Research Methods in Neurochemistry

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.