Further evidence of the presence of rat embryonic hypothalamic factors that induce the differentiation of gonadotrophic hormone-releasing hormone-containing secretory neurons.

We examined the presence of factor(s) in the embryonic medial basal hypothalamus (MBH) that may influence nasal placode (NAP)-derived luteinizing hormone-releasing hormone (LHRH) neurons in determining their secretory phenotype. In this study, we performed organotypic culture and transplantation of the NAP from 12.5-day-old embryos of rats and vomeronasal organ (VNO) from 14.5-day-old embryos. Surgical operations, however, were performed on 16.5-day-old embryos. The NAP and VNO were cultured singly or with the MBH obtained from the embryos of the same age and, further, in a medium with a nerve growth factor or fibroblast growth factors. Although LHRH neurons were derived from the NAP and VNO in all the cultures, judging from numbers and cellular morphologies, the MBH was most effective. The VNO was transplanted into the third ventricle of adult female rats singly or with the cerebral cortex, the mesencephalon-myelencephalon complex, or the MBH from 14.5-day-old embryos. All the grafts gave rise to LHRH neurons, but the number of the neurons was far greater in the grafts cotransplanted with the MBH, in which the neurons projected long processes to blood capillaries and formed neurovascular complexes, the feature of which may suggest the occurrence of the secretory activity in the fibers. The animals were examined 5 days after the surgical operations. In rhinoectomized embryos, LHRH neurons were distributed throughout the brain in the same pattern as found in intact rats, showing normal cellular morphology. In the encephalectomized rats, immunoreactive LHRH cells were present only in the terminal ganglia. These findings indicate that the embryonal MBH has a factor (s) that is essential to the development of secretory LHRH neurons.

Development of the hypothalamic luteinizing hormone-releasing hormone-containing neuron system in the rat: in vivo and in transplantation studies.

The development of the hypothalamic LHRH-containing neuron system was immunohistochemically investigated in vivo and in tissue transplantation using rat embryos aged from 12.5 to 17.5 days of gestation. The sera used were generated against rat gonadotropic hormone-releasing hormone-associated peptide (28-56) (rGAP) and LHRH. Immunoreaction for rGAP was first found in cells migrated from and in the vomeronasal organ on Days 13.5 and 14.5 of gestation. Immunoreactive cells seem to ascend along the terminal nerves, reaching the medial surface of the forebrain vesicles. Subsequently the cells occurred in the septum and further into their final position in the septopreoptic-diagonal band area on Days 16.5-17.5 of gestation; during this traverse the cells become secretory neurons after changes in morphology and in behavior. Intraventricular transplantation revealed that nasal epithelia of Day 12.5 embryos raised only a few cells immunoreactive both for LHRH and rGAP, but a great number of immunoreactive cells and fibers in the presence of the medial basal hypothalamus (MBH). The fibers formed a median eminence-like structure together with dense capillary plexus that had grown in the cografted MBH. The same phenomenon was apparently observed in the grafts obtained from older embryos of gestation, but not in the combined grafts of the anterior septum and the nasal epithelium or the MBH. We conclude that hypothalamic LHRH neurons originate from the nasal placode and acquire secretory behavior in the presence of the MBH.

Appearance of neurons with glucocorticoid receptors and neurovascular links in the embryonal rat hypothalamus grafted in the third ventricle.

We have investigated the appearance of the transmitter phenotypes of hypothalamic neurons in grafts transplanted into the third ventricle of adult female rats. The grafts were the mediobasal hypothalamus and the preoptic area of 12.5-day-old rat embryos, and were examined 40-100 days later. Wheat germ agglutinin (WGA) was injected into the jugular vein of several animals for the examination of the existence of neurovascular associations. Three days after the injection, WGA appeared to have been incorporated into the neurons in the paraventricular, periventricular, and arcuate nuclei of the host animals. In the grafts, WGA was also seen incorporated in certain neurons which were found immunoreactive for tyrosine hydroxylase (TH), rat corticotropin-releasing factor (rCRF), substance P (SP), or somatostatin (SRIH). Neurons immunoreactive for neuropeptide Y (NPY) and ACTH did not seem to incorporate WGA. These findings suggest that the neurons containing TH, rCRF, SP, or SRIH link with fenestrated capillaries developed in the grafts. The immunoreactivity for glucocorticoid receptor (GR) was detected mainly in the nucleus of certain neurons and glial cells in the grafts as well as in the host hypothalamic neurons. In the grafts, strong GR immunoreactivity was detected in the cells immunoreactive for TH, NPY, and rCRF as in the host animals. It is concluded that the undifferentiated hypothalamic neurons differentiate to synthesize GR as well as definitive peptides and TH in the grafts.