Metabotropic glutamate receptor 1 (mGluR1) is crucial for some forms of synaptic plasticity. A wild type mGluR1 (mGluR1α) is known to functionally couple to different types of G proteins, such as Gq, Gs and Gi/o, whereas a short form splice variant, mGluR1β, has been reported not to functionally couple with Gs. We have confirmed the regulation of dual signals via mGluR1α, Gs and Gq in CHO cells, by C-terminal tail and recently have demonstrated that interaction with a cytoskeletal protein, 4.1G, inhibited the Gs coupling but not Gq. The mGluR1β is reportedly known to exhibit different dual signals, Gi/o and Gq, in BHK cells. In this study, we observed that in HEK cells mGluR1α functionally couples with Gi/o and Gq, which were monitored by a combination of FRET based-cAMP indicator and Ca²⁺ indicator, as a decrease in intracellular cAMP concentration ([cAMP]_i) and an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), respectively. In addition, activation of mGluR1α evoked an inward current through co-transfected G protein-gated inwardly rectifying K⁺ (GIRK) channels, indicting the direct interaction of mGluR1α with Gi/o. The mGluR1β could not decrease the [cAMP]_i but could increase the [Ca²⁺]_i, which indicates that the dual signals of mGluR1 in HEK cells, activation of Gi/o and Gq, were also regulated by C-terminal tail. [J Physiol Sci. 2007;57 Suppl:S86]

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Sustained elevation of intracellular concentration of calcium ion ([Ca²⁺]i) in platelets induced by several stimuli such as collagen leads to the exposure of phosphatidylserine (PS) from the inner leaflet of the platelet plasma membrane to the outer leaflet. The exposed PS provides a catalytic surface for interacting coagulation factors, which in turn converts prothrombin into thrombin via prothrombinase complex, resulting in fibrin clot formation. However, we have not yet known whether these processes on a thrombus occur in the exactly same way inside blood vessel because of difficult manipulation of platelets outside blood vessel without provocation of platelet activation before an experiment. To overcome this drawback, we attempted to visualize thrombus formation induced by laser and examine its mechanisms in the GFP-expressing mouse mesenteric vein using intravital microscopy. We employed GFP-labeled platelets as a marker of platelet aggregation and the exposed PS as an initial maker of coagulation. We monitored change in fluorescence intensity of GFP-labeled platelets and of alexa-568 labeled annexin V (alexa-ANX), a specific binding protein for PS. The change in fluorescence intensity of alexa-ANX spatiotemporally increases at the site of a laser-induced thrombus, suggesting that the PS-exposed platelets propagate from the initial site of thrombus formation. Thus, sustained [Ca²⁺]i elevation in platelets leads to the exposure to PS, thereby promoting fibrin clot formation inside blood vessel. [J Physiol Sci. 2007;57 Suppl:S86]

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The diffusive water permeability of mouse parotid acinar cells was measured by ¹H nuclear magnetic resonance relaxation method using an extracellular relaxation reagent, gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). The rate constant for water efflux from the acinar cell suspension was estimated to be ca. 5 s⁻¹ at 28^oC which would be consistent with diffusive water permeability (Pd) of ca. 1.4 x 10⁻³ cm/s. This value is the same range of those obtained in perfused rat submandibular gland, Necturus gallbladder and human red blood cells (1.6 to 4.7 x 10⁻³ cm/s). Activation energy of water transport through the cell membrane (Ea) was estimated from temperature dependence (5-30^oC) of the rate constant for water efflux. From the slope of Arrenius plot, Ea value is estimated to be 4.6 ± 0.6 kcal/mol. This activation energy is much smaller than that obtained in the lipid bilayer vesicles (12- 14 kcal/mol), and is the same range of red blood cells with AQP1 (Ea = 5 kcal/mol). Thus, this indicates that water transport of acinar cells should be done by a channel-mediated pathway, and suggests contribution of AQP5. A potential inhibitor of AQP5 water channel, 50 uM HgCl₂, was applied to the acinar cells. The rate constant for water efflux from the cells did not change significantly, but only a tendency to decrease. Since 50 uM of HgCl₂ is a half of 100 uM used for perfused rat submandibular gland, it is possible that Hg²⁺ did not reach the effective concentration to inhibit AQP5. [J Physiol Sci. 2007;57 Suppl:S87]

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Exposure of humans to space flight condition resulted in numerous alterations in immunological and hormonal parameters. Similar changes are observed during head-down bed rest (HDBR). The mechanism and responsible factors of the changes in immunological parameters induced by space flight are not clear. The aim of this study was to investigate whether exposure to the "stress" environment are responsible for alterations in synthesis of this parameters and if the changes occur, there are possibility to use exercise to prevent this negative effect. In the study 12 volunteers were subjected for 20 days to a HDBR of -6 degree; 6 of them were exposure to physical exercise. The following parameters have been analyzed: noradrenaline (NA), adrenaline (Adr), dopamine, ADH, angiotensin II, aldosteron, ANP and BNP, renin plasma activity (PRA), peripheral white blood cells count (WBC), IL- 6, total serum protein (TP), CRP and α-1antichimotrypsin (ACT). In all subjects after HDBR: Adr, NA and PRA concentrations increased significantly, CRP decreased (ANOVA p<0.05).Concentrations of TP, ACT, CRP and WBC tended to be increase after 3 day of HDBR. Concentration of TP in non-exercise group was higher than in exercise group after 20 day of HDBR. We didnt observe differences between concentrations of other analyzed parameters in subjects maintained in these two groups. The results of this study indicate that several parameters are modulated by an antigravity posture during HDBR. [J Physiol Sci. 2007;57 Suppl:S87]

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Circadian rhythms are generated by endogenous clocks and regulate the daily fluctuations of biochemical, behavioral, and physiological rhythms. It has been shown that the disruption of circadian rhythms affects many physiological events including metabolism, however, the molecular mechanisms that connect circadian clocks and metabolism are poorly understood. The Nocturnin (Ccrn4l) gene encodes a deadenylase, which removes polyA tail and destabilizes target mRNA expression post-transcriptionally. The expression of mouse Nocturnin (mNoc) mRNA is rhythmic in many tissues, and peaks at early night. We found that the mice lacking mNoc gene were resistant to diet-induced obesity and remained lean when they were fed with High-Fat diet. The mutant mice do not exhibit increased activity or reduced food intake. Furthermore, KO mice had lower body temperatures, although metabolic rates of these mutant mice remained same as WT. Finally we have observed altered expression patterns of many genes known to be involved in regulation of lipid synthesis or utilization. Our data indicate that Nocturnin has an important role in the pathway that connects circadian rhythms and metabolism perhaps through changes in lipid uptake or storage. Given that mNoc is a deadenylase, the most probable explanation for this lean phenotype would be alterations in post-transcriptional regulation of mRNAs involved in these processes. [J Physiol Sci. 2007;57 Suppl:S87]

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When ambient temperature is decreased, mammals exert autonomic and behavioral thermoregulatory responses to keep their core temperatures. However, what molecules trigger these responses is still unclear. Recently, transient receptor potential melastatin-8 (TRPM8) was identified as cooling- and menthol-activated cation channel. We hypothesized that TRPM8 activation triggers autonomic and/or behavioral responses against cooling. All experiments were performed at 27ºC (room temperature). For TRPM8 activation, we used menthol instead of cooling. We applied 10% menthol in ethanol to mice skin, and measured core temperatures and autonomic and behavioral responses. Significant elevation of core temperatures (hyperthermia) was observed between 20 min and 2 h after menthol application. Pretreatment with diclofenac sodium, an antipyretic drug, did not affect this hyperthermia, indicating that the menthol-induced hyperthermia is not fever. Menthol application induced a rise in oxygen consumption, tail skin vasoconstriction and heat-seeking behavior, all of which are typical responses against cooling. We conclude that TRPM8 activation triggers autonomic and behavioral thermoregulatory responses against cooling. [J Physiol Sci. 2007;57 Suppl:S88]

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alpha-klotho was first identified as the gene responsible for premature ageing-like phenotypes in a short-lived mouse model. We found the molecular association of alpha-Klotho and Na+,K+-ATPase and subsequently a novel alpha-Klotho dependent pathway that recruits Na+,K+-ATPase to the plasma membrane. Low [Ca²⁺]e rapidly induces regulated PTH secretion, in an alpha-Klotho and Na+,K+-ATPase dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca²⁺ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings provide a paradigm for the fundamental roles of alpha-Klotho in the regulation of calcium metabolism. [J Physiol Sci. 2007;57 Suppl:S88]

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In order to determine the energy expenditure and heart rate during triathlon race, we tried to record ECG during triathlon competitions by using a newly developed waterproof Holter-electrocardiograph. Five college-level triathletes, 19-22 years old, 3 men and 2 women, volunteered in this study. ECGs in 4 subjects with Holter ECG (FM-180, Fukuda Denshi) and heart rates in 2 subjects by HR monitor (TZ-MAX50, Acumen) were recorded during triathlon competitions. Three incremental exercise tests until exhaustion in swimming, cycling and running were carried out in the laboratory and oxygen consumption and heart rate were monitered. Using the individual correlating equation between heart rate and oxygen consumption, energy expenditure during triathlon race was estimated. Except minimum artifacts during swimming, high quality recordings of ECG were obtained during triathlon race. No subject showed any arrhythmia except minimum numbers of premature beats. Mean competition time (±SD) for Olympic distance (1.5 km swimming, 40 km cycling and 10 km running) was 2h31m17s ±20m25s. Time for swim, bicycle and run were 27m41s ±4m23s, 1h13m05s ± 6m30s, and 50m31s ± 6m30s, respectively. Total heart beats and heart beats for swim, bicycle and run were 26539 ±3120, 4706 ±719, 1283 ±1079 and 8995 ±1610 beats, respectively. HR for race and HR for swim, bicycle and run were 175.9 ±6.5, 170.4 ±9.8, 175.8 ±6.3 and 179.0 ±6.4 bpm, respectively. [J Physiol Sci. 2007;57 Suppl:S88]

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Glaser and Shepherd (1961, 1963) suggested that man could be acclimatized simultaneously to both heat and cold. In this study, effects of heat- and cold-acclimation on thermoregulatory responses were examined in rats. Male Wistar rats aged 10, 22 and 53 wk were used. The rats were housed separately for 28 days under controlled conditions of ambient temperature (Ta) of 32°C from 9:00 to 21:00 and Ta of 5°C from 21:00 to 9:00, and 12 h light/dark cycle. Control rats were housed under constant Ta of 23°C for 28 days. After the acclimation period, thermoregulatory responses to the acute thermal stress were examined. Core temperature (Tc) and heart rate (HR) were continuously measured by using a telemetry system throughout the experiment. Rats aged 10 wk could regulate their Tcs within a narrow range during the acute thermal stress regardless of acclimated or not. Acclimated rats aged 10 wk showed a lower Tc and HR levels than those of unacclimated rats under warm Ta range (20 to 30°C). Tcs and HRs of acclimated rats aged 22 and 53 wk showed a lower levels than those of unacclimated rats of the same age under cold Ta range (0 to 10°C), but remarkable effect was not observed under warm Ta range. In summary, cold-acclimated effects were apparently observed in Tc and HR responses rather than warm-acclimated effects in warm- and cold-acclimated rats aged 22 and 53 wk. [J Physiol Sci. 2007;57 Suppl:S88]

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Resistin is a newly identified as a cysteine-rich adipose tissue-specific secretory adipokine that may link obesity and insulin resistance. We have studied the effect of resveratrol, a phytoalexne stilbene from grapes, on the regulation of resistin gene expression during 3T3-L1 adipogenesis. Resveratrol treatments attenuated adipocyte differentiation, which resulted in a dramatic decrease in resistin mRNA levels as well as its protein levels secreted into the culture medium in a dose-dependent fashion. In contrast, epigaro-catechin-3-gallate, a main polyphenoric compound of green tea leaf which has antioxidant potency same as resveratrol, did not repress resistin gene expression in the same dose-range. Transient transfection experiments with resistin promoter/luciferase reporter revealed that resveratrol reduced transcriptional activity of the resistin gene in a dose-dependent manner. These results indicate that the resistin gene expression is down-regulated by resveratrol through the reduction of gene transcription in 3T3-L1 adipocytes. [J Physiol Sci. 2007;57 Suppl:S89]

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To investigate whether stimulatory effect of ghrelin on food intake and inhibitory effect of leptin on gaining body weight are modified by cholecystokinin (CCK), levels of ghrelin, leptin and CCK in circulatory blood were measured by ELISA method at 7 and 38 weeks old of male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and its normal counterpart of male Long-Evans Tokushima Otsuka (LETO) rats. The averaged body weight of OLETF rats and levels of active ghrelin and leptin in OLETF rats were significantly increased as compared to those of LETO rats. Level of CCK of OLETF rats were significantly decreased as compared to those of LETO rats. Multiple linear regression analysis demonstrated that the ratio of active ghrelin to desacyl ghrelin was a significant determinant for food intake in OLETF rats and that leptin was a significant determinant for body weight in LETO rats. The ratio of the difference in the amount of food intake between the ages of 7 and 38 weeks to the difference in levels of active ghrelin between the ages was increased when CCK was decreased. The ratio of the difference in body weight between the ages to the difference in levels of leptin between the ages was increased when CCK was decreased. Thus, the decreased level of CCK in OLETF rats augment the effect of ghrelin on feeding and the inhibitory effect of leptin on gaining body weight, simultaneously. In OLETF rats, the effect of ghrelin may dominate that of leptin, resulting in obesity. [J Physiol Sci. 2007;57 Suppl:S89]

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Purpose: Dystrophin-glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures of muscle fibers which make a linkage between extracellular matrix (ECM) and intracellular cytoskeleton. They play a role of transmitting force from myofibrils to outside the fibers, but it is unknown how DGC and FAC are associated with contractile properties of skeletal muscles. The purpose of this study was to investigate whether the expression of dystrophin (component of DGC) and β1 integrin (component of FAC) have muscle type specificity in rat skeletal muscle. Methods: Soleus (SOL), plantaris (PLA), extensor digitorum (EDL), tibialis anterior (TA) muscles were dissected from male Wistar rats. The content of dystrophin and β1 integrin and the composition of myosin heavy chain isoforms were measured in these muscles. Immunohistochemical staining for dystrophin and β1 integrin in the cross-section of these muscles was also performed. Results: The dystrophin relative content was different among the muscles (SOL < PLA < EDL < TA), and β1 integrin was less contained in TA than in the other muscles. β1 integrin was expressed more strongly in type I and type IIa fibers than in type IIx and IIb, whereas dystrophin did not show fiber type specific expression. Discussion: Our data suggest that the expression of both DGC and FAC are contained more abundantly in the muscles which are recruited more frequently, increasing the efficiency of transmitting contractile force during daily physical activity. [J Physiol Sci. 2007;57 Suppl:S92]

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Rho-kinase (ROK)-mediated Ca²⁺-sensitization plays an essential role in abnormal VSM contraction such as vasospasm. We identified sphingosylphosphorylcholine (SPC) as upstream signaling molecule of ROK-mediated Ca²⁺-sensitization of VSM. Moreover, we found that SPC required activation of Src family tyrosine kinases (Src-TKs) to induce ROK-mediated Ca²⁺-sensitization. Among Src-TKs, Fyn and c-Src were found to be expressed in VSM tissue and cultured cells, and SPC induced the translocation of Fyn, but not of c-Src, from cytosol to plasma membrane of VSM cells. In order to obtain the direct evidence for the involvement of Fyn, we made recombinant Fyn proteins either in active or inactive form, using a baculovirus system and applied them to the cytosol of VSM whose membrane was permeabilized with β-escin. The constitutively active Fyn induced Ca²⁺-sensitization in VSM permeabilized with β-escin, but not with α-toxin, which was inhibited by a ROK inhibitor Y27632. The dominant negative Fyn (dn-Fyn) inhibited the contractions induced by SPC or a GPCR agonist U46619 +GTP. In addition, the SPC-induced contraction was about 50% of the maximum force and it was remarkably diminished to about 18% by applying dn-Fyn (n=4, P<0.01). These findings suggest that Fyn tyrosine kinase plays a pivotal role in the ROK-mediated Ca²⁺-sensitization of VSM contraction induced by SPC or U46619. [J Physiol Sci. 2007;57 Suppl:S92]

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Recent findings indicate that the sarcomere length (SL)-dependence of activation is modulated partly via a reduction in interfilament lattice spacing due to titin-based passive force. We tested the hypothesis that the Frank-Starling mechanism of the heart is regulated at the thin filament level, by the Ca²⁺ regulatory protein, troponin. We used skinned porcine ventricular muscle. Reconstitution of the preparations with fast skeletal troponin (from rabbit psoas muscle) increased Ca²⁺ sensitivity and attenuated SL-dependent increase in Ca²⁺ sensitivity. SDS-PAGE analysis showed that endogenous cardiac troponin subunits were clearly replaced with skeletal counterparts and titin was not degraded upon troponin exchange. Pimobendan, increased Ca²⁺ sensitivity similar to sTn; however this compound did not affect SL-dependent activation, suggesting that a simple increase in Ca²⁺ binding to troponin C does not underlie the attenuation of SL-dependent activation by sTn. Also, sTn diminished osmotic compression-induced increases in Ca²⁺ sensitivity. We found that the Ca²⁺-desensitizing effect of Pi was attenuated in sTn-reconstituted preparations, suggesting that cross-bridge formation is accelerated upon sTn reconstitution. It is likely that the proportion of detached cross-bridge is decreased as cross-bridge formation is accelerated. Therefore, sTn may attenuate SL-dependent activation via reduction in the proportion of detached cross-bridges that can potentially produce active force. [J Physiol Sci. 2007;57 Suppl:S92]

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Magnetic resonance imaging (MRI) is an important modality with no radiation exposure, used daily for clinical diagnosis. MRI is originally developed based on discovery the difference of relaxation time of water protons between tumor and normal tissues. Although MRI has rapidly spread clinically, the origin of the difference has not been resolved. Interpretation of signal intensity of MR image is commonly based on three water state model, that is, bulk, structural and bound water. But our recent experiment with nuclear magnetic resonance (NMR) on frog skeletal muscle revealed that tissue water is distinctly classified into 5 groups, whose characteristic relaxation time is T₂ > 0.4 s, T₂ around 0.15 s, 0.03 s < T₂ < 0.06 s, T₂ < 0.03 s and the fifth groups of T₂ shorter than the observable value with our pulse sequence. The water component with T₂ > 0.4 s was revealed as extracellular water, and the other four components were distinctly separated each other by their responses for perturbations of osmotic pressure. Based on our result obtained with frog muscle, we tried to reconstruct signal intensities in every pixel of human brain MR image as summation of signals from those five water components. Reconstructed model brain images were very similar to the original T₂-weighted images obtained by turbo spin echo sequence method. This means that water components classified in skeletal muscle are applicable to other human tissues. Analysis of MR image of other human organs will be discussed. [J Physiol Sci. 2007;57 Suppl:S93]

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Many of the troponin mutants which can cause familial cardiomyopathy have been reported so far. To elucidate the molecular mechanism of the cardiomyopathy related to troponin, we performed molecular dynamics study on the structure of troponin mutants which relate to familial hypertrophic cardiomyopathy (HCM). Two different troponin T mutant which relate to HCM, Glu244Asp and Lys247Arg were studied. Dynamics was calculated by the use of software Amber (vers. 9). Iteration was done in TIP3 water sphere with 0.5 or 1 fs time step in periodic condition at constant temperature (310 K) and pressure. Model structures of troponin mutants were constructed by introducing the mutation to the crystal structure of human cardiac troponin (core region of TIC complex) obtained from Protein Data Bank (ID number 1J1E). More than 4 trajectories of 1ns were obtained for wild and mutant structure. It was observed that electrostatic interaction between troponin I and troponin T which linked alfa helix of troponin T and troponin I in the wild was lost in the mutant. Furthermore, when a terminal residue of troponin I was pulled toward an actin molecule mimicking the intra-molecular force on activation, different structural change was observed in mutant compared with wild. The involvement of this difference between the wild and mutant in the tension regulation will be discussed. [J Physiol Sci. 2007;57 Suppl:S93]

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When Ca²⁺ activated skeletal muscle fibers are stretched with a moderate velocity, the force developed above the level of isometric force starts to decay after a sudden reduction of stretch velocity. To investigate behavior of the cross bridges to reduction of stretch velocity, we measured muscle fiber stiffness. Glycerinated single fibers from the rabbit psoas muscle were isometrically attached (Lo: 2.4um) between force transducer and servo motor. Sinuoidal lenght change of 0.1% p-p Lo at 2 kHz was applied to the fibers. When isometric force reached the steady state, the fibers were stretched 1.5%Lo at the velocity of 0.35 Lo/s and then further stretched 1.5% Lo at 0.035Lo/s. Muscle fiber stiffness and force were simultaneously recorded. The force increased by the first stretch decreased transiently with reduction of stretch velocity, and increased slowly, and after the completion of stretch it caused stress relaxation. On the other hand, muscle fiber stiffness started to decrease by the first stretch and then increased in the late phase pf the first stretch, and it increases slowly during the following slow stretch, after completion of stretch it settled in the level lower than the stiffness level under isometric force. Although number of attached cross bridge decreases during the first stretch from the above results, it is found that when stretch velocity decrease suddenly, large change in number of attached cross bridges is not seen even if it generates comparatively large force by lock-in cross bridges. [J Physiol Sci. 2007;57 Suppl:S93]

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In our research, we try to design a new transducer which using for force measurement during muscle contraction of myofibrils. We have used the principle of magnetic force of cubodial permanent magnets which exerts on metal materials in order to create a novel transducer that having T shape. The transducer is suitable for measurement of contractile force with 1µN resolution. In order to increase high resolution for measurement application, we have improved its natural frequency. As results shown, the natural frequency of our transducer have increased up to more than 100 Hz without reduce the weight. Beside that, we also found out the new way to manufacture very small steel needle with the size of 5 to 10 micrometer and even smaller. Natural frequency of the system can adjust by changing distance between two cubodial permanent magnets. Force measurement of biological application used laser reflection measurement by position sensitive detector circuit. We have successfully used this transducer to measure the force of glycerinated single fiber during its contractile and found that, it is useful for myofibrils level. In the future, we will continue to improve measurement system that will use for myofibril and/or myocyte measurement. [J Physiol Sci. 2007;57 Suppl:S93]

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Recently,it has been reported that the administration of granulocyte colony-stimulating factor (G-CSF) stimulated the angiogenesis and the regeneration of injured cardiac muscles by the mobilization of bone marrow stem cells(BMSCs).It has been suggested that BMSCs may participate in the regeneration of injured skeletal muscles.Therefore,the present study was performed to investigate the effects of G-CSF administration on the regenerative process of injured mammalian skeletal muscles in vivo.The effects of muscular activity-depression on the regeneration of skeletal muscles were also investigated. Male mice (C57BL/6J) were divided randomly into 7 groups:(1)cage control,(2)administration of G-CSF,(3)cardiotoxin(CTX)-injected(CX),(4)administration of G-CSF and CTX-injected(GX),(5)immobilized,(6)administration of G-CSF and immobilized,and (7)administration of G-CSF,CTX-injected and immobilized(GXI) groups.CTX was injected into tibialis anterior(TA) muscles of both limbs in CX,GX and GXI groups.We gave first injection of G-CSF 3 days before CTX-injection,and the administration was repeated for 8 consecutive days.TA muscles were dissected 7,14 and 28 days after CTX-injection.Results suggested that G-CSF may facilitate the regeneration of injured and immobilized skeletal muscles. [J Physiol Sci. 2007;57 Suppl:S94]

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Activation of muscle satellite cells (MSCs) plays a key role in muscle hypertrophy and regeneration. It is considered that MSCs are activated by muscle contraction, mechanical stretch,muscle injuries, heat-stress, etc. On the other hand,the denervation causes muscle atrophy. We hypothesized the denervation may depress MSCs activity and may delay the regenerative process of injured muscles. The purpose of this study was to investigate the effects of denervation on the regenerative process of injured skeletal muscle in mice. Male mice (C57BL/6J) were divided randomly into six groups: (1) cage control, (2) cardiotoxin (CTX)-injected (CX), (3) heat-stressed and CTX-injected, (4) denervated (DC), (5) CTX-injected and denervated (DX), and (6) CTX-injected, denervated,and heat-stressed (HDX) groups. In animals of DC, DX, and HDX groups, the denervation of hind limb muscles was performed by cutting the sciatic nerve at the gluteal region. Two weeks after the surgery, CTX was injected into tibialis anterior (TA) muscles of both limbs in CX, DX and HDX groups. TA muscles were dissected 14 and 28 days after CTX-injection. Responses of muscular protein contents and Pax7-positive muscle satellite cells during the regeneration were analyzed. Evidences suggested that heat-stress related facilitation of regeneration of injured muscle was delayed by denervation. [J Physiol Sci. 2007;57 Suppl:S94]

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During a very low load shortening, the intensity ratio of (1,0)/(1,1) equatorial x-ray diffraction (BL45XU at SPring 8), which indicates the mass transfer from myosin to actin filaments, does not decrease so much as during isometric contraction, that is, the amount of mass change is less during low load shortening. The (1,0) lattice spacing (d10) is one of the factors which affects the mass transfer. To examine the reduced mass transfer, the d10 during shortening is carefully studied in rat papillary muscle. The intensity of (1,0) equatorial diffraction is fitted by Gaussian equation, and its standard deviation (σ10) is calculated. Once σ10 is normalized by its mean value (ε10), it varies uniquely during isometric contraction compared with ε10 during low load shortening. The d10 increases more during low load shortening than during isometric contraction as is the results of sarcomere shortening. However the ε10 increases during isometric contraction, it flattens during low load shortening. Therefore, even if the d10 spread widely during low load shortening, the d10 disorder is less pronounced than during isometric contraction. It is suggested that the d10 disorder can be affected by the interaction between actin and myosin filaments caused by crossbridge formation and the amount of disorder may be associated with the strength of force generated by corssbridges. [J Physiol Sci. 2007;57 Suppl:S94]

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Responses of adductor longus muscle to hindlimb unloading followed by reloading were studied in 5-wk-old male Wistar Hannover rats. Hindlimb suspension was performed for 16 days. Half of the suspened group was sacrificed at the end of suspension and adductor longus was sampled. The remaining half was allowed to recover at 1-G environment for 16 days. Serial transverse cross sections (10-μm thick) were cut in a cryostat at -20 °C for immunohistochenical analyses. The% of each fiber phenotype and specific activities of succinate dehydrogenase and α-glycerophosphate dehydrogenase, as well as the fiber cross-sectional area were analyzed. Significant shift of fibers toward fast-twitch type and atrophy were noted in the suspended groups. The% of pure type I fibers was decreased and type I+II fibers was increased. After 16 days, the atrophy, not the phenotype, of fibers was recovered to the control revel. The responses of enzyme activities to unloading were not prominent. It is suggested that adductor longus of Wistar Hannover rat is very susceptible for gravitational unloading relative to other species, although the precise mechanism is still unclear. [J Physiol Sci. 2007;57 Suppl:S94]

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Effects of tenotomy (T) and/or denervation (D) on the properties of plantaris muscle fibers were studied in wild type (WT) and mdx mice. Distal tendons of the left plantarflexors were ablated in the T group. Sciatic nerve of the left limb was transected (–5mm) at the gluteal region in the D group. Both treatments were performed for T+D. The contralateral side was kept intact as the control. After 2 weeks, the plantaris muscles of both mice were sampled bilaterally and frozen in the liquid nitrogen-cooled isopentane. Subsequently, the serial cross sections were cut at 10 μm thickness and the immunohistochemical staining for the type I or II myosin heavy chain (MHC) or the nuclear staining was performed. The muscle was predominantly composed of fibers expressing only type II MHC (93%) in both mice. The remaining fibers (7%) were expressing type I+II MHC. The percent distribution of central-nucleated fibers was 58% in mdx mice, although few fibers contained the central-nuclei in WT mice. The percentage of the central-nucleated fibers was not affected by T, D, or T+D in both mice. The fiber cross-sectional area (CSA) was significantly decreased following D or T+D vs. contralateral side. Only in T group, CSA was significantly decreased in central nuclei fibers, but not in fibers without central nuclei. The magnitude of the response was similar between WT and mdx mice. The results suggested that the mechanism responsible for the plasticity of plantaris muscle is different from that of soleus muscle (Terada et al., J. Gravit. Physiol., 2005). [J Physiol Sci. 2007;57 Suppl:S95]

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Twelve weeks old male Wistar rats were separated into the pre- and post-experimental control, functional overload (FO), hindlimb-unloading (U), deafferentation (DA) and FO+DA groups. The tendons of the synergists of soleus muscle were transected in the FO rats. The continuous tail suspension was performed in the U rats. The dorsal roots of the L4-5 segmental levels of the spinal cord were transected in the DA rats. The sampling of the soleus muscle was performed before and 14 days after each treatments. Hypertrophy (+24%) of muscle fibers, associated with increased number of nucleoli within a single myonucleus, was induced by FO. The FO also increased the expression of the phosphorylated ribosomal protein S6 (S6) (+84%). Although the fiber size (-52%) and the expression of the phosphorylated S6 (-98%) was remarkably decreased following U, the nucleolar number was unchanged. Responses to DA were basically similar to those to U, although the magnitude of the effect was minor. The DA-related responses of these parameters were prevented by further addition of FO. FO+DA also resulted in the increase of the nucleolar number. These results suggested that the intranuclear number of nucleoli, which is stimulated by FO, may play an important role in the rate of protein synthesis and fiber size. [J Physiol Sci. 2007;57 Suppl:S95]

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Role of macrophage in the regulation of soleus muscle fibers in response to gravitational unloading followed by reloading was investigated in adult op/op (-/-) mice with deficient macrophage colony-stimulating factor, as well as heterozygous (+/-) and wild type (+/+) mice. They were separated into cage control and unloaded groups. The sampling of soleus muscle was performed before suspension, immediately after 10 days of hindlimb suspension, and 10 days after recovery. The fiber cross-sectional area (CSA), myonuclear number, and the number of quiescent and mitotic active satellite cells were measured in the single muscle fibers of soleus, which were isolated from tendon to tendon by collagen digestion. The mean length of muscle fibers and tibia of -/- mice was shorter than +/+ and +/- mice in both cage control and suspension groups, suggesting that the inhibition of longitudinal muscle growth in -/- mice was closely associated with the shorter bone length. The fiber CSA was decreased by unloading in all mice. However, the fiber CSA in -/- mice did not recover following 10 days of reloading, whereas the CSAs in +/+ and +/- mice were increased to the control level. Such irreversible effects were also observed in the myonuclear number and the number of quiescent and mitotic active satellite cells in -/- mice. It is suggested that the macrophages play one of the key roles during the recovery from muscular atrophy. [J Physiol Sci. 2007;57 Suppl:S95]

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Responses of single muscle fibers of human vastus lateralis to 8 weeks of either concentric or eccentric training were studied in 16 male subjects using a specially designed training machine with the compulsory rotation of pedals induced by electrical engine. The major difference of the machine from the traditional one was that the seat was not fixed firmly, but slid along an inclined beam. As a result, the lower extremities exerted the load which was a part of the subject's weight and depended on the angle of inclination. The machine was designed to choose either concentric or eccentric exercise of hip and knee extensors by changing the direction of pedal rotation. The aim of this experiment was to compare the effects of long-lasting training in concentric or eccentric regime with an equal external power. Fiber cross-sectional area (CSA), myonuclear number, myonuclear CSA, and myonuclear domain were analyzed in single muscle fibers. Myonuclear CSA and myonuclear number per mm of fiber length did not change after training in both regimes, although fiber CSA was increased significantly after eccentric training. Myonuclear domain size was also increased significantly by concentric and eccentric exercise. It was suggested that eccentric training was more effective for induction of hypertrophy of muscle fibers. However, these phenomena were not related to the increase of number and/or size of myonuclei. [J Physiol Sci. 2007;57 Suppl:S95]

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It has been well known that in skeletal muscle clenbuterol (CB, a beta-2-adrenergic agonist) promotes muscle hypertrophy as well as a fiber-type transition, whereas rapamycin (RAPA, a inhibitor of the mammalian target of rapamycin, mTOR) induces atrophy. To our knowledge, the interaction between the two drugs at the cellular level has not been studied. To gain more insight into the molecular mechanism of muscle growth and fiber-type transformations, we analyzed the effects of CB and/or RAPA on the diameter of muscle fibers as well as on the fiber-type composition in rat soleus muscle by using a immunohisochemical method with anti-fast and slow-type myosin heavy chain antibodies. As compared with control, the diameter of fast, slow and mixed fibers were significantly increased by the CB treatment (p<0.05) but decreased by the RAPA treatment (p<0.05), while that of these fibers did not change with the CB+RAPA treatment. The CB, RAPA and CB+RAPA treatments significantly (p<0.05) increased the relative contents of fast and mixed fibers at the expense of that of slow fibers. These results suggest that in rat soleus muscle the CB treatment as well as the RAPA treatment induce the slow-to-fast fiber-type transition, while the CB treatment induces the muscle hypertrophy, which is antagonized by rapamycin. [J Physiol Sci. 2007;57 Suppl:S96]

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To elucidate the mechanisms of relaxing effect of 2-Amino-4,4 α-dihydro-4 α-7-dimethyl-3H-phenoxazine-3-one (Phx-1) on the smooth muscle preparations, we investigated Phx-1 effects on the contraction of both intact and skinned (cell membrane peremeabilised) preparations from guinea pig taenia In intact preparations, Phx-1 dose dependently suppressed either the contraction induced by acetylcholine (ACh) or high-K⁺. Similar force inhibitory actions of Phx were observed in intracellular Ca store depleted preparations. In the cell membrane depolarized preparations in the absence of extra Ca, however, Phx-1 had little effects on the caffeine- or ACh-induced contraction. In skinned preparations, Phx-1 suppressed the Ca²⁺-induced contraction at only relatively higher concentrations. These results suggested that inhibition of the smooth muscle contraction by Phx-1 was due mainly to inhibition of Ca²⁺-influx, although Phx-1 seemed to have direct inhibitory effects on the activities of contractile apparatus [J Physiol Sci. 2007;57 Suppl:S96]

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In this study, we investigated whether or not disuse alters sarcomere length (SL) dependence of activation, using single skinned fibers of rat soleus muscle. Disuse was induced by hindlimb immobilization with casting tape, resulting in a reduction in wet weight of soleus muscle by ∼40%. We measured maximal Ca²⁺-activated force at varying sarcomere lengths (SL), from 2.0 up to 4.0μm. It was found that the descending limb of the relationship was shifted to the left upon disuse, with no change in the ascending limb. Electron microscopic observation demonstrated that the thick filament length was not uniform and varied in disused muscle, with shorter filaments coexisting with normal filaments, resulting in shortening of the average A-band length. The I-band length, on the other hand, tended to be shorter upon disuse, but not significant. These structural changes are likely to decrease the overlap between thick and thin filaments, and consequently reduces active force, especially at longer SLs. Our biochemical studies revealed that titin expression was reduced upon disuse, accompanying a reduction in passive force, and that the expression of other major sarcomere proteins was similar in muscles with and without disuse. It has been reported that titin may function as a molecular scaffold for thick filament formation during myofibrillogenesis. Therefore, a long-term reduction of titin expression may form myofibrils with altered sarcomere structure, resulting in the changes in SL-dependence. [J Physiol Sci. 2007;57 Suppl:S96]

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We investigated how hindlimb immobilization affected the contractile function of single soleus fibers from the rat. Hindlimb immobilization with casting tape reduced wet weight of soleus muscle by ∼40%. We found by using Triton X-100-treated single fibers that Ca²⁺ sensitivity and maximal Ca²⁺-activated force were reduced after disuse. A similar result was obtained with saponin-treated single fibers with intact sarcoplasmic reticulum function; that is, Ca²⁺ sensitivity as well as maximal force was reduced after disuse. Then, in order to investigate whether or not the reduction in Ca²⁺ sensitivity is due to possible isoform switches of troponin molecules, we reconstituted thin filaments of control and immobilized fibers with the identical troponin complex. It was found that Ca²⁺ sensitivity was still lower in immobilized fibers after troponin reconstitution, suggesting that troponin isoform switches, if at all, can not account for functional changes of immobilized fibers. Instead, our small-angle X-ray diffraction experiments revealed that interfilament lattice spacing was expanded by ∼6% in immobilized fibers. Dextran T-500 (MW, ∼500,000) 2% (w/v) reduced the lattice spacing to a level similar to that observed in control fibers and almost completely restored Ca ²⁺sensitivity. These results favor the interpretation that lattice expansion, but not troponin isoform switches, underlies the disuse-induced reduction in Ca²⁺ sensitivity. [J Physiol Sci. 2007;57 Suppl:S96]

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The longitudinal and transverse stiffness of myofibrils of rabbit skeletal muscle were examined under various physiological conditions to study the mechanical characteristics of the sarcomere structures of skeletal muscle fibers. Myofibrils were prepared from glycerinated fibers of rabbit psoas muscle. The longitudinal stiffness was examined for single myofibrils suspended between micro-needles based on the force responses of myofibrils to sinusoidal length changes. The transverse stiffness was examined for single myofibrils immobilized to the surface of aminosilane-treated coverslip based on force-distance curves obtained for the overlap regions of myofibrils by use of an atomic force microscope (AFM). By assuming that myofibrils are uniform rods, their Young's modulus was estimated. The Young's modulus of myofibrils was about 200-times greater in the longitudinal direction than in the transverse direction under various physiological conditions. The magnitudes of the longitudinal and transverse Young's modulus of myofibrils thus obtained linearly changed by changing the physiological conditions. The results obtained suggest that the overall mechanical strength of myofibrils is almost exclusively determined by the actomyosin filament networks linked by attached cross-bridges and that the mechanical anisotropy of myofibrils comes from the anisotropic nature of the actomyosin filament networks thus produced in the sarcomere structures. [J Physiol Sci. 2007;57 Suppl:S97]

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In a previous study, we have developed a method to determine the diameter of single myofibrils with the accuracy of 5 nm by use of an atomic force microscope (AFM). In the present study, we applied this method to examine the diameter changes of single skeletal myofibrils under various physiological conditions.< BR >Single myofibrils were prepared by homogenizing glycerinated muscle fibers of rabbit psoas muscle. AFM measurements were made as previously. Myofibrils were fixed on the surface of cover slip coated with aminosilane. Modified AFM cantilevers, having a small glass sphere (2 μm in diameter) glued to the tip of commercial AFM cantilever, were used. The diameter of myofibrils was determined as the difference between the height at the top surface of single myofibrils and that at the surface of cover slip next to the myofibrils.< BR >The diameter of single myofibrils thus obtained was 1.129 ± 0.064 μm in a relaxed state, 1.008 ± 0.075 μm in a rigor state and 1.011 ± 0.087 μm in a contracting state. The diameter of single myofibrils was nearly constant along the fiber axis ( < ±0.06 μm ) in each state. By the addition of Dextran, the diameter of myofibrils decreased in characteristic fashions; i.e. it decreased by 44% in a relaxed state and by 22% in a rigor state, at 8% Dextran. The diameter changes of single myofibrils thus obtained were consistent with the spacing changes of the actomyosin filaments lattice of muscle fibers under comparable conditions examined by X-ray diffraction studies. [J Physiol Sci. 2007;57 Suppl:S97]

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We investigated the mechanical properties of flexor carpi radialis muscle (FCR), one of the forelimb muscles in a male frog used for long-lasting mating posture, so-called "amplexus", by measuring the change in force and stiffness during contraction at 4ºC in breeding season. We found that this contraction has a characteristic 'long-lasting, more than 30 min, relaxation phase' after the end of stimulation. In the course of the relaxation, higher stiffness than in resting state was observed, indicating that some myosin molecules were binding to actin filaments. In the present experiment, we further measured the contractile force and the shortening velocity of FCR in breeding (Feb.-Mar.) and non-breeding seasons (Jul.-Sep.) and compared them. It was noted that the rate of force decrease in the relaxation in non-breeding season was remarkably faster than that in breeding season, while the shortening velocities on all loads were not significantly different in two seasons. Present results indicate that the extremely slow relaxation observed in breeding season was caused by the decrease in the very slow rate of force fall. Amplexus may be brought about by this phenomenon. Further it is inferred from the present experiment that the slow relaxation may not be induced by the change in muscle fiber types but by the change in the regulation of the intracellular Ca⁺⁺ concentration. [J Physiol Sci. 2007;57 Suppl:S97]

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We found that frog skeletal and cardiac muscles can survive for a long period in crude chicken egg white (CEW) . Any extra salts or solvents were not added to CEW. Resting potentials (RP) of skeletal muscle fibers immersed in CEW reduced to about -40 mV. This may be owing to high K⁺ of CEW. By return from CEW to normal Ringer's solution (NR), the fibers slowly but steadily recovered normal RP and soon conducted normal action potentials (AP). Hind leg amputated at the ankle and dissected whole heart were treated with CEW for a period of 18 days at 1–3 °C. Fibers of toe muscles isolated from the above hind leg were almost intact at a glance. After a day or two, their RP and heights of AP measured in NR at 15–17 °C were -95 ± 2.2 mV and 123 ± 4.5 mV (n = 50, mean±SD), respectively. Similarly, those of cells of ventricle isolated from the above heart were -92 ± 4.9 mV and 122 ± 9.4 mV (n = 46), respectively. The remaining atrium automatically beat about 20 times every min as if it had beaten in situ. Interestingly, some semitendinosus muscles treated with CEW for 3–12 days did not move an inch during twitches but developed almost normal caffeine contractures. Mechanism of this inhibition of E-C coupling is unclear, but such an inhibition was not observed in muscles treated with 190 K⁺ solution or 5% albumin NR (250 mOsm). Long-term survival of the muscles in CEW may be due to environmental conditions of (1) high viscosity, (2) low temperature, and (3) mild depolarization which act to minimize energy consumption of muscles. [J Physiol Sci. 2007;57 Suppl:S97]

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Serotonin (5-HT) is a key signaling molecule in both central and enteric nervous systems. We assessed The involvement of endogenous 5-HT receptors on pacemaker Ca²⁺ activity in gastrointestinal (GI). Cell cluster were prepared from the stomach and small intestine of WT mice (BALB/c), (including the enteric neurons) by enzymatic and mechanical treatments. After 2 days of culture, the fluo-3 AM (acetoxymethly ester of Fluo-3) was loaded to measure the intracellular Ca²⁺concentration [Ca²⁺]_i. In the presence of nifedipine a dihydropyridine Ca²⁺ channel antagonist, spontaneous [Ca²⁺]_i oscillations were observed within limited regions showing c-Kit-immunoreactivity, a marker of interstitial cells of Cajal (ICC). Application of 5-HT enlarged the region of pacemaker [Ca²⁺]_i activity via type 3 5-HT receptors. RT-PCR and immunohistochemical examinations revealed expression of serotonin receptors in ICC. On the other hand, in cell cluster preparations from the stomach and small in intestine of W/W^v mice failed to respond to serotonin. The results indicated that Kit-positive interstitial cells respond to serotonin. [J Physiol Sci. 2007;57 Suppl:S98]

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Low back muscle pain is problematic because the pathway and mechanisms still remain to be solved. The neuroanatomical pathway of nociception originating in low back muscles (erector spinae, ES) was examined in this study. The neurotracer true blue (2%, 10µl) injected into ES muscles just beside the spinous process L5 was retrogradely transported to dorsal root ganglia (DRG) cells mainly at L3, not at L5. Intramuscular injection of 5% formalin into ES muscles beside the spinous processes L4 and L5 induced c-Fos protein expression with wide craniocaudal distribution from L1-L5 of the spinal dorsal horn. The labeling with c-Fos was particularly marked in the most lateral part of the superficial dorsal horn. The supraspinal projection of the spinal nociceptive input was also examined. The neuronal tracer fluorogold (2%, 0.3µl) was injected into the lateral thalamus 6 days prior to the formalin injection. Fluorogold-positive neurons with c-Fos immunoreactive nuclei were rare in the spinal dorsal horn from L1-L5. These results indicate that 1) the input from ES muscles at L5 level mainly goes to DRG L3, 2) the craniocaudal distribution of nociceptive input from ES muscles is very wide with a well organized somatotopy in the spinal dorsal horn, and 3) the lateral thalamus is probably not a main target region for nociceptive information from ES muscles. [J Physiol Sci. 2007;57 Suppl:S98]

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Many reports have shown that there are sex differences in pain sensitivity, and that hormonal condition also modulates pain sensitivity. In the last year we reported that pressure pain threshold (PPT) was higher in males (n=10) than females (n=20, 10 in follicular and 10 in luteal phase), that females in follicular phase had a higher PPT than those in luteal phase, and that there was no difference in the time course of PPT changes (delayed onset muscle soreness) after lengthening contraction (LC). To make the results more solid, we recruited 10 more males and 25 females with clear basal temperature cycle, and we repeated measurements in some of the females who participated in the previous experiment and had clear cyclic change in the basal body temperature, but in the different phase of their menstrual cycle from the previous experiment. We confirmed that PPT was significantly higher in males than in females as previously reported, and that female PPT was higher in the follicular phase than in the luteal phase. PPT decreased 1 day after LC in all three groups and returned to the pre-exercise value 3 days after LC in luteal phase females, and 4 days after LC in follicular phase females and males. Present results showed clear sex and menstrual cycle differences in muscle pain threshold. [J Physiol Sci. 2007;57 Suppl:S98]

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Insulin-like growth factor-I (IGF-I) plays an important role in bulbo-epithelial interactions in the olfactory system. Translocation of TRPV2 (also known as VRL1 and GRC) to the plasma membrane is augmented by IGF-I. In this study, we explore the effect of IGF-I on the mouse olfactory sensory neurons (OSNs) via TRPV2 by measuring intracellular calcium levels in isolated OSNs by fluorecence microscopy with Fluo4-AM, a calcium indicator. The increase of intracellular calcium levels in OSNs was induced by the application of IGF-I (25 ng/ml), which was inhibited in the presence of SK&F 96365 (20μM). The increase of intracellular calcium levels was induced by 1 mM of 2-aminoethoxydiphenyl borate (2APB), an activator of TRPV1/2/3, which was also inhibited by SK&F 96365. The intracellular calcium levels were not elevated by the application of both IGF-I and 2APB when calcium ion was eliminated from extracellular solution. Immunohistochemistry revealed IGF-I receptor and TRPV2 were co-localized on mouse olfactory epithelium. These data suggest that TRPV2 contributes the elevation of intracellular calcium level in response to IGF-I in OSNs. [J Physiol Sci. 2007;57 Suppl:S98]

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There are sex differences in nociceptive behavior induced by formalin in rats. To determine whether these sex differences are the result of the sexual differentiation of the brain, that is masculinization and defeminization, some female rats were injected with testosterone propionate (TP, 100 mg/25 ml/rat) on the day of birth and on the following day. Other female rats and all male rats were injected with the same volume of sesame oil as controls. They were castrated at the age of 8 weeks, and implanted with a silicon tube containing 20% of 17b-estradiol or cholesterol. Two weeks after the implantation, rats were injected with 50 ml of 2% formalin in the right hind paw and their behavioral changes were observed for 1 h. In cholesterol-implanted rats, all rats exhibited 3 typical phases of pain response and there were no significant differences in the scores of nociceptive behavior. In 17b-estradiol implanted rats, female and TP-treated female rats had a significantly higher score of nociceptive behavior than male rats. These results indicate that estrogen produces sex differences in nociceptive behavior induced by formalin, and suggest that these differences are not due to the sexual differentiation of the brain, since the dose and the timing of the TP treatment effectively defeminize and masculinize female rats. Alternatively, sexual differentiation of the brain response to formalin-induced nociceptive behavior may be different from ordinary sexual differentiation. [J Physiol Sci. 2007;57 Suppl:S99]

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TRPV3 and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here, using in vitro co-culture systems, we show that ATP is such a candidate molecule released from keratinocytes upon heating (– 40°C). Using TRPV1 deficient DRG neurons, we show that increase in cytosolic Ca²⁺-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by PPADS (100 μM), a P2 purinoreceptor antagonist. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X2 cDNA to serve as a bio-sensor), we show that heat-activation of keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3 deficient mice. Release of serotonin and gulutamate was denied using the co-culture system with HEK293 cells expressing 5-HT3 and NMDA receptors, respectively. ATP-induced whole-cell currents were also observed in the co-culture of mouse keratinocytes and DRG neurons. This study provides direct evidence that ATP is a messenger molecule for TRPV3-mediated thermotransduction in skin. [J Physiol Sci. 2007;57 Suppl:S99]

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Parabens are commonly added in pharmaceutical, cosmetic and food products because of their wide antibacterial properties, low toxicity, inertness and chemical stability although the molecular mechanism of their antibacterial effect is not fully understood. Allyl isothiocyanate, cinnamaldehyde and allicin have strong antibacterial activities and are known to activate TRPA1 ion channel, which belongs to the TRP super family and is involved in nociception. Here we show that methyl p-hydroxybenzoate (methyl paraben) activates TRPA1 in both HEK293 cells expressing TRPA1 and in mouse sensory neurons with an EC₅₀ value of 4.4 mM, an easily attainable concentration in methyl paraben-containing products. Furthermore, methyl paraben caused pain-related behaviors in mice similar to those caused by allyl isothiocyanate, which was blocked by the TRP channel blocker, ruthenium red. These results indicate methyl paraben is able to activate TRPA1 and can cause pain sensation. As such, methyl paraben provides a useful tool for investigating TRPA1 function and development of antinociceptive agents acting on TRPA1. [J Physiol Sci. 2007;57 Suppl:S99]

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Electrophysiological properties of primary afferent neurons have been investigated by using dorsal root ganglion (DRG) neurons. It is well known that DRG neurons can be classified several subclasses on the basis of sensitivity to tetrodotoxin (TTX) and kinetic properties of Na⁺ currents. However, little is known about electrophysiological properties of gustatory neurons. In bullfrogs (Rana catesbeiana), afferents fibers transmitting gustatory information travel glossopharyngeal nerve. Cell bodies of glossopharyngeal nerve are located in glossopharyngeal nerve ganglion (GG). To investigate membrane properties of GG neuron, we applied patch clamp recordings to isolated GG neurons. GG neurons were retrogradely labeled with fluorescent dye Alexa Fluor dextran and whole cell patch clamp recordings were made from labeled neurons. In voltage-clamp recording condition, we recorded two subtypes of voltage-gated Na⁺ currents: TTX-sensitive and TTX-resistant Na⁺ currents. The neurons exhibiting TTX-sensitive Na⁺ currents had large cell membrane capacitance, whereas the neurons exhibiting TTX-resistant Na⁺ currents had small cell membrane capacitance. These results suggest that frog GG neurons consist of heterogeneous subpopulations. [J Physiol Sci. 2007;57 Suppl:S99]

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Quinine-sensitive units (Q-units) of the frog glossopharyngeal nerve respond to chloride-salts such as NaCl and choline Cl (Ch-Cl). Q and chloride-salts are quite different taste substances. Q-units show only a phasic response. In the present study, we investigated the specificity of and similarities between taste reception of Q and that of chloride salts. Anesthetized bullfrogs were used. Antidromic impulses of single Q-units were recorded from a single fungiform papilla that had been drawn into a suction electrode during chemical stimulation of adjacent papillae. The taste stimuli were delivered through a fine tube positioned close to the suction electrode. An electrical surge (onset of stimulus) appeared when the first drop of stimulant reached the tongue surface. The time between the onset of stimulus and appearance of the first impulse were measured as the latency of taste response. The latency decreased and the frequency of impulses increased with increasing concentrations of Q or Ch-Cl. In each Q-unit, the relation between the latency and the frequency of impulses elicite by Q at 0.001-1.0 mM are identical to that elicited by Ch-Cl at 1-500 mM, suggesting that Q and Ch-Cl use the same transduction pathway. However, Q-units could be excited by Ch-Cl after being desensitized by Q. It appears that Q and Ch-Cl interact with different receptor sites. The present results suggested that Q and Ch-Cl act on different receptors and use a common transduction pathway. [J Physiol Sci. 2007;57 Suppl:S100]

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We have shown that conditioning electrical stimulation of the amygdala has an inhibitory effect on nociceptive neurons of the medullary dorsal horn of the rat. The purpose of the present study is to determine whether the inhibitory effect depends on the activation of cell bodies or passing fibers by glutamate microinjection into the amygdala. The animals were anesthetized with N₂O-O₂ and 0.5% halothane and were immobilized with pancuronium bromide. A peripheral test stimulus (a single rectangular pulse of 2.0 msec in duration) was applied to the receptive field of nociceptive neurons, and ipsilateral amygdaloid conditioning stimuli to the recording site were trains of 33 pulses (100-300 μA) delivered at 330 Hz. Thirteen nociceptive neurons recorded in the superficial layer of the trigeminal caudal nucleus and the medial reticular nucleus, and the nociceptive responses of these neurons were inhibited by the conditioning electrical stimulation in the central, basomedial and basolateral amygdaloid nuclei. Microinjection of 0.5 M monosodium glutamate (5 μl) into these amygdaloid nuclei induced inhibition of 5 WDR neurons and 2 NS neurons. Most potent inhibition occurred at 5 min after start of injection, and the recovery of inhibition to 50% took place around 25 min. These findings suggest that the excitation of cell bodies in the amygdala contribute to the inhibitory effects on the nociceptive neurons in the medullary dorsal horn. [J Physiol Sci. 2007;57 Suppl:S100]

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In the present study, the role of sensory inputs from oral mucosal receptors in voluntary swallowing in humans was investigated. Twenty healthy volunteers were enrolled. A fine tube was inserted into the pharyngolaryngeal region (PL). In animal experiments, it has been reported that excitation of laryngeal water receptors elicit swallowing reflex and 0.3 M NaCl strongly inhibits water receptors. In this study, water or 0.3 M NaCl solution was delivered into the PL through this tube at slow infusion rates (0.2-5.0 ml/min). Each subject was instructed to repeat swallowing as fast as possible. The swallowing interval (SI) between two consecutive swallows in each infusion was measured. SIs were shorter in the case of water infusion than in the case of 0.3 M NaCl. Increasing infusion rate shortened SIs. The results suggest that sensory inputs from water and mechanical receptors can facilitate voluntary swallowing. Values of SI with surface anesthesia of the PL (diminution of sensation) by voluntary swallowing varied greatly in the subjects, suggesting that the ability of the swallowing center to perform repetitive voluntary swallowing varies among subjects. There was a linear relationship between SI with anesthesia and the sensory effect: the longer the SI with anesthesia was, the stronger was the sensory effect. It appears that sensory inputs in the PL strongly compensate for difficulty in swallowing in subjects showing a long SI. [J Physiol Sci. 2007;57 Suppl:S100]

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We report that the mRNAs of two serotonin receptor subtypes, 5-HT1B and 5HT2A, are specifically expressed in the primary visual cortex (V1) of macaque monkeys and that the strength of expression is controlled in an activity-dependent manner. To elucidate functional roles of these receptors in vivo, we performed electrophysiological recordings of visual responses from 101 V1 neurons with microiontophoretic administration of specific drugs for each receptor subtypes in anesthetized and paralyzed monkeys. The effects of activating these receptors seemed to be bidirectional depending on the activity levels of each neuron. The effect of agonist for 5-HT1B receptor, CP93129, was more facilitatory when the firing rate was high, but the effect was more suppressive when the firing rate was low. The effect of DOI, agonist for 5-HT2A receptor, was opposite in direction. That is, the effect of DOI was more suppressive when the firing rate was high, but more faciliatatory when the firing rate was low. Highly restricted expressions of these receptors and their neural-activity-dependent actions in V1 suggest that 5-HT1B and 5-HT2A receptors complementarily control the gain of input-output relation of V1 with an activity-dependent manner, which could modulate further processing in the cortical network. [J Physiol Sci. 2007;57 Suppl:S100]

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Mammalian inferior colliculus(IC) is an integrative auditory processing center of the midbrain. IC contains a large amount of GABAergic neurons and forms complex neural circuits. We investigated the response properties of IC neurons to sounds and their morphological characteristics by juxtacelluler staining method. Further, by GAD-67 immunohistological stainig, we classified the recorded neurons into two groups, GABAergic and non-GABAergic neurons, and compared the response properties to sound stimuli. [J Physiol Sci. 2007;57 Suppl:S101]

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One can perceive the motion of a visual object independent of eye movements that cause additional virtual motions on the retina. To understand the underlying neural mechanisms, we recorded neuronal responses in cortical MT/MST areas to a moving random dot pattern (-160 to 160deg/sec) that appeared briefly on the screen while monkeys performed smooth pursuit or stationary fixation. Although neurons in both areas exhibited substantial responses to the motion of the textured image in the preferred direction, the responses of the MST neurons were mostly correlated with the motion of the image on the screen, independent of pursuit, whereas the responses of MT neurons were mostly correlated with the motion of the image on the retina. When a check pattern of 0.5 cycles/deg was used instead of the random dots and its temporal frequency exceeded 20Hz (0.5 cycles/deg x 40 deg/sec) on the retina, neuronal responses in both areas decreased independent of pursuit eye movements. To confirm this observation, we replaced the check pattern with a different spatial frequency (0.25 cycles/deg). The stimulus speed that gave the best response was 80deg/sec on the retina independent of pursuit eye movements, and the temporal frequency of the visual stimulus was 20Hz (0.25 cycles/deg x 80 deg/sec). The decreased neuronal responses at higher temporal frequencies (>20Hz) supports the idea that the MST neurons code visual motion in the external world while compensating for the eye movements by utilizing the information of the visual motion on the retina and of the eye movements. [J Physiol Sci. 2007;57 Suppl:S101]

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To investigate how pheromonal information is processed in the rat accessory olfactory bulb (AOB), we optically imaged intrinsic signals and obtained high-resolution maps of activation induced by urinary stimulation. Application of volatile components in male rat urine induced activation exclusively in the anterior AOB (aAOB), irrespective of the recorded rats^, sex. Volatile components of female urine elicited activation not only in the aAOB but also to some extent in the caudal part of the posterior AOB (pAOB) of male rats, and no responses were recorded from female rats. Non-volatile components of both male and female rat urine induced activation mainly in the rostral part of the pAOB and to a lesser extent in the aAOB, irrespective of the recorded rats^, sex. In the present study, we further carried out single-unit recordings in the active region of the aAOB, where optical response to volatile urine was observed. We analyzed the firing activity of 46 neurons obtained from in nine animals. Of 13 neurons that responded to volatile stimulation (5% male urine, 5% female urine and/or 0.3% 2-heptanone) by increasing their firing rate significantly, 11 neurons responded to only one type of stimulation and 2 neurons responded to both female urine and 2-heptanone. By combining electrophysiological and intrinsic optical imaging techniques, we confirmed that the aAOB is activated by volatile urinary components. [J Physiol Sci. 2007;57 Suppl:S101]

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Mammalian taste bud cells (TBCs) express M1, one of five muscarinic acetylcholine receptor (mAChR) subtypes, M1-M5. However, the expression of the other four subtypes has not been examined. Also, it remained unknown if the expression of the subtype depends on the TBC type. In this study, we examined the expression of mAChR subtypes in mouse TBCs in fungiform papillae innervated by the chorda tympani nerve and those in circumvallate papillae innervated by the glossopharyngeal nerve. Here we show that acetylcholine (ACh) applied to the basolateral membrane of fungiform TBCs elevates the intracellular Ca²⁺ level in a concentration-dependent manner. The Ca²⁺ responses occur in the absence of extracellular Ca²⁺, and are inhibited by 100 nM atropine, a specific antagonist against mAChR subtypes, and 10 nM 4-DAMP, a selective antagonist against M3. RT-PCR studies suggest the expression of M3, but not the expression of the other subtypes of mAChR in fungiform TBCs. In fungiform papillae, immunohistochemical studies showed that TBCs show immunoreactivity for M3, but not for M1, and that M3 occurs on type II and type III TBCs. The expression of vesicular ACh transporter (vAChT), a transporter that packs ACh into synaptic vesicle was undetectable in fungiform TBCs. In circumvallate taste buds, RT-PCR and immunohistochemical studies showed the expression of M1, M3 and VAChT. The role of ACh on taste transductions and the innervation-dependent expression of mAChR subtypes are discussed. [J Physiol Sci. 2007;57 Suppl:S101]

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