We previously reported the negative interaction between excitatory P2X receptor (P2XR)- and nicotinic ACh receptor-channels in rat area postrema (AP) neurons. We now investigated whether there was also the interaction between excitatory P2XR and inhibitory GABA_A- or glycine-receptors (GlyR). At a holding potential of -70mV, the amplitude of the GABA- or Gly-induced current was significantly reduced in the presence of ATP, and there was an inverse correlation between the amplitudes of these responses. On the other hand, the ATP-induced current in the presence of GABA or Gly was only slightly reduced. As the GABA- or Gly-induced current desensitizes faster than the ATP-induced current, the weaker inhibition of the ATP-induced current by GABA or Gly could be due to the reduced amplitude of the GABA- or Gly-induced current at the time of ATP application. In fact, the current caused by the concomitant applications of ATP and GABA or Gly was smaller than the predicted sum of the individual currents.These results suggest that the negative interactions between the different receptors modify the strength of excitatory or inhibitory neurotransmission when plural transmitters are simultaneously released from presynaptic nerve terminals. [J Physiol Sci. 2006;56 Suppl:S155]

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We examined with a lipid bilayer method how the single channel currents of cardiac RyR channels are modified by pathophysiological metabolites from the sarcolemmmal membrane phospholipids. During the apoptosis and the hyperlipidemia, sphingosylphosphatidylcholine (SPC) is metabolized from sphingomyelin (SM) of a minor sarcolemmal phospholipid. (1-1) The cytoplasmic-side addition of SPC blocked the RyR channels at the μM level, while the SR luminal-side addition of SPC did not affect. (1-2) SPC unaltered the membrane capacitance. Thus, SPC could exert a specific effect via an intermolecular binding to the cytoplasmic domain of the RyR molecule, although SPC belongs to the lipid. Kinetics of a long-lived blocking state of the SPC-modified channels is characterized by an extremely low dissociation rate constant. During the cardiac ischemia, lysophosphatidylcholine (LPC) is produced from phosphatidylcholine (PC) of a major sarcolemmal phospholipid. (2-1) Both cytoplasmic-side and SR luminal-side additions of LPC activated the RyR channels at the μM level. (2-2) LPC increased the membrane capacitance. In contrast to SPC effects, LPC could thus exert an indirect effect via a fusion of LPC into the membrane lipids on the RyR channel. Here we propose that a second messenger metabolited from SM of sarcolemmmal membrane phospholipids specifically regulated in vivo cardiac RyR channel activities. [J Physiol Sci. 2006;56 Suppl:S155]

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Rat bone marrow stromal cells express G protein-coupled purinergic receptor (P2Y₂ receptor). We have investigated intracellular Ca²⁺ signals in these cells using fura-2 AM and found that UTP, an agonist of P2Y₂ receptor, induced not only Ca²⁺ rise but also Ca²⁺ oscillation. Removal of extracellular Ca²⁺ diminished UTP-induced Ca²⁺ oscillation. This fact indicates that the Ca²⁺ oscillation involves Ca²⁺ entry from extracellular space. SKF96365, a blocker for store-operated Ca²⁺ entry channel suppressed UTP-induced Ca²⁺ oscillation. Carbenoxolone (CBX), a gap junction blocker, also suppressed Ca²⁺ oscillation at higher concentration than 50μM, but enhanced at 10μM in some cells. At 10μM, CBX enhanced the peak amplitude of UTP-induced Ca²⁺ rise and kept high Ca²⁺ concentration until CBX was washed out. L-type voltage-dependent Ca²⁺ channel blockers, nifedipine or verapamil did not affect UTP-induced Ca²⁺ oscillation. These results suggest that UTP-induced Ca²⁺ oscillation may be regulated by a complex mechanism including Ca²⁺ entry through store-operated Ca²⁺ channel at plasma membrane and Ca²⁺-transport system via gap junction. [J Physiol Sci. 2006;56 Suppl:S155]

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Orexin A and B are a pair of neuropeptides which are implicated in the regulation of sleep-wakefulness and energy homeostasis. The regulatory mechanism of orexin neurons is poorly understood so far. In this study, we studied the effects of various neuropeptides on the activity of orexin neurons by calcium imaging using transgenic mice in which orexin neurons specifically express calcium sensing protein (Yellow Cameleon 2.1). We screened 21 neuropeptides and found that arginine-vasopressin (AVP), cholecystokinin-8s and oxytocin triggered a robust, concentration-dependent calcium increase in orexin neurons. We revealed the intracellular mechanisms and the subtype of AVP receptors involved in the AVP-induced activation of orexin neurons. The V1a AVP receptor antagonist, SR49059, inhibited AVP-induced activation of orexin neurons in a concentration-dependent manner, whereas the V1b and V2 receptor antagonists (SSR149415 and SR121463) had little effect. Removing extracellular calcium eliminated the AVP-induced increase in intracellular calcium concentration. These results suggested that the V1a receptor is involved in the AVP-induced activation of orexin neurons. This AVPergic excitatory input to orexin neurons might have an important role in the physiological regulation of sleep-wakefulness. [J Physiol Sci. 2006;56 Suppl:S156]

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Malignant hyperthermia (MH) is an inherited disorder caused by a missense mutation of type 1 ryanodine receptor (RyR1) of skeletal muscle. We have recently showed that Ca²⁺-induced Ca²⁺ release (CICR) activity of RyR1 in the SR is selectively stabilized by a probable interdomain interaction between the N-terminal and the central regions of RyR1 where many of mutated sites for MH are clustered (Murayama et al., Am. J. Physiol. 288; C1222-C1230, 2005). According to our hypothesis, a mutation within these regions will weaken the interdomain interaction, resulting in an increased CICR activity. We are presenting here further evidence to support this hypothesis using SR vesicles from skeletal muscles of wild type and MH pigs carrying the N-terminal Arg615Cys mutation in RyR1. Furthermore, we will show results using RyR1 channels carrying several human MH mutations including those in the C-terminal region that are stably expressed in HEK293 cells. [J Physiol Sci. 2006;56 Suppl:S156]

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Long QT syndrome (LQTS) results from mutations of several genes encoding cardiac ion channels. It was reported that in LQTS2, form associated with dysfunction of the rapid component of I_K (I_Kr), acute auditory stimuli could trigger the symptoms thus suggesting fast neural control of I_Kr. The aim of this study was by means of whole-cell patch-clamp method to investigate acute regulation of native I_Kr by α₁-adrenergic receptor (AR) in HL-1 cardiomyocytes.In the cells transiently transfected with AR, bath-application of 30 μmol phenylephrine (PHE) reversibly decreased I_Kr density by 29.4%, shifted activation curve (V_h from -17.6 to -9.2 mV) and accelerated deactivation. These effects remained in the presence of protein kinase C (PKC) inhibitor bisindolylmaleimide (200 nmol). In non-transfected cells 30 μmol PHE did not affect I_Kr. In HL-1 cells expressing muscarinic M1-receptor (known to be coupled to Gq–PLC pathway as AR), 10 μmol acetylcholine (Ach) suppressed I_Kr even more (37.2%).To confirm involvement of membrane PIP₂ breakdown in I_Kr modulation, HL-1 cells cotransfected with PH (PLCδ pleckstrin homology domain) -GFP and AR or M1-receptor were used for confocal microscopy. 30 μmol PHE or 10 μmol Ach induced translocation of PH-GFP fluorescence from the cell membrane to citosol, which was not observed in the cells transfected with PH-GFP alone.AR stimulation in HL-1 cells acutely suppressed I_Kr by depletion of membrane PIP₂ and was not dependent on PKC. This effect could explain onset of symptoms in the LQTS2 patients. [J Physiol Sci. 2006;56 Suppl:S156]

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In the ciliary muscle, tonic contraction requires a sustained influx of Ca²⁺ through the cell membrane. However, little has been known about the routes for the Ca²⁺ entry in this tissue that lacks voltage-gated Ca²⁺ channels. Recently we have shown by whole-cell voltage clamp experiments that in bovine ciliary muscle cells (BMCs) there are two types of non-selective cation channels (NSCCs) with widely different unitary conductances (35 pS and 100 fS), which are opened by muscarinic stimulation with carbachol (CCh). Here we examined effects of inhibitors of the NSCCs, La³⁺ and Gd³⁺, on CCh-induced changes of the intracellular Ca²⁺ concentration [Ca²⁺]_i. BMCs cultured for 18–48 hours in a serum-free media were used. The [Ca²⁺]_i was monitored by a Fluo-4 fluorescence method. Application of CCh to the BMCs in normal Krebs solution caused an initial phasic increase in the [Ca²⁺]_i followed by a plateau which was abolished by La³⁺ or Gd³⁺ (10-100 μM) as well as by removal of external Ca²⁺. The CCh-induced elevation of [Ca²⁺]_i was also completely inhibited by 100 nM of atropine or 4-DAMP. These results support the idea that the muscarinic receptor-operated NSCCs serve as entry pathway of Ca²⁺ during the sustained phase of contraction. We also conducted immunofluorescence microscopy of the plasma membrane of BCMs and thereby detected transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor-operated NSCCs. [J Physiol Sci. 2006;56 Suppl:S156]

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Purinergic receptor mechanisms have recently been implicated in peripheral (Rong et al 2000) and central (Hu et al 2002, Chiang et al 2005) nociceptive processing. Of the 7 iontotropic purinergic receptor subtypes (P2X receptor family), the expression of the P2X3 receptor is reported to be much higher than that of the other P2x receptor subtypes in trigeminal ganglia (Xiang et al 1998) and it has recently been found in TMJ tissues (Ichikawa et al 2004, Shinoda et al 2005). To clarify further the role of P2X receptors in TMJ-related functions, the application of P2X receptor agonist to the TMJ elicits nociceptive behaviors (Oliveira et al 2005, Shinoda et al 2005). The first aim of the present study was to test if the application of a P2X receptor agonist to the rat TMJ induces reflex activity in the jaw muscles and if this excitatory effect can be blocked by peripheral application of P2X receptor antagonist. The second aim was to test if blockade of peripheral NMDA receptors can influence the reflex jaw muscle activity evoked by ATP agonist. [J Physiol Sci. 2006;56 Suppl:S157]

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For the cytoplasmic domain (CD) of potassium channels, crucial roles for mediating intracellular stimuli and assembling subunits have been investigated. KcsA channel, with only 160 amino acid residues, possesses intracellular stretches in the C-terminus, which forms CD in the tetrameric channel. KcsA channels are activated by intracellular acidic pH, the mechanism of pH-sensing remains unsolved. Also only predicted structure is available for the CD. Present study investigated surface structure of the CD in KcsA channel by developing a novel approach. Single cystein was introduced into various parts of the channel and specific reaction between introduced cystein and a flat gold surface was evaluated by surface plasmon resonance signals. All mutations did not alter single-channel properties, such as single-channel current-voltage curves and the gating characteristics. In contrast to the closed channel at pH 7.5, various sites in the CD became exposed to the surface when channels were activated (pHi = 4.0). These observations indicate that the cytoplasmic domain takes several conformational states when the channel is actively gating. We have also investigated the effect of open channel blocker, tetrabutylammonium, on the conformational changes in the CD. [J Physiol Sci. 2006;56 Suppl:S157]

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We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif with significant homology to voltage-gated channels and a phosphatase domain just downstream of the transmembrane region. We showed that the voltage sensor functionally couples with the phosphatase domain (Nature, 2005). However, it remains unknown whether similar mechanisms for voltage sensing of voltage-gated channels operate in Ci-VSP.There are conserved negative-charged residues in S2 and S3 regions of both Ci-VSP and voltage-gated ion channels. These residues are known to contribute to the gating charge of the channels. We have previously shown that a mutation which neutralized the negative-charged residue in S2 altered voltage dependency of its gating current. In this mutant, voltage-dependence of the coupling was shifted in the same direction as the change of the Q-V curve. In this work, we systematically mutated negatively charged residues in S1-S3 regions of Ci-VSP. cRNAs encoding these mutants were expressed in Xenopus oocytes and the two-electrode voltage clamp recording was performed to record the gating current of these mutants. In addition, these mutants were co-expressed with GIRK2 channels and changes of the phosphatase activity with membrane potentials were detected by monitoring changes of ion currents through GIRK2 channels. We also analyzed the voltage dependence of phosphatase activity of the mutant constructs of which the properties of voltage sensor movement are altered. [J Physiol Sci. 2006;56 Suppl:S157]

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Paroxysmal dystonic choreoathetosis (PDC) is thought to be a hereditary channelopathy mapped to chromosome 2q32-36. By means of linkage analysis on a large Japanese family, we have narrowed the PDC locus that contains 32 candidate genes. Here, we report that a heterozygous mutation (A7V) in one of such genes, myofibrillogenesis regulator 1 (MR-1), is responsible for PDC in the Japanese family. This is consistent with the finding in American PDC families. We further report that there are several other polymorphisms in MR-1 in the Japanese PDC family. To characterize MR-1, we generated specific antibodies against MR-1 and performed the immunohistochemical analysis in rat brain. The results of the MR-1 localization will be discussed. Similar to other channelopathies such as epilepsy and migraine, PDC is characterized by involuntary movement attacks, and is presumed to be induced by abnormalities of ion channels. Although MR-1 may be associated with some ion channels, its physiological functions remain unclear. Further characterization of MR-1 including its molecular function and relationship to ion channels, may facilitate not only to understand pathophysiology of PDC, but also to develop effective therapies for paroxysmal neurological disorders. [J Physiol Sci. 2006;56 Suppl:S157]

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The amygdala plays significant roles in regulating emotional states and behaviors. Certain aspects of emotion are well known to be affected by the domaminergic projection system, of which targets includes the amygdala. Indeed, a large number of in vivo studies have shown that activation of dopamine (DA) receptors in lateral amygdala (LA) neurons alter emotional expression. For understanding DA-based modulation of emotion in the LA, it would therefore be beneficial to study effects of dopamine on intrinsic properties of the LA neurons, which remain largely unknown. In the present experiments, whole cell patch clamp recordings were carried out in rat brain slices to investigate DA effects on LA neurons. Application of DA depolarized resting membrane potential markedly, and induced slow afterdepolarization (sADP) in LA neurons. This sADP is induced in a voltage-dependent manner, and lasts for more than 5 seconds. D1, but not D2, receptor agonists induced the same type of sADP. Previous reports have repeatedly suggested that sADP in general is triggered by the calcium influx. Consistently, calcium channel blockers inhibited the present DA-induced sADP, but sodium channel blockers did not. Also, application of flufenamic acid (FFA), a calcium activated non-selective cation channel (CAN) blocker, inhibited the DA-induced sADP and canceled out the DA-induced depolarization as well. These results suggest that DA induces sADP in LA neurons by activating D1 receptors, and this sADP is attributable to activation of CANs. [J Physiol Sci. 2006;56 Suppl:S158]

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We have studied how endoplasmic reticulum and mitochondria communicate each other via Ca²⁺ and whether mitochondrial activity affects plasma Ca²⁺ entry. Intracellular Ca²⁺ concentration ([Ca²⁺]i) and mitochondrial membrane potential were measured by fluorometry in brown adipocytes in culture. FCCP, a protonophore, caused bi- or triphasic rises in [Ca²⁺]i. The first phase was accompanied by mitochondrial membrane depolarization. The second phase, whose rising phase paralleled mitochondrial membrane repolarization, was blocked by a Ca²⁺ free solution, indicating the activation of plasmalemmal Ca²⁺ entry. The third phase was blocked by a Ca²⁺ free, EGTA solution, but not by thapsigargin, and enhanced at pH 9, but not in a Na⁺-free solution, indicating the activation of store-operated Ca²⁺ entry (STOC). A blocker of phospholipase C, U73122, accelerated the decay of the first phase and enhanced the second phase. At a high [Ca²⁺]i under the effect of thapsigargin, FCCP produced a large rise in [Ca²⁺]i and subsequent reduction, or directly reduced [Ca²⁺]i for ten to tens of minutes. These results suggest that mitochondrial Ca²⁺ release and/or depolarization activates plasmalemmal Ca²⁺ entry different from STOC and Ca²⁺ release from ER, which leads to STOC activation, while Ca²⁺ release from ER activates Ca²⁺ accumulation in, or release from, mitochondria. [J Physiol Sci. 2006;56 Suppl:S158]

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In brown adipocytes, mitochondria and endoplasmic reticulum (ER) are found to couple each other via Ca²⁺, regulating plasmalemmal Ca²⁺ entry (Kuba et al., this meeting). To study how noradrenaline and glucagons that cause thermogenesis regulate this coupling and how it regulates plasmalemmal Ca²⁺ entry, intracellular Ca²⁺ concentration ([Ca²⁺]i) and mitochondrial membrane potential were measured by fluorometry in cultured rat brown adipocytes. Isoprotelenol and glucagon caused bi- or triphasic rises in [Ca²⁺]i. The first phase was accompanied by mitochondrial membrane depolarization. The second phase was paralleled by mitochondrial membrane repolarization and blocked by Ca²⁺ free solution, indicating plasmalemmal Ca²⁺ entry. The third phase was blocked by Ca²⁺ free, EGTA solution, but not by thapsigargin, a blocker of Ca²⁺ pump at ER, and enhanced at pH 9, but not in a Na⁺-free solution, indicating activation of STOC. A blocker of phospholipase C, U73122, enhanced the second and third phases of β₃-adrenergic and glucagon responses, while it blocked Ca²⁺ release by α₁-adrenoreceptor activation from ER and subsequent activation of store-operated Ca²⁺ entry. Thus, the activation of β₃-receptor and glucagons receptor causes multimodal plasmalemmal Ca²⁺ entry via changes in mitochondrial membrane potential and depletion of Ca²⁺ in ER via mitochondrial ER coupling. [J Physiol Sci. 2006;56 Suppl:S158]

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Myocardial gap junction channels (Gap) are indispensable to action potential propagation. The channel gate (chemical gating) is regulated by intracellular cations such as Ca²⁺, Mg²⁺ and H⁺, but its dynamic gating properties have been scarcely examined. In this study, we investigated effects of Mg²⁺ on the Gap conductance in paired cells dissociated from guinea-pig ventriculues.The two-electrode whole-cell patch-clamp technique was applied to one of the paired ventricular myocytes (cell1). The current response to±5 -mV voltage pulses was recorded every 400 ms. In order to apply Mg²⁺instantaneously, we perforated the membrane of the other pair of myocytes (cell 2), using a sealed pulsed nitrogen laser in the presence of a given concentration of Mg²⁺ in the bath solution. Under these conditions, the recorded current flowed from the cell 1 mostly through the Gap into the cell 2 whose interior was short circuited to the ground.The Gap conductance decreased in response to various Mg²⁺ concentrations ([Mg²⁺]_i) in a dose-dependent manner (Hill coefficient: 3.84 EC50: 0.603mM). The conductance decay was well fit by a single exponential function. The 1/τ–[Mg²⁺]_i relationship was almost linear over the range of [Mg²⁺]_i from 1 to 10 mM. Our results suggest that the Gap gating is regulated by multiple bindings of divalent cations, including one rate-limiting step. [J Physiol Sci. 2006;56 Suppl:S158]

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The regulator of G-protein signaling type 8 (RGS8) has a high affinity for Gαi and only a low affinity for Gαq. We previously reported that RGS8 decreased the amplitude of Gq-mediated response in a receptor type-specific manner and that RGS8S, a splice variant of N-terminus region, induced less inhibition. Although molecular mechanisms underlying receptor type-specific attenuation by RGS8 still remains unclear, recent evidences have raised a possibility that RGS may interact with certain GPCRs. Here we show by co-immunoprecipitation experiments that RGS8 directly binds to the third intracellular (i3) loop of M1- and M3-muscarinic AChR but not of M2, and that binding of RGS8S is weaker. We observed that a deletion of N-terminal 9 aa of RGS8 or substitutions of Arg-8 and Arg-9 of RGS8 for Ala reduced binding with M1i3, suggesting the importance of N-terminal region. To examine whether or not the interaction between RGS8 and M1 may occur in living cells, we performed BRET analysis. The results showed that RGS8 actually interacts with M1 and that the interaction of RGS8S is less clear. We next analyzed electrophysiologically the inhibitory effects of RGS8 w.t. and R8A/R9A mutant on Gq-mediated responses using Xenopus oocytes, and observed that the inhibitory effect of RGS8 was decreased by the mutations. These biochemical and electrophysiological results show that RGS8 inhibits M1-muscarinic AChR-mediated responses by a mechanism which involves direct interaction between N-terminus of RGS8 and i3 loop of M1. [J Physiol Sci. 2006;56 Suppl:S159]

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Articular chondrocytes play an important role in the formation of the cartilage in synovial joints, which is closely influenced by mechanical or osmotic stress. In the present study, whole-cell membrane currents were recorded from isolated rabbit articular chondrocytes during exposure to hyposmotic external solution, under conditions where Na⁺, Ca²⁺ and K⁺ channels and electrogenic transporters were minimized. Articular chondrocytes responded to a hyposmotic external solution (Na⁺ reduction to about 70% of control) with an osmotic cell swelling, which was consistently accompanied by the activation of an outwardly-rectifying Cl⁻ current (swelling-activated Cl⁻ current, I_Cl,swell). I_Cl,swell was practically time-independent at potentials negative to +30 mV but exhibited a gradual inactivation at more positive potentials. Bath application of arachidonic acid (AA) reversibly and concentration-dependently blocked I_Cl,swell with an IC₅₀ of 0.58 μM and Hill coefficient of 1.9. The maximal effect (100% block) was obtained with 10 μM AA. Neither cyclooxygenase inhibitor indomethacin (10 μM) nor lipoxygenase inhibitor nordihydroguaretic acid (NDGA, 3 μM) significantly affected the inhibitory action of AA. In addition, PGE₂, LTB₄ and LTD₄ did not have any appreciable effect on I_Cl,swell, suggesting that AA directly affected I_Cl,swell. The present study thus confirms the presence of I_Cl,swell which exhibits a high sensitivity to inhibition by AA in rabbit articular chondrocytes. [J Physiol Sci. 2006;56 Suppl:S159]

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There are six thermosensitive TRP channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Recently, we have demonstrated that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD⁺- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We have also reported that, even though cyclic ADP-ribose (cADP-ribose) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADP-ribose dramatically potentiates TRPM2 activity. Here we show that in rodent pancreatic islets, TRPM2 is co-expressed with insulin, and mild heating (around body temperature) of these cells evokes increases in both cytosolic Ca²⁺ and insulin release which is K_ATP channel-independent and cAMP-mediated. Heat-evoked response in pancreatic islets was significantly diminished by applying the known TRPM2 inhibitors; nonsteroidal anti-inflammatory drug flufenamic acid (FFA), anti-fungal reagent econazole or 2-aminoethoxydiphenyl borate (2-APB), and by treatment with TRPM2-specific siRNA. These results suggest that TRPM2 regulates Ca²⁺ entry into pancreatic β-cells at body temperature depending on production of cADPR-related molecules, thereby regulating insulin secretion. [J Physiol Sci. 2006;56 Suppl:S159]

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The plasma membrane Na⁺/Ca²⁺ exchangers (NCXs) are bi-directional transporter that catalyzes the exchange of Na⁺ for Ca²⁺ depending on the electrochemical gradients. Mammalian NCX forms a multigene family comprising NCX 1, NCX 2 and NCX 3. However, the expression and functional role of NCXs in mammalian osteoclasts are still unknown. The aim of present study is to clarify the expression of NCX and their functional role during bone resorption in mouse osteoclasts. We examined the expression of NCX using RT-PCR, immunocytochemical and Western blotting methods. The activation of NCX during bone resorption were assessed by measurement of intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 and the effect of NCX inhibitors on pit formation assay. Mouse osteoclasts were expressed NCX 1 and 3, not NCX 2 using RT-PCR, Western blotting and immunocytochemical methods. There are some isoforms in mouse osteoclasts; NCX 1.3 and 1.4 in NCX 1 and NCX 3.5 in NCX 3. Under the measurement of [Ca²⁺]_i, low or free extracellular sodium increased [Ca²⁺]_i in osteoclasts. The [Na⁺]_o free-induced [Ca²⁺]_i i increase was inhibited by NCX inhibitors. The NCX inhibitors also decreased in pit area resorbed by osteoclasts in dose dependent manner. These results suggest that NCXs are expressed in mouse osteoclasts and act as Ca²⁺ regulation during bone resorption. [J Physiol Sci. 2006;56 Suppl:S159]

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In the ciliary muscle, a smooth muscle under parasympathetic control, contraction is initiated by stimulation of muscarinic receptors of M₃subtype. It is established that the initial phasic component of the contraction is triggered by Ca²⁺ release from intracellular stores mediated by G_q-linked signalling pathway. The tonic component is also known to be highly dependent on Ca²⁺, but Ca²⁺ is now provided by influx through receptor-operated cation channels rather than by release from stores [Takai et al.(2005) J Physol 559, 899-922]. However, little is known about the signalling mechanism involved in this Ca²⁺ influx. Here we have examined effects of YM-254890, a putative specific G_q inhibitor on contraction and [Ca²⁺]_i elevation induced by carbachol (CCh). For mechanical experiments ciliary muscle bundles dissected from bovine eyes were vertically mounted in an organ bath continuously perfused with normal saline, and isometric tension was recorded using a U-gauge transducer. Bath application of 2 μM-CCh caused a contraction. Both phasic and tonic components of this response were abolished by YM-254890 (3-10 μM). Using a Fluo-4 fluorescence method, we observed that CCh (10 μM) induced an elevation of the [Ca²⁺]_i in dispersed bovine ciliary muscle cells. Both initial phase and sustained phase of this response were also abrogated by YM-254890 (3-10 μM). G_q appears to be critically involved in Ca²⁺ mobilization in tonic as well as phasic component of the contraction of bovine ciliary muscle. [J Physiol Sci. 2006;56 Suppl:S160]

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Disturbance of renal Na⁺ reabsorption develops hypertension in Dahl salt-sensitive (DS) rat. Aldosterone plays a critical role in controlling renal Na⁺ reabsorption by stimulating expression of epithelial Na⁺ channel (ENaC) and also activate an ENaC-regulating protein kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). Therefore, we studied how aldosterone regulates ENaC expression and SGK1 in DS rat. Aldosterone (1.5 mg/kg B.W.) was subcutaneously injected into adrenalectomized DS and Dahl salt-resistant (DR) rats kept with normal (0.3% NaCl) diet and saline for 2 weeks after adrenalectomy. RNA and protein were extracted from the kidney 6 hr after the aldosterone application. Aldosterone decreased mRNA expression of β- and γ-ENaC in DS rat unlike DR rat, while aldosterone increased α-ENaC mRNA expression in DS rat similar to DR rat. Further, we found that aldosterone did not affect SGK1 expression in DS rat but elevated it in DR rat. These observations indicate that ENaC and SGK1 are abnormally regulated by aldosterone in DS rats, suggesting that these abnormal responses to aldosterone would be one of factors causing salt-sensitive hypertension. Supported by JSPS 17390057, 17590191, 17790154. [J Physiol Sci. 2006;56 Suppl:S160]

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The disorder of dopamine (DA) system may be related to neurodevelopmental dysfunction. However, the action of DA on synaptic transmission during development is largely unknown. We studied the effect of DA on GABAergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from CA1 pyramidal cells. DA (100 μM) profoundly decreased the amplitude of GABA_A receptor-mediated postsynaptic currents (GABA_A-PSCs) to 32% in the first postnatal week, when GABA provides excitatory drive. DA also decreased the amplitude of AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) to 29% in the second postnatal week, when glutamate responses first appear. The DA-induced inhibition declined after these periods and became only partial after postnatal day 30. Further we identified the receptor subtype involved in the DA-induced inhibition as phosphatidylinositol (PI)-linked D1-like receptor, since SKF 83959, a selective agonist for PI-linked D1-like receptor, clearly mimicked the action of DA, and U-73122, an inhibitor of phospholipase C, significantly reduced the DA-induced inhibition. DA did not change the response to puff-applied GABA or kainic acid, nor miniature GABA_A-PSC or EPSC amplitudes. These results suggest that the activation of PI-linked D1-like receptor profoundly suppresses the excitatory transmission in the developing hippocampus by presynaptic mechanisms. [J Physiol Sci. 2006;56 Suppl:S160]

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The striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. Recent studies suggest the involvement of the dorsal striatum in the advanced stages of drug addiction. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the striatum and can control the excitability of medium-sized spiny (MS) neurons, which provide the striatal output. We investigated the effect of chronic nicotine treatment on GABAergic synaptic transmission in mouse MS neurons. Twice-daily subcutaneous injections of nicotine (1 mg/kg) for 10-15 days did not change the electrophysiological properties of MS neurons or of three types of interneurons. However, intrastriatal stimulation evoked multiphasic IPSCs more frequently in MS neurons of nicotine-treated mice than in those of PBS-treated mice. Multiphasic IPSCs consisted of early and late components, both of which were mediated by GABA_A receptors. However, the GABA_B receptor agonist SKF97541 suppressed the early but not the late IPSC, suggesting heterogeneity of GABAergic inputs to MS neurons. Furthermore, generation of the late IPSC required the activation of nicotinic acetylcholine receptors (nAChR) because dihydro-β-erythroidine, an antagonist of nAChR, suppressed only the late IPSC. These results suggest that chronic nicotine treatment enhances the cholinergic modulation of GABAergic synaptic transmission in the striatum and that the enhanced inhibitory inputs. [J Physiol Sci. 2006;56 Suppl:S161]

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Dorsal cochlear nucleus (DCN) is known to process complex sounds. The principal cells are known to integrate inputs from auditory nerve fibers (ANFs) and parallel fibers. Axons of granule cells form parallel fibers and convey multimodal information. Granule cells cluster around ventral cochlear nucleus and DCN, and have mutual synapses between inhibitory interneurons; Golgi cells. Thus, Golgi cells may have some modulatory effects on parallel fiber activities; however, little is known. We studied the excitability of Golgi cells and interpreted the roles played by Golgi cells in the neuronal activity of DCN. By depolarizing current injection, Golgi cells fired repetitively and the firing frequency increased with current injection. At higher current intensity (300 pA-400 pA), steep firing adaptation was observed. By hyperpolarizing current injection, a depolarizing voltage sag emerged due to h-current activation. EPSCs evoked by ANFs stimulation were of multiple-peaks suggesting inputs through polysynaptic pathway. Because some cholinergic projections were expected, we tested cholinergic agonists: Carbachol induced a membrane hyperpolarization, accompanied with a decrease in the input resistance; muscarine evoked similar responses. These indicate the activation of muscarinic receptors, which hyperpolarized Golgi cells through the activation of GIRK. Therefore, cholinergic innervation may contribute in modulation of parallel fiber activity through inhibitory Golgi cells. [J Physiol Sci. 2006;56 Suppl:S161]

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In the cerebellar cortex, previous reports indicated that noradrenaline (NA) enhances inhibitory synaptic transmission via β-adrenoceptor-pathways. However, the effects of α-adrenoceptor activation on cerebellar inhibitory postsynaptic currents (IPSCs) have not yet been fully understood. Therefore, we investigated the effects of the α₁- or α₂-adrenoceptor agonist on IPSCs recorded from mouse Purkinje cells (PCs). The selective α₁-adrenoceptor agonist phenylephrine (PE) increased both the frequency and amplitude of spontaneous IPSCs (sIPSCs). PE also enhanced the amplitude of evoked IPSCs (eIPSCs) and increased the frequency but not the amplitude of miniature IPSCs (mIPSCs). Moreover, PE decreased the paired-pulse ratio of eIPSCs and did not change GABA receptor sensitivity in PCs. Conversely, the selective α₂-adrenoceptor agonist clonidine significantly reduced both the frequency and the amplitude of sIPSCs. Neither eIPSCs nor mIPSCs were affected by clonidine. Furthermore, presynaptic cell-attached recordings showed that spontaneous activity of GABAergic interneurons was enhanced by PE, while reduced by clonidine. These results suggest that NA enhances inhibitory neurotransmitter release via α₁-adrenoceptors, which are expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of interneurons via α₂-adrenoceptors, which are expressed in presynaptic somatodendritic domains. Thus, cerebellar α-adrenoceptors play roles in a presynaptic dual modulation of GABAergic inputs from interneurons to PCs. [J Physiol Sci. 2006;56 Suppl:S161]

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Amphiphysin I, a member of the BAR (Bin-Amphiphysin-Rvsp) protein super family, plays a key role in clathrin-mediated endocytosis of synaptic vesicles. Amphiphysin I mediates invagination and fission of synaptic vesicles in cooperation with dynamin, which senses and facilitates membrane curvature by its BAR domain. In vitro, when present at higher concentrations, amphiphysin I can stabilize membrane curvature, generating lipid tubules, forming ring structure with dynamin, and increasing dynamin GTPase activity. In the present study, we found that amphiphysin I was cleaved to three fragments by treatment with high KCl (80 mM) in the mouse hippocampus slices. The cleavages were inhibited by pretreatment with calpain inhibitors. Calpain also cleaved amphihysin I to three fragments in vitro. We identified the three cleavage sites by mass spectrometry. Amphiphysin I was cleaved at the sites of 322, 349 and 386. Calpain-dependent cleavages of amphiphysin I can induce the liposome tubulation as the same as wild- type amphiphysin I, but it cannot form the ring structure with dynamin I under electron microscope. Moreover, transferrin uptake was inhibited by overexpression of the truncated form of amphiphysin I compared with that of wild-type amphiphysin I in COS-7 cell. These results suggest that amphiphysin I may be a substrate of calpain in presynaptic terminus and the cleavages are important for the regulation of clathrin-mediated endocytosis of synaptic vesicles. [J Physiol Sci. 2006;56 Suppl:S161]

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The efficacy of synaptic transmission is determined by the quantal parameters such as the number of readily releasable pool vesicles (N), release probability (p) and postsynaptic response to a single quantum of transmitter (q). After massive vesicle exocytosis, N is replenished by vesicle trafficking for maintaining synaptic efficacy. However the mechanism underlying vesicle dynamics remains unknown. As a first step, we investigated whether staurosporine, a general kinase inhibitor, affects quantal parameters at the calyx of Held synapse in the brainstem slices of 12- to 15-day-old rats. Pretreatment of slices with 2 μM staurosporine for 1h decreased the amplitude of evoked EPSCs and slowed their rise time, but had no effect on their decay time. Staurosporine also reduced the frequency of spontaneous miniature EPSCs without affecting their amplitude (q) or kinetics. Estimation of quantal parameters, using the tetanic stimulation protocol, revealed that staurosporine reduced Nq, but not p. Staurosporine increased the magnitude of synaptic depression during repetitive stimulation (1-100 Hz) supporting the depletion model of synaptic depression. Staurosporin slowed recovery from depression caused by 10-100 Hz-stimulation without affecting the fast phase of recovery time course after 100 Hz-stimulation. We conclude that staurosporine-sensitive protein kinases affect the size of readily releasable pool and specifically accelerate a slow component of vesicle mobilization thereby contributing to the replenishment of readily releasable vesicles. [J Physiol Sci. 2006;56 Suppl:S162]

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We have recently reported that a phospholipase A₂ (PLA₂) activator melittin reversibly enhances glutamatergic excitatory synaptic transmission in substantia gelatinosa (SG; lamina II of Rexed) neurons of the rat spinal cord. The SG neurons receive not only excitatory but also GABA- and glycine-mediated inhibitory transmission. In order to know the effect of melittin on the spontaneous inhibitory transmission, we applied the blind whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. Melittin (1 μM) superfused for 3 min gradually increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) at a holding potential of 0 mV, which were visible about 2 min after the beginning of its superfusion and subsided within 8 min after washout. These facilitatory actions of melittin were observed for GABAergic and glycinergic sIPSCs, which were recorded in the presence of a glycine-receptor antagonist strychnine (1 μM) and a GABA_A receptor antagonist bicuculline (10 μM), respectively, and were reduced in extent by a PLA₂ inhibitor 4-bromophenacryl bromide (10 μM). A voltage-gated Na⁺ channel blocker tetrodotoxin (TTX, 0.5 μM) had a tendency to inhibit the effect of melittin on the GABAergic but not glycinergic sIPSC. It is concluded that melittin enhances GABAergic and glycinergic inbibitory transmission in a pre- and postsynaptic manner through the activation of PLA₂ in the SG; a part of the effect of melittin on GABAergic transmission is due to its action on excitatory transmission. [J Physiol Sci. 2006;56 Suppl:S162]

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G-protein coupled proteinase-activated receptors (PARs) have a unique activation mechanism in that a proteolytically exposed N-terminal region acts as a tethered ligand. Four members of PARs such as PAR-1 and PAR-2, which were identified by molecular cloning, can be activated by synthetic peptides whose amino acid sequences correspond to the tethered ligands. Although PARs appear to be involved in nociceptive transmission in peripheral terminals of primary afferents, there is no report about involvement of PARs in the transmission in the spinal dorsal horn. In order to clarify a role of PARs in the nociceptive transmission, we examined how PAR-1 and PAR-2 agonist peptides affect glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in substantia gelatinosa (SG; lamina II of Rexed) neurons which are thought to play a pivotal role in regulating nociceptive transmission to the CNS. We applied the blind whole-cell voltage-clamp technique to the SG neurons in adult rat spinal cord slices. PAR-1 agonist peptide (SFLLRN-NH₂; 1 μM) reversibly increased the frequency of sEPSC without a change in its amplitude, while PAR-2 agonist peptide (SLIGKV-NH₂; 1 μM) had no effects on sEPSCs. Both peptides did not alter holding currents at -70 mV. These results indicate that PAR-1 but not PAR-2 agonist peptide enhances the spontaneous release of L-glutamate from nerve terminals in the SG. It is suggested that PAR-1s expressed in nerve terminals in the SG may play an important role in producing nociception. [J Physiol Sci. 2006;56 Suppl:S162]

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The periaqueductal grey (PAG) of the brainstem is a central site for the various physiological functions including cardiovascular control, defensive behavior and pain. Since the cholinergic modulation on PAG activity is still unknown, we used infrared-videomicroscopy in conjunction with whole-cell recordings to elucidate the effects of acetylcholine on PAG neurons in midbrain slices of juvenile rats. In current clamp mode, 40% of all PAG neurons examined showed depolarization of their membrane potential during the application of carbachol (CCh). On the other hand, 20% of all PAG neurons examined were hyperpolarized by CCh. The remaining PAG neurons (40%) were insensitive to CCh. Both the depolarizing and the hyperpolarizing action of CCh were atropine-sensitive, indicating that they were mediated by muscarinic receptors. The depolarizing response to CCh was abrogated by M1 antagonist, pirenzepine, while the hyperpolarizing response was abolished by M2 antagonist, gallamine. These results suggest that rat PAG neurons have functional muscarinic receptors. [J Physiol Sci. 2006;56 Suppl:S162]

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D-serine which is thought to facilitate the NMDA receptor, present in the forebrain and co-localize with N-methyl-D-aspartate (NMDA) receptor. We reported that the spatial learning in the water maze and tetanus induced hippocampal LTP in the slice preparations were facilitated in the mutant mice lacking D-amino-acid oxidase, an enzyme which metabolizes D-serine. In the present report, we compared NMDA dependent synaptic current and the effect of D-serine on NMDA dependent synaptic current in the hippocampal CA1 area between the wild-type mice and mutant mice lacking D-amino-acid oxidase, to clarify whether NMDA dependent synaptic current was enhanced in the mutant mice and whether the enhancement was D-serine dependent. Excitatory postsynaptic currents (EPSCs) were recorded in CA1 pyramidal cells using whole cell patch-clamp techniques by stimulating Schaffer collateral-commissural fibers of the hippocampal slices. The ratio of NMDA receptor-mediated EPSC amplitudes to non-NMDA receptor-mediated EPSC amplitudes was significantly larger in the mutant mice than in wild-type mice. The ratio of NMDA component to non-NMDA component of the wild-type mice was significantly increased to the levels observed in the mutant mice, when D-serine was added to perfusion medium. We suggest that D-serine increased in the mutant mice brain facilitates NMDA-mediated synaptic current. [J Physiol Sci. 2006;56 Suppl:S163]

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Extracellular recordings were made from CA1 regions in the rat hippocampal slice tissues. Superfusion of slice preparations with lidocaine at low concentrations (1–100 μM) induced a transient inhibition and subsequent augmentation of the maximal slope of the field excitatory postsynaptic potentials (fEPSPs). On the other hand, high concentrations (300–1000 μM) of lidocaine only suppressed the maximal slope of the fEPSPs. The amplitude of presynaptic volleys was simply suppressed by lidocaine (1–1000 μM) in a dose-dependent manner. Pretreatment of adenosine 1 (A₁) receptor antagonist, DPCPX (1 μM) diminished the transient inhibition of the fEPSPs. Intracellular recordings from CA1 neurons showed that lidocaine (3–100 μM) transiently suppressed the amplitudes of the evoked fast EPSPs and, of the fast and late IPSPs. Lidocaine at low concentrations (3–30 μM) also induced a DPCPX-sensitive transient hyperpolarization in the CA1 and CA3 neurons. In the presence of TEA (20 mM) and TTX (0.3 μM), lidocaine at the low concentrations reduced the amplitude and duration of Ca²⁺ spikes recorded from CA3 neurons. These results suggest that lidocaine at the low concentrations (3–100 μM) transiently inhibites the evoked fEPSPs via activation of A₁ receptors at both pre- and post-synaptic sites in the rat CA1 hippocampal neurons. [J Physiol Sci. 2006;56 Suppl:S163]

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To study differences in nature between synaptic and extrasynaptic AMPA-receptor in the adult brain, we recorded stimulus-evoked EPSC and AMPA-induced current response from hippocampal pyramidal neuron of rat brain slice under whole-cell patch clamp. Application of either NBQX or GYKI52466, AMPA antagonists, suppressed the both EPSC and AMPA-induced current response. Current(I)-voltage(V) relationship of the EPSC and AMPA-response intersected the voltage axis at 0 mV showing linear I-V curve. The AMPA-induced response was markedly augmented as much as twice of the control in the presence of cyclothiazide (CTZ), an inhibitor of desensitization of AMPA-receptor, whereas either EPSC or spontaneously evoked miniature EPSC was not affected at all by the CTZ. Furthermore, neither the amplitude nor the time course of the consecutive EPSCs evoked by repetitive stimulation of Schafer collateral at 100 Hz was not affected significantly by the presence of CTZ, although cumulative current responses to repetitive application of AMPA, which exhibited occlusion, were greatly augmented by CTZ in similar fashion to the single application of AMPA. The amplitude of the EPSC decreased in the presence of dihydrokainic acid (DHK), an inhibitor of GLT-1 glutamate transporter, but addition of CTZ to the DHK did not augment the EPSC amplitude. All these results suggest that synaptic AMPA receptor may lack the nature of desensitization unlike the extrasynaptic AMPA-receptor. [J Physiol Sci. 2006;56 Suppl:S163]

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We investigated the effects of fluid percussion injury (FPI) on the glutamatergic excitatory synaptic transmission of CA1 pyramidal neurons using conventional intracellular recording techniques. A moderate impact (3.8-4.8 atm) was applied to the left hemisphere of the parietal cerebral cortex by using a FPI device (HPD-1700, Dragonfly, Inc.). After a survival period of 7 days, horizontal brain slices containing hippocampus were cut. The slice preparations were devided into three groups (control, FPI-ipsilateral, and FPI-contralateral). The control group includes the data from normal and sham-operated rats. There were no significant differences in resting and acting membrane properties among three groups. However, the EPSPs evoked by stimulations of Schaffer collaterals in the presence of bicuculline showed steeper input-output relationship (I-O) in the FPI-ipsilateral group. The number of spikes evoked by EPSP in the presence of bicuculline was larger in FPI-ipsilateral group although there were no significant differences among three groups in the absence of bicuculline. The paired-pulse facilitation ratio in FPI groups was smaller than that of control group. Frequency of mEPSPs recorded in the presence of tetrodotoxin was higher than that of control while the amplitude of mEPSPs was not different. These results suggest that the post-traumatic facilitation of gultamatergic transmission in pyramidal neurons of rat hippocampal CA1 is mediated by presynaptic origin. [J Physiol Sci. 2006;56 Suppl:S163]

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GABA_Areceptor mediated two inhibition models, phasic and tonic. Midazolam, benzodiazepine, and a widely used anesthetic, propofol potentiate GABA_A receptor function. Bai et al. reported propofol, compared with midazolam, had a lower potency but higher efficacy for increasing the amplitude of the tonic current in the hippocampal CA1 pyramidal neurons. We compared the effect of midazolam and propofol on the tonic current in neocortex layer V pyramidal neuronsusing whole-cell patch clam techniques from 2- to 3- week old rats. Propofol and midazolam cause a concentration-dependent increase in the amplitude of the tonic current. In the neocortex, midazolam increased tonic current more than propofol. Picrotoxin blocked both midazolam and propofol effect on the tonic current. In contrast, bicuculline blocked tonic current induced by midazolam but not proporol. These suggest that these two drugs mediate tonic current via different subtypes of GABA_A receptors Referance (1)Molecular Pharmacology 2001;59:814-824 [J Physiol Sci. 2006;56 Suppl:S164]

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We showed the excitatory (glutamate) and inhibitory (GABA and glycine) synaptic inputs to the superior salivatory (SS) neurons innervating the submandibular salivary glands and tongue. However, the relationships between the higher and lower centers in the synaptic inputs have not yet been examined. In the present study, we studied electrophysiologically the inhibitory synaptic inputs in brainstem slices obtained from normal and decerebrate rats. The SS neurons were labeled by retrograde axonal transport of a fluorescent dye. Whole-cell patch-clamp recordings were made from the labeled neurons. The currents were evoked by agonists (GABA and Glycine) perfusion, and electrical stimulation near the recording cell. After decerebration, agonists perfusion induced larger currents, but their decay time constant were not altered as compared with those of normals. This increase may result from receptor up-regulation at postsynaptic membrane. By electrical stimulation, in 83% (n=34/41) neurons, enhanced IPSCs were evoked, suggesting that the neurons have the inhibitory inputs from both the higher and lower centers. In 17% neurons (n=7/41), no IPSCs were evoked, suggesting that the neurons have only the descending inhibitory inputs from the higher centers. It is suggested that all SS neurons have the descending inhibitory synaptic inputs from the higher centers. [J Physiol Sci. 2006;56 Suppl:S164]

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Glutamate transporters are essential to remove synaptically released glutamate in excitatory synapses. Glial glutamate transporters expressed in Bergmann glia remove the majority of glutamate at excitatory synapses in cerebellar Purkinje cells (PCs) at early times after transmitter release. The neuronal glutamate transporter, the excitatory amino acid transporter 4 (EAAT4), is concentrated at perisynaptic sites of PCs, where metabotropic glutamate receptors (mGluRs) are located. To clarify the contribution of EAAT4 to the regulation of mGluR activation, we recorded mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) in cerebellar slices of mice deficient in EAAT4 and compared them with those in wild-type (WT) mice. The amplitude of mGluR-EPSCs evoked by the stimulation of parallel fibers (PFs) was larger in EAAT4-deficient mice than that in WT mice. However, the amplitudes of PF-evoked mGluR-EPSCs in EAAT4-deficient and wild-type mice were similar in the presence of the glutamate transporter antagonist DL-threo-β-benzyloxyaspartic acid (TBOA). mGluR-EPSCs evoked by the stimulation of climbing fibers (CFs) were observed in EAAT4-deficient mice but not in WT mice in the normal saline. When the function of EAAT4 was inhibited by a pharmacological treatment, mGluR-EPSCs were elicited by the stimulation of CFs even in WT mice. These results indicate that EAAT4 plays a critical role in the regulation of the activation of perisynaptic mGluRs at both PF and CF synapses in PCs. [J Physiol Sci. 2006;56 Suppl:S164]

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Immature rat spinal motoneurons showed a slowly synchronized rhythmic activity (sSRA) in divalent cation- free solutions. This sSRA was attributed to rhythmic oscillations of extracellular potassium ions in spinal cord. So, possible occurrence of the same phenomena in the dorsal horn at this same special situation, was investigated in this experiment. In the isolated spinal cord preparation from newborn rats, extracellular neuronal activity was recorded from dorsal and ventral roots using suction electrodes. After removal of extracellular divalent cations, sSRA could be recorded from both the dorsal root (dorsal sSRA) and the ventral root (ventral sSRA). The dorsal and ventral sSRA had a mirror image, but ventral sSRA occurred slightly earlier. Both were recorded in rats soon after birth, but not in older rats. Sectioning the border between the dorsal and ventral horn eliminated ventral sSRA, but not dorsal sSRA. It is suggested that both dorsal and ventral sSRA depends on the activity of a rhythm generator located in the dorsal horn. Several characteristic features of these two sSRA will be discussed. [J Physiol Sci. 2006;56 Suppl:S164]

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It has been demonstrated that AMP is hydrolyzed to adenosine(ADO) in the synaptic cleft by 5^'-nucleotidase. This enzyme in the central nervous system is known to participate in neuroprotective properties. However, little is known about the relationship between 5^'-nucleotidase activity and nicotine effects. Nicotine significantly increased 5^'-nucleotidase in synaptosomes from hippocampus of adult rats. This activity was significantly inhibited by AMPCP which was a 5^'-nucleotidase inhibitor. Nicotine significantly increased the hydrolysis of ATP, ADP, AMP and ADO by analysis with HPLC. These hydrolysis of adenine nucleotides were completely blocked in EGTA Ca²⁺-free buffer solution. We also examined the effect of ovariectomy (OVX) and estrogen replacement therapy (ER) on the activity of the enzyme that degrade adenine nucleotides in female rats. The release of ATP, ADP, AMP and ADO from hippocampus slice preparations by nicotine application in the buffer solution tended to decrease in the OVX group when compared to a control group. ER reversed the inhibition of the release of these nucleotides observed in OVX rats. The regulation of enzymes that hydrolyze these nucleotides in the hippocampus is essential in the modulation of the processes of neuronprotective properties. Results suggest the presence of a strong relationship between 5^'-nucleotidase activity and nicotine effects to the ADO formation, and also to estrogen binding-sites. [J Physiol Sci. 2006;56 Suppl:S165]

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We have recently reported that there exist regional differences of benzodiazepine effects in GABA-mediated inhibitory synaptic transmission in vitro studies. In this study, we examined the effects of propofol, one of the most popular intravenous anesthetic agents, on the inhibitory postsynaptic currents (IPSC_s) in CA1 pyramidal cells (CA1-PC_s) and dentate gyrus granule cells (DG-GC_s) in rat hippocampal slices. The monosynaptic IPSC_s were evoked by electrical stimulation of GABAergic interneurons and recorded from CA1-PC_s and DG-GC_s by whole cell patch-clamp technique. The effects of specific concentrations of propofol (0.1, 1, 10 and 100μM) on the IPSC_s in CA1-PC_s and DG-GC_s were examined at varying membrane potentials (20 mV steps, from -120 to +40 mV). In the absence of propofol, at the clamped membrane potential of-120mV and +40mV, IPSC amplitudes and decay time constants in both CA1-PC_s and DG-GC_s were kept stable for at least 20 minutes. When tested within this stable period, propofol was observed to increase the amplitudes and prolonged the decay time constant of IPSC_s in CA1-PC_s. However, propofol changed neither the amplitude nor decay time constant of the IPSC_s in DG-GC_s. These results suggest that propofol possesses differential effects on IPSC_s in CA1-PC_s and DG-GC_s similar to benzodiazepines. The mechanism for these differential effects could be due to the different sensitivity to propofol of the GABA_Areceptor subtypes in the CA1-PC_s and DG-GC_s. [J Physiol Sci. 2006;56 Suppl:S165]

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The retrosplenial cortex is located at a critical juncture between the visual cortex and hippocampal formation. Herein we show how signals traveling from the visual cortex behave in local circuits of the retrosplenial cortex, using optical recording methods and application of caffeine to rat brain slices. Electrical signals evoked in the primary visual cortex penetrated into the deep layer of the retrosplenial granular a cortex (RSGa), and propagated further toward postsubiculum and upper layer. Non-NMDA receptor-dependent initial traveling signal from the visual cortex triggered NMDA receptor-dependent neural oscillation in the RSGa. Oscillatory signals originated from the local area in the deep layer of the RSGa, and the signal spread back and forth toward the visual cortex and postsubiculum, in addition to spreading toward the upper layer. From the perspective of the RSGa, extrinsic signal inputs from the visual cortex switched on neural oscillators in the RSGa that deliver NMDA receptor-dependent intrinsic signal outputs. Opening and strengthening of non-NMDA receptor-dependent input pathways from the visual cortex required NMDA receptor-dependent oscillatory neural activities. These input and output relationships indicate that the retrosplenial cortex may represent an important relay station between the visual cortex and hippocampal formation. [J Physiol Sci. 2006;56 Suppl:S165]

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Although I have analyzed the mechanism of muscarinic inhibition on the IPSC at substantia nigra pars reticulata GABA neurons of rat, the intracellular transduction mechanism is not clear yet. N-ethylmaleimide (NEM), a membrane permeable inhibitor of PTX-sensitive G-proteins, did not have any significant effect on the muscarinic inhibition at the concentration of 100 μM. NEM itself significantly increased in the amplitude of IPSC to 1.627 ± 0.166 of the control amplitude (n=5, mean ± S.E.M., p=0.019 by Student's t test paired). Muscarine (10 μM) reduced the IPSC amplitude to 0.420 ± 0.104 under the control condition and to 0.370 ± 0.050 of the increased IPSC in the solution with NEM, respectively (p=0.738). ω-Agatoxin TK (ω-Aga TK), a selective P-type Ca²⁺ channel blocker, exerted no inhibitory influence on the muscarinic inhibition. The amplitude of IPSC was significantly attenuated to 0.443 ± 0.139 (n=4, p=0.031) in the solution with ω-Aga TK (100 nM). The muscarinic inhibition ratios were 0.430 ± 0.089 in control and 0.326 ± 0.077 of the decreased IPSC in the solution with ω-Aga TK, respectively (p=0.386). These results mentioned above and those previously reported suggest that G_βγ subunits dissociated from G_q/11 linked with M₃-receptors reduce the GABA release through the direct effect on the release machinery, when M₃-muscarinic ACh receptors at the presynaptic terminal of a striato-nigral projection fiber is activated. Hopefully, these observations contribute to the new approach of drug therapy for the basal ganglia disturbance. [J Physiol Sci. 2006;56 Suppl:S165]

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Glutamate is an essential excitatory neurotransmitter in the central nervous systems. For the understanding of mechanisms underlying synaptic transmission, we developed a novel optical glutamate probe called S403C-OG which consists of a recombinant glutamate binding domain derived from GluR2 subunit of AMPA receptor and a fluorescent dye. To visualize synaptically released glutamate, we immobilized S403C-OG on the cell surface of the cultured hippocampal neurons and captured fluorescence images with CCD camera. With this maneuver, we successfully detected the glutamate release along active synapses in response to electrical stimuli. The amount of glutamate release considerably varied among locations within the same neuron, suggesting the spatial heterogeneity among the release sites. We also observed spontaneous and transient glutamate release events without electrical stimuli, which was stochastic, variable in amplitude and spatially confined in small regions. Neither application of tetrodotoxin nor removal of extracellular calcium blocked the spontaneous glutamate release. The frequency of the release increased upon application of high concentrations of sucrose which is known to increase the frequency of miniature EPSC. Furthermore, we succeeded in continuous monitoring of the changes in presynaptic activity induced by phorbol ester, indicating that our probe enable to directly visualize the presynaptic activity. In summary, S403C-OG is useful to address many fundamental issues related to glutamatergic synaptic transmission in the central nervous system. [J Physiol Sci. 2006;56 Suppl:S166]

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It is well known that wind-up is the phenomenon that the second order neurons increase in firing frequency following repetitive stimulation of C-fibers. Recently, several lines of evidences suggest that the phosphorylation of the extracellular signal-regulated protein kinase (ERK) is involved in the hyperexcitability of the nociceptive neurons. However, it is not known how the ERK is involved in the windup phenomena. The present study was designed to evaluate the change in wind-up of DH nociceptive neurons i.t. administration of MEK inhibitor, U0126. Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). When single neural activity was isolated, the left sciatic nerve was stimulated (0.5 Hz, 3 times higher than C-fiber threshold). After windup was observed, U0126 (2.5 mM, 25 mM and 250 mM) was applied to the DH. The increased firing was significantly depressed following application of U0126. DH nociceptive neurons were classified as WDR neurons. All WDR neurons were located in laminae I-II in the DH. We could not observe any clear dose dependent effect of U0126 on DH nociceptive neurons. The mechanical and thermal responses were not affected by U0126. The present findings suggest that the intracellular MAP kinase cascade is involved in the central sensitization of the DH nociceptive neurons without any effects on naturally evoked responses. [J Physiol Sci. 2006;56 Suppl:S166]

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Synaptophysin is an integral membrane protein abundant in the synaptic vesicle and is found in nerve terminals throughout the brain. Its function has been implicated in various aspects of synaptic vesicle cycling such as biogenesis of vesicles, the regulation of the SNARE complex formation, synaptic vesicle fusion with plasma membrane, endocytosis and recycling of synaptic vesicles. It was recently suggested that synaptophysin is also involved in the modulation of activity-dependent synapse formation under a competitive condition. In this study, we examined at the individual level whether tactile stimulation selectively influenced the synaptophysin mRNA expression level in the somatosensory cortex of rats. Anesthetized rats were caressed on the back by an experimenter' s palms for twenty minutes and the mRNA expression levels in the somatosensory cortex responsible for the back and in the visual cortices five minutes afterwards were determined using quantitative PCR methodology. The synaptophysin mRNA expression level was selectively higher in the experimental group than in the control group in the somatosensory cortex but not in the visual cortex. This result suggests that the mRNA expression level of synaptophysin induced by neuronal activity is related to the regulation of synapse formation or remodeling or both. [J Physiol Sci. 2006;56 Suppl:S166]

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Pregnenolone sulfate (PREGS) is an abundant neurosteroid in the brain. PREGS presynaptically facilitates glutamatergic synaptic transmission, but underlying mechanism is not known. At the giant synapse, the calyx of Held in the rat brainstem slices, PREGS potentiated nerve-evoked excitatory postsynaptic currents (EPSCs), and increased the frequency, but not the amplitude, of spontaneous miniature EPSCs (mEPSCs). The EPSCs potentiations by PREGS and those by forskolin or phorbol ester did not occlude with each other. In direct whole-cell recordings from presynaptic terminals PREGS accelerated activation kinetics of voltage-dependent Ca²⁺ channel currents. BAPTA (10 mM) loaded into the terminal only partially attenuated this PREGS effect, suggesting that the main effect is independent of intracellular Ca²⁺. PREGS had no effect on presynaptic voltage-dependent K⁺ currents or resting conductance. Ca²⁺ imaging at the nerve terminal showed that PREGS increased Ca²⁺ influx into the terminal at the resting membrane potential. Consistently the PREGS-dependent increment of the mEPSC frequency was attenuated by 300 μM Cd²⁺. The PREGS-induced Ca²⁺ current facilitation was reversed by the PREGS scavenger cyclodextrin applied from outside, but not from inside, of the nerve terminal. We conclude that PREGS, by acting from outside of the nerve terminal, activates Ca²⁺ channels, thereby increasing both evoked and spontaneous transmitter release at the calyx of Held. [J Physiol Sci. 2006;56 Suppl:S166]

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Cerebellar Purkinje cells are key elements regulating motor learning and motor coordination. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum, because of the obstacle of delivering genes into Purkinje cells. To overcome this, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector expressing GFP was injected into the cerebellar cortex. Seven days after the injection, GFP was predominantly expressed in Purkinje cells. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex, even in the deep cerebellar nuclei, pontine nuclei and inferior olivary complex, which are synaptically linked with Purkinje cells or granule cells. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells. [J Physiol Sci. 2006;56 Suppl:S167]

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Cbln1 is the prototype for a family of four brain-specific proteins (Cbln1-Cbln4) of unknown function that was first identified by virtue of its harboring a naturally occurring 16 amino acid peptide, cerebellin. Cbln1 is a cerebellum-specific protein and structurally related to the C1q and Tumor Necrosis Factor families of proteins. We show here that Cbln1 is secreted from cerebellar granule cells as a glycoprotein and is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and post-synaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and the induction of long-term depression at parallel fiber-Purkinje cell synapses. Interestingly, the behavioral, physiological and anatomical phenotype of cbln1-null mice precisely mimics loss-of-function mutations in the orphan glutamate receptor, GluR&delta2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a previously undocumented trans-neuronal signaling pathway that controls synaptic structure and plasticity. [J Physiol Sci. 2006;56 Suppl:S167]

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GABA is known to be a major inhibitory neurotransmitter in mature mammalian brains. However, the effect of GABA can be converted into depolarizing or even excitatory when the postsynaptic Cl⁻ concentration becomes relatively high. We have recently shown that seizure-like afterdischarge induced by tetanic stimulation in normal ACSF (post-tetanic afterdischarge) is mediated by GABAergic excitation in mature hippocampal CA1 pyramidal cells. Here we investigated the possible contribution of similar depolarizing/excitatory GABAergic input to seizure-like afterdischarge induced in a low extracellular Mg²⁺ condition, as another experimental seizure model (low-Mg²⁺ afterdischarge). Perfusion of GABA_A antagonists abolished low-Mg²⁺ afterdischarge in most cases. Each oscillatory response during low-Mg²⁺ afterdischarge was dependent on Cl⁻ conductance and contained an F⁻-insensitive depolarizing component in the pyramidal cells. Perforated patch-clamp recordings revealed that GABA responses were indeed depolarizing during low-Mg²⁺ afterdischarge. Moreover, interneurons in the strata pyramidale and oriens discharged in oscillatory cycles more actively than those in other layers. These results suggest that the depolarizing GABAergic input may facilitate oscillatory synchronization among hippocampal CA1 pyramidal cells during low-Mg²⁺ afterdischarge in a fashion similar to the expression of post-tetanic afterdischarge. [J Physiol Sci. 2006;56 Suppl:S167]

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Ca²⁺-induced Ca²⁺ release (CICR) takes place in response to Ca²⁺ entry via the activation of type-3 ryanodine receptors (RyRs) after its use- and Ca²⁺-dependent priming, and amplifies impulse-evoked transmitter release in frog motor nerve terminals. Since the activation of CICR occurs in less than 1 msec after a nerve impulse, the site of Ca²⁺ release is close to the high [Ca²⁺]_i microdomain, where the machinery of the exocytosis is activated. Then, the most possible Ca²⁺ stores, on which RyRs reside, would be synaptic vesicles. We studied here the effects of loading Ca²⁺ chelator into synaptic vesicles on the priming and induction of CICR. EGTA was loaded into synaptic vesicles by incubating preparations in a Ca²⁺-free, EGTA (1mM) and Mg²⁺ (1 or 10mM) solution for 20-30 min, in which endocytosis still took place following high frequency stimulation of the nerve in low Ca²⁺ (0.15-0.5mM), high Mg²⁺ (6-10mM) solutions. After loading EGTA, tetanus-induced rises in end-plate potential (EPP) amplitude and miniature EPP (MEPP) frequency, reflecting the priming and activattion of CICR, became slower in onset and smaller in amplitude and rate of rise. Results are in favor of the idea that synaptic vesicles are involved in the priming and activation of CICR and so synaptic plasticity. [J Physiol Sci. 2006;56 Suppl:S167]

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