Double retrograde neuronal labeling through divergent axon collaterals, using two fluorescent tracers with the same excitation wavelength which label different features of the cell.

Recent studies show that several fluorescent substances are transported retrogradely through axons to their parent cell bodies and label in different colors different features of the cell at the same 360 nm excitation wavelength. Thus, Bisbenzimide (Bb) and "Nuclear Yellow" (NY; Hoechst S 769121) produce green and golden-yellow retrograde labeling of the neuronal nucleus. "True Blue" (TB) and "Fast Blue" (FB) produce blue retrograde labeling of the neuronal cytoplasm. In the present study the possibility of retrograde double labeling of neurons by way of divergent axon collaterals using combinations of Bb or NY with TB or FB has been explored in rat and cat. The findings show that in these animals these tracer combinations are transported retrogradely through two axon collaterals to one and the same cell. Neurons which are retrogradely double-labeled with these tracer combinations display a blue fluorescent cytoplasm and a white or golden-yellow fluorescent nucleus at the same 360 nm excitation wavelength. Therefore, these tracer combinations can be successfully used to demonstrate the existence of divergent axon collaterals in the brain.