Yoshihito Okamura

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.

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.

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

SummaryAppearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.

CRF-containing neurons of the rat hypothalamus.

SummaryThe immunoreactive CRF-neurons of the rat hypothalamus have been examined immunohistochemically employing anti-rat CRF serum. These neurons are confined to the paraventricular nucleus, dorsomedial-lateral hypothalamic area, and suprachiasmatic nucleus, and are, respectively, also immunoreactive to anti-Met-enk, -alpha-MSH, and -VIP sera. Intraventricular administration of colchicine (50 μg/5 μl/rat) induces a dramatic enhancement of the immunostainability of the cell somata, and also accelerates the development of immunoreactivity of other stored peptides, especially in the paraventricular nucleus.The CRF-neurons respond to adrenalectomy by showing increased immunoreactivity and an increase in the number of cell bodies; in the dorsomedial-lateral area and suprachiasmatic nucleus, there is also an enhanced immunoreactivity for alpha-MSH and VIP, respectively. CRF-cells in the paraventricular nucleus become markedly hypertrophied, but do not show any enhanced immunoreactivity for Met-enk. Since the axons of the paraventricular neurons run to the median eminence, it is probable that they are involved with the endocrine control of hypophysial ACTH release. It is concluded that the CRF-containing neurons in rat hypothalamus consist of three types which are functionally and morphologically different.

Ontogenesis of hypothalamic immunoreactive ACTH cells in vivo and in vitro: Role of Rathke's pouch☆

The ontogenesis of immunoreactive (ir) ACTH cells and ir alpha-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir alpha-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir alpha-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathkes pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for alpha-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathkes pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH/alpha-MSH cells in the hypothalamus.

Hypothalamic substance P-containing neurons. Sex-dependent topographical differences and ultrastructural transformations associated with stages of the estrous cycle

Immunoreactive substance P (ir SP)-containing neurons were examined in rat hypothalamus. In untreated males, few if any ir perikarya were found in the arcuate nucleus, but after intraventricular colchicine administration, many appeared not only in the arcuate nucleus but also in the premammillary region, posterior hypothalamic, ventromedial, dorsomedial, subthalamic and paraventricular nuclei, and zona incerta. This was also the case in females treated with colchicine on the second day of diestrus. In untreated females, perikarya were seen only in the arcuate nucleus, varying in number and ultrastructure during the estrous cycle and being maximal in number in proestrus and estrus. The perikarya possessed well-developed Golgi bodies and lamellar bodies composed of many closely apposed cisternae of endoplasmic reticulum in proestrus, and showed stacks of two or three cisternae of endoplasmic reticulum as well as lysosomes in estrus. In diestrus, the perikarya had only a few granulated vesicles and fragmental cisternae of endoplasmic reticulum. In the perikarya of colchicine-treated males, many granulated vesicles and lysosomal bodies were noted. These findings suggest that, among the widely distributed SP neurons in the hypothalamus, some in the arcuate nucleus are involved in the hypothalamic-hypophysial-gonadal axis.

Ontogenetic studies on the topographical heterogeneity of somatostatin-containing neurons in rat hypothalamus

SummaryThe ontogeny of the somatostatin-containing neuron system was investigated by light-microscopic immunohistochemistry. During development, immunoreactive somatostatin-containing neurons arise from three discrete regions of the neuroepithelium of the third ventricle and show a chronological difference. The neurons are first evident within the third ventricle floor on day 12.5 of gestation; they move thereafter to the arcuate nucleus. The second generation occurs in the dorsal region of the arcuate nucleus during days 17.5–19.5; these neurons migrate sequentially into the arcuate-ventromedial nuclear region. The third generation is recognized in the neuroepithelial cell layer of the rostral hypothalamus on day 17.5 of gestation; these cells move to the periventricular area. This latter generation is most prominent during days 3–6 after birth, and some of the cells are seen sporadically even up to day 20. The first two generations give rise to the somatostatin neuron system in the arcuate-ventromedial nuclear region, while the latter gives rise to that in the rostral periventricular region in the adult rat hypothalamus.

Idoxifene and estradiol enhance antiapoptotic activity through estrogen receptor-beta in cultured rat hepatocytes.

Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen-receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in rat models of hepatic fibrosis and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on the antiapoptotic role of idoxifene and E2, and the functions of ER subtypes ER-α and ER-β in hepatocytes undergoing oxidative stress. Lipid peroxidation was induced in cultured rat hepatocytes with ferric nitrilotriacetate solution with idoxifene or E2. Oxidative stress-induced early apoptosis was linked to its ability to inhibit not only the expression of Bcl-2 and Bcl-XL but the production of antioxidant enzymes as well and to stimulate Bad expression. Hepatocytes possessed functional ER-β, but not ER-α, to respond directly to idoxifene and E2. Idoxifene and E2 suppressed oxidative stress-induced reactive oxygen species generation and lipid peroxidation, and their antiapoptotic effects on the activation of activator protein-1 and nuclear factor-κB, the loss of antioxidant enzyme activity, and Bcl-2 family protein expression in early apoptotic hepatocytes were blocked by the pure ER antagonist ICI 182,780. Our results indicate that idoxifene and E2 could enhance antiapoptotic activity through ER-β during oxidative damage in hepatocytes.

Synaptic regulation of hypothalamic neurons containing ACTH by substance P

Synaptic connections between substance P (SP)- and ACTH-containing neurons in the rat arcuate nucleus were studied by a technique of combined pre- and postembedding immunohistochemical staining. The results showed that immunoreactive (ir) SP fiber terminals form synapses with ir ACTH cell bodies. This strongly suggests that the activity of ACTH neurons is under neuronal regulation by SP neurons.

Development of hypothalamic neurons in intraventricular grafts: Expression of specific transmitter phenotypes

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathkes pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathkes pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.