Simultaneous anterograde labelling of two afferent pathways to the same target area with Phaseolus vulgaris leucoagglutinin and Phaseolus vulgaris leucoagglutinin conjugated to biotin or dinitrophenol.

Anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and PHA-L conjugated to either biotin or 2,4-dinitrophenol were used to simultaneously label two populations of axons converging onto the same target area. Using the rat hippocampus as a test system, the septohippocampal and contralateral hilar afferents were labelled with the tracers. Double immunohistochemical procedures and either nickel-enhanced 3,3'-diaminobenzidine (DAB) reaction (blue-black colour) or DAB alone (brown colour) were used to produce contrasting colours for the different tracers in the same section. Both the biotinylated PHA-L and PHA-L conjugated with dinitrophenol were used successfully as anterogradely transported axonal tracers. They produced extensive axonal labelling in the hippocampal formation. Excellent double labelling could be produced with the simultaneous application of PHA-L and biotinylated PHA-L. Biotinylated PHA-L was visualized by a sequence of avidin-biotinylated peroxidase complex (ABC), biotinylated goat antiavidin and ABC again using nickel-enhanced DAB as chromogen yielding a blue-black reaction endproduct. PHA-L alone was detected by the unlabelled antibody enzyme (PAP) method using goat antibodies to PHA-L, and DAB as chromogen for the peroxidase reaction, resulting in brown axons. The combination of biotinylated PHA-L and DNP-conjugated PHA-L gave similar results, although the sensitivity of detection by the latter procedure was inferior to that obtained with the other tracers. These protocols permitted visualization of axons of different origin, together with their terminals, either in a blue-black or brown colour, and also allowed the demonstration of overlapping inputs in strata radiatum and lacunosum moleculare of the hippocampus and stratum moleculare and hilus of the dentate gyrus.