Olfactory reception is unique in that the transduction current is generated by two types of ion channels; cyclic nucleotide-gated (CNG) channels and calcium-activated chloride (Cl(Ca)) channels in the nano-scale sensory cilia. Because of such sequential cascade in the nano-tube structure, quantification of individual channels is still limited. For long period of time, free manipulation of cytoplasmic factors in the cilia has been thought to be a technical limitation. However, recent achievements using caged compound have provided possibilities of overcoming these technical difficulties. In this work, we focused on manipulating the [Ca²⁺]i in the nano-tube cilia and on quantification of Cl(Ca) channels, by comparing with the odorant and caged cAMP responses. Such experiments enable us to examine the role of molecular elements in the sequential cascade step by step. We loaded the cilia with caged Ca (DM-Nitrophen) under whole-cell patch clamping, monitoring the Cl(Ca) current as an index of [Ca²⁺]i increase. Basic physiological properties of Cl(Ca) channels were examined; I-V curve, reversal potentials, [Cl]o dependence and dose-response relation showing non-linearity. The properties were consistent with the idea that the Cl current is included in the odorant- and cAMP-induced responses. Besides, adaptation and odorant-suppression that have been observed in the odorant- and cAMP-induced current were less observed in this current. We propose that Cl(Ca) channel is important for the non-linear amplification of the system and that the adaptation and suppression of the responses are controlled at the level of the CNG channel. [J Physiol Sci. 2008;58 Suppl:S166]

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In immature female mice, pheromones promote sexual maturations such the acceleration of an increase in uterine weight and the advance of the onset of first oestrus. Pheromones induce different effects on mature females such as Bruce effect and the suppression of oestrus, indicating that the role of pheromones on physical state develops with maturation. In the present study, we examined the changes in sensitivity to urinary pheromone excreted from males in female mice to explore the maturation of treatment of pheromonal information in the accessory olfactory bulb (AOB). After exposure to male urine, many Fos-immunorective (Fos-ir) cells appeared in the periglomerular cell (PGC), mitral/tufted cell and granule cell (GC) layers of the AOB of the immature female mice. The densities of Fos-ir cells in the immature female mice after exposure to male urine were higher than those in the mature females. Pheromonal information is modulated by GABAergic interneurons such as the PGC and GC. To explore the essential GABA receptors that modulate pheromonal information during maturation, we searched for GABA_A receptor subtypes expressed in the mouse AOB by RT-PCR analysis. In the PGC layer, amplification of fragment for subtype 1, 2 and 3 was clearly detected in mature females but not in the immature females suggesting that development of the inhibitory system in the AOB may regulate pheromonal sensitivity in the AOB. [J Physiol Sci. 2008;58 Suppl:S166]

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Most terrestrial vertebrates have at least two segregated chemosensory neurons in the nasal cavity: the olfactory receptor neurons (ORNs) and vomeronasal neurons (VNs). Recent studies have revealed that the receptive spectrum of molecular structure appears to be partially overlapping between ORNs and VNs. For example, MHC class I peptides which function directly as chemosignals, have been shown to activate both ORNs and VNs, in contrast to the widely held view that ORNs are stimulated by volatile chemosignals only. These observations give rise to a possibility that there may be volatile odorants which are detected by VNs to cause biological responses such as changes in hart rate and body temperature. As an initial step to explore the additional role of the vomeronasal system on animal behavior, we examined the sensitivity of the rat VNs to chemicals considered not to be related to pheromones by using Ca²⁺-imaging approach. In the present study, we observed that (±)-propane-1,2-diol (propireneglycol), a common organic solvent, elicited Ca²⁺ responses in a dose-dependent manner. We found detection thresholds for this compound at concentrations near or below 1 μM. These results reveal a sensitivity of the rat VNs for this chemical, which seems comparable to that of ORNs, supporting the idea mentioned above that some non-pheromonal volatile odorants may affect animals behavior via the vomeronsal system in rodent. [J Physiol Sci. 2008;58 Suppl:S166]

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By optical imaging of intrinsic signals, we have demonstrated a possible code for odor concentration in the anterior piriform cortex (PC). Lower concentrations activated the rostral region of the dorsal part of the anterior PC, whereas higher ones elicited caudally spreading activation, suggesting an important role of a rostro-caudal gradient in odor sensitivity among cortical neurons. These imaging results have also been confirmed with unit recordings, which indicated a rostro-caudal gradient in odor sensitivity among cortical neurons. In this study, we used optical imaging of voltage-sensitive dye signals to study spatiotemporal spread of signal propagation in the endopiriform nucleus (EPN), one of the main output targets from PC, using the horizontal slice preparations including both EPN and PC. Electrical stimulation of EPN evoked an excitation that propagated slowly (about 150-200 mm/s) to both the rostral and caudal directions from the stimulation site in EPN. On the other hand, stimulation of layer I in PC induced signal propagation from PC to EPN. Excitation in EPN, then, propagated slowly (160-250 mm/s) from rostral to caudal, but the rostral propagation was weak. These findings suggest that a rostro-caudal slow propagation in EPN is involved in further processing of information from the anterior PC, which may be associated with odor concentration. [J Physiol Sci. 2008;58 Suppl:S166]

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With aging, there is a generalized decrease in mucosal sensitivity. Such decreased sensitivity may contribute to swallowing problems in the elderly. This study was designed to compare the effects of stimulation of the pharyngolaryngeal region (PL) on voluntary swallowing between young (23-37 years, n = 18) and elderly (60-84 years, n = 23) healthy subjects. Water or 0.3 M NaCl solution was delivered into the PL through a fine tube at 0.2-5.0 ml/min. Water stimulates water receptors in the PL and 0.3 M NaCl inhibits them. Each subject was instructed to repeat swallowing as fast as possible. Swallowing intervals (SIs) between two consecutive swallows were measured. Values of SI with weak stimulation (0.3 M NaCl at 0.2 ml/min slow infusion rate) varied greatly in subjects and the values in the elderly did not differ from those in the young, suggesting that the ability of swallowing center to perform repetitive voluntary swallowing varies among subjects and that the ability to swallow in the elderly does not differ from that in the young. In the young and elderly, SIs were shorter in the case of water infusion than in the case of 0.3 M NaCl (water effect), and increasing infusion rate shortened SIs (mechanical effect). Sensory effects (water and mechanical effects) decreased in the elderly. Diminution in sensitivity of the PL with increasing age might be a contributing factor in the development of dysphagia and aspiration in the elderly. [J Physiol Sci. 2008;58 Suppl:S167]

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Taste signals are transmitted from taste cells to gustatory nerve fibers through chemical synapses. Taste cells are morphologically classified into four types: I, II, III and basal cells. Among them, chemical synapses are observed only in type III cells with putative sour receptors. However, sweet, bitter and umami receptors are expressed in type II cells, and the mechanisms have not been elucidated. Recent reports have highlighted the role of ATP as a key neurotransmitter. Here we tried to detect ATP release from single taste cells with action potentials in mouse fungiform papillae. The action potentials were recorded with the electrode basolaterally attached to a cell. When an increase in the firing rate was observed in response to a tastant, the electrode solution was applied for luciferase assay to determine the ATP. Type II and III cells express gustducin and glutamic acid decarboxylase (GAD67), respectively. We identified these cell types with transgenic mice expressing GFP in gustducin- or GAD67-positive cells. When Type II cells responded to saccharin or quinine, ATP was detected in a firing rate-dependent manner. When Type III cells increased the firing rate by application of HCl, ATP was below the detection limit. The results suggest that the amount of ATP released from taste cells differ with the response properties, or that type III cells release another neurotransmitter. [J Physiol Sci. 2008;58 Suppl:S167]

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Sweet taste responses in receptor cells are known to occur through a common signaling pathway involving T1R2/T1R3 heterodimeric receptors, G-αgustducin (Ggust) and TRPM5 channels. In mice, sweet responses of the chorda tympani (CT) nerve are classified into two components; one is inhibited by gurmarin [Gur-sensitive (GS)] and the other is not [Gur-insensitive (GI)]. Recently, TRPM5 is found to be a temperature-denpendent increase (TDI) in response to sweeteners. In the current study, we examined GS and GI components to sucrose (Suc), glucose (Glc), saccharin (Sac), SC45647 (SC) in the CT nerve at 15, 25, 35°C of temperature in T1R3-, Ggust- and TRPM5-KO mice. Compared to those in wild type mice, the CT nerve responses of three types of KO mice to Suc, Glc, Sac were either greatly reduced, or abolished in case of SC at every temperature. In T1R3-KO mice, responses to Suc and Glc exhibited TDI and GS. In Ggust-KO mice, responses to Suc and Glc exhibited only TDI. In TRPM5-KO mice, responses to Glc exhibited TDI and GS. The responses to Sac did not exhibit TDI and GS in all KO mice. These results suggest that (1) the GS component may occur through not only TRPM5-dependent (temperature-sensitive) pathway, but also other unknown pathway which may be TRPM5-independent (temperature-sensitive) in Glc responses, and (2) it is possible that Ggust may play an indispensable role in the GS transduction pathway for sweet taste responses in mice. [J Physiol Sci. 2008;58 Suppl:S167]

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Monosodium L-glutamate (MSG), a sodium salt of amino acid L-glutamate (Glu), elicits umami taste. Recent studies suggest the post-oral effect of Glu. Consumption of Glu is decreased in gastric vagotomized rats. Gastric vagal afferent fibers specifically respond to the intragastric (luminal) administration of Glu, but not to the other amino acids. These results raised hypotheses that ingested Glu is detected by chemoreceptors in the gastrointestinal tract and the signal is transmitted to the brain via vagus nerve. The goal of present study was to demonstrate spatiotemporal activation of the brain following intragastric Glu administration (in the absence of taste) using functional magnetic resonance imaging. Rats were anesthetized with α-chloralose and 60 mM MSG solution was infused in the stomach for 10 min. To exclude effects with sodium ion, NaCl (60 mM) was used as control. Second, we assessed a role of the vagal afferent nerve in rats under subdiaphragmatic total vagotomy (TVX). The results showed that only MSG elicited significant activation on several brain regions including the medial preoptic area, dorthomedial hypothalamus, habenular nucleus and amygdala. Both MSG and NaCl activated the anterior cingulate, caudate-putamen, insular cortex and hippocampus. TVX eliminated substantially the MSG or NaCl-induced activation. Our data are the first evidences demonstrating that post-oral Glu can activate the brain via vagus nerve. The brain activation caused by post-oral Glu may link to various physiological responses. [J Physiol Sci. 2008;58 Suppl:S167]

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Vertebrate taste buds contain diverse types of cells. Recent studies have shown that type II cells are likely to be the sensory receptor cells for bitter taste stimuli. However, type III cells form synapses with nerve fibers, whereas type II cells do not form morphologically identifiable synapses. Hence, it has been proposed that type II cells communicate with type III cells via gap junctions or paracrine secretions. If so, bitter responses from single taste fibers would be modulated by other taste stimulation. In the frog glosopharyngeal nerve, there are quinine-sensitive fibers (Q-fibers) and calcium-sensitive fibers (Ca²⁺-fibers). In this study, we examined whether quinine (Q) responses are altered by Ca²⁺ taste stimulation. Bullfrogs (Rana catesbeiana) were used. Antidromic unitary impulses of Q-fibers or of Ca²⁺-fibers were recorded from a single fungiform papilla drawn into a suction electrode. Q at 0.5 mM elicited a phasic response, whereas CaCl₂ at 1 mM elicited a sustained response. Q responses (the latency between onset of stimulation and appearance of the first impulse, and the frequency of impulses) were not altered by appearance of neural response from Ca²⁺-fibers. The present results suggest that Q and Ca²⁺ taste reception do not use a common pathway. In another experiments, we have shown that Q excites type II cells in the frog taste disc, whereas Ca²⁺ excites type III cells. Therefore, it is likely that bitter taste information from type II cells is directly communicated to the Q-fibers. [J Physiol Sci. 2008;58 Suppl:S168]

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The frog fungiform papilla (taste disc) contains morphologically distinct cells, type I, II, III and IV. Since only type III cells show synaptic-like contacts with afferents, type III cells have been thought to be taste receptor cells. In the frog glossopharyngeal nerves, it has been reported that Ca²⁺-sensitive fibers (Ca²⁺-fibers) and quinine-sensitive fibers (Q-fibers) are present. Ca²⁺-fibers do not respond to quinine-HCl (Q), while Q-fibers do not respond to CaCl₂. These findings suggest that Q and CaCl₂ taste stimuli are received by the respective types of taste cells. In this study, we recorded changes in membrane currents and intracellular Ca²⁺ concentration ([Ca²⁺]_i) of type identified taste cells of the bullfrog (Rana catesbeiana) elicited by puff application of 10 mM Q or 40 mM CaCl₂ to the apical portion of taste cells. Q elicited inward current and [Ca²⁺]_i increase in type II cells, but not in type III cells. CaCl₂ elicited inward current and [Ca²⁺]_i increase in type III cells, but not in type II cells. We found that [Ca²⁺]_i increase in type II cells is due to release from intracellular store and that [Ca²⁺]_i increase in type III cells is due to influx through voltage-gated Ca²⁺ channel. We could not find cell-cell communication between type II and III cells. These results suggest that quinine taste information from type II cells is directly transmitted to Q-fibers via unconventional mechanisms and Ca²⁺ taste information from type III cells is transmitted to Ca²⁺-fibers via conventional synaptic mechanisms. [J Physiol Sci. 2008;58 Suppl:S168]

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Taste bud cells (TBCs) fire action potentials in response to taste stimuli. Recent studies showed that the action potentials opened hemichannels to release ATP, a neurotransmitter. Voltage-gated Na⁺ channels consist of α and β subunits. Ten genes encoding α subunits and four genes encoding β subunits have been identified. We investigated the expression of mRNA encoding α and β subunits and electrophysiological properties of voltage-gated Na⁺ currents in mouse fungiform TBCs. RT-PCR studies using mRNA extracted from taste buds suggested the expression of Nav 1.3, Nav 1.5, and Nav 1.6 α subunits and β1 subunit. In whole-cell clamp experiments, the midpoint of activation and inactivation was ∼-25 mV and ∼-65 mV, respectively. The recovery from inactivation was well fitted with double exponential with tau1 of ∼20 ms and tau2 of ∼800 ms. A subset of TBCs exhibited the Na⁺ currents in the presence of 1 μ M TTX. The TTX-resistant component was ∼6% of total voltage-gated Na⁺ currents recorded on membrane depolarization from -70 mV to -20mV. These results suggest that TBCs can be classified into at least two groups based on TTX-sensitivity. One group of TBCs expresses only TTX-sensitive Na⁺ channels (Nav 1.3 and/or Nav 1.6). Another has a little amount of TTX-resistant Nav 1.5 channels in addition to TTX-sensitive ones. Supported by a COE program (center #J19) granted to Kyushu Institute of Technology by MEXT of Japan. [J Physiol Sci. 2008;58 Suppl:S168]

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After acquisition of conditioned taste aversion (CTA), hedonics of a taste solution as the conditioned stimulus (CS) changes from palatable to aversive. Previous studies suggest that the opioidergic system in the ventral pallidum (VP) is involved in taste hedonics. To elucidate the role of μ-opioid receptors in the VP in a hedonic shift accompanying CTA, we examined the effects of stimulation of μ-opioid receptors on the intake of CS after acquisition of CTA. Rats were conditioned with a pairing of 5 mM saccharin solution (CS) and 0.15 M lithium chloride (20 mg/kg: unconditioned stimulus). Two days after the conditioning, the rats received bilateral microinjections of μ-opioid receptors agonist D-Ala²-N-Me-Phe⁴-Glycol⁵-enkephalin (DAMGO) (10 or 100 g/0.25 µl) or vehicle (Ringer solution) just before the presentation of CS. The consumption of CS was measured at a 30 min interval for 120 min. There were no significant differences in the intake of the CS between the 10 ng DAMGO and vehicle groups, indicating that lower dose of DAMGO did not affect aversion to the CS. On the other hand, the 100 ng DAMGO group showed significantly higher level of CS consumption than the vehicle group at 90 and 120 min after CS presentation. These results suggest that higher dose of DAMGO might change taste hedonics of the CS from aversive to palatable. It is plausible that the μ-opioid receptors in the VP play a role in a hedonic shift in CTA. [J Physiol Sci. 2008;58 Suppl:S168]

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A number of reports showed that epigenetic mechanisms have an important role in synaptic plasticity and memory formation. Particularly as cellular mechanisms, chromatin structures are remodeled by modification of histone acetylation during the early stages of long-term memory formation. In the hippocampus decrease in acetylation of histone is induced by inhibition of the MEK-MAPK/ERK cascade. Preweanling young rats prior to eye opening depend on somatosensory and olfactory function for survival, as they can learn their dam's odor and approach her without visual information. In order to establish olfactory learning, the pairing of odor and somatosensory stimulation is crucial. We have previously shown that synaptic plasticity in the OB underlies aversive olfactory learning. Behavioral pharmacology shows that long-term, but not short-term, olfactory memory required activation of the transcription factor, CREB. Western blot analyses reveal that expression of P-MAPK/ERK was increased for 1 hour after odor-shock training, followed by increase of P-CREB lasting for 6 hours. Therefore we examined if intrabulbar infusion of the histone deacetylase (HDAC) inhibitor, sodium butyrate (SB) has a facilitatory effect on aversive olfactory learning in young rats. Animals infused with SB 1 or 2 hours after odor-shock training show aversion in the odor preference test 2 days later, but control rats no longer show aversive responses. These results show that HDAC inhibition consolidates aversive olfactory learning in young rats. [J Physiol Sci. 2008;58 Suppl:S169]

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Taste buds consist of different cell types such as type II and III cells. Type II cells have taste receptor molecules for sweet, bitter, and umami substances, but have no synaptic contact with taste nerves. By contrast, type III cells have none of such taste receptor molecules, but have synaptic contacts with taste nerves. Recent studies thus considered the interactions among these taste bud cells via neurotransmitters. We investigated the expression of neurotransmitter receptors by in-situ Ca²⁺ imaging. The application of 10 μM ACh and ATP to the basolateral membrane of taste bud cells increased intracellular Ca²⁺ concentration, but that of 100 μM glutamate, 5HT, and noradrenalin had no effect. All taste buds examined responded to ATP, and all cells in these taste buds were apparently ATP-sensitive. The Ca²⁺ response to ACh occurred in ∼15% of taste buds examined, and the number of responsive cells in these taste buds was ∼2.4 cells per taste bud. The application of 100 nM atropine completely inhibited the Ca²⁺ response, but 10 μM d-tubocurarine had no effect, showing that expressed ACh receptors are muscarinic. RT-PCR studies suggested the expression of muscarinic ACh receptor subtypes, M1, M3, M4 and M5. These results suggest that purinergic receptors and muscarinic ACh receptors are involved in the interaction among taste bud cells. Supported by a COE program (center #J19) granted to Kyushu Institute of Technology by MEXT of Japan. [J Physiol Sci. 2008;58 Suppl:S169]

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Umami is a taste quality exemplified by taste of MSG and evoked also by some amino acids and purine nucleotides. There is an evidence that a heterodimer of T1r1 and T1r3 proteins functions as an umami taste receptor in humans and rodents. A splice variants of a metabotropic glutamate receptor, mGluR1 and mGluR4, were also proposed as a taste receptor for glutamate. Taste sensitivity to umami substances varies among individual humans. However, little is known about association of genetic variation with umami taste responses in both human and rodents. In this study, therefore, we investigated possible relationships of individual differences in umami sensitivity with single nucleotide polymorphisms (SNPs) of T1r1, T1r3 in human. The working sample consists of non-obese 200 adults of both sexes (110 men, 90 women), aged 19-35. Taste recognition thresholds for MSG, IMP and MSG plus 0.5 mM IMP were determined by blind-test, series of graded aqueous solutions of each product. Distributions of MSG and IMP taste recognition thresholds showed a normal distribution curve, while, that for MSG plus IMP showed a tri-modal distribution curve. In sequencing analysis, 5 SNPs (amino acid positions: E12H, T139M, N191S, E347K, T372A) and 3 SNPs (T716K, R757C, R825S) with amino acid substitutions were identified in T1r1 and T1r3, respectively. We have analyzed the detail association between a diversity of umami sensitivity and these SNPs. [J Physiol Sci. 2008;58 Suppl:S169]

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We have developed the coculture system of the vomeronasal organ (VNO), which detects the pheromonal chemosignals, and the accessory olfactory bulb (AOB), the first relay station of the vomeronasal system, to investigate the regulatory mechanisms for vomeronasal development. The rat vomeronasal neurons can not mature in the singly cultured VNO, but morphologically show the maturational characteristics in the coculture with the AOB. To investigate whether morphological changes are accompanied by functional maturation, we applied charged compounds in urine iontophoretically into the cultured VNO using a microelectrode and analyzed responses by a Ca²⁺ imaging method with or without cultured AOB neurons. The rat VNO cocultured with the AOB neurons could respond to rat urine, which was ejected into the cocultured VNO with a negative current. These VNO responses were not observed without AOB neurons, by urine-ejection with a positive current or by the ejection of artificial urine. Interestingly, the cocultured VNO could respond to not only rat but also mouse urine. Moreover, all of a western blot, a RT-PCR and an immunofluorescence analyses clearly indicated that the expression of two representatives (VR1 and VR4) from different subfamilies of the V2R family of vomeronasal receptors was significantly enhanced in co-culture. These results indicate that vomeronasal neurons result in expressing vomeronasal receptors by interacting with AOB neurons in co-culture, and then acquiring responsiveness to negatively charged compounds in urine. [J Physiol Sci. 2008;58 Suppl:S169]

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A large positive potential in the endolymph, named the endocochlear potential (EP) has been considered to occur at cochlear stria vascularis and this DC potential is essential for the transduction of sound by hair cells. In the previous paper, we examined the regulation of EP by studying the L-type Ca²⁺ channels in the basolateral membrane of marginal cells in the stria vascularis (JPS, 2007). In this study, we examined the contribution of TRP channels to the regulation of EP, measuring the transient asphyxia- or furosemide-induced decrease in the EP (TAIDEP or FUIDEP) with or without inhibitors of Ca²⁺-permeable channels. 1) Endolymphatic administration of 100 μM SKF96365, an inhibitor of both L-type Ca²⁺ and TRPC channels, inhibited almost completely the TAIDEP and the FUIDEP. 2) Administration of 1 μg/ml (3 μM) nifedipine, a specific inhibitor of L-type Ca²⁺ channels, into the endolymph significantly inhibits the TAIDEP, but slightly the FUIDEP. 3) The administration of 300 μM EGTA-tetraacetoxymethyl ester (EGTA-AM), a membrane-permeable Ca²⁺ chelator, to the endolymph significantly but partially suppressed the TAIDEP or almost completely the FUIDEP. These findings suggest that both the TRPC channels and the L-type Ca²⁺ channels play an important role in the regulation of the EP by affecting the cytosolic Ca²⁺ concentration of the endolymphatic surface cells (marginal cells) in the stria vascularis. [J Physiol Sci. 2008;58 Suppl:S170]

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The endocochlear potential (EP) of +80 mV is essential for audition. Although regulation of the K⁺ concentration in various compartments of the cochlear stria vascularis is crucial for formation of the EP, the mechanism remains uncertain. We have used multi barreled electrodes to measure the potential, K⁺ concentration ([K⁺]), and input resistance in each compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two epithelial layers, the marginal cell layer and that composed of intermediate and basal cells. The fluid in the IS exhibits a low [K⁺] and a positive potential, called the intrastrial potential (ISP). We found that the input resistance of the IS was high, indicating that this space is electrically isolated from the neighboring extracellular fluids. This arrangement is crucial for maintaining the positive ISP. Inhibiting the K⁺ transporters of the stria by anoxia, ouabain, or bumetanide caused the [K⁺] of the IS to increase and the intracellular [K⁺] of marginal cells to decrease, reducing of both the ISP and the EP. Calculations indicate that the ISP represents the K⁺ diffusion potential across the apical membranes of intermediate cells through Ba²⁺ sensitive K⁺ channels. The K⁺ diffusion potential across the apical membranes of marginal cells also contributes to the EP. Therefore the EP depends upon two K⁺ diffusion potentials and an electrical barrier in the stria vascularis. [J Physiol Sci. 2008;58 Suppl:S170]

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Inferior colliculus (IC) is a center of integration for auditory processing, receiving inputs from ascending, descending and intrinsic connections. The 20% of all the IC neurons are GABAergic and thought to play an important role in sound feature detection of IC. However their activities in vivo have not been not elucidated. For investigating the activity characteristis of IC GABAergic neurons in vivo, we combined juxtacellular labeling with immunocytochemistry to reveal the electrophysiologically recorded neurons are GABAergic or not and analyzed the response properties to sound stimuli and morphological characteristics of the neurons identified as GABAergic, comparing them with those of non-GABAergic neurons. [J Physiol Sci. 2008;58 Suppl:S170]

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Loop connections between thalamic and cortical cells are anatomical basis for interactive bottom-up and top-down streams of sensory information processing. The thalamic reticular nucleus (TRN), receiving thalamic and cortical inputs, plays a pivotal role in modulating loop connectivity by providing inhibition to thalamic cells. Previously, we have revealed the tonotopy-related topographies of TRN projection to the medial geniculate nucleus (MG) and cortical projections to the TRN and MG in the rat. In the present study, we further determined features of MG projection to the TRN, based on axonal projections of single auditory cells that were labeled with biocytin using juxta-cellular recording and labeling techniques. We isolated two types of auditory cells (S and L cells) that exhibited unit discharges (single and/or burst) to noise burst stimuli (duration, 100 ms) with short (<50 ms) and long (>100 ms) response latencies. Of particular interest is that the projections of S cells were topographic along the dorsoventral neural axis while those of L cells were topographic along the rostrocaudal axis. Since tonotopy is primarily organized along the dorsoventral axis in the MG and TRN, it is suggested that S cells, directly receiving ascending auditory inputs, activate the TRN with regard to tonotopic organization and L cells, most likely receiving corticofugal inputs, activate the TRN for other information processing less relevant to tonotopic organization. [J Physiol Sci. 2008;58 Suppl:S170]

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Recognition of human vowels depends on the first and second formants. We have reported that discrimination learning of synthetic vowels with multiple formants requires the presence of the auditory association cortex in rats. In the present study, characteristics of the synthetic vowel discrimination were investigated. Vowel-like sounds with two formants were synthesized in an all-pole model. Water-deprived rats were trained to discriminate between two vowels. Licking a spout during presentation of one (S+) was rewarded with water while the other (S-) was not. Either S+ or S- was presented randomly in a trial, which was repeated every minute throughout a test session of 12 hours on consecutive 4 days. Percentage of trials in which rats licked the spout was calculated separately for S+ and S-, and test performance was estimated as the difference. Discrimination characteristics, which were analyzed using synthetic vowels with various formant frequencies, appeared to parallel those in humans. We also investigated discrimination of voiced stop consonants /b, d, g/, which are characterized by transition of formants. Bilateral lesions of the auditory association cortex impaired discrimination learning between stop consonants with multiple formant transitions, while discrimination between single formant transitions was not. These results suggest that discrimination learning between synthetic speech sounds in rats may serve as an animal model for investigating speech sound recognition in humans. [J Physiol Sci. 2008;58 Suppl:S171]

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Primary auditory cortex (AI) neurons exhibit qualitatively distinct responses to successive signals depending on the inter-stimulus intervals (ISIs). We investigated the neural mechanisms of such ISI-dependent responses with single unit recording. We specifically examined 105 AI cells of awake cats, which exhibited only onset/offset responses to tone signals. When click trains were delivered with ISIs >30 ms, the majority of the cells ("synchronization cells") exhibited stimulus-locking responses. Analysis of excitability during and after the click trains revealed the involvement of short-term plasticity. Single-click stimulation elicited, at maximum, four distinct responses in the following order: 1st excitation, 1st suppression, 2nd excitation then 2nd suppression. Interplay of the 1st excitation and 1st suppression (i.e., post-activation suppression) occurred exclusively in the synchronization cells. Among the synchronization cells, those showing the 2nd suppression had a band-pass temporal modulation transfer function (tMTF) whereas those with low-pass tMTF never showed the 2nd suppression. A single cell model that incorporated AMPA, GABA_A, NMDA and GABA_Breceptors as well as short-term plasticity captured experimentally observed AI responses. Overall, these findings suggest that responses of a subset of AI cells are configured through the temporal interplay of excitation and suppression (inhibition) along with short-term plasticity. [J Physiol Sci. 2008;58 Suppl:S171]

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The processing of spectral and temporal information in the core fields of the left and right auditory cortices of the guinea pig was investigated using optical imaging with a voltage-sensitive dye (RH795). Eleven guinea pigs were anesthetized with ketamine (80mg/kg) and xylazine (40mg/kg). In order to compare the functional difference between the left and the right auditory cortex, optical imaging patterns to repeated sound ( tone, click, upward and downward FM sound ) at different repetition rates (4-14 Hz) were recorded from core auditory fields (primary (AI), dorsocaudal (DC) fields) of both sides. Repetition rate transfer functions (RRTF) in field AI of the left cotex were low-pass showing a sharp drop-off in evoked activity per upward FM sound at 4-6 Hz but RRTFs of the right cortex were bandpass with the peak of 6 Hz. On the other hand, the cut-off frequencies of RRTFs per downward FM sounds in field AI of the left cortex were 6-8 Hz and those of the right cortex were 6 Hz. We discuss the functional difference between the left and right auditory cortices of guinea pigs. [J Physiol Sci. 2008;58 Suppl:S171]

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Excitotoxic neuronal death is thought to be a fundamental process in stroke and traumatic brain injury. In the present study, we examined effects of visual input on the recovery of visual cortex following excitotoxic lesion. Adult male Long Evans rats, aged postnatal days 84 to 277, received small injections of ibotenic acid in bilateral primary visual cortex with or without binocular eye enucleation. After survival period of 7 days, the rats were perfused with fixative and cortical sections including the lesioned area were prepared. The extent of lesioned areas was determined by nissl staining. Neighboring sections were stained with antibodies to an immediate early gene, zif268 to evaluate recovery of neural activity in the lesioned area. We found that maximum size of the lesion tended to be larger in eye-enucleated group than that in normal eye group. In addition, the expression of zif268 remarkably decreased around the lesioned area in the eye-enucleated group. These results suggest that neural activity caused by visual inputs might prevent an expansion of cortical lesion in acute phase and facilitate functional recovery from brain damage. [J Physiol Sci. 2008;58 Suppl:S171]

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Direction of gaze is an important, socially communicative signal between humans. When a gaze directed toward us exists in our visual field, our eyes are frequently and automatically captured by such a gaze. This implies that a gaze has salience and is processed in a parallel search strategy under the bottom-up attentional control. Previous studies have shown two different types of behavioral strategies in visual search, a parallel search strategy under the bottom-up attentional control and a serial search strategy under the top-down attentional control. In this study using visual search diagnostics, first, we investigated whether a gaze directed toward the subject can "pop out" or not (Exp.1). Second, we analyzed eye movements (saccades) during the visual search, to characterize differences in visual behavior between subjects with the "pop-out" effect for gazes and without the effect (Exp. 2). Finally, we investigated the effect of eye fixation (suppression of saccades), on visual search strategy which was taken by the subjects (Exp. 3). As the results show, (1) In about 40% of the subjects, a gaze toward them "popped out", indicating a parallel search strategy under the bottom-up attentional control (Exp.1). (2) Frequency of saccades occurred during visual search was smaller in the subjects with the "pop-out" effect for gazes than in those without the effect (Exp. 2). (3) Eye fixation biased the search strategy from top-down (serial) toward bottom-up (parallel) (Exp. 3). Taken together, the results indicate that in visual search for gazes, eye movement (saccade) is a critical constraint of search strategy taken by the subject. [J Physiol Sci. 2008;58 Suppl:S172]

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The multiple-site optical recording from the cerebral cortex is essential approach to analyze the brain function, but in situ measurements is difficult because of large artifacts resulting from the heartbeat. This pulsation artifact can be reduced by subtracting neural activity-free control signal aligned to ECG. Since the cortex has spontaneous neural activities, to select the activity-free signals as controls, simultaneous electrocorticogram (ECoG) recording was necessary. However, the conventional electrode and its lead wire are usually opaque and interrupting the light path when placed on the optical sampling area. Therefore, we have newly developed a transparent electrode. To fit the curvature surface of the cortex, we used a semicircular plano-concave lens as the electrode base to cover and record from a broad area. Ga doped ZnO film, a transparent conductor, was deposited on the surface of the electrode base by radio-frequency magnetron sputtering and insulated with SiO₂ except for a hole ca 300 μm in a diameter, which hole eventually served as an electrode. Using this transparent electrode, we succeeded in recording ECoG at a site within the optical recording area of the rat somatosensory cortex without disturbing optical recording. After the pulsation artifacts in optical signal, recorded in a single sweep, were largely reduced using ECoG trace and software, spontaneous and/or evoked neural activities can be analyzed in detail. [J Physiol Sci. 2008;58 Suppl:S172]

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Local thermotherapy reduces mental stress, but its mechanisms are still poorly understood. In this study, we investigated effects of a heat- and steam-generating sheet (HSG sheet) on the cerebral hemodynamics and autonomic nervous activity using near infrared spectroscopy (NIRS) and electrocardiograms (ECGs). Ten healthy young subjects (mean age= 26.2) participated in this experiment. Each experimental session consisted of 4 phases; 1) application of HSG sheet or simple eye-mask (control) to the eye region for 120 sec, 2) rest for 60 sec, 3) mental arithmetic for 60 sec, and 4) rest for 120 sec. Each subject received 5 sessions. During the sessions following parameters were monitored; changes in brain oxygenated-Hb concentration (oxy-Hb), high frequency (HF) and low frequency (LF) components of heart rate variability (HRV). The results indicated that the HSG sheet decreased oxy-Hb concentration in the prefrontal cortex, while it increased hemodymamic responses to mental arithmetic. Furthermore, parasympathetic activity increased while sympathetic activity decreased. These results suggest that the application of the HSG sheet to the eye region induced relaxation while it increased activity of the prefrontal cortex during mental works. [J Physiol Sci. 2008;58 Suppl:S172]

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Williams syndrome which characterized by a unique cognitive and behavioral pattern is caused by hemizygous deletion of 7q11.23. This region contains the gene for HPC-1/syntaxin1A (STX1A) which is believed to regulate synaptic transmission. Previously, we have produced STX1A knockout mice and reported its behavioural abnormality including visual discrimination task. To evaluate effects of the loss of STX1A on peripheral nervous system, we examined structure of retinas in homozygote (KO) and heterozygote (HT). We found a case with abnormal layer structure in the HT that showed deficit in the visual discrimination task. Normal retinas have three nuclear layers and two plexiform layers. The abnormal animal had only two nuclear layers and one plexiform layer. To determine the lost nuclear layer, we examined the existence of photoreceptors by applying in situ hybridization with the RNA probes that recognise rhodopsin. We could not find any rhodopsin-positive cells in the case. From these results we suggest that the lost layers were photoreceptor layer and its adjacent plexiform layer, outer plexiform layer. Although we could not determine whether the loss was innate deficit or developmental deficit, this aberration may play critical role for the deficit in visual discrimination. Thus, this case, just a case study though, suggests a possibility that STX1A has effects on development of retinal structure and function. [J Physiol Sci. 2008;58 Suppl:S172]

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Midorikawa et al. (2007) have recently demonstrated that the goldfish giant bipolar (Mb1) cell with cone and rod inputs sends a transient signal through the ribbon-associated synapses and a sustained signal through the ribbon-free synapses. This raised a question whether mammalian rod bipolar cells also have the ribbon-free synapses. We examined 23 rod bipolar cells in the serial electron micrographs of a mouse retina. Rod bipolar cells could be morphologically classified into three groups, the axon terminal of which ramified at a slightly different but widely overlapping IPL depth. RB1 had direct contacts with the somas of ganglion or displaced amacrine cells and 70.7 ± 5.3% (mean ± SD, n=9) of their ribbon-associated synapses received reciprocal input from amacrine cells. RB2 had no direct contact with those somas and 85.7 ± 3.5% (n=7) of their ribbon-associated synapses received reciprocal input. We also recognized the intermediate group (81.9 ± 3.0%, n=7). RB1 and RB2 might correspond to the physiologically classified groups (Pang et al., 2004). RB1 had the ribbon-free synapses that showed similar architecture to those observed in the goldfish Mb1 cell. These synapses were occasionally found near a ribbon-associated synapse. However, the ratio of the ribbon-free (1.6 ± 1.5, n=7) to ribbon-associated (55.9 ± 7.6, n=7) synapses was about 0.03, much smaller than the goldfish Mb1 cell (about 1.0). RB2 had no ribbon-free synapses. Thus the ribbon-free synapse in the mouse RB1 cell terminal might be a rudimentary structure in phylogeny. [J Physiol Sci. 2008;58 Suppl:S173]

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Oxidation of rod outer segment (ROS) membrane lipids of photoreceptors may be involved in light-induced retinal degeneration. All-trans retinal is a major candidate of photooxidizer. We have examined the phototoxicity of physiologically generated all-trans retinal in isolated living rod photoreceptors. Lipid peroxidation in isolated living cells was measured with fluorescence imaging from the oxidation of internalized BODIPY C-11. Photooxidation was measured before, immediately after, and 1 hour after the bleaching of rhodopsin. Photooxidation was highest in rod outer segments without ellipsoid and 1 hour after the bleaching of rhodopsin. Rod outer segments without ellipsoid could not efficiently convert the all-trans retinal to retinol through reduction by the retinol dehydrogenase. Separating the outer segment from the rest of the cell body suppresses all-trans retinol formation. The separation renders the rod outer segment metabolically compromised. In conclusion, All-trans retinal released from bleached rhodopsin can mediate light-induced lipid peroxidation in isolated living rod photoreceptors. The removal of all-trans retinal through reduction to all-trans retinol by retinol dehydrogenase protects photoreceptors against light-induced damage. [J Physiol Sci. 2008;58 Suppl:S173]

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We have reported that P2X2-purinoceptors selectively expressed in the OFF-pathway but not in the ON-pathway. In the present study, we immunohistochemically examined whether such a pathway specific expression of P2X2-purinoceptors is generated by the intrinsic mechanisms. In the mouse kept in normal light cycle (12 h light, 12 h dark), immunoreactivity for P2X2 became detectable at 2 weeks after birth. At 2-3 week, there were no significant differences of immunoreactivity between ON- and OFF-pathways. After 3 weeks, immunoreactivity in the ON-pathway became weak. In the mouse retina kept in complete darkness or brightness until 8 weeks after birth, we also observed the similar developmental change of the expression pattern of P2X2-purinoceptors. Our data indicate that the OFF-pathway specific expression of P2X2-purinoceptors is a result of extinction of the immunoreactivity of ON pathway during the development. The extinction process in the ON pathway might be determined by the intrinsic mechanisms and not by the environmental condition. [J Physiol Sci. 2008;58 Suppl:S173]

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ON-OFF type transient amacrine cells (TACs) are intensely connected with each other by gap junctions, forming a syncytium with a wide receptive field. We studied effects of external pH (pH₀) on the control of cell functions. Photoresponses of TACs in isolated retinae were recorded intracellularly. A slit of light was used as photostimuli, which simplified the estimation of the current flow in the cellular network into a one-dimensional problem. Under slightly alkaline pH₀ conditions of the superfusate, by raising only 0.2 pH units from the baseline of 7.60, we found a remarkable increase of the conduction velocity (40%) as well as a reduction of the space constant (15%) of the receptive field, besides depolarization of the resting potential and enhancement of photoresponses. Acidic conditions, by lowering 0.2 pH units, had opposite effects, decreasing the conduction velocity by 20-25%, increasing the space constant (15-20%). Based on our theoretical model, combined with experimental measurements of conduction velocity and space constant, we could estimate both the gap junctional and the non-gap junctional conductances of the cell. Thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gap junctions. [J Physiol Sci. 2008;58 Suppl:S173]

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We measured fraction of open channels in gap junctions of retinal horizontal, amacrine (J Intgra Neurosci, 2005) and ganglion cells (J Neurosci, 2004). The open fraction in retinal neurons expressing connexins was on the order of 0.1 to 1. The open fraction is small, when we consider whether electrotonic transmission acts to synchronize action potentials and extends the size of receptive fields beyond the diameter of dendritic fields (space length constant λ > 1.9 mm). Although our study showed that similar properties of electrical synapses are present in amacrine cells expressing the specific connexin, it is very likely that different types of amacrine cells make differential inhibitory synapses. Electrical current spread through connections of specific amacrine cell types is expected to synapse certain inhibitions. Here I investigated cellular components in several amacrine cells performing lateral electrical interactions in the inner plexiform layer. I measured cells' receptive fields by microelectrode technique and gap junction conductance between these cells by dual patch-clamp methods. In high-voltage electron microscopy (Hitachi H-1250M, NIPS, 2007HVEM-13) and freeze-fracturing, dendritic location, size and packing density of gap junction connexons were measured. This study shows that current decay through electrical synapses of amacrine cells was correlated with a total number of gap junction connexin channels localized on their dendritic tips. [J Physiol Sci. 2008;58 Suppl:S174]

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It has been hypothesized that proton mediates the negative feedback from horizontal cells (HCs) to cone photoreceptors, and the proton-mediated feedback is responsible for forming antagonistic center-surround receptive field in the outer retina. We have shown that bafilomycin A1, a specific inhibitor of V-ATPase (H⁺ pump), suppressed depolarization-induced acidification of the external surface of isolated HCs, suggesting the existence of V-ATPase in the HC membrane of goldfish retina. We studied the localization of V-ATPase by immunohistochemical analyses of the fish and monkey retinal slice by using an antibody against V-ATPase under the fluorescence microscopy and the immuno-electron microscopy. In the fish retina a very strong immunoreactivity (IR) was seen at the cone terminals. IR was also observed in the membrane of external HCs. IR was seen in the outer plexiform layer where HC dendrites invaginate into the photoreceptor cell terminals. In the invaginating synapse, IR was located at the tips of HC dendrites. In the monkey retina the V-ATPase IR was seen also in HCs. These results suggest that HC dendrite is the active site of proton extrusion. [J Physiol Sci. 2008;58 Suppl:S174]

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Synchronous Ca²⁺ oscillation plays essential roles in early neuronal development. The Ca²⁺ oscillation occurs at various parts of the central nervous system including retina even before synapse formation. The Ca²⁺ transients regulate proliferation, axon guidance and neurotransmitter expression. However, it remains unknown how the synchronous Ca²⁺ rises are generated without synaptic inputs. Here I show in embryonic retina that the Ca²⁺ rises are caused by releases of Ca²⁺ from intracellular Ca²⁺ stores, of which synchronization is mediated by high frequency fluctuations in the membrane potential of Ca²⁺ stores. Synchronized Ca²⁺ rises may be caused by synchronous action potentials and ensuing Ca²⁺ influx. However, this was not the case at the early stage of neurodevelopment because, 1) the resting membrane potential of undifferentiated neuroepithelial cells, which show synchronous Ca²⁺ oscillation, was stabilized by lowering input resistance through gap junctions, 2) newborn ganglion cells showed Ca²⁺ influx-dependent repolarization after depolarizing stimuli without any evidence for action potentials, 3) synchronous Ca²⁺ oscillation occurred even in the absence of extracellular Ca²⁺. Fluorescence measurement of store membrane potential with DiOC₅(3) revealed that synchronous voltage fluctuations burst periodically with Ca²⁺ rises. Close apposition of store membranes and plasma membranes in an epithelial structure seemed to allow electrical coupling across the cells. [J Physiol Sci. 2008;58 Suppl:S174]

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It has been shown that a certain level of noise can help to improve detection of sensory stimuli in a non-linear system. In our previous study we have shown that addition of noise improved contrast detection sensitivity in humans (Sasaki et al., Neurosci. Lett. 2006, 408, 94-97). Here we examined whether pattern discrimination can be improved by addition of noise. In six undergraduate students (five male and one female, aged 19–22 years), discrimination of vertical and horizontal lines of light was tested using a two-alternative forced-choice paradigm. Using slits and three white LEDs arraigned either vertically or horizontally produced the lines of light. The luminance was modulated by sin waves at 1 Hz. Noise was random flickering light driven by white noise through a low-pass filter at 50 Hz. Noise intensity was varied from -20 to 20 dB, with a step of 10 dB, referring the noise threshold as 0 dB. A session was consisted of 63 trials, ten trials for each of five noise intensities, and one control without noise. In addition, three catch trials were performed, in order to ascertain the validity of the subject's response. Control correct response rate was 58.3±4.0%, whereas the response rate increased to 86.7±4.2% (p<.05) and 86.7±4.9% (p<.01) with noise intensity at -10 dB and 0 dB, respectively. These findings suggest that visual pattern discrimination can be improved by addition of noise. [J Physiol Sci. 2008;58 Suppl:S174]

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OBJECTIVE: Increments of local blood flow and vasodilatation induced by acupuncture (acp) and moxibustion (mox) have been known as axon reflex of unmyelinated afferent fiber receptors such as polymodal receptors. In this study, effects of application of local anesthetic patch on mechanical or heat pain threshold of the skin and axon reflex induced by acup and mox were examined. METHODS: Sixteen healthy volunteers with informed consent (32 forearms allocated anesthetic or sham group, double blinded manner) were used and pin-prick pain and heat pain thresholds were measured. The flare reactions were induced by acupuncture (diameter 240 μ m) or an indirect moxibustion (peak temperature was about 50 degree, Kamaya Co. Ltd, Japan), and the vasodilatation was measured by blood flow meter. A piece of anesthetic (lidocaine 18 mg) or sham patch (30.5 × 50 mm) was applied to the skin surface where pain tests and blood flow measurements were done. RESULTS: Mechanical (pin-prick) pain threshold was significantly reduced (P<0.01, Wilcoxon t-test) 60 min after the application of anesthetic patch, whereas, thermal pain threshold and flare reactions produced by both acp and mox did not change. CONCLUSION: The facts that acp and/or mox induced flare reactions were not blocked by the lidocaine application, might suggest the involvement of lidocaine-insensitive and heat sensitive afferents and/or receptors channels in the signal transduction processes of acp and mox stimulation. [J Physiol Sci. 2008;58 Suppl:S175]

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The aim of the present study is to probe candidate genes which were involved in the electroacupuncture (EA) analgesia and to understand the molecular basis of the individual difference of EA analgesia in rats. We compared hypothalamus transcriptional profiles of responders with those of non-responders after 1 Hz EA treatment at ST36 acupoint for 1 hour by using oligonucleotide microarray. Responders and non-responders were determined by tail flick latency (TFL). A real-time quantitative RT-PCR was applied to validate the differential expressed genes. Our study provided a global hypothalamus transcriptional profile of EA analgesia in rats. We found that 63 and 3 genes were up- and down-regulated in the responder group, respectively. Half of the differentially expressed genes were classified into 9 functional groups which were (1)ion transport, (2)sensory perception, (3)synaptogenesis and synaptic transmission, (4)signal transduction, (5)inflammatory response, (6)apoptosis, (7)transcription, (8)protein amino acid phosphorylation and (9)G-protein signaling. Glutamatergic receptors, ghrelin precursor, melanocortin 4 receptor (MC4-R) and neuroligin 1 were found to be up-regulated in the responder group which may become new targets for nociceptive study and deserve further investigation for developing new acupuncture therapy and intervention of pain modulation. [J Physiol Sci. 2008;58 Suppl:S175]

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Recent studies have implicated that spinal GIRK channels play an important role in thermal nociception and the analgesic action of morphine or other related agents. In this study, we have shown that spinal GIRK channels are activated by an endogenous neurotransmitter using whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons in adult rat spinal cord slices. Although repetitive stimuli applied to the dorsal root did not induce any slow responses, ones focally-applied to the spinal dorsal horn produced slow inhibitory postsynaptic currents (IPSCs) at a holding potential of -50 mV in about 30% of SG neurons recorded. The slow IPSCs increased in amplitude and duration with increasing number of the stimuli and significantly decreased by the removal of Ca²⁺ from external Krebs solution. The slow IPSC was associated with an increase in membrane conductance and reversed its polarity at a potential close to the equilibrium potential for K⁺, calculated from the Nernst equation. The slow IPSC was blocked by the addition of GDP-b-S into patch-pipette solution, reduced in amplitude in the presence of Ba²⁺, and significantly suppressed in the presence of tertiapin-Q, a selective antagonist for GIRK channels. Moreover, somatostatin produced an outward current in a subpopulation of SG neurons and the slow IPSC was occluded during the somatostatin-induced outward current. These results suggest that endogenously-released somatostatin may induce slow IPSCs through the activation of GIRK channels in SG neurons. [J Physiol Sci. 2008;58 Suppl:S175]

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BACKGROUND: It has been proposed that atrial natriuretic peptide (ANP), which promotes renal NaCl excretion, is secreted when the heart dilates. However, it is not easy to determine whether this occurs with land animals, without measuring [Na⁺]_o. The present work, therefore, has been carried out to examine the relation between [Na⁺]_o and the cardiac function before stepping into the study of regulation of ANP secretion by [Na⁺]_o. METHODS: Two types of preparations were subjected to experiments, 1) cultured cardiac myocytes obtained from fetal or new-born mice (to exclude effect of cardiac stretch), and 2) adult mouse whole-heart. Intracellular [Na⁺] or [Ca²⁺] of myocytes in culture was monitored with an image analyzer, and myocardial contraction with a video camera or a strain gauge. RESULTS & DISCUSSION: When [Na⁺]_o was increased from a normal concentration (140 mM) to 165 or 190 mM, [Na⁺]_i was raised and followed by an increase in [Ca²⁺]_i. Concomitantly, myocardial contractility was decreased and arrhythmias were induced. Similar phenomena were observed when the myocardial tissue was exposed to an inhibitor of mitochondrial function (10 μM CCCP). These effects were mimicked by applying a Na⁺ ionophore (10 μM monensin) but not by increasing osmolarity of the solution with mannitol. The results indicate that myocardial function is perturbed by changing [Na⁺]_o, and a possible target of increased Na⁺ is mitochondria. Such effect may be sufficient to lead to an alteration in the ANP secretion. [J Physiol Sci. 2008;58 Suppl:S175]

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We investigated whether pp60^src increases L-type Ca²⁺ channel current (I_Ca(L)) in cultured rat VSM cells (A7r5). A7r5 cells were transfected with a plasmid of wild type mouse src cDNA/pUSE expression vector using lipofectamine. Negative control cells were prepared by transfection with an empty vector, (-)/pUSE. After transfection, the cells were treated with 1 mg/ml of G418 to select the positive cells stably expressing src cDNA or (-)/pUSE. I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier for recording of I_Ca(L). Western blot demonstrated that expression of pp60^src was increased by 34-fold and that β₃ subunit of L-type Ca²⁺ channel current was significantly increased in src cDNA/pUSE expressing cells as compared with (-)/pUSE expressing cells. Current/voltage relationships demonstrated that although voltages for peak amplitude of I_Ca(L) were 20 mV for both src cDNA and (-)/pUSE expressing cells, peak amplitude of I_Ca(L) in src cDNA/pUSE expressing cells was increased by 2-fold (src, 6.1 ± 0.96; (-), 3.1 ± 0.38 pA/pF). From steady-state inactivation and activation analyses of I_Ca(L), voltages for half-activation and -inactivation and slope factors did not differ between src cDNA and (-)/pUSE expressing cells. Thus, overexpression of pp60^src increases I_Ca(L) through upregulation of β₃ subunit of L-type Ca²⁺ channel without affecting voltage sensors of L-type Ca²⁺ channels. [J Physiol Sci. 2008;58 Suppl:S176]

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Oxcytocin (OXT) and [Arg⁸]-vasopressin (AVP), neurohypophyseal hormones, have recently been implicated in cell growth, differentiation, and contraction in the heart. However, effects of these hormones on cardiomyocytes by means of ion channel expression and contraction modulation have not completely elucidated. This study was designed to investigate the effect of OXT and AVP on transcriptional regulation of L-type Ca²⁺ channel expression and cardiac contractility using neonatal rat ventricular cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with or without 0.01-1 μM OXT or AVP for 24-72 h. Expression of Ca²⁺ channel isoforms (Ca_v1.2, Ca_v1.3) and cardiac transcriptional factors (Csx/Nkx2.5, CREB, and NFATc4) was assessed by real-time PCR, while Ca²⁺ channel currents were measured with the patch clamp technique. Stimulation with OXT but not AVP decreased the expression of cardiomyocyte Ca_v1.2 mRNA in a dose- and a time-dependent manner with a reduction of L-type Ca²⁺ channel current. In addition, these three transcriptional factors were also down-regulated by OXT stimulation for 24-48 h. In conclusion, we found that OXT may play a role in modulation of intracellular Ca²⁺ homeostasis through a down-regulation of the L-type Ca²⁺ channel at the gene transcriptional level in cardiomyocytes. [J Physiol Sci. 2008;58 Suppl:S176]

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Heart rate is well known to be dependent on physical activity. However, its underlying signaling pathway of cardiac adaptation in response to voluntary exercise remains unclear. In this study, we investigated the role of the L-type Ca²⁺ channel in the heart of SPORTS rats, a novel hyper-running rat strain. Heart rate was significantly higher in SPORTS rats than those in control rats at sedentary condition by using the telemetric ECG recording technique. Cardiac hypertrophy was also observed in SPORTS rats. On the other hands, patch clamp studies revealed that L-type Ca²⁺ current was significantly larger in isolated neonatal SPORTS rat ventricular cardiomyocytes than in control myocytes. We confirmed the expression of L-type Ca²⁺ channel isoforms (Ca_v1.2, Ca_v1.3) mRNA in adult rat atrial and neonatal myocytes were significantly larger in SPORTS rat than those in control rat by 2.3-10 folds by quantitative real-time PCR. These results suggest that the L-type Ca²⁺ channel is a key modulator in congenital and exercise-adapted cardiac function in SPORTS rat featured by hyper-running activity. [J Physiol Sci. 2008;58 Suppl:S176]

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It is well known that cardiac KCNQ1 channel assembles with KCNE1 β-subunit to generate the slow I_Ks current that plays a major role in repolarization of action potential. However, it is also plausible that other KCNE members (i.e. KCNE2-5) may interact with cardiac KCNQ1 to form K⁺ channels with different properties because they are also substantially expressed in hearts. Here, we investigated the potential roles of KCNE1, KCNE2 and KCNE3 in cardiac KCNQ1 channel function. Transfection of KCNE1 into CHO cells stably expressing KCNQ1 resulted in evocation of slowly activating outward current that resembles I_Ks, whereas expression of either KCNE2 or KCNE3 induced a constitutively active K⁺ current. When KCNE1 and KCNE2 were cotransfected, the cell displayed I_Ks-like current whose characteristics substantially differed from those of the KCNQ1/KCNE1 current. On the other hand, the cell expressing both KCNE1 and KCNE3 exhibited an ensemble of the KCNQ1/KCNE1 and KCNQ1/KCNE3 currents. These results suggest that both KCNE2 and KCNE3 can participate in regulation of KCNQ1 channel even in the presence of KCNE1. In guinea-pig cardiomyocytes, transfection of siRNA directed against KCNE1 or KCNE2 reduced the amplitude of I_Ks and prolonged the action potential duration (APD). On the other hand, knockdown of KCNE3 resulted in a prolongation of APD without changing I_Ks amplitude which suggests that KCNE3 contributes to the determination of APD independent of I_Ks modulation. Our data suggests that not only KCNE1 but also KCNE2 and KCNE3 may play a physiological role in cardiac repolarization. [J Physiol Sci. 2008;58 Suppl:S176]

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Cardiac excitation is dynamically controlled by the autonomic nervous system, i.e., sympathetic and parasympathetic nerves, through G-protein coupled receptor-dependent modulation of membrane ion channels. The signaling mechanisms are composed of cardiac GPCRs (β-adrenergic and m2-muscarinic receptors), G proteins (Gs and PTX-sensitive GK), intracellular cell-signaling molecules and the target ion channels (L-type Ca²⁺ channel and G protein-gated K⁺ channel). For quantitative understanding of the neural control of cardiac excitation, we developed mathematical models for parasympathetic deceleration of heart beat. Muscarinic activation of KG channels has been modeled based on our own experimental data. KG channels are activated directly by the βγ subunits of GK coupled to m2 receptors. The model is composed of two parts: one is for the interaction between G_βγ and KG channel, and the other is for m2 receptor and G proteins. Our recent finding on the role of RGS proteins in the control of GK activity has allowed us to construct the models reasonably reproducing temporal behavior of ACh-activation of KG channels. This model may be useful for quantitative understanding of neural control of cardiac excitation. [J Physiol Sci. 2008;58 Suppl:S177]

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By finding a suggestive evidence that Ca²⁺ repletion in the junctional sarcoplasmic reticulum (JSR) occurs with a common JSR Ca²⁺ content versus time relationship after various degrees of JSR Ca²⁺ release, we have developed a method to estimate a time course of the JSR Ca²⁺ repletion after JSR Ca²⁺ release and JSR Ca²⁺ release/content relation by analyzing short-term mechanical restitution in cardiac muscle of murine hearts (Tameyasu, et al, Jpn J Physiol 2004). Though murine heart muscle is useful to study pathophysiology of heart failure because of development of model animals for heart disease, its SR handles Ca²⁺ in a different way from human cardiac muscle. In the present study, we applied our method to rabbit heart, the mechanical property of which resembles human heart, by measuring the left ventricular pressure. After the Ca²⁺ content of the SR in whole heart preparation isolated from rabbit was increased to a maximum level by 10 current pulses at 5 Hz at 37C in the presence of acetylcholine to suppress automaticity, double pulses with a various interval were applied externally to produce left ventricular systolic pressure of a desirable magnitude, which was followed by a test pulse to determine restitution of systolic pressure. Restitution of the left ventricular systolic pressure after systoles of various magnitudes was thus determined. The JSR Ca²⁺ release/content relation deduced from restitution of the left ventricular systolic pressure was somewhat different from that in rat cardiac muscle. [J Physiol Sci. 2008;58 Suppl:S177]

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Cardiomyocytes respond to a mechanical stretch with an intracellular calcium increase. Extensive stretch of the cardiomyocyte may induce pathological condition to the heart, such as arrhythmia. However, the molecular mechanism responsible for the mechanosensitivity of the cardiomyocyte is not fully understood. We have studied the role of the mechanosensitive ion channels, TRPCs and TRPV2, in the stretch-induced calcium response of the rat cardiomyocyte cell line H9c2. The cells were cultured on a flexible silicone chamber and were loaded with the calcium sensitive dye Fura2. The intracellular calcium concentration increased in proportion to the extent of the chamber stretch. This calcium response was completely blocked by a bath application of calcium free solution. Furthermore, neither 5 uM-thapsigargin nor 2 uM-ryanodine altered the stretch-induced calcium response, suggesting that the calcium increase is mainly caused by Ca²⁺ influx from external space probably via channel mechanism. Bath application of the TRPV2 channel blocker, ruthenium red (50 uM), inhibited the stretch-induced calcium increase by 70%. In contrast, the TRPC channel blockers, BTP2 (100 uM) and SKF-96365 (50 uM), showed no effect on the stretch-induced calcium response. These results suggest that the stretch-sensitivity of the rat cardiomyocyte H9c2 is attributable to the mechanosensitive ion channel TRPV2. [J Physiol Sci. 2008;58 Suppl:S177]

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To elucidate the roles of intracellular Ca²⁺ dynamics in sinoatrial (SA) node pacemaking, we investigated the effects of SR Ca²⁺ cycling dynamics on bifurcation structures of mathematical models for the rabbit SA node cells. Modified versions of our model (Kurata et al. Am J Physiol, 2002) with various SR Ca²⁺ uptake and release rates were used. Equilibrium points (EPs), periodic orbits, stability of EPs, and bifurcation points were calculated for the model cells during changes in bifurcation parameters. Structural stability to applications of constant bias currents (I_bias) or hyperpolarizing loads was also evaluated. In all the model cells, blocking L-type Ca²⁺ current (I_Ca,L) caused EP stabilization and cessation of pacemaking via a Hopf bifurcation, and the unstable EP region determined with I_bias applications shrunk and finally disappeared as I_Ca,L diminished. All the model cells exhibited essentially the same bifurcation structures, regardless of whether or not intracellular Ca²⁺ dynamics significantly affects the action potential dynamics. Changing SR Ca²⁺ uptake and release rates only slightly altered the bifurcation structures of the model cells. These results suggest that the effect of intracellular Ca²⁺ dynamics on dynamical properties of SA node pacemaking is relatively small, and that the membrane clock plays a pivotal role in SA node pacemaking. [J Physiol Sci. 2008;58 Suppl:S177]

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Ca²⁺ overloading of cardiac muscle leads to abnormal E-C coupling such as a delayed onset of Ca²⁺ transient and an alternating occurrence of Ca²⁺ transient at electrical stimulation. In this study, we determined membrane potential signals as well as Ca²⁺ signals at the cellular level in cardiac muscles to examine a possible involvement of altered membrane excitability in this abnormal E-C coupling. Papillary muscles were dissected from guinea pig ventricles and loaded with rhod-2 and/or Di-4-ANEPPS. Muscles were field stimulated and sequential two-dimensional images of cells were acquired from an area of 0.3x0.15mm² every 8–40 ms using a laser-scanning confocal microscope. Under control condition, action potential signals were regular and simultaneous in all cells in the field of view. To induce abnormal Ca²⁺ signals, muscles were prestimulated at 2–3 Hz under hypoxic condition. In muscles showing alternating Ca²⁺ transients, action potentials were usually regular, showing no alternation. In cells showing delayed Ca²⁺ transients, onset of action potentials also delayed. Similar delayed action potentials were also observed in the presence of heptanol. These delayed onsets of action potentials can be interpreted by impaired gap junctions. In summary, there are two underlying causes for abnormal Ca²⁺ transients in Ca²⁺ overloaded heart muscles; an abnormal E-C coupling and an abnormal action potential propagation/generation. [J Physiol Sci. 2008;58 Suppl:S178]

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We have introduced multiple-site optical recording of transmembrane voltage activity, using a fast merocyanine-rhodanine voltage-sensitive dye (NK2761) and a multi-element (16 X 16) photodiode array, to monitor action potentials in the isolated rat atrium. The atrial wall preparation including the right or left auricle was dissected from the adult rat heart. The preparation was then stained with a voltage-sensitive dye. For suppression of optical artefacts due to contractile movements, a bathing medium containing 2,3-butanedione monoxime (BDM: 20 mM) was used. The spread of excitation was assessed optically by timing the initiation of the action potential-related extrinsic absorption changes. The pacing stimulations were applied by a bipolar electrode at 0.5 Hz. We have optically mapped the spatiotemporal patterns of the spread of excitation. When the octanol (0.1–0.2 mM) was applied to the bath, the conduction velocity of the excitation spread was reversibly decreased. The decrease in the conduction velocity was relatively nonuniform in the preparation, and spatial nonuniformity of the conduction velocity was observed. The block of the spread of excitation was sometimes observed. Furthermore, the amplitude and the rate of rise of the optical action potential also reversibly decreased. These results suggest that octanol reversibly increased the resistance of the gap junction, and decreased the electrical coupling between atrial muscle cells. [J Physiol Sci. 2008;58 Suppl:S178]

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