

1. Introduction ............................................. 223 II. Lipid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 223

III. Assay of Total Plasmalogen ............................... 224 IV. Determination of the Phospholipid Composition

Including the Alkenyl Acyl and Alkyl Acyl Components . . . . . .. 225 A. Two-Dimensional Thin-Layer Chromatography ............ 225 B. Assays of Diacyl and Alkyl Acyl GPE ................... 227 C. Phosphorus Assays .................................... 228

V. Isolation of Ethanolamine Phosphoglycerides ................ 228 VI. Preparation of Derivatives for Gas-Liquid

Chromatography ......................................... 229 References ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 23 J

Chapter 10

Methods for Separation and Determination of Gangliosides

L. S. Wolfe

I. Introduction ............................................. 233 II. Isolation and Purification .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 236

A. Notes on Alternate Procedures .......................... 237 B. Purification of the Crude Mixed Ganglioside Fractions ..... 238

III. Resolution ............................................... 239 A. Column Chromatography .............................. 239 B. Thin-Layer Chromatography on Silica Gel ................ 240

IV. Analytical Procedures ..................................... 242 A. Determination of Sialic Acids ........................... 243 B. Determination of Hexoses and Hexosamines

in Gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 244 C. Isolation of Sialyloligosaccharides from Gangliosides . . . . . .. 245

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D. Analysis of Ganglioside Oligosaccharides by Gas-Liquid Chromatography ........................... 246

E. Analysis of the Ceramide Part of Gangliosides ............ 246 F. Mass Spectrometry of Gangliosides .......... :........... 247

References .................................................... 247

Chapter II

Mucopolysaccbarides and Glycoproteins

Richard U. Margolis and Renee K. Margolis

I. Introduction ............................................. 249 II. Extraction of Mucopolysaccharides and Glycoproteins ........ 250

A. Fractionation and Characterization of M ucopolysaccharides .................................. 253

B. Fractionation and Characterization of Glycopeptides and Glycoproteins ..................................... 257

III. Analytical Methods ........................... . . . . . . . . . . .. 259 A. Hexosamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 259 B. N-Acetylhexosamines .................................. 262 C. N-Sulfated Hexosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 262 D. Uronic Acids ......................................... 263 E. Sulfate ............................................... 264 F. Total Neutral Sugar ................................... 265 G. Methylpentoses ....................................... 266 H. Hexoses .............................................. 267 r. Sialic Acids ................................. . . . . . . . . .. 268 J. Column Chromatographic Separation of Neutral

Sugars ................................................ 269 K. Gas Chromatographic Methods ......................... 270 L. Paper Chromatography and Electrophoresis. " ............ 271

IV. Enzymic Analysis of Mucopolysaccharides and Glycoproteins ............................................ 273 A. Bacterial Chondroitinases and Chondrosulfatases .......... 273 B. Testicular Hyaluronidase ............................... 275 C. Other Mucopolysaccharidases ........................... 276 D. Glycosidases Acting on Glycopeptides and

Glycoproteins ................................... . . . . .. 278 References .................................................... 280

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Section IV. BIOLOGICALLY ACTIVE AMINES

Chapter 12

Assay of Biogenic Amines and Their Deaminating Enzymes in Animal Tissues Solomon H. Snyder and Kenneth M. Taylor

J. Introduction ............................................. 287 II. Spectrophotofluorometric Assay of Catecholamines

and Related Compounds .................................. 288 A. Materials ............................................. 289 B. Procedure ............................................ 290 C. Discussion .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 296

III. Spectrophotofluorometric Assay of Tissue Serotonin by the Ninhydrin Procedure ............................... 297 A. Procedure ............................................ 297

IV. Sensitive and Specific Enzymic-Isotopic Assay for Tissue Histamine ......................................... 299 A. Materials ............................................ 300 B. Procedure ............................................ 301

V. Microassay for Histamine ................................. 303 VI. Sensitive Fluorometric and Radiometric Assays for

Monoamine Oxidase and Diamine Oxidase .................. 309 A. Fluorometric Assay .................................... 309 B. Radiometric Assays for Monoamine Oxidase and

Diamine Oxidase ...................................... 312 C. Monoamine Oxidase Assay ............................. 312 D. Diamine Oxidase Assay ................................ 313

References .................................................... 314

Chapter 13

Enzymes Involved in the Catalysis of Catecholamine Biosynthesis M. Goldstein

I. Introduction ............................................. 317 II. Properties and Purification oflndividual Enzymes ............ 318

A. Tyrosine Hydroxylase .................................. 318 B. Dihydropteridine Reductase ............................ 321 C. Aromatic L-Amino Acid Decarboxylase (Dopa

Decarboxylase) ....................................... 321 D. Dopamine-,B-Hydroxylase ............................... 325 E. Phenylethanolamine N-Methyltransferase ................. 329

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III. Cellular Localization of Enzymes Involved in Catecholamine Biosynthesis ................................ 331 A. Cellular Localization of Dopamine-p-Hydroxylase ......... 333

References .................................................... 339

Chapter 14

Detection and Quantitative Analysis of Some Noncatecholic Primary Aromatic Amines Alan A. Boulton and John R. Majer

I. Introduction ............................................. 341 II. Chromatographic Techniques .............................. 343

A. Materials, Methods, and Results ........................ 344 B. Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 349

III. Mass Spectrometry ....................................... 349 A. Materials. Methods. and Results ........................ 350 B. Comment ............................................ 353

Acknowledgment ............................................... 355 References .................................................... 355

Index ........................................................ 357

ResearCh Methods In NeuroChemistry

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