Analysis of collateral projections with a double retrograde labeling technique.

A method is described whereby collateral innervation by a single cell type may be determined by double retrograde labeling with horseradish peroxidase (HRP) and tritiated bovine serum albumin ([(3)H]BSA). The projections from the entorhinal area to the hippocampal formation were analyzed as a model system, since layer III pyramidal cells project bilaterally to the hippocampus. Following HRP injections into the hippocampus of one side, and [(3)H]BSA injections into the opposite hippocampus, double labeled cells were found in layer III of the entorhinal cortex. These doubly labeled cells could best be viewed in plastic embedded material, whereas frozen sections were found to be inadequate for clearly distinguishing between silver grains and HRP reaction product.