Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog ran a ridibunda. Immunocytochemical evidence for co-localization of thyrotropinreleasing hormone and mesotocin in fibers innervating pars intermedia cells

Abstract

The localization of thyrotropin-releasing hormone-immunoreactive structures was investigated in the hypothalamo-hypophyseal complex of the frog, Rana ridibunda, by light and electron microscopy using the conventional indirect immunoperoxidase technique and the immuno-gold technique, respectively. The localization of mesotocin-, vasotocin- and neurophysin-immunoreactive elements was compared to that of thyrotropin-releasing hormone either by comparing homologous fields on serial sections or by staining the same section with two different antibodies. Thyrotropin-releasing hormoneimmunoreactive perikarya occurred mainly in the anterobasal periventricular area and dorsal extension of the preoptic nucleus and in the lateral zone of the infundibular nucleus. In the anterobasal preoptic nucleus, the distribution of thyrotropin-releasing hormone-immunoreactive perikarya partially over-lapped that of vasotocin- and mesotocin-containing neurons; however, co-localization of thyrotropin-releasing hormone with either nonapeptide could not be detected there. In contrast, in the caudal extension of the preoptic nucleus, thyrotropin-releasing hormone- and mesotocin-like immunoreactivities were frequently co-localized in the same neurons. In the external zone of the median eminence, abundant networks of thyrotropin-releasing hormone- and vasotocin-immunoreactive nerve fibers were found in the vicinity of portal capillaries, while mesotocin-immunoreactive axons were only found in the internal zone. Using the immuno-gold technique at the electron microscopic level, three distinct thyrotropin-releasing hormone-immunoreactive systems were identified in the median eminence-neurointermediate lobe complex. (1) In the external zone of the median eminence, a conspicuous population of pericapillary endings contained 100-nm dense core vesicles immunoreactive solely for thyrotropin-releasing hormone. (2) In the neural lobe of the pituitary, thyrotropin-releasing hormone immunoreactivity occurred on secretory vesicles in a subpopulation of the mesotocinergic axons containing 160-nm secretory granules; colocalization with vasotocin was never seen. (3) In the intermediate lobe, thyrotropin-releasing hormone- and mesotocin (or neurophysin I)-immunoreactivities were systematically found in the same 120-nm dense core vesicles; these thyrotropin-releasing hormone-/mesotocin-immunoreactive axon terminals frequently made synaptic contacts with melanotropic cells. The possible modulatory effect of mesotocin on thyrotropin-releasing hormone-induced alpha-melanocyte-stimulating hormone secretion was investigated using perifused frog neurointermediate lobes. Administration of graded doses of mesotocin (from 10 −10 to 10 −5 M) did not affect the spontaneous release of alpha-melanocyte-stimulating hormone. In addition, mesotocin (10 −7 and 10 −6M) did not modify thyrotropin-releasing hormone-evoked alpha-melanocyte-stimulating hormone release. Taken together, our data indicate that thyrotropin-releasing hormone-immunoreactivity is present in part of the mesotocin-immunoreactive axons of the neural lobe and that thyrotropin-releasing hormone-immunoreactive and mesotocin-immunoreactive material systematically co-exist in nerve terminals of the pars intermedia. Although thyrotropin-releasing hormone and mesotocin are co-sequestered in the same dense core vesicles of the intermediate lobe (indicating that the two regulatory peptides are likely released together), we could not demonstrate any modulatory effect of mesotocin on thyrotropin-releasing hormone-induced alpha-melanocyte-stimulating hormone secretion.