title: Multiple anterograde tracing, combining Phaseolus vulgaris leucoagglutinin with rhodamine- and biotin-conjugated dextran amine.
abstract: The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of afferent systems in a particular brain area. However, a simple routine procedure to apply multiple anterograde tracers in conjunction with their simultaneous visualization is still missing. We report an easy and straightforward application of three sensitive anterograde tracers: Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers can be visualized simultaneously and permanently through a triple-staining procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projections from the nucleus reuniens thalami and entorhinal cortex. Both projection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right entorhinal cortex, respectively; a third tracer was injected in the nucleus reuniens. This combination of injections provided a good opportunity 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 projection systems in great detail. The triple-staining protocol yielded an excellent, simultaneous detectability of the three tracers with a remarkably low background level. Thus, the combination of the anterograde tracers PHA-L, RDA and BDA, in conjunction with the triple-staining procedure, offers a very attractive approach for neuroanatomical research.
authors.affiliation: Graduate School Neurosciences Amsterdam, Faculty of Medicine, Department of Anatomy and Embryology, Vrije Universiteit, Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Dolleman-Van der Weel
authors.forename: F G
authors.initials: FG
authors.lastname: Wouterlood
authors.forename: M P
authors.initials: MP
authors.lastname: Witter
mesh_terms: D000818:Animals; D001692:Biological Transport; D001710:Biotin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D001808:Blood Vessels / Q000166:cytology; D002560:Cerebrovascular Circulation; D003429:Cross Reactions; D003911:Dextrans / Q000493:pharmacokinetics; D005260:Female; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D008032:Limbic System / Q000502:physiology; D008722:Methods; D009434:Neural Pathways / Q000378:metabolism; D010835:Phytohemagglutinins / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines / Q000493:pharmacokinetics; D012680:Sensitivity and Specificity; D009435:Synaptic Transmission; D013787:Thalamic Nuclei / Q000502:physiology
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D010835:Phytohemagglutinins; D012235:Rhodamines; C076397:biotinylated dextran amine; C100156:leukoagglutinins, plants; C051311:rhodamine dextran; D001710:Biotin