A new double-labeling method demonstrates transmitter-specific projections

We report a method which combines retrograde transport of the fluorescent dye, diamidino yellow dihydrochloride (DY), with peroxidase immunocytochemical staining for glutamic acid decarboxylase (GAD), an enzyme essential for the synthesis of gamma-aminobutyric acid (GABA). Cells exhibiting both retrograde fluorescent label and GAD-positive immunoreactivity were observed in the cerebellar cortex, the striatum and the ventrobasal complex following injections of DY into the superior vestibular nucleus, substantia nigra and dorsal thalamus. The method, which can in principle be applied to any antigen, takes advantage of the differential nuclear/cytoplasmic distribution of the two stains. By using appropriate filter combinations and balanced epi- and transillumination, double-labeled cells are readily identifiable.