MULTIPLE ANTEROGRADE TRACING, COMBINING PHASEOLUS-VULGARIS-LEUKOAGGLUTININ WITH RHODAMINE-CONJUGATED AND BIOTIN-CONJUGATED DEXTRAN-AMINE

The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of aff erent systems in a particular brain area. However, a simple routine pr ocedure to apply multiple anterograde tracers in conjunction with thei r simultaneous visualization is still missing. We report an easy and s traightforward application of three sensitive anterograde tracers: Pha seolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers c an be visualized simultaneously and permanently through a triple-stain ing procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projecti ons from the nucleus reuniens thalami and entorhinal cortex. Both proj ection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right en torhinal cortex, respectively; a third tracer was injected in the nucl eus reuniens. This combination of injections provided a good opportuni ty to compare the three tracers in one and the same animal. PHA-L, RDA and BDA, injected in either of the injection sites, turned out to be equally sensitive and revealed the morphology of the involved projecti on systems in great detail. The triple-staining protocol yielded an ex cellent, simultaneous detectability of the three tracers with a remark ably low background level. Thus, the combination of the anterograde tr acers PHA-L, RDA and BDA, in conjunction with the triple-staining proc edure, offers a very attractive approach for neuroanatomical research.