[Tyrosine hydroxylase and dopa decarboxylase expression in the neurons of a mediobasal hypothalamic transplant]. / Ekspressiia tirozingidroksilazy i dopadekarboksilazy v neironakh transplanta mediobazal'nogo gipotalamusa.

Specific antibodies were used for studying the expression of tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC) in neurons of rat embryonic mediobasal hypothalamus (MBH), whose fragments were implanted into the third brain ventricle of the adult female Wistar rats. Two months after surgery, most immunopositive neurons expressed TH, smaller number of neurons expressed both TH and DDC, and few cells contained only DDC. As during development in situ, immunopositive neurons formed nuclei-like clusters located in the ventral region of the graft. This data suggest that MBH grafts contain a population of dopaminergic neurons and a population of TH-containing neurons that probably are not dopaminergic.

[The development of a graft of embryonic mediobasal hypothalamus in the 3rd cerebral ventricle of the adult rat]. / Razvitie transplantata émbrional'nogo mediobazal'nogo gipotalamusa v 3-m zheludochke mozga vzrosloi krysy.

Development of the mediobasal hypothalamus from 15-day rat foetuses and 8-week human foetuses transplanted in the 3rd ventricle of the adult rat brain has been morphologically analyzed. The graft was shown to fill the ventral area of the 3rd ventricle and integrate with the host brain, as was especially distinct in the region of optic chiasma. The graft was abundantly vascularized and its vessels connected with the host brain vessels. The graft neurons were normally differentiating in situ. Some neurons migrated in the host brain. The graft neuropile ultrastructure was characterized by the abundance of synaptic contacts. Some graft neurons expressed dopaminergic phenotype by synthesizing tyrosine hydroxylase and DOPA-decarboxylase and displaying specific capture of 3H-dopamine. Dopaminergic axons of the neurons were spreading both within the graft and penetrating in the host tissue, especially in the region of optic chiasma and tracts. Unlike allotransplantation in rats, survival of xenotransplants of the human embryonic nervous tissue in the 3rd ventricle of the adult rat brain was possible only under the conditions of constant immunosuppression.

Migration of LHRH neurons derived from the olfactory placode in rats.

Using the olfactory placode of 12.5- and 14.5-day-old (E12.5, E14.5) rat embryos, we examined the migration of LHRH neurons by in vivo intraventricular transplantation and in vitro organotypic culture systems. In the transplantation, the olfactory placode of E12.5 embryos was co-transplanted with the cerebral cortex and also with medial basal hypothalamus (MBH). LHRH neurons that had migrated into the co-transplanted brain tissues were fusiform, but those that had moved into the neuro-mesenchymal tissue were polyhedral. The migration occurred most conspicuously in the MBH. In our in vitro studies, we used E14.5 embryos; their vomeronasal organ was cultured with MBH, the olfactory cortex, and the septum of the telencephalon in two systems (piled-culture with an intervening transferrable membrane and co-culture). Among these brain tissues, the MBH was the most effective in inducing the development and migration of LHRH neurons. We further found synaptic junctions of immunonegative nerve fibers on immunoreactive LHRH neurons located in the septum of E16.5 and 17.5 embryos. These findings suggest that the MBH may lead the intraseptal migration of LHRH neurons by yielding certain substances after introducing the neurons into the medial aspect of forebrain vesicles. The early development of the neuronal connection may further promote the migration of LHRH neurons.

Effects of transplants of fetal mediobasal hypothalamus on luteinizing hormone pulses impaired by hypothalamic deafferentation in adult ovariectomized rats.

Pulsatile luteinizing hormone (LH) secretion is impaired after posterior anterior-hypothalamic deafferentation (PAD), which separates the anterior part of the arcuate nucleus from the mediobasal hypothalamus (MBH). In the present study, we examined whether transplants of fetal brain tissue could prevent the effects of PAD. The brain tissue containing the MBH or the cerebral cortex taken from the fetal brain was transplanted into the third ventricle of ovariectomized rats. Four weeks after the brain transplantation, animals with or without the brain transplantation were subjected to PAD. One week after PAD, blood samples were collected every 6 min for 3 h through an indwelling atrial cannula. Rats bearing PAD without transplantation showed irregular pulsatile fluctuation of plasma LH, whereas LH pulses were maintained in rats bearing transplantation of the fetal MBH tissue. In rats which had been transplanted with the cerebral cortex, LH pulses were less apparent after PAD than in the MBH-transplanted or sham-deafferentated animals. No cell bodies of LH-releasing hormone (LHRH) neurons were found immunohistochemically in the MBH grafts. These results suggest that the graft containing the fetal MBH tissue maintains regular LH pulses after PAD and that the LHRH pulse generator may consist, at least in part, of a group of neurons in the MBH other than LHRH-producing neurons.

Transplantation of fetal growth hormone-releasing factor-immunoreactive neurons into the ventricular system of adult MSG-treated rats.

Fetal (17-18 days of gestation) mediobasal hypothalamic tissue (MBH) was transplanted into the third ventricle of adult, male rats which had been treated neonatally with monosodium glutamate (MSG). MSG treatment caused a marked reduction of growth hormone-releasing factor-like-immunoreactive (GRF-i) perikarya in the arcuate nucleus and GRF-i fibers in the median eminence (ME), as compared to littermate controls. When normal fetal MBH was transplanted into the third ventricle of MSG recipients, numerous GRF-i perikarya were located within the graft four weeks following surgery. GRF-i fibers in the ME of MSG-treated rats were enhanced when MBH grafts were in close contact with the ME, but not when transplants were located dorsally or rostrally in the third ventricle without making contact with the recipient's ME. Fetal cerebral cortex, which was grafted as a control tissue, did not contain GRF-i neurons. These immunohistochemical results suggest that grafted fetal GRF-i perikarya may contact the recipient's ME to increase the content of GRF previously depleted by exposure to MSG.

Modification of nerve growth factor and sex steroid in development of hypothalamic neurons.

The medial basal hypothalamic (MBH) tissues were taken from rat fetuses at day 17-18 of gestation and transplanted into the third ventricle of adult female rats. 2.5 S mouse nerve growth factor (NGF) was infused into the third ventricle of some hosts for 2 weeks using a mini-osmotic pump. The other hosts were implanted silastic capsules containing estradiol. Three weeks after the operation, the transplants were immunocytochemically stained with anti-tyrosine hydroxylase (TH) antibody. A number of immunoreactive neurons was obtained in all of NGF-treated, estrogen-exposed and control transplants. Immunoreactive axons distributed densely in NGF-treated transplants. However, the distribution of immunoreactive axons was less dense in estrogen-exposed and control MBH transplants. These results suggest that NGF infused into the host ventricle stimulates the growth and/or regeneration of axons of hypothalamic neurons. In contrast, action of estrogen on transplanted hypothalamic tissues was not evident in the present study.

Development of hypothalamic neurons in intraventricular grafts: expression of specific transmitter phenotypes.

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's 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 Rathke's 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.

Ultrastructural and immunohistochemical analysis of fetal mediobasal hypothalamic tissue transplanted into the aged rat brain.

MBH tissue, which included the hypothalamic arcuate nucleus of fetal or neonatal rats, was transplanted into the third ventricle of aged (21-30-month-old) female rats. The brain and ovaries of each recipient were examined histologically 3 or 4 weeks after transplantation. Four grafted MBH tissues were examined ultrastructurally and immunohistochemically 4 weeks after transplantation. The appearance of the MBH grafts was similar to that of normal neural tissue. The neuropil in the grafts was fully occupied with numerous axons, dendrites, and glial processes. A number of axodendritic shaft and spine synapses were observed in the neuropil. Immunohistochemical analysis with antiserum to TH revealed stained (immunoreactive) neuronal perikarya and processes in the grafts. TH-immunoreactive processes originating from the TH-positive neurons in the grafts could be seen to extend across the graft-host interface. The ovaries of six out of nine females that received MBH grafts exhibited follicles of various sizes and healthy appearing corpora lutea. On the other hand, some follicles and masses of interstitial cells were prominent in the ovaries of the intact animals or controls that had received cortical grafts. In the females that received MBH grafts, the ovarian weight was significantly greater than that in the controls. These results suggest that the neural substrates in fetal MBH tissue can survive and develop well in the aged rat brain and that MBH grafts may play some role in the recovery of declined ovarian function in aged female rats.