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Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a 35S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2-3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against alpha-melanocyte-stimulating hormone (alpha MSH), gamma 3MSH and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of alpha MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the alpha MSH content of the explants and alpha MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in alpha MSH secretion, which was significant after 3 days in culture, indicating that dopamine D2 receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.

Immunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plastic-embedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH-immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.

Distribution of the gamma-aminobutyric acid (GABA)ergic and dopaminergic innervations was studied in the rat neurointermediate lobe using antibodies against GABA and tyrosine hydroxylase. In light microscopy, immunoperoxidase staining revealed similar distribution patterns of the axons reacting with both antibodies. Diffusely scattered in both lobes, they were more concentrated along the marginal zone of the neural lobe. Application of a double, recto-verso, immunogold labelling method in electron microscopy revealed systematic colocalization of GABA and tyrosine hydroxylase (TH) immunoreactivities in the axons innervating the intermediate lobe; in the neural lobe, almost all GABA-immunoreactive axons were also labelled for TH. Thus, GABA and dopamine, hitherto reported to occur in distinct axons, in fact colocalize in the axonal systems which innervate the pituitary neurointermediate lobe. These observations suggest possible interactions (pre- or postsynaptic) of both transmitters in the functional regulation of the intermediate and neural lobes.