Research Methods in Neurochemistry

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 Pituitary Hormone from Rat Hemipituitaries in Vitro During Two Successive Incubations.- C. Stimulation of TSH Release in Vitro from Sheep and Goat Pituitaries.- D. Stimulation of TSH Release in Vitro from Cultures of Dispersed Rat Pituitary Cells.- E. Stimulation of TSH Secretion in Organ Cultures of Rat Anterior Pituitaries.- F. The Release of 131I from Thyroid Glands of Mice Treated with Codeine and Thyroxine (T4).- G. Release of 125I or 131I from Thyroid Glands of Mice Treated with a Low Dose of Triiodothyronine (T3).- H. RIA for TRH.- III. Assay for LH-RH and FSH-RH.- A. Release of LH or FSH from Rat Pituitaries in Vitro.- B. Release of LH and FSH from Cultures of Rat Pituitary Cells.- C. Elevation of Serum LH in Ovariectomized Estrogen-Progesterone-Treated Rats.- D. Elevation of Serum LH and FSH in Immature Male Rats by Prolonged Infusion of LH-RH/FSH-RH.- E. Induction of Ovulation in Rabbits, Rats, and Hamsters.- F. Determination of LH-RH by Radioimmunoassay.- IV. Assay for CRH.- A. ACTH Release from Rat Pituitaries in Vitro.- B. ACTH Release in Vivo.- V. Assay for GH-RH and GH-RIH.- A. GH Release from Rat Pituitary Fragments in Vitro.- B. GH Release from Cultured Rat Pituitary Cells.- C. GH Release by Infusion of GH-RH into a Hypophyseal Portal Vessel of the Rat.- D. Other in Vivo Methods for GH-RH Using Rats.- VI. PRIH or PIF and PRH.- A. In Vitro Method for PRIH and PRH.- B. In Vivo Assays for PRIH and PRH.- VII. Assay for MRIH (MIF) and MRH.- A. In Vivo Assay for MRIH Using Nembutal, Morphine, and Ether-Treated Rats.- B. In Vivo Assay for MRIH by Direct Application of MRIH Preparations to Exposed Frog Pituitaries.- VIII. Binding Assay for Hypothalamic Hormones.- References.- 13 Structure—Activity Relationship of LH and FSH Releasing Hormone.- I. Introduction.- II. Isolation Methods for LH-RH.- III. Synthesis of LH-RH.- A. Solid-Phase Syntheses.- B. Classical Syntheses.- IV. Analogs of LH-RH.- A. The 1-Pyroglutamic Acid Position.- B. The 2-Histidine Position.- C. The 3-Tryptophan Position.- D. The 4-Serine Position.- E. The 5-Tyrosine Position.- F. The 6-Glycine Position.- G. The 7-Leucine Position.- H. The 8-Arginine Position.- I. The 9-Proline Position.- J. The 10-Glycine Position.- K. Peptides Shorter than the Decapeptide.- L. Peptide Inhibitors of LH-RH.- References.- Section IIC Components of Neural Tissues: Amino Acids.- 14 Assay Procedures for Polyamines and GABA in Animal Tissues with Special Reference to Dansylation Methods.- I. Introduction.- II. Analytical Procedures for Polyamines and GABA.- A. Chemical Methods.- B. Biological Assays.- C. Enzymatic Methods.- III. Principles of the Dansylation Reaction.- IV. Quantitative Analysis of GABA and Polyamines.- A. Reaction of Polyamines and GABA with Dansyl-Cl.- B. Chromatographic Separation.- C. Quantitative Evaluation of Thin-Layer Plates.- V. Conclusion.- References.- 15 The Use of Dansyl-Chloride for the Detection of Amino Acids and Serotonin in Nervous Tissue.- I. Introduction.- II. Procedures.- A. Microanalysis Using [14C]dansyl-Chloride.- B. Fluorescence Spectrophotometry.- C. Mass Spectrometry.- III. Use of the Dansyl-Chloride Technique in Neurochemical Studies.- A. Free Amino Acids and Serotonin in Isolated Snail Neurons Using [14C]Dansyl-Chloride.- B. Studies on “Turnover” of Glucose and Glutamic Acid in Isolated Snail Neurons.- C. Amino Acid Content of Vertebrate and Invertebrate Nervous Tissue.- D. Effects of Drugs on Isolated Snail Neurons.- E. Amino Acids and Serotonin in Different Anatomical Regions of the Rat Brain.- IV. Use of the Dansyl-Chloride Technique in the Analysis of Peptides and Proteins.- A. End-Group Determinations.- B. “Fingerprinting” Techniques.- V. Conclusion.- References.