Neuroanatomical Techniques : Insect Nervous System

Most neurobiological research is performed on vertebrates, and it is only natural that most texts describing neuroanatomical methods refer almost exclusively to this Phylum. Nevertheless, in recent years insects have been studied intensively and are becoming even more popular in some areas of research. They have advantages over vertebrates with respect to studying genetics of neuronal development and with respect to studying many aspects of integration by uniquely identifiable nerve cells. Insect central nervous system is characterized by its compactness and the rather large number of nerve cells in a structure so small. But despite their size, parts of the insect eNS bear structural comparisons with parts of vertebrate eNS. This applies particularly to the organization of the thoracic ganglia (and spinal cord), to the insect and vertebrate visual sys­ tems and, possibly, to parts of the olfactory neuropils. The neurons that make up these areas in insects are often large enough to be impaled by microelectrodes and can be injected with dyes. Added to advantages of using a small eNS, into which the sensory periphery is precisely mapped, are the many aspects of insect behaviour whose components can be quan­ titized and which may find both structural and functional correlates within clearly defined regions of neuropil. Together, these various features make the insect eNS a rewarding object for study. This volume is the first of two that describe both classic and recent methods for neuroanatomical research on insect eNS.

1 The Methylene Blue Technique: Classic and Recent Applications to the Insect Nervous System.- I. Historical Review.- II. Methods.- III. The Staining Schedule.- IV. Recent Applications of the Method.- V. Further Developments of the Method.- 2 Permanent Staining of Tracheae with Trypan Blue.- I. Essentials of the Technique.- II. Practical Procedure.- III. Results and Comments.- 3 Toluidine Blue as a Rapid Stain for Nerve Cell Bodies in Intact Ganglia.- I. Stain.- II. Differentiator and Fixative.- III. Dehydration and Mounting.- IV. Results.- V. Special Applications.- 4 Demonstration of Neurosecretory Cells in the Insect Central Nervous System.- I. Methods for NSC Detection.- II. Comparisons of Present-Day Neurosecretory Staining Methods.- III. Conclusions.- 5 Histochemical Demonstration of Biogenic Monoamines (Falck-Hillarp Method) in the Insect Nervous System.- I. General Considerations.- II. Procedure.- III. Fluorescence Microscopy.- IV. Microspectrofluorometry.- 6 The Bodian Protargol Technique.- I. Practical Procedure.- II. Factors Affecting Staining.- III. Choice of Staining Conditions.- IV. Fault Tracing.- 7 Reduced Silver Impregnations Derived from the Holmes Technique.- I. Basic Strategies of Silver Impregnation.- II. Fixation.- III. Embedding and Sectioning.- IV. A Basic Schedule for Holmes-Blest Impregnations.- V. The Holmes-Rowell Method.- VI. Weiss’s Method.- VII. Pretreatment with Dilute Nitric Acid.- VIII. Cobalt-Mercury Mordanting.- IX. Varying the Processing Strategies.- X. General Comments on Handling Sections.- XI. Results.- 8 Reduced Silver Impregnations of the Ungewitter Type.- I. Mordanting: The Use of Mercury Pretreatment.- II. Basic Schedule for a Urea-Silver Nitrate Impregnation.- III. Choice of Variants.- IV. Comparative Conclusions.- 9 The Golgi Method: Its Application to the Insect Nervous System and the Phenomenon of Stochastic Impregnation.- I. Historical Background.- II. Use of the Method and Its Selectivity.- III. Summary of the Basic Procedures.- IV. Golgi Procedures for Insect Central Nervous System.- V. The Golgi-Colonnier and Golgi-Rapid Procedures.- VI. The Nature of Random Impregnation.- VII. Possible Events During Silver Impregnation.- VIII. Golgi Methods Using Mercury Salts.- IX. Conclusions.- X. Appendix I: A Model of Random Impregnation.- XI. Appendix II: Methods.- 10 Electron-Microscopic Methods for Nervous Tissues.- I. Fixation.- II. Embedding.- III. Sectioning.- IV. Staining Ultrathin Sections.- V. Appendix I: Buffer Solutions.- VI. Appendix II: List of Suppliers.- 11 Methods for Special Staining of Synaptic Sites.- I. Methodology.- II. Phosphotungstic Acid Technique.- III. The Bismuth Iodide-Uranyl Acetate-Lead Citrate Technique.- IV. Zinc Iodide-Osmium Tetroxide Stain.- V. Conclusions and Outlook.- VI. Addendum.- 12 Experimental Anterograde Degeneration of Nerve Fibers: A Tool for Combined Light- and Electron-Microscopic Studies of the Insect Nervous System.- I. Choice of Animal.- II. Experimental Lesions to the Nerve Cord: Controlled Surgery.- III. Timing of Postoperative Survival.- IV. Preparation for Light and Electron Microscopy.- V. Structural Aspects of Anterograde Degeneration.- VI. Light Microscopy.- VII. Ultrastructural Observations.- VIII. Physiologic and Chemical Correlates of Anterograde Degeneration.- IX. Concluding Remarks.- X. Addendum.- 13 Simple Axonal Filling of Neurons with Procion Yellow.- I. The Immersion Method.- II. The Two-Bath Method.- III. The Two Methods Compared.- 14 Intracellular Staining with Fluorescent Dyes.- I. Procion Dyes.- II. Procion as a Recording Electrode.- III. Injection of Procion.- IV. Fixation.- V. Viewing.- VI. Photography.- VII. Lucifer Yellow.- 15 Intracellular Staining of Insect Neurons with Procion Yellow.- I. Recording.- II. Dye Injection.- III. Histology.- IV. Fluorescence Microscopy.- V. Reconstruction.- VI. Fly Interneurons.- VII. Concluding Remarks.- 16 The Use of Horseradish Peroxidase as a Neuronal Marker in the Arthropod Central Nervous System.- I. History and Chemical Nature of Horseradish Peroxidase.- II. Materials and Methods.- III. Results and Discussion.- IV. Uptake and Transport of HRP by Nerve Cells.- V. Technical Considerations.- VI. Concluding Remarks.- 17 Cobalt Staining of Neurons by Microelectrodes.- I. General Methods.- II. Applications and Interpretations.- 18 Nonrandom Resolution of Neuron Arrangements.- I. The Method.- II. Injection and Diffusion Parameters.- III. Species-Specific Variations of the Methods.- IV. Peripheral Fillings via Micropipettes.- V. Cobalt Precipitation.- VI. Resolution of the Cobalt Reservoir and Nerve Cells.- VII. Patterns of Uptake by Neurons.- VIII. A Simple Comparison between the Golgi Method and the Cobalt Injection Technique.- 19 Filling Selected Neurons with Cobalt through Cut Axons.- I. Methods for Introducing Cobalt Chloride into Cut Axons.- II. Filling Selected Neurons.- III. Processing after Filling.- IV. Examination and Analysis.- V. Errors and Artifacts: Cautions for Interpretation.- VI. Tactics for Identifying the Origin of a Neuron or Tract.- VII. Conclusions.- VIII. Addendum.- 20 Silver-Staining Cobalt Sulfide Deposits within Neurons of Intact Ganglia.- I. Silver Intensification Procedure.- II. Methodology.- III. General Considerations.- 21 Intensification of Cobalt-Filled Neurons in Sections (Light and Electron Microscopy).- I. Timm’s Sulfide-Silver Intensification Method for Cobalt.- II. Light Microscopy.- III. Electron Microscopy.- IV. Sensitivity of Timm’s Method.- References.