Intracellular and extracellular labeling of neurons in fresh slice preparations combined with multiple tract tracing

We describe methods for studying axo-dendritic projections, one of the forms of neural connection involved in the complex circuits of the central nervous system, including brainstem auditory pathways. This form of neural connection is often difficult to visualize by conventional tract tracing techniques. Retrogradely identified cells were filled intracellularly with a mixture of fluorescent Lucifer yellow and nonfluorescent HRP in live slice preparations to reveal the detailed morphological features of these cells with special attention to the distal dendrite that may receive projections from suspected or known input axons. Extracellular or intracellular labeling of cells with axons that project to the distal dendrite of the identified cells was accomplished in the same live slice preparation. Using a live slice rather than a fixed slice allows accurate, visually controlled placement of anterograde tracer, which requires living axons for transport, into the source of input to the identified cells within the slice. Live slices also permit one to characterize the identified cells electrophysiologically. Intracellular labeling of cells in a potential source of local input to the identified cells also provides conclusive information concerning with connections of the cells involved.