A survey of current neuroanatomical tracing techniques

Abstract

This chapter provides a systematic description of neuroanatomical tracing methods, with a brush of history. Tracing can be based on uptake and transport of tracer in living neurons but can also be based on physical diffusion in living neurons after intracellular injection of tracer or, in fi xed tissue as is the case of Golgi silver staining, based on complex anorganic chemical reactions. Because of the special fi xation status of human brain tissue, the physicochemical methods are prominent with this kind of nervous tissue. Nowadays, the transport methods enjoy popularity in animal connectivity models because they produce fast and decisive results in terms of specifi c connectivity of functional systems. Transport-based tracing methods are best suited to visualize long-axon projections. For the study of short projection axons and interneurons, more sophisticated methods need to be applied such as pericellular injection or intracellular fi lling with dye after neurophysiological recording in living slice preparations or in vivo. The chapter discusses the current neuroanatomical tracing methods and its equipment and procedures, starting with the "mother of retrograde tracing methods": the technique of injection, uptake, and transport of the enzyme horseradish peroxidase. It continues with fl uorescent dye tracing. The fl uorescent compounds have two advantages: they are extremely stable and they can easily be combined with immunofl uorescence to determine the neurochemical identity of the labeled neurons. The most commonly used anterograde tracers are Phaseolus vulgaris -Leucoagglutinin (PHA-L), which requires immunohistochemical detection, and biotinylated dextran amine (BDA), which is detected via reaction with streptavidin conjugated to a reporter molecule. Single and multiple fl uorescence methods receive much attention because they so perfectly combine with modern laser scanning microscopes and digital image processing.