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Times of dramatic progress in brain research have often been correlated with the development of new and powerful techniques that have changed the kinds of questions one can ask. An historical example may illustrate the point. More than 50 years ago, Nissl studies (Ferraro, 1928) showed that extensive forebrain lesions resulted in chromatolysis and cell loss in the sub stantia nigra; thus, it was suggested that the substantia nigra gave rise to projections into the basal forebrain. In the late 1950s, another clue emerged, this time linking observations from the field of neuropathology with a dis covery in experimental neuropharmacology (Carlsson, 1959a,b; Ehringer and Hornykiewicz, 1960). It had long been recognized that patients with Par kinson's disease suffered neuronal loss in the substantia nigra and that their symptoms were somehow related to striatal dysfunction. Thus, when flu orescent catecholamine assays were developed and combined with pharma cological and neuropathological studies of Parkinson's disease, the dopamin ergic nature of the illness was shown. A bit later, Falck and Hillarp (Falck et at. , 1962) developed a fluorescent histochemical method to visualize mono amine-containing cells in the brain; this technique was soon applied to show that the rich dopaminergic terminal field in the striatum derived from neu rons in the substantia nigra (Anden et at. , 1964). In the following decade, refinements in the histofluorescent method and the development of sensitive silver impregnation methods permitted a detailed light microscopic explo ration of the dopaminergic nigrostriatal system.

Neuroanatomical Tract-Tracing Methods

1 Experimental Neuroanatomy: General Approaches and Laboratory Procedures.- I. Introduction.- II. Tract-Tracing Methods.- A. Types of Methods.- B. Choice of Method.- III. Practical Problems.- A. The Experimental Animal.- B. The Tissue.- IV. Analysis.- A. Amount of Material.- B. Analytical Approach.- C. Normal Material.- D. Mapping and Reconstruction.- V. The Neuroanatomical Laboratory.- A. Animal Quarters and Surgery.- B. Optical Equipment.- C. Laboratory Precautions.- VI. Appendix.- A. Encasing of Tissue for Preparation of Frozen Sections.- B. Preparation of Frozen Sections on Sliding Microtome.- C. Special Staining Procedures.- D. Graphic Reconstructions.- References.- 2 Methods for Selective, Restricted Lesion Placement in the Central Nervous System.- I. Introduction.- II. Stereotaxic Technique.- A. Theoretical Background.- B. The Stereotaxic Atlas.- C. Verification of the Coordinates.- III. Nonselective Lesion Techniques.- A. Mechanical Lesions.- B. Injected, Nonselective Toxins.- C. Alterations of Cerebral Vasculature.- D. Radioisotopic and Related Methods.- E. Ultrasound Lesions.- F. Thermal Lesions.- G. Electrolytic Lesions.- IV. Evaluation of the Electrolytic Lesion.- V. Selective Lesion Techniques.- A. Kainic Acid and Glutamate Derivatives.- B. Neurotoxic Catecholamine and Indolamine Derivatives.- VI. The Interpretation of Lesion Effects.- VII. Appendix: Stereotaxic Atlases.- A. Rat Brain Atlases.- B. Cat Brain Atlases.- C. Primate Brain Atlases.- D. Dog Brain Atlases.- References.- 3 Methods for Delivering Tracers.- I. Introduction.- II. Pressure Injection.- A. Microsyringe Injection.- B. Micropipette Injection.- III. Iontophoretic Injection.- A. General Considerations.- B. Extracellular Injection.- C. Intracellular Injection.- IV. Appendix.- A. Microelectrode Preparation.- B. Iontophoresis Assays.- References.- 4 Silver Methods for the Impregnation of Degenerating Axoplasm.- I. Introduction.- II. Theoretical Considerations.- A. When to Use the Silver Methods.- B. The Choice of Silver Method.- III. Practical Aspects.- A. Postoperative Survival Time.- B. Fixation and Sectioning.- IV. General Characteristics of the Silver Methods.- A. The Nauta-Laidlaw Method.- B. The Fink-Heimer Method.- C. The Cupric Silver Method.- D. Comparison among the Nauta-Laidlaw, the Fink-Heimer, and the Cupric Silver Methods.- E. Other Silver Methods.- V. Interpretation of Degenerating Fibers and Terminal Degeneration.- A. Axonal Degeneration.- B. Terminal Degeneration.- VI. Other Degenerative Neuronal Phenomena.- A. Degeneration of Cell Bodies and Dendrites.- B. Indirect Wallerian Degeneration.- C. "Retrograde Dust" in Thalamus.- VII. Sources of Error.- A. Neuronal Deposits.- B. Spontaneous, Accidental, and Infectious Degeneration.- C. Dark Neurons of Cammermeyer.- D. Glial Elements and Connective Tissue.- E. Artifacts in the Olfactory Bulb.- VIII. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- IX. Prospects for the Future.- X. Appendix.- A. The Nauta-Laidlaw Method.- B. The Fink-Heimer Procedures.- C. The Cupric Silver Method.- D. The Application of Silver Degeneration Techniques to the Human Brain (M.-M. Mesulam).- References.- 5 The Autoradiographic Tracing of Axonal Connections in the Central Nervous System.- I. Introduction.- II. The Principles of the Method.- III. Methodology.- A. Selection of the Radioactive Tracer.- B. Injection of the Tracer into the Brain.- C. Survival Time.- D. Perfusion and Fixation.- E. Cutting and Mounting the Sections on Glass Slides.- F. Coating the Mounted Sections.- G. Exposure of the Emulsion.- H. Development and Fixation of the Emulsion.- I. Staining of the Tissue.- IV. Analysis of the Data.- A. Definition of a Labeled Pathway.- B. Common Artifacts.- V. Electron Microscopic Autoradiography.- VI. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- VII. Appendix.- A. Paraffin Embedding Schedule for Cat Brain.- B. Darkroom Equipment Needed for Emulsion Coating.- C. Cresyl Violet Staining for Cat and Rat Paraffin Sections.- References.- 6 Horseradish Peroxidase: The Basic Procedure.- I. Introduction.- II. Basic Applications.- III. Incorporation and Transport of HRP.- A. Characteristics of HRP.- B. Diffusion of HRP.- C. Incorporation of HRP by Neurons.- IV. Methodology.- A. Choice of Anesthetic.- B. Methods of Extracellular Delivery.- C. Survival Time.- D. Fixation and Sectioning.- E. Potentiation of Uptake and Transport of HRP.- V. General Characteristics of the Different HRP Methods.- A. The DAB Method.- B. The o-Dianisidine Method.- C. The BDHC Method.- D. The TMB Method.- VI. Results and Interpretations.- A. The Site of Injection.- B. Labeling of Cell Bodies.- C. Labeling of Axons and Terminals.- D. Sources of Error.- VII. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- VIII. Appendix.- A. The 3,3?-Diaminobenzidine (DAB) Method (LaVail).- B. Benzidine Dihydrochloride (BDHC) Method (de Olmos).- C. Tetramethylbenzidine (TMB) Method (de Olmos).- D. Tetramethylbenzidine (TMB) Method (Mesulam).- References.- 7 Horseradish Peroxidase: Intracellular Staining of Neurons.- I. Introduction.- II. Methods.- A. Preparation Procedures.- B. Recording and Injection.- C. Animal Perfusion.- D. Histological Processing.- E. Analysis of the Data.- III. Application of the Technique.- IV. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- V. Appendix.- A. Chemicals for HRP Histological Processing (Intracellular Staining).- B. An Alternative Approach using Retrograde Golgi-like Labeling of Neuronal Populations (D. Keefer).- References.- 8 Horseradish Peroxidase and Fluorescent Substances and Their Combination with Other Techniques.- I. Introduction.- II. The Tracing of Collateral Projections.- A. Retrograde Double-Labeling Procedures Using HRP in Different Combinations.- B. Double Labeling with Fluorescent Substances.- C. Collateral Transport of HRP.- III. HRP and Anterograde Tracing Methods.- IV. HRP and Transmitter-Related Histochemical Procedures.- V. HRP and 2-Deoxyglucose Procedures.- VI. Prospects for the Future.- VII. Appendix.- A. Procedures for Retrograde Double Labeling with HRP and [3H]-BSA.- B. Procedures for Retrograde Double Labeling with HRP and [3H]-apo-HRP (A. Rustioni).- C. Procedure for Retrograde Double Labeling with Fluorescent Substances (H. G.J. M. Kuypers).- D. Procedures for Simultaneous Demonstration of HRP and AChE.- E. 2-Deoxyglucose Autoradiography and HRP Histochemistry (O. Steward).- F. A Note on the Combination of Retrograde Fluorescent Tracers with Transmitter Histochemistry (T. Hökfelt).- References.- 9 The Golgi Methods.- I. Introduction.- II. The Rapid Golgi Method.- A. Preparatory Steps.- B. Fixation.- C. Silver Impregnation.- D. Sectioning the Tissue.- E. Dehydrating and Clearing.- F. Mounting.- G. A Note on Perfusion Fixation.- III. Analysis of the Data.- A. Cell Location.- B. Cell Processes.- IV. Presentation of the Data.- A. Golgi Drawings.- B. Photography.- V. Variations of the Golgi Method.- A. Double and Triple Impregnations.- B. Golgi-Kopsch Method.- C. Golgi-Cox Method.- D. Golgi Method for Embryonic Tissue.- VI. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- VII. Appendix.- A. Recipe for Perfusion Technique.- B. Embedding of Rapid Golgi Blocks in Nitrocellulose.- C. Rapid Golgi Method for Use on Aldehyde-Fixed Material.- D. Stabilizing and Counterstaining Rapid Golgi Sections.- E. Variations of the Golgi-Kopsch Method.- F. The Golgi-Cox Procedure.- G. Golgi Method for Embryonic Tissue.- References.- 10 Electron Microscopy: Preparation of Neural Tissues for Electron Microscopy.- I. Introduction.- II Basic Procedures for Fixation and Embedding.- A. Anesthesia.- B. Surgical Procedure.- C. Dissection and Postfixation.- D. Dehydration and Embedding.- III. Variations.- A. Artificial Respiration.- B. Ventilation with O2-CO2.- C. Pressure of Perfusion.- D. Temperature of Perfusates.- E. Vascular Rinsing Solution.- F. Composition of the Primary Fixative.- G. Double Perfusion.- H. Buffer Wash and Postfixative.- I. Stabilization with Uranyl Acetate.- J. Phosphate Precipitate.- K. A Procedure for Myelin.- IV. Evaluation of Results with the Light Microscope.- V. Cutting and Staining Ultrathin Sections.- VI. Synthesis.- VIII. Appendix.- A. Vascular Rinse.- B. 0.4 M Phosphate Buffer Stock.- C. First Perfusion Fixative.- D. Second Perfusion Fixative.- E. Phosphate Buffer Wash.- F. Double-Strength Osmication Buffer.- G. 4% Osmium Tetroxide Stock Solution.- H. Osmium Tetroxide Postfixative.- I. Osmium Ferrocyanide Postfixative.- J. Acetate Buffer Wash.- K. Uranyl Acetate Block Treatment.- L. Epoxide Embedding Mixture.- M. Mounting and Staining of Semithin Sections.- N. Staining Thin Sections.- References.- 11 Electron Microscopy: Identification and Study of Normal and Degenerating Neural Elements by Electron Microscopy.- I. Introduction.- II. Bridging the Gap between Light and Electron Microscopy.- III. Practical Guidelines for Electron Microscopy.- A. Selecting Sections.- B. Topography.- C. Scanning at Low Magnification (1000-4000x).- D. Scanning at Intermediate Magnification (5000-12,000x)..- E. Scanning at High Magnification (15,000-25,000x).- IV. Identification of Neuronal Elements.- A. Axons.- B. Dendrites.- C. Axoniform Dendrites.- D. Synthesis.- V. Ultrastructure of Degenerating Nerve Fibers.- A. Patterns of degeneration.- B. Synthesis.- VI. Morphometry.- A. Measures.- B. Sampling.- C. Synthesis.- References.- 12 Tract Tracing by Electron Microscopy of Golgi Preparations.- I. Introduction.- A. Purpose and Potentialities.- B. Technical Approach.- II. General Description of Techniques.- A. Fixation.- B. Osmification and Chromation.- C. Silver Impregnation.- D. Primary Thick Sectioning.- E. Deimpregnation.- F. Embedding and Remounting of Primary Sections.- G. Cutting Thick Sections of Tissue Embedded in Resin.- H. Ultramicrotomy.- III. Summary of Advantages and Limitations.- A. Advantages.- B. Limitations.- IV. Concluding Comments and Troubleshooting.- V. Appendix.- A. Osmification and Chromation.- B. Infiltration of Blocks with Glycerol.- C. Thick Sectioning of Impregnated Tissue.- D. Gold Toning.- E. Photochemical (UV) Method without Gold.- F. Photochemical Method with Gold.- G. "Interrupted Golgi Impregnation".- H. Flat Embedding of Primary Sections.- I. Cutting Thick Sections of Plastic.- J. A Method for Remounting Sections.- K. Monitoring Ultrathin Sectioning.- L. Protection of Impregnation with Silver Chromate.- References.- 13 Fluorescence Histochemical Methods: Neurotransmitter Histochemistry.- I. Introduction.- II Chemical Basis of the Fluorescence Histochemical Methods.- A. Introduction.- B. The Falck-Hillarp Method (Formaldehyde Condensation).- C. The Glyoxylic Acid Method.- III. Equipment.- IV. Methods Using Formaldehyde or Glyoxylic Acid Condensation.- A. Introduction.- B. The Falck-Hillarp Method.- C. Methods Using Glyoxylic Acid.- V. The Selection of Fluorescence Histochemical Methods.- VI. Advantages and Limitations of the Fluorescence Histochemical Method.- A. Advantages.- B. Limitations.- C. Conclusion.- VII. Appendix.- A. Fluorescence Microscopy and the Fluorescence Microscope.- B. Freeze-Dryers.- C. Vibratome.- D. Cryostat.- References.- 14 Immunocytochemical Methods.- I. Introduction.- A. Immunologic Basis.- B. Rationale.- II. Types of Immunocytochemical Techniques.- A. Direct Method.- B. Indirect Methods.- C. Summary.- III. The Peroxidase-Antiperoxidase (PAP) Technique.- A. Fixation.- B. Sectioning.- C. Immunolabeling.- D. Light Microscopy.- E. Electron Microscopy.- IV. Variations in PAP Technique.- A. Fixation.- B. Sectioning.- C. Reagents.- V. Specificity of the PAP Technique.- VI. Use of the PAP Technique in the Demonstration of Catecholaminergic Neurons.- A. Light Microscopic Pathways.- B. Ultrastructural Localization of Tyrosine Hydroxylase.- VII. Use of the PAP Technique in the Localization of Neuropeptides.- A. Light Microscopy.- B. Ultrastructural Localization of Peptides in Axon Terminals.- C. Synaptic Interactions Between Peptidergic Axons and Catecholaminergic Neurons.- VIII Summary of Advantages and Limitations of the PAP Technique.- A. Advantages.- B. Limitations.- IX. Conclusions.- References.- 15 The 2-Deoxyglucose Method.- I. Introduction.- II. Basic Principles of the Method.- III. General Applications of the Method.- IV. Methodology for [14C]-2DG.- A. Injection of Deoxyglucose and Methods for Determining Metabolic Rates of Glucose.- B. Fixation and Sectioning.- C. Preparation of Autoradiograms.- V. Methodology for [3H]-2DG.- VI. Data Analysis.- A. Qualitative.- B. Quantitative.- VII. Advantages and Limitations.- A. Advantages.- B. Limitations.- VIII Appendix.- A. Fixation.- B. Thionin Stain for 2DG Sections.- C. Staining Procedure for [14C]-2DG Sections.- References.- Epilogue: Some General Advice to the Young Investigator.- Author Index.