Research Methods in Neurochemistry

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 neurochem­ istry? 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 ques­ tions 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 path­ how it can open new fields, just as the microscope founded histology. Heter­ ogeneity 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 ele­ ments to the whole. The problem is not only physical separation-clearly methods are essential to study complex structures in situ.

Section I. Ultrastructure and Fragmentation of Neural Tissues.- 1 Nervous System Cell Preparations: Microdissection and Micromanipulation.- I. Introduction: A Brief History.- II. Preparation by Microdissection.- A. Freeze-dried Tissue.- B. Fresh Tissue.- III. Preparation of Neuronal Perikarya by Micromanipulation Using Cell Suspensions.- IV. Handling Neuronal and Glial Perikarya for Electron Microscopy and Adjunct Techniques.- V. Preparation of Instruments Used in the Techniques.- Appendix: Sources of Materials.- Acknowledgments.- References.- 2 The Bulk Separation of Neuroglia and Neuron Perikarya.- I. Introduction.- II. The Isolation of Neuronal Perikarya and Astrocytes from Rat Brain.- III. The Isolation of Oligodendroglia from Bovine White Matter.- IV. Miscellaneous Applications and Modifications.- V. General Comments.- VI. Cell Properties.- Acknowledgment.- References.- 3 Separation of Myelin Fragments from the Central Nervous System.- I. Introduction.- II. Recommended Procedure.- A. Step 1.- B. Step 2a and 2b.- C. Step 3.- D. Step 4.- E. Step 5.- F. Steps 6 and 7.- III. Alternative Methods for the Isolation of Myelin.- IV. Criteria for Purity.- V. Yield of Myelin.- References.- 4 Principles for the Optimization of Centrifugation Conditions for Fractionation of Brain Tissue.- I. Introduction.- II. Factors Affecting Subcellular Separations.- A. Properties of Subcellular Particles.- B. Properties of the Centrifuge Rotor.- III. Techniques for Preparing and Evaluating Subcellular Separations.- A. Operation of the Zonal Centrifuge System.- B. Evaluation of Subcellular Separations.- IV. Application of Zonal Centrifugation to Brain Separations.- A. Measurements of S20,w Values for Mitochondria and Synaptosomes.- B. Use of Sedimentation Coefficients for Determining Separation Conditions.- V. Analytical Differential Centrifugation.- VI. Conclusions and Future Applications of Centrifugation to the Study of Brain.- VII. Summary.- VIII. Appendix.- Acknowledgment.- References.- 5 Brain Ribosomes.- I. Introduction.- II. General Considerations.- III. Preparation of Microsomal Fractions.- A. Large and Small Microsomes.- B. Total Microsomes.- IV. Preparation of Ribosomal Fractions.- A. Mixed Ribosomes.- B. Polyribosomes.- C. Stripped Ribosomes.- V. Ribosomal Amino Acid-Incorporating Systems.- A. The pH 5 Enzyme Preparation.- B. Ribosomal Factor Protein.- C. Amino Acids.- D. Ribosomes.- E. Cerebral Messenger RNA.- F. Other Additives.- G. Measurement of Incorporation.- VI. Amino Acid-Incorporating Properties of Cerebral Ribosomal Systems.- A. Microsomal and Mixed Ribosomal Systems.- B. Polyribosomal Systems.- C. Purified Ribosomal Systems.- VII. Conclusions.- Acknowledgments.- References.- 6 Isolation of Brain Cell Nuclei.- I. Introduction.- II. Equipment.- A. Homogenizer.- B. Centrifuges.- C. Centrifuge Rotors.- III. Solutions.- IV. Nuclear Isolation Procedures.- A. Whole Brain.- B. Brain Regions.- C. Nuclear Isolation in Combination with Cell Fractionation.- D. Nuclear Isolation without Triton.- V. Determining Purity of the Isolated Nuclear Pellet.- A. Light Microscopy.- B. Electron Microscopy.- C. Enzymic Determinations of Purity.- VI. Chemical Analysis of the Isolated Nuclei and Nuclear Yield Procedure.- VII. Other Methods for Isolating Brain Cell Nuclei.- VIII. Separation of Nuclear Types.- IX. Properties of Cell Nuclei.- A. Energy Metabolism.- B. Protein Synthesis.- C. RNA Synthesis.- D. DNA Synthesis.- E. Modification of Nuclear Macromolecules.- F. Nucleocytoplasmic Interactions.- X. Uses of Isolated Brain Clel Nuclei.- A. Steroid Hormone-Binding Macromolecules in Brain Cell Nuclei.- B. Study of Other Nuclear Components after in Vivo Labeling.- C. Study of Nuclear Enzymes.- D. Study of Biosynthetic Reactions in Isolated Brain Nuclei.- XI. Summary of Some of the Factors Affecting Nuclear Isolation and the Choice of Isolation Procedures.- Acknowledgments.- References.- Section II. Properties of Intact Neural Tissues.- 7 Ventriculocisternal Perfusion as a Technique for Studying Transport and Metabolism within the Brain.- I. Introduction.- II. Method.- A. General Surgical Procedures.- B. Placement of Inflow and Outflow Needles.- C. Perfusion Solution.- D. Procedure for Perfusion.- E. Duration of Perfusion.- F. Tissue Sampling.- G. Analysis of Data.- H. Use of Drugs.- III. Equipment Needs.- IV. Variations of the Technique.- V. Conclusions.- Acknowledgments.- References.- 8 The Estimation of Extracellular Space of Brain Tissue in Vitro.- I. Introduction.- II. Estimation of the Extracellular Space from the Penetration of “Extracellular Space Markers” in Vitro.- A. General Considerations.- B. Extracellular Spaces and Markers.- C. Recommended Markers and Conventions.- III. Method for Determining Marker Space in Brain Slices.- A. Procedure.- B. Notes.- C. Computations and Bases for Expressing Marker Spaces.- IV. Other Methods.- A. From Steady-State Efflux Kinetics of a Marker.- B. From Marker Spaces in Vivo.- C. By Electron Microscopy.- D. From Electrical Resistance of Tissue.- V. Concluding Remarks.- VI. Addendum.- Acknowledgments.- References.- Section III. Components of Neural Tissues.- 9 Ethanolamine Plasmalogens.- I. Introduction.- II. Lipid Extraction.- III. Assay of Total Plasmalogen.- IV. Determination of the Phospholipid Composition Including the Alkenyl Acyl and Alkyl Acyl Components.- A. Two-Dimensional Thin-Layer Chromatography.- B. Assays of Diacyl and Alkyl Acyl GPE.- C. Phosphorus Assays.- V. Isolation of Ethanolamine Phosphoglycerides.- VI. Preparation of Derivatives for Gas-Liquid.- Chromatography.- References.- 10 Methods for Separation and Determination of Gangliosides.- I. Introduction.- II. Isolation and Purification.- A. Notes on Alternate Procedures.- B. Purification of the Crude Mixed Ganglioside Fractions.- III. Resolution.- A. Column Chromatography.- B. Thin-Layer Chromatography on Silica Gel.- IV. Analytical Procedures.- A. Determination of Sialic Acids.- B. Determination of Hexoses and Hexosamines in Gangliosides.- C. Isolation of Sialyloligosaccharides from Gangliosides.- D. Analysis of Ganglioside Oligosaccharides by Gas-Liquid Chromatography.- E. Analysis of the Ceramide Part of Gangliosides.- F. Mass Spectrometry of Gangliosides.- References.- 11 Mucopolysaccharides and Glycoproteins.- I. Introduction.- II. Extraction of Mucopolysaccharides and Glycoproteins.- A. Fractionation and Characterization of Mucopolysaccharides.- B. Fractionation and Characterization of Glycopeptides and Glycoproteins.- III. Analytical Methods.- A. Hexosamines.- B. N-Acetylhexosamines.- C. N-Sulfated Hexosamine.- D. Uronic Acids.- E. Sulfate.- F. Total Neutral Sugar.- G. Methylpentoses.- H. Hexoses.- I. Sialic Acids.- J. Column Chromatographic Separation of Neutral Sugars.- K. Gas Chromatographic Methods.- L. Paper Chromatography and Electrophoresis.- IV. Enzymic Analysis of Mucopolysaccharides and Glycoproteins.- A. Bacterial Chondroitinases and Chondrosulfatases.- B. Testicular Hyaluronidase.- C. Other Mucopolysaccharidases.- D. Glycosidases Acting on Glycopeptides and Glycoproteins.- References.- Section IV. Biologically Active Amines.- 12 Assay of Biogenic Amines and Their Deaminating Enzymes in Animal Tissues.- I. Introduction.- II. Spectrophotofluorometnc Assay of Catecholamines and Related Compounds.- A. Materials.- B. Procedure.- C. Discussion.- III. Spectrophotofluorometnc Assay of Tissue Serotonin by the Ninhydrin Procedure.- A. Procedure.- IV. Sensitive and Specific Enzymic-Isotopic Assay for Tissue Histamine.- A. Materials.- B. Procedure.- V. Microassay for Histamine.- VI. Sensitive Fluorometric and Radiometric Assays for Monoamine Oxidase and Diamine Oxidase.- A. Fluorometric Assay.- B. Radiometric Assays for Monoamine Oxidase and Diamine Oxidase.- C. Monoamine Oxidase Assay.- D. Diamine Oxidase Assay.- References.- 13 Enzymes Involved in the Catalysis of Catecholamine Biosynthesis.- I. Introduction.- II. Properties and Purification of Individual Enzymes.- A. Tyrosine Hydroxylase.- B. Dihydropteridine Reductase.- C. Aromatic L-Amino Acid Decarboxylase (Dopa Decarboxylase).- D. Dopamine-?-Hydroxylase.- E. Phenylethanolamine N-Methyltransferase.- III. Cellular Localization of Enzymes Involved in Catecholamine Biosynthesis.- A. Cellular Localization of Dopamines-Hydroxylase.- References.- 14 Detection and Quantitative Analysis of Some Noncatecholic Primary Aromatic Amines.- I. Introduction.- II. Chromatographic Techniques.- A. Materials, Methods, and Results.- B. Comments.- III. Mass Spectrometry.- A. Materials, Methods, and Results.- B. Comment.- Acknowledgment.- References.