KcsA, found in prokaryotic Streptomyces lividans, is a tetrameric potassium channel gated by cytosolic pH. High-resolution structure of KcsA channel for the transmembrane domain has brought some insights for understanding the mechanism of ion selectivity and permeation through the pore. Recently, structure of the cytoplasmic domain and its functional roles have been elucidated for other types of potassium channels, such as for Shaker channels. However, a detailed structure and role of cytoplasmic domain of KcsA channel remain unsolved. In this study, we prepared site-directed cystein mutants of KcsA channel and accesibility of gold to cystein residues were investigated by using surface plasmon resonance (SPR) signals. These results provided information about geometry around the mutated site and surface structure of cytoplasmic domain of KcsA channel. In addition, single-channel recordings of each mutant channel reconstituted in planar lipid bilayer were performed. Effect of mutation and its modification by gold particle on the gating properties were analyzed. Structural information from each mutated position and relationship to their channel function will be discussed. [Jpn J Physiol 55 Suppl:S135 (2005)]

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[Background] Arsenic trioxide (As₂O₃), a newly introduced agent for acute promyelocytic leukemia, has been shown to induce QT prolongation of the electrocardiogram (ECG), thereby potentially causing life-threatening ventricular arrhythmias. The purpose of the present study was to elucidate the electrophysiological mechanisms that underlie the arrhythmogenic effects of As₂O₃. [Methods and Results] We obtained ECG signals by a radio transmitter from control and the As₂O₃-treated rats. The QT intervals were significantly increased in the As₂O₃-treated rats (control 45.3 ± 0.2 ms versus As₂O₃ 58.5 ± 0.4 ms, p<0.01). Action potentials (AP) of rat papillary muscle were recorded by using intracellular microelectrode technique. As₂O₃ significantly prolonged AP durations (APD) measured at 90% of repolarization (APD₉₀; control 54.4 ± 9.6 ms versus As₂O₃ 91.3 ± 36.3 ms, p<0.01). Furthermore, whole-cell patch clamp studies show that As₂O₃ significantly depressed inward rectifier K⁺ currents (I_K1) (control -15.3 ± 1.1 pA/pF versus As₂O₃ -11.1 ± 1.0 pA/pF at -120 mV, p<0.01) and increased Ca²⁺ currents (I_CaL) (control -6.8 ± 0.7 pA/pF versus As₂O₃ -9.9 ± 0.3 pA/pF at 0 mV, p<0.01). [Conclusion] The results of this study indicate that inhibition of I_K1 and an increase in I_CaL by As₂O₃ may underlie AP prolongation in cardiac myocyte and thereby contribute to prolonged QT intervals observed in patients. [Jpn J Physiol 55 Suppl:S136 (2005)]

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There are seven mammalian thermo-sensitive transient receptor potential channels (thermoTRPs). TRPM2 is a non-selective Ca²⁺-permeable cation channel in the TRP family of ion channels. Among the thermoTRPs, TRPM2 has been known to be activated by elevated temperature with a threshold of near 35°C, nicotinamide adenine dinucleotide (β-NAD⁺) and adenosine 5'-diphosphoribose (ADP-ribose). The current responses have been reported to be potentiated by combination of each ligand with heat. Furthermore, cyclic ADP-ribose (cADP-ribose) with heat also activates TRPM2 although cADP-ribose alone cannot activate the channel. To date, it is known that non-steroidal anti-inflammatory drug, flufenamic acid (FFA) and antifungal agents, econazole and clotrimazole inhibit TRPM2. However, the inhibition was gradual and irreversible. Here we show that 2-aminoethoxydiphenyl borate (2-APB) exhibits instantaneous blocking of TRPM2 channel activity with a reversible fashion using a patch-clamp technique in HEK293 cells expressing human TRPM2. 2-APB blocked TRPM2 responses evoked not only by its ligand but also by ligand with heat. Thus, 2-APB seems to be a more powerful and effective tool in studying the role of TRPM2. [Jpn J Physiol 55 Suppl:S136 (2005)]

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The streaming potential (V_stream) is an outcome of the electroosmosis that arises from coupled flux of ion and water in the pore. V_stream gives quantitative information on the coupling ratio of ion-water flux and, thus, it is prerequisite for investigating ion permeation mechanisms. V_stream is evaluated as a shift of the reversal potential (V_rev) upon changes in osmotic gradient. Measurements of V_stream are, however, technically challenging. High osmotic gradient must be imposed across the membrane and planar bilayer method has been used exclusively. Moreover, the value of V_stream is so small that precise evaluation of V_rev is necessary. Here, we developed a novel method for measuring V_stream under the whole-cell patch-clamp configuration. Two features are essential in this method. Firstly, exposure time to high osmotic gradient was defined for short period of time by the ultra-fast solution-exchange system, thus circumvented cellular damages and subsequent responses. Secondly, V_rev was evaluated with great accuracy from macroscopic current measurements upon a series of ramp command. Whole time courses of current traces during a ramp voltage were used for optimizing the parameters of an equivalent membrane circuit. We applied this method to HERG potassium channel expressed in HEK293 cells and revealed that the ion-water coupling ratio changed with potassium concentration. [Jpn J Physiol 55 Suppl:S136 (2005)]

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Mechanosensitive channels (MSCs) have been implicated to play key roles in mechanotransduction of a variety of cells. Yet little is known on their physiological functions. One of major reasons is a lack of inhibitors specific to MSCs. Gadolinium and aminoglycosides have been used as potential MSC inhibitors, however, they have relatively high Kds over 10 uM and inhibit several ion channels other than MSCs. Very recently the peptide GsMTx-4 from a spider (Grammostola spatulata) venom was reported to be a promising candidate for a specific inhibitor of MSCs. We tested if GsMTx-4 inhibits Stretch-Activated and Ca-activated K channel (SAKcaC) and its loss-of-mechanosensitivity mutant by use of the back-fill method with excised inside-out patches. Open probability (Po) of SAKcaC was significantly reduced by nM range of GsMTx-4. Analysis of the effect of GsMTx-4 on the Po-V curve of SAKcaC revealed that the peptide acts as a gating modifier rather than a channel blocker. In contrast GsMTx-4 scarcely inhibited the loss-of-mechanosensitivity mutant, suggesting that GsTMx-4 would work as an inihibitor selective to MSCs. Inspired by these results, we designed and synthetized a variety of peptide mimetics of GsMTx-4. Among them a decamer peptide, which shares the sequence with the flexible loop2 in GsMTx-4, was found to inhibit SAKcaC in the same manner as GsMTx-4 with a lower Kd. This result opens up a promising way to synthetize a new class of MSC-specific inhibitors. [Jpn J Physiol 55 Suppl:S136 (2005)]

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The activation of β₃-adrenoreceptor in brown adipocytes hydrolyses triglycerides to free fatty acids, which increase the electrochemical potential for H⁺ via TCA cycle and activate an uncoupling protein, UCP-1, and thermogenesis. α₁-adrenoreceptor activation elicits a phasic rise in intracellular Ca²⁺ ([Ca²⁺]_i) via Ca²⁺ release through IP₃ receptors from endoplasmic reticulum (ER) and subsequent store-operated Ca²⁺ entry (STOC). To study how the β-action regulates [Ca²⁺]_i and how the α- and β-actions interact with each other, [Ca²⁺]_i and mitochondrial membrane potential (V_mito) were measured by fluorometry in cultured adipocytes. Isoprotelenol caused a biphasic rise in [Ca²⁺]_i . The first phase was blocked by FCCP, a protonophore, and accompanied by a transient decrease in V_mito, while the second 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 plasmalemmal Ca²⁺ entry. At a high basal [Ca²⁺]_i via STOC activation by thapsigargin, isoprotelenol reduced [Ca²⁺]_i for ten to tens of minutes. Likewise, STOC activated by α-receptor activation was blocked by β-receptor activation, vice versa. Thus, the activation of β-receptor activates or suppresses plasmalemmal Ca²⁺ entry via changes in mitochondrial membrane potential, when the basal level of [Ca²⁺]_i is low or high, respcetively. [Jpn J Physiol 55 Suppl:S137 (2005)]

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Drosophila genome displays two NMDA receptor (NR) subunit homologous genes, dNR1 and dNR2. We have previously found that either dNR1 or dNR2 is essential for functional NMDA receptors. Because Mg²⁺ block of the NR is released by depolarization of the postsynaptic cell, NRs has been considered as a coincidence detector for correlated synaptic inputs and may be crucial for learning acquisition. However, interestingly, Drosophila mutant for dNR1 showed severe disruption in protein-synthesis-dependent long-term memory (LTM) but not much in acquisition (LRN) after olfactory Pavlovian conditioning. Ion blockade of glutamate receptors are governed by residue located at the apex of the M2 loop that is called the N/Q site. The NR subunits have Asn (N) residue at the N/Q site, whereas non-NMDA (AMPA and kainate) receptors which do not show Mg²⁺ block have Gln (Q) residue at this site. In contrast to mammalian NR subunits and dNR1, N/Q site of dNR2 is occupied by Q. Despite of this N/Q site of dNR2, NMDA receptors formed in combination of dNR1 and dNR2 exhibited significant Mg²⁺ block property under physiological Mg²⁺ concentration (20 mM). When N/Q site of dNR1 was substituted to Q from N, dNRs consist of mutated dNR1 (dNR1-N631Q) and dNR2 did not display the Mg²⁺ block property. Similar to dNR1 mutant, transgenic flies overexpressing dNR1-N631Q in neuronal cells displayed significant impairment in LTM but not in LRN. These results suggest that Mg²⁺ block of NRs are important for LTM but not in acquisiton [Jpn J Physiol 55 Suppl:S137 (2005)]

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Endoplasmic reticulum (ER) stores and releases Ca²⁺ through IP₃ receptors in response to stimuli. Resultant Ca²⁺ depletion activates store-operated Ca²⁺ entry (STOC). Mitochondria also accumulates Ca²⁺ via a large membrane potential (V_mito), acting as local Ca²⁺ buffers for the close location to ER and plasma membranes. To examine how Ca²⁺ release from mitochondria affects STOC, intracellular Ca²⁺ ([Ca²⁺]_i) and V_mito were measured by fluorometry in brown adipocytes cultured from rats pre-exposed to the cold. Thapsigargin, a blocker of Ca²⁺ pump at ER, produced a sustained rise in [Ca²⁺]_i, which was abolished by a Ca²⁺ free, EGTA solution. FCCP, a protonophore, caused a biphasic rise in Ca²⁺]_i. The first phase was accompanied by a transient decrease in V_mito, while the second 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 plasmalemmal Ca²⁺ entry. At a high basal [Ca²⁺]_i due to STOC activation by thapsigargin, FCCP reduced [Ca²⁺]_i for ten to tens of minutes. Likewise, STOC activated by noradrenaline was blocked by FCCP or thapsigargin. These results suggest that reduction of mitochondrial membrane potential activataes or inactivates plasmalemmal Ca²⁺ entry in brown adipocytes, when the basal level of [Ca²⁺]_i is low or high, respectively. [Jpn J Physiol 55 Suppl:S137 (2005)]

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We have recently demonstrated that type 1 ryanodine receptor (RyR1) exhibits lower gain of Ca²⁺-induced Ca²⁺ release (CICR) activity than type 3 (RyR3) in mammalian skeletal muscle sarcoplasmic reticulum (SR). The lowered gain was reversed by CHAPS and DP4, a peptide corresponding to the Leu²⁴⁴²-Pro²⁴⁷⁷ region of rabbit RyR1, indicating hypothesized interactions between the N-terminal and the central domains of RyR might underlie channel dysfunction in malignant hyperthermia (MH) which shows mutations within these domains. In this study, we tested this hypothesis by comparing [³H]ryanodine binding to the SR vesicles from wild-type (WT) and MH-susceptible (MHS) pigs carrying RyR1 Arg⁶¹⁵Cys mutation. Caffeine-induced Ca²⁺ release was more enhanced with the SR from MHS as compared to those from WT. RyR1_MHS exhibited gain 8-fold greater than RyR1_WT with minor change in sensitivity to endogenous ligands including Ca²⁺, Mg²⁺, and ATP. Thus, the enhanced activity of RyR1_MHS is primarily due to a greater gain of the mutated channel. DP4 markedly activated RyR1_WT, whereas little activation with RyR1_MHS. These findings support the notion that increase in the gain of RyR1 by weakened interdomain interactions underlies MH. [Jpn J Physiol 55 Suppl:S137 (2005)]

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Application of adenosine (Ade) induces K⁺-current response in the follicular cells surrounding a Xenopus oocyte under voltage clamp. This Ade-induced response is produced by activation of Gs, subsequent adenylate cyclase, cAMP-dependent protein kinase, and by opening of the ATP-sensitive K⁺ channels. Application of 20 nM insulin initially augmented the Ade-induced response, and subsequently depressed. The depressing effect of insulin on the K⁺-current response was irreversible. Application of 100 μM cromakalim (Cro) induces a K⁺-current response similar to the Ade-induced response. The Cro-induced response was not depressed significantly by prior application of 20 nM insulin for the same period as the case of Ade-induced response. Prior application of 30 nM Lavendustin A (Lav.A), an inhibitor of protein tyrosine kinase, markedly blocked the effect of insulin on the Ade-induced response. Application of Lav. A alone to the cells did not affect the Ade-induced response significantly. Application of 1 μM PAO, an inhibitor of protein tyrosine phosphatase, alone depressed the Ade-induced response but did not depress the Cro-induced response significantly. These results suggested that the depressing effect of insulin on the Ade-induced response might be regulated by activation of protein tyrosine kinase through the stimulation of insulin receptor. [Jpn J Physiol 55 Suppl:S138 (2005)]

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[Purpose] To characterize the electrophysiological properties of the voltage-gated sodium channel in bovine ciliary muscle cells. [Materials and methods] Smooth muscle cells enzymatically dispersed from the bovine ciliary body were used for the whole cell-voltage clamp experiments. The bath solution used was HEPES-Krebs (pH 7.4, 30°C), and pipette solution contained 100 mM-Cs⁺ 70 nM-Ca²⁺ and 200 μM-GTP (pH 7.0). The following test pulse protocols were used: (1) ramp pulses of ascending and descending directions between -120 and +80 mV from a holding potential (H_p) of -50 mV at a rate of 1 V/s (200 ms duration); (2) depolarizing steps of 100 ms duration from H_p=-80 mV to a voltage between -110 and +80 mV; and (3) pre-conditioning by a long (5 s) step pulse from a H_p of -80 mV to a voltage between -110 and +80 mV followed by an abrupt switch to 0 mV. [Results] Ascending ramp-pulses evoked an inward current having a peak of -40 to -200 pA at -10 to 0 mV, which was not observed when the descending ramp-pulse protocol was used. The current was dose-dependently inhibited by superfusion of tetrodotoxin (K_i=3.1 nM, n=21). The voltage-current relationships obtained by both step and ramp pulse protocols showed a peak at -10 mV and a polarity reversal at about +14 mV. A 50% inactivation by conditioning pulses occurred at -46±2 mV (n=13). [Conclusion] The bovine ciliary muscle possesses a voltage-dependent sodium channel similar to those distributed in neurones and skeletal muscle. [Jpn J Physiol 55 Suppl:S138 (2005)]

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In bovine ciliary muscle cells, carbachol (CCh) evokes inward currents resulting from opening of two types of muscarinic receptor-operated non-selective cation channels (NSCCL & NSCCS) with very different unitary conductances (35 pS & 100 fS)¹. Here we compared the dependence of their openings on the agonist concentration. Whole-cell membrane currents were recorded at 30°C under voltage clamp at -50 mV in myocytes freshly isolated from the bovine ciliary body with collagenase. The cells were superfused with HEPES-buffered physiological saline (PSS; pH 7.4). Pipettes were filled with K⁺-free solution containing 100 mM-Cs aspartate, 5 mM-BAPTA and 100 μM-GTP (pH 7.0). To record NSCCL current selectively, the free Ca²⁺ concentration in the pipette [Ca²⁺]_p was set slightly high (200 nM) so as to suppress opening of NSCCS¹. For selective measurement of NSCCS currents, Na⁺, K⁺, Ca²⁺ and Mg²⁺ in PSS were isotonically replaced with Ba²⁺, [Ca²⁺]_p was kept at 70 nM, and GTP was omitted from the pipette¹. The total amounts of charge carried by the CCh-evoked currents (0.1-100 μM) for 30 s were estimated by numerical integration. The present experiments estimated the concentration of CCh required for half-maximal activation and the Hill coefficient to be 0.5 ± 0.1 μM and 0.97 ± 0.08 for NSCCS (n=37) and 8.5 ± 2.1 μM and 1.1 ± 0.1 for NSCCL (n=43). The difference of the agonist sensitivities suggests the link of the channels with M₃-receptor through different signalling pathways. 1. Takai et al (2004) J. Physiol. 559, 899-922 [Jpn J Physiol 55 Suppl:S138 (2005)]

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We examined the kinetic properties of gap junction channels (GJCs) responsible for the outward rectification and low-pass filtering. When dual whole-cell recordings were made from two mesencephalic trigeminal neurons coupled via GJCs, both depolarizing and hyperpolarizing responses evoked by current pulse injections into one cell under current-clamp conditions were markedly attenuated in the other cell. Depolarizing responses were more markedly attenuated in the other cell than hyperpolarizing responses, suggesting the nature of outward rectification of GJCs. Furthermore, the action potential was more markedly attenuated than the sustained depolarization, suggesting the low-pass filter property of GJCs. When current responses to the symmetrical positive- and negative-going ramp pulses were recorded by using 0.2 mM EGTA internal solution, the maximal outward currents evoked by negative-going ramp pulses were smaller than those to positive-going ramp pulses. This became more prominent when the ramp rate was decreased. However, when 10 mM BAPTA was used instead of EGTA, the difference in the current amplitude became less clear. These results suggest that the apparent outward rectification may be mediated by the voltage dependence of the junctional conductance, which may be modified by [Ca²⁺]_i changes. Furthermore, it is likely that the apparent nature as low-pass filter may be mediated by the time-dependent activation of GJCs, which is determined by the channel-opening rate constant that decreases with membrane depolarization. [Jpn J Physiol 55 Suppl:S138 (2005)]

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Lucifer Yellow CH (LY), a fluorescence dye, has been injected into cells examined electrophysiologically in order to visualize them. We had reported that LY had blocked the inactivation of voltage-gated Na⁺ channel currents in a light-dependent manner and suggested that LY radicals generated by illumination modified channel proteins (J. Physiol, 2003). In this study, we investigated the effect of LY on other voltage-gated currents (delayed rectifier, inward rectifier K⁺, Cl⁻, HVA-Ca²⁺ currents) and ligand-gated currents (NMDA, AMPA, kainite, 5HT₃, and GABA_A receptor currents) of cultured mouse hippocampal neurons under voltage-clamp conditions. LY applied inside cells irreversibly blocked the steady state inactivation of AMPA receptor currents upon a few minutes exposure to light of usual intensity for microscopy. The pre-treatment of cells with 1 mM dithiothreitol antagonized the LY effect. The reversal potential of the steady state inactivation currents remained unchanged, indicating LY hardly changed the ionic selectivity of AMPA receptors. LY also increased the magnitude of delayed rectifier and inward rectifier K⁺ currents that a little inactivated in a light-dependent manner. LY had no effects on other voltage-gated and ligand-gated currents, including rapid inactivating 5HT₃ receptor currents. These results suggest that LY radicals produced by the exposure oxidize the cytoplasmic loops of AMPA receptors responsible for the steady state inactivation with a moderate selectivity. [Jpn J Physiol 55 Suppl:S139 (2005)]

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We investigated the characteristics of UTP-induced currents in rat small pulmonary artery smooth muscle cells, using whole cell patch-camp technique. In the condition of high Ca²⁺ bath solution (100 mM Ca²⁺) and Cs⁺ rich pipette solution, 100 μM UTP elicited inward current at holding potential -60 mV. However, UTP failed to evoke the inward current in NMDG-Asp extracellular solution. The induced inward currents were not inhibited by 100 μM niflumic acid. U-73122 (5 μM), a specific PLC inhibitor, attenuated the UTP-induced current but U-73343 (5 μM), an inactive form of U-73122, had no effect. Treatment with 2APB (50 μM) and Xestospongin C (10 μM) inhibited the induced current. These results suggest that UTP elicits inward current independent of Cl⁻ current in high Ca²⁺ solution. In addition, the current activation mechanisms may involved in PLC activation. [Jpn J Physiol 55 Suppl:S139 (2005)]

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Cold sensitive channel TRPA1 is activated by Noxious cold stimuli or pungent compounds, irritant drugs like isothiocyanate. But its active site is unkwon.Therefor, we measured the activity of variety of isothiocyanate to TRPA1 by using inner Ca²⁺ion concentration measurements,and study quantitative structure-activity relationship(QSAR) method for estimate activity site.Each compound induce [Ca²⁺] rise with dose dependent manner.Ec50 and Emax of those compound showed various value.We applicate QSAR method both EC50 and Emax values and search activity site and physical property. [Jpn J Physiol 55 Suppl:S139 (2005)]

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Homomeric and heteromeric isoforms of the human inward-rectifier K⁺ channels Kir4.1 and Kir5.1 are essential to K⁺ regulation in the kidney and the brain. This study establishes the current-voltage relationship, inward rectification, sensitivity to pH and known Kir channel blockers, K⁺ selectivity, and several single-channel properties of these channels using the patch-clamp technique. Identical experiments were conducted with cells expressing either Kir4.1 alone or co-expressing Kir4.1 and Kir5.1 (mass ration 1:5), as homomeric Kir5.1 is non-functional. Our data present Kir4.1 and Kir4.1/5.1 channels as functionally distinct proteins, consistent with previous work on orthologous channels. In contrast to strongly-rectifying Kir4.1/5.1 heteromers, homomeric Kir4.1 channels rectify only moderately, have a higher open probability, lower (non-burst) single-channel conductance, and are less susceptible to rundown. Sensitivity to Kir channels blockers (BaCl₂ >> CsCl) and extracellular pH was comparable. Based on this functional separation and recent reports of distinct distribution and expression onset in the brain, we propose that Kir4.1 and Kir4.1/5.1 channels engage in distinct physiological functions that make them likely candidates for excitability disorders in humans. Supported by The Kanehara Foundation (CL), the Japanese Ministry of Education, Science, Sports, and Culture: Grant-in-aid for specific research in a priority area (HH, YK). [Jpn J Physiol 55 Suppl:S139 (2005)]

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Acid-sensing ion channels (ASICs) are formed by diverse combinations of ASIC subunit proteins that exhibit distinct sensitivity, kinetics and permeability. To better understand proton signalling in nociceptors, we characterized the pH-dependent activation, kinetics and Ca²⁺ permeability of proton activated currents. Whole cell patch recordings were obtained from dorsal root ganglion (DRG) neurons (n= 40) that were acutely dissociated and classified by electrophysiological signature into 6 uniform types (type 3, 5, 6, 7, 8 and 9) of neurons known to express ASICs, but differentially express SP and/or CGRP (Petruska et al., 2000; 2002). Increasing proton concentrations had distinct influences on DRG cells. Non-nociceptive (type 3) and nociceptive groups (type 5, 6, 7, 8, 9) had similar thresholds (pH 6.8) but varied in potency (EC₅₀; type 3 and 5 > 6, 7, 8 and 9; p<.05), and reactive range (type 3 (0.4 pH unit) < type 5, 6, 7, 8, 9 (0.7 to 1.4 pH unit), p<.05). Decay kinetics did not covary with nociceptive function (155.7 ± 22.7 to 1463.2 ± 131.6 msec). Because a portion of ASICs are Ca²⁺ permeable, ratiometric Ca²⁺ imaging was examined in peptidergic nociceptors with ASIC1a-like kinetics. Ca²⁺ permeability was observed in both type 5 and 8 cells (pH 6.0; n=14). Ruthenium red (5 μM) did not block Ca²⁺ entry. In conclusion, nociceptors and non-nociceptors have distinct proton sensitivity. Ca²⁺ entry via ASICs may support release of peptides into tissues or promote transmitter release at synapses. Supported by NS39874. [Jpn J Physiol 55 Suppl:S140 (2005)]

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Correlated neuronal activity plays a fundamental role in the development of the nervous systems. Using a multiple-site optical recording technique with a fast voltage-sensitive dye (NK2761), we found large-scale correlated activity in the embryonic rat brain. Stimulation applied to the region caudal to the obex elicited widely-spreading depolarization waves in the whole brain-spinal cord preparations dissected from E15-E17 rat embryos. The depolarization wave traveled rostrally to the forebrain and caudally to the lumbosacral spinal cord, suggesting that functional cell-to-cell communication systems mediated by the depolarization wave are widely generated in the embryonic rat central nervous system. Stimulation applied to the cranial nerves, such as the trigeminal and vagus nerves, evoked depolarization waves with similar spatio-temporal distribution patterns, indicating that the wave activity is triggered by multiple sources of external inputs. Furthermore, wave activity spontaneously occurred. Ca²⁺-imaging with a Ca²⁺ indicator (Ca green 1-AM) showed that the depolarization wave was accompanied by a Ca²⁺-wave. The result suggests that the wave activity provides not only electrical synchrony, but also biochemical signals associated with [Ca²⁺]i elevation, which might regulate developmental processes of the mammalian central nervous system. [Jpn J Physiol 55 Suppl:S140 (2005)]

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Using a multiple-site optical recording technique with a voltage-sensitive dye (NK2761), we found a widely-spreading depolarization wave in the embryonic rat central nervous system. In this study, we analyzed pharmacological characteristics of the depolarization wave. The depolarization wave was eliminated by Cd²⁺, Ca²⁺-free solution and tetrodotoxin. In experiments using antagonists of chemical neurotransmitters, it was shown that the depolarization wave was partly inhibited by glutamate-, acetylcholine-, GABA- and glycine-receptor antagonists. With combined application of these blockers, the wave was eliminated. These results suggest that chemical synaptic networks mediated by multiple neurotransmitters are responsible for wave propagation. In addition to chemical synaptic antagonists, the depolarization wave was inhibited by gap junction blockers such as octanol and 18β-glycyrrhetinic acid. In immunohistochemical studies, we observed abundant expression of connexin26 and connexin32 in E16 rat brainstems. These results suggest the possibility that a dual network of gap junctions and chemical synapses mediates the propagation of the depolarization wave in the embryonic rat brain. [Jpn J Physiol 55 Suppl:S140 (2005)]

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The hippocampal network presents various types of oscillatory activities subserving diverse physiological functions including learning and memory. An intrinsic synchronous bursting of the interneurons appearing during early postnatal period in a form of giant depolarizing potentials (GDPs) is proposed to be involved in the maturation of the interneuronal networks (Ben-Ari et al., 2004), however, the molecular mechanism of its generation remains poorly understood. In juvenile rats and mice, extracellular ATP excites inhibitory interneurons and increase pyramidal IPSCs through activation of interneuronal P2Y1 receptors in the CA3 (Kawamura et al., J Neurosci 49 2004). Here we examined whether ATP-mediated signaling in the interneurons affects the GDPs. The spontaneous GDP activities were recorded from CA3 pyramidal neurons in the coronal hippocampal slice of the rats (P4-8). MRS2179, a specific antagonist to P2Y1 receptors abolished GDP and significantly reduced its frequency in a reversible manner. PPADS (a non-selective P2 receptor antagonist), but not TNP-ATP (a blocker of P2X receptors) and U-73122 (an inhibitor of phospholipase C), also significantly reduced the GDP frequency. 2meSADP, a selective P2Y1 receptor agonist, first increased GDP frequency, which was followed by a reduction. These results point to a possible involvement of ATP release and activation of interneuronal P2Y1 receptors underly the generation of intrinsic oscillatory activities in the rat hippocampus. [Jpn J Physiol 55 Suppl:S141 (2005)]

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Soluble amyloid β_1-42 (Aβ_1-42) inhibits cholinergic synaptic transmission by reducing ACh release from rat superior cervical ganglion neurons in long-term culture, whereas ACh synthesis and the uptake into synaptic vesicles (SVs) are not affected in the presence of Aβ_1-42. To further examine possible mechanisms for Aβ_1-42 inhibition of ACh release, effects of Aβ_1-42 on SV use and refilling of vesicles pools in the presynaptic terminals were analyzed. The inhibition of neurotransmitter release by Aβ_1-42 at 1 μM in the bath occurred in a neural activity-dependent fashion. When SVs were cycled in presynaptic nerve terminals by repetitive stimulation with action potentials (0.2 Hz), Aβ_1-42 decreased the size of the readily-releasable pool (RRP) of SVs measured by focal application of a hypertonic solution (0.5 M sucrose), however, the refilling rate of the RRP was not affected after depletion of this pool. After depletion of both the RRP and the reserved pool (RP) of SVs by train of action potentials (5 Hz for 3 min), the refilling rate of the RRP was not affected, but the rate of vesicle entry into the RP was significantly reduced by Aβ_1-42. To examine the Aβ_1-42 on the classical endocytic vesicle trafficking, a dynamin peptide, the interacting site with amphiphisin, at 1 mM in the pipette, was microinjected into presynaptic neurons along with Aβ_1-42. The decrease in EPSP amplitudes was comparable with the dynamin peptide alone. Together, these results suggest that soluble Aβ_1-42reduces ACh release by affecting the classical endocytic site in the cholinergic presynaptic terminal. [Jpn J Physiol 55 Suppl:S141 (2005)]

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Using the combination of a voltage-sensitive fluorescent dye and high luminance light emitting diodes (LEDs) for a light source, we have developed an optical recording system for neural activity from 1020-site in the intact rat cerebral cortex. Green LEDs provide an excitation light, and the fluorescent light from the stained preparation formes a magnified real image by a tandem lens optic system. The fluorescent light intensity at the image plane is detected simultaneously by a 34 x 34-element photodiode array (PDA). The outputs from PDA are amplified, multiplexed and digitized at a time resolution of 1 msec for each pixel. Data are sent to a computer and stored in a high-speed hard disk unit. Since the data storing speed is greater than the data transferring speed, we succeed in continuous recording on hard disk in real time. Now, the longest recording time is about 1000 sec. This restriction is simply owing to the maximum size of one file, 2 GB. We applied this system to the rat primary somatosensory cortex (Sm1). The cortex was stained with RH414 and placed under the optical apparatus. When the foot was electrically stimulated, optical signals from the hindlimb region had a small deflection with similar latency to that of the evoked potential. We consider this signal as representing the neural response of Sm1. Though the artifact derived from heartbeat was large, we could detect the neural response optically in a single sweep, if the stimulating pulse was given between the pulsations where the artifact was relatively small. [Jpn J Physiol 55 Suppl:S141 (2005)]

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GABA plays an essential role in the regulation of GnRH neurons and melatonin is involved in reproductive functions. Besides, melatonin is recently reported to modulate the current through GABA_A receptor (GABA_A current) in certain central neurons. We, therefore, decided to examine the effect of melatonin on GABA_A current in rat GnRH neurons. Transgenic rats with EGFP tagged to GnRH neurons were used. After enzymatic digestion, EGFP-GnRH neurons were cultured overnight with Neurobasal-A medium supplemented with L-glutamine and B-27. The currents were recorded with perforated patch clamp configuration. GABA (30 μM) was applied by puff and melatonin (10-1000 nM) was applied through perfusion. Melatonin reduced the GABA_A current in adult females while augmented the current in adult males. These effects of melatonin were blocked either by melatonin receptor antagonist luzindole (1 μM) or by pretreatment with pertussis toxin (100 ng/ml). There was no apparent effect of melatonin on GABA_A current in infantile GnRH neurons in both sexes. In conclusion, GABA_A current in rat GnRH neurons was modulated by melatonin via melatonin receptor. The modulation was developmentally and sexually regulated. [Jpn J Physiol 55 Suppl:S141 (2005)]

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In contrast to the excitatory synaptic plasticity, the modulation of GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) that modify excitatory synaptic transmission has not been sufficiently characterized. Although post tetanic depression of electrically-evoked IPSCs has been described by several investigators, relatively few studies were made on potentiation of the IPSCs. In present experiments, changes in evoked and spontaneous GABA_Aergic synaptic transmission after tetanic stimulation were examined using acute slices of rat hippocampus. IPSCs were recorded from CA3 neurons, by patch-clamp recording method. Spontaneous IPSCs were recorded in the presence of specific inhibitors against excitatory glutamatergic currents. High frequency stimulation was applied with microelectrode. Transient facilitation of miniature and spontaneous IPSCs were detected after intensive tetanus. As to evoked IPSC, suppression, rather than facilitation, was observed after tetanization under similar conditions. By fluorescent imaging, changes of calcium concentration during IPSC recording were also followed in hippocampal slice preparation. Increment of intracellular calcium concentration was demonstrated upon high frequency stimulation. Contributions, to the IPSC modulation, of extra-cellular calcium as well as calcium elevation in post synaptic neuron were examined. In addition, effect of several calcium mobilizing reagents was studied, on the efficacy of inhibitory synapse. [Jpn J Physiol 55 Suppl:S142 (2005)]

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Midazolam (MDZ), a benzodiazepine analogue, that is clinically used to induce sedation, relieve anxiety, and to impair memory of preoperative events has some unique actions. to explore unknown effects of MDZ on presynaptic GABA release, have not been studied. Thus we examined the effects of MDZ on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in layer V pyramidal neurons in somatosensory cortex by using whole-cell patch-clamp technique for 2-3 weeks-old rat brain slices. MDZ increases the frequency of GABAergic mIPSC via insertion of α7 nicotinic acetylcholine receptors (nAChRs) at presynaptic GABAergic terminals. MDZ also increased the number of α-bungarotoxin-bound boutons as revealed by confocal imaging. To elucidate the mechanism of nAChR translocation, we examined whether PKC are involved in the action of MDZ by using PKC inhibitors. Bath application of the cell-permeable and specific PKC inhibitor calphostin C (1 μM) for 20 min did not change the mIPSC frequency but blocked its increments by MDZ. Another PKC inhibitor, bisindolylmaleimide (5 μM), gave similar results. Thus, PKC activity is essential for the effect of MDZ. [Jpn J Physiol 55 Suppl:S142 (2005)]

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Repetitive Ca²⁺ entries through voltage-gated Ca²⁺ channels gradually prime β type-ryanodine receptors (RyRs) and activate Ca²⁺-induced Ca²⁺ release (CICR) which enhances both spontaneous and evoked transmitter exocytosis. The time course of priming, subsequent activation and inactivation of CICR was seen as a slow increase and subsequent decrease (hump) in the frequency of miniature end-plate potentials (MEPPs) during continuous tetanus. MEPP-hump was reduced in magnitude by reducing external Ca²⁺, and abolished in a Ca²⁺ free solution. In this study, hypertonicity produced a similar slow rise and fall of MEPP frequency even in a Ca²⁺ free, EGTA solution containing a small amount of Mg²⁺. These changes in MEPP frequency were blocked by ryanodine (20 μM), thus being equivalent in property to MEPP-hump induced by tetanus. The hypertonicity-induced MEPP-hump increased in magnitude with the extent of hypertonicity, but decreased with a rise in Mg²⁺ concentration and abolished at more than 2 mM Mg²⁺. The results suggest that hypertonicity facilitates spontaneous activation of Ca²⁺ release via RyRs even at the low basal Ca²⁺ level. Since Murayama et al. (1998) reported that hypertonicity increased the binding of ryanodine to α- and β-RyR of frog skeletal muscle, it may be that hypertonic solution sets up the condition for the activation of RyRs even at a low cytosolic Ca²⁺ level. [Jpn J Physiol 55 Suppl:S142 (2005)]

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Neurons in nucleus laminaris (NL) act as coincidence detectors of binaural synaptic inputs for sound source localization. In NL, neurons are tonotopically organized, and characteristic frequencies (CF) decrease from rostro-medial (high-CF) to caudo-lateral (low-CF) direction within the nucleus. The acuity of coincidence detection is different along the tonotopic axis; the acuity is higher in the high- and middle-CF neurons than that in the low-CF neurons. Previously, we showed that a predominant expression of Kv1.2 channels in these higher-CF neurons accelerates the EPSP time course and improves the coincidence detection. On the other hand, the process of transforming EPSPs into action potentials is also distinct along the tonotopic axis in NL; the amplitude of action potential is smaller in the higher-CF neurons (about 20 mV) than in the low-CF neurons. The small spikes in the higher-CF neurons would be due to a small Na⁺ conductance, as these spikes exhibited small maximum rate of rise. Furthermore, it is reported that the initial segment and the axon hillock in the high-CF neurons are myelinated. These results suggest a possibility that the action potential is generated at a remote site from cell soma in the higher-CF neurons, which should contribute to improve the coincidence detection. In this study, we evoked orthodromic and antidromic spikes under the whole-cell slice-patch recordings, and examined the properties of action potential generation in NL neurons. We also evaluated the distribution of sodium channels in NL neurons. [Jpn J Physiol 55 Suppl:S142 (2005)]

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It is well established that the Ca²⁺-influx through voltage-dependent Ca²⁺ channels plays a key role in promoting the depolarization-induced release of neurotransmitters from nerve terminals. In contrast, it has also been suggested that membrane potential itself has a decisive role in controlling neurotransmitter release. Here, we report that the presynaptic depolarization in the absence of extracellular Ca²⁺ increases the glycine release at rat spinal dorsal horn neurons. In the Ca²⁺-free solution, the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from mechanically dissociated rat dorsal horn neurons was increased by raising extracellular K⁺ concentration and by application of 100 μM 4-aminopyridine. The high K⁺-induced increase in spontaneous IPSC frequency under Ca²⁺-free conditions was markedly inhibited by pretreatment of BAPTA-AM, but not by nifedipine and ryanodine. Thapsigargin, a blocker of Ca²⁺-pump of internal Ca²⁺-stores, increased the spontaneous IPSC frequency even in the absence of extracellular Ca²⁺ and reduced the high K⁺-induced increase in IPSC frequency. The present results suggest that presynaptic depolarization increases the release of Ca²⁺ from internal stores, which in turn increases the glycine release in rat spinal dorsal horn. The regulation of Ca²⁺-release by depolarization per se provides a new mechanism by which neuronal activity regulates the cytosolic Ca²⁺ concentration in nerve terminals. [Jpn J Physiol 55 Suppl:S143 (2005)]

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Subfornical organ (SFO) is a brain center which controls body fluid homeostasis. Injection of noradrenaline (NA) into SFO caused a increase of water intake. Hemorrhage increased NA release in SFO area. Accordingly, noradrenergic activation on SFO neurons suggests to regulate body fluid balance. In the present study, the effects of NA and its analogues on SFO neurons in rat slice preparations were investigated by using whole cell patch clamp recording. In the current clamp mode, the application of NA at 10-100 mM produced membrane depolarization (64%, 14 responsive neurons /22 neurons tested) and hyperpolarization (27%, 6/22 neurons). In the voltage clamp mode, NA application at 1-100 mM produced inward currents (63%, 33/52 neurons) and outward currents (27%, 14/52 neurons). These currents were not affected by the presence of TTX. In most of the neurons (15/21 neurons) showing inward currents in the presence of NA, the membrane conductance was not changed by voltage ramps or hyperpolarizing pulse stimulation. Similar responses were obtained by the application of the α1 agonist phenylephrine. The phenylephrine-induced inward currents were inhibited by the α1 antagonist prazosin. In addition, the α2 agonist clonidine decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (4/10 neurons). The results suggest that SFO neurons in rats are activated postsynaptically through α1 adrenoceptors, and that the activation is enhanced by suppressing GABAergic inhibitory synaptic inputs through presynaptic α2 adrenoceptors. [Jpn J Physiol 55 Suppl:S143 (2005)]

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At the mossy fiber synapses of hippocampus activity-dependent induction of long-term potentiation (LTP) is dependent on cyclic AMP and protein kinase A (PKA). Activation of PKA turns on inactive synapses to active in hippocampal CA3-CA1 neuronal culture whereas it up-regulates the release probability of active synapses without changes the number of functional synapses in cultured hippocampal interneurons. Here we investigated if new synapses are formed along the mossy fiber axons during PKA activation. Transverse hippocampal slices were made at 400μm from a P14-21 mouse (C57BL/6). The granule cells were identified at the dentate gyrus and labeled with DiI. After incubation for 3 hours at 34°C, the 3-dimensional structure of a mossy fiber was examined at CA3 region under confocal microscopy. We found that the structure of mossy fiber and their terminals little changed in morphology during the experimental period of up to 2 hours. When slices were treated with forskolin (50μM) and 3-isobuthyl-1-methyl xanthine (IBMX, 100μM) for 20 minutes, synapse-like structures were newly formed as early as in 30 minutes. The number of synapse-like structures become about 3 times of the control in 120 minutes and the difference of their densities was statistically significant (P<0.01, Kolmogorov-Smirnov test). The structural changes were almost completely blocked by KT5720 (10μM), a selective PKA inhibitor. It is suggested that the early neurogenesis was induced by cAMP and the subsequent activation of PKA. [Jpn J Physiol 55 Suppl:S143 (2005)]

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Extracellular recordings were made from CA1 regions in the rat hippocampal slice tissues. Superfusion of slice preparations with various concentration (1–1000 μM) of lidocaine induced a transient inhibition and subsequent augmentation of the maximal slope of the field excitatory postsynaptic potentials (fEPSPs). High concetrations (300–1000 μM) of lidocaine suppressed the maximal slope of the fEPSPs. The amplitude of presynaptic volleies was simply suppressed by lidocaine in a conaentration-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 suppressed the amplitudes of the evoked fast EPSPs and, of the fast and late IPSPs dose-dependently; the IC₅₀ value for the fast EPSPs was 68 μM and, that for the fast IPSP was 7 μM. Neither exogenous glutamate-induced depolarization nor GABA-induced hyperpolarization was affected by the administration of lidocaine (30 or 300 μM). These results suggest that the lidocaine-induced transient inhibition of the fEPSPs is mediated by activation of A₁ receptors. The subsequent augmentation of the fEPSPs is due to the depression of the IPSPs by the low concentrations of lidocaine. [Jpn J Physiol 55 Suppl:S143 (2005)]

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Little is known about factors to control critical period for developmental plasticity. In vitro model of critical period for activity-dependent plasticity would be expected to make a substantial contribution to elucidate this problem. Corticospinal synapses were reconstructed in vitro by co-culturing the sensorimotor cortex and spinal cord slices of P0 Wister rats. Recordings of field EPSPs along 100μm-interval lattice in the spinal gray matter evoked by the stimulation of deep cortical layer allowed evaluation of spatial distribution of synapse formation quantitatively. Corticospinal synapses are formed diffusely throughout the spinal gray matter, but later the synapses in the ventral side are eliminated. This synapse elimination was blocked by APV, suggesting that this process is NMDA-dependent. Using this in vitro system, we made a detailed quantitative analysis on the time course of the sensitivity to APV. By changing the application period of APV, we found that 6-11DIV application is necessary and sufficient for a full and irreversible block of synapse elimination. Application of a specific blocker of NR2B containing NMDA-R, ifenprodil (10μM) during the critical period blocked the synapse elimination. However, a blocker of NR2A containing NMDA-R, NVP-AAM077 (0.4μM) showed no effect. These suggest that NMDA-dependent corticospinal synapse elimination depends on NR2B containing NMDA-R. It is possible that there is a shift of glutamate receptor subtype from NR2B to 2A or NR2B-NMDA-R to AMPA-R. This shift may explain the end of the critical period. [Jpn J Physiol 55 Suppl:S144 (2005)]

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In mammalian dorsal cochlear nucleus (DCN), principal cells receive synapses not only from auditory nerve fibers but also from parallel fibers, which consist of granule cells' axons and are known to convey non-auditory information. As in the cerebellum, granule cells have mutual synapses between inhibitory interneurons; Golgi cells. Cerebellar Golgi cells play important roles in motor coordination. So, Golgi cells in DCN may similarly play some roles in modulation of parallel fibers' activity. However, little is known about the properties of Golgi cells because of their small population. Since Golgi cells are known to express mGluR2, we used transgenic mice that express GFP under the control of mGluR2 promoter to facilitate identification of the neuron. By immunohistochemical studies against vesicular Ach transporter and GFP, we demonstrated a presence of some cholinergic projections on Golgi cells, therefore Golgi cells may be a target of cholinergic modulation. We further studied electro-physiological properties of the neuons, demonstrating that carbachol induced a hyperpolarization of the membrane, accompanied with a decrease in the input impedance in current-clamp recordings. Muscarine evoked similar responses, indicating an activation of mAchR. Under a voltage-clamp recording, I-V relation of muscarine-induced currents showed an inward rectification and the reversal potential of the muscarine sensitive current was close to E_K. These results may indicate that Golgi cells are suppressed through the activation of GIRK activated by mAchR. [Jpn J Physiol 55 Suppl:S144 (2005)]

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Previous studies have revealed that the stimulation of the rostral ventromedial medulla (RVM), including raphe magnus inhibits nociceptive transmission in the spinal doral horn and multiple neurotransmitters besides 5-HT are shown to be involved in the raphe-spinal system (RSS). To elucidate the inhibitory mechanisms of the RSS, we analyzed IPSCs of the substantia gelatinosa neurons (SG) in the adult rat spinal cord elicited by glutamate injection and electrical stimulation of the RVM (RVM-GI and RVM-ES) using in vivo patch-clamp technique. In the voltage-clamp mode (V_H, 0 mV), the RVM-GI and RVM-ES facilitated both of frequency and amplitude of IPSCs in the SG neurons. The RVM-ES evoked IPSCs were considered to be monosynaptic and conduction velocities of the evoked IPSCs were calculated as over 3 m/sec (3.1-20.7 m/s), suggesting that the IPSCs were mediated by myelinated fibers. Furthermore, the evoked IPSCs had either long or short decay time courses and were blocked by bicuculline and strychnine, respectively, but not by 5-HT antagonists. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in 63% of the neurons tested (10/16). These findings suggest that the RSS includes the direct GABAergic and glycinergic descending inputs to the SG. And these non-serotonergic descending inhibitory systems may exert distinct modulatory effects on the excitatory noxious transmission in the spinal dorsal horn. [Jpn J Physiol 55 Suppl:S144 (2005)]

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GABA_A receptor-mediated responses are often depolarizing in immature neurons, which was mainly due to a high [Cl⁻]_i. Therefore Cl⁻ transporters that change [Cl⁻]_i may affect synaptic responses and synaptic development of immature neurons. To investigate the roles of Cl⁻ transporters in synaptic development, we cultured rat neocortical neurons for 2 weeks in the presence of NKCC (Na⁺-K⁺-Cl⁻ cotransporter) inhibitor bumetanide and examined the sEPSCs and sIPSCs.Chronic treatment with 10 μM bumetanide during cultivation decreased the soma size of neurons. The frequency of sIPSC but not of sEPSC decreased after chronic treatment with bumetanide. These results suggest that cation-chloride cotransporters such as NKCC affect synaptic activities and synaptic development presumably by attenuating depolarized GABA responses. [Jpn J Physiol 55 Suppl:S144 (2005)]

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We have previously reported Cre/loxP recombination system which enables us region-specific expression of synaptopHluorin (SpH)¹, one of green fluorescent protein-based sensors for vesicular exocytosis. Here we report another series of SpH transgenic lines in which SpH expression was regulated by the neuron specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in broad spectrum of brain regions in 4 of them whereas in others it was expressed in the specific regions of hippocampus and cerebellum. SpH was preferentially expressed in the presynaptic elements of neurons. The SpH fluorescent intensity was somewhat decreased by acidic buffer surperfusion and greatly increased by vesicular neutralization of pH, indicating that they are mainly distributed in the synaptic vesicles. SpH fluorescence was transiently increased by neuronal activation. These transgenic lines are suggested to be useful for the morphological and physiological studies of presynaptic terminals in the brain.1. Araki, R. et al. Neuroscience Research 50S1 (2004) S124. [Jpn J Physiol 55 Suppl:S145 (2005)]

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It has been reported that an anandamide (endocannabinoid)-transport inhibitor AM404 activates cloned vanilloid TRPV1 (which is also known as VR1) receptors. In order to address this issue in the spinal dorsal horn, we examined the effect of AM404 (30μM) on spontaneous glutamatergic excitatory transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by use of the whole-cell patch-clamp technique. In 84% of the SG neurons examined (n = 44), AM404 reversibly increased the frequency of spontaneous excitatory postsynaptic current (sEPSC; by 68 ± 7% at 2 min after the beginning of AM404 superfusion) in a manner similar to that of capsaicin. AM404 was without actions in the presence of a TRPV1 receptor antagonist capsazepine (10μM). On the other hand, sEPSC amplitude and also a response of SG neurons to AMPA (5μM) were not affected by AM404. These results indicate that AM404 enhances the spontaneous release of L-glutamate by activating TRPV1 receptors in the SG. We conclude that when AM404 is used as an anandamide-transport inhibitor in the SG, its TRPV1 receptor activation also have to be taken into consideration. [Jpn J Physiol 55 Suppl:S145 (2005)]

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GABA has been believed to be a sole transmitter in spinal presynaptic inhibition. Recently, transmitter switching was reported in the lower central nervous system. That is, transmitter for IPSP was initially GABAergic, and was changed to be glycinergic during early development. This experiment was planned to elucidate the possibility of this transmitter switching in primary afferent depolarization ( PAD ), which is closely related to presynaptic inhibition. In the isolated spinal cord preparation from newborn rats, the dorsal root ( L4 or L5 ) was split into two or three rootlets. Using suction electrodes, one rootlet was stimulated electrically and PAD was recorded from the adjacent rootlet. PAD consisted of fast and slow portions. The slow PAD was usually hidden, and appeared only when bicuculline or ( /and ) strychnine was applied. The fast PAD was glycinergic and GABAergic, and the slow PAD was glutaminergic. In this report, the fast PAD will be discussed. In 0-day-old rat, strychnine had little effect, and additional bicuculline eliminated fast PAD. In 7-day-old rat, strychnine enhanced fast PAD, and additional bicuculline eliminated it. In older rats, strychnine depressed fast PAD to a little extent, and additional bicuculline eliminated it. These results suggested that PAD at birth was dependent predominantly on GABA, and that glycinergic component increased until 7th day. In older age, glycinergic component decreased. It is concluded that a part of transmitter in related to PAD, changed from GABA to glycine, but this change is only transient in the course of development. [Jpn J Physiol 55 Suppl:S145 (2005)]

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Application of either serotonin (5-HT) or dopamine (DA) induces a slow Na⁺-current response in the identified neurons of Aplysia ganglia under voltage clamp. We previously reported that this type of response is produced by the activation of cholera toxin-sensitive G-protein. The Na⁺-current response to 5-HT or DA was gradually and irreversibly depressed after intracellular injection of Clostridium difficile toxin A or toxin B, which are known to mono-glucosylate and inactivate all Rho family G-proteins RhoA-C, Rac and Cdc42. Furthermore, intracellular injection of Clostridium botulinum exoenzyme C3, which is known to specifically ADP-ribosylate the monomeric G-protein, RhoA-C, and uncouple their effectors, irreversibly depressed both the 5-HT- and DA-induced responses. In contrast, intracellular injection of L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane. Application of active form of Rac, or Cdc42 in the Rho-family G-protein did not have any effect either on the 5-HT- and DA-induced current responses or on the resting membrane. However, intracellular application of inhibitors for Rho-dependent kinase did not have any significant effect at all. These results suggest that both the 5-HT- and DA-induced Na⁺-current responses may be commonly facilitated by the activation of monomeric G-protein RhoA. [Jpn J Physiol 55 Suppl:S145 (2005)]

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Sensory neural inputs during developmental period from outer world are important for maturation of brain functions. Paying attention to oral sensory system, sensory inputs from teeth and tooth-related organs may also be important for maturation of oral sensory system. Here we investigated whether multiple tooth losses during the developmental period disturb age-dependent appearance of the network oscillation in the neocortex, by applying caffeine to rat brain slices. Electrical stimulation was delivered to the white matter and field potentials were recorded from layer II/III. In control animals, synchronized population oscillation at the frequency of alpha-range appeared during postnatal weeks 2 to 3, and was observed in mature periods in both oral somatosensory cortex and visual cortex. In multiple tooth loss rats, in contrast, the oscillation appeared during postnatal weeks 2 to 3 in the visual cortex, but did not appear in the oral somatosensory cortex even in mature periods. Induction and maintenance of the oscillation were dependent on NMDA receptor activities. Thus, sufficient neural information from the teeth may be important for development of neural activities in the oral somatosensory cortex, suggesting that the brain has the critical period for tooth-related sensory system. [Jpn J Physiol 55 Suppl:S146 (2005)]

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Although activity-dependent changes in synaptic strength as a cellular substrate for leaning and memory have been extensively studied, activity-dependent maintenance of basal synaptic strength is not well understood. We examined the effect of chronic in vivo inhibition of synaptic inputs on the strength of parallel fiber (PF)-Purkinje cell (PC) synapses in the adult mouse cerebellar cortex. To suppress the PF activity, the NMDA-type glutamate receptors on cerebellar granule cells was blocked by continuous infusion of APV from an ethylene-vinyl acetate copolymer implant placed over the cerebellar cortex on postnatal day 23. Three to seven days later, PF-evoked excitatory post-synaptic currents (EPSCs) were recorded from PCs in acute slices. The chronic APV application resulted in increase in the paired-pulse ratio and the coefficient of variation of EPSC amplitude, suggesting a decrease in the probability of neurotransmitter release from PFs. On the other hand, the postsynaptic sensitivity to transmitters seemed unchanged. Furthermore, persistent suppression of one of the postsynaptic signaling pathways, namely the type-1 metabotropic glutamate receptor (mGluR1)-IP₃ signaling pathway, also decreased the probability of transmitter release from PFs. These results suggest that the strength of PF-PC synapses is presynaptically maintained by the PF activity, and that the mGluR-IP₃ signaling pathway in PCs is involved in a retrograde regulation of the presynaptic function. [Jpn J Physiol 55 Suppl:S146 (2005)]

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In the present study we examined the expression of neurotransmitter receptors on human astrocytoma cell lines, U251-MG, U87-MG, and KINGS-1. Those cells were loaded with fura-2 AM, and subjected to Ca²⁺ imaging using a specific imaging device. The cells which showed the increase in [Ca²⁺]_i during the administration of a neurotransmitter were determined as positive. We tested the effects of histamine, acetylcholine, noradrenaline, dopamine, GABA, serotonin, glutamate, and ATP on each cell line. Those cells showed the significant increase in [Ca²⁺]_i during the administration of histamine, acetylcholine, noradrenaline, and ATP, but each cell line showed characteristic Ca²⁺ responses to those agonists. The amplitude of Ca response was dose-dependent (10^–6∼10^–3 M), and significantly blocked by each specific antagonist. Surprisingly, none of those cells responded to glutamate (100 μM), which had been reported to be an important mediator between glial and neuronal cells, cause significant increase in [Ca²⁺]_i. Since recent studies on astrocytes have demonstrated the important participation of those cells on regulation of synaptic transmission, the expressed neurotransmitter receptors on those cells and the regulatory systems for expression of those receptors will provide us important clue for elucidating the glia-neuron inter-communication. [Jpn J Physiol 55 Suppl:S146 (2005)]

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Small molecule G-protein Arf family has a major role in regulation of intracellular trafficking of the proteins from Golgi apparatus to endoplasmic reticulum. However, it has recently been reported that it also regulates the activity of ionic channels in epithelial cells. To clarify possible involvement of Arf in G-protein coupled receptor (GPCR)-induced current responses, we examined the effects of several reagents specific to Arf1 or Arf6 on the K⁺- current responses induced by dopamine (DA) and Phe-Met-Arg-Phe-NH₂ (FMRFamide) in the ganglion cells of Aplysia by using conventional two-electrode voltage clamp method. Intracellular injection of Brefeldin A, a specific blocker of class I Arf, significantly depressed the K⁺-current responses to both DA and FMRFamide without affecting the Na⁺-current responses to acetylcholine in the same cell. The K⁺-current responses to DA and FMRFamide were also depressed by intracellular injection of 2-(4-fluorobenzoilamino)-benzoic methyl ester (Exo1), which is known to facilitate the GTPase activity for Arf1. Intracellular application of N-terminal peptide of Arf1, a competitive inhibitor of Arf1, markedly depressed the both K⁺-current responses. In contrast, injection of similar N-terminal peptide of Arf6 had no significant effect on the responses. These results suggest that Arf1 may commonly facilitate the production of the K⁺-current responses to both DA and FMRFamide. [Jpn J Physiol 55 Suppl:S146 (2005)]

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We previously reported that, when continued for 15 minutes, 2 Hz stimulation induced NMDA receptor-independent LTP at excitatory synapses in rat visual cortex. This LTP occurred during the critical period for visual response plasticity, but not in adulthood. LTP induction was blocked by 50 μM Ni²⁺, suggesting that it requires T or R type Ca²⁺ channel activation. To determine which one of these channels is responsible for the induction, we used blockers for T and R type Ca²⁺ channels. Extracellular field potentials evoked by layer 4 stimulation were recorded from layer 2/3 in visual cortical slices prepared from rats at postnatal 21-30 days. LTP was blocked by 3 μM Mibefradil, which blocks T type Ca²⁺ channels potently although it also affects some other channels including R type Ca²⁺ channels. Kurtoxin (250 nM), which blocks T type, but not R type, Ca²⁺ channels, also abolished LTP induction. In contrast, in the presence of a high dose (500 nM) of the R type Ca²⁺ channel blocker SNX-482, LTP occurred in the same way as in control. In addition, we recorded Ca²⁺ channel currents from layer 2/3 pyramidal cells under a pharmacological blockade of P/Q, N and L type Ca²⁺ channels. Ca²⁺ currents evoked by voltage steps from -100 to -40 mV were sensitive to 50 μM Ni²⁺ but not 10 μM Cd²⁺, indicating that they were generated by T type Ca²⁺ channels. We found that Ni²⁺-sensitive Ca²⁺ currents decreased developmentally in parallel with LTP incidence. These results suggest that T type Ca²⁺ channels are responsible for the induction of NMDA receptor-independent LTP. [Jpn J Physiol 55 Suppl:S147 (2005)]

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To clarify modulatory synaptic transmission in autonomic neurons in the brain, neurons in slices(200 μm in thickness) of the area postrema (AP) from rats (aged 12-24 days) were studied using whole-cell patch clamp techniques. When glycine (100 μM) or GABA (10 μM) was applied to the AP neurons from a ‘Y-tube’, large outward currents were induced (average peak amplitudes of about 230 pA and 170 pA at -10 mV, respectively). Both of the currents showed reversal potential of -67 mV, which was approximately the estimated Cl⁻ equilibrium potential. At a holding potential of -10 mV, application of high K⁺ (20 mM) to the AP neurons evoked massive inhibitory synaptic currents (IPSCs) in the neurons. Most of the evoked currents were sensitive to bicuculline but resistant to strychnine, indicating that GABAergic but not glycinergic transmissions were prevalent in AP neurons. When nicotine (5-100 μM) was applied to AP neurons, robust IPSCs were evoked, indicating GABAergic identity. Interestingly, a fraction of nicotine-induced IPSCs still appeared in the presence of tetorodotoxin (1 μM) but were blocked in the presence of 5 mM Mg²⁺, indicating that functional nicotinic ACh receptors were present at GABAergic presynaptic terminals and induced Ca²⁺ influx after activation of the receptors. By application of cytisine, DMPP and ACh around an AP neuron, similar presynaptic facilitation of GABA release was observed. The observations revealed that presynaptic facilitation of GABA release by nicotine occurs in AP neurons of the rat. [Jpn J Physiol 55 Suppl:S147 (2005)]

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We have examined the effects of peripheral administration of cannabinoids in the paraventricular (PVN) and the supraoptic nuclei (SON) of rats. Intraperitoneal (Ip) administration of CP 55,940 (CB1 and 2 agonist), anandamide (CB1 and 2 agonist) and noladin ether (selective CB1 agonist) caused a marked induction of the c-fos gene expression in the PVN and the SON. These effects were dose-dependent. Plasma corticosterone and oxytocin levels were significantly increased by ip administration of cannabinoids, but arginine vasopressin level was not changed. Furthermore, to determine the process of modulating activities of the SON neurons via CB-1 receptors, we have investigated the effects of CP 55,940 on excitatory and inhibitory synaptic transmission in the rat SON. Whole-cell patch clamp recordings were performed from magnocellular neurons in the SON in in vitro brain slice preparation. CP 55,940 significantly reduced both excitatory and inhibitory postsynaptic currents, and these changes were potently reversed by subsequent application of AM 251 (CB1 antagonist). These results suggest that cannabinoids may be involved in the regulation of the neuronal activity in the PVN and the SON, and at least in the SON, modulate the activity of the neurosecretory cells via presynaptic inhibition of excitatory and inhibitory synaptic transmission. [Jpn J Physiol 55 Suppl:S147 (2005)]

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The synaptic vesicles keep recycling by the processes of endocytosis and exocytosis to maintain the normal synaptic transmission. The synaptic vesicles are classified as the readily releasable pool (RRP) and the reserve pool (RP). In the endocytosis process, calcineurin (CaN), a Ca²⁺/calmodulin-dependent protein phosphatase, has been shown to play important roles. However, it is unclear about its roles in different vesicle pools. Here, we investigated the role of CaN in the regulation of vesicle recycling in the RRP and RP. Vesicle recycling was monitored by using fluorescent dyes FM1-43 and FM4-64 in the primary cultures of hippocampal neurons. Inhibition of CaN by FK506 and cyclosporin A suppressed the endocytosis in the RP, but not in the RRP. Inhibition of CaN also restrained the exocytic process triggered by 10 Hz stimulation, but had no effect on 3-5 Hz stimulation-induced exocytosis. FK506 also reduced the total vesicle pool size in the synaptic terminals. A synthesized CaN inhibitory peptide showed the similar effects as FK506 and cyclosporin A. These results revealed a novel mechanism that CaN plays critical roles in the distinct vesicle recycling processes. [Jpn J Physiol 55 Suppl:S147 (2005)]

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Quantal packets of transmitter do not normally saturate postsynaptic AMPA receptors at the calyx of Held, a fast relay synapse in the auditory brainstem, of mature rats (postnatal day (P) 13-29; Ishikawa et al., 2002; Yamashita et al., 2003). However, this may not be the case at the immature calyx of Held (P7), where an increase in extracellular Ca²⁺ concentration does not increase the amplitude of evoked EPSCs. To test this possibility, we infused transmitter L-glutamate into the nerve terminal through whole-cell patch pipettes, and examined its effect on quantal and evoked EPSCs at the immature calyx of Held (P7-9). The endogenous glutamate concentration in the presynaptic terminal, estimated by matching quantal size between calyceal terminals with or without loading L-glutamate, was 1-10 mM, similar to the mature calyx of Held. Infusion of 100 mM L-glutamate into the calyceal terminal increased the mean amplitude of quantal EPSCs, but had no effect on the amplitude of evoked EPSCs. When the release probability was lowered by reducing extracellular Ca²⁺ concentration, decay time of evoked EPSCs became faster, indicating that multiple quanta are released at a single release site. We conclude that a single quantal packet of transmitter does not saturate postsynaptic AMPA receptors, whereas simultaneously released multiple packets of transmitter overlap and eventually saturate AMPA receptors at the immature calyx of Held. [Jpn J Physiol 55 Suppl:S148 (2005)]

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Mice homozygous for targeted disruption of the α1A subunit of the voltage gated Ca channel (PQKO) show fatal ataxia and dystonia. We investigated properties of the cerebellar Purkinje cell (PC) in PQKO (P14-20) using acute cerebellar slices. PCs were patch-clamped at whole-cell mode and electrophysiologic/Ca imaging studies were performed in response to synaptic stimuli and somatic depolarization as well.In comparison with wild type littermates (WT), the magnitude of Ca transient (CaT) in PQKO PC induced by a train of parallel fiber stimuli (PF) was much smaller, whereas CaT evoked by climbing fiber (CF) was as large as that in WTs.Pharmacological studies revealed that compared to WT PF- and CF-EPSC in PQKO were more sensitive to w-conotoxin, an N-type Ca channel blocker, whereas deporalization-induced dendritic CaT was more sensitive to low-dose Ni²⁺, a T-type channel blocker. Overall, contribution of L-type channel was very little in PQKO and WT. These data suggest that the defect in P/Q-type Ca channel is not well conpensated at least in part, resulting in compromised Ca signaling in PC dendrites, and that the compensation mechanism is different between pre- and post synaptic sites in PCs. [Jpn J Physiol 55 Suppl:S148 (2005)]

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