Adrenomedullin 2 (AM2)/Intermedin, as a novel member of calcitonin/calcitonin gene-peptide (CGRP) family, has been reported to play diverse physiological roles. The previous studies have showed that intravenously and intracerebroventricular (ICV) administrations of AM2 can produce strong influences on cardiovascular activities. To examine the physiological role of AM2 in the nucleus tractus solitarius (NTS) to regulate cardiovascular activities, we directly microinjected AM2 into the NTS. In 36 urethane-anesthetized male Wistar rats, AM2 (10 pmol, 20 pmol and 40 pmol/100 nl) was unilaterally microinjected into the NTS, where L-glu microinjection induced typical depressor and bradycardic responses, resulting in significant transient increases on mean arterial pressure (MAP) and heart rate (HR). Microinjection of vehicle (artificial cerebrospinal fluid, 100 nl) had no significant effects on MAP and HR. This study demonstrates that AM2 acting in NTS induces significant cardiovascular responses. In addition, to clarify further whether the actions of AM2 in NTS are related to the CRLR/RAMP receptor complexes, we are examining the expression of these receptors in NTS using real time PCR and immunohistochemistry analysis. [J Physiol Sci. 2008;58 Suppl:S153]

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We have shown previously that freezing behavior, which induced by exposing animals to loud white noise, was associated with an increase in renal sympathetic nerve activity (RSNA), a decrease in heart rate (HR), and no change in lumbar sympathetic nerve activity (LSNA) in rats. The present study was designed to examine the potential contribution of arterial baroreflex to the differential responses of the sympathetic outflows and HR observed during freezing behavior. Wistar male rats were instrumented chronically with electrodes for measurements of RSNA, LSNA, and electrocardiogram, and with arterial and venous catheters. At least three days after the surgery, rats were exposed to white noise with 90dB over 10 min following a 60 min control period. The baroreflex curve for RSNA, LSNA and HR was determined by changing systemic arterial pressure using rapid intravenous infusions of vasoactive drugs and then fitted to an inverse sigmoid function curve. During the freezing behavior, the baroreflex curve for RSNA was shifted upward with a significant (P<0.05) increase in the response range of 115% compared with control level; while the baroreflex curve for LSNA remained unchanged during freezing behavior. The baroreflex curve for HR was shifted leftward with a significant (P<0.05) decrease in the midpoint pressure of 11mmHg relative to the control level. These data suggest the differential responses of RSNA, LSNA and HR observed during freezing behavior may be attributed to, in part, the differential shifts in baroreflex curves for RSNA, LSNA and HR in rats. [J Physiol Sci. 2008;58 Suppl:S153]

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Salivary gland function is regulated by the autonomic nervous system, which innervates both salivary acinar and ductal cells. In both the salivary acinar and ductal cells, cholinergic and adrenergic stimulations increase intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, there is no systematical study comparing sensitivity to the cholinergic and adrenergic drugs between parotid acini and ducts. In the present study, cholinergic and adrenergic drugs-induced changes of [Ca²⁺]_i were investigated in fura-2-loaded rat parotid acini and ducts by using Ca²⁺-imaging technique. Carbachol (CCh) at 1 μM induced stronger Ca²⁺ response in parotid acini than ducts whereas noradrenaline (NA) at 1 μM conversely induced lesser response in acini than ducts. CCh-induced Ca²⁺ responses in both acini and ducts were completely blocked by a M3 receptor selective antagonist (4-diphenylacetoxy-N-methylpiredine methiodide: 100 nM). On the other hand, although NA-induced Ca²⁺ response in acini was completely blocked by a α1 selective antagaonist (phentolamine: 100 nM), the response in ducts was partially inhibited by the antagonist. The phentolamine-resistanse response in ducts was completely blocked by a β selective antagonist (propranolol: 1 μM) and a β selective agonist (isoproterenol: 10-100 μM) induced increment of [Ca²⁺]_i in ducts, but not acini. These results suggest that there are different sensitivitiies to cholinergic and adrenergic agonists between parotid acini and ducts. [J Physiol Sci. 2008;58 Suppl:S154]

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The superior salivatory (SS) nucleus , the primary parasympathetic center for the submandibular 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 (higher centers) and lower brainstem (lower centers). Recently, we have electrophysiologically shown that many SS neurons receive GABAergic and glycinergic inputs from higher and lower centers. In the present study, we aimed to examine the distribution of GABAergic neurons innervating SS neurons. We carried out double-labeling of the GABAergic neurons projecting to SS neurons by injection of retrograde fluorescent tracer, FluoroGold (2%, 0.025 μl), into SS nucleus, and by immunohistochemical staining for glutamic acid decarboxylase, a GABA synthetic enzyme. In the higher centers, double-labeled neurons were mainly observed in the lateral hypothalamic area and central nucleus of the amygdala. In the lower centers, double-labeled neurons were mainly found in the parabrachial nucleus, nucleus of the solitary tract and its surrounding reticular formation. These findings imply that these higher and lower centers are involved in the inhibitory salivary control. [J Physiol Sci. 2008;58 Suppl:S154]

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Previous studies in our laboratory have shown that electrical stimulation of both cervical sympathetic trunks (CSTs) produces an initial-increase and a late-decrease of regional cerebral blood flow (rCBF) in the neocortex, by an activation of β- and α-adrenergic receptors, respectively. The responses reached about 10% in both adult and aged rats. Neurodegenerative disorders such as Alzheimer's disease are known to involve early olfactory deficits and typical histopathological lesions in the olfactory bulb. Thus, it seems important to determine nature and aging of the sympathetic neural regulation of rCBF in the olfactory bulb. In the present study, the effects of repetitive electrical stimulation of CSTs for 1 min at various frequencies and supramaximum intensity on rCBF in the olfactory bulb were studied using laser Doppler flowmetry in adult (4-6 mo) and aged (18-21 mo) male Wistar rats anesthetized with urethane. In adult rats, stimulation at 0.5-30 Hz produced frequency-dependent decreases in rCBF, by up to 31±4% of the prestimulus control flow. In aged rats, the decreased response was produced at 10-30 Hz, by up to 14±2%. The responses were abolished by an i.v. administration of the α-adrenergic blocker phenoxybenzamine, in both age groups. These results indicate that, in the olfactory bulb, blood vessels are constricted by sympathetic nerve fibers via activation of α-adrenergic receptors; the effectiveness of this regulation declines with age. [J Physiol Sci. 2008;58 Suppl:S154]

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We studied that effect of intravesical administration of noradrenaline (NA) for micturition reflex in alpha_1A-knockout (KO) mice and alpha_1D-KO mice using the filling cystometry(CMG). Urinary bladder was infused with saline(0.4ml/h) or NA (1μg-100μg/ml). NA (10μg/ml) infusion in WT (wild type) mice shortened ICI (intercontraction interval) from 649s to 391s (60% of control, P<0.05, n=7). But NA did not altered ICI in alpha_1A-KO mice (from 1047s to 1044s, 99.7%, n=7) or alpha _1D KO mice (from 663s to 746s,113%, n=7). NA shortened ICI in WT mice dose dependent fashion (1μg-100μg/ml,n=5). NA did not altered MVP (maximal voiding pressure) in WT mice, alpha_1A-KO mice or alpha_1D-KO mice. Intravesical administration of NA did not facilitate micturition reflex in alpha_1A-KO mice and in alpha_1D-KO mice. ICI in alpha _1A-KO mice was longer than WT mice or alpha_1D-KO mice. Silodosin (selective alpha_1A-adrenocepter antagonist) infusion followed by NA infusion did not altered the ICI from 363s to 370s (101% of control, n=4). Alpha_1A-receptor and alpha_1D-receptor in urothelium could activated by NA and facilitate the micturition reflex in mice. These data suggest that NA might cause hyper refexia in micturition pathway. [J Physiol Sci. 2008;58 Suppl:S154]

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In the present study we examined an effect of application of Eye-Steam-Sheet in comparison with a sham eye mask. The Eye-Steam-Sheet produces heat and steam of about 40°C, when applied on the skin. After the sheet applied on the bilateral eyes of healthy subjects for 10min, the following parameters were measured: Palmar sweating measured by the finite-difference ventilation capsule method; palmar skin blood flow measured using a laser-doppler flowmeter; shoulder tissue hemoglobin measured by near infrared-spectroscopy; pupillary light reflex measured by the closed loop method. After application of the Eye-Steam-Sheet, we found that the palmar sweating decreased, the palmar skin blood flow increased, the shoulder tissue hemoglobin level increased, the miosis rate after light stimulation increased, and sensitivity of the baro receptor reflex elevated. These results suggest that application of Eye-Steam-Sheet on the bilateral eyes of healthy subjects produces parasympathetic predominance and improves peripheral hemodynamics. [J Physiol Sci. 2008;58 Suppl:S155]

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We examined the sympathetic nervous system (SNS) on the chronic pain model rats, induced by 2-week unilateral limb-immobilization, which indicated bilateral long-lasting pain behavior for over 10 weeks. We measured systolic blood pressure (SBP) and heart rate (HR) under the free moving over time along the pain behavior at the resting state and at the stimulating state in the cold exposure(9±1°C). At the resting state, SBPs increased during cast-immobilization and then decreased after cast-removal compared with normal level. These decreases lasted for the 1-10 weeks after cast-removal. We analyzed the middle frequency (MF) component calculated by power spectrum analysis of SBP, known as a sympathetic index. The MF components significantly correlated with SBPs through the whole periods. The fluctuation of HRs during and after immobilization was similar to that of SBPs. However, the cold exposure caused the different effects on SBP and HR: SBPs tended to increase as much as them of normal rats, but HRs did not. Our results suggest that, in this model, sympathetic activation increased by immobilizing, then decreased below normal level after removing, and this deactivated state lasted while persisting pain behavior continued. And the degree of the rise of HR in the cold exposure decreased in the pathological state, which indicates lowering of SNS. [J Physiol Sci. 2008;58 Suppl:S155]

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To evaluate the role of serotonin and the vagal afferent nerves on preinflammatory responses (such as fever and feed-suppression) induced by inflammation broken out within the peritoneal cavity, we observed core temperature (TC) and food-intake (FI) in the Wistar strain rat after an administration of lipopolysaccaride (LPS) with or without ramosetron (a serotonin-3 type receptor antagonist). An administration of 100 μg/kg LPS elevated TC and suppressed FI with increase in afferent vagal nerve activity. Such LPS-induced fever and feed-suppression were completely abolished by the combined vagotomy in hepatic and bilateral gastric branches. Furthermore, the LPS-induced preinflammatory responses were prevented significantly by a pretreatment with 30 μg/kg ramosetron given into the peritoneal cavity before the pyrogenic challenge. These data suggest 1) that vagal afferent nerves convey an inflammatory signal to the central nervous system via the vagal hepatic and gastric branch and 2) that LPS was recognized by immune sensitive cells (e.g., dendritic cells) or testing cells (e.g., basal-granulated cells) when LPS-evoked preinflammation broke out in the peritoneal region and 3) that 5HT may transmit the LPS-evoked preflammatory signal via the 5HT-3 type receptors located in vagal afferent nerve terminals. [J Physiol Sci. 2008;58 Suppl:S155]

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Simultaneously, we measured dynamic movements of a stomach by ultrasonography (ECHO) and electrical signals by electrogastrography (EGG). A man (29 aged) was participated in this study for the purpose of studying the relations between dynamic movements and electrical phenomena of the stomach. He was measured before breakfast in the morning during 10 minutes at rest, and 5 minutes in drinking and eating and 10 minutes at rest in supine position. Nine electrodes were attached to the abdomen and electrical signals were recorded (BIOTOP-6R12, NEC san-ei Ins., Japan, band pass filter from 0.016 to 30Hz, sampling clock: 250Hz, reference electrode: right leg). And, a probe of ECHO (TOSHIBA Co., Aplio, Japan, TOSHIBA PVM-375AT probe) was put on the upper right abdomen between the electrodes. Movements of the stomach for ECHO were obtained measuring the pylorus diameter for every one second by observation. Results of the fast Fourier transform (FFT) showed a peak about 3 cycles per one minute for both ECHO and EGG. We think that dynamic movements and electric signals were correlated, so only EGG measures the stomach movement. In the future, we clarify the relation to the movement of the stomach by only EGG to various tasks as evaluation index. [J Physiol Sci. 2008;58 Suppl:S155]

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Morphine is thought to inhibit gastrointestinal transit by the modulation of neural control and/or the direct action to the gut. Our previous studies suggest that morphine inhibits the colon motility through the activation of sympathetic nervous system. The aim of this study was to determine if morphine-induced inhibition on the colonic motility is due to either direct action on the colon or the modulation of autonomic nervous system. Male adult Wistar rats (300-350 g) were anesthetized under mixture of urethane and α-chloralose. To measure the colon motility, a catheter with a balloon sensor was inserted into the colon 5-6 cm from the anus for the in vivo study. Morphine (10 mg/ml/kg) hydrochloride or saline (1 ml/kg) was injected (i.v.) to the experiment group and to the control group respectively. The longitudinal segment dissected from the colon 5 cm from the anus was suspended in Kreb's solution to measure the motility for the in vitro study. As the results, intravenous administration of morphine significantly depressed the colonic motility in vivo compared to the control group. The inhibitory effect lasted for about 4 hours. Contrary, the colonic motility in vitro restarted after the dissection and did not show significant depression. These results suggest that the mechanism of the inhibitory effect of morphine (i.v.) on the colonic motility involves indirect action; possibly of the central or autonomic nervous system and it might be the activation of sympathetic nervous system. [J Physiol Sci. 2008;58 Suppl:S156]

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Claudins are identified at least 24 members as component proteins of the tight junction (TJ). Each family member varies in the expression and the barrier function. In this study, we investigated whether claudin-1, -2, -3 and other TJ component proteins: ZO-1 and occuludin, were expressed in ENS by immunohistochemistry and RT-PCR. As a result, we found out that only claudin-4 was presence in the enteric neurons. The immunoreactivity for claudin-4 was detected in cell bodies and axons of almost all neurofilament-positive neurons of the submucosal and myenteric plexus, however, quite separated from GFAP, a marker protein of glia. In the myenteric plexus of whole-mount preparations, the stained neurons were characterized by Dogiel morphological classification. The claudin-4 more intense stained neurons showed Dogiel type II, while the less stained ones showed Dogiel type I morphology. The results of mRNA expression analysis of muscle plus submucosa preparation, which contains the submucosal and myenteric plexuses, supported immunohistochemical data. The physiological function of claudin-4 in enteric neurons is still unclear, however, the colocalization of claudin-4 and neurofilament was displayed also in rat small intestine, cecum, and human colon. It is suggested that claudin-4 in enteric neurons might play roles in neural activity, for example, as insulation between each neurofiber to prevent interaction of neuro-transmissions. [J Physiol Sci. 2008;58 Suppl:S156]

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We have demonstrated that nNOS activity in the diencephalon was significantly lower in hypertensive Dahl salt-sensitive (DSS) rats than in normotensive Sprague-Dawley (SD) rats. On the contrary, no difference was found in nNOS activity of the brainstem between the two rat-groups. In order to discuss the differences in the nNOS neuronal system between hypertensive and normotensive rats, we compared the number of nNOS neurons in the hypothalamus and brainstem in this study. DSS rats were fed 8% NaCl foods (DSS8%) and SD rats were fed 0.4% NaCl foods (SD0.4%). Immunohistochemical staining using anti-nNOS antibody revealed that the number of nNOS neurons in the paraventricular nucleus (PVN) was significantly smaller in DSS8% than that in SD0.4% rats. This might reflect the result of nNOS activity in the diencephalon. Although no difference was found in brainstem nNOS activity, the number of nNOS neurons in the lateral parabrachial nucleus (LPB), rostral ventrolateral medulla (RVLM), and nucleus tractus solitarius (NTS) was significantly greater in DSS8% rats compared with those in SD0.4% rats. These results indicated that in hypertensive DSS rats, nNOS neurons mediated-sympathoinhibition in the PVN remained downregulated, whether that in the LPB, RVLM, or NTS was upregulated in order to compensate high blood pressure. As a whole, those central nNOS-mediated sympathoinhibition might be not enough to lower high blood pressure. [J Physiol Sci. 2008;58 Suppl:S156]

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There is direct evidence for dendritic vasopressin (AVP) release, which is regulated independently of secretion into the peripheral circulation, in the hypothalamus. Dendritically released AVP seems to act on AVP neurons to modulate its activity with an action of inhibitory or stimulatory. We showed how central AVP system has an effect on the neuronal activity in the paraventricular nucleus of the hypothalamus (PVN) after central salt loading pretreated with i.c.v. AVP V₁ receptor antagonist, OPC-21268 (250 μg/kg, Otsuka Pharmaceutical). The neuronal activity evaluated by Fos-like immunoreactivity (FLI) seemed to be suppressed in the PVN, especially in the parvocellular parts. Then, we make a hypothesis that the results of FLI in the parvocellular neurons might relate to the autonomic outflows. To settle this hypothesis, we recorded renal sympathetic nerve activity (RSNA) after central salt loading. The suppression of RSNA was attenuated significantly by pretreatment with i.c.v. OPC-21268. These results suggest that the suppression of RSNA after central salt loading ascribe to the decrease of neuronal activity in the parvocellular PVN via the inhibitory action of the central AVP system. [J Physiol Sci. 2008;58 Suppl:S156]

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There are contradictory reports concerning the effects of stimulation in the dorsal raphe nucleus (DRN) on the blood flow in the frontal cortex. In this connection, it is known that DRN consists of two distinct neuronal populations. ; serotonergic (5-HT) neurons are mainly located in the rostral, dorsal part of DRN (rdDRN) , while GABAergic neurons are located in the caudal, ventral part of DRN (cvDRN). In the present study we measured changes in blood flow of the orbito-frontal cortex (OFC-BF) using laser Doppler flowmeter when we gave chemical stimulations (L-glutamate) in DRN in anesthetized spontaneously respiratory rats. As a results, rdDRN stimulation caused the increase in OFC-BF, whereas cvDRN stimulation elicited the decrease in OFC-BF. These results suggest rdDRN 5-HT neurons may be essential for regulation of OFC-BF. [J Physiol Sci. 2008;58 Suppl:S157]

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We recently reported the presence of parasympathetic vasodilator fibers in the rat masseter muscle. During the course of studies for neural mechanisms underlying parasympathetic vasodilation evoked by activation of these fibers, we found that cholinergic system would be more involved in the parasympathetic vasodilation in the masseter muscle in females than males. However, it is still questionable whether this is a specific for masseter muscle or not. We explored this question to compare parasympathetic vasodilations in the masseter muscle and lower lip, and increases in blood flow of common carotid artery (CCABF) derived from the orofacial area evoked by the trigeminal-mediated reflex in anesthetized male and female rats. Parasympathetic vasodilations in the masseter muscle and lower lip were much more reduced by atropine in female than male rats. Pretreatment with atropine significantly reduced the CCABF increase in female rats, but not that in male rats. The present study indicates that 1) parasympathetic vasodilations would depend on more cholinergic system in female than male rats, and 2) cholinergic parasympathetic vasodilations accounts for the CCABF increase in female rats. These results suggest that cholinergic system would be more involved in the parasympathetic vasodilation in the orofacial area in females than males. [J Physiol Sci. 2008;58 Suppl:S157]

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Microdialysis was employed to examine whether osmotic stimulation causes alterations in the release of noradrenaline (NA) in the median preoptic nucleus (MnPO), and whether γ-aminobutyric acid (GABA) receptor mechanisms are involved in the modulation of the stimulus-induced alteration in the NA release in the MnPO. In urethane-anesthetized male rats, intraperitoneal injection of hypertonic saline (0.3 M NaCl, 1 ml) significantly decreased dialysate NA concentration in the MnPO area. The decrease in the NA level elicited by the osmotic stimulation was significantly attenuated by perfusion with phaclofen (10 μM), a GABA_B receptor antagonist, but not by perfusion with bicuculline (10 μM), a GABA_A receptor antagonist, through a microdialysis probe. Microinjection of the local anesthetic lidocaine (2%, 0.2 µl) into the organum vasclosum of the lamina terminalis (OVLT) significantly attenuated the decrease in the NA release in the MnPO area. We conclude, in conjunction with the previous findings that the OVLT exerts inhibitory influences on the NA release in the MnPO area through GABA_B receptor mechanisms, that the decrease in the NA release in the MnPO area elicited by the osmotic stimulation may be mediated in part by GABAergic inputs from the OVLT. [J Physiol Sci. 2008;58 Suppl:S157]

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The effect of stimulation of the magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM), which projects cholinergic fibers diffusely to the cerebral cortex, on the extracellular release of nerve growth factor (NGF) in the cerebral cortex in the parietal lobe was examined in halothane-anesthetized rats. NGF in the cortical extracellular fluid was collected every 100 min for 700 min using a microdialysis method and was measured by high sensitivity ELISA. The NGF concentration in the cortical perfusate in the resting condition was approximately 20 pg/ml, and this concentration was stable throughout the experiment when the NBM was not stimulated. When focal electrical stimulation was applied monolaterally to the NBM for 100 min, NGF in the perfusate of the cerebral cortex ipsilateral to the stimulated side did not change significantly during stimulation, but was increased by up to 66% at 200-500 min after the end of stimulation. These results show that stimulation of the NBM produces an increase in extracellular release of NGF in the cerebral cortex. The increased NGF may contribute to the maintenance of neuronal organization in both the cerebral cortex and the NBM. [J Physiol Sci. 2008;58 Suppl:S157]

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To investigate the effects of muscarinic receptor agonisits on area postrema (AP) neuronal activity, we performed whole-cell recordings from neurons in rat brain slices. Excitatory responses were found in 44% (22/50) of cells tested during the bath application of cevimeline (M3 receptor selective agonist, up to 500 μM). Voltage-clamp recordings showed a marked inward current without an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) (n=18). Such responses were found in either major electrophysiological cell classes, i.e. cells displaying I_h and cells not displaying I_h. Current-clamp recordings showed cevimeline-induced depolarization of membrane potential that modulated the frequency of action potentials (n=4/10). In 3 cells showed cevimeline-induced inward currents, bath-applied pilocarpine (a partial agonist at the M3 and M5 receptors) elicited a decrease in the frequency of mEPSCs (from 1.0 ± 0.2 Hz to 0.5 ± 0.1 Hz). These results suggest that M3 receptors are present in the post- and/or extrasynaptic regions of AP neurons and M5 receptors are present in the presynaptic nerve terminals. A side effect of cevimeline for dry mouth patient, such as nausea and vomiting, may be triggered by the activation of M3 receptors in AP neurons. [J Physiol Sci. 2008;58 Suppl:S158]

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A large portion of the monkey thalamic parafascicular nucleus (Pf) receives vagus afferent input (Ito & Craig; J Neurophysiol, 2005) and projects to the associative part of the striatum (Ito & Craig; J Comp Neurol, 2008). To elucidate the origin of this sensory input, its afferent projections were investigated using fluorescent dextrans or biotinylated dextran amine. Cynomolgus monkeys were anesthetized with pentobarbital. The cervical vagus nerve was electrically stimulated and evoked potential responses were mapped in the thalamus with a microelectrode. Tracers were injected into the vagus evoked potential focus in the parafascicular nucleus either iontophoretically or by pressure, and after survival of two to three weeks retrogradely labeled cells in the hindbrain were examined. Ipsilateral parabrachial nucleus (PB) was the most densely and consistently labeled area in the hindbrain. Most cells were in the ventral part of the medial subnucleus (PBm), spreading into Kolliker-Fuse (KF). Only a few cells were labeled in the lateral subnucleus (PBl). Contralateral PB also contained labeled cells, albeit fewer. A few cells were labeled in the solitary tract nucleus (NTS); labeled cells were sparsely located laterally in or around the nucleus at caudal, middle and rostral levels. Consistent labeling was also found in the contralateral deep cerebellar (dentate) nucleus. These results suggest that viscerosensory input to the parafascicular nucleus from vagal afferents mainly ascends by way of the ipsilateral parabrachial nucleus. [J Physiol Sci. 2008;58 Suppl:S158]

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Histological studies in rats have shown that the ovary receives autonomic nerves via the superior ovarian nerves (SON) and the ovarian plexus nerves (OPN). It is well known that hypothalamus-pituitary hormones regulate ovarian estradiol secretion. However, it has not yet been clarified whether the autonomic nerves innervating the ovary regulate ovarian estradiol secretion. In the present study, we examined the effects of electrical stimulation of SON and OPN on the ovarian estradiol secretion in rats. Rats were anesthetized on the day of estrous, and ovarian venous blood samples were collected through a catheter inserted into the ovarian vein. Estradiol concentration of ovarian venous plasma was measured using EIA. Electrical stimulation of SON at a supra-maximal intensity for C-fibers produced decreases in plasma flow rate (by about 26 ± 4%), estradiol concentration of ovarian venous plasma (by about 19 ± 8%), and estradiol secretion rate (by about 50 ± 6%). In contrast, electrical stimulation of OPN at supra-maximal intensity for C-fibers produced decrease in plasma flow rate (by about 35 ± 3%) but no changes in estradiol concentration of ovarian venous plasma and estradiol secretion rate. From these results, it is concluded that ovarian estradiol secretion is directly regulated by C-fibers in the SON in rat on the day of estrous. [J Physiol Sci. 2008;58 Suppl:S158]

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To understand regulation circuits controlling swallowing, we used fish brain since fish swallowing simply consists of upper esophageal relaxation and pharyngeal contraction. However, regulation of esophageal relaxation has not been clear even in fish. Recently we have reported that spontaneous activities of motoneurons innervating the upper esophageal sphincter (UES) in the middle part of the glossopharyngeal-vagal motor complex (mGVC, i.e. the nucleus ambiguus) are suppressed by catecholamines (CAs). In the present study we explored catecholaminergic inputs to the mGVC neurons in eel. The eel brain sections retrogradely stained with a neuronal tracer injected into the UES were immunostained against tyrosin hydroxylase (TH). Around retrogradely labeled mGVC neurons, TH-immunoreactive fibers were predominantly distributed and a source of the fibers was traced back the commissural nucleus of Cajal (NCC, i.e. the nucleus of solitary tract). To examine the neuronal connection between the NCC and the mGVC in eel, electric field stimulation was applied to the NCC and the response was recorded from the mGVC extracellularly. Spontaneous firings of the mGVC neurons were transiently inhibited by the NCC stimulation. Such inhibition was suppressed after treatment with prazosin. Since prazosin is shown previously to block the inhibitory effects of CAs in the eel GVC, this result indicates that the NCC innervates the mGVC catecholaminergically. [J Physiol Sci. 2008;58 Suppl:S158]

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The effects of moxibustion-like heat stimulation (MHS) on the skeletal muscle blood flow (MBF) were examined in anesthetized, artificially ventilated rats. MBF in gastrocnemius muscles was measured by using a laser-Doppler flowmeter. MHS was generated by using a heat stimulator for therapeutic use of moxibustion treatment. Three times per minute of MHS (each heating duration of 7.5 s, 41-51°C) was applied to the cutaneous region lying on the gastrocnemius muscle by using an applicator (5 mm in diameter) for 1 min. In addition, to estimate the effects of vasoactivors on changes in MBF induced by MHS, several vasoactivator blockers were intravenously administered. In the intact rats, decreased changes in MBF were observed during the MHS, and increased changes in MBF were observed after the MHS offset. Blood pressure did not show any change accompanying the MHS. In the experimental group under the sciatic nerve cut, MBF did not show increased changes after MHS offset, but showed the decreased changes during the MHS. The time-course changes in MBF accompanying the MHS were not affected by the spinalization at the level of L2-3. The decreased changes in MBF were abolished under the presence of α-adrenergic blocker (phentramine, i.v.), while the increased changes in MBF were abolished under the presence of CGRP blocker (hCGRP_8-37). These results suggest that MHS can affect a local MBF without evoking systemic change. Moreover, these results also suggest that MHS induces vasodilation in muscle vasclature via action of CGRP. [J Physiol Sci. 2008;58 Suppl:S159]

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We have recently reported that both non-noxious and noxious mechanical stimulation (brushing and pinching) of the ipsilateral hindpaw increases L4–6 dorsal spinal blood flow (SCBF) in anesthetized rats. In the present study we employed electro-acupuncture stimulation (EAS) with various frequencies and intensities for investigating contribution of Group II–IV afferent nerves to the SCBF responses. Regional spinal cord blood flow was measured with a laser Doppler flowmeter probe placed on the left dorsal surface of the spinal cord in the L4–6 regions. EAS was applied to the left hindpaw for 30 s at frequencies of 0.1- 100 Hz and at intensities of 0.05–10.0 mA. EAS (2 mA) above 0.5 Hz increased the SCBF in a frequency-dependent manner, reaching maximum increases at 20–50 Hz. On the other hand, the EAS (20 Hz) above 0.2 mA, threshold intensity of Group II afferents of the hindpaw, produced significant increases in the SCBF. With further increases in intensity up to 10 mA, larger increases in the SCBF were produced. The increases of SCBF were disappeared after severance of the dorsal roots. These results indicate that the SCBF responses to EAS of the ipsilateral hindpaw were dependent upon activation of sensory afferent nerves. Furthermore, it was demonstrated that increases in the SCBF were augmented by recruiting Group II, III and IV afferent nerves. [J Physiol Sci. 2008;58 Suppl:S159]

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It is generally accepted that manual acupuncture reduces heart rate via autonomic nerves in humans and in anesthetized animals. However, it is also known that sometimes it is ineffective in humans. To test the hypothesis that the mental state of the subject influence the acupuncture-induced autonomic response of the heart rate, we examined the effects of acupuncture on heart rate at normal condition and stressful condition in healthy adult subjects. We performed mental arithmetic load to induce a stressful condition. A 40-mm-long needle with a diameter of 0.18 mm was inserted to a depth of about 15 mm at the Tsusanli acupoint (ST-36) of the right leg. Sparrow-pecking stimulation was performed at a stimulation frequency of 1 Hz for 4 minutes. The heart rate was calculated using R-R intervals obtained from an electrocardiogram. Acupuncture significantly decreased the heart rate in normal condition. The maximum mean decrease for all subjects was 6.1 + 3.0 beats/min. A mental arithmetic load increased heart rate. The maximum mean increase for all subjects was 4.3 + 2.5 beats/min. In a stressful condition, the acupuncture-induced decrease in heart rate was significantly attenuated. Therefore, we conclude that the reaction caused by acupuncture stimulation is affected by the mental state of the subject. [J Physiol Sci. 2008;58 Suppl:S159]

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Although electroacupuncture (EA) at several acupoints including Zusanli is known to have sympathoinhibitory effect, inter-individual differences and time-dependent changes in the responsiveness make the quantitative application of EA difficult. We examined if we could control the stimulus intensity of EA based on its effect on arterial blood pressure (BP). In five anesthetized cats, we identified the transfer function from EA to BP using a white noise analysis, and designed a feedback controller of the EA stimulus intensity based on a proportional integral controller. The controller adjusted the stimulus current (1-5 mA) to minimize the difference between measured and target BP values. A nonlinear threshold effect of the BP response to EA was avoided by adjusting the stimulus frequency simultaneously (0-10 Hz). When we set the target level at 20 mmHg below baseline BP, the developed system was able to reduce BP within a minute with a steady-state error of 1.2±0.2 mmHg (mean±SE) for 10 min. During the period of the feedback control, renal sympathetic nerve activity was remained suppressed at 78±5% of the baseline level. The quantitative application of EA to suppress sympathetic nerve activity would contribute to treat several cardiovascular diseases accompanying sympathetic overactivity. [J Physiol Sci. 2008;58 Suppl:S159]

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We examined the effects of the amount of fluid ingestion on the renal concentrating ability during moderate exercise in a hot environment. Untrained subjects (n=6) performed 100 min of intermittent exercise on a cycle ergometer at 60% VO₂max in a climatic chamber (32°C, 60% RH). The subjects were tested under the following three conditions: 1) no fluid ingestion (NF), 2) fluid ingestion in amounts equal to one-half of dehydration (FL-1%), 3) fluid ingestion in amounts equal to dehydration (FL-2%). During the NF and FL-1% trials, urine flow rate, osmolar clearance, and creatinine clearance decreased significantly (P<0.05) during exercise compared with the pre-exercise level. In addition, free water clearance was significantly (P<0.05) higher during exercise than the pre-exercise level in the NF and FL-1% trials. During the FL-2% trial, there were no significant differences in urine flow rate and osmolar, free water, and creatinine clearances during exercise compared with the pre-exercise level. On the basis of these observations, it is likely that the decrease in the tubular water reabsorption and the increase in the tubular sodium reabsorption were attenuated in the FL-2% trial. Thus, we suggest that fluid ingestion in amounts equal to dehydration is capable of attenuating the decline in the renal concentrating ability during moderate exercise. [J Physiol Sci. 2008;58 Suppl:S160]

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We examined the sex difference in the relationship between muscle blood flow and muscle tissue oxygenation level following arterial occlusion with and without static handgrip exercise. Healthy young subjects (9 females and 9 males) participated in the present study. We measured brachial arterial blood flow by ultrasound Doppler flowetry before occlusion and after cuff release following arterial occlusion for 2, 4, or 6 min. We also measured muscle tissue oxygen saturation (StO₂) before, during and after occlusions with near infrared spectroscopy, and determined the relationship between StO₂ just before cuff release and muscle blood flow response. The relationship between StO₂ and muscle blood flow following occlusion with static handgrip exercise at 20% maximal voluntary contraction for 2 or 3 min was also determined. StO₂ decreased during occlusion in dependence upon the duration of occlusion and exercise. Brachial blood flow increased with decrease in StO₂, and the increase in brachial blood flow at a given decrease in StO₂ was larger following occlusion with exercise than without exercise, suggesting that exercise augments the blood flow response to increased oxygen demand. The decrease in StO₂ was much larger in males than females in all conditions, while percent increase in brachial blood flow in males was not different from females. These suggest that there exists sex difference in muscle blood flow regulation. The increase in muscle blood flow at a given decrease in StO₂ was larger in females than males. [J Physiol Sci. 2008;58 Suppl:S160]

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The purpose of this study is to examine the usefulness to evaluate a muscle fatigue in three kinds of the surface electromyography(bipolar-lead, monopolar-lead and source derivation method). The subjects were 8 healthy college students (19.5±0.5 years; mean±S.D.). In an attempt to cause a muscle fatigue, the subjects were forced to maintain a forearm levelly for 120 seconds in having the iron array (2kg, 5kg, 7kg, 10kg) in their hands and the surface electromyograms (sEMG) of the biceps brachii were recorded with three kinds of leads (bipolar-lead, monopolar-lead and source derivation method) at the same time. The powere spectrums of each sEMGs were gotten by Fast Fourier Transform (FFT). The center frequencys and integral values of the power spectrums were compared in sEMG recorded by each derivations. It was more remarkable of both of the decline of the center frequency elapse and the increase of the integral value of power spectrums with a muscle fatigue in the sEMG by the source derivation (SD) method than in sEMG by other leads(p<0.05). When judging a muscle fatigue from the power spectrum of sEMG from these results, it thought that the SD method was a valid method. [J Physiol Sci. 2008;58 Suppl:S160]

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We have reported the effectiveness of resistance exercise against skeletal muscle atrophy caused by hindlimb unloading (HU). This study was undertaken to examine an association of ubiquitin proteasome activity with this exercise effect. Adult F344 female rats were placed into either weight-bearing control (n=7) or HU group (n=14). Rats in the HU group had their hindlimbs suspended for 3 weeks. The HU group was subdivided to a group with (n=7) or without (n=7) an intermittent resistance exercise (IRE), in which the rats were put in a cylindrical wire mesh inclined 60 or 80 degree with a weight of 50-70% of body mass hung from the tail. The IRE was performed for 10 min, three times a day (total 30 min/day) throughout the HU. After the HU period, the soleus (SOL) and medial gastrocnemius (MG) muscles were dissected out, weighed, and then homogenated. The protein expression levels of two ubiquitin ligases, MAFbx-1 and MuRF-1 were determined using a western blotting method. The HU resulted in significant atrophy in the SOL (-47%) and MG (-31%). The IRE attenuated the atrophy of the SOL (37%) and MG (54%). The HU resulted in significant increases in the expression levels of MAFbx-1 and MuRF-1. However, IRE attenuated up-regulations of MAFbx-1 and MuRF-1 with HU in both muscles. These results show that down-regulation of ubiquitin ligase protein is account for the inhibitory effect of IRE against muscle atrophy. [J Physiol Sci. 2008;58 Suppl:S160]

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Adipocytes are known to release adipokines which are related with the pathogenesis of metabolic and cardiovascular complications of obesity. The expressions and releases of adipokines are shown to depend on adipocyte size. In order to prevent obesity-related metabolic and cardiovascular complications, therefore, it will be important to reduce adipocyte size in obese individuals. To this end, exercise training (ET) is used to reduce adipocyte size. However, the effect of ET on relationship between fat cell size and the adipokines expressions remains unknown. In the present study, we tried to clarify this issue. The rats (Male Wistar, SLC, Japan) in the ET group were subjected to exercise on a treadmill5 days/week for 9 weeks. The sedentary control animals (C) were not subjected to running. Adipocytes were isolated from the epididymal adipose tissue. The adipocytes were filtered through some meshes of which each pore size is different, and then the different size of adipocytes was collected. Total RNA was prepared and mRNA expressions were determined by quantitative RT-PCR. All experiments were approved by the Animal Care Committee of our institute. The adipose tissue mass and adipocyte size were significantly less in ET than in C. Either leptin or adiponectin mRNA expression was well correlated with adipocyte size in both groups. Thus, mRNA expressions of both adipokines would depend on adipocyte size. Consequently, it is possible that ET reduces adipocyte size, thereby resulting in size-dependent changes in leptin and adiponectin mRNA expressions. [J Physiol Sci. 2008;58 Suppl:S161]

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Cutaneous vascular responses to mental tasks and physical exercise were studied independently and combined at the different levels of baseline skin blood flow (SkBF). We hypothesized that combined mental and physical stresses produce a synergistic interaction in the cutaneous vascular responses at any levels of SkBF. We studied the responses of cutaneous vascular conductance (CVC) to 2 min of static handgrip (HG), mental arithmetic (MA), and the combined (COM) stimuli in random order in 9 healthy subjects. SkBF was monitored from the calf and foot by laser-Doppler flowmetry. Local temperature around the sites of SkBF measurement was maintained at 29, 34 and 39°C to change the baseline levels of SkBF. CVC was calculated as the ratio of SkBF to blood pressure and expressed as percent change from the baseline control value at 34°C. The baseline values of CVC in calf and foot decreased by 60-70% at 29°C and increased by 140-160% at 39°C compared with those at 34°C. HG, MA and COM significantly decreased CVC in each skin site, and the stress-induced decrease of CVC increased with increasing baseline levels of SkBF. In each level of SkBF, the decrease of CVC in calf and foot during HG or MA did not differ from those during COM. The sum of the CVC responses during HG and MA was greater than those during COM. Hence, MA and HG induce the vasoconstrictor responses in glabrous and nonglabrous skin, resulting in a nonadditive effect of these two stresses regardless of the baseline SkBF. [J Physiol Sci. 2008;58 Suppl:S161]

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Objective Characteristic motions during putting on and taking off underwear and pants-type diapers were clarified by a comparison between young and elderly persons. Methods (Evaluation items)The following were performed during the activity of putting on and taking off underwear and pants-type diapers on standing (putting on and taking off)–Subjective evaluation –Motion analysis–Electromyography Results (Putting on) The levels of the wrists were significantly lower in the elderly pants-type diaper group. Shoulder movement was more marked in the diaper group in both the young and elderly subjects. The highest and lowest positions of the head were both lower in the diaper group in the elderly subjects. On subjective evaluation, both the young and elderly subjects felt it difficult to wear the diaper. The muscle load was increased in the diaper group in both the young and elderly subjects. (Taking off) The distance of the anteroposterior movement of the wrists was slightly reduced in the elderly diaper group. The highest and lowest positions of the head were slightly lower in the elderly diaper group. On subjective evaluation, the young subjects felt it difficult to take off the pants-type diaper. Summary It was predicted that lowering the head and wrist positions places a heavy burden on elderly persons because their body flexibility, muscle strength, and balance are reduced. It is important to design pants-type diapers that reduce the senile body-specific burden. [J Physiol Sci. 2008;58 Suppl:S161]

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Purpose: To establish an experimental numbness model in humans, the evoked subjective sensation and CDT (current detection threshold) by compression of upper arm were examined. Methods: Healthy volunteers with informed consents were used (n=8, 25.5±4 years). Ischemic compression was applied to the arm with tourniquet (200mmHg, 15-20min). Nature of the subjective sensation was recorded and its intensity was monitored by electronic VAS device continuously. The CDT was measured by three different frequencies (5, 250, 2000 Hz) of sine waves applied to the index finger with a pair of surface gel electrodes. The frequency was randomly changed at intervals of 30 sec, and the current intensity was recorded (Geosense, Japan). Results: Clear reproducible subjective numbness sensation was evoked after the cessation of the compression and it lasted for about 15 min in VAS. The baseline CDTs were 5 <250 <2000Hz. After the compression, significant increases of CDT were observed in 250 and 2000 Hz (baseline vs. after, P<0.01, Dunnett's multiple test). Discussion: The sine wave stimulation of 5, 250 and 2000 Hz were assumed to activate C, A-delta and A-beta fibers, respectively. The production of numb sensation was accompanied with the significant increases of CDT of 250 and 2000Hz after the releases of compression. These results might be explained, at least in part, by the interaction of ectopic firing elicited by re-perfusion and current stimuli in the A fibers. [J Physiol Sci. 2008;58 Suppl:S161]

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Itch is one of unpleasant sensations but is clearly distinct from pain sensation. It provokes scratching behavior while painful stimulation provokes withdrawal reflexes. Although numerous studies have examined nociceptive signal transduction in primary afferent fibers, the mechanism of itch sensation remains largely unknown. In order to clarify how itch sensation is conveyed in primary afferent fibers, we developed an in vivo intracellular recording technique from dorsal root ganglion (DRG) cells in adult rats. Under urethane-anesthesia, lumbar laminectomy was performed and DRG at lumbar levels was exposed. Microelectrodes were inserted into DRG neurons and action potentials (APs) elicited by cutaneous stimulation were recorded. In behavior analysis, 5-HT applied to the skin provoked scratching. The 5-HT-induced scratching behavior was inhibited by naloxone. DRG neurons examined were classified with Aβ, Aδ and C fibers based on their conduction velocities calculated with latency of APs elicited by cutaneous electric stimulation. 5-HT applied to the skin elicited APs in a subpopulation of C fibers, but not in Aβ and Aδ fibers. The 5-HT-sensitive C fibers also responded to mechanical stimulation applied to the skin. The time course of the 5-HT responses was quite similar to that of 5-HT-induced scratching in behavioral analysis. These results suggest that selective activation of a subpopulation of C fibers by 5-HT may be essential to elicit itch sensation. [J Physiol Sci. 2008;58 Suppl:S162]

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We developed chronic pain model with the cast-immobilization to reveal mechanism of chronic pain without clear nerve injuries. This model produced the long-lasting (weeks) and widespread hyperalgesia were seen not only in immobilized side, but also in contralateral side and in tail without nerve injury. Furthermore, nerve blockade in the sciatic nerve 3 weeks after the cast removal had no effect on the contralateral hyperalgesia in the hindpaw. It is suggested that the cast-immobilization causes the central plasticity. In this study, we investigated changes in spinal glial cells with immunohistology (OX42, GFAP). We found the activation and increase in the number of microglia in L4 dorsal horn following the cast removal in the acute phase when hindpaw hyperalgesia began, while in the chronic phase that showed maximum pain behaviors we found the activation of astroglia in L4 dorsal horn and microglia in coccygeal dorsal horn. In the end of chronic phase that showed attenuated pain behaviors, both glial cells activations tended to reduce. It is suggested that microglia and astroglia are differently activated in the course of pain development in this cast-immobilization model. [J Physiol Sci. 2008;58 Suppl:S162]

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Response of membrane potential to substance P (SP) has well been examined in spinal dorsal horn neurons, but little is known about those to CGRP and somatostatin. Therefore, we addressed these issues by the blind patch clamp technique used freshly sliced spinal cord of the rat (3-4 weeks). Of the dorsal horn neurons (32/51 cells) in the deep lamina (III-VI), about 60% displayed the slow inward current by the bath application of SP (1 μM). This current has been considered to be evoked though G protein-coupled SP receptor on the patched cells. Many these neurons simultaneously increased excitatory post-synaptic currents (EPSCs). About 30% of deep dorsal horn neurons (9/27 cells) showed the slow inward current by the application of CGRP (1 μM), as well as SP. The responsive neurons to CGRP always displayed the inward current by SP. However, the amplitude of the inward current by CGRP was smaller than that by SP, and a little number of the neurons increased EPSPs. On the other hand, the application of somatostatin (1 μM) evoked the slow outward current in about 30% of the patched deep dorsal horn neurons (7/22 cells). Many these neurons also showed the inward current by SP. The outward current was not suppressed by the repetitive application of somatostatin, although the inward currents by SP and CGRP were desensitized. The present study suggests that CGRP and somatostatin, in addition to SP, play an important role in synaptic transmission to deep dorsal horn neurons. [J Physiol Sci. 2008;58 Suppl:S162]

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Although central sensitization is assumed to be an essential factor causing chronic low back pain (CLBP), the underlying spinal mechanisms are poorly understood. In the present study, we recorded activities of spinal dorsal horn neurons receiving input from low back structures (defined as "LB neurons") in rats, and examined the chronic sensitization effect of complete Freund's adjuvant (CFA) on these neurons. Extracellular recording was performed in the spinal segments L2 and L3 in non-treated control animals (CTR), and animals after intramuscular injection of CFA into the multifidus (MF) muscle bilaterally 6 or 12 days prior to the experiment. Mechanical withdrawal threshold following stimulation of the MF significantly decreased 6 and 12 days after CFA injection, indicating mechanical allodynia. The proportion of LB neurons 6 days after CFA was significantly higher than that in the CTR in the segments L2 and L3 (9/76 neurons in the CTR vs. 16/61 neurons in the CFA, p < 0.05, Fisher's exact test). In addition, LB neurons which had convergent inputs from different tissues and regions of the body were more frequent in the CFA group than in the CTR (6/9 neurons in the CTR vs. 16/16 neurons in the CFA, p < 0.05, Fisher's exact test). These results show that the responsiveness of LB neurons is increased during a pathologic alteration of the MF. The findings may explain the allodynia of some cases of chronic low back pain in patients. [J Physiol Sci. 2008;58 Suppl:S162]

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Melittin is the main toxin of bee venom. Previously, we have reported that intradermal injection of melittin into the volar aspect of forearm in humans produces a temporary pain and a subsequent sustained neurogenic-inflammation-skin temperature increase. Furthermore, not only subcutaneous melittin but also subcutaneous glutamate produced neurogenic inflammation on the rats' hindpaw. Aim of the present study was to confirm the involvement of NMDA receptors on melittin-induced neurogenic inflammation. We examined the neurogenic inflammation-skin temperature increase and glutamate release after melittin was injected subcutaneously into the hindpaw of pentobarbital-anesthetized rats. Skin temperature increase was analyzed using the computer-assisted-thermography. Subcutaneous glutamate was collected through a microdialysis probe, and glutamate level was measured using the HPLC-ECD method. Furthermore we examined the effect of NMDA receptor agonist and antagonist on the neurogenic inflammation-skin temperature increase. Intraplantar injection of melittin increased both skin temperature and glutamate level in subcutaneous microdialysate. Intraplantar injection of NMDA produced significant skin temperature increase compared with vehicle injection. Co-injection of MK-801 dose-dependently suppressed melittin-induced skin temperature increase. These findings suggest that melittin-induced neurogenic inflammation is enhanced through activation of peripheral NMDA receptors by peripherally released glutamate. [J Physiol Sci. 2008;58 Suppl:S163]

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TRPA1 is expressed in small diameter DRG neurons and hair cells, and is proposed to be a receptor for noxious cold (<17 °C) and pungent ingredients such as cinnamaldehyde (CA) and allyl isothiocynate (AI). Recent studies have shown that TRPA1 acts on presynaptic terminals in the substantia gelatinosa of the dorsal horn to increse the glutamate release. It is not fully understood, however, how synaptic transmission through C fibers is eventually modified by the activation of TRPA1 in the SG which exhibits a heterogeneous population of cells. We investigated the effects of CA and AI, on spontaneous and dorsal root-evoked EPSCs in SG neurons, and relationship between the modulatory effects on synaptic transmission and morphological features of recorded neurons. Under voltage-clamp conditions, CA (and AI) dose-dependently increased the frequency and amplitude of sEPSCs while it increased only the frequency of mEPSCs observed in the presence of TTX. On the other hand, CA reversibly inhibited the amplitude of C fiber- but not A δ fiber-evoked monosynaptic EPSCs. In combination with morphological analyze of recorded cells, the excitatory effect of CA was observed in radial and vertical (excitatory) cells but not in islet and central (inhibitory) cells. These results suggest that CA and AI act on TRPA1 receptors expressed in the presynaptic terminals of C fibers to facilitate miniature release in excitatory interneurons in the SG, but inhibit the C fiber-evoked release. [J Physiol Sci. 2008;58 Suppl:S163]

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We have reported that the extensor digitorum longus muscle (EDL) of rat 2 days after lengthening contraction (LC) is mechanically hyperalgesic (delayed onset muscle soreness, DOMS) (Taguchi et al, J Physiol 564) and that the C-fiber receptors of this hyperalgesic muscle increased their sensitivity to mechanical stimulation (Taguchi et al, J Neurophysiol 94). The mechanism for this increased sensitivity to mechanical stimulation has not been known yet. In the present study, we used PCR-based cDNA subtraction method, "suppression subtractive hybridization" procedures to define genes upregulated in dorsal root ganglion cell (DRG) of SD rat with DOMS. LC of the right EDL was induced by electrical stimulation of the common peroneal nerve while the muscle was being stretched. The right DRGs (L3-L4) were dissected in the control group and 2 days after LC in the DOMS group. Using the cDNA subtraction method, we detected 65 different genes upregulated. Among these 65 genes were annexin A2 and calbindin 1, which were recently reported to be involved in nociception. We quantified expression levels of these 2 genes in DRGs form the DOMS group and the control group using quantitative PCR assay. Expression levels of these two genes relative to an internal standard (glyceraldehyde-3-phosphate dehydrogenase) were significantly increased in the DOMS group compared to the control group (p<0.05, n=5 each). This is the first demonstration that annexin A2 and calbindin 1 were upregulated in DRG from rat with DOMS. [J Physiol Sci. 2008;58 Suppl:S163]

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Previously we showed the existence of muscular mechanical hyperalgesia (DOMS) after lengthening contraction (LC) in rats. The mechanical withdrawal threshold of EDL muscle underwent LC decreased 1 day to 3 days after LC. It returned to the pre-LC value 4 days after LC (Taguchi et al, J. Physiol., 2005). In addition, we showed that administration of selective COX-2 inhibitors, Zaltoprofen and Celecoxib, before LC, but not after LC, blocked the development of the mechanical hyperalgesia elicited by LC. Meanwhile, it was reported that intramuscular injection of NGF induced lasting tenderness (Svennson et al, Pain, 2003). Therefore, we examined the expression of COX-2 and NGF mRNA in EDL muscle immediately after LC (0hr), 3, 6, 12 hrs and 1, 2, 3, 5 days after LC, and effects of anti-rat β NGF antibody on DOMS. EDL muscle without LC was used as a control. Expression of COX-2 mRNA increased 0-12 hrs and returned to the control value 1day after LC. Expression of COX-2 protein determined by western blotting also increased 0-12 hrs after LC. On the other hand, expression of NGF mRNA increased a little later, namely, 12 hrs-2 days after LC. To know if NGF is involved in DOMS, we examined effects of anti-rat β-NGF antibody (R&D systems). Rats were administered anti-NGF antibody (10 μg/ 5 μl, i. m.) or saline 6 hrs after LC under light halothane anesthesia. Anti-NGF completely blocked the development of the mechanical hyperalgesia. These results suggest that COX-2 acts as a trigger of the development of mechanical hyperalgesia and NGF was involved later in DOMS than COX-2. [J Physiol Sci. 2008;58 Suppl:S163]

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Living things survive in a very narrow range of pH, and possess physiological detection and defense mechanisms when ambient pH becomes out of the narrow range. Regarding pain sensation evoked by exposure to acidic pH, sensory neurons sense the reduction in pH by specific ion channels such as TRPV1 and ASIC. Here we show that alkaline pH activates TRPA1 ion channel, which belongs to the TRP super family and is involved in nociception, using calcium-imaging and patch-clamp methods. Furthermore, it became clear that increase in the intracellular, but not extracellular pH causes TRPA1 activation, in the experiment where ammonium chloride with pH7.4 was used, which causes intracellular alkalization. Activation of TRPA1 by intracellular alkalization was supported by the single-channel recordings in outside/out excised membrane patches. In addition, alkaline solution-evoked whole-cell currents exhibit similar electrophysiological properties to those observed in HEK293 cells expressing TRPA1 in response to AITC application. [J Physiol Sci. 2008;58 Suppl:S164]

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The neurons of the trigeminal subnucleus caudalis (Sp5C) play an important role in the processing pain and sensory information from the cranio-facial area. In the present study, we investigated the characteristics of GABAergic and non-GABAergic neurons in the Sp5C using neonatal transgenic mice (P2-6) in which green fluorescent protein (GFP) is specifically expressed in GABAergic neurons. We recorded activities of GFP-positive (GABAergic, n=20) and GFP-negative (non-GABAergic, n=33) neurons in a perforated whole-cell patch clamp mode in the sagittal slice of brainstem attaching the trigeminal nerve rootlet. Electrical stimulation to the trigeminal nerve evoked excitatory postsynaptic potentials (EPSPs) in all of the recorded GFP-positive neurons and in 31 GFP-negative neurons. Inhibitory postsynaptic potentials (IPSPs) were evoked in 3 GFP-positive and in 13 GFP-negative neurons. In morphological study, most of GFP-positive neurons had multipolar-shaped somata (n=8/10) and GFP-negative neurons had bipolar (n=5/10) or triangular (n=5/10) somata. The difference of somatic size between GFP-negative and GFP-positive neurons was not significant. These results indicate that GABAergic neurons in the Sp5C are characterized by multipolar-shaped somata, and receive exclusively excitatory postsynaptic inputs from the cranio-facial area. [J Physiol Sci. 2008;58 Suppl:S164]

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It is well known that patients with orofacial cancer suffer from cancer-induced pain. To understand mechanisms of pain by facial cancer, we have recently created a rat facial cancer model by inoculation with Walker carcinosarcoma 256 B-cells into the right vibrissal pad. The aim of the present study was to investigate changes of pain-related behavior and ingestive behavior in the model, along with c-Fos expression in the trigeminal subnuclei caudalis which is a center of facial pain sensation, by using both behavioral and immunohistochemical techniques. Mechanical allodynia and thermal hyperalgesia developed on the inoculated facial side on days 4 and 7 post-inoculation. After day 10, the sensitivities of the rats to mechanical and thermal stimuli were reduced, and there were physical difficulties in ingestion. Furthermore, the periods of facial grooming were prolonged post-inoculation, as compared with those in the sham group. In the trigeminal subnuclei caudalis, the level of c-Fos immunoreactivity of the ipsilateral side was increased significantly on days 4, 7 and 10 post-inoculation, as compared with those of the contralateral side and in the sham group. However, substance P and calcitonin gene-related peptide levels in the trigeminal subnuclei caudalis were not changed in the facial cancer model whereas they were increased in facial inflammation model of rat. These results suggest that neuropathic pains in the facial cancer model are probably caused by different pathway of inflamation-induced. [J Physiol Sci. 2008;58 Suppl:S164]

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It is well known that capsaicin application to the facial skin causes a decrease in pain threshold in humans. We also observed that the application of capsaicin to the monkey face causes a decrease in cold detection threshold. However, the neural mechanisms of the change in cold pain threshold following capsaicin treatment are still remained unclear. The aim of present study is to clarify the neural mechanisms underlying capsaicin-induced cold allodynia in face. The phosphorylation of the extracellular signal-regulated protein kinase (ERK) was precisely analyzed in the trigeminal spinal subnucleus caudalis (Vc) neurons in pentobarbital anesthetized rats (sodium pentobarbital: 50mg/kg, ip.) after the topical application of capsaicin (10mM) to the facial skin. A large number of pERK-LI cells was observed in the superficial laminae of Vc after heat stimulation of the face in vehicle treated rats, but not after cold stimulation. However, the number of pERK-LI cells was significantly increased after cold stimulation of the face in the rats with capsaicin treatment compared with vehicle treated rats as well as heat stimulation. The present findings suggest that the sensitization of TRPV1 channels is involved in capsaicin-induced cold allodynia in the facial skin. [J Physiol Sci. 2008;58 Suppl:S164]

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Extracellular signal-regulated kinases (ERK1 and ERK2) are members of the serine/threonine protein kinases implicated in the transduction of neurotrophic and neurochemical signals from the cell surface to the nucleus, which play important roles in synaptic plasticity and memory formation. The locus coeruleus (LC) sends noradrenergic projection to the spinal dorsal horn and contributes to pain modulation. In the present study, the activation of ERK in the LC following the injection of complete Freund's adjuvant (CFA) into the rat hindpaw was examined in order to clarify the mechanisms underlying the dynamic changes in the descending pain modulatory system after peripheral inflammation. Phospho-ERK -immunoreactive (p-ERK-IR) neurons were observed in the LC and the adjacent area. CFA injection induced the activation of ERK in the LC, with a peak at 2 minutes, which was transient. In the LC, the number of p-ERK-IR neurons in rats killed at 2 min after CFA injection (77.4±12.1) was significantly higher than that in the naive rats (4.5±1.3) [p<0.05]. However, there was no significant difference in the number of p-ERK-IR neurons in the LC between CFA and saline groups. The number of p-ERK-IR neurons in the handling group (no injection) was significantly lower than those in both CFA and saline groups. These findings suggest that activation of ERK in the LC may be induced by transient noxious stimulation, such as injection, not by peripheral inflammation. [J Physiol Sci. 2008;58 Suppl:S165]

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Descending influence from the locus coeruleus/subcoeruleus (LC/SC) on cutaneous and visceral nociceptive transmission in the spinal cord was investigated in the anesthetized rat. Extracellular recordings were made from the L6-S2 segmental level using a carbon filament glass microelectrode (4-6 Mω). Colorectal distention (CRD) was produced by inflating a balloon inside the descending colon and rectum. All neurons tested responded to both CRD and to cutaneous pinch (a force of 613 g/mm²), indicating that nociceptive signals from visceral organs and nociceptive signals from the cutaneous receptive field converge to a neuron. These neurons were divided into two groups based on their response to CRD: short latency-abrupt and short latency-sustained neurons. Electrical stimulation of the LC/SC (30 or 50 μA, 100 Hz, 0.1 ms pulses) failed to inhibit CRD-evoked responses in 10 of 52 short latency-abrupt neurons, whereas cutaneous pinch-evoked responses were inhibited in these convergence neurons. In all short latency-sustained neurons tested (n=15), electrical stimulation of the LC/SC significantly inhibited both cutaneous pinch-evoked responses and CRD-evoked responses. The present result that cutaneous nociceptive transmission was independently inhibited without inhibition of visceral nociceptive transmission may be explained by a synaptic arrangement in which a nerve ending of a descending LC/SC neuron terminates on a cutaneous primary afferent terminal (presynaptic inhibition). [J Physiol Sci. 2008;58 Suppl:S165]

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Sex differences in the response to nociceptive stimuli have long been recognized in many species. For example, in rats, females are more sensitive to formalin-induced nociceptive stimuli than males, as revealed by behavioral test. However, the neural mechanism for sex differences in the response to nociceptive stimuli was still unclear. In the previous study, we showed that females were more sensitive to formalin-induced nociceptive stimuli than males, as revealed by behavioral test. We looked for a possible sex difference in the response of the brain to formalin-induced nociceptive stimuli by checking the expression of phosphorylated cAMP response element-binding protein (pCREB) as a marker of neural activity. We found that, in the bed nucleus of the stria terminalis lateral subdivision (BSTL), the number of cells expressing pCREB was increased 5-10 min after formalin injection in female rats but not in male rats. In the present study, we examined the function of CREB in the BSTL. We found that the expression of a dominant negative form of CREB (mCREB) using adenovirus vector, significantly attenuated the response to formalin only in females but not in males, suggesting that CREB in the BSTL play a role for controlling pain-related behavior only in females. [J Physiol Sci. 2008;58 Suppl:S165]

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Conditioning electrical stimulation of the amygdala has an inhibitory effect on two types of nociceptive neurons, NS and WDR neurons, of the rat medullary dorsal horn. The purpose of the present study was to determine whether the activation of cell bodies by glutamate microinjection into the amygdala induces inhibition of both types of nociceptive neurons. The nociceptive neurons were recorded in the medullary dorsal horn of the rat anesthetized with N₂O-O₂ and 0.5% halothane and immobilized with pancuronium bromide. A peripheral test stimulus (a single rectangular pulse of 2.0 msec in duration) was applied to the facial area, and ipsilateral amygdaloid conditioning stimuli to the recording site were trains of 33 pulses (100-300 μA) delivered at 330 Hz. Glutamate microinjection was conducted through the same electrode as that used for electrical stimulation. Four WDR and three NS 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 maximal inhibition of these nociceptive neurons (85.4±9.3% for WDR neurons; 66.4±16.6% for NS neurons) at 5 min after injection, and the recovery of inhibition to 50% occurred at about 20-25 min. These findings suggest that excitation of neurons in the amygdala influences sensory-discriminative and motivational-affective components of pain experience. [J Physiol Sci. 2008;58 Suppl:S165]

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