Research Methods in Neurochemistry. Volume 2.

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.