The availability of neuronal tract-tracing techniques has been fundamental to the development of the neurosciences. While most of the previously described methods are performed in vivo, in the present paper, detailed protocols are reported for tracing neuronal connections in an in vitro preparation. This technique, tested in various neural systems of the teleost brain, allows precise application of tracer substance(s) under visual control. After the isolation of the brain, the tissue is kept alive by superfusion with oxygenated artificial cerebrospinal fluid in a slice chamber. Neuronal connections are traced by the application of crystals of biocytin or dextran-tetramethyl-rhodamine to the region of interest. Following intracellular transport over 8-18 h, the tissue is fixed and processed histochemically for visualization of structures filled with the tracer substance. This method can readily be modified for double labelling. Step-by-step procedures are outlined for (a) the simultaneous detection of two tracer substances in the same tissue sample, (b) the combination of tract tracing with the immunohistochemical identification of various biochemical markers such as 'classical' transmitters and neuropeptides, and (c) the visualization of both traced structures and mitotically active cells labelled with the thymidine analogue 5-bromo-2'-deoxyuridine. By exhibiting a high degree of efficiency, the described in vitro tract-tracing technique represents also a significant contribution towards a reduction of living animals in neurobiological experimentation.
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