Yasuaki Kagotani

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.

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.

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).

Immunohistochemical evidence for synaptic connections between neuropeptide Y-containing axons and periventricular somatostatin neurons in the anterior hypothalamus in rats

By employing a pre-embedding double immunolabeling technique, we examined light and electron microscopically synaptic associations between neuropeptide Y (NPY)-containing axons and somatostatin (SRIH)-containing neurons in the anterior periventricular area (APV) of the rat hypothalamus. For light microscopy, the immunoreactions for NPY and SRIH were visualized with silver-gold and diaminobenzidine (DAB), respectively, and the reverse labeling was used for electron microscopy. Light microscopy disclosed many brown SRIH perikarya surrounded by several black beads of NPY fibers in the APV. In electron microscopy, immunoreactive SRIH neurons revealed silver-gold particles scattered throughout the cytoplasm and accumulated in the Golgi area and the secretory granules. SRIH perikarya and dendritic processes indicated synaptic associations with DAB-labeled NPY fiber terminals and immunonegative fibers. NPY presynaptic terminals possessed numerous small clear vesicles and a few dense core vesicles; vesicular membranes and cores were labeled with DAB chromogen. Both the pre- and postsynaptic membranes were thickened equally to be a symmetric synapse. These findings suggest that NPY neurons are involved in the regulation of growth hormone secretion from the pituitary by affecting periventricular SRIH neurons.

Synaptic associations between oxytocin-containing magnocellular neurons and neurons containing corticotropin-releasing factor in the rat magnocellular paraventricular nucleus

In the paraventricular nucleus (PVN) of the rat hypothalamus, we determined synaptic associations between oxytocin (OXT)-containing magnocellular neurons and parvocellular neurons containing corticotropin-releasing factor (CRF) by using a double immunolabeling technique in 7 animals. In single vibratome sections of the hypothalamus, immunoreactive CRF and OXT were labeled with silver-gold particles and diaminobenzidine (DAB) chromogen, respectively. By light microscopy CRF-containing fibers appeared to be black dots, some of which encircled magnocellular perikarya labeled with brown DAB chromogen in the PVN. By electron microscopy we discriminated OXT neurons having fine DAB-chromogen particles distributed throughout the cytoplasm and on large secretory granules from CRF neurons having dense coarse particles of silver-gold. Occasional CRF axons terminated on perikarya or dendritic processes of OXT neurons, making synaptic contacts. The terminals which were characterized by having clusters of small clear vesicles and a few dense core vesicles showed equal thickenings of pre- and postsynaptic membranes at the synaptic junctions.

Light- and electron-microscopic evidence of costoring of immunoreactive enkephalins and substance P in dorsal horn neurons of rat

SummaryThe topographical localization of substance P (SP) and methionine-enkephalin-octapeptide (Enk-8) was examined immunohistochemically in the surface layer of the dorsal horn of rat cervical spinal cord. Although a few neurons were immunoreactive for Enk-8 in the intact animals, after an intracisternal administration of colchicine, immunoreactive Enk-8 neurons were numerous, and half of them indicated immunoreactivity also for SP. Some immunoreactive SP neurons appeared to show no immunoreactivity for Enk-8. Immuno-reactive nerve fibers, on the other hand, were numerous, and many of them contained both peptides. Electron-microscopic examination of the nerve fibers in tissue prepared by a freeze-drying procedure and stained by a postembedding procedure, revealed the costoring of both peptides in the same cored vesicles. The physiological significance of this costoring is discussed.

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.

Intragranular co-storage of neuropeptide Y and arginine vasopressin in the paraventricular magnocellular neurons of the rat hypothalamus

SummaryCertain populations of arginine vasopressin (AVP) neurons in the magnocellular paraventricular nucleus became immunoreactive for neuropeptide Y (NPY) when rats were treated with colchicine or monosodium glutamate (MSG). The co-storage of these peptides was examined by empooying a post-embedding electron-microscopic immunohistochemistry technique using goldlabeled antibodies to the two peptides. In colchicinetreated rats, the neuronal perikarya contained numerous secretory granules showing co-storage of the two peptides. The cells of the MSG-treated rats were characterized by having well-developed Golgi bodies with the granular structures also co-storing the two peptides, although the secretory granules in the perikarya were rather fewer than in the colchicine-treated rats. It is concluded that the destruction of the arcuate nucleus by MSG-treatment may potentiate the synthesis of NPY in AVP neurons, the synthesis of which is latent in intact animals.