Research Methods in Neurochemistry: Volume 6

ISBN-13: 9781461577621 / Angielski / Miękka / 2012 / 372 str.

Neville Marks

Opis

More than ever, the introduction of new methods or techniques serves to stimulate progress into understanding the structure and function of the nervous system. This axiom is exemplified by recent techniques that have revolutionized several branches of neurochemistry and promise to remain dominant for many years. Such developments underscore the need to remain abreast of new research strategies and provide further justification for the present series. The use of high performance liquid chromatography combined with bioassay methods provides a powerful technique for iso- lation and assay of trace amounts of neuropeptides. Two chapters in the present volume deal with this subject: one (Stenn and Lewis) describes the assay of enkephalins, and the other (Chretien and Seidah), on lipo- tropic peptides, includes procedures for structural analysis by microse- quencing. These methods rival earlier ones for peptide separations in speed, sensitivity, and cost and have general applicability in most labo- ratories. High performance liquid chromatography has also largely sup- planted earlier and more tedious procedures for the assay of catechola- mines, as described in Chapter 7 by Causon. As in earlier volumes, we have striven to retain a balance between studies on intact tissues and those on subcellular components.

Spis treści

Section I Ultrastructure and Fragmentation of Neural Tissues.- 1 Techniques for Neurochemical Research on the Retina.- I. The Retina as a Neurochemical Model.- II. Structure, Function, and Species Differences.- A. General Structure and Function.- B. Retinal Vascularization and the Blood-Retinal Barrier.- C. Photoreceptor Cells and the Fovea.- D. Photoreceptor Outer Segment Turnover and Phagocytosis by the Pigment Epithelium.- E. The Visual Pigments.- F. The Visual Cycle.- III. Functional Stimulation of the Retina.- A. Photoreceptor Function: Stimulating Rods and Cones.- B. Monitoring Retinal Function: The Electroretinogram.- C. Working with a Dark-Adapted Retina.- IV. Studying the Retina in Vivo.- A. Intravitreal Injection.- B. Vitreal Perfusion.- C. Retinal Superfusion.- V. The Retina in Vitro.- A. Enucleation.- B. Eyes and Eye-Cups in Vitro.- C. Isolating the Retina.- D. Quantifying the Retina.- E. Maintenance of the Retina in Vitro.- F. Retinal Culture.- G. Techniques for Isolating and Studying the Retinal Pigment Epithelium.- VI. Fractionation of the Retina.- A. Retinal Fractionation by Cell Degeneration.- B. The Separation and Isolation of Retinal Cells.- C. Subcellular Fractionation of the Retina.- D. Tangential Sectioning of the Retina.- VII. Rhodopsin.- A. Assaying Rhodopsin.- B. Optimizing Rhodopsin and Quantifying Opsin.- References.- 2 Isolation of Cells from Frozen Brain Tissue and Storage of Isolated Cells in the Frozen State.- I. Introduction.- II. Events Occurring during Freezing of Cells: The Use of Cryopreservatives to Minimize Freezing Damage.- III. Isolation of Cells from Frozen Brain Tissue and Freezing of Isolated Oligodendroglia.- A. Isolation of Oligodendroglial Perikarya from Whole Brain Stored at -30 or -80°C.- B. Isolation of Cells from Cryopreservative-Treated Gray and White Matter.- C. Freezing of Isolated Cells in the Presence of Cryopreservative.- IV. Properties of Isolated Cells: Assessment of Preservation of Cytoplasm.- A. Morphology of Isolated Cells.- B. Biochemistry of Isolated Cells.- V. Conclusions and Future Developments.- References.- Section II Properties of Intact Neural Tissues.- 3 The Deoxyglucose Method for the Measurement of Local Glucose Utilization and the Metabolic Mapping of Functional Neural Pathways in the Central Nervous System.- I. Introduction.- II. Theoretical Basis of Radioactive Deoxyglucose Method.- III. Procedure.- A. Preparation of Animals.- B. Administration of [14C]Deoxyglucose and the Sampling of Arterial Blood.- C. Analysis of Arterial Plasma for [14C]Deoxyglucose and Glucose Concentrations.- D. Processing of Brain Tissue.- E. Preparation of Autoradiographs.- F. Densitometric Analysis of Autoradiographs.- G. Calculation of Rate of Glucose Utilization.- IV. Theoretical and Practical Considerations.- A. Rate Constants.- B. Lumped Constant.- C. Role of Glucose-6-phosphatase.- D. Influence of Varying Plasma Glucose Concentration.- E. Animal Behavior during the Experimental Period.- V. Rates of Local Cerebral Glucose Utilization in the Normal Conscious State.- VI. Effects of General Anesthesia.- VII. Relationship between Local Functional Activity and Energy Metabolism.- A. Increased Functional Activity—Experimental Focal Seizures.- B. Decreased Functional Activity—Visual Occlusion.- VIII. Computerized Color-Coded Image Processing.- IX. The Use of the [14C]Deoxyglucose Method for Metabolic Mapping of Functional Neural Pathways.- X. Microscopic Resolution.- XL [18F]Fluorodeoxyglucose Technique.- References.- 4 Continuous-Injection Methods for the Measurement of Flux across the Blood-Brain Barrier: The Steady-State, Initial-Rate Method.- I. Introduction.- II. Principle of the Method.- III. Development of a Procedure to Measure Flux across the Blood-Brain Barrier by the Steady-State, Initial-Rate Method.- A. Background to the Problem.- B. Improvements in Technique.- C. Derivation of an Injection Schedule to Maintain a Steady Level in the Circulation.- IV. Preliminary Preparation.- A. Preparation of the Animal.- B. Preliminary Considerations.- C. Assessing Rate of Tracer Disappearance from the Bloodstream.- V. Devising a Suitable Injection Program.- VI. Implementation of the Injection Program.- A. An Apparatus Suitable for Giving Electronically Controlled Injections.- B. Checking the Effectiveness of the Injection Program.- C. Empirical Adjustment of the Infusion Program to Meet Altered Conditions.- VII. Measurement of Flux across the Blood-Brain Barrier.- A. General Considerations.- B. Tissue Sampling and Tracer Assay.- VIII. Monitoring the Time Course of Tissue Tracer Uptake.- IX. Testing for Saturability of the Transport System.- X. Testing for Competitive Inhibition.- XI. Discussion.- XII. Advantages.- XIII. Precautions.- References.- Section III Components of Neural Tissues—Peptide Hormones and Amines.- 5 Methods for Isolation, Characterization, and Sequence Analysis of Enkephalin Precursors.- I. Introduction.- II. Preliminary Purification Steps.- A. Chromaffin Granule Isolation.- B. Extraction Procedure.- C. Size-Exclusion Chromatography.- D. Assays.- III. Purification of Enkephalin-Containing Polypeptides: Reverse-Phase HPLC of Peptides and Proteins.- A. Instrumentation.- B. High-Performance Liquid Chromatography Methods.- C. Application of Instrumentation and Methods.- IV. Chemical Analysis of Enkephalin-Containing Polypeptides.- A. Amino Acid Analysis.- B. Tryptic Mapping.- C. Sequencing.- V. mRNA-cDNA Cloning.- VI. Summary.- References.- 6 Microsequence of Polypeptide Hormones: Its Usefulness to Monitor the Isolation of Novel Molecules.- I. Introduction.- II. Microsequencing.- A. General Comments.- B. Characterization from Pulse and Pulse-Chase Experiments.- III. Application to Monitoring Purification of a New Pituitary Glycoprotein.- A. Methods.- B. Results.- IV. Chemical Characterization.- A. Methods.- B. Results.- V. Conclusion.- References.- 7 High-Performance Liquid Chromatographic Separation and Determination of Catecholamines.- I. Introduction.- A. High-Performance Liquid Chromatography.- B. Analysis of Catecholamines.- II. Procedures.- A. Extraction and Concentration.- B. High-Performance Liquid Chromatography Systems.- C. Catecholamines Determined by HPLC with EC Detection.- D. Method for Human Plasma and Urine.- III. Recent Developments.- IV. Conclusions.- References.- Section IV Components of Neural Tissues—Enzymes and Proteins.- 8 Purification of Brain Carbonic Anhydrase by Preparative and Immunologic Techniques.- I. Introduction.- II. Enzyme Assay.- A. Solutions.- B. Supplies.- C. Procedure.- III. Extraction of Soluble and Membrane-Bound Carbonic Anhydrase from Rat Brain.- A. Solutions.- B. Supplies.- C. Procedure.- IV. Preparation of Affinity Columns.- A. Solutions.- B. Supplies.- C. Procedure.- V. Affinity Chromatography.- A. Solutions.- B. Supplies.- C. Procedure.- VI. Analysis of Purified Material.- VII. Analytical Methods for the Isolation of Brain Carbonic Anhydrase.- A. Antibody Production.- B. Preparation of Immunoadsorbants.- C. Application of Immunoadsorbants.- VIII. Summary.- References.- 9 Research Methods in Studies with the P2 Basic Protein.- I. Introduction.- II. Isolation and Characterization of P2 Protein.- A. Isolation and Purification Procedures.- B. Chemical Characterization of P2.- C. Characteristics of the P2 Molecule.- III. Immunochemical Techniques in Studies of P2 Protein.- A. Immunochemical Methods.- B. Localization of the Protein in Nervous System Tissue.- IV. P2 in Studies of Experimental Allergic Neuritis.- A. Disease Induction Studies.- B. Neuritogenic Domains of P2 Protein.- C. Immune Response to P2.- D. Protection against EAN.- V. Concluding Remarks.- References.- 10 Methods for the Identification and Characterization of Glycoproteins in Central and Peripheral Myelin.- I. Introduction.- II. Isolation of Myelin and Myelin-Related Fractions.- A. Isolation of Myelin.- B. Subfractions of Myelin and Myelin-Related Membranes.- III. Methods for Detecting Glycoproteins of Myelin.- A. Polyacrylamide Gel Electrophoresis.- B. Staining Glycoproteins with Periodic Acid-Schiff Reagents.- C. Labeling Glycoproteins in Myelin with Radioactive Precursors.- D. Binding of Radioactive Lectins to Myelin Glycoproteins on SDS Gels.- E. Tritium Labeling of Glycoproteins with Tritiated Borohydride.- IV. Distinguishing between Components That Are Genuine Components of Myelin Sheaths and Those That Are in Contaminants of the Isolated Myelin.- V. Purification and Characterization of Specific Glycoproteins.- A. P0 Glycoprotein.- B. Myelin-Associated Glycoprotein.- C. Analytical Methods Used for Chemical Characterization.- VI. Immunologic Procedures.- A. Preparation of Antibodies to MAG and P0.- B. Detection and Characterization of Antibodies and Antigents.- VII. Quantitation of Glycoproteins in Myelin.- A. Determination of Total Protein-Bound Carbohydrate in Myelin.- B. Densitometric Measurement of Individual Glycoproteins on Polyacrylamide Gels.- C. Radioimmunoassay for the Myelin-Associated Glycoprotein.- VIII. General Comments and Conclusions.- References.