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Biomedical subjects

H Kiyama

Publications and source records attributed to H Kiyama.

At least 109 records · Page 6Linked to original sources
PubMed1995

Colchicine induces the GAP-43 gene expression in rat hypothalamus.

Injection of colchicine, a mitogen inhibitor, in the dorsal third ventricle induced the expression of the growth associated protein-43 (GAP-43) mRNA in some groups of cells of the adult rat brain. These mRNAs were detected by in situ hybridization histochemistry using an alkaline phosphatase labeled oligonucleotide probe. A substantial up-regulation of GAP-43 mRNA was noticed by the increase of both the number of positive cells and the intensity of the hybridization signal. These changes were observed in the hypothalamic nuclei located near the ventral third ventricle, namely the preoptic area, the supraoptic nucleus, the peri- and the paraventricular nuclei of the hypothalamus, the dorsal subnucleus of the ventromedial nucleus, the arcuate nucleus and the posterior part of the peri-mammillary region. Such abundant GAP-43 mRNA positive cells have not been observed in control adult rat hypothalamus. Since the positive cell number and shape initially suggested that these were neurons or astrocytes, double labeling in situ hybridization using both radioactive (for the detection of GFAP mRNA as a marker of astrocyte) and non-radioactive (for the detection of GAP-43 mRNA) probes was carried out. This demonstrated that these GAP-43 mRNA positive cells were not astrocytes. In addition enhanced GAP-43 mRNA expression was also found in some neuronal component, particularly in neurosecretory magnocells of the pareaventricular and the supraoptic nuclei. This up-regulation was further confirmed by the Northern blot analysis. About five fold increase in GAP-43 mRNA in the colchicine-treated hypothalamic tissue was shown.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Dexamethasone enhances level of GAP-43 mRNA after nerve injury and facilitates re-projection of the hypoglossal nerve.

Application of dexamethasone was found to induce an enhanced expression level of mRNA encoding the growth associated protein (GAP-43) after peripheral nerve injury. Following hypoglossal nerve axotomy, a dexamethasone releasing pellet (1.5 mg released in 3 weeks) was placed near the transected nerve. GAP-43 mRNA was detected in the hypoglossal nucleus by non-radioactive in situ hybridization histochemistry using an alkaline phosphatase-labeled oligonucleotide probe. A significant elevation of GAP-43 mRNA level was observed 2 weeks after the transection in dexamethasone treated animals. This induction was not observed in the dorsal motor nucleus of vagus which expresses moderately high levels of GAP-43 mRNA even without nerve injury. Although dexamethasone did not alter the maximum level of GAP-43 mRNA in the hypoglossal nucleus after nerve injury, it prolonged the period in which the mRNA expression remained elevated. This may be due to post-transcriptional effect by the glucocorticoid. Dexamethasone treatment also caused a slight facilitation of reprojection. This may be due to the enhancement of GAP-43 mRNA level by the glucocorticoid.

Animals↗
PubMed1995

GAP-43 mRNA suppression by the ribozyme in PC12 cells and inhibition of evoked dopamine release.

We constructed a ribozyme designed to cleave the GAP-43 mRNA and which contained a bacteriophage T7 transcription terminator at its 3' site to maintain stability. The ribozyme was overexpressed in PC12 cells by pEF-BOS, a powerful mammalian expression vector. Consequently, PC12 cells overexpressing the GAP-43 ribozyme revealed a drastic decrease in the levels of GAP-43 mRNA expression, and the evoked dopamine release was significantly suppressed in these cell lines. These results support the previous observations that GAP-43 is associated with Ca-dependent dopamine release in PC12 cells, and the ribozyme expression system used in the present study was demonstrated to be useful for suppression of the functions of specific mRNAs and exploration of specific gene products.

Animals↗
PubMed1995

Role of amino acids in salivation and the localization of their receptors in the rat salivary gland.

The distribution of gamma-aminobutyric acid (GABA) receptor subunits such as GABAAR-gamma 1 and GABAAR-gamma 2, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type receptor subunits such as GluR-1, GluR-2/3 and GluR-4, and N-methyl-D-aspartic acid (NMDA) type subunits such as NR1 were investigated by immunocytochemistry. Furthermore, the roles of these amino acids, GABA and glutamate, on salivation were analyzed in the rat submandibular and sublingual glands. Some similarities were observed in the distribution patterns of GABAA type receptors and AMPA receptors. In the submandibular ganglion cells, collecting ducts and striated ducts, these subunits were expressed strongly; however, there were some differences in their expression patterns between the submandibular and sublingual gland acinar cells. Since these receptor subunits were expressed in the acinar cell bodies of the submandibular gland, they were not expressed in the acinar cells but were expressed in the myoepithelial cells in the sublingual gland. On the other hand, no NR1 expression was observed. To examine the roles of GABA and glutamate in salivation, the submandibular and sublingual glands were perfused partially with Ringer's solution via a facial artery to avoid systemic influence, and substrates were infused into the perfusion solution. No salivary secretion was evoked by GABA or glutamate infusion in the absence of electrical stimulation (2-3 V, 5 ms, 20 Hz). Salivary flow evoked by electrical stimulation of the chorda-lingual nerve caused significant inhibition by GABA (10(-6), 10(-5), 10(-4) and 10(-3) M) and the GABAAR agonist muscimol 10(-3) and 10(-6) M) (n = 6, P < 0.05). Such GABA-induced inhibition was antagonized by the GABAAR antagonists bicuculline (BCC; 10(-6) and 10(-3) M) and picrotoxin (PTX; 10(-6) and 10(-3) M). On the other hand, salivary flow evoked by electrical stimulation (8-10 V, 5 ms, 20 Hz) of the superior cervical ganglion (SCG) was not affected by GABA. While high doses of glutamate (10(-1) M) and NMDA (10(-1) M) showed no effects on salivary flow despite application of electrical stimulation, AMPA at a high concentration (10(-1) M) significantly inhibited salivary secretion (n = 6, P < 0.05). These studies revealed that inhibitory and excitatory amino acid receptors such as GABAA and AMPA type receptors are coexpressed in the rat salivary glands, and that GABA inhibits salivary secretion via GABAA receptors which may act with acetylcholine. However, the role of glutamate in salivation remains unclear despite the presence of AMPA type receptors. The present findings suggest that glutamate does not act alone but with other substances such as peptides and/or other amino acids.

Animals↗
PubMed1995

Expression patterns of a glutamate-binding protein in the rat central nervous system: comparison with N-methyl-D-aspartate receptor subunit 1 in rat.

Using radioactive in situ hybridization histochemistry, we examined the topographical patterns of expression of the messenger RNA encoding a glutamate-binding protein (N-methyl-D-aspartate receptor glutamate-binding protein in rat; NMDARgbs) in the central nervous system of the rat. Expression patterns of N-methyl-D-aspartate receptor glutamate-binding protein were compared with those of N-methyl-D-aspartate receptor subunit 1 (NMDAR1) of the N-methyl-D-aspartate receptor on adjacent sections. N-methyl-D-aspartate receptor glutamate-binding protein is not expressed in glial cells. The expression of both N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor subunit 1 was observed in virtually all neurons throughout the central nervous system. The mean level of N-methyl-D-aspartate receptor subunit 1 expression was higher than that of N-methyl-D-aspartate receptor glutamate-binding protein. Similar topographical patterns of expression of N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor were observed in most regions, except in discrete thalamic, hypothalamic and brainstem nuclei. Concomitantly for N-methyl-D-aspartate receptor glutamate-binding protein and N-methyl-D-aspartate receptor subunit 1, the highest expression levels were distributed in the mitral layer of main and accessory olfactory bulbs, granule cell layer of the dentate gyrus, polymorphic and pyramidal layers of CA1-3 fields of Ammon's horn. A slightly less prominent expression was observed in the glomerular and granule cell layers of main and accessory olfactory bulbs, anterior olfactory nucleus, layer 2 of piriform cortex, olfactory tubercle and taenia tecta. In the cerebellum, the prominent level of N-methyl-D-aspartate receptor glutamate-binding protein expression was slightly higher in the Purkinje cell layer than in the granule cell layer, an opposite pattern being observed for N-methyl-D-aspartate receptor subunit 1. A moderately high expression level of both messenger RNAs was observed in the medial septal nucleus, nucleus of the diagonal band of Broca, dorsal part of the endopiriform nucleus, and in the anteroventral and anterolateral parts of the bed nucleus of the stria terminalis. In the neocortex, the mean expression level of N-methyl-D-aspartate receptor glutamate-binding protein is moderate, while the mean level of N-methyl-D-aspartate receptor subunit 1 expression is high. With both probes, layer IV is slightly less labeled than the other layers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The messenger RNAs encoding metabotropic glutamate receptor subtypes are expressed in different neuronal subpopulations of the rat suprachiasmatic nucleus.

Glutamate is the principal transmitter of retinal projections to the rodent suprachiasmatic nucleus, a circadian clock synchronized with the light-dark cycle through the activation of glutamate receptors of the ionotropic type. In vitro, an intracellular mobilization of calcium can be induced by glutamate within cells of the suprachiasmatic nucleus maintained in a calcium-free medium, suggesting a participation of metabotropic glutamate receptors coupled to phospholipase C. Using in situ hybridization histochemistry, we examined the expression of messenger RNAs encoding the mGluR1 and mGluR5 subtypes of metabotropic glutamate receptors in the suprachiasmatic nucleus of the adult rat and during postnatal development. In the adult, mGluR1 was expressed in a small subset of neurons segregated caudally within the ventrolateral subdivision of the nucleus, while mGluR5 was mainly expressed in ventrolateral neurons within the middle third of the nucleus. Both subtypes were expressed in morphologically similar small cells, but mGluR5 was also solely expressed in a small population of larger neurons located at the dorsalmost aspect of the ventrolateral subdivision. In addition, with mGluR1 probe silver grain clusters exhibiting a grain density close but below the significant level were observed throughout the ventrolateral subdivision of the nucleus. At birth, mGluR1 and mGluR5 were similarly expressed throughout the caudal half of the nucleus. The expression of mGluR1 increased during early postnatal development and exhibited an adult pattern at postnatal day 21. The expression of mGluR5 increased from postnatal day 7 and reached the adult pattern at postnatal day 45. These observations suggest that each subtype of metabotropic glutamate receptor coupled to phospholipase C underlies specific roles within the rat suprachiasmatic nucleus during postnatal development and in the adult. In the adult, ionotropic and metabotropic receptors likely co-expressed within neuronal subsets located in the retinal terminal field may have interactive and/or additive effects on intracellular calcium concentration. Metabotropic receptors may thus participate in the mediation of photic information conveyed to a subset of neurons. During postnatal development, metabotropic receptors may play a role in the maturation of glutamatergic synapses associated with the retinal input.

Animals↗
PubMed1995

Increased F1/GAP-43 mRNA accumulation in gerbil hippocampus after brain ischemia.

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

Animals↗
PubMed1995

Changes in Fos and Jun expression in the rat brainstem in the process of vestibular compensation.

By means of immunohistochemical technique, we examined the changes in Fos and Jun expression after unilateral labyrinthectomy (UL) in the rat brainstem. We observed Fos-like immunoreactivity (-LIR) in the ipsilateral medial vestibular nucleus (ipsi-MVe), the contralateral prepositus hypoglossal nucleus (contra-PrH) and the contralateral inferior olive beta subnucleus (contra-IOb) 1 h after UL. Then, Fos expression in the contra-PrH and the contra-IOb disappeared 3 days after surgery. However, we still found the residual expression in the ipsi-MVe, which disappeared within 7 days. On the other hand, no Jun-LIR was detected in the vestibular or vestibular-related nuclei before or after the operation. Fos expression in the MVe, PrH and IOb was induced immediately after UL. Then, the Fos expression disappeared in accordance with the development of the vestibular compensation. These findings suggest that the transient Fos expression in the vestibular and vestibular related nuclei is a trigger of vestibular compensation.

Animals↗
PubMed1995

Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus.

A novel murine cDNA which encodes a protein designated neuropsin was cloned. Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1-3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons. The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.

Amino Acid Sequence↗
PubMed1995

Nerve injury enhances rat neuronal glutamate transporter expression: identification by differential display PCR.

An increase in neuronal glutamate transporter expression after nerve injury was demonstrated by means of differential display PCR (DD-PCR) coupled with in situ hybridization. DD-PCR was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the rat. The expression of several gene fragments were found to be increased following nerve injury; the full length cDNA corresponding to one fragment was cloned by subsequent rat cDNA library screening. The close homology of glutamate transporters with our rat cDNA led us to conclude that this clone corresponds to the rat neuronal glutamate transporter (rat EAAC1). We speculate that the upregulation of this glutamate uptake system may increase the resistance of these cells against neurotoxic glutamate accumulation during the process of nerve regeneration.

ATP-Binding Cassette Transporters↗
PubMed1994

Neurotensin transcription is oppositely regulated during development and regeneration.

Our previous work demonstrated that neurotensin mRNA expression was transiently up-regulated during development in the hypoglossal motoneurone. The present study was carried out to examine how neurotensin mRNA expression changes during nerve regeneration. Following either unilateral resection or crushing of the hypoglossal nerve, mRNA expression was detected by non-radioactive in situ hybridization histochemistry using an alkaline phosphatase-labelled oligonucleotide probe. A reduction in neurotensin mRNA was observed on the operated side after the nerve injury. It took around 2 months for NT mRNA levels to return to normal. Similar down-regulation was observed following crushing, but recovery was more rapid than that observed with sectioning, probably due to the extent of nerve damage. This finding indicates that neurotensin transcription is up-regulated during development but, on the contrary, down-regulated during regeneration.

Animals↗
PubMed1994

The production of CINC/gro, a member of the interleukin-8 family, in rat anterior pituitary gland.

We investigated the possibility of detection of CINC/gro, which is a IL-8-like neutrophil chemoattractant, immunoreactivity in rat normal anterior pituitary gland by immunohistochemistry, western blot analysis and an ELISA. We first ascertained the possibility of detection of CINC/gro immunoreactivity in the anterior pituitary gland by immunocytochemistry. In the anterior lobe of the pituitary gland, a few CINC/gro-like immunoreactive cells were observed (1-3% of all cells in the anterior pituitary). The positive cells were middle or large in size and looked angular in shape. Intense immunoreactivity was observed in the cytoplasm but not in the nucleus. Analysis by immunoblotting with anti-CINC/gro antiserum gave a characteristic single CINC/gro band with a molecular weight of 6.3 kDa. CINC/gro immunoreactivity was also detected in 3-h conditioned medium of normal anterior pituitary cells by an ELISA, and that immunoreactivity increased significantly in a time-dependent manner during 24-h incubation. This immunoreactivity could be induced by TNF-alpha in a dose-dependent manner. These findings indicate that CINC/gro is produced in pituitary gland and also suggests the possibility that CINC/gro may play some role asa modulator of anterior pituitary function, especially in the cross-talk mechanism between the immune and neuroendocrine systems.

Analysis of Variance↗
PubMed1994

Expression of GABAA receptor gamma 1 and gamma 2 subunits in the peripheral vestibular system of the rat.

The distribution of GABAA receptor gamma 1 and gamma 2 subunits in the rat vestibular ganglion cells and end-organs was examined by using immunohistochemical techniques. Both gamma 1 and gamma 2 subunit-like immunoreactivities were observed in most vestibular ganglion cell bodies and peripheral terminal endings in the vestibular sensory epithelia. These results indicate that GABAA receptors are expressed in the vestibular afferent terminal endings and therefore suggest that GABA in addition to glutamate is a neurotransmitter which effects on vestibular afferents.

Afferent Pathways↗
PubMed1994

Occurrence of galanin-like immunoreactivity in vestibular and cochlear efferent neurons after labyrinthectomy in the rat.

The origins of the vestibular and cochlear efferent systems lie in the lower brainstem and innervate the labyrinth. In the present study we investigated the changes in galanin-like immunoreactivity (GAL-IR) in the vestibular and cochlear efferent neurons in control and labyrinthectomized rats. In control animals, no GAL-IR was noticed in these neurons. However, in response to unilateral labyrinthectomy, similar GAL-IR was bilaterally expressed in the two systems. GAL-immunostained cells appeared on postoperative day 3 and reached a peak of intensity and number at postoperative week 2. Retrograde tracing by fluorogold combined with GAL immunohistochemistry demonstrated that, except for the cells of the contralateral lateral superior olivary nucleus (LSO), GAL-IR neurons project into the lesioned labyrinth.

Animals↗
PubMed1994

Up-regulation of ERK (MAP kinase) and MEK (MAP kinase kinase) transcription after rat facial nerve transection.

Up-regulation of ERK (extracellular signal-regulated kinase or MAP kinase) and MEK (ERK kinase or MAPK kinase) expression after rat facial nerve injury was demonstrated by in situ hybridization histochemistry and immunohistochemistry. These two enzymes play roles in one of the major intracellular signal cascade pathways involving receptor tyrosine kinase common to growth factor receptors, and transcription factors. Significant increases in ERK1 mRNA levels were observed from day 3 after facial nerve transection, with the highest level of expression from 1 to 2 weeks after the operation. This high level of mRNA expression then decreased gradually to the normal level. ERK1-like immunoreactivity showed a similar time course to that of its mRNA expression; however, the decay profile was more prolonged. The up-regulation of MEK, the ERK kinase/MAPK kinase, was also detected by immunohistochemistry. The protein expression profiles were almost equivalent, but the MEK expression was slightly advanced, suggesting that the observed up-regulation of MEK was not due to that of ERK. The receptor tyrosine kinase signal transduction pathway via MEK-ERK located downstream of growth factor receptors seems vital as a regulator of the synthesis of molecules that play important roles in the recovery process following injury or/and regeneration.

Animals↗
PubMed1994

Gene transfer and the expression of a foreign gene in vivo in post-mitotic neurons of the adult rat brain using the hemagglutinating virus of the Japan-liposome method.

Neurons in the adult rat brain were transfected in vivo with a simple plasmid that harbored the gene for beta-galactosidase from Escherichia coli under control of a chicken beta-actin promoter by use of the hemagglutinating virus of Japan (HVJ) and liposomes. Cells that expressed beta-galactosidase were detected only in the target area of the central nervous system for 10 days by light microscopic analysis. Since electron microscopic analysis revealed that the products of the histochemical reaction were predominantly associated with the nuclear membrane and the endoplasmic reticulum of positive cells, it appeared that the products were translated endogenously and had not been entrapped by endocytosis. Furthermore, the products were observed in typical neuronal cells with a large, round, and pale nuclei, and with direct axo-somatic and axo-dendritic synaptic contacts. This report suggests the possibility of introducing functionally significant genes into neurons in targeted areas of the adult central nervous system.

Animals↗