Research Methods in Neurochemistry: Volume 1

ISBN-13: 9781461577508 / Angielski / Miękka / 2012 / 368 str.

Neville Marks

Opis

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."

Spis treści

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.