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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) gamma-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.

Neuroanatomic comparison of the binding capability of 2-[125I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[125I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.

The distribution of gamma-aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cells bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.

The distribution of serotonin (5-HT) immunoreactive structures has been investigated in the brain of the crested newt by means of indirect immunofluorescence, and unlabeled antibody peroxidase-antiperoxidase-complex (PAP) or biotin-avidin-system (BAS) techniques. In the newt, the bulk of the serotoninergic system extends from the raphe region of the medulla oblongata, through the isthmus, toward the mesencephalic tegmentum, and is characterized by pyriform neurons mainly located in a subependymal position, close to the midline. Also in the caudal hypothalamus, in addition to some 5-HT-positive adenohypophysial cells, many immunoreactive CSF-contacting neurons are found lining the paraventricular organ and the nucleus infundibularis dorsalis. A rich serotoninergic innervation was observed in the preoptic area and in the habenular complex. Concerning the telencephalon, immunopositive nerve fibers are encountered in the dorsal pallium, primordium hippocampi, striatum and olfactory bulbs. The general organization of serotoninergic systems in the newt brain exhibit close similarities to that described in higher vertebrates.

The localization of neurons, fibers, and terminals containing tyrosine hydroxylase (TH)-like immunoreactivity was studied in the brain of the crested newt by using an antiserum to rat phaeochromocytoma tyrosine hydroxylase. Immunoreactive cells and fibers were found in the spinal cord, the medulla oblongata (lateral periventricular areas), and the acousticolateral area. In the tegmentum mesencephali, two bilateral clusters of labelled cells were localized in the ventrolateral periventricular gray extending toward the caudal hypothalamus. In the hypothalamic tuberal lobes, the TH-like reactive neurons, frequently of CSF-contacting type, lined the dorsal wall of the lateral infundibular recesses. A thick network of TH-like reactive nerve fibers and terminals was observed in the perivascular zone of the median eminence and in the adenohypophysial pars intermedia. A number of labelled cell bodies were also found in the dorsal thalamus (pars intercalaris diencephali), the paraventricular organ, and the ventral wall of the preoptic recess. In the telencephalon, immunoreactive innervation was identified in the striatum, together with immunopositive cell bodies in the olfactory bulbs. The pattern of organization of TH-immunoreactive systems in the newt showed, except for some peculiarities (e.g., the labelled cell bodies in dorsal thalamus), close similarities to the arrangement typical of mammals.