VISUALIZATION OF NEURONS FILLED WITH BIOTINYLATED-LUCIFER YELLOW FOLLOWING IDENTIFICATION OF EFFERENT CONNECTIVITY WITH RETROGRADE TRANSPORT

A new method is described that allows identification of neurons by retrograde transport, intracellular injection of biotin-labeled Lucifer Yellow, and a histochemical reaction of the biotin. Cells in the inferior colliculus that project to the ipsilateral thalamus are identified by injection of rhodamine-labeled latex beads into the medial geniculate body and retrograde transport in vivo. Several days later, the brains are fixed with aldehydes, and the inferior colliculus is cut on a vibratome. The rhodamine-labeled cells are observed with epi-fluorescent optics, impaled with intracellular pipettes under visual control, and injected with 9% Lucifer Yellow dilithium salt and 1% Lucifer Yellow cadaverine biotin-X. The biotinylated Lucifer Yellow is visualized by incubation of the slice overnight in avidin-biotin-HRP complex in the presence of 0.1% Triton X-100 at 4-degrees-C. A diaminobenzidine reaction with simultaneous cobalt and nickel intensification follows. This method produces well-filled neuronal cell bodies, dendrites, and spines for light microscopic analysis. The non-fluorescent reaction product in these intracellularly filled cells may permit transmitter immunohistochemistry and synaptic fine structure to be studied in combination with neural connections and dendritic morphology.