Multiple axonal tracing: simultaneous detection of three tracers in the same section.
Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.
Lanciego
J L
JL
Departamento de Anatomía, Facultad de Medicina, Universidad de Navarra, Pamplona, Spain. jlanciego@unav.es
Wouterlood
F G
FG
Erro
E
E
Giménez-Amaya
J M
JM
D000373:Agglutinins; D000818:Animals; D001370:Axonal Transport; D001710:Biotin / Q000031:analogs & derivatives; D003911:Dextrans; D005260:Female; D005456:Fluorescent Dyes; D009474:Neurons / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013266:Stilbamidines
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D000373:Agglutinins; D003911:Dextrans; D005456:Fluorescent Dyes; D013266:Stilbamidines; C076397:biotinylated dextran amine; D001710:Biotin