The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling in various cell types. In neuronal cells, we previously showed that primary cultured hippocampal or cortical neurons express functional VSOR channels. Here, by using a whole-cell patch-clamp technique, we examined whether or not hippocampal and cortical pyramidal neurons express the VSOR channel in a more physiological preparation, that is, in the slice preparation. Whole-cell voltage-clamp recordings revealed that chloride currents were gradually activated upon cell swelling induced by application of hypotonic solution. This current was inhibited by chloride channel blockers, IAA-94, NPPB and DIDS, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in primary cultured neurons. It is concluded that the VSOR channel is functionally expressed in hippocampal and cortical pyramidal neurons in the slice preparation. There is a possibility that the neuronal activities are affected by its activation. [J Physiol Sci. 2007;57 Suppl:S230]

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KCNQ1 is a voltage-dependent K⁺ channel whose gating property is dramatically changed by association of auxiliary KCNE proteins. KCNE1, which is mainly expressed in heart, drastically decelerates the activation kinetics of KCNQ1. However, the question if the voltage-sensing S4 domain moves differently with KCNE1 has not been revealed yet. To address this point by MTSET accessibility analysis, we systematically introduced cysteine mutations one at a time to the first half of S4 domain of human KCNQ1 gene. These mutants were expressed in Xenopus oocytes and analyzed under two-electrode voltage clamp. Before the MTSET experiments, we first characterized the properties of mutants. Unexpectedly, we observed that some of these mutants (I227C, R228C, G229C, I230C, F232C, L233C) showed extremely slow deactivation, and as a result, they eventually became constitutively-active after several times of depolarization. However, this phenotype was waned or completely gone with co-expression of KCNE1 except in G229C, which became constitutively active more easily than without KCNE1. For the analysis of MTSET accessibility, KCNQ1 mutants were incubated in 98 mM K⁺ extracellular solution with 1 mM MTSET for 30 min. When KCNE1 was co-expressed, G229C and I230C became constitutively active by MTSET while they were only partially activated by MTSET in the absence of KCNE1. Taken together, these results suggest that association of KCNE1 change molecular environments of S4 domain of KCNQ1. [J Physiol Sci. 2007;57 Suppl:S230]

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Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are the major anions in the large intestinal lumen, and are known to have a variety of physiological and pathophysiological effects on the intestine. This study examined the distribution of SCFA receptors, GPR41 and GPR43, by RT-PCR, Western blotting, and immunohistochemistry in rat intestine, including duodenum, jejunum, terminal ileum, cecum, proximal colon, and distal colon. Protein and mRNA expressions of GPR41 and GPR43 were analyzed by dividing into mucosa and submucosa-muscle layers in addition to whole wall. Moreover we investigated the contractile effects of SCFAs on rat intestinal smooth muscle preparations with mucosa in vitro. As a result, GPR43 was expressed in mucosa of distal intestine, especially terminal ileum and distal colon. In the physiological experiments, propionate and butylate induced both phasic contractions and tonic contractions respectively, moreover propionate increased the frequency of spontaneous contractions in circular muscle of distal colon. [J Physiol Sci. 2007;57 Suppl:S230]

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TRPV4, a member of TRPV subfamily, is a nonselective cation channel that is activated by hypotonic stimulus, warm temperatures (about 25-34 °C) or chemical compounds such as 4α-PDD, and is expressed in various tissues. To investigate TRPV4 function in the epithelial tissue, we screened a cDNA library from epithelial cells to identify TRPV4 interacting protein using a yeast two-hybrid system. Sequence analysis revealed that one of the positive clones encodes a protein containing one PDZ and three LIM domains. We found that the N terminal region of TRPV4 bound to LIM domains of the PDZ-LIM protein. When both TRPV4 and the PDZ-LIM protein were co-expressed in HEK293 cells, patch-clamp analysis showed that 4α-PDD-evoked currents were larger than those observed in cells expressing TRPV4 alone. However, the increase in the 4α-PDD-evoked currents was profoundly decreased by PMA treatment in the cells expressing TRPV4 with the PDZ-LIM protein. These results suggest that the PDZ-LIM protein regulates TRPV4 activity by PKC activation. [J Physiol Sci. 2007;57 Suppl:S231]

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Using electrophysiological and immunohistochemical methods, we investigated the role of L-type Ca²⁺ channel in the regulation of endocochlear potential (EP) in the stria vascularis. 1) Administration of 1 μg/ml nifedipine into endolymph, which produced a gradual increase in EP, inhibits significantly transient asphyxia induced decrease (TAID) in the EP. TAID in EP was inhibited by endolymphatic perfusion with nifedipine (0.001-10 μg/ml) in dose dependent manner. Administration of 30 μg/ml nifedipine via vertebral artery suppressed significantly TAID in EP, whereas it produced the gradual decrease in EP at a control level. These results suggested that L-type Ca²⁺ channel in marginal cells or intermediate cells may participated in the regulation of both control level and TAID in EP. 2) A positive staining for L-type Ca²⁺ channel was presented in the luminal and basolateral membrane in marginal cells. 3) Administration of (-)-Bay K 8644, a L-type Ca²⁺ channel activator, into the endolymph caused the large decrease in EP, whereas administration of (+)-Bay K 8644, a L-type Ca²⁺ channel blocker, produced the increase in EP and suppressed the TAID in EP. These findings support the view that L-type Ca²⁺ channel in the marginal cells regulates the EP in both control and asphyxia conditions. [J Physiol Sci. 2007;57 Suppl:S231]

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Over 30 missense mutations in human skeletal muscle Na channel (SCN4A) have been identified in families with either myotonia or periodic paralysis, or both. A 38-years-old woman with positive family history was manifested cold-aggravated myotonia without weakness from childhood. She was clinically diagnosed as potassium-aggravated myotonia, and screened SCN4A gene. A novel missense change of a residue located at intracellular C-terminal region (Q1633E) was identified. The functional effect of this mutation was assessed by recording whole-cell sodium currents from transiently transfected HEK293t cells using calcium phosphate method. Recordings were made with Axopatch 200B amplifier using pClamp 9 program. Fast inactivation was impaired for Q1633E channel, as demonstrated by approximately 10 mV rightwards shift in voltage dependence compared with wild type channel. There was no significant difference in the activation. We conclude that the observed defect of fast inactivation in mutant sodium channel leads myotonic symptom. Furthermore, our results suggest that the C-terminal region in SCN4A also plays an important role in fast inactivation. [J Physiol Sci. 2007;57 Suppl:S231]

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The inwardly rectifying Kir4.1 channels are predominantly expressed on astrocytes in the brain, where they play a crucial role in the spatial potassium buffering. We previously reported that the tricyclic antidepressants such as nortriptyline produce a voltage-dependent inhibition of Kir4.1 channel currents. Here we extended the evaluation for various antidepressants to further characterize their actions on Kir4.1 channels, using a whole-cell patch-clamp technique. Kir channels were expressed heterogeneously in HEK293T cells and the step pulse-induced Kir currents were measured. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), reversibly inhibited Kir4.1 currents in a concentration-dependent manner. The inhibitory effect of fluoxetine was more potent than that of nortriptyline and virtually voltage-independent. When compared with other Kir channel subtypes, fluoxetine inhibited Kir4.1/Kir5.1 channels, but barely Kir1.1 or Kir2.1 channels. Other SSRIs, sertraline and fluvoxamine, also inhibited Kir4.1 channels whereas the actions of tetracyclic (i.e., mianserin) or 5-HT_1A-related (i.e., buspirone) antidepressant were negligible. The present study demonstrates for the first time that SSRIs, such as sertraline and fluoxetine, potently block astroglial K⁺-buffering Kir4.1 channels. These actions might be involved in therapeutic and/or adverse reactions of the antidepressants. [J Physiol Sci. 2007;57 Suppl:S231]

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The detergent-resistant membrane (DRM) microdomains scaffold various molecules and orchestrate signal-pathways in many cells. Although it is shown that brain astrocytes possess abundant DRM microdomains, their physiological significance is largely unknown. Here we report a novel functional role of the DRM microdomains in astroglial cells. Biochemical analysis of brain protein revealed that the K⁺-buffering inwardly rectifying Kir4.1 channels and the water channels AQP4, both of which are expressed specifically in astrocytes, were enriched in DRM fractions. Caveolae isolated by the caveolin-antibody did not contain either channel proteins. When coexpressed in cultured astrocytes, the immuno-staining study showed that the Kir channels and AQP4 existed in a very close vicinity and sometimes overlapped on the cell membrane. Treatment of the transfected HEK cells with methyl-β-cyclodextrin, which depletes membrane cholesterol, resulted in disappearance of Kir channels from DRM fractions and also in loss of channel-activity. In contrast, the same treatment did not affect either distribution or function of AQP4. These results may indicate that the astroglial non-caveolar DRM comprises at least two distinct components, cholesterol-dependent and -independent microdomains, each of which transports K⁺ or water by expressing either K⁺ or water channels and controlling their functionality. [J Physiol Sci. 2007;57 Suppl:S232]

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Inward rectifier K⁺ (Kir) channels can be functionally categorized into two groups: those that are constitutively active and those that are constitutively inactive, with examples such as Kir2.x and Kir3.x, respectively. Their cytoplasmic regions are thought to be critical for control of channel gating, but a structural basis for this hypothesis is not known. In this study, we report a structure for the cytoplasmic region of a G protein-gated Kir channel, Kir3.2, and compare it with those of Kir3.1 and Kir2.1 channels. The isolated cytoplasmic region of Kir3.2 forms a tetrameric assembly in solution and also in the crystal. While the secondary structure arrangement and the subunit interface of the Kir3.2 crystal structure are found to be nearly identical to those of Kir3.1 and Kir2.1, it is quite different at and around loops between βC- and βD-strands and between βH- and βI-strands. These structural elements are located at the interface with the plasma membrane and therefore could associate with the Kir channel transmembrane helices. These structural elements could therefore be involved in the regulation of Kir channel gating. [J Physiol Sci. 2007;57 Suppl:S232]

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HERG channel is blocked by many drugs, which cause life threatening cardiac arrhythmia. However, nifekalant is effective in suppressing tachyarrhythmias without induction of serious life-threatening arrhythmia. In addition to block, some drugs such as nifekalant stimulate HERG at low potentials by shifting its activation curve to hyperpolarizing direction, which is called facilitation. We investigated the mechanism of the drug-HERG interaction by electrophysiological approach. We examined its effects on HERG mutants substituted systematically to alanine in its pore region. We found that S649 is sensitive for nifekalant-induced facilitation. Thus, it supposed that binding nifekalant at closely S649 caused the channel to the facilitation. We found that nifekalant causes facilitation of HERG current by interacting with a serine residue at a top of pore cavity by modeling of homologymodeling and docking simulation. This effect of facilitation is thought one of reasons why nifekalant is effective in suppressing ventricular tachyarrhythmias. Further studies on the detailed mechanism for facilitation should be useful for the design of clinically effective Ikr blockers with reduced proarrhythmic effects. [J Physiol Sci. 2007;57 Suppl:S232]

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The primary sensory neuron in the mesencephalic trigeminal nucleus (MTN) is unique in expressing various receptors including glutamate receptors (GluRs). To characterize these GluRs, we performed the whole-cell voltage-clamp recording from MTN neurons in slice preparations. At the holding potentials ranging from -110 to +90 mV, the current responses to puff application of glutamate were recorded. When the peak current amplitudes were plotted against the holding potentials, an N-shaped I-V relationship crossing near 0 mV was obtained. This N-shaped relationship was distinct from those previously seen in inwardly rectifying type II and outwardly rectifying type I AMPA channels. Furthermore, at negative holding potentials, the biphasic responses consisting of inward and outward current components were often observed. The outward component seen at < 0 mV was sensitive to Cs⁺ and ZD7288, indicating an involvement of h-current in generating the glutamate-induced outward current. Provided that GluR channels shared the microdomain with h-channels in MTN neurons, it is possible that the apparent reversal potential of glutamate-induced current is shifted negatively due to the co-localization of the two active channels that have different Na⁺/K⁺ permeability ratio of 0.2 and 1. Furthermore, a transient increase in [Na⁺]_i in such a microdomain would further shift the reversal potential negatively, leading to a generation of biphasic responses where the N-shaped I-V relationship can be seen. [J Physiol Sci. 2007;57 Suppl:S232]

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Voltage-gated L-type Ca²⁺ channel current (I_Ca) can be enhanced by preceding strong depolarization. Although this phenomenon is well recognized as voltage-dependent facilitation, its fundamental mechanisms are still unknown. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been reported to play a key role in this process. Therefore we aimed to further clarify these regulation using the whole-cell mode of patch-clamp technique in HEK 293 cells expressing Ca_V1.2_b, β2 and CaMKII. Voltage-dependent facilitation was Ca²⁺-dependent, and was prevented by CaMKII-inhibitor KN93 and autocamtide-2-related inhibitory peptide. Sixteen putative phosphorylation sites in Ca_V1.2_b and β2 subunits according to the consensus sequences which were previously reported. Ca_V1.2_b subunit truncated at amino acid 1728 did not affect voltage-dependent facilitation of I_Ca. Replacement of Ser¹⁵¹² and/or Ser¹⁵⁷⁰ in Ca_V1.2_b subunit by Ala markedly attenuated the voltage-dependent facilitation of I_Ca, while other mutants had no effects. These results indicate that voltage-dependent facilitation of I_Ca is CaMKII-dependent and is generated by phosphorylation of Ca_V1.2_b at Ser¹⁵¹² and/or Ser¹⁵⁷⁰. [J Physiol Sci. 2007;57 Suppl:S233]

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We examined patch clamp recordings in murine macrophage-like J774-A.1 cells after the uptake of IgG-opsonized latex beads. First, we prepared isolated phagosomes from the macrophages by cutting the plasma membrane with a sharp glass pipette. Single channel recordings of the isolated phagosomes revealed that large conductance (230pS) chloride selective channels were present in the phagosomes. These channels had the characteristics of the current frequenty observed in excised patches of the plasma membranes (but very rarely in whole-cell recordings). Second, we combined whole-cell capacitance and volage-clamp recordings to evaluate phagosome membrane potentials. Exocyotis of the phagosomes by the activation of G proteins were measured in the capacitance step and transient current. We found that phagosomes have lumen negative membrane potentials. In some cases, 200-300pS channels emerged immediate after the capacitance steps, showing that large conductance chlolide channels were incorporated to the cell membrane. These data indicate that large conductance chloride channels regulate the membrane potentials of the phagosomes in macrophages. [J Physiol Sci. 2007;57 Suppl:S233]

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Responsive properties of principal cells in the pineal gland (pinealocytes) of rodents (ddy mice and Wistar rats) were studied using a whole-cell patch-electrode. The pineal gland is one of the circumventricular organs in the brain and thus possesses an incomplete blood-brain barrier. When external saline containing 10 microM nicotine was applied from a "Y tube" to pinealocytes, which were whole-cell clamped at -50 mV, definite inward currents were induced. Other nicotinic agonists, including ACh (10 microM), choline (1 mM), lobeline (10 microM) and cytisine (10 microM), also induced inward currents. However, time courses and amplitude as well as recovery from desensitization of the agonist-evoked currents showed variation among cells, indicating heterogeneity of functional nicotinic ACh receptors expressed in these cells. For nicotine-induced currents in the pinealocytes of ddy mice, the I-V relationship measured with a saw-tooth voltage clamp showed reversal potential near -5 mV. Notably, in previous morphological studies on mammalian pineal glands, the presence of cholinergic fibers innervating pinealocytes was found. To determine whether nicotinic synaptic transmission in pinealocytes occurs in vivo, external saline containing 20 mM KCl was applied from a "Y tube" to pinealocytes, but no typical excitatory postsynaptic currents were detected from the preparations of ddy mice. (Supported by a grant from Smoking Research Foundation, Japan) [J Physiol Sci. 2007;57 Suppl:S233]

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Gonadotropin-releasing hormone (GnRH) neurons discharge action potential to release GnRH from their nerve terminals in the median eminence. It is, therefore essential, to know how the excitability of GnRH neurons is regulated. A variety of ion channels are involved in membrane excitability. Here we focus on large-conductance Ca²⁺ and voltage-activated K⁺ (BK) channels. BK channels modulate neuronal signaling by contributing to action potential repolarization, mediating the fast phase of afterhyperpolarization, and establishing a feedback loop between membrane potential and cytosolic Ca²⁺ that regulates release of hormones and transmitters. Overnight culture of GnRH neurons were prepared from GnRH-EGFP transgenic rats. The whole-cell currents were measured by means of the perforated patch-clamp technique at room temperature. The cell was held at -90 mV and the membrane potential was stepped to various voltages for 50 ms at 0.2 Hz. The K⁺ currents were activated from -30 mV and reached around 2000 pA at +60 mV. These K⁺ currents decreased around 30% by blocking Ca²⁺ channels with Ni²⁺ and Cd²⁺. The Ni²⁺ and Cd²⁺ sensitive currents were designated as the BK currents and the remained as the delayed rectifier K⁺ currents. In addition, BK channel blocker charybdotoxin attenuated the currents. Futhermore, RT-PCR of GnRH neurons revealed an expression of αsubunit of BK channels. Taken together, the results indicate a functional expression of BK channels in rat GnRH neurons. [J Physiol Sci. 2007;57 Suppl:S234]

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Two kinds of gustometry electrogustometry (EMG) and filter paper disk (FPD method) were used for evaluation of human taste disorder frequently. In these gustometries, a sense of a subject was required. These gustometris consumed much time of examiner. The taste receptor gene expression was used for new gustometry method as evaluation methods of taste disorder. And the technique was performed in BMS (Burning mouth syndrome, glossodynia) patient. BMS is a medical disorder characterized by a burning sensation in oral cavity. However, BMS usually lacks mucosal physical abnormalities in an oral cavity and medical underlying disease, and taste disorder is found in some cases of BMS. It is thought that many cases of BMS develop it from a psychological problem such as cancer phobia. Taste receptors (T2R3, T2R8, T2R9, T2R10, T2R13, T2R16) in scrape smear specimen from foliate papillae in BMS patient was detected by RT-PCR method. BMS patient appealed for a pain of a tongue severe at first. And the evaluation of EMG and FPD method showed severe taste disorder. 12 weeks later, BMS patient did not appeal for a pain of a tongue and the evaluation of EMG and FPD method showed an approximately normal level. In scrape smear specimen of BMS patient, five kinds of taste receptors (T2R9, T2R10, T2R13, T2R16) were detected. These results suggest that evaluation of taste receptor expression in scrape smear is useful for a diagnosis of taste disorder. [J Physiol Sci. 2007;57 Suppl:S234]

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We found that the fetal variants, ASI(-) of the ryanodine receptor 1 (RyR1) which lacks residue 3481-3485 and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1b which differs at the C-terminal, were significantly increased in skeletal muscles from Myotonic dystrophy type 1 (DM1) patients and the transgenic mouse model of DM1. Channel open probability was significantly decreased in ASI(-) than in adult isoform of RyR1, ASI(+). To determine how this aberrant splicing affects the activity of RyR1 channels, we tested whether the splicing region is involved in an inter-domain interaction using synthetic peptides. Both peptides corresponding to the Thr³⁴⁷¹ -Gly³⁵⁰⁰ around the ASI region in the presence (ASI(+)) and the absence (ASI(-)) of exon ASI increased RyR1. The results suggest that ASI(-) peptide interrupts an inhibitory interdomain interaction in the native RyR1 more strongly than the ASI(+) peptide. We therefore suggest that ASI(-) region may interact more tightly with other domains and produce stronger inhibition of ASI(-) RyR1, resulting in reduced activity of the ASI(-) RyR1. Moreover, peptide ASI+/- includes highly positively charged residues similar to A region of dihydropyridine receptor II-III loop which has been reported to activate RyR1. We further tested if these highly positively charged residues in peptide ASI(+/-) are essential for the activation and if peptide A and peptides ASI(+/-) activates RyR1 competitively. [J Physiol Sci. 2007;57 Suppl:S234]

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In skeletal muscle, malignant hyperthermia (MH) has been reported mainly due to increased Ca-induced Ca release (CICR) activity induced by type 1 ryanodine receptor (RyR1) mutation. There are three hot spots for mutation; the N-terminal, central and C-terminal regions. A 36 residue peptide of the central domain (DP4, Leu2442-Pro2477) activates RyR1 channel (Ikemoto & Yamamoto, 2002; Murayama et al., 2005). Recently, we also found that the weakened interdomain interaction between the N-terminal and central regions stimulates RyR1 channel activity and suggested that this abnormal channel dysfunction produces MH. On the other hand, our previous results demonstrated that RyR1 channel activity is regulated by redox states of the channel protein. Therefore, we investigated here whether RyR1 channel dysfunction induced by interdomain interaction is affected by intracellular redox states. We will show results using hydrogen peroxide, a weak oxidant for SR protein, and glutathione (GSH/GSSG) buffer on channel activity observed with or without DP4 exposure to RyR1. [J Physiol Sci. 2007;57 Suppl:S234]

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Stretch activated and Ca²⁺-activated big K channel (SAKcaC) identified by our group, is one form of splice variants of the BKca channel encoded by slo-1 gene, and has the STREX-sequence, a 59 amino-acid splice insert located in the cytoplasmic side of the channel, which may responsible for the membrane stretch sensitivity of SAKcaCs. Recently, we found that most SAKcaCs exhibited simple diffusion on the plasma membrane but that approximatly 25% of the channels was immobilized on or near focal contacts (FCs), suggesting that SAKcaCs are associated with the FC components such as adhesion molecules and cytoskeletons to form a mechano-sensing device. However, the mechanisms of such channel immobilization on FC remains unclear. To address this issue, we examined the role of STREX on the channel immobilization. We expressed GFP-tagged STREX and observed using the single fluorophore video imaging technique. Simultaneous observation with RFP-tagged paxillin showed that STREX molecules were recruited and diffused rapidly on the plasma membrane but significant fraction of them was immobilized on FCs, indicating that STREX itself could associate with certain FC components. However, STREX-deleted SAKcaCs were also immobilized on or near FCs, indicating that SAKcaCs could be associated with FCs via STREX and/or other sites of the channel. [J Physiol Sci. 2007;57 Suppl:S235]

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Although angiotensin II (Ang II) has been reported to increase L-type Ca²⁺ channel current (I_Ca(L)) in vascular smooth muscle (VSM) cells, the mechanism how Ang II stimulates I_Ca(L) is not clarified. Since pp60^src is activated by Ang II, we investigated whether activation of pp60^src increases I_Ca(L) in cultured rat VSM cells (A7r5). A7r5 cells were transfected with a plasmid of wild type mouse src cDNA/pUSE expression vector using lipofectamine. Negative control cells were prepared by transfection with an empty vector, (-)/pUSE. After transfection, the cells were treated with 1 mg/ml of G418 to select the positive cells stably expressing src cDNA or (-)/pUSE. I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier for recording of I_Ca(L). Western blot demonstrated that expression of pp60^src was increased by 34-fold in src cDNA/pUSE expressing cells as compared with (-)/pUSE expressing cells. Current/voltage relationships demonstrated that although voltages for peak amplitude of I_Ca(L) were 20 mV for both src cDNA and (-)/pUSE expressing cells, peak amplitude of I_Ca(L) in src cDNA/pUSE expressing cells was increased by 2-fold (src, 6.1±0.96; (-), 3.1±0.38 pA/pF). From steady-state inactivation and activation analyses of I_Ca(L), voltages for half-activation and -inactivation and slope factors did not differ between src and (-)/pUSE expressing cells. Thus, overexpression of pp60^src increases I_Ca(L) without affecting voltage sensors of L-type Ca²⁺ channels. [J Physiol Sci. 2007;57 Suppl:S235]

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We examined the effect of antiallergic drugs, azelastine and epinastine, on the expression of FcεRIα, β, and γ chains and phosphorylation of the γ chains in rat basophilic leukemia cells (RBL-2H3). The cells were cultured for 24 h with IgE treatment in the presence of azelastine or epinastine at the concentration of 10⁻⁵ M. The FcεRIα, β, and γ chains mRNA expressions were determined by northern blot analysis or RT-PCR. The protein level of FcεRI expressed on the plasma membrane was examined after 24 h-treatment with IgE by immunoprecipitation with anti-IgE light chain, followed by western blot analysis with anti-γ chain of FcR. Azelastine and epinastine had no effect on the FcεRIα, β and γ mRNA levels. Although the amount of γ chain assembled into IgE-bound FcεRI was not changed by treatment with azelastine nor epinastine, phosphorylation levels of γ chains of IgE-bound FcεRI were inhibited by azelastine. The inhibitory effect of the antiallergic drugs, especially azelastine, on the IgE-stimulated function of FcεRI protein is not due to their inhibition of mRNA and protein expression, but due to abrogating phosphorylation of the γ chains, which is important for initiation of FcεRI signaling cascade elicited by IgE interaction. [J Physiol Sci. 2007;57 Suppl:S235]

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To clarify the mechanism by which high concentration of cAMP decreases L-type Ca²⁺channel current (I_Ca(L)) in vascular smooth muscle (VSM) cells (A7r5), I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier to record I_Ca(L). Forskolin (FSK, 100 μM) decreased I_Ca(L) by 30%. When measured FSK-induced inhibition of I_Ca(L) after pretreatment with H-89 (PKA inhibitor) or KT5823 (PKG inhibitor), H-89 partially abolished the FSK inhibition by 30% and KT5823 further abolished by 77%. When the expression of type Iβ protein kinase G (PKG Iβ) was abolished using antisense oligodeoxynucleotides (ODN) against PKG Iβ (AS-PKG), dibutyryl-cAMP (db-cAMP, 3 mM) failed to decrease I_Ca(L). Furthermore, the expression of β subunit of L-type Ca²⁺ channel was abolished by antisense ODN against common sequence of β2a, β2b and β3, db-cAMP (3 mM) also failed to decrease I_Ca(L). Thus, β subunit of L-type Ca²⁺ channel may be necessary for inhibition of I_Ca(L) by cAMP/PKG Iβ cross-activation in VSM cells. To further explore the role of β subunit in regulation of L-type Ca²⁺ channel by phosphorylation, A7r5 cells were transfected with β2a cDNA/pcDNA3 or β3 cDNA /pRc/CMV expression vectors using lipofectamine and I_Ca(L) will be tested for further inhibition in the presence of FSK, db-cAMP or db-cGMP. [J Physiol Sci. 2007;57 Suppl:S235]

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Ghrelin is a recently identified endogenous legand for the grows hormone secretagogue receptor (GHS-R). In addition to GH-releasing activity, ghrelin enhances feeding. Since swallowing is early step of feeding behavior, ghrelin might modify swallowing. In the present study, we examined the effects of ghrelin on the reflex swallowing using anaesthetized rats. Ghrelin, which was dissolved in Ringer^,s solution, was administered into the fourth ventricle. Swallowing was induced by the electrical stimulation (20 Hz, 0.2 mA, 20 sec) of the central cut end of the superior laryngeal nerve (SLN) and was monitored by recording the electromyogram of the mylohyoideus muscle. The number of swallowing during the electrical stimulation of the SLN decreased after the administration of ghrelin in a dose-dependent manner. The latency of the response tended to be longer in the presence of ghrelin. Simultaneous administration of ghrelin and GHS-R antagonist ([D-Lys³] GHRP-6) did not change the number of swallowing induced by the electrical stimulation of the SLN. After sectioning both vagi at the cervical level below where the SLN entered the vagal trunk, the inhibitory response of swallowing induced by ghrelin was still observed. Neither mean blood pressure nor mean heart rate changed after the administration of ghrelin. These results suggest that ghrelin inhibits reflex swallowing modifying the neural activities of the dorsal medulla where swallowing center is present. [J Physiol Sci. 2007;57 Suppl:S236]

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The superior salivatory (SS) nucleus, the primary parasympathetic center for salivary secretion, is located in the lateral reticular formation of the medulla oblongata. Many neuroanatomical studies suggest that the neurons innervating SS neurons are distributed in the forebrain and brainstem. Our electrophysiological studies showed that most SS neurons receive GABAergic and glycynergic inhibitory inputs from higher and lower centers. In the present study, we examined distribution of GABAergic and glycinergic neurons innervating SS neurons immunohistochemically. The neurons innervating SS neurons were labeled by the injection of retrograde tracer FluoroGold into SS nucleus. To examine the distribution of GABAergic and glycinergic somata, double immunostaining was performed using anti-GABA and anti-glycine antibodies, and was observed under the fluorescence microscope. GABA-immunoreactive neurons were mainly observed in higher centers including the lateral hypothalamic area, paraventricular nucleus of hypothalamus, central nucleus of amygdala, bed nucleus of stria terminalis and preoptic area. Glycine-immunoreactive neurons were observed mainly in lower centers including the parabrachial nucleus, sensory trigeminal nucleus, nucleus of solitary tract. These findings imply that these higher and lower centers are involved in the inhibitory salivary control. [J Physiol Sci. 2007;57 Suppl:S236]

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We showed the glutamatergic, GABAergic and glycinergic synaptic inputs to superior salivatory (SS) neurons which is the primary center of submandibular salivary secretion. This glutamatergic input is considered to derive from the forebrain and brainstem. In the present study, we studied how SS neurons receive the glutamatergic inputs from the forebrain and brainstem in rats. The SS neurons innervating the salivary glands were labeled by retrograde axonal transport of a fluorescent dye. Subsequently some rats were decerebrate. Whole-cell patch-clamp recordings were performed from the labeled cells in slices. Excitatory postsynaptic currents were evoked by electrical stimulation near the recording cell. As compared with normal SS neurons, decerebrate SS neurons showed 3 types of the responses: enhanced responses, similar responses, no responses. The SS neurons which showed enhanced EPSCs receive the excitatory inputs from forebrain and brainstem. Decerebration induced denervation-hypersensitivity in the glutamate receptors. Enhanced EPSCs may be evoked by stimulation of glutamatergic inputs from brainstem. The SS neurons displayed similar responses have mainly excitatory inputs from the brainstem. The SS neurons which displayed no responses produced larger currents by the application of glutamate, suggesting that this type has excitatory inputs exclusively from the forebrain. [J Physiol Sci. 2007;57 Suppl:S236]

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We examined role of the feeding center on salivation during feeding of various foods, using normal and feeding center-destroyed rats. For this examination rats were placed on a food deprivation schedule and allowed access to various food for 3 hours per day for one week. The foods were pellets, power diet, and 3 types of mashed diet (power diet : water = 1:1.5, 1:1, and 1:0.5). After training, the animals were anesthetized and the duct of left submandibular gland was cannulated for recording salivation, and a pair of stainless electrodes was inserted into the masseter muscle for recording EMG activities. The feeding center (lateral hypothalamus) was destroyed by passing of DC current. After recovery from anesthesia, the recording experiment was performed and following results were obtained: 1) In normal rats, the average salivary flow rates for 3 min were in the order power diet > hard mashed diet > medium mashed diet > food pellet > soft mashed diet; 2) total EMG activities for 3 min were similar in various foods; 3) when the ipsilateral feeding center was destroyed, salivation during feeding of various foods decreased by about 80%; 4) destruction of the contralateral feeding center yielded about 40% decrease in salivary flow rates; 5) no changes were observed in total chewing cycles or total EMG activities for 3 min. These results suggest that descending effects from the feeding center are essential for salivation during chewing, and involved in main neural network to evoke the masticatory-salivary reflex. [J Physiol Sci. 2007;57 Suppl:S236]

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Area postrema (AP) is well known to be the chemoreceptor trigger zone for emesis. Previous studies have reported that tachykinin NK1 receptor and sustance P (SP) played an important role for emesis. Immunohistochemical studies have identified the presence of tachykinin receptors in the area postrema, however, the effects of tachykinins on the neuronal excitability of AP neurons remain to be determined. In the present study, we sought to clarify the responses of AP neurons to tachykinin receptor activation using a patch-clamp technique in rat brain slices (150-200 µm thick) obtained from Sprague-Dawley rats (7-21 days postnatal). Voltage-clamp recordings were performed in the presence of TTX (1 µM) at a holding potential of -50 mV. We found excitatory responses of AP neurons to SP (up to 1 µM), Sar⁹-Met¹¹(O₂) substance P (SM-SP : selective NK1 receptor agonist, up to 1µM) and αneurokinin (αNK : selective NK2 receptor agonist, up to 1 µM). SP, SM-SP and αNK induced a marked inward currents. In some cells, these drugs elicited the significant changes in the frequency of miniature EPSCs. SP induced larger inward currents than those induced by SM-SP and αNK. These results indicate that tachykinin receptors are present both pre- and post- and/or extrasynaptically in the AP. [J Physiol Sci. 2007;57 Suppl:S237]

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Our recent anatomical study shows that the sensory nerve travels in both the sympathetic and parasympathetic nerve supplies to the submandibular gland of the rat. In the present study we analyze afferent neural activities in the sensory nerve from the submandibular gland in urethane-anesthetized rats. The following results were obtained: 1) The afferent activity could be recorded from both sympathetic and parasympathetic routes; 2) both afferents responded to mechanical pressure applied onto the gland; 3) when back pressure was applied from the main excretory duct, both afferents showed tonic impulse discharges at pressure of higher than 100 mmHg; 4) the threshold pressure was little lower than the maximal secretory pressure which was induced by electrical stimulation of the parasympathetic secretory nerve (the chorda tympani); 5) the sensory nerve in the parasympathetic, but not sympathetic, nerve supply responded to mechanical pressure on the main excretory duct; 6) the sensory nerve in the sympathetic nerve supply discharged after ligation of the artery to the gland. Our results suggest that the sensory nerves in the parasympathetic nerve supply innervate the submandibular gland and its main duct, whereas those in the sympathetic nerve supply innervate the gland and its artery. The nerves innervating the duct are responsive to relatively strong duct pressure compared to the maximum secretory pressure. The nerves innervating the artery may contribute control of blood flow changes. [J Physiol Sci. 2007;57 Suppl:S237]

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To understand the integrative roles of the paraventricular nucleus (PVN) in the central regulation of autonomic and endocrine systems, we simultaneously recorded the single unit in the PVN, arterial blood pressure, electrocardiogram (ECG), electroencepharogram (EEG) and electromyogram (EMG) of cervical muscle in conscious freely-moving rats. The miniature drivable apparatus with eight microwires was implanted to the skull for the recording of single unit. Three groups of neurons were revealed by the cross-correlation analysis between heart rate (HR) and firing rate of unit activity. Group 1 neurons showed a positive correlation. The firing rate started to increase 2-4 sec prior to increasing HR. Group 2 neurons showed a negative correlation at similar lag. Group 3 did not show a consistent relationship between HR and firing rate. Some of this type showed rhythmic phasic activity pattern. All groups did not showed a reflex changing in firing associated with perfusion of vasoactive drugs. However group 1 and 2 neurons increased and decrease in firing rate, respectively, in response to the moderate stressor. Sleep-wakefulness states were discriminated based on EEG and EMG. The simultaneous firing of neuron pairs not only across groups but also within group was subjected to additional cross-correlation analysis. The relationship was also compared between sleep-wakefulness states. These findings suggested that the combination of neurons in the PVN contribute to reciprocal control of autonomic nervous system. [J Physiol Sci. 2007;57 Suppl:S237]

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Orexins were initially reported as regulators of food intake. More recent reports suggest that they might play roles in the multiple functions of neuronal systems, causing medical conditions such as narcolepsy, a sleep disorder. Orexins and their receptors (OX1R and OX2R) are distributed in the brain regions involved in the autonomic and neuroendocrine control. Within the hypothalamus, orexins fibers and orexins receptors, especially OX2R, are found extensively in the paraventricular nucleus (PVN) of the hypothalamus. The PVN is an integrative center of the autonomic nervous system and neuroendocrine system. To examine the effects of orexin-A on the neural activity of the PVN neurons, we simultaneously recorded the single unit activities in the PVN, arterial blood pressure (ABP), and heart rate (HR) in conscious rats. Intracerebroventricular (i.c.v.) administration of orexin-A (0.3nmol) elicited increases in ABP and HR, as previously reported. Of irregularly active neurons (n=12), 6 neurons were excited, 2 neurons were inhibited, and 4 neurons were non-responsive. Majority of the neurons affected by orexin-A also responded to perturbation in ABP and systemic administration of cholecystokinin-8 (CCK). One phasically active neuron (putative vasopressin neuron) was excited by orexin-A. Thus orexin-A may modulate the neural activity of PVN neurons, which are involved in autonomic and neuroendocrine responses to intero-receptive stressors. [J Physiol Sci. 2007;57 Suppl:S237]

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To test the hypothesis that renal afferents send feedback signal to the central nervous system to modulate the cardiopulmonary baroreflex, we have studied the role of the renal afferents on the cardiopulmonary baroreflex during acute saline volume expansion in anesthetized rats. We measured the reflex change in efferent renal sympathetic nerve activity (ERSNA) in response to saline volume expansion (2% of body weight with a rate of 0.5 ml/min) before and after crush of the renal afferents at the distal side of the recording site. ERSNA measured from the left renal nerve decreased to 27% of the baseline control during volume expansion in the intact kidney (n=4). After crushing the left renal afferents, ERSNA reduced to 47% of the baseline control during volume expansion, which became smaller than before the renal nerve crush. In 4 nephrectomized rats, ERSNA measured from the left renal nerve decreased to 71% of the baseline control during volume expansion. The decrease in ERSNA was abolished by crushing all renal afferents. Thus the reflex response in ERSNA during volume expansion was attenuated depending upon the extent of renal afferent ablation. We conclude that renal afferents play an important role in modulating the cardiopulmonary baroreflex during saline volume expansion in anesthetized rats. [J Physiol Sci. 2007;57 Suppl:S238]

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We have shown that electroacupuncture (EA) stimulation to the tibialis anterior muscle improves plasma glucose response to insulin in streptozotocin (STZ)-induced diabetic rats. In the present study, we aimed to clarify whether the effect of EA stimulation on the insulin sensitivity is mainly due to contraction of the muscle or excitation of the somatic afferents. Rats were treated with STZ two weeks prior to experiments. Experiments were performed under urethane-chloralose anesthetized, artificially ventilated condition. EA stimulation was delivered for 10 min at 10 mA, 20 Hz via two stainless steel needles which were inserted perpendicularly about 5 mm into the right tibialis anterior muscle. In rats whose right sciatic and femoral nerves were severed, EA stimulation to the tibialis anterior muscle failed to improve plasma glucose response to insulin. These results suggest that in STZ rats EA stimulation can facilitate glucose uptake in response to insulin by reflex mechanism, i.e., via excitation of the somatic afferents. [J Physiol Sci. 2007;57 Suppl:S238]

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We have shown that electroacupuncture (EA) applied to the area over the abdomen and hindlimb modulates ovarian blood flow (OBF) in anesthetized rats. The present study aimed to further elucidate the role of the site and the frequency of EA stimulation and the influence of the estrous cycle on the OBF response using urethane-anesthetized rats. EA stimulation was applied to the abdominal or the hindlimb muscles at three different frequencies (2, 10, and 80 Hz) during the estrus or diestrus phase. Abdominal EA stimulation at 10 Hz increased OBF, while hindlimb EA stimulation at 10 Hz and abdominal and hindlimb EA stimulation at 80 Hz decreased the OBF; 2-Hz EA caused no OBF response. The OBF response to abdominal EA was more pronounced in the estrus than the diestrus phase. The OBF response to abdominal and hindlimb EA stimulation at both 10 Hz and 80 Hz was almost abolished, both after severance of the sympathetic nerves and after spinal cord transection. In conclusion, the OBF response to both abdominal and hindlimb EA stimulation was mediated as a reflex response via the ovarian sympathetic nerves, and the response was controlled via supraspinal pathways. Furthermore, the OBF response to segmental abdominal EA stimulation was frequency dependent and amplified in the estrous phase. [J Physiol Sci. 2007;57 Suppl:S238]

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We examined the possible involvement of the putative neurotransmitters, GABA and serotonin (5-HT), in raphe-induced facilitatory or inhibitory effects on the respiratory activity of rats. We observed that an intravenous (i.v.) injection of (+)-bicuculline, a GABA(A) receptor antagonist, significantly reduced respiratory inhibition induced by electrical stimulation of the raphe magnus (RM) or the raphe obscurus (RO). On the other hand, an i.v. injection of methysergide, a broad-spectrum 5-HT receptor antagonist, significantly reduced the respiratory facilitation induced by stimulation of the raphe pallidus (RP) or RO. We further investigated the spinal projection of GABAergic and serotonergicneurons from the caudal raphe nuclei to the phrenic motor nucleus (PMN) using a combined method of retrograde labeling of raphespinal neurons utilizing Texas Red injected into the PMN region and immunohistochemical labeling of GAD, a GABA synthesizing enzyme, or 5-HT positive cells. We observed that GAD and Texas Red double-labeled neurons were predominantly localized in the RM, and additionally in the RO. However 5-HT and Texas Red double-labeled neurons were predominantly localized in the RP, and additionally in the RO and RM. These findings suggest that RM-, or RO-induced inhibitory effects are transmitted (at least in part) to the PMN via a direct GABAergic descending pathway. The RP-, or RO- induced facilitatory effects however, are transmitted via a serotonergic descending pathway. [J Physiol Sci. 2007;57 Suppl:S238]

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We have previously shown that the defense response against stressor was attenuated in prepro-orexin knockout mice (Am J Physiol 285:R581, 2003) and orexin neuron-ablated mice (Am J Physiol 290:R1654, 2006) and proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. It is still open question, however, how information of stressor activates the orexinergic neurons. In this study, we examined possible contribution of the amygdala and bed nucleus of stria terminalis (BNST) as one of the afferent nuclei to activate orexinergic neurons. In urethane-anesthetized mice, a GABA-A receptor antagonist, bicuculline methiodide (0.1-10 mM in 20 nl), was microinjected into the amygdala or BNST, of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiratory frequency. Bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated mice and wild-type controls. However, dose-response curve was rightward shifted in the orexin neuron-ablated mice. In an immunohistochemical study, we confirmed that microinjection of bicuculline to the amygdala or BNST induced c-fos expression in the orexinergic neurons. We conclude that the amygdala and BNST constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. [J Physiol Sci. 2007;57 Suppl:S239]

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Decrease of calcium in the cerebrospinal fluid (CSF) raises blood pressure whereas increase of calcium lowers it. Although these phenomena have been known for more than 50 years, precise mechanism has not fully been understood. We examined whether the extracellular calcium-sensing receptor (CaSR), that was originally cloned from the parathyroid cells and later found in the brain, contributes to the blood pressure regulation by the calcium in the CSF. Cardiovascular regulatory neurons were identified by immunohistochemical detection of c-fos, a marker for cellular activation, in response to a rise in blood pressure by intravenous infusion of phenylephrine for 1 hr. Distribution of CaSR was identified using double immunostaining technique in the same brain section. Many neurons in the caudal ventrolateral medulla (CVLM) showed both of c-fos and CaSR-like immunoreactivity. These results indicate that an increase of calcium in the CSF may activate depressor neurons in the CVLM through CaSR on them. [J Physiol Sci. 2007;57 Suppl:S239]

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Objective: To examine the development of subjective symptoms and heart rate variability (HRV) during motion sickness induced by virtual reality (VR). Methods: Subjects were 10 healthy young volunteers. During VR immersion, subjects were immersed in a visual-vestibular conflict produced by VR. The levels of the subjective symptoms were assecced by Graybiel's and Hamilton's criteria. HRV was determined by measuring microvascular blood flow or electrocardiogram. Results: Subjective symptoms evaluated by Graybiel's and Hamilton's criteria were gradually worsened during VR. Power spectrum analysis of HRV demonstrated a gradual increase in the low frequency but no change in the high frequency during VR. In this study, individual subjective symptoms were not correlated with the individual result of power spectrum analysis. Conlusion: These findings indicate that there was an increase in sympathetic nervous acitivity, but no change in parasympathetic nervous activity during motion sickness induced by VR. Given the large inter-individual variability and the reliability of subjective measures, it is not surprising that there is scarcely a relation between the subjective symptoms and the results of power spectrum analysis. [J Physiol Sci. 2007;57 Suppl:S239]

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Our previous experiments using rats suggested that autonomic responses accompanying with nausea were controlled by neurons near the nucleus ambiguus (NA) of medulla oblongata, and these responses were produced via neurokinin 1 receptor (NK1R) activation. In this study, the changes of activities in the neurons near NA to tetanus stimulation and NK1 antagonist and/or agonist were investigated. Whole-cell recording was applied to the neurons near the NA in coronal slice preparations of brainstem isolated from infant rat (6-12 days). The frequency of spontaneous activities of neurons (n=18) was increased (39%) or decreased (28%) by perfusion of substance P (SP, 1μM), a selective agonist of NK1R. The changes of amplitude of excitatory postsynaptic currents (EPSCs) were recorded in the 29 neurons near NA. EPSCs evoked by electrical stimulation of adjacent area to the recording neuron were enhanced (28%) or inhibited (21%) by perfusion of SP. Furthermore, in the several neurons, tetanus stimulation (100Hz) induced the augmentation of amplitude of EPSCs and these augmentation were canceled by perfusion of NK1R antagonist, sendide (1μM).These results suggest that SP acts as neuromediator, which plastically changes the reactivity of neurons near NA via NK1R activation and hence might participate in generating autonomic responses accompanying with nausea. [J Physiol Sci. 2007;57 Suppl:S239]

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To evaluate the effect of glucocorticoid on fever induced by inflammation within the peritoneal cavity, we observed core temperature (Tc) in Wistar strain rats given LPS or cytokines (IL-1, TNF, IFN) with or without methylpredonisolone succinate (MP). Tc was recorded in free moving animals using a telemetry system. Vagal afferent nerve activities in response to an administration of LPS (100 μg/kg) increased markedly in intact rats under urethan anesthesia. When animals were given LPS, a marked increase in nocturnal Tc was found. The LPS-induced hyperthermia was abolished completely by the combined hepatic vagotomy with bilateral gastric vagotomy. Tc in rats given not only IL-1 but also TNF increased significantly.Such fever induced by not only LPS but also IL-1 or TNF were prevented completely by a pretreatment with 30mg/kg MP given into the peritoneal cavity before the pyrogenic challenge. These data suggest 1) that afferent signals from the hepatic and gastric branch of the vagus nerves to the brain play an important role in the transmission of an inflammatory signal concerning febrile response, and 2) that MP ameliorate the LPS-evoked fever within the peritoneal region, and 3) that fever induced by either IL-1 or TNF as well as the LPS-evoked fever might be triggered by glococorticoid-sensitive inflammatory processes via another febrific cytokines or prostaglandins yielded within gastrointestinal region. [J Physiol Sci. 2007;57 Suppl:S240]

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Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have the same sequence of amino acid residue in 68%. PACAP is known to regulate gastrointestinal secretion and motility. Indeed, electrophysiological studies of the myenteric plexus neurons of the guinea-pig ileum have shown that PACAP depolarized AH neurons (96%) and S neurons (36%). In the present study, we investigated actions of PACAP on the postsynaptic membrane and on evoked synaptic potentials of cecum submucosal neurons. Intracellular recordings were made from submucosal neurons in vitro with glass microelectrodes. PACAP applied by superfusion (3-300 nM) caused membrane depolarizations in the submucosal neurons (92%) in a dose-dependent manner (EC₅₀ = 6.3 nM), often accompanied by repetitive firings (45%). PACAP reduced the amplitude of fast EPSPs by ACh and slow IPSPs by noradrenalin. Since PACAP did not affect the postsynaptic sensitivity to ACh, it was suggested that PACAP inhibited the presynaptic release of ACh; effect of PACAP on the neuronal sensitivity to noradrenalin has to be tested. It was concluded that PACAP excited postsynaptically ganglion neurons and modulated presynaptically ganglionic transmission in the submucosal plexus of the guinea-pig cecum. [J Physiol Sci. 2007;57 Suppl:S240]

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Barrington's nucleus, or pontine micturition center, has been shown to project directly to the sacral area using neuron tracing technique: the area involves preganglionic neurons (PGN) that innervate the bladder muscle. We examined using electrophysiological technique whether this nucleus connects directly to sacral PGNs, and whether the efferent inputs from this nucleus are modulated by micturition cycle, i.e., voiding phase and relaxation phase, in cats anesthetized with α-chloralose. 1. Intracellular recordings were made from sacral PGNs that were identified by antidromic activation from the sacral ventral root. Single to three electrical shocks to Barrington's nucleus (up to 100 μA, 200Hz) evoked weak or no excitatory postsynaptic potentials (EPSPs) in PGNs during relaxation phase. On the other hand, clear EPSPs with latencies of 9-48 ms were evoked during voiding phase. Latencies of EPSPs were not fixed in any PGNs, indicating that EPSPs were evoked polysynaptically. 2. Stimulous effects of Barrington's nucleus on bladder contraction were examined. Stimulation was much more effective during voiding phase: a few train pulses were enough to evoke further bladder contraction. While during relaxation phase, 30-50 train stimulation was needed to evoke bladder contraction. The observations suggest that there is no direct connection between Barrington's nucleus and sacral PGNs, and that this descending pathway to sacral PGNs is facilitated during voiding cycle. [J Physiol Sci. 2007;57 Suppl:S240]

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Recently we showed that regional cerebral cortical blood flow (rCBF) responses induced by somatosensory stimulation in anesthetized cats with a transected spinal cord were quite different from those reported in rats: vasodilative responses in cats were restricted to the primary somatosensory cortex contralateral to the stimulated forelimb, while those in rats were bilateral and widespread. The responses in rats were due, in part, to the activation of the basal forebrain-originating cholinergic vasodilator fibers projecting to the cerebral cortex. Therefore, in the present experiments, we aimed to examine whether stimulation of the basal forebrain affects rCBF in the primary somatosensory cortex in cats. We recorded rCBF in anesthetized cats with a transected spinal cord at the first thoracic level to eliminate the influence of changes in blood pressure. The rCBF in the primary somatosensory cortex that was increased by stimulation of the contralateral forepaw was also increased by focal electrical stimulation of the ipsilateral basal forebrain. The response was the largest when the tip of the electrode was located within the area known to contain the cholinergic magnocellular neurons sending axons to the primary somatosensory cortex.These results suggest that neurons originating in the basal forebrain and projecting to the primary somatosensory cortex have the vasodilative function in cats, as previously found in rats. [J Physiol Sci. 2007;57 Suppl:S240]

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We have previously reported that wearing of mouth guard (MG) improves dynamic visual acuity (DVA) during head rotation. Some subjects, however, showed the opposite effect of the MG wearing on DVA. To test a hypothesis that unpleasant feeling caused by MG affects DVA, we monitored pupillary responses induced by flash stimulation and evaluated the autonomic nervous activity. The difference between the initial pupil diameter and that at 2.4 seconds after flash stimulation was used as sympathetic nervous activity, and the maximum velocity of pupil constriction was used as parasympathetic nervous activity (Yamaji et al., Syst Comput Jpn. 31:22-31, 2000). Seven healthy subjects (21-46 years) wore a comfortable (compact and thin) MG or an uncomfortable (large and thick) MG for 20 minutes and pupillary flash responses were monitored before, during and after MG wearing. We found that the autonomic nervous activities were clearly modulated by the uncomfortable MG. We will discuss the relationship between MG wearing and the autonomic nervous activity. [J Physiol Sci. 2007;57 Suppl:S241]

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The gastric emptying is regulated by the coordination of contraction and relaxation in antrum, sphincter and duodenum. The role of purinoceptors and nitric oxide (NO) in the regulation of neurotransmission in the gastric emptying remains unclear. We investigated KCl- and ATP-induced contractile responses in antrum, sphincter and duodenum isolated from rats, and distribution of P2X receptors in these three regions. Sodium nitroprusside, and the nicotinic agonist dimethylphenylpiperazinium iodide caused relaxation in sphincter. ATP caused contraction followed by relaxation in sphincter. Lower (5-20 mM) KCl caused relaxation in sphincter and duodenum but not in antrum, and these relaxations were inhibited by N(G)-nitro-L-arginine methyl ester. NO synthase activity was recognized in sphincter and duodenum. Major expression of P2 purinoceptor was P2X4, and minor expression was P2X6 in these three regions. The P2Y2 subtype was expressed strongly in antrum , less in sphincter and least in duodenum. Amount of P2Y2 receptor expression on these three regions may be associated with loss of contractile activity. The content of ATP was 15 times more in antrum than that in duodenum. Distribution of P2 purinoceptors and nucletides metabolism may be associated with a shift in contractile activity from contraction to relaxation. The phenotype change of P2 receptors in these three regions is associated with weakening of the ATP-induced contraction upon purinoceptor activation and may be a causal factor in the gastric emptying. [J Physiol Sci. 2007;57 Suppl:S241]

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We have demonstrated that the central nNOS neuronal system in normotensive Dahl rats was downregulatd compared with normotensive Sprague-Dawley (SD) rats. In accordance with development of hypertension, the above system especially in the brainstem became upregulated to the level in SD rats, although the nNOS activity in the diencephalon remained downregulated. Since tissue blocks used in the enzyme assay might be too large to discern alterations in nNOS in small populations of cells if they were present, in this study we tried to compare the number of nNOS neurons within the diencephalon between Dahl rats and SD rats. The Dahl salt-sensitive (DSS) and salt-resistant (DSR) rats were fed 8% NaCl (DSS8% and DSR8%) or 0.4% NaCl (DSS0.4% and DSR0.4%). SD rats were fed only 0.4% NaCl foods (SD0.4%). Immunohistochemical staining using anti-nNOS antibody revealed that the distribution of nNOS neurons in the hypothalamus is highly localized in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). The number of nNOS neurons in the SON was higher in DSS8% and SD0.4% than the other normotensive rat groups (DSS0.4%, DSR8% and DSR0.4%). While that in the PVN was higher in SD0.4% than all Dahl rat groups. These results indicated that in hypertensive DSS rats, downregulation of nNOS neuronal system in the PVN could not be normalized, although that system in the brainstem was upregulated to compensate high blood pressure. Thus, overall NO-mediated sympathoinhibition might not be enough to normalize high blood pressure. [J Physiol Sci. 2007;57 Suppl:S241]

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Although masseter muscle disorders are well known to be more prevalent in females than males, the precise reason for the sex difference remains unknown. We have recently suggested that there are parasympathetic cholinergic and non-cholinergic vasodilator fibers in the rat masseter muscle and that these fibers would be involved in the hemodynamics of jaw muscles. Estrogen has been reported to influence cholinergic activity in the heart and brain. The present study was thus designed to examine whether there are sex difference for the effect of atropine on the parasympathetic vasodilatation (PSV) in the masseter muscle and on blood flow in common carotid artery (CCABF) derived from orofacial tissues in anesthetized male and female rats. Electrical stimulation of the central cut end of the lingual nerve elicited intensity- and frequency-dependent PSV and CCABF increase in both the male and female. The PSV was much more reduced by atropine in females (>90%) than males (<30%). Pretreatment with atropine significantly reduced the CCABF increases in females, but had no effect in males. These results indicate that the PSV in the masseter muscle seems to be largely due to cholinergic mechanism in female than in male, and the cholinergic PSV in female accounts for the CCABF increase, and suggest that there are abundant parasympathetic cholinergic vasodilator fibers, which would be regulated by estrogen, in the orofacial area in females. [J Physiol Sci. 2007;57 Suppl:S241]

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The area postrema is well known to be a chemoreceptor trigger zone for emesis. Central administration of nicotine has been found to induce vomiting in cats (Beleslin & Krstic, 1987) and shrews (Rudd et al., 1999 ). Recently, emetic effects have been found in patients who were using M3 muscarinic receptor agonist for the treatment of dry mouth. To investigate the mechanism of emetic effects induced by cholinergic drugs, we performed the patch-clamp recording from area postrema neurons in the brain slices. Postsynaptic membrane currents and membrane potentials were recorded during the application of nicotine (10-50 μM) and carbachol (10-50 μM). Bath applied nicotine and carbachol caused excitatory responses in 21 of 27 cells and 3 of 14 cells, respectively. All three cells that responded to carbachol simultaneously responded to nicotine. Carbachol induced the mEPSCs without effects on the membrane potential in a cell. In the other cell, carbachol induced the marked depolarization of the membrane potential with generation of action potentials. In many cells that responded to nicotine, responses to carbachol were not observed. From these results, we considered the multiple mechanisms of muscarinic modulation of area postrema neuronal excitability. [J Physiol Sci. 2007;57 Suppl:S242]

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Recently, we showed that luminal glutamate triggered the vagal afferent activation via NO and 5-HT transduction pathways in the rat gastric mucosa (Uneyama et al., Am. J. Physiol: 2006, 291: G1163-1170). In the present experiment, we examined the existence of the functional couplings of NO and vagal nerve endings in the rat gastric mucosa. Male Sprague-Dawley rats were anesthetized with urethane and the afferent nerve discharges from nerve bundle of the left gastric were monitored. Blood pressure and body temperature were also continuously monitored during experiments. Gastric vagal afferent nerve was activated both by luminal and intra-vascular applications of NO donors such as sodium nitroprusside (SNP). SNP-evoked gastric afferent nerve discharges were abolished by pre- and post-treatment with serotonin type3 antagonist, granisetron. The SNP-evoked afferent nerve discharge was depressed by the treatment with a mucosal serotonin depletor, p-chlorophenylalanine. With receptor pharmacology using specific 5-HT receptor subtypes, it is revealed that the 5-HT receptor subtype of the gastric vagal nerve endings is dominantly type3. These results strongly indicate that a chemical perception pathway of NO-triggered 5-HT release to the vagus exists in the rat gastric mucosa. [J Physiol Sci. 2007;57 Suppl:S242]

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The aim of this study was to investigate the effect of electro acupuncture (EA) on erectile function in anesthetized male rats. Intracavernous pressure (ICP) during penile erection that was induced by electric stimulation to the cavernous nerve was measured. ICP index (filling, mean ICP, emptying, and mean ICP/mean blood pressure ratio) were calculated in before and after EA. EA was performed to the sacral area, which was penetrated 10 mm subcutaneously. In the results, ICP did not change after EA with electricity at 2 mA. ICP index, however, was significant decreased after EA at 6 mA. To determine the role of the sympathetic nerve on this result, it was denervated by 6-hydroxydopamine. After sympathetectomy, both of filling and emptying were not decreased after EA at 6 mA. Still the changes of means of ICP and ICP/mean blood pressure ratio were obtained after EA. Decrease of ICP index was disappeared after proximal denervation of the cavernous nerve on site of electrical stimulation. These results suggest that the EA stimulation affects erectile function via the sympathetic nerve pathway. And it is considered that parasympathetic and/or non adrenergic-non cholinergic nervous system might be related the changes of mean ICP. [J Physiol Sci. 2007;57 Suppl:S242]

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Aim. What factors contribute to the under internationalization of the Japanese domestic research environment ? Introduction. Among industrialized countries Japanese university environments are conspicuous by the lack of foreign scientists. This is well recognized by the Japanese government (http://job.yomiuri.co.jp/news/jo_ne_05041103.cfm). In excess of 98,000 full-time Professors and Assistant Professors are employed at Japanese universities (http://www.mext.go.jp/english/statist/index11.htm). In 2004 JSPS offered 80 long-term fellowships for senior foreign scientists to work in Japan. In 2005 this number was reduced to 70. Results. Table comparing employment conditions for senior Japanese and foreign scientists. Japanese. Foreigner. Length of contract 5-10 yrs. 10 months. Renewal upon suitable performance. No. Promotion upon suitable performance. No. Title Assistant, Associate, Professor. Guest scientist. Salary 45-60 man yen/month. 45 man yen/month. Research Funding university dependent. 200 yen/day. After expiry of JSPS funding, inequality of zaibatsu funding oportunities, a miniscule market for English language science lecturers and discriminatory government pension policy combine to force foreign scientists out of the Japanese research community. Conclusion. As the Japanese scientific population ages and young scientists seek lucrative employment in America, JSPS policy ensures that the domestic Japanese research environment is under internationalized. [J Physiol Sci. 2007;57 Suppl:S242]

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