CHARACTERIZATION OF PARS-INTERMEDIA CONNECTIONS IN AMPHIBIANS BY BIOCYTIN TRACT TRACING AND IMMUNOFLUORESCENCE AIDED BY CONFOCAL MICROSCOPY

Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in or der to analyse the neurochemical characters of some central neuronal p rojections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin inse rtions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brain stem in both species. Some scattered biocytin-labelled neurons were ob served in the preoptic area. Moreover, working on the same sections, i mmunofluorescence revealed a number of codistributions and, in some ca ses, colocalization in the same neurons of biocytin labellings and imm unopositivity for(1)tyrosine hydroxylase in the suprachiasmatic hypoth alamus and the locus coeruleus of Rana and Triturus, (2) gamma-aminobu tyric acid in the suprachiasmatic hypothalamus of Rana and Triturus an d (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-cha nnel confocal laser scanning microscopy analysis.