Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy

Abstract.

Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin insertions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brainstem in both species. Some scattered biocytin-labelled neurons were observed in the preoptic area. Moreover, working on the same sections, immunofluorescence revealed a number of codistributions and, in some cases, colocalization in the same neurons of biocytin labellings and immunopositivity for (1) tyrosine hydroxylase in the suprachiasmatic hypothalamus and the locus coeruleus of Rana and Triturus, (2) γ-aminobutyric acid in the suprachiasmatic hypothalamus of Rana and Triturus and (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-channel confocal laser scanning microscopy analysis.