Photoconversion of fluorescent retrograde tracers

Photoconversion of fluorescent dyes, retrogradely transported through axons to their parent cell bodies, into a stable diaminobenzidine (DAB) reaction product was tested in the nigrostriatal and thalamocortical systems of rats. Satisfactory results were obtained with Propidium Iodide (PI), Fluoro-Gold (FG), Fast Blue (FB), Diamidino Yellow (DY), and rhodamine-labeled latex microspheres (RLM); some photoconversion was also observed in Evans Blue (EB)-labeled neurons. The red fluorescent tracers PI, EB and RLM were photoconverted under the excitation wavelength appropriate for eliciting their fluorescent emission. With the yellow or blue fluorescent tracers FG, FB, and DY satisfactory results could instead be obtained using an excitation wavelength which did not elicit visible fluorescent emission. This finding indicates that the latter is not a critical factor for obtaining photoconversion. The features and subcellular localization of photoconverted DAB were different from those of the fluorescent labeling: photoconversion resulted in the appearance of brown granules of DAB reaction products in the cytoplasm, independently from the occurrence of fluorescent labeling in the neuronal cytoplasm or nucleus. Photoconversion may enable new applications of fluorescent retrograde tracing and, in particular, its electron microscopic visualization.