Neuronal Tract Tracing with Light and Electron Microscopy

Abstract

AbstractNeuronal tract tracing technique was developed based on two principles, Wallerian degeneration and axon flow theory, and has been enhanced by knocking in an enhancer or inhibitor to a chain of neurons in studying specific pathways with molecular biological functionality. The tract tracing, including anterograde, retrograde, and transganglion tract tracing, can be achieved through axon transportation of tracers. Based on the visibility of the tracers, they are classified as fluorescent and nonfluorescent tracers. As this chapter focuses on the methods that benefit both light microscopy (LM) and electron microscopy (EM) studies, the protocols of fluorescent dye tract tracing are not discussed. Among the nonfluorescent tracers, horseradish peroxidase (HRP) is a marvelous tracer that can be used in all anterograde, retrograde, and transganglion tract tracing; therefore, the protocol of histochemical visualization of HRP is presented herein. Cholera toxin subunit B (CTB) is also well suited for both anterograde and retrograde labeling. Biotinylated dextran amine (BDA), a predominant anterograde tracer, is broadly used in combination with other tracers for identification of pathway connections or convergent innervations. Phaseolus vulgaris Leucoagglutinin (PHA-L) is also a super anterograde tracer that can be applied together with BDA to study convergent projections or combined with any retrograde tracer to explore recipient of the convergent innervations. Fluoro-gold (FG) is a retrograde tracer that can be viewed directly with conventional fluorescent LM or by using anti-FG antibody to modify it and to make any combination as well. The protocols for double labeling either of HRP and BDA, BDA and CTB, or BDA and PHA-L, as well as different combinations of triple labeling with immunostaining of neuroactive substances are also presented. These protocols also summarize methods of histochemical and fluorescent illumination, as well as tissue processing, embedding, and immunostaining methods for EM studies. Two major double labeling combinations used in EM observation are delineated: TMB-ST (Tetramethyl benzidine–sodium tungsten) visualization of HRP combined with ABC (Avidin-biotin-complex) histochemical stain and ABC histochemical stain combined with silver-gold pre-embedding method. Each protocol is supplemented with images to illustrate the expected results by following the related protocols.KeywordsAxonal flew-based neuronal tracerAnterograde, retrograde, and transganglion tract tracingHistochemical visualization of tracer labelingDouble or triple labelingImmuno-electron microscopy