MULTIPLE AXONAL TRACING - SIMULTANEOUS DETECTION OF 3 TRACERS IN THE SAME SECTION

Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons . Evaluation of the question as to whether two specific fiber inputs c onverge on a particular, identified population of projection neurons r equires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The prese nt report deals with a combination of tracing methods using anterograd ely transported Phaseolus vulgaris leucoagglutinin and biotinylated de xtran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substra tes (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector(R) VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projectio ns arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.