The ventral pallidum (VP) is suggested to control feeding behavior. The functions of the ventral pallidum are considered to be modulated by GABAergic projection from the nucleus accumbens. Previous studies have shown that blockade of GABA receptors in the VP increased food intake. Our previous study has also demonstrated that lesions of the VP decreased the intake of palatable sucrose solution. However, it is unclear whether GABA is critically involved in the ingestion of palatable taste stimuli. To address this issue, we examined the effects of pharmacological manipulations of GABAergic, dopaminergic, glutamatergic, or opioidergic neurotransmission within the VP on the intake of taste solutions in rats. Injection of bicuculline, a GABA_A receptor antagonist, into the VP significantly increased the intake of sweet solution, but not distilled water and bitter solution. On the contrary, injection of muscimol, a GABA_A receptor agonist, into the VP resulted in intensive aversive reactions to sweet solution and distilled water. Although solution intakes were not significantly changed by injection of dopamine or glutamate antagonist, saccharin intake tended to decrease by injection of both antagonists. Injection of [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO), a μ-selective opioid agonist, increased palatable sucrose intake. These results suggest that the GABAergic, but not dopaminergic and glutamatergic, transmission in the VP critically modulated the intake of palatable solution. [Jpn J Physiol 55 Suppl:S200 (2005)]

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In mammals, the circadian oscillation in the suprachiasmatic nucleus (SCN) is believed to base on an autoregulatory feedback loop associated with clock gene expression and their protein products (core loop). Previously, we reported that DEC1 and DEC2, bHLH transcription factors, consist an additional feedback loop which interlocks with the core-loop. Although the expression patterns of Decs and Pers are not identical in the SCN, functional differences are not yet known. In the present experiment, we examined the circadian expression and light responsiveness of Dec1, Dec2 and Per1 in the rat SCN by in situ hybridization using Digoxigenin labeled cRNA probes, and constructed four-dimensional maps of gene expression. Photomicrographs of coronal SCN sections were sectionalized into 75×75 μm squares, and number of mRNA positive cells in each area was counted. The circadian expressions of three genes were peaked at the subjective noon, but the wide expression of Dec2 throughout the SCN was contrasted with restricted expression of Dec1 and Per1 in the medial SCN. In response to light pulse, Per1 dramatically increased only in the ventral SCN, while Dec1 induction extended more dorsally. In addition, after peaking the circadian and light-induced expressions, Per1 signals disappeared faster than Dec1. The results suggest differential roles of Dec1, Dec2 and Per1 in rhythm generation and light entrainment of the circadian clock in the SCN. [Jpn J Physiol 55 Suppl:S200 (2005)]

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In mammals, the circadian pacemaker in the suprachiasmatic nucleus (SCN) is entrained by photic time cues. Explants of peripheral organs also exhibit circadian rhythms in clock gene expression, indicating the presence of peripheral clocks. However, the mechanisms are not known how the peripheral clocks entrain to the circadian pacemaker in the SCN. Cultured fibroblasts treated by high concentration of serum or dexamethasone (Dex) exhibit circadian rhythms in clock gene expression, which is considered as a model system of the peripheral clock. In the present experiment, we constructed a bioluminescent reporter gene Bmal1-LUC which expresses firefly luciferase under the control of clock gene Bmal1, and continuously monitored the luciferase activity from the stable Rat-1 fibroblast cell line expressing Bmal1-LUC. Rat-1 fibroblasts showed a type 0 phase-response curve to Dex pulses. In addition, a similar type 0 phase-response curve was obtained by vehicle pulses. These results suggest that the cellular oscillators in the rat fibroblasts are desynchronized without stimulation and become arrhythmic as a whole system, which can, however, be reset and synchronized to each other by a single stimulus. [Jpn J Physiol 55 Suppl:S200 (2005)]

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When animals drink a taste solution (conditioned stimulus, CS) and are followed by malaise (unconditioned stimulus, US), the animals acquire aversion to the taste solution. This is a kind of fear learning referred to as conditioned taste aversion (CTA). Palatability shifts from ingestive to aversive occur after the acquisition of CTA. Recently, we found that the GABAergic system in the ventral pallidum, a part of reward system, is involved in taste palatability. To elucidate the role of the VP on the expression of CTA, we examined the effects of microinjections of a GABA_A receptor antagonist, bicuculline, on the intake of CS in the retrieval test. Rats received a pairing of palatable saccharin (CS) with an i.p. injection of 0.15 M lithium chloride (US). After this conditioning, vehicle or bicuculline (50, 100 or 200 ng) was bilaterally infused into the VP immediately before the re-exposure to saccharin solution. The microinjections of bicuculline significantly increased the saccharin intake. However, when the rats acquired aversion to innately aversive quinine hydrochloride (QHCl) as the CS, the microinjections of bicuculline had no effects on the intake of QHCl. These results may be explained by our previous finding that the blockade of the GABAergic transmission in the VP increases the intake of sweet solution. [Jpn J Physiol 55 Suppl:S201 (2005)]

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To gain better insights into the mechanisms for estrogen-induced attenuation of food intake, we examined the effect of estradiol replacement in ovariectomized rats on diurnal feeding and hypothalamic c-Fos expression patterns, and on feeding and hypothalamic c-Fos expression responses to 48-h fasting. Seven-week-old rats were ovariectomized and were assigned into estradiol- (E2-T) and placebo-treated (Pla-T) groups. The daily food intake and body weight gain in the E2-T group were significantly less than the Pla-T group. Food intake during both light and dark phases in the Pla-T group was larger than the E2-T group, but the difference between the two groups was more prominent during light phase. In addition, the fractional food intake during light phase was 28% in E2-T rats, and 39% in Pla-T rats. The number of c-Fos ir cells in suprachiasmatic nucleus (SCN) in the Pla-T group was not different from the E2-T group at the beginning of dark phase, but was less than the E2-T group at the beginning of light phase. The number of c-Fos expressing cells at the arcuate nucleus (ARC) in E2-T rats was less than Pla-T rats in both light and dark phases. The feeding response and c-Fos expression at ARC following 48 h fasting were not different between the two groups. These results suggest that 1) estrogen tonically inhibits ARC neuronal activity and feeding, 2) estrogen might play a role in the generation of circadian SCN neuronal activity rhythm and attenuation of feeding during light phase, and 3) estrogen modulates spontaneous feeding but does not influence fasting-induced feeding response. [Jpn J Physiol 55 Suppl:S201 (2005)]

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Cilostazol, an antiplatelet agent, increases cerebral blood flow and can be expected to improve brain function. We investigated effect of Cilostazol on learning disability by transient cerebral ischemia in the mouse. Subjects (14 male C57BL/6 mice) were trained for reacting to illuminated key as soon as possible using operant conditioning technique. After the reaction time became stable, the mice were subjected bilateral common carotid artery occlusion for 10 min followed by reperfusion. From the next day of the operation, a half of the subjects was administrated orally 50 mg/kg Cilostazol with Methyl Cellulose solution and the other half was exposed to the Methyl Cellulose solution as a control group. From a week after the operation, subjects were re-trained and the reaction time and the correct response ratio were compared between these two groups. In the control group, the correct response ratio was decreased sharply at soon after the operation and then recovered quickly. From around 50 days after the operation, temporal decrease of the correct reaction was observed. In the Cilostazol group, although the fall of the correct reaction ratio was steeper than that in the control group, there was no temporal decrease in the chronic phase observed in the control. In the both groups, the reaction time and the correct reaction ratio were not recovered to those of pre-operation level by re-training for three months. From these results we suggested that Cilostazol may diminish the learning disability in the chronic phase. [Jpn J Physiol 55 Suppl:S201 (2005)]

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CCK modulates dopamine release in the nucleus accumbens via the CCK-A receptor (R). The dopaminergic neurotransmission between the ventral tegmental area and the limbic forebrain is a critical neurobiological component of alcohol and drug self-administration. We previously found that the CCK-AR gene -81A/G polymorphism was associated with alcohol dependence (Alco & Alco 2004). In the present study, the role of CCK-AR in ethanol ingestion was examined using CCK-AR gene deficient (KO) mice. The two-bottle choice protocol (ethanol, saccharin, and quinine) was conducted and the righting reflex was examined. The protein level of dopamine 2 receptor (D2R) in the nucleus accumbens as well as dopamine content in the brain were determined. The spontaneous activities were recorded at 1-minute intervals using a biotelemetry system. CCK-ARKO mice consumed more ethanol than wild-type mice, and showed no aversion to high concentrations of ethanol solution. However, the difference was actually in the total fluid consumption and alcohol preference remained unchanged. Ethanol metabolism and dopamine content did not differ significantly between the groups. D2R expression in the nucleus accumbens was significantly higher in CCK-ARKO mice than in wild-type mice. The activity during the night-time was significantly higher in CCK-ARKO mice than in wild-type mice. The difference in voluntary ingestion of ethanol might be attributable in part to different levels of D2R expression in the nucleus accumbens as well as to different levels of spontaneous activities. [Jpn J Physiol 55 Suppl:S201 (2005)]

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Wild-type human tyrosine hydroxylase (TH) type 1 and 4 mutants (del-52, the first 52 amino acid residues deleted; del-157, the first 157 amino acid residues deleted; RR-EE, Arg³⁷-Arg³⁸ replaced by Glu³⁷-Glu³⁸; and S40D, Ser⁴⁰ replaced by Asp⁴⁰) were expressed in AtT-20 mouse neuroendocrine cells in order to clarify how deeply the N-terminus of TH is involved in the efficient production of dopamine (DA) in mammalian cells. The amounts of DA that accumulated in AtT-20 cells expressing these human TH type 1 (hTH1) phenotypes were in the following order: del-52 = del-157 = RR-EE > S40D > wild-type, although the enzyme activities of del-52 and del-157 were lower than those of wild-type, RR-EE, and S40D. The observation on immunoblot analyses that the N-terminus-deleted hTH1 mutants were much more stable than wild-type can reconcile the discrepant results. Computer-assisted analysis of the spatial configuration of hTH1 identified 5 newly recognized PEST motifs, one of which was located in the N-terminus sequence of Met1-Lys12 and predicted that deletion of the N-terminus region would alter the secondary structure within the catalytic domain. We report that the high stability of the N-terminus-deleted hTH1 mutants can be generated by the loss of a PEST motif in their N-termini and the structural change in the catalytic domain, which would promise an efficient production of DA in mammalian cells expressing N-terminus deleted hTH1. [Jpn J Physiol 55 Suppl:S202 (2005)]

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Cytokines and catecholamines are known to constitute a significant portion of the regulatory neuroimmune networks involved in maintaining homeostasis in the central nervous system (CNS). Although we have already reported the increase of norepinephrine (NE) turnover and the upregulation of proinflammatory cytokines within the locus coeruleus after the intraperitoneal injection of lipopolysaccharide (LPS), the relationship between these increase remains unclear. In order to clarify it, we examined the effect of NE on the expression level of proinflammatory cytokines in the primary cultured microglia. Microglia, which are one of the major cell types that produce proinflammatory cytokines in the CNS, were isolated from mouse neonate brains. Simultaneous incubation of microglia with LPS and NE enhanced the expression of IL-1β at both mRNA and protein levels, but reduced the mRNA and protein levels of TNF-α. These data support the hypothesis that NE negatively regulates the expression of proinflammatory cytokine expression, at least in the case of TNF-α, which contributes to the observed anti-inflammatory properties of NE. The investigation to elucidate the regulatory mechanism of cytokine expression in the cells is currently in progress in our laboratory. [Jpn J Physiol 55 Suppl:S202 (2005)]

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Olfactory bulb (OB) is one of the few structures in adult mammalian CNS in which there is a continuous supply of newly generated neurons in subventricular zone. Therefore, the balance between the supply of new cells and apoptosis in OB probably determines olfactory function. In this study, the effect of the propagation of innate immune reaction triggered by peripheral lipopolysaccharide (LPS) on OB of C3H/HeN mice (eight weeks old, male) was examined, because LPS-induced tumor necrosis factor (TNF)-a is known to trigger the apoptotic cascade mediated by TNF/TNF receptor (TNFR) pathway. Within 2 h after the intraperitoneal injection of 50 mg LPS, mRNA expression levels in OB of the genes encoding IkB, TNF-a, and TNFR type 1 were significantly enhanced in the LPS-injected mice. Double immunofluorescence microscopy confirmed that almost all TNF-a-immunopositive cells in OB were in the granule cell layer (GCL) and that they overlapped with those immunostained with antibody against glial fibrillary acidic protein, an antigenic marker of astrocyte. The number of TUNEL-positive cells exclusively identified in GCL was significantly increased in LPS-injected mice sacrificed at 24 h after the injection. These results suggest that peripheral LPS injection may cause a disequilibrium between the supply and the disappearance of the cells in OB, which may lead to olfactory dysfunction. [Jpn J Physiol 55 Suppl:S202 (2005)]

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Pleiotrophin (PTN) and GDNF expressions are enhanced in the dopamine (DA)-depleted striatum, and PTN as well as GDNF specifically promotes the survival of DAergic neurons in vitro. To investigate whether PTN promoted the survival of grafted DAergic neurons and how PTN effected on those cells, ventral mesencephalic donor cells were treated with PTN (100 ng/ml) and/or GDNF (500 ng/ml) during all procedure in cell preparation, and then they (3.0 x 10⁴) were grafted into the striatum of hemi-parkinson model rats. In both PTN-treated and GDNF-treated groups, recovery of motor function (methamphetamine-induced rotation test) was better than non-treated controls. There were more surviving TH-positive cells in the striatum in PTN-treated group (188.4 ± 21.7, n=11) and GDNF-treated group (230.6 ± 18.0, n=9) compared to control (133.9 ± 12.3, n=15). In co-treated (PTN and GDNF) group, more prominent motor recovery and cell survival (273.9 ± 23.6, n=7) were found, showing that effects of PTN and GDNF on grafted DAergic neurons were additive. Activation of caspase-3 during cell preparation was inhibited in PTN-treated group (68.8 ± 7.4% of control, n=3). Even that cells were treated with PTN just before transplantation, recovery of motor function was better in PTN-treatment than non-treated controls. Data suggested that PTN had effect on donor cells for neural transplantation both in the phase of cell preparation and in the phase just after the grafting. [Jpn J Physiol 55 Suppl:S203 (2005)]

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Macrophages and microglia are implicated in spinal cord injury but their precise role is not clear. In the present study, activation of these cells was examined in a spinal cord injury (SCI) model using two different antibodies against ED1 and ionized calcium binding adaptor molecule 1 (Iba1). Male Sprague-Dawley rats were used to prepare the model. After anesthetizing, the animals were fixed on a surgical table and surgery was performed. A 2-mm-diameter metal rod weighing 30g was gently placed centrally onto the dura of the spinal cord on the dorsal side for 10min. Samples were obtained at 1 week, 4 weeks, 8 weeks and 12 weeks after SCI and compared with sham operated controls. For immunohistological examination, ED1 was used as a marker for macrophages and Iba1 was used as a marker for microglia. Immunohistochemical analysis was done by ABC Kit and fluorescent analysis was done by confocal laser scanning microscope. Results showed that activation was observed in both dorsal funiculus and ventral white matter area in spinal cord at 5mm rostral to the epicenter of injury. For both cells, there was a gradual increase in activation from 1 week to 4 weeks followed by down-regulation up to 12 weeks. We could stain macrophages by ED1 and microglia by Iba1. Macrophages may play a role in the phagocytosis of denatured dendrites after SCI, while microglia may have some cooperative functions since they were found scattered near macrophages. [Jpn J Physiol 55 Suppl:S203 (2005)]

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Galectin-1 belongs to the galectin family of lectins, and is expressed in a variety of tissues including placenta, liver, skeletal and smooth muscle. This protein is thought to be a versatile modulator of cell function, and binds lectin only when it is in a reduced form. Once galectin-1 is oxidized, it shows an axonal regeneration-promoting activity though it loses its ability to bind lectin (Inagaki et al., 2000). Although we have demonstrated that the oxidized galectin-1 promotes axonal regeneration via macrophage after peripheral nerve axotomy (Horie et al., 2004), intracellular signaling pathway remains unknown. Therefore, in order to explore the pathway involved in oxidized galectin-1 via receptor, we have performed proteomic analysis of macrophages stimulated with oxidized galectin-1, galectin-1 mutant in which all six cysteine residues were replaced by serine (CS-Gal), or bovine serum albumin (BSA) using 2-dimensional gel electrophoresis. We found that expression levels of some proteins were changed in macrophages exposed to oxidized galectin-1 as compared with BSA. Those proteins were analyzed and identified with LC/MS/MS and the findings suggest to be related with some kinase pathways. Now we are analyzing relationships between these proteins. [Jpn J Physiol 55 Suppl:S203 (2005)]

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We have been developing the methods of measuring in vivo brain metabolism using 2T ¹³C NMR (nuclear magnetic resonance) spectroscopy (¹³C-MRS) research system. These methods make it possible to measure the synthesis rates of metabolites such as glutamate in the hemispheres simultaneously by using the ¹³C, stable isotope, as a tracer. In this study, we have used these newly developed methods to determine the rate of glutamate synthesis in the hemispheres of monkey (Macaca fascicularis crab-eating macaque) brain after transient (60 min) middle cerebral artery occlusion (MCAO). Animals had no behavioral anomaly induced by MCAO throughout this study. ¹³C-MRS measurements were performed for 2 hours at 1, 3, 8, 16 and 80 days after the onset of MCAO with intravenous injection of [1-1¹³C] glucose (1 g/ kg). The amounts of 13C incorporation into glutamate in the ipsilateral region were significantly decreased at 3 and 8 days after the insult as compared with the collateral region (P < 0.05). These data may suggest that MCAO induces metabolic dysfunction involved in mitochondrial abnormality and demonstrate that ¹³C-MRS can provide information that is useful for assessing brain metabolism non-invasively. [Jpn J Physiol 55 Suppl:S203 (2005)]

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Cyclin-dependent serine/threonine protein kinases (CDK) are regulated by activating protein called cyclin. CDKs play physiological roles in regulating cell cycle progression. CDK5, unlike other CDKs, is active only in post-mitotic neurons in the CNS. Recently, CDK5 and its activators are suspected to be a risk factor of Alzheimer's disease (AD). Truncation of p35, a CDK5 activator, into p25 modulates CDK5 activity, and produces an intensive phosphorylation at Ser202 and Thr205 of tau protein. We hypothesize that phosphorylation of these closely-packed sites induces conformation change of tau that allows access of a wider variety of tau kinases to the possible phosphorylation sites located in the assembly domain. The resulting hyperphosphorylation of tau could play a pivotal role in the pathogenesis of AD. If so, an inhibition of CDK5 may effectively rescue neuronal cell death in AD patients brain. We have set off a search for CDK-antagonizing flavonoids using water-dispersible propolis (WDP), and found that WDP inhibited cell-growth of rat C6 glioma cell line at a reasonable concentration. The result also demonstrates that WDP, unlike the ethanol extract of propolis, is suitable for in-vitro experiments. [Jpn J Physiol 55 Suppl:S204 (2005)]

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We have previously demonstrated that the peripheral vasopressin (AVP) system participates in the control of renal sympathetic nerve activity (RSNA), and suppresses Fos-like immunoreactivity (FLI), as a neuronal marker, in the paraventricular nucleus (PVN) of the hypothalamus, via the vasopressin V₁ receptor during central salt loading in conscious rats. The present study examined the effects of endogenous vasopressin on AVP and oxytocin (OT) mRNA production in the PVN and supraoptic nucleus (SON) of the hypothalamus in response to central salt loading. Either vasopressin V₁ receptor antagonist, OPC-21268, or vehicle were administered into lateral ventricle 30 min before the central salt loading (0.67M NaCl at 1 μl/min for 20 min). In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes were used to detect the distribution of AVP and OT mRNA. The PVN was divided into three areas, including the medial magnocellular part (PVM), the lateral magnocellular part (PVL), and the posterior portion (PVPO). In the groups pretreated with OPC-21268, the distribution of the hybridization signal on the x-ray films obtained from AVP and OT probe were decreased in the SON and PVL, and in the SON, respectively. These results suggest, at the cellular level, that central vasopressin stimulates its own synthesis in the PVN and SON, and also affects the synthesis of OT in the SON. [Jpn J Physiol 55 Suppl:S204 (2005)]

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Ca²⁺ regulates numerous cellular functions. The intracellular Ca²⁺ concentration of free Ca²⁺ have been measured by using synthetic fluorescent chelators such as FURA2. The chelators are easily imaged, but they are hard to be targeted to specific intracellular locations, and they gradually leak out of the cell. We report here properties of a fluorescence resonance energy transfer (FRET)-based calcium indicator protein. We designed a tandem fusion of cyan- and yellow- fluorescent protein and N-terminal icosapeptide (palmitoylation signal) of growth-associated protein-43 (GAP43). This fusion protein associates to cell membrane by palmitoylation signal. Two fluorescent protein was combined with the calpain cleavage site of fodrin. Calpain is known as a calcium-dependent protease widely distributed in mammalian cells. The construction of this calpain sensitive fusion protein is the same as Vanderklish et. al reported except for N-terminal signal. They reported that the fusion protein was cleaved by calpain and marked synaptic activity, but our fusion protein was not cleaved by calpain, surprisingly. Purkinje cells from rat cerebellum were transiently infected with virus encoded this protein using Sindbis virus system, and then fluorescent image analysis was performed. The fluorescence signal from this protein was reversible and was repeatedly changed when intracellular Ca²⁺ concentration was altered. Basically the same results were acquired in vivo infection. These properties of this protein is useful to be a Ca²⁺ indicator. [Jpn J Physiol 55 Suppl:S204 (2005)]

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We studied the effect of exposing hippocampal slice cultures to 6 μM bicuculline (BiC) for 6 days in vitro. The slices were prepared from Wistar rat hippocampus at postnatal day (P) 2, 8 or 21, and developmental and regional difference of neuronal vulnerability was investigated. Cellular propidium iodide (PI) uptake and immuno-histochemical staining of activated caspase-3 were used to evaluate neuronal cell damages. BiC induced PI uptake only in CA2-CA3 sectors after 12-24 hours exposure in slice cultures prepared from P8 or P21 rat hippocampus. No PI uptake was observed in slices from P2. In some cases, caspase-3 positive cells co-localized with the PI uptake positive cells. However, cells immunoreactive to c-fos antibody seldom co-localized with PI uptake positive cells. These results suggest that (1) BiC induces neuronal cell death in age- and region-dependent manner. (2) Both necrosis and apoptosis pathways are involved in the neuronal cell death. [Jpn J Physiol 55 Suppl:S204 (2005)]

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We investigated the structural changes in asparagine-linked sugar chains of rat sciatic nerve glycoproteins during aging. High-mannose-type sugar chains were abundant in younger animals, whereas sialylated sugar chains were increased by aging. We previously reported that two sialylated/sulfated oligosaccharides, OIBA1 and OIBA2, increased during maturation. The molecular weights (Mw) of de-sialylated OIBA1 and de-sialylated OIBA2 were the same and so were their retention times on HPLC, indicating that the core structures of OIBA1 and OIBA2 are the same. The common structure of both the OIBA1 and the OIBA2 was determined to be a bi-antennary complex-type oligosaccharide; Galβ1-4GlcNAcβ1-2-Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAc. Sulfate residue linked to the 6-O-GlcNAc on the C3 antenna. Non-sialylated form of OIBAs was also found in the oligosaccharides from nerve tissues. Neuraminidase digestion experiments indicated that OIBA2 is di-sialylated in tandem at the non-reducing terminal of the C6 antenna. Neuraminidases digestion experiments and time-of-flight (TOF) mass spectrometric analyses reveled that there were many complex-type oligosaccharides containing di/oligo sialylic acids in aged rats. [Jpn J Physiol 55 Suppl:S205 (2005)]

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Chemokines and chemokine receptors play important roles in differentiation, migration and localization of immune cells. In this study, we introduce the profile of chemokines and its receptors gene expression induced by lipopolysaccharide (LPS) in rat microglia (MG) using a cDNA microarray. When MG were isolated from the primary mixed glial cultures (MGC) prepared from newborn rats and incubated with LPS for 24 h, the upregulation of CXCL7, CXCL15, and SDF-2 and the downregulation of CCL25, CCL28, and CX3CR1 were detected in response to LPS. When MGC were treated with LPS for 24 h, MG in the MGC changed their expression more divergently than the isolated MG, increasing CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCR5 expression and decreasing CCL25, CCR3 and CX3CR1 expression. These results suggested that the microenvironment surrounding MG, for example the glial network, is an important factor for the expressional regulation of microglial chemokines and its receptors in the immune responses. Treatment of MG in the MGC with LPS for 2 h increased the expression of 13 chemokines and 16 chemokine receptors, suggesting that acute and transient production of chemokines and its receptors in LPS-stimulated MG might be required at the early immune responses. Taken together, these evidences indicated that the divergent expression of chemokines and its receptors in MG might mediate a series of immune reactions induced by microglial activation. [Jpn J Physiol 55 Suppl:S205 (2005)]

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Semaphorins are a family of secreted and membrane-bound proteins, known to control axonal pathfinding. It was recently demonstrated that Semaphorin4A (Sema4A) is crucially involved in immune cell activation. However, the role of Sema4A in the nervous system has not yet been clarified. Our growth cone collapse assay with recombinant Sema4A disclosed that Sema4A induced a significant growth cone collapse in primary hippocampal neurons in a Rho signal-dependent manner. The study also suggested the presence of a receptor on hippocampal neurons that interacts with Sema4A. To identify a high-affinity receptor specific for Sema4A, we firstly prepared molecular probe consisting of the extracellular domain of Sema4A fused to secreted alkaline phosphatase (Sema4A-AP). Then it was examined whether Sema4A-AP could bind as a ligand to COS cells expressing different candidate receptors among plexin family members, by revealing cell-bound AP activity. As a result, Sema4A was found to bind to cells expressing plexin-B1, plexin-B2 or plexin-B3 with high affinity. Sema4A barely bound to cells expressing either plexin-C1 or plexin-D1. Thus the result indicates that Sema4A preferentially binds to members of the plexin-B family, B1-B3. We are currently examining which member of the plexin-B family is functional for Sema4A-induced growth cone collapse in primary hippocampal neurons. [Jpn J Physiol 55 Suppl:S205 (2005)]

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Ghrelin is an gut-brain peptide and its endocrine activities are mediated by GH secretagogue receptor (GHSR)-1a. Des-acyl ghrelin does not activate GHSR-1a and is devoid of endocrine activities. While the microinjection of ghrelin into rat nucleus tractus solitarius (NTS) elicited hypotensive effects, this was not the case upon injection into GHSR-expressing caudal ventrolateral medulla or rostral ventrolateral medulla. To make clear the reason of the discrepancy between receptor distribution and neuronal responses, we examined the cardiovascular response of rats microinjected with des-acyl ghrelin into NTS. Intra-NTS injection of des-acyl ghrelin significantly reduced mean arterial pressure and heart rate (80pmol, -13±2 mmHg and -34±6 bpm, p<0.05). The hypotensive and bradycardic activity evoked by des-acyl ghrelin was not significantly different from that of native ghrelin (80 pmol, -13±2 mmHg and -23±4 bpm). These results suggest that des-acyl ghrelin contribute to the regulation of cardiovascular control and that a receptor other than GHSR-1a exists in NTS. [Jpn J Physiol 55 Suppl:S205 (2005)]

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Neuronal nitric-oxide synthase (nNOS) has been shown to function as signal generators controlled by electrons supplied by NADPH and Ca²⁺-dependent binding of calmodulin (CaM). In the present study, we established that nNOS is directly inhibited through the phosphorylation of Thr1296 in NG108-15 neuronal cells. The treatment of calyculin A or okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, resulted in a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr1296 residue in NG108-15 cells expressing nNOS. Cells expressing a phosphorylation-deficient mutant in which Thr1296 was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation at Thr1296 by introduction of a Thr to Asp mutation resulted in a 40-50% decrease in nNOS enzyme activity and 20-fold increase in the Km for NADPH, with little change in the Kact for CaM relative to the wild-type enzyme. The mutant and Thr1296 -phosphorylated nNOS showed decrease of ADP-agarose binding ability relative to wild-type and un-phosphorylated enzymes, respectively. These data suggests that phosphorylation of nNOS at Thr1296 may involve the attenuation of NO production in neuronal cells through the decrease of NADPH-binding to the enzyme. [Jpn J Physiol 55 Suppl:S206 (2005)]

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It has been suggested that carbohydrate, widely expressed on cell surface, could participate in regulating several cellular functions, inflammation, differentiation, proliferation and adhesion, but the precise mechanisms of carbohydrates on neural stem cell surface remain unknown. In this study, we have analyzed the carbohydrate expression in the embryonic mouse brain and participation in the cell adhesion in vitro neuronal culture system. We have found that positive staining of the binding proteins for Asn-linked complex type carbohydrates in the embryonic mice brain, especially in the subventricular zone and the lateral ganglionic eminence, and that staining was co localized with nestin, marker for neural stem cells.To determine the relation between the adhesions with the complex type carbohydrates, the dissociated brain cells was incubated for 30 min onto the carbohydrate-binding protein-treated well, and attached cells after vigorous wash were analyzed by immunocytochemistry. Interestingly, among several binding proteins for the carbohydrates, complex type-binding protein able to attract embryonic brain cells at greater extent comparing to the other carbohydrate-binding proteins. Vast majority of these attracted cells by carbohydrate express nestin antigen, and form neurosphere after 5 days in culture. These results suggest that complex type carbohydrates could play significant role in cell adhesion, and could be of assistance to prospective separation of neural stem cells from embryonic brain. [Jpn J Physiol 55 Suppl:S206 (2005)]

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It is well known that activation of glutamatergic NMDA receptors in brain areas such as the cerebellum stimulates syntheses of nitric oxide (NO) and cGMP, leading to cooperation in forming synaptic plasticity or promoting neurotransmission. However, existence of such action modes in the anteroventral third ventricular region (AV3V), a pivotal area for autonomic function, is not certain. The aim of this study was to address the issue by experiments in conscious rats. Local infusion of NMDA to the AV3V caused dose-related rises of plasma ADH (VP), osmolality, glucose and arterial pressure (AP). These phenomena were blocked by preadministering its antagonist MK-801 (MK). AV3V injections of the NO substrate L-Arg and a NO donor sodium nitroprusside (SNP) were also potent to provoke those phenomena. The effects of L-Arg and SNP were inhibited, respectively, by the pretreatments with a NO synthase inhibitor L-NAME (NAME) and a NO scavenger hemoglobin. However, plasma osmolality did not increase after the AV3V infusions of the NO-producing drugs, and the infusion of a cGMP analogue did not elevate plasma VP. Moreover, none of the NMDA-evoked responses were not blunted by preapplicating NAME. Furthermore, the VP response to i.v. load of hypertonic NaCl was prevented by the AV3V injection of MK, but not by that of NAME. These results suggest that, despite its potent local actions, NO may not play mediatory or cooperative roles when AV3V NMDA receptors may operate to activate autonomic functions. [Jpn J Physiol 55 Suppl:S206 (2005)]

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The hypothalamic-pituitary-adrenal (HPA) axis plays a pivotal role in the stress responses. It has been established that certain cytokines, particularly interleukin-1β (IL-1β), can potently stimulate HPA activity. Recent studies have shown that 11,12-EET, a metabolite of cytochrome P-450 (CYP-450) of arachidonic acid cascade, attenuates fever induced by IL-1β in the rat. In this study, we investigated the involvement of CYP-450 metabolite in the plasma ACTH responses to IL-1β administration or immobilization stress. Male Wistar rats were subjected to repeated (daily for 3 days) and single immobilization (2h) or IL-1β administration (5μg/kg, ip). Blood samples were collected immediately after their last immobilization or IL-1β administration. CYP-450 inhibitor, econazole (15 mg/kg ip), or their vehicle was injected 30 min before the stress. Single and repeated immobilization stress or IL-1β administration caused marked increase in the plasma ACTH level. Although econazole has no effect on the IL-1β administration groups, the CYP-450 inhibitor suppressed plasma ACTH elevation in the immobilization stressed groups. These results may indicate that CYP-450 metabolite potentiates HPA axis activity and the involved different mechanisms depend on quality of stressors. [Jpn J Physiol 55 Suppl:S207 (2005)]

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Green odor emitted by green leaves is presumed to show refreshing actions on mammals, besides roles of communicating signals between plants. We have demonstrated that green odor attenuates elevation of plasma ACTH level induced by an immobilization stress. Here, we determine whether green odor attenuates elevation of plasma corticosterone (CORT) level and whether these effects are adrenal hormone-mediated, using bilateral adrenalectomized and CORT pellet replaced (ADX + CORT) rats. Male Wistar rats of 8 weeks old were used. In ADX + CORT experiment, rats were adrenalectomized bilaterally and implanted 100mg CORT/cholesterol pellet subcutaneously under pentobarbital anesthesia (50 mg/kg ip). The rats were allowed to recover for more than 10 days before experiment. The day of experiment, rats were exposed to immobilization for 120 min (10:00 to 12:00). Plasma ACTH and CORT responses to the immobilization stress with or without green odor (mixture of 0.03% cis-3-hexenol and 0.03% trans-3 hexenal) were measures at end of and 2 days after the stress. Green odor decreased plasma CORT levels at the end of and 2 days after the stress. In ADX + CORT rats, neither the immediate nor the long-lasting plasma ACTH response was affected by green odor exposure. These results suggest that green odor affects on plasma ACTH and CORT levels via feedback mechanisms of HPA axis. [Jpn J Physiol 55 Suppl:S207 (2005)]

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We asked whether CACNA1C is expressed in parathyroid cells in patients with secondary hyperparathyroidism (SHPTH). Immunohistochemical staining gave positive results for CACNA1C in the parathyroid cells. Parathyroid glands surgically removed from these patients were treated with the digestion buffer to isolate and culture the cells. PTH measurements confirmed secretion of PTH from the cultured cells. Cytoplasmic Ca²⁺ concentration of the fluo-3 AM loaded cells was measured with a modified Nipkow laser scanning confocal microscope system (CSU-21, Yokogawa Electric Company, Japan) attached to an inverted microscope (Olympus IX70). When the fluo-3 loaded cells were depolarized by exposure to 150 mM K⁺ solution, a transient increase in fluo-3 fluorescence was detected only in the presence of extracellular Ca²⁺. The fluo-3 Ca²⁺ transients were found to be sensitive to Ca²⁺ channel agonist, FPL64176 and a Ca²⁺ channel antagonist, nitrendipine. Depolarizing the cells under the whole-cell configuration also evoked inward Ca²⁺ currents, which showed current-voltage relationship similar to that of L-type Ca²⁺ channels (peak current around 0 mV). These results suggest that CACNA1C is expressed in parathyorid cells in patients with SHPTH. [Jpn J Physiol 55 Suppl:S207 (2005)]

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Two primary systems are particularly involved in setting on the physiological stress response, HPA and sympathetic-adrenomedullary (SAM) system. The present study compared the responses of both the HPA and SAM activities to the acute psychological stress between high and low anxious subjects groups. Healthy subjects at the upper (score: >55) and lower (score: <45) limits of the normal range of a trait anxiety scale in the Spielberger’s Trait Anxiety Inventory. The video of corneal surgery was served as the stressor for 15 min. Unstimulated whole saliva was collected every 3 min throughout the session. Salivary cortisol and amylase were assayed as indexes of the HPA and SAM activities, respectively. Salivary β-endorphin was also assayed as a possible index of HPA activity. There were no differences of all the resting salivary parameters levels between high and low anxious groups. As expected, during the stressful video viewing, all the salivary parameters were significantly increased in both the groups. The high anxious group exhibited lower levels of salivary amylase than low anxious group. However, there were no differences of cortisol and β-endorphin levels between the groups. Thus, high anxious subjects exhibited lower HPA hormone release during stressful video viewing than low anxious subjects. Our findings suggest that high trait anxiety may be associated with an inability to respond with sufficient activation of HPA to acute psychological stress. [Jpn J Physiol 55 Suppl:S207 (2005)]

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Estrogen plays critical roles in the regulation of reproductive neuroendocrinology and behavior in female rats, through separate actions on the preoptic area (POA) and ventromedial hypothalamus (VMH). We obtained separate samples of total RNA from the POA and VMH of 6-week old ovariectomized rats, treated with or without estrogen. cDNA microarrays designed for evaluation of estrogen effects were used for the analyses. Genes up-regulated 4 times or more or down-regulated one forth or less were selected based on the microarray profiles. The transcriptional regulation of the selected genes was further examined by RT-PCR and real-time PCR techniques. According to the transcriptional analyses, we focused on four neuronal system-related genes, GABA transporter 2, GABA-A receptor delta, serotonin receptor 6 and cholinergic receptor. The regulation of the selected genes by estrogen was examined at the translational level by Western-blotting and immunohistochemistry. The results showed that the regulation of these genes by estrogen differed between POA and VMH, providing molecular bases for site-specific, differential effect of estrogen on neuroendocrinology and behavior. [Jpn J Physiol 55 Suppl:S208 (2005)]

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Tight glycaemic control in patients with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycaemia mainly target ATP-sensitive K⁺ (K⁺_ATP) channel of pancreatic β-cells to increase insulin secretion. However, these current approaches are often associated with the side effect of hypoglycaemia. In the present study, we showed that inhibition of cyclin-dependent kinase 5 (Cdk5)/p35 activity enhanced insulin secretion under conditions of stimulation by high glucose but not in low glucose in MIN-6 cells and rat pancreatic islets. The role of Cdk5 in regulation of insulin secretion was also confirmed in pancreatic β-cells of p35-deficient mice. The kinase inhibition enhanced the inward whole-cell Ca²⁺ channel current and increased Ca²⁺ influx across the L-type voltage-dependent Ca²⁺ channel (L-VDCC) upon high glucose stimulation in β-cells, but had no effect on Ca²⁺ influx without glucose stimulation. The molecular mechanism of the Cdk5-dependent enhancement of insulin secretion was that Cdk5 phosphorylated loop II-III of L-VDCC at Ser783, and this phosphorylation reduced the binding to SNARE proteins, resulting in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a potential drug target for the control of hyperglycaemia without the side effect of hypoglycaemia. [Jpn J Physiol 55 Suppl:S208 (2005)]

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We sought to examine the physiological functions of the OTR in mediating the neuroendocrine responses following immune challenges. Hypothalamic neuroendocrine responses were characterized in the OTR deficient mice, that we recently generated by genetic engineering, and comparison was made with those in wild type animals. Three groups of mice deficient in OTR (–/–)(groups 1, 2, 3) and 3 groups of wild type counterparts (+/+)(groups 4, 5, 6) were used. Groups 1 and 4 were naive groups, and these mice were decapitated without any challenge. Lipopolysacchride (LPS) at a dose of 10 mg/kg b.w. was injected i.p. into the animals in groups 2 and 5, and after 2 hr, these mice were sacrificed. 0.9% saline was injected instead of LPS in groups 3 and 6. Ten μm serial brain sections were made by cryostat and CRH mRNA and OT mRNA were detected by in situ hybridazation. CRH mRNA increased significantly in the paraventricular nucleus of the hypothalamus (PVN) after LPS injection both in OTR (–/–) and (+/+) group, but no statistical difference was observed between the two genotypes. OT mRNA increased in the caudal lateral division of the PVN in OTR (+/+) animals after LPS injection. However, this increase was not observed in OTR (–/–) animals. Thus, OTR may mediate autonomic activation via OT neurons in the PVN following immune challenge, since OT neurons in this division project to the sympathetic premoter neurons. [Jpn J Physiol 55 Suppl:S208 (2005)]

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Neural stem cells (NSCs) are defined as self-renewing, multipotent progenitor cells that give rise to the diverse types of neurons, astrocytes and oligodendrocytes in the central nervous system. The cell-lineage-specific fate is defined by spatial and temporal gene expression regulated by various transcription factors and intermediary co-regulators designated as coactivators and corepressors. We recently demonstrated that steroid receptor coactivator (SRC-1), which was identified to be nuclear proteins interacting with DNA-bound steroid receptors to potentiate their transcriptional efficiency, was highly expressed in the central nervous system at embryonic stages and the disruption of SRC-1 caused the delayed Purkinje cell development in the mouse cerebellum. To further examine the SRC-1 function in the brain development, we investigated the localization of SRC-1 in mouse NSCs derived from the ganglionic eminence at embryonic day 12.5 and the expression profiles during differentiation of NSCs. As a result, SRC-1 expression was detected in nestin-positive neural stem cells. Following to differentiation, SRC-1 expression was increased in neural lineage cells but not in glial lineage cells. These results raised the possibility that SRC-1 may be involved in gene regulation for neuron specific development from NSCs. [Jpn J Physiol 55 Suppl:S208 (2005)]

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Recently, we have cloned and characterized a coactivator activator (CoAA) using Sos-Ras Yeast Two-Hybrid screening and thyroid hormone receptor (TR)-binding protein (TRBP)-C-terminus as bait. Interestingly, we obtained a shorter splicing variant, named coactivator modulator (CoAM). Both CoAA and CoAM contain RNA-recognition motifs (RRMs) in their N-termini. An In vitro PolyA RNA-protein interacting assay showed a broad specificity of CoAA/CoAM with native RNAs. Transient transfection-based reporter assays showed that the glucocorticoid rceptor (GR)-mediated transcription activated by a series of coactivators were completely repressed by adding CoAM. CoAM suppressed AP-1 -mediated transcription in CV-1 cells, suggesting that CoAM functions as a general repressor. A histone deacethylase (HDAC) inhibitor trichostatin A (TSA) did not affect on the CoAM-mediated transcriptional repression through thyroid hormone response element (TRE) in CV-1 cells, suggesting that the repression by CoAM may not due to recruitment of HDACs. On the other hand, twice as much as CoAM did not fully repressed the CoAA activated transcription. These results suggest that a ratio of CoAM versus CoAA in nucleus might determine the level of transcription. Resent study revealed that CoAA functions not only as a transcriptional coactivator but also as a splicing activator. Taken together, the ratio of CoAM versus CoAA may regulate the level of protein product by controlling gene expression and processing at multiple levels. [Jpn J Physiol 55 Suppl:S209 (2005)]

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In the companion abstract, we showed that cardiac-like L-type Ca²⁺ channel is expressed in hyperparathyroid cells. We then posed the question whether this channel could be activated under physiological conditions. Cytoplasmic Ca²⁺ concentration was measured with a Ca²⁺-sensitive fluorescent indicator probe, fluo-3. The cells were immersed in Hepes-buffered solution containing 1-2 mM Ca²⁺. Local application of the same solution via a small-tipped pipette resulted in no change in the fluo-3 fluorescence, suggesting that stretch-activated Ca²⁺ permeable channels were not activated under our experimental conditions . When Ca²⁺ concentration of the pipette solution was raised to 2.5-3 mM, however, application of the high Ca²⁺ solution for 10 s evoked a transient increase in fluo-3 fluorescence (a fluo-3 transient). In the presence of 10 μM nitrendipine, the fluo-3 transient was markedly reduced. These results could be explained by the hypothesis that increase in extracellular Ca²⁺ concentration induces Ca²⁺ entry, at least in part, through the dihydropyridine-sensitive L-type Ca²⁺ channels. In addition, neither skeletal myotubes nor cardiac myocytes showed fluo-3 transients upon exposure to 10 mM extraccelular Ca²⁺. [Jpn J Physiol 55 Suppl:S209 (2005)]

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There is circumstantial evidence that long-term, but not short-term, exercise affects the function of endocrine systems and increases beta-endorphin levels in both hypothalamus and plasma in rat experimental model. There is, however, little information about the relationship between exercise strength and changes in beta-endorphin levels. Therefore, the present study was carried out to examine the relationship between exercise strength and beta-endorphin levels using rat experimental model.F344 male rats, 6 weeks of age, exercised in treadmill at 5 to 35 m/min. After one hour, hypothalamus was obtained and beta-endorphin levels in water extracts of hypothalamus were measured by ELISA.Exercise at 5 to 25 m/min did not cause changes in beta-endorphin levels: beta-endorphin levels in experimental rats were nearly identical to those observed in controls. On the other hand, exercise at more than 30 m/min affected the endocrine system and significantly increased in beta-endorphin levels as compared with those in control.Since beta-endorphin is recognized to produce or secrete in hypothalamus in response to several types of stress, these results suggest that exercise at higher speed acts upon rat endocrine systems as a stressor. [Jpn J Physiol 55 Suppl:S209 (2005)]

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Dioxins (PCDD, PCDF, coplaner-PCB) are known as the highly toxic environmental chemicals on the growth and maintenance of many organs. On the other hand, thyroid hormone (TH) is important factor for normal brain development. Previous study has reported that TCDD induced an increase in the number of TSH-producing cell without changing TH (T3) level. Recently, we reported that PCB and its metabolites (hydroxylated-PCB: OH-PCB) suppressed TH receptor (TR)-mediated transcriptional activation. Thus, dioxins may also partly affect TH system to disrupt brain development and function. To investigate the effects of dioxins on TR, we performed transient cotransfection experiments. Interestingly, 2,3,7,8-TCDD and PCDFs did not affect on TR-mediated transcription. When we used coplanar-PCBs, (PCB77, PCB126) they suppressed TR-mediated transcription. However, these repressions were weaker than that of OH-PCBs. Furthermore, various effects were observed with OH-PCBs. These results suggest that toxicities of dioxins to TH systems are different among each compound and the effect of TR-mediated transcription of dioxins may not always be related to their toxicities. [Jpn J Physiol 55 Suppl:S209 (2005)]

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The deletion of RORα, a retinoic acid receptor-related orphan nuclear receptor induces a characteristic severe cerebellar ataxia and early death around postnatal 3 weeks in Staggerer(sg) mouse. The vital function of RORα during biological processes is questioned. RORα action is mediated by binding to DNA sequence called ROR response element (RORE) to regulate expression of the target gene. Interestingly, the morphology of Purkinje cell in sg appears almost similar to that of hypothyroid mouse. Thus, we hypothesized that there may be an interaction between RORα and thyroid hormone receptors (TRs). To understand whether mRNA levels of neurotrophins and their receptors in sg mouse are similar to those in hypothyroid mouse or not, we investigated the gene expression patterns in the developing cerebellum of sg mouse by RT-PCR. An evident alteration in expression levels of NGF, BDNF, NT-3 and their receptors TrkA, B, C in cerebellum due to RORα mutation was observed. NGF, TrkA and TrkB showed significantly higher levels in homozygous sg mouse than those in both heterozygote and wild at postnatal 15 days. The expression levels of BDNF and NT-3 were lower at postnatal 15 or 21 days compared to those of wild type animal. Our results indicate that the expression patterns of neurotrophins and their receptors during cerebellar development of both sg mouse and hypothyroid mouse are very much alike. The cross talk between ROR and TR could exist in vivo. [Jpn J Physiol 55 Suppl:S210 (2005)]

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Platelet-activating factor (PAF) is a highly potent stimulator of the secretion of cortisol and corticosterone. We previously reported that PAF-receptor antibody recognized by western blot an about 38 kDa protein of the guinea pig adrenal gland. PAF-receptor immunoreactivity was localized in zona glomerulosa cells of guinea pig and human. Zona fasciculata and reticularis showed no immunoreactivity. In the present study, we studied the role of PAF on aldosterone secretion from perfused adrenal gland and dispersed adrenocortical cells. 1) Adrenal glands of the guinea pig were perfused with Krebs Ringer bicarbonate glucose solution equilibrated with 95%O₂-5%CO₂. The administration of 100nM PAF significantly stimulated the secretion of aldosterone. The concurrent administration of 1nM angiotensin II (ANG II) with 1 nM PAF significantly inhibited the secretion of aldosterone to ANG II. 2) Dispersed adrenocortical cells were prepared using collagenase (10U/ml) and trypsin (10U/ml). Incubation of cells and effectors was carried out with shaking for 90 min under an atmosphere of 95%O₂-5%CO₂. The response to PAF increased significantly at 10 nM. The response to PAF at 100 -1000nM declined as compared with that at 10 nM. When concurrent applied with 1nM ANG II, 0.01-100nM PAF attenuated the ANG II-induced increase in aldosterone secretion. These results implicate that in adrenal glands of the guinea pig, PAF regulates the cortisol and aldosterone secretion via endogenous PAF receptor-mediated mechanism. [Jpn J Physiol 55 Suppl:S210 (2005)]

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The effect of peripheral hypertonic stimulation on the neurons of hypothalamic paraventricular nucleus (PVN) was investigated in the present study with both electrophysiological and immunocytochemical methods. The discharge frequency of the phasic activity neurons in PVN could be increased by intraperitoneal (ip) injection of hypertonic saline (HS, 1.5M NaCl) (from 2.8±0.5 to 5.4±0.9 Hz, P<0.001). The Fos expression in PVN could be enhanced (from 21.2±12.9 to 217.3±38.5 Fos-positive neurons, P<0.001) by ip HS and the majority of AVP-positive neurons expressing Fos (91.7±3.6%) was in magnocellular subdivision of PVN. After intracerebroventricular (icv) injection of losartan, angiotensin II type 1 (AT1) receptor antagonist (5µg/µl), the excitatory effect of peripheral hypertonic stimulation on phasic activity neurons of PVN was inhibited significantly, and the number of the neurons co-expressing Fos and AVP in PVN decreased significantly (P<0.001) as well. The result demonstrated that the vasopressinergic neurons in PVN could be excited by peripheral hypertonic stimulation, and this excitation might be mediated by angiotensin II fibers projecting from periventricular area to PVN. [Jpn J Physiol 55 Suppl:S210 (2005)]

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Adrenomedullin 2 (AM2), a 47-amino-acid neuropeptide, was first isolated from pufferfish and later from mammals. In the present study, we examined the effects of intracerebroventricular (icv) administration of AM2 on neuroendocrine and autonomic nervous functions in rats. Immunohistochemistry for c-fos protein (Fos) revealed that Fos-like immunoreactivity (LI) was observed in various brain areas, including the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the nucleus of the tractus solitarius (NTS) after icv administration of AM2 in conscious rats. Dual immunostaining for oxytocin (OXT)/Fos and arginine vasopressin (AVP)/Fos showed that Fos-LI was mainly observed in OXT-secreting neurons in the PVN and the SON after icv administration of AM2. Plasma concentrations of OXT were significantly increased 5 min after icv administration of AM2 and in a dose-related after icv administration of AM2. The mean arterial pressure and heart rate were increased potently for an extended period in a dose-related manner, and the concentrations of plasma adrenaline and noradrenaline were significantly increased after icv administration of AM2 in urethane-anesthetized rats. These results suggest that centrally administered AM2 activates OXT-secreting neurons in the PVN and the SON predominantly with elevation of OXT level in plasma, and causes an increase of sympathetic outflow. [Jpn J Physiol 55 Suppl:S210 (2005)]

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It has been reported that the constitutive nitric oxide synthase located in all kinds of endocrine cells in the rat islet of Langerhans (Alm, et al., 1999). We directly visualized NO production in A- and B-cells of the rat (Ishikawa, et al., 2004). In the present study, we attempted to demonstrate the production of NO in the pancreatic polypeptide-producing cells (PP-cells) of rat pancreatic islets. Cells of the pancreas were dispersed by the collagenase method. The islets were collected and superfused with Krebs solution under the fluorescence microscope. After loading with diaminofluorescien-2 diacetate (DAF-2 DA), a bright green fluorescence indicating production of NO was developed within a large number of islet cells. The islet was then fixed and stained by immunohistochemistry for PP. The stained islets were observed with a confocal laser microscope (FV500, Olympus). The green fluorescence was observed in most of PP-immunoreactive cells in the islet. The result demonstrates that NO is spontaneously produced in PP-cells of the islet, suggesting that NO modulates the secretion of PP. [Jpn J Physiol 55 Suppl:S211 (2005)]

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Prolactin-releasing peptide (PrRP) is known to play a role in the function of the hypothalamic-pituitary-adrenal axis. To analyze the function of PrRP as a stress mediator, we studied the effects of acute inflammation and nociceptive stimulus on the PrRP gene expression in the rat brain, using in situ hybridization histochemistry. Adult male rats (200-250 g) were used in all experiments. As an acute inflammatory stress, rats were intraperitoneally (ip) injected with lipopolysaccharide (LPS) from E. coli at a dose of 5 mg/kg body weight. LPS was dissolved in pyrogen-free 0.9% saline with 1 mg/ml. Control rats were ip injected with 1 ml of pyrogen-free 0.9% saline. All rats were decapitated at 3, 6, 12, 24 and 48 hr after ip injection. As a nociceptive stress, rats were subcutaneously (sc) injected with 100μl of 5% formalin in 0.9% saline into the dorsal surface of each hind paw. Control rats were sc injected with 100μl of 0.9% saline. All rats were decapitated at 3, 6, 12 and 24 hr after sc injection. After an acute inflammatory stress, PrRP mRNA levels in the nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM) were significantly increased. After a nociceptive stress, PrRP mRNA levels in the VLM were significantly increased. These results suggest that acute inflammation and nociceptive stimulus may be potent stimulants to up-regulate the PrRP gene expression in the rat brain. [Jpn J Physiol 55 Suppl:S211 (2005)]

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We have generated transgenic (Tg) rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and the suprachiasmatic nucleus (SCN) in the hypothalamus of Tg rats. In this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine Na⁺ concentration, and body weight after drinking 2% saline for 5 days in Tg rats. There were no difference between Tg rats and Tg(-) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine Na⁺ concentration, and body weight after salt loading. Salt loading for 5 days caused marked increase of eGFP fluorescence in the SON, the PVN and hypothalamo-neurohypophyseal tract but not the SCN. These results suggest that the expression of AVP-eGFP transgene does not disturb water balance in Tg rats and may be up-regulated by salt loading. [Jpn J Physiol 55 Suppl:S211 (2005)]

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Neural substances mediating the negative feedback effects of estrogen on LHRH release in the stalk-median eminence (S-ME) were studied using the in vivo push-pull perfusion method in female rhesus monkeys. Twelve ovariectomized animals were implanted with Silastic capsules containing 17β-estradiol two weeks prior to the experiments and on the day of the experiment estradiol benzoate (EB, 50 μg/kg, sc) or oil was injected. Perfusate samples from the S-ME were collected in 10-min fractions from 4 h before to 18-20 h after EB or oil. LHRH and neuropeptide Y (NPY) levels in the same perfusates were measured by RIA, and glutamate and γ-amino-butyric acid (GABA), also in the same perfusates, were assessed by HPLC. The results indicate that EB suppressed LHRH release (p<0.005) starting within 2 h after EB, and continued until the end of the experiment. Pulse analysis suggested that estrogen suppressed the pulse amplitude, but not pulse frequency, of LHRH release. In contrast, EB did not alter any parameters (mean release, pulse amplitude or frequency) of pulsatile NPY release throughout the experiment. HPLC analysis further suggested that neither glutamate nor GABA levels in the S-ME were changed with estrogen-induced LHRH suppression. Oil treatment did not alter LHRH, NPY, GABA or glutamate levels. In conclusion, estrogen induces suppression of pulsatile LHRH release within 2 h, but negative feedback effect of estrogen does not appear to be mediated by NPY, GABAergic, or glutamatergic neurons, at least, at the level of S-ME. [Jpn J Physiol 55 Suppl:S211 (2005)]

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We have generated transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of estrogen receptor α (ERα) gene promoter 0/B. In the adults of this trait, fluorescence of EGFP was observed in the brain, particularly in the preoptic area (POA)-bed nucleus of the stria terminalis (BNST), amygdala, midbrain central gray, hippocampus and cortex. In the POA-BNST and the medial amygdala, 70% of EGFP-labeled cells were ERα immunoreactive suggesting the usefulness of the transgenic rats for physiological studies on estrogen signaling in the adult brain. Here we report the developmental expression of EGFP during fetal and neonatal life. Sporadic EGFP fluorescence were detected on embryonic day 15; the expression gradually increased and the adult pattern of distribution was established in early postnatal period. ERα/EGFP double-labeled cells were more numerous in the neonates than adults. Only in the neonates, double-labeled cells were detected in the midline thalamus. EGFP labels were observed clearly in fibers in the fornix and anterior commissure, and surrounded the ventromedial hypothalamic nucleus and suprachiasmatic nucleus without penetrating into the nuclear core. EGFP was also expressed in the sexually dimorphic nucleus of the POA. These results suggest that the action of ERα gene promoter 0/B is regulated developmentally in the rat brain. [Jpn J Physiol 55 Suppl:S212 (2005)]

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The anterior pituitary gland contains five types of endocrine cells. The mixed constitution sometimes obstructs the direct analysis on a single population. Although pituitary cell lines that consist of a homogenous cell population have been extensively used, these cell lines are differently regulated from normal pituitary cells. Therefore, for the physiological and biochemical analysis in a single population of pituitary cells, it is necessary to identify and collect specific endocrine cells from a mixed population. In this study, we generated transgenic (TG) rats expressing the enhanced green fluorescence protein (EGFP) gene under the control of a rat prolactin promoter region of about 3.3 kb. We obtained 13 founders that carried EGFP gene as detected by PCR and established an appropriate line of TG rats, MI-11. About 57% of anterior pituitary cells of the TG rats expressed EGFP with markedly varying amounts while 54% of the pituitary cells were lactotrophs, indicating additional EGFP expression in the cells other than lactotrophs. Indeed, the fluorescence of EGFP was detected partly in somatotrophs and S-100-immunoreactive folliculostellate cells. However, because pituitary cells expressing great amounts of EGFP were exclusively lactotrophs, cell sorting successfully collected EGFP expressing cell-enriched populations that contained lactotrophs up to 90%. This enrichment system provides a useful model for analysis based on the single population of lactotrophs. [Jpn J Physiol 55 Suppl:S212 (2005)]

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Compensatory hyperinsulinemia associated with peripheral insulin resistance is a well-recognized metabolic disturbance. However, the mechanism underlying the induction of hyperinsulinemia is unclear. Resistin, a small cysteine-rich protein secreted from adipocytes, is one of the major risk factor of insulin resistance in obesity. The present study employed adenovirus-mediated resistin gene transfer in muscle of the ICR male mice and examined whether acute increases in circulating resistin could cause hyperinsulinemia. In addition, we examined the effect of resistin on pancreatic β-cell function in vitro. Mice expressing resisitin showed increased plasma level of resistin by 2-5 fold, decreased insulin sensitivity and impaired glucose tolerance, accompanied with hyperinsulinemia. In the liver and muscle of resisitin-expressing mice, insulin-induced phosphorylation of Akt was suppressed. Resistin increased 8.3 mM glucose-induced insulin secretion in mice pancreatic islets. Resistin also enhanced glucose-induced cytosolic Ca²⁺ oscillations in islets, whereas the enhancement was not observed in single β-cells. In addition, resistin inhibited insulin-induced phosphorylation of Akt in islets. These results indicate that over-expression of resistin causes insulin resistance in pancreatic islets, as well as liver and muscle, which may attenuate the autocrine negative-feedback action of insulin on b-cells and thereby enhances insulin secretion. [Jpn J Physiol 55 Suppl:S212 (2005)]

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During lactation the suckling stimulus exerts profound influences on the structures and functions of the central nervous system of nursing rats. We have examined the acute effect of pup removal on estrogen-induced LH surge. Lactating and nonlactaing, cycling female rats were given an estradiol-containing capsule after ovariectomy and were bled with an indwelling cather for serum LH determinations. In ovariectomized, lactating rats with pups, estrogen treatment induced an afternoon LH surge with an amplitude and timing comparable to those seen in nonlactating rats. Removal of pups from the lactating rats at 0900, 1100, or 1300 h, but not at 1500 h, abolished the estrogen-induced surge that would occur in the following afternoon. The inhibitory effect of pup removal at 0900 h was reversed when the animals were resuckled by 1400 h. However, pup removal was ineffective in abolishing the enhancing effects of progesterone at low and high doses on estrogen-induced LH surge. Double immunohistochemical staining for gonadotropin-releasing hormone (GnRH) and c-Fos, a marker for neuronal activation, revealed a concomitant decrease in the number of c-Fos-immunoreactive GnRH neurons in the preoptic region with the blockade of LH surges in pup-removed lactating rats.These results suggest that neural mechanism for the GnRH surge generator is altered during lactation, and that GnRH neurons require an additional stimulus that is not involved in the surge in nonlactating, cycling female rats. [Jpn J Physiol 55 Suppl:S212 (2005)]

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We recently showed that the expression of progesterone receptor (PR) mRNA in the neocortex and hippocampus was significantly affected by estrogen in adult ovariectomized rats. To gain a better understanding of sex differences in brain functions, we examined whether the expression of PR mRNA in these areas of male rats was affected by estrogen or not. Adult orchiectomiaed (ORX) rats were given a subcutaneous injection of 200 μl of sesame oil containing 10 μg or 100 μg of 17β-estradiol and were killed 6, 12, and 24 h after the injection. Some rats were also killed after injection of sesame oil alone as control. At each time point, the frontal cortex, occipital cortex, temporal cortex, and hippocampus were collected, and the expression of PR mRNA in each brain area was measured by Northern blot. The results showed that estrogen had only minor effects on the expression of PR mRNA in the neocortex and the hippocampus of ORX rats; 100 μg of 17β-estradiol slightly but significantly increased the expression of PR mRNA in the frontal cortex and hippocampus, but had no significant effect in the occipital cortex and temporal cortex. Ten μg of 17β-estradiol had no significant effect on the expression of PR mRNA in areas we examined. Together with results in ovariectomized rats that either 10 μg or 100 μg of 17β-estradiol increased the expression of PR mRNA in the frontal cortex and hippocampus, we suggest that estrogen stimulates the frontal cortex and hippocampus by increasing the expression of PR mRNA only in female rats but not in male rats. [Jpn J Physiol 55 Suppl:S213 (2005)]

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