Mayumi Nishi

Effects of near-infrared low-level laser irradiation on microcirculation.

Recently, there has been an increase in the clinical application of low‐level laser irradiation (LLLI) in various fields. The present study was conducted to explore the effects of LLLI on microcirculation.

Localization of nuclear coactivators p300 and steroid receptor coactivator 1 in the rat hippocampus

Cofactors (coactivators and corepressors) play an important role in mediating ligand-dependent transcription by steroid hormone receptors, but little has been reported about the distribution of coactivators in the brain. The present study represents the first immunohistochemical attempt to elucidate the localization of coactivators p300 and steroid receptor coactivator 1 (SRC-1) in the hippocampus of the rat. p300- and SRC-1-immunoreactivities were observed in CA1-CA4 pyramidal cell layers of the hippocampus and the granular cell layer of the dentate gyrus, and their localization was in the cell nucleus. Double-labeling immunohistochemistry of p300 and SRC-1 indicated that both coactivators were colocalized in the same cell. Antibodies against neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were used to identify whether neuron and glia contained p300 or SRC-1. Most p300- or SRC-1-containing cells showed NSE-immunoreactivity, but a limited number of these cells showed GFAP-immunoreactivity. These findings suggest that in the hippocampus p300 and SRC-1 synergistically play an important role in activating ligand-dependent transcription by steroid hormone receptor.

REAL-TIME IMAGING OF GLUCOCORTICOID RECEPTOR DYNAMICS IN LIVING NEURONS AND GLIAL CELLS IN COMPARISON WITH NON-NEURAL CELLS

To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP‐GR) was prepared. We transiently transfected GFP‐GR into primary cultured rat hippocampal neurons, cortical glial cells, and non‐neural cells, e.g. COS‐1 cells and CV‐1 cells, and compared the dynamic changes in subcellular localization of GFP‐GR in these cells. When GFP‐GR was expressed in the cells, GFP‐GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm‐to‐nuclear translocation of GFP‐GR induced with 10–7 m DEX, a specific agonist of GR, at 37u2003°C was completed within 30u2003min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP‐GR into the nucleus from the cytoplasm was induced in a ligand‐specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX‐induced GFP‐GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time‐lapse imaging analysis for 1u2003h at 37u2003°C. The present findings suggest that the subcellular localization of GFP‐GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.

Enhanced synaptophysin immunoreactivity in rat hippocampal culture by 5-HT1A agonist, S100b, and corticosteroid receptor agonists

Serotonin (5‐HT) has been shown to modulate brain maturation during development and adult plasticity. This effect in the whole animal may be due to activation of 5‐HT1A receptors and a corresponding increases in S100b and corticosterone. Synaptophysin, an integral protein of the synaptic vesicle membrane that correlates with synaptic density and neurotransmitter release, is reduced by depletion of 5‐HT in the cortex and hippocampus of the adult rat. Injections of a 5‐HT1A agonist or dexamethasone can reverse the loss of synaptophysin immunoreactivity (IR). In this study we used morphometric analysis of synaptophysin‐IR to study the effects of the 5‐HT1A agonist, ipsapirone, and the neuronal extension factor, S100b on hippocampal neurons grown in a serum and steroid free media. Both compounds increased the synaptophysin‐IR at doses previously established to be highly specific. Ipsapirone (10−9 M) was more effective on neuronal cell bodies staining and S100b (10 ng/ml) was more effective in increasing the number of synaptophysin‐IR varicosities on neuronal processes. In addition both types of corticosteroid receptor agonists, at previously established specific doses, Ru28362 (10−8 M) and aldosterone (10−9 M) produced smaller increases compared to control groups in both the cell body staining and the number of varicosities. The effect of these differentiating factors on the expression of synaptophysin‐IR suggests multiple regulation sites for producing and maintaining pre‐synaptic elements in the brain.

Steroid hormones and their receptors in the brain.

Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the brain was examined. Second biphasic effects of glucocorticoid on the hippocampus was described and particular emphasis was given on the apoptosis. Third the significance of estrogen receptor in the sexually dimorphic areas was discussed. These results suggest that steroids modulate the gene expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.

Trophic interactions between brain-derived neurotrophic factor and S100β on cultured serotonergic neurons

Brain-derived neurotrophic factor (BDNF) and S100beta stimulate serotonergic neurons in fetal rat raphe primary cultures grown under serum-free conditions. BDNF (50 ng/ml) treatment for 3 h enhanced S100beta immunoreactivity in both raphe and hippocampal glial cells. Combined treatment with BDNF and S100beta for 3 days increased the soma area of 5-HT neurons, but not the neurite length. Our results suggest that BDNF and S100beta, which regulate different signal transduction cascades, interact to exert complimentary effects on neuronal maturation by acting sequentially, not concurrently.

S100β promotes the extension of microtubule associated protein2 (MAP2)-immunoreactive neurites retracted after colchicine treatment in rat spinal cord culture

S100beta, a glial derived calcium-binding protein with neurotrophic activity in the central nervous system, stimulates neurite extension of fetal raphe, cortex, spinal cord, and dorsal root ganglion neurons. The effects of S100beta on neurite length and microtubule associated protein2 (MAP2) immunoreactivity (IR) after microtubule disruption with colchicine were investigated in primary rat spinal cord culture. The incubation with S100beta (20 ng/ml) for 3 h after exposure to colchicine (10(-6) M) for 30 min altered the distribution of MAP2-IR. The length of MAP2-IR neurites increased by 65% compared to that in colchicine treatment alone. MAP2-IR intensity in the cell body was reduced by 26% compared to that in colchicine treatment alone. These results indicate that neurites shrink when the microtubular cytoskeletal system is disrupted and S100beta rapidly promotes re-assembly and/or stabilization.

Intracellular dynamics of steroid hormone receptor

Steroid hormones substantially influence brain development, reproduction sexual differentiation and emotion. These effects are mediated by steroid hormone receptors and cofactors, which directly regulate gene expression. Deciphering how and where these transcriptional activators occur in a cell provides the groundwork for elucidating the influence of these small hydrophobic signal molecules on various brain functions. This paper describes some of the recent investigations into the subcellular localization of steroid hormone receptors and cofactors using GFPs and other immunocytochemical methods.

AGONIST- AND ANTAGONIST-INDUCED PLASTICITY OF RAT 5-HT1A RECEPTOR IN HIPPOCAMPAL CELL CULTURE

We examined the response and regulation of 5‐HT1A receptor on hippocampal cultured fetal neurons grown in the absence of serotonin and steroids using three experimental designs: 1) functional response using an antibody against phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB); 2) transcriptional regulation using in situ hybridization; and 3) translational expression using antipeptide 5‐HT1A receptor antibody. Pretreatment of cultured hippocampal cells with the agonist 8‐hydroxy‐2‐(di‐N‐propylamino)‐tetralin (8‐OH‐DPAT) (10−8 M) or ipsapirone (IPS) (10−9 M) for 10 min blocked the forskolin‐stimulated increase in pCREB immunoreactivity. In situ hybridization radioautography revealed that IPS (10−9 M) decreased the 5‐HT1A receptor mRNA expression (−33%) after a 24‐h treatment. The decrease in 5‐HT1A receptor mRNA was accompanied by a change in protein immunoreactivity using a 5‐HT1A receptor antipeptide antibody. Computer‐assisted morphometric analyses showed a reduction in the 5‐HT1A receptor immunoreactive (IR) intensity as compared to control 24 h after treatment with 8‐OH‐DPAT (10−7–10−12 M) and IPS (10−9 M). Thus, fetal hippocampal neurons have a functional 5‐HT1A receptor that is downregulated at both the transcription and translation levels. In addition, we found increased 5‐HT1A receptor‐IR intensity (+17%u2009∼u2009+39%) 24 h after treatment with the antagonist N‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl) cyclohexane carboxamide (WAY 100635) (10−7–10−12 M). Our results indicate that the 5‐HT1A receptor is sensitive to both agonists (downregulation) and antagonists (upregulation) in hippocampal fetal neurons grown in the absence of serotonin and steroids. Synapse 31:186–195, 1999.

5-HT1A receptor expression is modulated by corticosteroid receptor agonists in primary rat hippocampal culture

The effects of corticosteroid receptor agonists on the expression of 5-HT1A receptor mRNA were measured in rat hippocampal cultures using in situ hybridization histochemistry. In our normal culture system, grown in serum and steroid-free media, moderate to heavy signal for 5-HT1A mRNA transcripts were detected in hippocampal neurons and glial cells. Aldosterone, a type I corticosteroid receptor agonist (10(-9) M), significantly reduced the expression of 5-HT1A mRNA both in neurons and glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. The type II corticosteroid receptor agonist, Ru28362 (10(-8) M), also significantly decreased neuronal 5-HT1A mRNA expression. However, it was not as effective as aldosterone in reducing the label over GFAP-IR cells. These data indicate that corticosteroids may directly regulate the expression of hippocampal 5-HT1A receptors at the mRNA level in cultured hippocampal cells.