[Background] We have previously shown that allograft-induced macrophages (AIM) in C57BL/6 (H-2D^bK^b) mice were essential for allograft (e.g., BALB/c skin and Meth A tumor; H-2D^dK^d) rejection. Recently, we reported the isolation of two cDNAs, which encoded novel receptors on AIM for allogeneic MHCs (H-2D^d and H-2K^d), respectively, by using anti-AIM monoclonal antibodies (mAbs; R15 and R12) and allogeneic MHC tetramers (H-2D^d and H-2K^d). We named these two receptors macrophage MHC receptor (MMR) 1 and MMR2, respectively. In the present study, we explored the percentages of MMR1⁺ or MMR2⁺ AIM by staining with R15 or R12 mAb and investigated the homologues of MMR1 and MMR2 proteins in human and other species. [Results] There were no AIM expressing both MMR1 and MMR2. The R15⁺MMR1⁺ and R12⁺MMR2⁺ AIM were ≈76% and ≈8%, respectively. Human homologues of MMR1 (95% homologous) and MMR2 (71% homologous) were found by a search for sequence homology (BLAST http://blast.genome.ad.jp/). Also, the MMR1 and MMR2 protein sequences were considerably conserved in other animal species such as chicken, blowfish, xenopus, C.elegans, zebrafish and drosophila. [Conclusion] Most of AIM expressed H-2D^d receptors, whereas H-2K^d receptor-positive cells were the minor population of AIM. Homologues of MMR1 and MMR2 were seen in human and other animal species, implying that the allorecognition systems by MMR1 and MMR2 on AIM might be conserved among various species of animals. [J Physiol Sci. 2007;57 Suppl:S127]

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An increases in extracellular K⁺ concentration ([K⁺]_o) inhibits Ca²⁺ entry in rat submandibular acinar cells. The high [K⁺]_o induced inhibition of TG-stimulated Ca²⁺ entry is thought to be induced by decreases in the driving force for Ca²⁺ entry. In the present study, we examined the effects of increasing [K⁺]_o on Ca²⁺ entry in TG-stimulated and ionomycin (IM)-stimulated rat submandibular acinar cells. Cells were first perfused with Ca²⁺-free control solution and were stimulated with TG (2 µM) for 10 min. Then Ca²⁺ (1.5 mM) was added (reintroduction of Ca²⁺). The introduction of Ca²⁺ increased [Ca²⁺]_i. Increases in [Ca²⁺]_i induced by the reintroduction of Ca²⁺ were examined in TG-stimulated submandibular acinar cells perfused with various [K⁺]_o. As increment of [K⁺]_o, [Ca²⁺]_i increases induced by the reintroduction of Ca²⁺ were suppressed. Similar experiments were performed using IM (5 µM). In IM-treated cells, the introduction of Ca²⁺ also increased [Ca²⁺]_i. However, as increment of [K⁺]_o, [Ca²⁺]_i increases induced by the reintroduction of Ca²⁺ were not suppressed. IM-induced [Ca²⁺]_i increases were also inhibited by Gd³⁺ (1 µM). The final [Ca²⁺]_is were similar in various [K⁺]_o, although the rate of [Ca²⁺]_i increase in the high K⁺ solution was slow compared with that in the control solution. The present study demonstrated that the driving force for Ca²⁺ entry is sufficient in the high K⁺ solution in rat submandibular acinar cells. A high K⁺ solution which depolarizes membrane potential appears to modulate Ca²⁺ entry pathways in rat submandibular acinar cells. [J Physiol Sci. 2007;57 Suppl:S128]

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Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood.We investigated whether CXCL12 regulate invasion into type I collagen and degradate of type I collagen. CXCL12 enhanced the invasion into type I collagen on NK cells. GM6001, a brode synthetic MMP inhibitor, significantly inhibited CXCL12-stimulated enhanced invasion. Furthermore, TIMP-2, recombinant tissue inhibitor of metalloproteinases, also significantly inhibited CXCL12-stimulated invasion into type I collagen. Fresh NK cells were incubated in the presence or absence of CXCL12 and plated on the DQ-collagen I, intramolecular-quenched fluorescent protein substrate, coated plates. CXCL12 stimulated cells significantly enhanced degradation of type I collagen on NK cells. This enhanced degradation was significantly inhibited by GM6001 or TIMP-2, although there was residual degradation of type I collagen.These results indicate that MMPs play a key role in CXCL12-stimulated NK cell invasion into type I collagen, and CXCL12 enhanced NK cell surface proteolysis of type I collagen in an MMP-dependent manner, thereby promoting NK cell invasion into the matrix. [J Physiol Sci. 2007;57 Suppl:S128]

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The most straightforward approach to the amplitude of cross-bridge powerstroke in muscle is to record ATP-induced cross-bridge movement in living thick filaments, using the gas environmental chamber(Sugi et al. PNAS 94:4378-4382,1997). To determine the direction of cross-bridge movement with respect to the center of the thick filament, large bipolar thick filaments are prepared by slowly polymerizing rabbit skeletal muscle myosin. Gold particles are attached to the cross-bridges to serve as position markers. Filament images are recorded on the imaging plate under a magnification of 7,000-10,000 diameters. Position of the gold particles does not change appreciably with time, indicating that the time-averaged cross-bridge position is constant. Oniontophoretic ATP application, the cross-bridges are found to move by 7-8nm parallel to the filament axis. [J Physiol Sci. 2007;57 Suppl:S128]

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Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) of rat ventricular myocytes were measured with the fluorescent indicator furaptra (mag-fura 2). To estimate the rates of Mg²⁺ efflux by the Na⁺/Mg²⁺ exchange in deenergized cells, we treated the cells with a metabolic inhibitor in the Na⁺ and Ca²⁺-free conditions, and analyzed the initial rate of decrease in [Mg²⁺]_i upon introduction of extracellular Na⁺ at 25°C. Treatment of the cells with 1 μ M FCCP, an uncoupler of mitrochondria, significantly increased [Mg²⁺]_i for 10 min, on average, from 0.94 mM to 2.47 mM (probably due to breakdown of ATP and release of Mg²⁺ from Mg²⁺ATP) and shortened the cell length (probably due to formation of rigor crossbridges). Similar effects were observed after application of 5 mM KCN for 60-90 min. The Mg²⁺ efflux rate was largely reduced, by 78±5.1% (n=6) or 47±15.9% (n=4) in FCCP- or KCN-treated cells, respectively. The slowed Mg²⁺ efflux is unlikely attributed to intracellular acidosis caused by metabolic inhibition, because equilibration of intracellular and extracellular pH at 7.15 by addition of 10 μM nigericin, a proton ionophore, did not reverse the inhibition of the Mg²⁺ efflux. In FCCP-treated cells, the decrease in [Mg²⁺]_i was initially slow (above), but was often markedly accelerated in later periods after variable latency. As the late decrease in [Mg²⁺]_i was observed even in the absence of extracellular Na⁺, this late component seems unrelated to Mg²⁺ efflux by Na⁺/Mg²⁺ exchange. We conclude that Na⁺/Mg²⁺ exchange was largely inhibited by metabolic inhibition, presumably due to depletion of intracellular ATP. [J Physiol Sci. 2007;57 Suppl:S128]

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We tested the effects of exposure to a time-varying magnetic field on functions of cytoskeletal protein in bovine adrenal chromaffin cells. Transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) stimulated by acetylcholine (ACh) in Ca²⁺-free medium was inhibited by 2 hr-exposure to the magnetic field. The exposure also affected oxygen consumption, mitochondrial membrane potential and actin protein. Adrenal chromaffin cells exposed to the magnetic field showed a regulatory volume decrease (RVD) after replacement to hypotonic medium, and similar volume regulation was also seen in 2 hr-exposed cells. But, in exposed cells, the cell volume was increased at a slower rate and reached a peak of higher value than control, and recovery to the original value was also delayed. It is suggested that this effect by the exposure on the volume regulation is caused as a result of changing the content or structure of F-actin in chromaffin cells. [J Physiol Sci. 2007;57 Suppl:S129]

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Myristoylated alanine-rich C kinase substrate (MARCKS) is a major cellular substrate for protein kinase C (PKC). Involvement of phosphorylation of MARCKS in secretory function has been reported in pancreatic islets and chromaffin cells. In rat parotid acinar cells, the activation of β-adrenergic receptor provokes exocytotic amylase release. We investigated the involvement of phosphorylation of MARCKS in β-adrenergic agonist-induced amylase release in rat parotid acinar cells. Rat parotid acinar cells were prepared using trypsin and collagenase. Membrane and cytosolic fractions were isolated by ultracentrifugation. Secretory granules were purified using Percoll gradient. Protein expression was determined by western blotting using anti-MARCKS and anti-phosphorylated MARCKS (p-MARCKS) antibodies. Amylase activity in the medium was measured by Bernfeld's methods. In western blotting analysis, MARCKS and p-MARCKS were detected in membrane and cytosolic fractions and secretory granule membrane. The β-adrenergic agonist isoproterenol (IPR) stimulated phosphorylation of MARCKS time-dependently. This agonist had no effect on the total amount of MARCKS. Myristoylated PKC peptide inhibitor partially inhibited IPR-induced phosphorylation of MARCKS and amylase release. These results suggest that phosphorylation of MARCKS is involved in regulation of amylase release in rat parotid acinar cells. [J Physiol Sci. 2007;57 Suppl:S129]

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It is well-known that the oxidative stress activates melanogenesis which relates with DNA damage. On the other hand, it was reported that tyrosinase, the enzyme that controls the synthesis of melanin, was regulated by the extra-cellular glucose level. First we culture B16 cells in high/low glucose conditions and confirmed that the melanogenesis was suppressed in low-glucose conditions. Then we compare this little-known melanogenesis induced by low-glucose conditions and that by the oxidative stress. Peroxide, typical oxidizing agent, increased melanosome production, and broke mtDNA detected with LA-PCR. On the other hand, B16 cells cultured in the low-glucose medium showed the melanogenesis without mtDNA fragmentation. Expression of tyrosinase, TRP1 and TRP2 were measured by real-time RT-PCR. Since glucose enters into cells across glucose transporters (GLUTs), we analyzed the expression of them. Anti-oxidants can suppress melanogenesis caused by peroxide, on the other hand some anti-oxidants failed to suppress the melanogenesis in low glucose conditions. From these results, we suggest that melanogenesis in low glucose conditions was enhanced by the different mechanism from that by oxidative stress. [J Physiol Sci. 2007;57 Suppl:S129]

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UV irradiation is known to induces necrotic cell death in the acute phase, while the mechanisms of the cell death is not completely understood. On the other hand we have investigated the mechanisms of cell death caused by the membrane permeability increase. Here we compare these two types of cell death. As the experimental model, we used human corneal epithelial cells (HCEC) and applied UV irradiation or membrane-pore forming reagent, amphotericin B (amB). We observed the fluorescence increase in the nucleus by the UV irradiation in the presence of extracellular DAPI under the fluorescent microscope. For the amB experiments, we used propidium iodide for the fluorescent indicator. The fluorescence increase by the UV irradiation was suppressed by the anti-oxidant reagents. On the other hand, extracellular low Cl⁻- conditions, which blocked amB-induced cell death, did not suppress UV-induced cell death. From these results, we conclude that the mechanism of the UV-induced cell death is different form the amB-induced cell death. [J Physiol Sci. 2007;57 Suppl:S129]

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Experiments were performed in a newly established mouse macula densa cell line (NE-MD) (Jpn J Physiol, 2005) to investigate whether metabolic acidosis may modulate the nNOS protein expression and activity. In the present study, NE-MD cells were incubated in the presence of furosemide (12 μM) for 2 hours with or without amiloride (or its analogue), an inhibitor of the Na⁺/H⁺ exchangers. The nNOS protein expression was analysed by Western blotting. L-arginine (Arg)-induced NO generation was measured by using an NO-sensitive electrode. Intracellular pH (pH_i) of NE-MD cells was monitored by the BCECF assay. We found that both L-Arg-induced NO generation and furosemide-induced nNOS protein expression were significantly decreased by EIPA. Further, L-Arg-induced NO generation was significantly higher when NE-MD cells were incubated with low (1/10) Cl⁻ solution, but not with low (1/10) Na⁺ solution. Moreover, L-Arg-induced NO generation was significantly lower at pH=7.1, compared with control (pH7.4). These results indicate that nNOS protein expression and activity may be sensitive to an acidic condition. In conclusion, low [Cl⁻] and acidic urine may increase and decrease, respectively, NO generation in NE-MD cells. [J Physiol Sci. 2007;57 Suppl:S130]

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We investigated the influence of reactive oxygen species and free radicals on axonal transport in cultured mouse dorsal root ganglion neurons. Video-microscopy was used for observation of movement of organelles. The nitric oxide (NO) donor NOC18 (100 μM) and the hydrogen peroxide (H₂O₂) donor tert-butyl hydroperoxide (100 μM) significantly decreased anterograde and retrograde axonal transport of organelles. High concentration (1 mM) of tert-butyl hydroperoxide immediately induced swelling of mitochondria and cell death. The superoxide dismutase (SOD) inhibitor diethyldithio carbamate (100 μM) significantly decreased anterograde and retrograde axonal transport. High concentration (10 mM) of diethyl carbamate induced swelling of growth cones and vacuole formation in the cell body. The singlet oxygen scavengers L-ascorbic acid (25 mM) and L-histidine (10 mM) and the NO scavenger PTIO (30 μM) slightly but significantly increased axonal transport. In contrast, the hydroxyl radical scavenger dimethyl thioirea (5 mM) had no effect. These results indicate that exogenous and endogenous reactive oxygen species and free radicals impair axonal transport. This may lead to irreversible axonal degeneration and neuronal cell death, which are observed in neurodegenerative diseases. [J Physiol Sci. 2007;57 Suppl:S130]

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We have previously shown that amyloid β-protein (Aβ) inhibits axonal transport via actin aggregation. In the present study, we determined the effects of angiotensin II (Ang II) on axonal transport and whether Ang II enhances the effects of Aβ. Axonal transport of neurites in cultured rat hippocampal neurons was investigated using video-microscopy. Application of Ang II decreased axonal transport of organelles. We also found that Ang II reduced the neurite diameter. These effects were mimicked by the angiotensin AT1 receptor agonist L-162,313. Aggregation of actin filaments were frequently observed in Ang II-treated neurons, and the effects of Ang II were blocked by the actin filament depolymerizer latrunculin B. These effects of Ang II were similar to those of Aβ, and the impairment of axonal transport by Aβ was enhanced by Ang II. The present in vitro study suggests that the neurotoxicity of Aβ is enhanced by Ang II through intracellular actin transformation. Angiotensin II may be involved in the progression of Alzheimer's disease. [J Physiol Sci. 2007;57 Suppl:S130]

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The purpose of this study was to examine whether Na⁺-K⁺ ATPase, MR, and major renal Na⁺ transporters expressed in NE-MD cells. It has been proposed that instead of Na⁺-K⁺ ATPase, apical colonic H⁺-K⁺ ATPase in concert with Na⁺-H⁺ exchanger (NHE2) may account for Na⁺ exit from MD cells (Peti-Peterdi et al, 2002). In the present study, expression of Na⁺-K⁺ ATPase subunits and related genes was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in newly established NE-MD cells (Yasuoka et al, 2005). Alpha₁,₃, beta₁,₃ subunits expression was positive, but Alpha₂, beta₂, and gamma (a,b,c) subunits expression was negative. Further, NHE1,2 expression was positive, whereas NHE3,4 expression was negative. MR and GR expression was positive. Moreover, colonic H⁺-K⁺ ATPase was positive, but gastric H⁺-K⁺ ATPase was negative. These data suggest that although colonic H⁺-K⁺ ATPase and NHE2 mRNAs were expressed, Na⁺-K⁺ ATPase may regulate cytosolic [Na⁺] to create a driving force for NaCl entry at the apical membrane in MD cells, and that the gamma subunit may not be essential for functional Na⁺-K⁺ ATPase in the MD. [J Physiol Sci. 2007;57 Suppl:S131]

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We analyzed a number of 2-DE protein spots in Long-Evans Cinnamon (LEC) rats as an animal model for human Wilson's disease using matrix-assisted laser desorption/ionization (MALDI)-time of flight mass spectrometry (TOF-MS). Livers from LEC and WKAH/Hkm (control) rats of 12 and 16 weeks of age were separately homogenized and performed with 2-DE. Protein spots of 2-DE were analyzed by using image analysis software to compare the changes in protein quantities. Seventeen protein spots were found to be different between the two strains of 12 and 16 weeks of age. In detail, four protein spots increased by 1.5 times in LEC rats, compared with control. The two protein spots were determined as fructose-1-6-bisphosphatase and argininosuccinate-synthase. On the other hand, 12 protein spots decreased by 2 times (n=8) and by 1.5 times (n=4) in LEC rats. All identified proteins (10 of 12) were belonging to a group of superoxide dismutase (SOD), such as catalase, glutathione-transferase, peroxisome. Interestingly,one protein spot of 35 kDa was completely disappeared in LEC rats of 4-16 weeks of age, whereas it was positive in control. Finally, increase and decrease patterns of the 17 protein spots identified in 12 and 16 weeks LEC rats were similarly observed in the younger LEC rats of 4, 8 and 10 weeks of age. These results suggest that decrease in the expression of SOD proteins may be related with a cause and/or progress of Wilson's disease. [J Physiol Sci. 2007;57 Suppl:S131]

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In brown adipocytes, α1 adrenoceptor activation elicits IP3-induced Ca²⁺ release and store-operated Ca²⁺ entry (SOC) from the ER, while β3-receptor activity activates lipolysis and uncoupling proteins in mitochondria, producing heat. Uncoupling of mitochondrial respiration by FCCP activates mitochondrial Ca²⁺ release, non-SOC Ca²⁺ entry, Ca²⁺ release from the ER and SOC. How this mitochondria-ER-plasmalemma Ca²⁺ coupling is regulated by an endogenous activator, we have measured Ca²⁺ in the ER ([Ca²⁺]ER) using cameleon expressed in the ER as well as intracellular Ca²⁺ ([Ca²⁺]i) and mitochondrial potential. The first phase of isoprotelenol-induced rises in [Ca²⁺]i was accompanied by mitochondrial depolarization. The second phase was paralleled by mitochondrial repolarization, accompanied by a rise in intracellular Mg2+ and partially blocked by removing external Ca²⁺ and fully by oligomycin. The second and third phases were blocked by removing external Ca²⁺ and U73122. FCCP as well as cyclopiazonic acid (CPA) transiently reduced [Ca²⁺]ER. Likewise, isoprotelenol decreased [Ca²⁺]ER for a period much longer than those by FCCP and CPA. Thus, β3-receptor activation causes uncoupling of mitochondrial respiration that depolarizes the mitochondrial membrane, releases Ca²⁺, activates uncoupling-associated non-SOC Ca²⁺ entry and activates Ca²⁺ release from the ER via activation of phospholipase C and subsequently SOC. [J Physiol Sci. 2007;57 Suppl:S131]

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Brown adipose tissue is a thermogenic organ and plays important roles in maintenance of energy balance and body temperature. α1 adrenoceptor activation elicits IP3-induced Ca²⁺ release and store-operated Ca²⁺ entry (SOC) from the ER, while β3-receptor activity activates lipolysis and uncoupling proteins in mitochondria, producing heat. Uncoupling of mitochondrial respiration by FCCP activates mitochondrial Ca²⁺ release, non- SOC Ca²⁺ entry, Ca²⁺ release from the ER and SOC. We have studied how bile acid intake prevents obesity in high fat-fed mice. Changes in intracellular Ca²⁺ ([Ca²⁺]i), mitochondrial membrane potential and heat production were recorded by fluorometry and thermography. Mice were fed with high fat diet, high fat diet with cholic acids and chow for 7-9 weeks. Cholic acids diets prevented increases in body weight, white adipose tissue and whitish changes in brown adipose tissue caused by high fat diet. Cholic acids-fed mice showed greater FCCP-, noradrenaline- and isoprenaline-induced rises in [Ca²⁺]i in brown adipocytes than high fat diet- or chow-fed mice and produced smaller mitochondrial depolarization accompanying the initial rise in [Ca²⁺]i and greater heat production from brown adipose tissues. Thus, cholic acid intake prevents high fat diet-induced obesity by increasing energy expenditure as the result of increased responses of brown adipose tissues to sympathetic nerve activity. [J Physiol Sci. 2007;57 Suppl:S132]

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Differentiation to dopamine(DA)ergic neurons from ES cells was induced by 5 steps: stage 1, maintenance of ES cells; stage 2, embryoid body formation; stage 3, selection of nestin-positive cells; stage 4, expansion of nestin-positive neural stem cells (NSCs); stage 5, induction to DAergic neurons. To investigate how to induce DAergic differentiation from ES-derived NSCs, physiological low oxygen (3.5% O₂) in development or increased factors in the DA-depleted striatum (cytokine cocktail: IL-1β, IL-11, LIF, GDNF) was treated to NSCs. Treatment of cytokine cocktail or 3.5% O₂ increased the number of tyrosine hydroxylase (TH)-positive cells as compared to controls (2.20 -fold or 1.83-fold respectively). However, combination of low O₂ with cytokine cocktail did not show any additive effect. Furthermore, treatment of IL-1β and LIF showed most of the effect of cytokine cocktail. To investigate how cytokine cocktail induces DAergic differentiation, we focused on the effect of hypoxia inducible factor (HIF)-1α. Cytokine cocktail increased the expression of HIF-1α that is known as an activator of TH promoter. Inhibition of HIF-1αexpression by antisense treatment to LIF and IL-1β-treated NSCs decreased the number of TH-positive cells. Data suggest that enhanced differentiation to DAergic neurons from ES-derived NSCs by physiological low O₂ in development or increased cytokines in injured brain was mediated by increased expression of HIF-1α. [J Physiol Sci. 2007;57 Suppl:S132]

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We have shown that pre-treatment with deferoxamine (DFO: cell cycle blocker in G1/S phase) increase the number of neurons from neural stem/progenitor cells (NPCs) with relation to prolonged enhancement of p27^kip1 mRNA expression, and transfection of p27^kip1 induced neuroD promoter activation and increase of the number of β-tubulin III-positive cells. To investigate the molecular mechanism of neuronal differentiation by cell cycle blocker in detail, we focused on the relation between p27^kip1 and cyclin-dependent kinase 5 (cdk5). As cdk5 is known to bind with p27^kip1 and an cdk5 activator p35, the expression of cdk5 or p35 was investigated at the end point of DFO treatment for 8 hours and at 24 hours after differentiation to neurons. Expression of cdk5 mRNA was significantly elevated after the differentiation, although it was decreased during DFO treatment. On the other hand, expression of p35 mRNA was reduced during DFO treatment, but did not alter after differentiation. Data suggest that enhancement of cdk5 expression following to transient decrease during treatment would be included in the mechanism of neuronal differentiation followed by cell cycle arrest. [J Physiol Sci. 2007;57 Suppl:S132]

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The detachment of integrin in focal contacts (FCs) is crucial for this morphological change. This study investigated how mechanical stimuli regulate the detachment of integrin in cultured HUVECs. Mechanical stimulation was made by displacing laterally a fibronectin coated glass bead attached on the apical cell surface for a few micrometers. As the FCs spotaneously formed underneath the bead were linked to the FCs at the basal surface of the cell via actin stress fibers, the tension in the actin fibers generated by the bead displacement was transmitted to the basal FCs. HUVECs were stained with an Alexa-conjugated antibody against the extracellular domain of the β1 integrin of which dynamics was live-imaged by a multi-mode fluorescence microscope. When the bead was displaced, integrin clusters constituting the basal FCs gradually disappeared within 10 min. These integrins appeared to be associated with clathrin and internalized into the cytoplasm. They were also associated with certain structures that were stained with FM4-64, a fluorescent endocytosis marker. The disappearance of integrin clusters was inhibited by the depletion of external K⁺, which procedure is known to inhibit the clathrin-dependent endocytosis. These results suggest that mechanical stimuli to the FCs via actin stress fibers facilitate clathrin-dependent endocytosis of integrins in the basal FCs. [J Physiol Sci. 2007;57 Suppl:S132]

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Cell type conversion by genetic reprogramming is ubiquitously observed in neoplasia and also in non-tumor cells under pathological condition. Although mechanisms, by which a cell converts, has remained to be resolved, the conditions for critical transcriptional switching must be the central issue. On the other hand, the regenerative cell or ESC therapy becomes more and more important to treat a vast diseases, such as Parkinson's disease, spinal cord injury, and diabetes. However, the ethical difficulty and the tissue rejection problem are main obstacles against the use of ESC. The generation of the differentiated or pluripotent cells from the patients' own cells by the conversion is one way to circumvent the problems. We attempted and partly succeeded to transdifferentiate or dedifferentiate mouse fibroblast cell line Balb3T12/3 into skeletal muscle, neuron-like cell, and stem-cell-like cell, when MyoD, neurogenin, and Nanog cDNA were forcibly expressed respectively. The differentiated cell fates were identified by detecting the expression of skeletal myosin and L-type Ca channel, MAP2, and alkaline phosphatase and specific morphology respectively with immunocytochemical and electrophysiological methods. Further the transdifferentiation into muscle or neuron-like cell was quantitatively facilitated with histone deacetylase inhibitor, chromatin configulation modifier. While, Nanog expression dedifferentiated the fibroblast into stem cell like, providing the possibility for multidirectional cell type conversation. [J Physiol Sci. 2007;57 Suppl:S133]

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We investigated whether elevations of intracellular cyclic nucleotides (cAMP and cGMP) or elevation of intracellular Ca²⁺([Ca²⁺]_i) inhibits cell proliferation and migration of cultured rat vascular smooth muscle cells (A7r5). Cell proliferation activity was measured by incubation of A7r5 cells with WST-1 reagent for 1 hour, followed by measurement of absorbance at 415 nm. Cell migration activity was measured by counting migrated cells through pores of cell culture insert (8-μm size) at 24 hour after feeding cells. Dibutyryl cAMP (db-cAMP, 0.03 to 3.0 mM) significantly inhibited cell proliferation in a dose-dependent manner, whereas db-cGMP (0.03 to 3.0 mM) had no effect. Olprinone hydrochloride (OPN, type 3 phosphodiesterase inhibitor) inhibited cell proliferation and enhanced db-cAMP (0.3 mM)-induced inhibition of cell proliferation. Elevation of [Ca²⁺]_i by KCl (70 to 80 mM) and Bay K 8644 (above 30 μM) significantly inhibited cell proliferation. Low concentrations of Bay K 8644 (3 to 30 μM) stimulated cell proliferation. Nifedipine (1 to 100 μM) inhibited dose-dependently cell proliferation. Cell migration activity was significantly inhibited by db-cAMP (3 mM), but not by db-cAMP (0.3 mM) nor by OPN (1 mM). Db-cAMP (0.3 mM) plus OPN (1 mM), however, significantly inhibited cell migration activity. Thus, elvation of cAMP inhibits cell proliferation and cell migration of VSM cells and elevation of [Ca²⁺]_i may have biphasic effect on cell proliferation. [J Physiol Sci. 2007;57 Suppl:S133]

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To evaluate the effect of expression of latent membrane protein (LMP)1 encoded by Epstein-Barr virus (EBV) on Cε mRNA expression, mRNA levels were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) or northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cε mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cε mRNA levels were decreased in the splenocytes derived from the immunized rats. Cε mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen. [J Physiol Sci. 2007;57 Suppl:S133]

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We have recently reported that the extracellular loop 12, the extracellular loop between transmembrane domain (TM) 12 and TM13, is a part of the sugar translocation pathway of Na+/glucose cotransporter type 1 (SGLT1) by making a chimera: the loop of Xenopus SGLT1-like protein (xSGLT1L) was substituted with the homologous region of rabbit SGLT1 (BBA 1758, 747-754, 2006). The chimera showed the increased apparent affinities for D-glucose (Glc) and myo-inositol (MI) compared to wild type xSGLT1L and transported both substrates equally. Next we made the second chimera in which the substituted region was expanded to both sides: the substituted region included about one-third of TM12 and almost of TM13. The second chimera transported Glc better than MI, this order was the reverse of the wild type xSGLT1L. From our results we will discuss about the important amino acids in the sugar translocation pathway of SGLT1. [J Physiol Sci. 2007;57 Suppl:S133]

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Ameloblasts are the cells responsible for enamel formation (amelogenesis). Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins has been well documented, Ca²⁺ transport mechanisms during amelogenesis have not been fully elucidated. In order to clarify Ca²⁺ transport mechanisms in rat ameloblasts, we investigated presence and functional characteristics of Na⁺/Ca²⁺ exchangers (NCX). NCX activities were measured with fura-2 and patch clamp technique. RT-PCR analysis was performed to identify the presence of transcripts for the NCX isoforms. For Immunohistochemistry, cryostat sections were prepared by slicing of the mandible including incisor. These sections were incubated with monoclonal antibodies against NCX, and labeled using fluorescent secondary antibodies. Ca²⁺ influx via reverse exchange showed dependence on extracellular Ca²⁺ concentration (Kd₅₀ = 0.25 mM), and was blocked by a NCX inhibitor, KB-R 7943, in a concentration dependent manner (IC₅₀ = 4.34 μM). Ca²⁺ efflux via forward exchange showed dependence on extracellular Na⁺ concentration. RT-PCR analysis revealed expression of NCX1 and 3 mRNA in ameloblasts. Immunohistochemistry showed localization of NCX on the ameloblast plasma membranes. Our results indicate significant expression of NCX in ameloblasts. Na⁺/Ca²⁺ exchangers may serve as a directional Ca²⁺ transporting pathway to the enamel mineralizing front from circulation. [J Physiol Sci. 2007;57 Suppl:S134]

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We examined the expression of Na/K-ATPase α-subunit isoforms in salivary glands by RT-PCR. mRNA of α1 isoform was abundant in all 3 major salivary glands. The α2 isoform was expressed in submandibular gland (SMG) and sublingual gland (SLG), and faintly in the parotid gland (PG). In contrast, α3 was expressed in the SLG alone. The α3 mRNA was also detected in dispersed cells that had been prepared from the SLG to remove nerve termini. The Na/K-ATPase α3 protein was detected in the membrane preparation from SLG with anti-α3 isoform monoclonal antibody by Western blot analysis. These results indicate that SLG actually expressed the α3 isoform. Furthermore, we also found the presence of antisense RNA of Na/K-ATPase α3 isoforms in rat SLG by conducting RT-PCR with gene-specific forward primers. The first strand cDNA was synthesized with specific forward primers targeted to the CDS region or the promoter region of upstream from the transcription start site. PCRs with these 1st-strand cDNAs revealed the reasonable PCR products of Na/K-ATPase α3 isoform. The antisense RNA was disrupted by treatment of the total RNA preparations with RNase prior to synthesis of 1st strand cDNAs. These results suggest the presence of antisense RNA of Na/K-ATPase α3 isoform in rat SLG. The level of the sense RNA of α3 isoform was higher than that of antisense RNA of the enzyme. The α3 antisense RNA as well as the α3 sense RNA was abundant in SLGs of female rats. [J Physiol Sci. 2007;57 Suppl:S134]

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Williams syndrome which exhibited unique neuropsychological profile including partial autistic-like behavioral property is caused by hemizygous deletion of 7q11.23. This region contains the gene for HPC-1/syntaxin1A (STX1A) which is believed to regulate synaptic transmission. Previously, we have produced STX1A knockout mice. Interestingly, consolidation and extinction of conditioned fear memory was impaired in homozygote (KO), but was normal in heterozygote (HT). In this study, we have examined if STX1A knockout mice exhibited abnormal behavior. KO mice exhibited almost normal learning properties in a discriminated operant task, unlike consolidation of conditioned fear memory test. However, latent inhibition of cued fear memory (LI) was attenuated both in HT and KO mice. The attenuation of LI in these mutant mice was recovered by administration of SSRI, Fluoxetine, implying serotonergic disturbance. In novel object exploration test, HT and KO mice explore the familial object for longer time than WT mice. Behavioral abnormality in HT and KO mice were also observed in social interaction test. These results suggested that haploinsufficiency of STX1A might cause for unique neuropsychological profile in WS. In addition, STX1A knockout mice revealed autistic-behavior and could be a model animal of autism. [J Physiol Sci. 2007;57 Suppl:S134]

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It has been well established that glucocorticoid plays a key role in the formation of hippocampal dependent spatial memory in rodents. However, behavioral studies on the effect of only glucocoricoid (without other adrenal steroid hormones) on the animal^,s spatial cognitive function have been few reported. Here, we investigated whether the chronically administration of corticosterone (25μg/ml) dissolved in daily drinking fluid (CORT) affects the spatial learning and memory in adrenalectomized (ADX) rats. Male Sprague-Dawley rats were divided into three groups (ADX, ADX+CORT and sham-ADX) and assigned to two water maze tasks to assess the abilities of rat^,s spatial learning and memory retrieval. Escape latency, the time that animals locate the submerged platform under water surface, declined across every trial. Two-way analysis of variance (ANOVA) for rat^,s learning performance revealed no differences between all groups. In memory retrieval test, they searched in target quadrant longer than others. Fischer^,s PLSD test showed that ADX+CORT group significantly remembered platform location compared to ADX group. These data suggest that rats with ADX could also learn and retrieve similar to sham-ADX group, single administration of corticosterone to ADX rats reinforces spatial memory retrieval. [J Physiol Sci. 2007;57 Suppl:S134]

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Pilocarpine, a muscarinic receptor agonist, is a typical sialagogue to treat hyposalivation. Although peripherally applied pilocarpine makes the oral cavity wet by increasing saliva, it is well-known in animal experiments that it also induces water intake. Mechanisms underlying the relationships between these events are unknown. Intracerebroventricularly injected pilocarpine induced water intake but not salivary secretion. Intracerebroventricularly applied atropine, a muscarinic receptor antagonist, suppressed the increased water intake by the intraperitonially and intracerebroventricularly applied pilocarpine. We tested which parts of brain were involved in these responses by using c-Fos immunohistochemistry. Intraperitonially injected pilocarpine increased the number of c-Fos immunopositive cells in some nuclei of the circumventricular organs, hypothalamus and medulla, which are related to thirst sensation. Intracerebroventricularly applied atropine suppressed the increased number of c-Fos immunopositive cells by the intraperitonially and intracerebroventricularly applied pilocarpine. We conclude that peripherally injected pilocarpine affects the parotid glands and the thirst center in the central nervous system. [J Physiol Sci. 2007;57 Suppl:S135]

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Rodents exposed to alcohol prenatally have been shown to exhibit impairments of memory and attention. In this study, we investigated whether ethanol-induced attention deficits evaluated by use of operant behavior tests for rat offspring were ameliorated by treatments of several psychotropic drugs. Pregnant Wistar rats were separated into three groups from gestation day 8 through 20: 20% ethanol treated (6g/kg/day), 30% sucrose treated and saline treated via intragastric intubation. The sucrose and saline groups received the same volume of ethanol, respectively. Operant experimental chambers that have two lever and two lights were used in this test. Ability of attention in these offspring was evaluated using choice reaction time (CRT) and percent correct. Rats were trained on the CRT task with 100 trials per day for 20 days. Prenatal ethanol exposure resulted in a siginificantly longer CRT than those of the other two groups, however, there were no effects on percent correct. Moreover, in ethanol exposed rats, oral treatments of methylphenidate and fluvoxamine, which are inhibitors of dopamine and serotonin transporters, there was a significantly shortened CRT compared with solvent treatments 20 min before the tasks, but donepezil, a cholinesterase inhibitor, had no effect. It was found that attention deficits caused by prenatal ethanol exposure were ameliorated by methylphenidate and fluvoxamine, but not donepezil. These findings suggest that rats exposured to ethanol prenatally were likely to suffer impairment in their dopaminergic and serotoninergic rather than their cholinergic neuronal systems. [J Physiol Sci. 2007;57 Suppl:S135]

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We have established an evaluation method by which domestic chick social behavior can be categorized and quantified. A test chick was left alone in a novel test cage and after a few minutes exposed in sensory-modality specific manner to chicks which were grouped together in another test cage. The test chick raised distress call when left alone, but made food/joyful call, approached, and walked along the cage wall facing to the grouping chicks. The chick called back with a different call pattern to unfamiliar and familiar chicks, suggesting the capability of cognition of familiarity. This grouping behavior, however, was induced only in the case of group-reared chick. A chick reared in socially deprived condition escaped or froze in the same test context. We addressed a question what kind of sensory-motor interaction is crucial for the development of the grouping behavior. Chicks were reared for two weeks in various conditions where a limited social cue was allowed. We found that an integration of auditory and visual cues was crucial and surprisingly experience of feeding together only 10 min per day was enough to develop the grouping behavior. In contrast, no grouping behavior developed without food/water. This result suggested the importance of mutual sensory-motor interaction with a common motivation is important for development of social behavior. [J Physiol Sci. 2007;57 Suppl:S135]

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Cyclosporine is widely used in immunosuppressive therapy for organ transplantation and autoimmune disease. However, cyclosporine administration causes neuropsychological side effects such as depression, anxiety, confusion, and tremor in some patients. We have previously shown that cyclosporine induces anxiety-related behaviour and decreases social behaviour in mice, and that clozapine partially restored social behaviour in cyclosporine-injected mice. The prefrontal cortex has been shown to control anxiety-related and social behaviour. In the present study, we examined effects of cyclosporine and clozapine upon release of dopamine and serotonin within the prefrontal cortex. Microdialysis probes were inserted in the prefrontal cortex of urethane anaesthetized mice, and contents of dopamine and serotonin in the perfusates were measured by HPLC with electrochemical detection. Release of dopamine and serotonin was reduced in the cyclosporine-treated mice. In the mice that received both cyclosporine and clozapine, dopamine release was not reduced. All these data are consistent with the hypothesis that cyclosporine disturbs social behaviour as a result of reduced dopamine release in the prefrontal cortex. [J Physiol Sci. 2007;57 Suppl:S135]

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To study the cardiac function and its development of newborn mice, I first measured the heart rate (HR-PZT) of free-moving newborn C57BL/6 mice using a piezoelectric transducer (PZT) sensor, from immediately after birth (postnatal day 0: P0) to P14, and compared the HR-PZT with the heart rate (HR) obtained by ECG (HR-ECG). The HR-PZT increased age-dependently from 323±38 b/m at P0 (n=5) to 674±56 b/m at P14 (n=9), while the HR-ECG measured immediately after attaching ECG electrodes was over 57 b/m lower than the HR-PZT in the mice aged P3 to P14 (P<.0001-.0022, n=6-15). During 5 minutes of HR-ECG measurement, the HR-ECG did not change and remained lower than the HR-PZT in mice aged P3 to P8, while it recovered to a value comparable to the HR-PZT in the mice aged P9 to P14 and it slowly but significantly decreased in the mice aged P0.5 to P2. The present study demonstrates that the PZT system is the non-invasive and practical method for the measurement of HR in free-moving newborn mice in contrast to attaching ECG electrodes which is invasive and influences HR. Furthermore, I found that all mice aged P0 to P14 restrained by attaching ECG electrodes suddenly stopped their motion after several seconds of struggle just as tonic immobility at death feigning. Further study is needed to elucidate the mechanisms of the diverse changes in HR-ECG induced by attaching ECG electrodes to mice and the death-mimicking-like behavior in a neonatal period, during which the central nervous system dramatically develops. [J Physiol Sci. 2007;57 Suppl:S136]

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Students of today suffering from psychosomatic complaints (e.g. drowsiness and dullness) have problems in their basic lifestyle habits. The aim of our study was to clarify the physiological sources underlying their psychosomatic complaints. We examined 14 healthy students ( 4 men, 10 women ) of 21-22 years old. To evaluate the level of psychosomatic symptoms, we developed a self-assessment Psychosomatic Complaints Questionnaire for this study consisting of two categories: 5 items on physical symptoms and 5 items on mental symptoms. Subjects estimated their psychosomatic conditions twice a day (08.00 h and 20.00 h). As cortisol and melatonin have well-documented circadian pattern, in this study we assessed these circadian secretion pattern by FEIA (Fluorescence Enzyme Immunoassay). Salivary samples were taken from each subjects four times a day (08.00 h, 12.00 h, 20.00 h and 24.00 h) in Salivette tubes at home. The samples were centrifuged and frozen at -20 degrees until analysed. Physiological variables were assessed by means of a standardized score. Our results showed that the self-assessment scores were related to level of cortisol and melatonin concentration, though in the limited number subjects. Subjects of low self-assessment score in the morning tended to display low peak in cortisol and melatonin concentration. These results suggest that the psychosomatic symptoms might be associated with the physiological hormone rhythm related to their basic lifestyle habits. [J Physiol Sci. 2007;57 Suppl:S136]

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Olfactory neural messages are sent to the hypothalamus and induce various autonomic reactions depending on the kind of odor stimulus. Since the hypothalamus is known to produce feeding-related neuropeptides, odors may affect the production of these neuropeptides. In Wistar rats, we examined how the orexin level was affected by odor stimuli. Among several stimuli tested, the odor of Osmanthus fragrans flowers lowered the level of prepro-orexin mRNA in the hypothalamus. Rats fed with food containing the Osmanthus odor showed less food intake and body weight than those fed with non-odor food or food with milk odor used as a comparative odor. In the human experiment, 10 healthy females ranging from 20 to 40 years old were divided into 2 groups with or without exposure to Osmanthus odor and all the subjects were presented with the same menus of meals for 12 days. The results showed that the body weight of the odor group reduced more with better mental and physical status than the control group. Locomotion and rearing were reduced under Osmanthus odor in rats, and the larger contingent negative variance of EEG was observed under Osmanthus odor than under milk odor in humans: both of these results suggest some sedative effects of Osmanthus odor. Collectively, these results suggest that Osmanthus odor affects autonomic, appetitive and mental activities at least through reducing the orexin level. [J Physiol Sci. 2007;57 Suppl:S136]

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The finding that orexin deficiency causes narcolepsy suggested that these hypothalamic neuropeptides play a critical role in regulating sleep/wakefulness states. The actions of orexins are mediated via two receptors named orexin 1 (OX1R) and orexin 2 (OX2R) receptors. Some reports have indicated that the effect of orexin on wakefulness is largely mediated by activation of the histaminergic system through OX2R. Consistently, OX2R knockout mice exhibit a narcoleptic phenotype, while OX1R knockout mice show almost normal sleep/wakefulness behavior. However, it is important not to disregard the importance of this receptor in the regulation of sleep/wakefulness states, because the phenotype of double receptor knockout mice is more severe than OX2R knockout mice, supporting an important but less significant contribution of OX1R. In this study, we analyzed phenotype of OX1R/histamin H1R -double deficient mice to further examine the importance of the histaminergic pathway as the downstream effecter of orexin. OX1R/HX1R -double deficient mice do not show cataplexy or direct transition from wakefulness to REM sleep. However, these mice showed severe fragmentation of wakefulness period, while both OX1R knockout mice and H1R knockout mice show almost normal sleep-waking behavior. These finding suggest that 1) the histaminergic pathway is important for stabilization of wakefulness states. 2) OX2R in regions other than TMN histaminergic cells are important for REM sleep regulation. 3) OX1R is also important for stabilization of wakefulness states. [J Physiol Sci. 2007;57 Suppl:S136]

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Control of the stability of protein is thought to be involved in various biological processes, such as cell cycle and circadian clock. Endogenous ubiquitinated (Ub-) BMAL1 accumulated with MG132, a reversible inhibitor for proteolysis of Ub-proteins during Dexamethazone (Dex)-pulse treatment-induced cellular clock synchronization. This might be due to inhibition of BMAL1 degradation via Ub-proteasome. BMAL1 ubiqutination was suppressed by U0126, a phosphorylation inhibitor via ERK pathway. Inhibition of Ub-Proteasome during Dex-pulse significantly damped out-put circadian rhythm of luciferase-reporting Per2 gene expression (Per2-luc rhythm). BMAL1 contains conserved ERK phosphorylation sites in its PEST-like sequence, which is thought to be involved in the controlling protein stability. Transient transfection of GFP-BMAL1/PEST protein, which is expected to be a competitive inhibitor for ERK phosphorylation during Dex-pulse resulted in dramatically damped Per2-luc rhythm. We propose that controlling stability of BMAL1 via ERK phohorylation-dependent ubiquitination in its PEST-like sequence that has essential role in generating circadian clock oscillation. [J Physiol Sci. 2007;57 Suppl:S137]

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We simultaneously recorded hippocampal and amygdaloid field activity during REM sleep in rats. In the amygdala, intermittent rhythmic activity, whose frequency was about 8 Hz, was recorded during REM sleep. This activity was found to be synchronized with hippocampal theta activity. We also recorded pontine phasic potential, which is called P wave. This potential is known to relate to the cholinergic neuronal activity in the brainstem. We found that i) the frequency of theta wave in both the amygdala and hippocampus increased associated with increase in the P wave density, ii) duration of theta in the amygdala increased as P wave density increases iii) synchronization between theta wave in the amygdala and hippocampus became stronger with the increase in the P wave density. Because cholinergic neurons in the brainstem send projections to the amygdala and hippocampus, these results suggest that the brainstem cholinergic neurons activate theta activity both in the amygdala and hippocampus. [J Physiol Sci. 2007;57 Suppl:S137]

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Music is known to be able to elicit emotional changes, including anxiolytic effects. The gonadal steroid hormones estradiol and progesterone have been reported to be associated with anxiety levels. In the present study, we examined whether the effect of music on anxiety is related to ovarian steroid in female mice. Behavioral paradigms measuring anxiety were tested in gonadally intact (SHAM) and ovariectomized (OVX) female mice chronically treated with either placebo (OVX/Placebo), 17β-estradiol (OVX/E), or progesterone (OVX/P). In the elevated plus maze, light-dark transition, and marble burying tests, SHAM and OVX/P mice exposed to music showed less anxiety than those exposed to white noise or silence, while OVX/placebo mice did not show these effects at all. OVX/E mice showed the anxiolytic effect of music only in the marble burying test. Furthermore, pretreatment with progesterone's metabolite inhibitor completely prevented the anxiolytic effect of music in behavioral tests, while pretreatment with a progesterone receptor blocker did not prevent the anxiolytic effect of music. These results suggest that exposure to music reduces anxiety levels, and ovarian steroids, mainly progesterone, may be involved in the anxiolytic effect of music observed in female mice. [J Physiol Sci. 2007;57 Suppl:S137]

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Homeostatic mechanism of sleep is thought to have an essential role for rest and maintenance of neural function. Recent report has suggested that vitamin A (retinol and its derivatives) is involved in the electroencephalogram (EEG) delta oscillation during sleep. However, the evidence is not confirmed by other study. In this study, we attempted to record sleep EEG and behavior, and to quantify striatal monoamines for mice that fed vitamin A deficient (VAD) diet during four weeks, in order to clarify the linkage between delta oscillation and vitamin A. VAD mice demonstrated a significant decrease of delta power in EEG and spontaneous activity throughout 24-hour. And then, striatal dihydroxyphenylacetic acid, which is a metabolite of dopamine, was significantly decreased in VAD mice. Our results suggest that deficiency of vitamin A induces the attenuation of spontaneous activity and dopaminergic dysfunction. This decrease of spontaneous activity is considered to suppress the delta power in homeostatic manner, because 6-hour forced awaking caused the recovery of delta power in VAD mice up to that in the control. [J Physiol Sci. 2007;57 Suppl:S137]

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The neural mechanism of discriminative taste preference learning was examined using the modified procedure of flavor-postingestive consequence learning paradigm in C57BL/6 male mice. Mice under a food restriction schedule were given six two-bottle training sessions with the four exposures of 0.15 M sucrose (Suc) paired with intragastric (IG) 0.5 M Suc infusion and those of 5 mM saccharin (Sacch) paired with IG water infusion with an alternative sequence. The intake of Suc significantly increased with decreasing of that of Sacch, and the increasing of a preference for Suc was suppressed when the mice received the simultaneous IG infusion of 2 mM phloridzin dehydrate, a blocker of Na-glucose transporter at the intestine. Lesions of the amygdala with ibotenic acid impaired development of a preference for Suc. These results suggest that learned preference for Suc is mediated by the absorption of glucose produced from the injected sucrose at the intestine, and the amygdala plays a role in discriminative association of hedonically positive visceral signal of glucose with the sweet taste quality of Suc. [J Physiol Sci. 2007;57 Suppl:S138]

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The present study was conducted to evaluate the role of the septal neurons for modulation of penile erections during various states of vigilance in rats. A chronic preparation was made fixing a U-shape plate over the skull for semi-restraining the animal in un-anesthetized state. The single units were recorded from the septum simultaneously with recordings of penile erections and EEG. Neurons, which could be recorded, at least during 2 states of vigilance and erectile event were analysed. Erection consisted of a slow increase in corpus spongiosum penis pressure (CSP) with several sharp peaks superimposed on it. Out of 43 neurons recorded during erection across different stages sleep-wakefulness, 26 neurons were related to the erectile activity. The erection related neurons, which showed phasic firing in relation with CSP pressure peaks were grouped as E-phasic (n=15). Two neurons showed increased firing during erection (E-tonic type) while 9 neurons showed decreased firing during erection (I type). Of the 26 erection related neurons, 22 showed relation with erection during REM sleep, while 4 did with erection during waking. These results suggest that a state dependent regulatory mechanism for penile erections is present within this forebrain limbic structure. [J Physiol Sci. 2007;57 Suppl:S138]

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[Introduction] Circadian rhythms in clock gene expression have been reported to show photoperiodic responses in the suprachiasmatic nucleus(SCN) of nocturnal rodents. On the other hand, different parts of the SCN were demonstrated to response differentially to photoperiods. In the present study, we examined photoperiodic responses of circadian rhythms in Per1 and Per2 expression in the rat SCN. [Material and Methods] Male Wister rats were exposed to either to a long (LD18:6) or to a short (LD6:18) photoperiod for more than 3 weeks. The brains were obtained at 3 hours intervals under darkness. Coronal sections of 30μm in thickness were made from the rostral to the caudal portions of the SCN and in situ hybridization was performed to measure Per1 and Per2 expressions in individual cells with digoxigenin-labeled probes. The number of positive cells were counted in regular squares of 75μm length, and the circadian rhythms in gene expressions were obtained in discrete areas of the SCN. [Results and Conclusions] Under a long photoperiod, the circadian rhythm in Per1 expression cells showed a bimodal pattern in the rostral part, whereas it showed one peak in the caudal. On the other hand, the circadian rhythms in Per2 expression showed essentially a single peak thoughout the SCN. These findings indicate that there are more than two oscillating cell populations in the rat SCN, which respond differentially to different photoperiod. [J Physiol Sci. 2007;57 Suppl:S138]

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Photoperiodic time measurement by the circadian clock is one of the strategies by which animals know the time of year. In nocturnal rodents, circadian behavioral rhythms are photoperiod-dependent. The activity bands are compressed and decompressed in long and short photoperiods, respectively. Two separate oscillators, evening (E) and morning (M) ones, have been proposed to explain photoperiodic responses of behavioral rhythms, the location of which is not yet identified. We exposed transgenic mice carrying a Per1-luciferase reporter gene to three different photoperiods, and compared circadian parameters of behavioral rhythms and Per1 expression rhythms in single cells as well as tissues of serial coronal slices of the suprachiasmatic nucleus (SCN). Furthermore, those of horizontal slice were examined. Irrespective of photoperiods, the circadian peak in the posterior SCN was phase-locked to the end of activity, suggesting the localization of M oscillator, whereas that in the anterior SCN, to the activity onset, suggesting that of E oscillator. In the longest photoperiod, two Per1 peaks were detected only in the anterior SCN. Single cell analyses revealed two separate cell groups in the anterior SCN. The long photoperiod seem to separate the third component in the anterior SCN responding light-on from that responding light-off. We further analyze the three oscillating cell networks, two in the anterior and one in posterior SCN, which couple differentially to dawn and dusk using horizontal slices with intact rostro-caudal connections. [J Physiol Sci. 2007;57 Suppl:S138]

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Behavioral rhythms in mice were desynchronized from light-dark (LD) cycle under chronic methamphetamine(MAP) treatment. Our previous findings indicate that clock gene expression rhythms in the caudate-putamen(CPU) and parietal cortex(PC) are in phase with MAP-induced behavioral rhythms, whereas those in the SCN remains to entrain to LD cycle. These findings suggested that behavior rhythms in MAP treated animals were driven by an oscillator(s) outside the SCN. However, it is not known whether or not the CPU and PC are the site of oscillator. To identify the location of MAP-induced circadian oscillator, we examined Per1 expression rhythms in several regions of brain culture from mice under chronic MAP treatment. Transgenic mice expressing luciferase under the control of Per1 promoter (Per1-Luc) were treated with 0.005% MAP through drinking water until their spontaneous locomotor rhythms desynchronized from LD cycle. They were decapitated on the day when their activity onset reached to the light on phase, and the coronal brain slices of 300μm thick were cultured in the medium containing 0.1mM luciferin. Bioluminescence rhythms were continuously measured for 10 days. Comparing with un-treated control mice, MAP-treated mice exhibited significant phase differences in substantia nigra(SN). This result suggests that the MAP-induced circadian oscillation was generated in the SN. [J Physiol Sci. 2007;57 Suppl:S139]

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Most organisms show circadian rhythms in their physiological functions, which were driven by the pacemaker located, in mammals, in the suprachiasmatic nucleus of the hypothalamus (SCN). In rodents, the fetal SCN(s) already show circadian rhythmicity which is entrained by their maternal rhythms. The maternal signals are also dominant to entrain the pups' circadian pacemaker for several days after birth, which, however, are gradually replaced by the photic signal. In this study, we compared the responses to non-photic stimuli between newborn and adult SCN to examine the development of clock function in the SCN. [Method] Adult (8-42 weeks old) and newborn (6 days old) Bmal1-luc transgenic mice were used, in which firefly luciferase was expressed under control of clock gene Bmal1 promoter. Their SCN slices were cultured in serum-free medium containing 0.1mM luciferin, and bioluminescence was monitored continuously. Culture medium was exchanged at different phases of Bmal1-luc rhythms and the phase responses to medium exchange were examined. [Result and Discussion] In adult SCN, the phase of Bmal1-luc rhythm was not influenced by medium exchange at any phase, whereas, in newborn SCN, medium exchange induced phase-dependent phase shifts. In addition, about 25% of new born SCN exhibited bimodal rhythms in a day, which merged during the course of culturing. Such bimodality was never seen in adult SCN. These results can be explained in terms of coupling among neuronal oscillators which was much weaker in newborn SCN than in adult SCN. [J Physiol Sci. 2007;57 Suppl:S139]

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An auto-feedback loop of clock gene transcription and translation is believed to generate the circadian oscillation. Two clock genes, Per2 and Bmal1 are involved in interlocking two molecular feedback loops, and expressed anti-phasically. To investigate the dynamics of interlocked feedback loops, we developed a dual bioluminescence reporter system with two different Phrixothrix luciferases (PTGR/RED). We established stable transformants in fibroblast cell lines transfecting two plasmids, which consisted of Per2 and Bmal1 promoter fragment (390bp/915bp) with PTGR and RED luciferase of green-light emitting and red-light emitting wavelength, respectively. These clones showed anti-phasic circadian rhythm in Per2 and Bmal1 gene expressions after dexamethasone treatment. To find out critical steps in the feedback loop, we perturbed the feedback loop by changing the concentration of constitutional components, and determined the circadian period. When Cry1,2 gene expressions were enhanced, the circadian period was not changed significantly, but the expression of Per2 was reduced by 80%. The result suggests that its suppression of Per2 gene expression is not a critical step to determine the lapse of loop rotation, or that the critical amount of Per2 expression is enough to maintain the auto-feedback loop. We'll also examine the effects of other loop elements such as Bmal-Clock complex, Per1,Per2, and orphan nuclear receptor RORα. [J Physiol Sci. 2007;57 Suppl:S139]

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Palatability of food is one of strong motivations to feeding behavior. Favorite food works as a natural reward, many reports suggest that the ventral tegmental area (VTA) is involved with hedonic responses to food. We studied the effects that lesions of dopamine cells in the VTA would have on preferences of tastes. After bilateral injections of 6-OHDA into the VTAs of rats (VTA rats), we tested the difference of preferences between VTA rats and sham surgical rats (sham rats) by the two-bottle choice test for 2 days with following taste fluids: saline (0.9% of NaCl), Sucrose (30, 60 and 120 mM) and umami substances monosodium glutamate (MSG, 30, 60, and 120 mM), inosine monophosphate (IMP, 15, 30 and 60 mM) and guanosine monophosphate (GMP, 15, 30 and 60 mM). There is no significant difference in% preference, only the consumption of sucrose was significantly decreased in the VTA rats, but the consumption of saline and umami tastants was not significantly. Second, we tested whether VTA rats could detect lysine (Lys) when they fed Lys deficient diet, in this condition Lys could have strong reward value for rats. After one week with a Lys deficient feed, both VTA and sham rats chose a Lys solution. These results suggest that dopaminergic cells in the VTA are related to sugar consumption but not to umami consumption. The consumption of an essential amino acid is not affected with the reward system even if it has a high reward value. The intake of essential nutrients might be regulated by another brain regions except the reward system. [J Physiol Sci. 2007;57 Suppl:S139]

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Modulation of behavioral and neurochemical activities in the mesolimbic and nigrostriatal dopaminergic systems by estradiol (E2) is well known. Dense expression of cocaine- and amphetamine-regulated transcript (CART) mRNA and peptide in the nucleus accumbens (NA) and striatum (ST) are also reported. Whether E2 modulate the stimulatory effects of CART peptide in the mesolimbic and nigrostriatal dopaminergic (DA) systems in female Sprague-Dawley rats was examined in this study. DA neuronal activities were determined by measuring the concentration of DOPAC (3,4-dihydroxyphenylacetic acid), the major metabolite of DA, in the NA and ST by HPLC-ECD. Intracerebroventricular administration of CART peptide increased the DOPAC content of NA and ST in ovariectomized (OVX) priming E, but not in OVX only and E2-BSA (the membrane-impermeable form of E2) treated female rats. However, two days of injections of E2-S (the water-soluble form of E2) can restore the stimulation of CART peptide on NA and ST DOPAC content. Only E2 antagonist blocked the E2 effects, but testosterone antagonists did not. These findings indicate that E2 play a regulatory role in stimulation of the CART peptide in mesolimbic and nigrostriatal DA systems, and suggest that E2 acts through intracellular genomic rather than extracellular non-genomic mechanisms. [J Physiol Sci. 2007;57 Suppl:S140]

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The mechanism of cisplatin, anti-cancer drug,-induced delayed emesis has not been resolved clearly yet. We supposed that dynorphin, an endogenous ligand of κ-opioid receptor, could be a candidate for substances relating to cisplatin-induced delayed emesis because it appears in the central nervous system by various noxious stimuli and also relates to feeding behavior. Firstly we established a model of cisplatin-induced delayed emesis by means of pica meaning a response that increases an intake of a non-nutritional diet such as kaolin after administration of toxic stimulus. Consequently the dose of 5 mg/kg body weight was considered to be a suitable dose for the experimental model. Following the behavioral experiment, we immunohistochemically examined the induction of dynorphin A in the area postrema (AP), which is the chemoreceptor trigger zone for emesis, at 24, 48 and 72 h after the administration of 5 mg/kg cisplatin. Dynorphin A accumulates in the neurosoma of the AP neurons gradually by cisplatin. The numbers of positive AP neurons at 48 and 72 h after the administration of cisplatin were significantly higher than those after the administration of the same volume of 0.9% NaCl. These findings suggest that dynorphin A is associated with cisplatin-induced delayed emesis. [J Physiol Sci. 2007;57 Suppl:S140]

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We have identified occ1 as a gene enriched in the primary visual cortex (V1) of macaque monkeys. We have also reported that the occ1 mRNA expression in excitatory neurons of V1 is strongly downregulated by monocular deprivation (MD) by intraocular TTX injection in macaques, and that the V1-enriched expression pattern of occ1 is restiricted to primates among mammals. In the present study, we show that the expression patterns of occ1 mRNA were similar between macaques and mice, despite the distinct pattern in V1. Furthermore, we examined differences of activity-dependency in occ1 mRNA expression between macaques and mice, comparing those of immideate-early genes (IEGs), c-fos and zif268. MD dramatically reduced mRNA expression of occ1 and IEGs in macaque V1. The same treatment in mice decreased IEG expression both in the visual cortex and dLGN. The mRNA expression of occ1 in mouse visual cortices and dLGN was, however, scarcely affected by MD. Moreover, cochleotomy and olfactonomy neither affected occ1 mRNA expression in the cochlear nucleus nor in the olfactory bulb, respectively. These observations illustrated that whereas the strong activity-dependent regulation of IEG expression is common throughout regions and species, occ1 mRNA expression in primate V1 is evolutionarily unique. [J Physiol Sci. 2007;57 Suppl:S140]

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