Mika Chikamori-Aoyama

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.

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.

Differential immunolabeling for electron microscopy of diverse peptidergic neurons.

We describe a simple and reliable method for differential immunolabeling of pre- and post-synaptic signal peptides at the ultrastructural level. Hypothalamic tissues of rats, including the suprachiasmatic nucleus, were cut on a Vibratome. Visualization of the immunolabeling of somatostatin (SRIH) and vasoactive intestinal polypeptide (VIP) was performed with avidin-biotin-peroxidase-diaminobenzidine (DAB). The end product of the DAB to VIP was further silver-intensified in a physical processing using silver nitrate, and the silver grains were finally substituted for gold. DAB-labeled SRIH fibers synapse on gold-labeled VIP perikarya and dendrites in the suprachiasmatic nucleus.

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

SummaryBy applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptie membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.

Reciprocal synaptic relations between CRF-immunoreactive- and TRH-immunoreactive neurons in the paraventricular nucleus of the rat hypothalamus

By double immunoelectron microscopy, we studied synaptic relations between corticotropin-releasing factor (CRF)-immunoreactive (ir) and thyrotropin-releasing hormone (TRH)-ir neurons in the paraventricular nucleus (PVN) of the rat hypothalamus. CRF-ir and TRH-ir neurons made reciprocal synaptic connections in the medial and periventricular parvocellular regions. These results may suggest that both the parvocellular neurons interplay on their hypophysiotropic functions within the PVN.

Vitamin B6 suppresses growth and expression of albumin gene in a human hepatoma cell line HepG2

The effect of vitamin B6 on the growth of a human hepatoma cell line HepG2 in culture was studied. The growth of HepG2 cells and protein synthesis were almost completely inhibited in medium supplemented with 5 mM pyridoxine. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed no inhibitory action. The growth inhibition of HepG2 cells by pyridoxine was accompanied by a marked inhibition of secretion of plasma proteins, particularly albumin. Northern blot analysis of albumin mRNA showed that pyridoxine caused a rapid decrease in the expression of albumin gene. The electron-microscopic examination of pyridoxine-treated HepG2 cells revealed a smoothing of nuclear membrane, a decrease in the number of nucleoli, and an appearance of aggregated heterochromatin structures. These morphological features are compatible with the depressed transcriptional activity in the pyridoxine-treated cells. The mechanism by which vitamin B6 exerts its inhibitory effect was discussed in terms of our recent finding that vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding proteins. Binding of pyridoxal phosphate with tissue-specific transcription factors may reduce the capacity of these factors to interact with the regulatory region of albumin gene, resulting in the inhibition of the gene expression.

Synaptic association between enkephalin-containing axon terminals and proopiomelanocortin-containing neurons in the arcuate nucleus of rat hypothalamus

By employing an electron microscopic dual immunolabeling technique, a synaptic association between neurons containing immunoreactive adrenocorticotropin (ACTH) and axonal terminals containing immunoreactive methionine-enkephalin octapeptide (Enk-8) was found in the arcuate nucleus of the rat hypothalamus. The axonal terminals contained many small clear vesicles and some large cored vesicles. At the synaptic portions, membrane specialization was asymmetric.

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.

Somatostatin-like immunoreactive axon terminals on oxytocin-like immunoreactive neurons in the paraventricular nucleus of the rat hypothalamus

By double label immunohistochemistry, we studied the synaptic relation between somatostatin (SS) and oxytocin (OT) or vasopressin (VP) neurons in the paraventricular nucleus (PVN) of the rat hypothalamus. In the light microscopic observation, SS-immunoreactive (ir) fibers were seen very frequently in contact with OT-ir neurons, but not with VP-ir neurons. Immunoelectron microscopic analysis revealed that somata and their dendrites of OT-ir magnocellular neurons were in synaptic contacts with SS-ir axon terminals. No SS-ir synaptic terminals were found on VP-ir magnocellular neurons. The results suggest that OT-containing magnocellular neurons in the PVN are under regulatory influences of SS-containing neurons.

Alteration of energy substrates utilized by small and large thymocytes in resting and stimulating state

Abstract Glucose and glutamine are well known as a major energy source for proliferating rat thymocytes. Nevertheless, it is still unclear whether there is a difference in utilization of energy substrates between the mature and immature thymocytes. In the present study, we prepared small and large thymocytes from male Wistar rats by Percoll density-gradient centrifugation to answer these questions. The average diameter of the large thymocytes was approximately 1.6 times that of the small thymocytes. By flow cytometry analysis, the most of small thymocytes was immature T cells (CD4 + 8 + cells) and about 20% of the large thymocytes was CD4 + 8 − T cells. After phytohemagglutinin (PHA) and concanavalin A (ConA) stimulations in vitro , large thymocytes showed significantly higher incorporation of [ 3 H]thymidine. In contrast, small thymocytes did not exhibit a proliferative response at all. After 48 hours culture of large or small thymocytes with or without ConA stimulation, the alterations in the medium concentrations of glucose, pyruvate, lactate, ketone bodies, free fatty acids (FFA), and amino acids were analyzed. The resting large thymocytes showed significantly higher consumption of glucose and production of lactate, which were further elevated in response to ConA. In contrast, the resting small thymocytes showed slight consumption of glutamine and production of glucose. When small thymocytes were stimulated with ConA, a change to glucose consumption and lactate production was observed without an increase in either total substrate consumption or proliferative response. These results suggest that the substrates utilized by thymocytes vary from immature to mature cells and that these changes of energy substrates utilized by thymocytes are closely related to the maturation and function of thymic T lymphocytes.