A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques

A simple method for the simultaneous localization of an antigen and a retrogradely transported fluorescent dye within single neurons is described. The method is based upon: (1) the efficiency of retrograde neuronal labeling with the fluorescent marker 'true blue'; (2) the near-quantitative persistence of retrogradely transported true blue localizations after subsequent processing of the tissue for immunohistochemistry; and (3) the ability to distinguish clearly between true blue- and immunohistochemically-stained cells by simply using appropriate excitation wavelengths for each. First we examined the characteristics of two fluorescent tracers which are effectively transported over long distances in the rat. The results confirm that true blue and bisbenzimide are transported from terminal fields to parent cell bodies and that both tracers are taken up and transported by damaged fibers and by undamaged fibers-of-passage. No evidence for transneuronal transport of either dye in the anterograde or in the retrograde direction was found. Next, using the projection of the paraventricular nucleus of the hypothalamus (PVH) to the spinal cord as a test system, it was found by direct cell counts that a considerably greater percentage of cells in a specific subdivision of the nucleus was labeled following true blue injections into the spinal cord (88%) than was labeled after comparable injections of bisbenzimide (58%), or horseradish peroxidase (HRP) (24%), or after HRP-polyacrylamide gel implants (39%). A comparison of cell counts of true blue-labeled cells in normal material and in series of adjacent sections that were processed for immunohistochemistry suggested that only about 5% of the true blue-labeled cells are no longer detectable following the immunohistochemical procedures employed. Finally, by coupling the fluorescent retrograde tracing method with an indirect immunofluorescence technique, we have been able to localize reproducibly both retrogradely transported true blue and an antigen in individual neurons. The perfusion and staining method employed provided adequate staining of cell bodies that cross-reacted with antisera to one or another of the 9 peptides and enzymes tested. The results indicate that true blue is a versatile and highly sensitive marker for retrograde tracing studies that can be used alone, or in conjunction with respect to their biochemistry as well as their efferent connections.