Research Methods in Neurochemistry

This second volume of Research Methods in Neurochemistry develops themes featured in the first volume and also introduces new approaches to neurochemical methodology. A further chapter on cell separation tech­ niques seemed justified in view of its general importance to the study of neurone-glia relationships and the complexity of the methodological prob­ lems involved. Biogenic amines also receive more attention in chapters on the assay of monoamine oxidase, catechol-o-methyltransferase and pineal indoles; subcellular fractionation features again in chapters on methods for the isolation and study of brain mitochondria and synaptic vesicles. Among the new subjects dealt with in this volume, the chapter on cell culture per­ haps represents the most distinctive departure from the themes of the first volume. Culture of neural tissue and cells has, of course, been studied by neurobiologists for several decades, but it is only recently that the neuro­ chemical potential of this approach has begun to be exploited. We believe that neurochemists wishing to enter this field will find Dr. Herschman's extensive treatment of the subject in this volume of particular interest. Readers familiar with Volume 1 will note that the balance of subjects in this volume is broadly similar. Thus it has proved convenient to group the chapters under the same section headings as before, with the inclusion of chapters on amino acids in Section IV.

Section I Ultrastructure and Fragmentation of Neural Tissue.- 1 Bulk Separation of Neuronal Cell Bodies and Glial Cells in the Absence of Added Digestive Enzymes.- I. Introduction.- II. Bulk Isolation Procedures Requiring No Added Digestive Enzyme(s).- A. The Procedure Developed in the Authors’ Laboratory.- B. The Procedure of Nagata et al. (1971).- C. The Procedure of Iqbal and Tellez-Nagel (1972).- D. The Procedure of Jones et al. (1971).- III. General Procedural Comments.- IV. Cell Yield and Biochemical Characterization.- V. Applications in Cellular Neurochemistry.- A. Centrifugal Fractionation of Isolated Nerve Cell Bodies.- B. Metabolic and Enzyme Studies.- C. Miscellaneous Applications.- Acknowledgments.- References.- 2 Isolation of Brain Mitochondria.- I. Introduction.- II. Isolation of Brain Mitochondrial Fractions by Differential Centrifugaron.- A. General Aspects.- B. Homogenization Techniques.- C. Isolation Media.- D. Method I: Isolation of Bovine Brain Mitochondria (Stahl et al., 1963).- E. Method II: Isolation of Rat Brain Mitochondria (Ozawa et al., 1966).- III. Fractionation of Crude Mitochondrial Fractions by Density Gradient Centrifugation.- A. General Aspects.- B. Method III: Isolation of Rat Brain Mitochondria (Tanaka and Abood, 1963).- C. Method IV: Isolation of Rat Brain Mitochondria in a Continuous Gradient by Isopycnic Centrifugation (van Kempen et al., 1965).- IV. Criteria of Purity of Mitochondrial Fractions.- A. General Aspects.- B. Electron Microscopy.- C. Enzymes as Markers for Mitochondrial Fraction from Brain.- References.- 3 Preparation of Brain Mitochondrial Membranes.- I. Introduction.- A. Definitions and Abbreviations.- B. Membrane Markers.- II. Methods for Fragmentation.- A. Hypotonic Procedures.- B. Hypotonic Procedures with Sonication.- C. Phospholipases.- D. Digitonin.- III. Enzymatic and Other Criteria.- Acknowledgments.- References.- 4 Isolation and Study of Synaptic Vesicles.- R. M. Marchbanks.- I. Introduction: Types of Vesicles.- II. General Comments on Isolation Methods.- A. Homogenization.- B. Separation of Synaptic Vesicles.- C. Monitoring the Synaptic Vesicle Fraction.- D. Determination of Bound Transmitter.- III. Procedures for the Isolation of Synaptic Vesicles.- A. Synaptic Vesicles from Cerebral Cortex and Related Tissues.- B. Cholinergic Vesicles from Electric Organ.- C. Adrenergic Vesicles from Heart, Vas Deferens, and Spleen.- IV. Studies on Isolated Synaptic Vesicles.- A. Biochemical Manipulations.- B. Morphological Examination.- References.- Section II Properties of Intact Neural Tissues.- 5 Culture of Neural Tissue and Cells.- I. Introduction.- II. Historical Background.- III. Methods of Primary Culture.- A. General.- B. Expiant Cultures of Neural Tissue.- C. Dissociated Cell Cultures of Neural Tissue.- D. Reaggregation Cultures of Neural Tissue.- IV. Methods for Clonal Cultures of Differentiated Tumors of the Nervous System.- A. Induction of Neural Tumors.- B. Primary Culture of Differentiated Neural Tumors.- C. Animal Passage of Primary Culture.- D. Subculture of Cultured Cells.- E. Cloning of Cultured Cells.- F. Culture Media.- G. Hybridization of Cultured Cells.- V. Studies with Primary Cultures.- A. Primary Expiants.- B. Dissociated Cells in Culture.- C. Reaggregation Cultures.- VI. Studies with Clonal Cells.- A. Glial Cells.- B. Neuroblastoma Cells.- VII. Conclusions.- Appendix: Preparation of Rat Tail Collagen.- Acknowledgments.- References.- 6 Measurement of Arteriovenous Differences Across Rat Brain.- I. Introduction.- II. General Considerations.- III. Procedures for Measurement of Arteriovenous Differences..- A. Anesthesia.- B. Cannulation of the Femoral Artery and Vein.- C. Cannulation of the Confluens Sinuum.- D. Blood Sampling and Metabolite Extraction.- E. Consideration of Blood Flow.- IV. Raising and Maintaining Blood Metabolite Concentrations.- A. General Considerations.- B. Approximation of the Priming Dose.- C. Maintenance of an Elevated Concentration.- D. Measurement of Metabolite Removal Rates.- E. General Comments.- F. Acid-Base Changes.- Acknowledgment.- References.- 7 Brain Blowing: A Technique for In Vivo Study of Brain Metabolism.- I. Introduction.- II. Procedures.- A. Description and Operation of the Brain Blower.- B. The Brain Tissue Sample: Region Removed, Sample Size, and Appearance.- C. Treatment of the Brain Sample and Extraction of Metabolites.- D. Measurement of Metabolites.- E. Extracellular Contamination.- F. Troubleshooting.- III. General Comments.- A. Speed of Brain Removal and Freezing.- B. Comparison of the Brain Blower with Other Methods.- C. Indicators of Adequate Freezing Speed.- Acknowledgments.- References.- 8 Determination of Transport Rates In Vivo.- I. Introduction.- II. Definitions and Concepts.- III. Tracer Administration Routes.- A. Feeding.- B. Intramuscular and Subcutaneous Injections.- C. Intraperitoneal Injections.- D. Intravenous Injections.- E. Intravenous Infusions.- F. Intraventricular, Subarachnoidal, and Intracerebral Injections.- IV. Choice of Tracer.- V. Theoretical Model vs. Actual Experiment.- A. Construction of the Compartment Model.- B. Two-Compartment Closed System.- C. Further Aspects of Two-Compartment Systems.- D. Simple Mammillary System.- E. Further Aspects of Mammillary Systems.- F. Use of Analog and Digital Computers.- G. Curve Fitting.- VI. Criticism of Current Methods.- A. Why Tracer Kinetic Studies?.- B. Non Steady State vs. Steady State.- C. Recirculation of the Label.- D. Variation Between Animals.- E. Incomplete Mixing of the Tracer in Compartments.- F. Selection of Molecules for Transport.- VII. Epilogue.- References.- Section III Components of Neural Tissues.- 9 Isolation of Myelin Basic Proteins.- I. Introduction.- A. Myelin Basic Protein and Experimental Allergic Encephalomyelitis.- B. Early Isolation Procedures.- C. Variability of Myelin Basic Proteins.- II. Isolation of Myelin Basic Protein from Central Nervous System Tissue.- A. Preparation of Tissue.- B. Defatting.- C. Acid Extraction.- D. Purification of Myelin Basic Protein.- E. Rat Myelin Basic Proteins.- F. Yields.- III. Isolation of Basic Protein from Purified Myelin.- A. Extraction from Whole Myelin.- B. Extraction from Myelin Dissolved in Organic Solvents.- IV. Isolation of Myelin Basic Proteins from Peripheral Nerves.- V. Purity of Myelin Basic Protein.- A. Introduction.- B. Gel Electrophoresis.- C. Microheterogeneity.- VI. Isolation of Encephalitogenic Peptides from Myelin Basic Protein.- Acknowledgments.- References.- 10 Methods of Isolation and Identification of Neurophysin Proteins.- I. Introduction.- A. Synopsis.- B. Nomenclature.- II. Characterization of Neurophysin Proteins.- III. Preparation of Neurophysins.- A. A Brief History.- B. Preparation of Bovine Neurophysins by Chromatographic Procedures.- C. Preparation of Porcine Neurophysins by Chromatographic Procedures.- D. Preparation of Neurophysins as Lipoproteins.- E. Isolation of Neurophysins by Preparative Discontinuous Electrophoresis and Isoelectric Focusing.- Acknowledgments.- References.- 11 Methods for the Neurochemical Study of Microtubules.- I. Introduction.- II. In Vivo Manipulation of Microtubules.- A. Ablation Experiments.- B. Colchicine and Vinblastine.- C. Other Pharmacological Agents.- D. Physical Factors.- III. Purification of Tubulin from Brain.- A. Biochemical Fractionation.- B. Tubulin Purification by Vinblastine-Induced Precipitation.- C. Purification of Stabilized Microtubules.- D. In Vitro Assembly of Microtubules and Purification of Tubulin by Assembly and Disassembly.- IV. Assays and Purity.- A. Colchicine Binding.- B. Gel Electrophoresis.- V. Conclusion.- Acknowledgments.- References.- 12 Determination of Glycogen in Nervous Tissue.- I. Introduction.- II. Fixation of the Brain to Prevent Postmortem Autolysis of Glycogen.- A. Treatment of Rapidly Frozen Tissue.- III. Partial Purification of Brain Glycogen.- A. Effect of Alcohol Concentration and Electrolytes.- IV. “Bound” and “Free” Brain Glycogen.- V. Methods of Glycogen Determination.- A. Chemical Methods.- B. Enzymatic Methods.- VI. Lack of Purity of Glycogen Isolated from the Brain.- VII. Recommended Method for Isolation and Determination of Brain Glycogen.- A. Procedure.- Acknowledgments.- References.- Section IV Amino Acids and Biologically Active Amines.- 13 Methods for the Isolation and Determination of Glutamate, Glutamine, Aspartate, and ?-Aminobutyrate in Brain.- I. Isolation of Amino Acids.- II. Extraction of Amino Acids from Brain Tissue.- A. Introduction.- B. Procedure.- III. Isolation of Amino Acid Fraction.- A. Ion-Exchange Resin.- B. Procedure.- IV. Isolation of ?-Aminobutyrate.- A. Ion-Exchange Resin.- B. Procedure.- V. Isolation of Glutamate and Aspartate.- A. Materials and Reagents.- B. Procedure.- VI. Isolation of ?-Aminobutyrate, Glutamate, and Aspartate by One-Step Procedure.- A. Procedure.- VII. Isolation of Glutamine.- A. Introduction.- B. Isolation of Glutamine Using a Long Column.- C. Isolation of Glutamine Using a Short Column.- D. Determination of Glutamine After Hydrolysis.- VIII. Quantitative Determination of Amino Acids.- A. Removal of Interfering Compounds.- B. The Method of Yemm and Cocking (1955).- C. The Method of Rosen (1957).- D. The Autoanalyzer Method.- IX. Measurement of Radioactivity.- A. Scintillation Solutions.- B. Sources of Error in the Assay.- References.- 14 Isolation and Determination of N-Acetyl-l-Aspartic Acid and Related Compounds in Brain.- I. Introduction.- II. Column Chromatographic Separation of NA-Asp.- III. Gas Chromatographic Separation of NA-Asp.- IV. Enzymatic Assay of NA-Asp.- A. Preparation of Acylase II.- B. Spectrophotometric Assay of NA-Asp.- C. Fluorometric Assay of NA-Asp.- D. Assay of Acylase II.- V. Identification of NA-Asp.- VI. Crystallization of NA-Asp.- VII. Conclusions.- Acknowledgments.- References.- 15 Methods for Analyzing Monoamine Oxidase and Catechol-O-methyltransferase in Nervous Tissue.- I. Introduction.- II. Procedure.- A. Preparation of Homogenates.- B. Radiometric Assays of MAO.- C. Radiometric Assay of COMT.- III. Discussion.- A. Assay for MAO.- B. Assay for COMT.- IV. Applications.- References.- 16 The Pineal Organ.- I. Introduction.- II. Pineal Indoles.- A. Methods for Measurement of Melatonin and Other Pineal Indoles.- B. Methods for Assaying Pineal Enzymes Involved in Indole Metabolism.- C. Methods for Studying Indole Metabolism in Pineal Organ Cultures.- D. Synthesis and Metabolism of Radioisotopic Melatonin.- III. Nonindolic Pineal Compounds.- Acknowledgments.- References.