Shigeo Daikoku

An immunohistochemical study on the cytogenesis of adenohypophysial cells in fetal rats.

Abstract An immunohistochemical study was undertaken in fetal rats to determine the time of differentiation of various types of adenohypophysial cells and the site where they first appear and proliferate during development. A cell count was carried out on the first and second days of cytodifferentiation in each type of cell. Both the time and the site of cytodifferentiation were peculiar to respective cell types. ACTH cells appeared on Day 15 of gestation in the ventral region of the pars distalis where it faces mesenchymal tissue. While TSH cells first appeared exclusively in the posterior half on Day 16, further proliferation of this cell type also occurred predominantly in the posterocentral portion of the gland. LH and FSH cells appeared on Days 17 and 19, respectively; both cell types showed a similar localization, i.e., they were almost concentrated in the ventral region in the anterior half and posteriorly they were distributed sparsely and homogeneously. GH cells appeared on Day 18 in the central region of the pars distalis. Prolactin cells failed to be seen in the fetal adenohypophysis, and even in newborns 0–1 days of age, this type of cell was not consistently seen, although sometimes a few cells were encountered in the central region of the gland. Of these cell types, TSH, LH, and FSH cells persisted to be concentrated even after birth in the area where they first appeared. These observations are discussed in relation to data previously reported by other investigators.

Immunohistochemical demonstration of LHRH neurons and their pathways in the rat hypothalamus.

LRHR neurons and fiber tracts have been studied immunohistochemically in rats. After the intraventricular infusion of colchicine, a number of immunoreactive LHRH neurons were consistently evident in the preoptico-septal area and in the diagonal band of Broca. The immunoreactive fiber tracts originating in those areas were classified into two groups: preoptico-terminal and preoptico-infundibular tracts. The former terminated in the OVLT. The latter was further divided into the mediobasal and laterobasal tracts; both were found to terminate in the median eminence. The mediobasal tract ran posteriorly through the ventral portion of the lateral periventricular area. The laterobasal tract ran posteriorly in the medial forebrain bundle and was accompanied by several immunoreactive neurons. The immunoreactive cell bodies were further scattered in the base of the tuberal hypothalamic area. An anterior bilateral Halász cut in the retrochiasmatic region was followed by a great reduction of the immunoreactive materials in the median eminence and by the appearance of the immunoreactive perikarya and in the preoptico-septal area and in the diagonal band of Broca. A horizontal hypothalamic cut, on the other hand, which disconnected the tubero-infundibular tract between the median eminence and the arcuate nuclei, caused neither reduction of the immunoreactive materials in the median eminence nor appearance of the immunoreactive perikarya in the arcuate nuclei. Those results indicate that the bulk of the immunoreactive LHRH is synthesized in the anterior hypothalamic area and transported by the preoptico-terminal and -infundibular tracts but a minor part is produced in the cell bodies located in the basal tuberal hypothalamus and transported to the median eminence.

Suprachiasmatic nucleus neurons immunoreactive for vasoactive intestinal polypeptide have synaptic contacts with axons immunoreactive for neuropeptide Y: An immunoelectron microscopic study in the rat

An electron microscopic study showed by using a dual immunolabeling technique that in the suprachiasmatic nucleus of the rat, axon terminals immunoreactive for neuropeptide Y (NPY) made synaptic contacts upon neurons immunoreactive for vasoactive intestinal polypeptide (VIP). Diaminobenzidine (DAB)-labeled NPY axon terminals made synaptic contacts on silver-gold-labeled VIP perikarya and dendritic processes. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles labeled with DAB chromogen. At the synaptic portion, a symmetrical thickening of the pre- and post-synaptic membranes was evident.

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.

Immunohistochemical studies of intrahypothalamic somatostatin-containing neurons in rat

Intrahypothalamic somatostatin-containing neurons were investigated immunohistochemically. In intact rats, immunoreactive cell bodies appeared in the rostral periventricular area, and immunoreactive beaded fibers were observed to terminate in the median eminence and to form delicate networks surrounding immunonegative cell bodies within the medial preoptic, suprachiasmatic, arcuate, ventromedial and premammillary nuclei. Intraventricular colchicine infusion resulted in the appearance of immunoreactive cell bodies in the arcuate, ventromedial and suprachiasmatic nuclei, and an increase in the number of cell bodies seen in the periventricular area. Complete deafferentation of the medial-basal hypothalamus excluding the rostral periventricular area caused the immunoreactive structures in the median eminence to disappear and enhanced the staining of periventricular cell bodies. In the arcuate and ventromedial nuclei, the immunoreactive fiber networks were left intact and the immunoreactive cell bodies were occasionally recognized. Horizontal knife cut between the arcuate nuclei and median eminence did not alter immunoreactivity in either region. Neonatal administration of MSG caused only the disappearance of arcuate nuclei. The results indicate that two kinds of somatostatin neuronal systems exist in rat hypothalamus: one is involved in the production of hormonal somatostatin and the other serves for the regulation of neuronal activities in restricted hypothalamic nuclei.

Morphological evidence for neuronal regulation of luteinizing hormone-releasing hormone-containing neurons by neuropeptide Y in the rat septo-preoptic area

Using a preembedding double immunolabeling technique, synaptic contacts were found between luteinizing hormone-releasing hormone (LHRH)-containing neurons and neuropeptide Y-containing axonal fibers in the rat septo-preoptic area. In demonstrating LHRH neurons, we used mainly an antiserum generated against rat gonadotrophic hormone-releasing hormone-associated peptide. Although many diaminobenzidine-labeled neuropeptide Y-containing fibers were seen around silver-gold-labeled LHRH cell bodies, synapses with synaptic membrane specialization were scarce. The fiber terminals usually contained many small clear vesicles and some large cored vesicles. The synapses were characterized with the presynaptic accumulation of the small clear vesicles and symmetric thickenings of the synaptic membranes.

Development of the neuronal system containing neuropeptide Y in the rat hypothalamus

In the rat hypothalamus, neuropeptide Y‐containing neurons first appeared on day 14.5 of gestation in the arcuate nucleus and in the dorsolateral hypothalamic area. Until birth neuropeptide Y‐containing cell bodies increased in number in the arcuate, dorsomedial‐lateral and paraventricular nuclei, but disappeared thereafter, but some cells remaining in the arcuate nucleus. In animals treated neonatally with monosodium l‐glutamate to destroy the arcuate nucleus, neuropeptide Y‐immunoreactivity became evident in many cells scattered in the magnocellular paraventricular and dorsomediallateral hypothalamic nuclei on day 16 but not on days 60 and 120. These neuropeptide Y‐immunoreactive neurons which appeared in the paraventricular nucleus were also vasopressin‐positive. Neuropeptide Y fibers, on the contrary, remarkably diminished in number on day 16, particularly in the paraventricular and dorsomedial‐lateral nuclei, and the medial preoptic area, but made a considerable recovery on days 60 and 120. Hence it is probable that, in normal ontogenetic progress, the development of the neuropeptide Y fibers in these areas is inhibitorily affected by that of arcuate neuropeptide Y neurons.

Ultrastructural evidence for neuronal regulation of growth hormone secretion.

The morphological substrate for the central mechanisms that control growth hormone (GH) release in the rat hypothalamus was investigated immunohistochemically by light and electron microscopy. In electron-microscopic studies, a dual immunolabeling technique was employed to demonstrate pairs of peptides, i.e. rat hypothalamic growth hormone-releasing factor (rhGRF) and somatostatin (SRIH), rhGRF and substance P (SP), and rhGRF and methionine-enkephalin-Arg6-Gly7-Leu8 (Enk-8), in different neuronal structures. Immunoreactivity of rhGRF was detected as silver-gold particles and those of the other substances as diaminobenzidine products by preembedding immunostaining procedures. In the external layer of the median eminence, axonal terminals immunolabeled for rhGRF and for SRIH showed the same pattern of distribution and close proximity. The neuronal inputs to GRF cell bodies in the arcuate nucleus were examined, and SRIH, SP and Enk-8 fibers with varicosities were found to form dense networks around the perikarya of GRF neurons, suggesting the presence of synaptic associations. Axonal terminals immunolabeled for SRIH, SP or Enk-8, and unlabeled terminals appeared to form coincidental synaptic junctions on GRF perikarya. These findings suggest that the central regulation of GH release occurs at the levels of the median eminence and the cell bodies.

Ontogenesis of immunoreactive tyrosine hydroxylase-containing neurons in rat hypothalamus

By employing anti-tyrosine hydroxylase (TH) serum, the ontogenesis of hypothalamic dopamine (DA) neurons was immunohistochemically examined with special attention to the medial basal hypothalamic area. DA neurons first appeared in the lateral hypothalamic walls on day 13.5 of gestation and in the anterior periventricular region and arcuate nucleus on day 15.5-16.5. In the arcuate nucleus, the appearance of the neurons was confined to the ventrolateral (VL) region, but extended to the periventricular region thereafter. About day 10 postnatally, the population of the arcuate DA neurons conjoins anterodorsally with the cell population in the anterior periventricular region. Concomitant with this, DA neurons in the VL region of the nucleus diminished in number and in stainability, becoming barely visible. Interestingly enough, the latter neurons reappeared after an anterolateral deafferentation of the medial basal hypothalamus. This did not occur in pregnant and lactating rats. Although most of the arcuate DA neurons were retarded by neonatal administration of monosodium glutamate, the immunoreactive fibers remained almost intact in the medial portion of the median eminence. It is concluded that in the periventricular-arcuate complex, DA neurons seem to play different roles relating with their ontogenetic heterogeneity.

Localization of glucocorticoid receptor in neuropeptide Y-containing neurons in the arcuate nucleus of the rat hypothalamus.

Glucocorticoid receptor (GR) was localized immunohistochemically in nuclei of neurons in the arcuate nucleus of the rat hypothalamus. The double immunostaining method further revealed that about half of the GR-positive neurons in the arcuate nucleus were also immunoreactive for neuropeptide Y (NPY).