Combined Immunocytochemistry and Tracing of Neural Connections

Protocol
Part of the Neuromethods book series (NM, volume 101)

Abstract

Neuronal tracing (neurotracing ) using anterograde and retrograde tracers is widely used to study the projections between different brain regions and the wiring between individual neurons . Neurotracing is a technique essential not only for examining the connectivity of complex neuronal networks but also for providing the neuroanatomical basis for electrophysiological , pharmacological and behavioral experiments. If neurotracing is combined with immunocytochemical labeling, the combined technique can characterize the neurochemical properties, postsynaptic targets and innervation modes of neurons. The utility and versatility of this approach can be further extended by adopting appropriate cellular and subcellular markers for immunocytochemistry , by applying the approach to animal models generated by advanced gene-manipulation technology, and by using single-cell labeling techniques, e.g., after viral transfection of fluorescent proteins or in utero /in vivo electroporation . In this chapter, we introduce the methods for combined immunocytochemistry and neurotracing at both light and electron microscopic levels. We have developed and employed these combined approaches to study the mechanisms underlying the development and refinement of climbing fiber mono-innervation in cerebellar Purkinje cells . Therefore, we present some examples of the images obtained in this experimental context.

Key words

Climbing fiber Purkinje cell Anterograde labeling Immunohistochemistry Lentivirus Green fluorescentprotein 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of AnatomyHokkaido University School of MedicineSapporoJapan

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