Research Methods in Neurochemistry

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.

Contenu

Section I Properties of Intact Neural Tissues.- 1 Use of the Isolated Canine Brain in Studies of Cerebral Metabolism, Metabolite Transport, and Cerebrovascular Physiology.- I. Introduction.- II. Methods.- A. Procedure for the Isolation of the Canine Brain.- B. Anesthesia.- C. Perfusion Fluid.- D. Blood Gases.- E. The Perfusion System.- F. Criteria of Viability.- III. Experimental Procedures.- A. Net Metabolite Flux.- B. Unidirectional Metabolite Influx.- C. Intermediary Metabolism.- D. Vascular Physiology.- IV. Initial Equipment Needs.- References.- 2 Axoplasmic Transport.- I. Introduction.- II. Criteria.- III. Central Nervous System.- A. The Optic Pathway.- B. Nonvisual CNS Systems.- IV. Peripheral Nervous System.- A. Radioisotopic Methods.- B. Histochemical and Enzymic Methods.- C. Bidirectional and Retrograde Axoplasmic Flow.- V. Invertebrate Preparations.- VI. Radioautography.- VII. Conclusions.- A. Isolated Systems.- B. In Vivo Systems.- References.- 3 Transport of Amino Acids in Brain Slices.- I. Introduction.- II. Current Transport Mechanism Concepts.- A. Diffusion.- B. Carrier Transport.- C. Simultaneous Transport Mechanisms.- D. Solute Interactions in Carrier Transport.- III. The Study of Influx.- A. General Technical Procedures.- B. Determination of the Compound Transported.- C. Swelling or Shrinking of Slices During Incubation.- D. Intracellular and Extracellular Spaces.- E. Data Analysis.- F. Evaluation of Influx Constants.- G. A Method for Studying the Influx of Phenylalanine.- IV. Studies on Efflux.- A. Data Analysis.- B. A Method of Studying the Efflux of Tryptophan.- V. Equilibrium Experiments.- A. Incubation Conditions.- B. Data Analysis.- VI. Conclusions.- References.- 4 Glycine Enzymes and Uptake Systems.- I. Introduction.- II. Enzymes of Glycine Metabolism.- A. General Considerations.- B. Serine Hydroxymethyltransferase.- C. Glycine Transaminase.- D. 3-Phosphoglycerate and Glycerate Dehydrogenases.- E. Glycine Cleavage System.- III. Uptake Systems.- A. Uptake into Tissue Slices.- B. Uptake into Subcellular Particles.- References.- Section IIA Components of Neural Tissues: Enzymes.- 5 Assay and Purification of Brain Monoamine Oxidase.- I. Introduction.- II. Assay Methods of MAO.- A. Histochemical Localization of MAO.- B. Histochemical Localization on Polyacrylamide Gels.- C. Spectrophotometric Assay.- D. Fluorometric Determination Assays.- E. Oxygen Polarographic Assay.- F. Ammonia Determination.- G. Radioactive Assay.- H. In Vivo Assay of MAO.- I. Estimation of MAO by Titration with Irreversible 14C Inhibitors.- III. Preparation of Particulate and Soluble Brain MAO.- A. Distribution.- B. Intracellular Distribution.- C. Preparation of Crude Mitochondrial-Synaptosomal Fraction by Differental Centrifugation.- D. Density Gradient Centrifugation.- E. Purification of Brain MAO.- F. Electrophoretic Separation of Multiple Forms of MAO.- G. Immunological Identity of the Multiple Forms of Mitochondrial MAO.- IV. Future Developments.- V. Guide to Monoamine Oxidase Assay Procedures.- A. Histochemical Localization.- B. Histochemical Localization on Gel.- C. Spectrophotometric Assay.- D. Radioactive Assay.- E. Oxygen and Ammonia Determination.- F. In Vivo Assay.- G. Titration of MAO with 14C Inhibitors.- References.- 6 Acetylcholinesterase.- I. Introduction.- II. Assay Methods.- A. Biochemical Assays.- B. Histochemical Techniques.- III. Purification.- A. Conventional Techniques.- B. Affinity Chromatography.- IV. Methods of Molecular Characterization.- A. Gel Filtration.- B. Electrophoretic Techniques.- C. Density Gradient Centrifugation.- D. Analytical Ultracentrifugation.- E. Active Site Titrations.- F. Amino Acid Analysis.- G. Electron Microscopy.- V. Concluding Remarks.- References.- 7 Radiochemical Assays for Choline Acetyltransferase and Acetylcholinesterase.- I. Introduction.- II. Choline Acetyltransferase.- A. Enzyme Preparation.- B. Assay Methods.- C. Procedures for Choline Acetyltransferase Assay.- III. Acetylcholinesterase.- A. Procedure.- Appendix-Special Reagents.- References.- 8 Phosphate-Activated Glutaminase in Brain.- I. Introduction.- II. Assay of Glutaminase.- A. Determination of Ammonia.- B. Determination of Glutamate.- III. Purification of Phosphate-Activated Pig Brain Glutaminase.- A. Acetone Powder Preparation.- B. Sodium Sulfate Fractionation.- C. Repeated Solubilizations and Precipitations in Tris-HCl Buffer and Phosphate-Borate Buffer, Respectively.- D. Purity Tests.- IV. Properties.- A. Molecular Properties.- B. Kinetic Properties and Metabolic Regulation.- References.- Section IIB Components of Neural Tissues: Lipids, Proteins, and Polylipids.- 9 Analysis of Phospholipids by Sequential Chemical Degradation.- I. Introduction.- II. Factors Involved in the Procedures.- A. Optimum Conditions.- B. Prevention of Side-Product Formation.- C. Partition of Products.- D. Fractionation of Products.- III. Procedure.- References.- 10 Analysis of Free and Esterified Fatty Acids in Neural Tissues Using Gradient-Thickness Thin-Layer Chromatography (GT-TLC).- I. Introduction.- II. Design Features.- III. General Precautions.- IV. Lipid Extraction.- A. Lipid Extraction from Whole Brain Tissue.- B. Lipid Extraction from Subcellular Fractions, Membranes, and Incubation Media.- C. Lipid Extraction from Frozen Neuroanatomical Regions.- D. Lipid Extraction with Recovery of Acid-Soluble Precursors.- V. Preparation of Gradient Layers.- VI. Quantitative Techniques.- VII. Preparative Techniques.- VIII. Preparation of Derivatives.- IX. Gas-Liquid Chromatography (GLC).- References.- 11 Methods for Studying Protein Phosphorylation in Cerebral Tissues.- I. Introduction.- II. Determination of Phosphoproteins in Neural Tissue.- A. Alternative Approaches to Determining Protein-Bound Phosphorus.- III. In Vitro Study of Protein Phosphorylation in Intact Preparations of Cerebral Tissue.- A. Experimental Design.- B. Determination of [32P] Protein Phosphorus.- C. Expression and Interpretation of Data.- IV. Determination of Protein Kinase Activity.- A. Protein Kinase Activity Toward Extrinsic Substrates.- B. Intrinsic Protein Kinase Activity.- V. Determination of Protein Phosphatase Activity.- A. Available Methods.- B. Protein Phosphatase Activity Toward Extrinsic Substrates.- VI. Conclusions.- References.- 12 Assays of Hypothalamic Releasing and Inhibiting Hormones.- I. Introduction.- II. Assay for TRH Activity.- A. Stimulation of TSH in Vitro in Short-Term Incubation of Rat Pituitaries.- B. Release of the Pitu…