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. Hokfelt).- 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.