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. We have shown that the function of Amphiphysin I is regulated by Cdk5- and calcineurin-dependent phosphorylation and dephosphorylation. Cdk5-dependent phosphorylation of the protein inhibits the association with the binding proteins such as β-adaptin. 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 treatment with ALLM, a potent calpain inhibitor. The high K+ stimulation also induced the cleavage of α-spectrin, a physiological substrate of calpain. Treatment with FK506, a potent calcineurin inhibitor, increased the extent of the KCl-induced cleavages of Amphiphysin I in the hippocampal slices. In contrast, treatment with roscovitine, a Cdk5 inhibitor, inhibited the cleavages. These results suggest that Amphiphysin I may be a substrate of calpain in presynaptic terminals and the cleavages may be regulated by the phosphorylation states. And the calpain-induced cleavages of Amphiphysin might be an important step to prevent neuron from abnormal excitement caused by high K stimulus. [Jpn J Physiol 55 Suppl:S148 (2005)]

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Tramadol is a clinically-used, orally-active drug which is considered to act as an analgesic in the CNS. In order to provide a cellular basis for this action, we examined a current response induced by a tramadol metabolite, mono-O-dimethyl-tramadol (M1), in substantia gelatinosa neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 41% of the neurons examined, superfusing M1 induced an outward current at a holding potential of -70 mV. M1 current hardly declined and persisted for > 30 min after its washout. The M1 current reversed its polarity at a potential which is close to the equilibrium potential for K⁺. This M1 current correlated in amplitude with current produced by μ-opioid receptor agonist DAMGO in the same neuron, and suppressed in amplitude in the presence of μ-opioid receptor antagonist CTAP but not α₂-adorenoceptor antagonist yohimbine. The amplitude of the M1 response, relative to that of the DAMGO response, exhibited an EC₅₀ value of 300 μM. We conclude that M1 produces a persistent hyperpolarization by activating μ-opioid receptors in adult rat substantia gelatinosa neurons. This could contribute to at least a part of pain alleviation produced by tramadol. [Jpn J Physiol 55 Suppl:S148 (2005)]

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Recent studies demonstrated that chloride efflux via anion channels is involved in progression of apoptosis in various cell types. We have recently shown that the volume-sensitive outwardly rectifying (VSOR) chloride channel serves as the apoptotic chloride efflux pathway in human epithelial cells (PNAS 101, 6770, 2004). In the present study, we tested the neuroprotective effect of VSOR channel blockers on delayed neuronal cell death induced by transient forebrain ischemia. Whole-cell recordings revealed that cultured hippocampal neurons respond to osmotic swelling with activation of outwardly rectifying chloride current, which is sensitive to DIDS (500 μM) and genistein (100 μM), but not to daidzein (100 μM). Protective effects of these blockers on delayed neuronal cell death were examined in mice subjected to transient common carotid artery occlusion (12 min) by histological assessments. Pre-administration of DIDS (0.48, 12, 60 mg/kg) and genistein (0.24, 6.0, 30 mg/kg) via jugular vein followed by intraperitoneal administrations (once per day) after reperfusion reduced the number of damaged neurons in the CA1 region of hippocampus and cytochrome c release therefrom. These results suggest that VSOR chloride channels are involved in induction of delayed cell death in hippocampal neurons after transient forebrain ischemia and that blockers of this channel may provide a therapeutic target for the treatment of stroke. [Jpn J Physiol 55 Suppl:S149 (2005)]

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The intermediate gray layer (SGI) of the superior colliculus (SC) send descending motor command for orienting behaviors. SGI neurons receive cholinergic inputs from the midbrain cholinergic nuclei. In rats, we reported that cholinergic stimulation induced mostly fast inward + slow inward currents or fast inward + slow inward-outward currents in SGI neurons. Fast inward current is mediated by nicotinic receptors and both slow inward and slow outward currents by muscarinic receptors. We report the cholinergic responses of identified GABAergic SGI neurons in GAD67-GFP knock-in mice (17-24 days old) by whole-cell recordings in SC slices. Current responses to 30 μM carbachol were investigated in the presence of TTX. The most common response pattern was fast inward current alone (26/54), found mainly in fast spiking GABAergic neurons (25/29). The second common pattern was fast inward + slow inward current (21/54), found in burst spiking (18/22), late spiking (2/2) and fast spiking (1/29) GABAergic neurons. These results suggest that the cholinergic inputs may exert facilitatory effects on SGI GABAergic neurons in the mouse SC. [Jpn J Physiol 55 Suppl:S149 (2005)]

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GLAST and GLT-1 are glial glutamate transporters that regulate the concentration of glutamate in the synaptic cleft. In the Bergmann glial processes ensheathing excitatory synapses on Purkinje cells in the cerebellum, GLAST is expressed 6 times more abundantly than GLT-1. Unexpectedly, no change was detected in the kinetics of EPSCs evoked by climbing fiber (CF) stimulation in cerebellar slices in GLAST-deficient mice as compared with those in the wild-type mice (WT). This suggested that GLT-1 contributed to the uptake of glutamate in CF-Purkinje cell synapses. Here we aimed to clarify the role of GLT-1 in both WT and GLAST-deficient mice using dihydrokainate (DHK), a selective GLT-1 blocker. DHK caused no change in the kinetics of CF-EPSCs in WT. In the presence of cyclothiazide (CTZ), that not only reduces the desensitization of AMPA receptors but also increases the affinity of glutamate to the receptors, however, DHK significantly prolonged the decay time constant of CF-EPSCs in WT. Although there was no change in the decay time constant of CF-EPSCs between WT and GLAST-deficient mice in the normal saline, it was significantly longer in the mutant mice in the presence of CTZ. Taken together, these results indicate that GLT-1 plays a partial role in the uptake of glutamate in WT, and that it compensates a large portion of the function of GLAST in GLAST-deficient mice although the compensation is incomplete. The GLAST-deficient mice were kindly provided by Prof. Kohichi Tanaka. [Jpn J Physiol 55 Suppl:S149 (2005)]

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Long-term depression (LTD) of the parallel fiber (PF)–Purkinje cell (PC) synapse is an underlying mechanism by which motor coordination and learning are regulated. Elevation of cytoplasmic calcium in the PC dendrites is a critical event for LTD induction. To investigate the relationship between the calcium signal pattern and the LTD induction, we performed calcium imaging from PCs in acute cerebellar slices. LTD was induced by ten trains of 50Hz x 5 bursts of parallel fiber stimuli at 1Hz in PCs from 2 weeks old mice, but not from 4 weeks old mice. This PF short burst stimuli (50Hz x 5) produced a biphasic calcium transient in dendrites of PCs, first peak of which reflects calcium influx and second one does calcium release from intracellular calcium stores. By comparing amplitudes of the first and second calcium transients, we found a correlation between calcium release activity and LTD amplitude in PCs from 2 weeks old mice. This finding unveils an importance of calcium release especially in synaptic maturation in the developing cerebellar cortex. [Jpn J Physiol 55 Suppl:S149 (2005)]

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We examined the effects of temperature increase on the resting membrane potential (RMP) and the excitatory postsynaptic potential (EPSP) evoked by stimulation of Schaffer collaterals in rat hippocampal CA1 neurons in vitro. The temperature of the external solution of the rat hippocampal slice was initially kept at 36°C for 30min. The mean RMP was -67.4±1.2mV (n=13) at 36°C. The increase of the temperature from 36 to 40°C for 5∼8 min produced a membrane hyperpolarization (3.2±0.8mV, n=8) associated with a decrease in input resistance. After returning the temperature to 36°C for 20min, the RMP recovered. The EPSP was reversibly depressed to 40% of the control by the temperature increase from 36 to 40°C for 5∼8 min. Consecutive exposure of high temperature (40°C) hyperpolarized neurons more promptly than that obtained by the first exposure to 40°C, and also depressed the EPSP. When the temperature was maintained at 40°C, a rapid and large depolarization to nearly 0mV occurred in about 50∼90 min. A rapid and large depolarization was produced within 10min by the temperature increase from 36 to 42°C. The EPSP was completely depressed immediately with the onset of the large depolarization. The RMP and the EPSP hardly recovered by returning the temperature to 36°C, after the onset of the large depolarization. These results suggest that high temperature rapidly depresses the excitatory synaptic transmission and that long-lasting high temperature may insult the membrane structures of CA1 neurons by an unknown mechanism. [Jpn J Physiol 55 Suppl:S150 (2005)]

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Membrane traffic is one of the important functions in a variety of animal cells, which includes vesicular transportation, secretion and morphologic transformation of organelles. To investigate roles of the membrane traffic on the neuronal functions, we analyzed pharmacological effects of Brefeldin-A, a drug which is known to disassemble Golgi apparatus, on synaptic responses in the CNS neurons. Hippocampal slices were prepared from 3-5 weeks old mice, and field EPSPs (fEPSPs) were recorded from stratum radiatum of the CA1 region. Brefeldin-A was solved into methanol, and then into normal ACSF at a final concentration (0.1-1μg/ml). The drug solution was continuously applied 10 min during a record session. Five to fifteen minutes after application, maximum slope of fEPSP transiently decreased to 50-70% and then increased to 150-200% of control. This augmentation maintained at least 2 hrs in all slices examined, and was inhibited in the presence of BAPTA-AM, an intracellular Ca²⁺ chelator. After drug application, paired-pulse facilitation ratio of the fEPSP was decreased because the slope of first fEPSP was increased although the second one did not increase. These results suggest that Brefeldin-A enhance excitatory synaptic transmission in the hippocampal CA1 region. An increase in intracellular Ca²⁺ at pre- and/or postsynaptic sites is assumed to be a factor in the induction of potentiation. [Jpn J Physiol 55 Suppl:S150 (2005)]

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Cerebellar Purkinje cells highly express B-type G-protein-coupled γ-aminobutyric acid receptors (GABA_BR). However, the functional contribution of GABA_BR is not fully elucidated in this cell type. Here we identified a GABA_BR agonist (baclofen)-induced inwardly rectifying current (I_bacl) in cultured mouse Purkinje cells, using a ruptured-patch whole-cell technique. I_bacl is coupled to GABA_BR via G_i/o-proteins as it is not inducible in pertussis toxin-treated cells. I_bacl is carried by K⁺ but not Na⁺. I_bacl is blocked by Ba²⁺, Cs⁺, and tertiapin-Q. I_bacl is activated rapidly upon hyperpolarization with a double-exponential time-course (time constants, 0.8 and 80 ms). These results indicate that I_bacl is mediated by G-protein-coupled inwardly rectifying K⁺ channels. Perforated-patch recordings demonstrate that I_bacl hyperpolarizes the resting potential and the peak level of glutamate-evoked potentials initiated in the dendrites. Moreover, cell-attached recordings in cerebellar slices demonstrate that I_bacl slows down the rate of spontaneous firing. I_bacl activation via GABA_BR reduces the postsynaptic and intrinsic excitability of Purkinjes and may thereby influence the integration of motor information conveyed by excitatory parallel and climbing fibers. [Jpn J Physiol 55 Suppl:S150 (2005)]

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Cajal-Retzius cells in marginal zone (MZ) play important roles in the construction of the cerebral cortex. Details of synaptic transmission in MZ are yet to be elucidated. Several reports have proved excitatory GABAergic neurotransmission in the MZ. Tangential slices were made from MZ of postnatal 0-3 rats and optical imaging using voltage-sensitive dye (JPW1114) was performed. Action potentials evoked by single electrical stimulations spread out radially over MZ. The spread of excitation were completely blocked by TTX and Ca²⁺-free. They were not affected by glutamate receptor blockers (AP5, CNQX). On the other hand, they were inhibited by either GABA_A receptor antagonists (bicuculline, picrotoxin) or glycine receptor antagonist (strychnine). Co-application of GABA_A and glycine receptor blockers completely abolished evoked EPSPs and spread of excitation. A Na⁺, K⁺-2Cl⁻ cotransporter inhibitor (bumetanide) also reduced the spread of excitation. By using the microdialysis with HPLC analysis, we have proved that taurine, a glycine receptor agonist, but not glycine was released by stimulation and that TTX reduced the basal level of taurine. The results indicate that the excitatory action of taurine based on high [Cl⁻]_i plays an important role in neurotransmission in the MZ. [Jpn J Physiol 55 Suppl:S150 (2005)]

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DBA/2 mice exhibit audiogenic seizures in response to strong sound stimuli, although the underlying cellular mechanisms are unknown. In the present study, we investigated cortical activities during strong sound stimuli in DBA/2 mice using flavoprotein autofluorescence imaging. DBA/2 mice (4–6 weeks old) were anesthetized by urethane. The skin was incised and the skull was exposed. The surface of the skull was covered with clear acrylic resin for keeping the skull transparent. Three days after the operation, the skull of the mice recovered from the operation were fixed under an epifluorescence binocular microscope with a CCD camera, and cortical activities during sound stimuli at 13 kHz for 60 s were visualized as autofluorescence changes. Electrocorticogram (ECoG) was also recorded. Autofluorescence responses were observed in the sensorimotor areas approximately 40 s after the onset of the sound stimuli. The maximal amplitude of lambdaF/F was lager than 5.0% and lasted for more than 90 s. Epileptic discharges recorded in ECoG were observed only in the sensorimotor areas, in which autofluorescence responses were observed. The present results suggest that epileptic foci for audiogenic seizures may be present in the sensorimotor cortex of DBA/2 mice. [Jpn J Physiol 55 Suppl:S151 (2005)]

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In the development of cerebral cortex, cells derived from the ventricular zone (VZ) migrate in radial direction (radial migration), and form cortical plate (CP). It is well known that role of GABA is important for early CNS development. The radial migration may also be affected by such GABA actions. In GAD67-GFP knock-in mouse, the GABA contents in the brain decrease compared with the wild type. Therefore, in this study, we investigate the influence of the circumferential GABA concentration by using GAD67-GFP knock-in mice. To identify the cells in VZ at E14, HcRed was transfected into VZcells by means of in uteroelectroporation. Three days after the electroporation (E17), HcRed-labeledcells located in CP, intermediate zone, and VZ were counted. There was no significantdifference in distribution among genotypes. GABA_A-R mediated currentswere recorded by whole-cell recording from HcRed-expressingneurons at three days after the electroporation.Although spontaneous GABA currents were not observed, when GABA was apllied, GABA_A-R mediated current were evoked ina dose-dependent manner. There were no differences in dose-response propertiesof GABA-evoked currents among genotypes. These results suggest that the cells generatedin the VZ possess rather equivalent GABA_A-Rs and migrate radially independent of circumferential GABA concentration. [Jpn J Physiol 55 Suppl:S151 (2005)]

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Synaptic plasticity such as long-term potentiation (LTP) or long-term depression (LTD) is the cellular mechanism underlying learning and memory. In sensory cortices, cortical plasticity has also an important role in the development of neural circuits. However, the differential roles and mechanisms for various types of LTP and LTD are not clear. In the present study, we investigated supragranular LTP and two types of supragranular LTD in the slices obtained from the rat auditory cortex, and compared their properties. Frontal cortical slices (400 μm thick) were prepared from male Wistar rats (4-6 weeks old). Supragranular field potentials elicited by the stimulation applied to layer IV-V were recorded through a metal electrode. Trans-synaptic components sensitive to glutamate receptor antagonists exhibited marked LTP after tetanic stimulation (100 Hz for 1 s) applied to layer IV-VI. The same field potential components exhibited LTD after low-frequency stimulation (1 Hz for 900 s) applied to layer VI. LTD of supragranular field potentials were also induced by tetanic stimulation (100 Hz for 1 s) applied to supragranular layers near the recording site. Induction of LTP and the two types of LTD was completely abolished in the presence of 50 μM APV, an NMDA receptor antagonist, indicating that the induction of these three types of supragranular plasticity is dependent on NMDA receptors. [Jpn J Physiol 55 Suppl:S151 (2005)]

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The mechanism underlying a presynaptic GABA_B receptor-mediated inhibition of transmitter release has been characterized for a variety of synapses in the central nervous system. These studies have suggested a range of transduction mechanisms, including the activation of adenylate cyclase and the closure of K⁺ and Ca²⁺ channels via G-coupled proteins. We have examined the intracellular signaling pathways underlying baclofen-induced inhibition of transmitter release in autaptic synapses of isolated mouse hippocampal neurons by using patch-clamp recording methods. Baclofen, a selective GABA_B receptor agonist, reversibly decreased both the amplitude of evoked postsynaptic currents (ePSCs) and the frequency of spontaneously occurring miniature postsynaptic currents (mPSCs). During the inhibition of mPSCs frequency, the mean amplitude of mPSCs did not show any significant alternations. These observations suggest that baclofen inhibits synaptic transmission by presynaptic mechanism. To clarify the intracellular mechanisms of the observed presynaptic inhibition by baclofen, the effects of Ca²⁺-free extracellular solution were studied. In the absence of extracellular Ca²⁺, baclofen blocked ePSC and reduced the frequency of mPSCs. The results might suggest that baclofen inhibits neurotransmitter release downstream of Ca²⁺ entry and may directly affect the exocytotic machinery at the presynaptic terminal. [Jpn J Physiol 55 Suppl:S151 (2005)]

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AMPA type glutamate receptor (AMPA-R) trafficking is thought to be one of the underlying bases of synaptic plasticity in cerebellum, however, detailed characteristics of AMPA-R trafficking has not yet been revealed. We have reported several characteristics of constitutive endocytosis of AMPA receptors in parallel fiber (PF) synapse in cerebellar Purkinje cell (PC) by means of Tetanus Toxin (TeTX) infusion. Recent studies have reported that activity of PF-PC synapse shows bidirectional plasticity, LTP or LTD, through the postsynaptic Ca²⁺ concentration, and that Nitric oxide (NO) produces induction for LTP in PF-PC synapse postsynaptically, through cGMP-PKG independent pathways. We therefore examined the role of NO on constitutive trafficking of AMPA-R in cerebellar Purkinje cell. Whole-cell voltage clamp recordings were made from a Purkinje cell held at -70 mV in cerebellar sagittal slice (300 μm). Bath-application of NO donor, both NOC-7 (5 μM, half life = 5 min) and NOR3 (30 μM, half life = 30 min), induced 50-60% potentiation of PF-evoked EPSCs amplitude. Intracellular infusion of TeTX suppressed this potentiation of PF-evoked EPSCs amplitude. These results suggest that NO-induced potentiation of PF-evoked EPSCs amplitude is caused by enhancement of surface-expressed AMPA-R number through exocytosis. [Jpn J Physiol 55 Suppl:S152 (2005)]

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Fluctuation in the amplitude of a synaptic response were first reported at the neuromuscular junction (NMJ) and then these phenomenon were observed at the synapses in the central nervous system (CNS). These fluctuations were led to the ‘quantal’ hypothesis of transmitter release and has been used for many studies of synaptic function and plasticity. On the other hand, nonstationary noise analysis were developed to estimate the single channel conductances both in the excitable and postsynaptic membrane of the neurons. Furthermore, this analysis for estimating single channel conductances has been extended to the amplitude-variance of synaptic responses around the mean-value of synaptic currents. The experimental, graphical and analytical approach, which is less sensitive to recording noise than previous techniques, was developed by others under fewer assumptions. We also developed a non-stationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. In the present study, the principle of this modified version of nonstationary fluctuation analysis is described and experimental results obtained from inhibitory cholinergic synapses in dissected buccal ganglia of Aplysia are discussed applying this analytical method. [Jpn J Physiol 55 Suppl:S152 (2005)]

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Various presynaptic modulations involve changes in the presynaptic membrane potential. However, it is not entirely clear how presynaptic membrane potential determines synaptic efficacy. We addressed this issue using simultaneous pre- and postsynaptic whole-cell recordings at the calyx of Held in rat brainstem slices. Gradual decrease in the presynaptic action potential amplitude by tetrodotoxin (TTX, 20-40 nM) had no effect on the EPSCs amplitude as far as the action potential overshoot exceeded +10 mV, but further reduction below this level steeply diminished EPSCs. In the absence of TTX, presynaptic depolarization (by 10-20 mV) increased the EPSC amplitude despite a reduction in the action potential amplitude, whereas presynaptic hyperpolarization had no clear effect on EPSCs. Presynaptic Ca²⁺ currents, recorded after blocking sodium and potassium conductance, underwent facilitation when the nerve terminal was depolarized (15-20 mV, 2 sec). Although this magnitude of depolarization did not evoke detectable Ca²⁺ currents, Ca²⁺ measurements using fluo-4 showed a clear increase in the Ca²⁺ concentration in the nerve terminal. These results suggest that presynaptic depolarization increases Ca²⁺ influx into the nerve terminal thereby facilitating transmitter release via mechanisms involving Ca²⁺ -dependent facilitation of Ca²⁺ currents. [Jpn J Physiol 55 Suppl:S152 (2005)]

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Hippocampal mossy fiber pathway contains the highest concentration of BDNF in the CNS. It has been suggested that BDNF in the mossy fiber pathway regulates granule cell morphology and susceptibility to temporal lobe seizures and that it also anterogradely regulates the synaptic plasticity. Recently it is shown that BDNF is localized to the giant mossy fiber boutons. Here we report that BDNF is released from the giant mossy fiber boutons by the activity-dependent manner. The sindvis virus vector containing a fusion protein constract of BDNF and GFP derivative, Venus, was made and stereotaxically injected in the dentate gyrus of mouse hippocampus (C57BL/6, P14-21). After 2-3 days, hippocampus was removed, sliced at 300 mm and observed under confocal microscopy. We found that BDNF-Venus fluorescence was accumulated in the large MF boutons. The fluorescence was decreased in intensity by the high-frequency stimulation of MF axons. This response depended on extracellular calcium, and disappeared by either removing extracellular calcium or treating with NEM. These observations would provide direct evidences that BDNF is released from the giant mossy fiber boutons through exocytosis. [Jpn J Physiol 55 Suppl:S152 (2005)]

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Electrophysiological properties of jaw-closing motoneurons (JCMNs) in developing rats aged 4-27 days were investigated in slice preparations using intracellular recording techniques. The input resistance of JCMNs from 4-9-day-old rats was larger than that of JCMNs from older rats. The action potential half-duration progressively decreased with age, whereas the incidence of post-spike afterdepolarization (ADP) increased during postnatal development. The amplitude and half-duration of the medium-duration afterhyperpolarization decreased with age. Firing frequency adaptation was observed during the first four spikes in a spike train. The subsequent firing pattern was classified into two groups, constant firing (type I) and rapid acceleration (type II). Percentage of type I neurons increased with age. In more than 50% of the neurons of each age group, an extra spike was produced from the ADP of the 1st spike in a spike train, leading to a high firing rate for the first inter-spike interval (1st ISI). Incidence of an extra spike also increased with age. The firing rate for the steady state was also high in JCMNs even at 4-9 days of age. About 50% of the neurons of this age fired at higher than 50 Hz. These findings demonstrate that JCMNs can fire at a high rate even in the early period after birth and firing rate for the 1st ISI further increases with age. [Jpn J Physiol 55 Suppl:S153 (2005)]

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We have reported that exposure to environmental sounds induces neural potentiation in the auditory cortex using transcranial flavoprotein autofluorescence imaging in the mouse auditory cortex in vivo. However, the location of targets for the effect of acoustic environment is unknown. To elucidate possible involvement of intracortical circuits in the potentiation, we reared C57BL/6 mice in a sound proof room with or without exposure to environmental sound stimuli at 10 kHz for several weeks, and cortical slices including the auditory cortex were prepared from the mice. Cortical images of green autofluorescence of flavoproteins in blue light were recorded. To evaluate the function of intracortical circuits, repetitive electrical stimulation at 10 Hz for 0.5 s was applied to supragranular layers in the auditory cortex, and the resulting horizontal spreading of cortical activities were evaluated with autofluorescence imaging. The size of the response areas in the mice exposed to environmental sound stimuli was significantly larger than that in the mice not exposed to sound stimuli. The present results confirm the previous results that acoustic environments are required for the development of neural activities in the auditory cortex in vivo. Furthermore, it is clearly demonstrated that intracortical circuits are one of the responsive sites for the effect of acoustic environment. [Jpn J Physiol 55 Suppl:S153 (2005)]

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Endocannabinoids (eCB) mediate retrograde signal and modulate transmission efficacy at various central synapses. Release of eCB is induced by either depolarization or activation of G_q/11-coupled receptors. However it is markedly enhanced by the coincidence of depolarization and receptor activation. Phospholipase C (PLC) is involved in biosynthesis of the major eCB, 2-arachidonoylglycerol. PLCβ requires both activation of G_q/11-coupled receptors and Ca²⁺ for its activation. These results suggest that PLCβ is likely to mediate the enhancement of eCB release. To examine this possibility, we used cultured hippocampal neurons and recorded cannnabinoid-sensitive synaptic currents. We confirmed that the receptor-driven eCB release was absent in PLCβ1-knockout mice. We found that this PLCβ1-mediated eCB release was dependent on physiological levels of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and markedly enhanced by depolarization-induced [Ca²⁺]_i elevation. We measured PLCβ1 activity in intact neurons by using exogenous TRPC6 channel as a biosensor for the PLC product diacylglycerol. The receptor-driven TRPC6 currents were absent in PLCβ1-knockout mice, showed a similar [Ca²⁺]_i dependence to that of receptor-driven eCB release and were augmented by depolarization-induced [Ca²⁺]_i elevation. These results indicate that PLCβ1 serves as a coincidence detector through its Ca²⁺ dependency for triggering eCB release in hippocampal neurons. [Jpn J Physiol 55 Suppl:S153 (2005)]

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We previously showed both in vivo and in vitro that the corticospinal (CS) synapses are at first distributed in the whole spinal cord but eliminated from the ventrolateral side later. In this study we investigated the daily progress of the synapse elimination from P7 to P11 in vivo rat by electrophysiological and histological methods. Field EPSPs were evoked by stimulation of the medullary pyramid and recorded from the lower cervical cord (C7). They were widely distributed at P7, and eliminated from the ventrolateral side of spinal cord at P8. Their distribution was limited to the dorsomedial area at P10 and P11. To label the CS terminals anterogradely, biotin dextran amine (BDA) was injected to the sensorimotor cortex. Sections prepared from the C7 spinal cord studied morphologically. First, peroxidase reaction with diaminobenzidine was used for counting CS fibers in the ventrolateral area. The CS fibers spread to the entire spinal cord in fan-shape at P7. Terminals in the ventrolateral area began to regress as early as at P8 and further decreased at P10 and P11. This regression of the CS terminals paralleled closely with field EPSPs change. Second, localization of presynaptic structure on the labeled terminals in the ventrolateral area was confirmed by double immunostaining of BDA and synaptophysin, which was observed with laser confocal microscope. These findings suggest that the CS synapses are formed in the whole spinal cord at P7, but immediately after this the elimination begins in the ventrolateral area. The start of elimination is much earlier than previously reported. [Jpn J Physiol 55 Suppl:S153 (2005)]

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Interaural time difference (ITD) is an essential cue for the sound source localization along the horizontal axis. In birds, ITDs are first calculated in neurons of nucleus laminaris (NL) by detecting the coincidence of binaural synaptic inputs. Previously, we recorded neurophonic potential (NP) in NL of anesthetized chicken (posthatch 1-6 days), using a tungsten electrode inserted into a glass pipette: The NP changed periodically with ITDs. The characteristic frequencies were tonotopically arranged, and the ITD sensitivity varied orderly as the recording site shifted. NP was specifically reduced by CNQX, and this may indicate that NP originates in EPSCs and action potentials from NL. However, the relationship between the NPs in NL and the activities of NL neurons is not known. In this study, we made single-unit recordings with a sharp electrode to understand the ITD processing in NL. The basic properties of NL spikes agreed with those of NP, in that they were phase-locked to pure-tone stimulus, and sensitive to frequency and ITD of stimulus sound. This suggests that the NPs recorded in NL would reflect the activities of NL neurons. As the sound intensity increases, binaural responses for favorable ITD largely increased, but those for unfavorable ITD did not. Furthermore, responses for unfavorable ITD were sometimes smaller than spontaneous ones. These results suggest a presence of some inhibitory system in NL. This inhibition may play an essential role for accurate ITD detection. [Jpn J Physiol 55 Suppl:S154 (2005)]

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Cholinergic neurons in the basal forebrain nuclei (BF) which project to various nuclei in CNS receive glutamatergic inputs from several brain regions. A whole-cell patch-clamp study using slice preparations of the rat brain was performed to elucidate the developmental change in calcium channel subtypes involved in non-NMDA glutamatergic synaptic transmission onto BF cholinergic neurons. In the present study, a novel fluorescent marker, Cy3-192IgG was stereotaxically injected into the lateral ventricle to selectively label live rat cholinergic neurons in the BF region, then 4-6 days later slices were cut. Pharmacologically isolated non-NMDA glutamatergic postsynaptic currents (EPSCs) were evoked by focal stimulation. The magnitude of inhibition of EPSCs by bath application of ω-CgTX (3 μM), an N-type calcium channel blocker, was gradually decreased from 49% to 23.9% during the postnatal stages between P18 and P34. On the other hand, application of both ω-CgTX (3 μM) and ω-Aga-TK (200 nM) inhibited the EPSCs by 96-98% throughout the postnatal stages. These results suggest that the contribution of N-type channels to the synaptic transmission declines with age, indicating any physiological implication. [Jpn J Physiol 55 Suppl:S154 (2005)]

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The neurosteroid 17b-Estradiol (E2) has been reported to be against global ischemia-induced neuronal cell death by preventing apoptotic processes in the hippocampal CA1. It remains unknown, however, whether E2 treatment can also protect CA1 neurons from functional deficit caused by moderate ischemia. To address this issue, animals were subjected to a transient cerebral ischemia, which will lead to impairment in LTP induction, and the effect of E2 against this deficit was examined. Adult male Wistar rats were subjected to a transient four-vessel occlusion (4VO) by clamping the bilateral common carotid arteries. It was found that 10 min but 20 min clamping induced no observable cell death in CA1. We employed the 10 min ischemic animals and examined the electrophysological properties of their Shaffer collateral-CA1 synapses 7 days after the 4VO. Brains were cut into slices, stained with the voltage sensitive dye RH482 and subjected to real-time optical recordings. The ischemic brain showed a decreased synaptic transmission and an impairment of LTP induction but no alteration in paired-pulse facilitation (PPF), indicating that these dysfunctions occurred in postsynaptic neurons but not in presynaptic terminals. Administration of E2 (1-4 mg/kg) 3h before 4VO was found to be able to protect CA1 neurons from these ischemia-induced synaptic dysfunctions. Above results suggest that E2 can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. [Jpn J Physiol 55 Suppl:S154 (2005)]

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We have previously shown that glutamate induces the translocation of drebrin and F-actin from dendritic spines to the parent dendrites. We demonstrated that postsynaptic Ca²⁺-influx via NMDA receptor was needed to the translocation. However, the mechanisms of the translocation have not been clarified yet. In the present study, we analyzed the effect of blebbistatin, an inhibitor of myosin II ATPase activity, on the glutamate -induced translocation in cultured hippocampal neurons. We prepared the low-density cultures of rat hippocampal neurons from 18-day embryo. After 21 days in vitro, we pretreated the cultured neurons with 100 μM blebbistatin for 60 min. Then, we applied 100 μM glutamate for 10 min and analyzed the location of drebrin and F-actin immunohistochemically. Blebbistatin completely inhibited the glutamate-induced translocation of drebrin and F-actin. These data demonstrate that myosin II activity is required for translocation of drebrin and F-actin from dendritic spines to parent dendrites in hippocampal neurons. [Jpn J Physiol 55 Suppl:S154 (2005)]

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Neuronal networks in the mammalian brain can generate various frequencies of synchronized membrane potential oscillations. It has been reported that synchronized oscillation of excitatory synaptic potentials at 5-10Hz can be evoked in neocortex slices under three different conditions: 1) with low concentrations of Mg²⁺ in ACSF, 2) with blockade of GABAa receptors, or 3) under bath-applications of caffeine. Synchronized activities evoked under all these conditions exhibited similar waveforms and, once induced, were able to survive replacement of the medium by normal ACSF. The latter result suggests involvement of long-term changes in neural or network properties. Antagonists of NMDA receptor inhibited both induction and expression of synchronized activities. On the other hand, IP3R antagonists inhibit expression of these synchronized activities, but not the induction. Furthermore, these synchronized activities were not inhibited by blockade of any of the followings: ryanodine receptors, sarcoplasmic endplasmic reticulum Ca²⁺-ATPase(SERCA), PLC, group I mGluRs, muscarinic acetylcholin receptors and store operated calcium (SOC) channels. These findings suggest that expression of the present synchronized activities requires IP3Rs, which may play different roles than calcium release channels. [Jpn J Physiol 55 Suppl:S155 (2005)]

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Reduction of transmitter release by activation of presynaptic G protein-coupled receptors (GPCRs) is a major mechanism in the presynaptic inhibition. In many neuronal structures, GPCR agonists, such as adenosine, exert a presynaptic inhibitory effect through inhibiting N-type voltage-dependent calcium channels (VDCCs). To understand how the functional coupling between G proteins and VDCCs is regulated, we analyzed the effect of adenosine on the synaptic transmission from the primary afferents to the second-order neurons in the nucleus tractus solitarii in the slices from the mice lacking α_1B subunit of N-type VDCCs (Ca_V2.2^−/−). Both in the wild type and Ca_V2.2^−/− mice, adenosine decreased the amplitude of evoked EPSC with similar magnitude and time course. This effect was mimicked by CPA, blocked by DPCPX and accompanied by an increase in the paired-pulse ratio, indicating an involvement of presynaptic A₁ receptors. Whereas ω-conotoxin GVIA occluded the inhibitory effect of adenosine in the wild type mice, adenosine significantly reduced EPSC amplitude even in the presence of ω-conotoxin GVIA, ω-agatoxin IVA and NiCl₂ in Ca_V2.2^−/−. These results imply an unidentified mechanism enabling the lack of the G protein target at the presynaptic terminal to lead to a compensatory replacement of another target so that the function of GPCR in regulating transmitter release could be conserved. [Jpn J Physiol 55 Suppl:S155 (2005)]

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I had suggested that muscarine presynaptically inhibited the GABAergic IPSC at a putative striato-nigral “direct” pathway on a substantia nigra pars reticulata (SNr) GABA neurons through the activation of M₃-muscarinic acetylcholine (ACh) receptor, as 4-DAMP abolished the inhibition. However, GF109203X (1 μM, a previous result), ryanodine (200 nM & 20 μM, n=4 in each concentration), thapsigargin (5 μM, n=3) and apamin (1 μM, n=4) did not have any significant inhibitory effects on this IPSC inhibition by muscarine (10 μM). These results might indicate that the M₁-, M₃- or M₅-receptors activation did not induce the inhibition. I had already reported that a M₂-receptors antagonist, AF-DX116 (300 nM), did not affect the inhibition. Therefore, the relation between the M₄-receptor activation and this inhibition was investigated, in this time. Forskolin (10 μM) significantly decreased this IPSC inhibition by muscarine (p<0.005, n=6). In the solution with a M₄-receptor selective antagonist, muscarine toxin-3 (MT-3), the IPSC inhibition was dose-dependently decreased. The inhibition ratio, which was calculated by the equation of “1 – an amplitude of IPSC in the muscarine solution / a control IPSC amplitude”, was decreased from 0.56 to 0.32 in the solution with 30 nM MT-3. These results may suggest that the activation of the M₃-muscarinic ACh receptor dose not induce the presynaptic inhibition of a putative striato-nigral GABAergic IPSC at SNr GABA neurons by muscarine, but the M₄-receptor activation induces it. [Jpn J Physiol 55 Suppl:S155 (2005)]

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We applied the intrinsic optical imaging technique to the human primary somatosensory cortex during brain tumor/epilepsy surgery for nine patients. The cortical surface was illuminated with a Xenon light through an operating microscope, and the reflected light, which passed through a 605nm bandpass filter, was detected by a CCD camera-based optical imaging system. Individual electrical stimulation of five digits induced changes in the reflected light intensities. Visualizing the intrinsic optical responses, we constructed maps of finger representation in Brodmann's area 1. In the maps, response areas of Digit I to V were sequentially aligned along the central sulcus in the crown of the postcentral gyrus from the latero-inferior region (Digit I) to the medio-superior region (Digit V). The neighboring response areas partially overlapped each other, as previously described in the monkey somatosensory cortex. Similar results were obtained in the face region with stimulation of the three branches of the trigeminal nerve. These results suggest that the overlap of the response areas is a common feature in the somatosensory cortex not only in monkeys, but also in humans. [Jpn J Physiol 55 Suppl:S156 (2005)]

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We examined whether elevation of cAMP or activation of macrophage can make crushed axons of retinal ganglion cells (RGCs) to regenerate into the cat optic nerve (OpN). The left OpN was crushed with a 6-0 thread pulled with 0.2 N, 60 sec (microcrush). Forskolin (30 min before OpN crush), zymozan (60 m after the crush), or oxidized galectin-1 (60 m after the crush), was injected into the vitreous. At day 12, WGA-HRP was injected to anterogradely label regenerated axons. On day 14, deeply anesthetized cats perfused with fixative. The excised OpN was longitudinally cut on a cryostat, labeled axons were visualized with TMB reaction. Number of Regenerated Axons. The OpN with saline-injection (control) contained very few labeled axons elongating beyond the crush site, whereas OpN with injections of forskolin (0.1 mg), zymozan (50 μg), or galectin (0.5-1000 ng) had many labeled axons elongating beyond the crush site. Mean numbers (N=2 to 4) of labeled axons of 0.5 mm or longer were 24 (control), 341 (forskolin 0.1 mg), 275 (galectin 0.5 ng), 2755 (galectin 100 ng), 1355 (galectin 1000 ng), 601 (zymozan), respectively. Similar tendency was found in the mean axon numbers at 1.0 mm. Longest Axons. We measured length of the longest axons, and found no statistically significant difference among values from experiments. Crushed axons of adult cats regenerated into the OpN when a macrophage activator was injected or intracellular cAMP was elevated. The elongation rate of regenerated axons was estimated as less than 1 mm/ wk. [Jpn J Physiol 55 Suppl:S156 (2005)]

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Orexins are synthesized specifically by neurons in the hypothalamus and contribute to multiple physiological functions. Orexin fibers innervate many regions of the CNS, which include the spinal dorsal horn and areas involved in descending control of pain. We examined the possible role that orexin may play in endogenous modulation of pain transmission using orexin knockout mice. We utilized two conditions that are known to activate endogenous pain modulation systems, i.e. persistent pain and stress, and compared the changes in pain thresholds induced by these conditions in knockout and wild type mice. Inflammation was induced by carrageenan injection to produce persistent pain and electric foot shocks were used to apply stress. Pain thresholds were determined by the paw withdrawal or tail flick test. Baseline pain thresholds of knockout mice were not different from wild type mice. Knockout mice presented greater degree of hyperalgesia induced by peripheral inflammation and less stress-induced analgesia than wild type mice. Furthermore, in an immunohistochemistry study, we examined whether these two conditions were able to induce activation of orexin neurons. Double-staining of orexin and c-Fos in the hypothalamus of wild type mice revealed that orexin neurons were activated under the two conditions. The results suggest that both persistent pain and stress activate orexin pathways which act to inhibit pain transmission. [Jpn J Physiol 55 Suppl:S156 (2005)]

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Behavioral responses to visually moving stimulus in rats are not fully investigated. Here we determined the lowest velocity of motion stimulus effective for the shuttle box avoidance task. Four groups of male Wistar rats were trained to avoid electrical shocks from a grid floor using a different velocity of sin-wave gratings as a conditioned stimulus (CS; 40, 20, 1.5 or 0.5 deg/s). Sin-wave gratings with a spatial frequency of 0.5 c/deg was presented on a CRT display placed on the top of the shuttle-box, and moved for a 10 sec. The foot shocks were delivered at 10 sec after CS onset, unless the animal crossed the central barrier to move the adjacent compartment of the box. An inter-trial interval was random with a mean of 60 sec (45-75 sec). A daily session consisted of 50 trials. Avoidance rate increased as training proceeded in all four groups and reached at an asymptote level of 50-70% after 3-4th sessions. The 0.5 deg/s group showed a higher avoidance rate than those in the other groups after 3rd session. However, inter-trial responses (ITRs) in the 0.5 deg/s group were significantly higher than those in the other groups (5.6 vs. 3.0/trial), indicating that 0.5 deg/s group did not learn the task. At the 5th session ITRs were highest in 0.5 deg/s group (N=6) and lowest in 20 deg/s group (N=4). The ITRs in 0.5 and 40 deg/s groups (N=6 and N=4, respectively) were similar. These results suggest that rat can perceive motion most easily with a velocity of 20 deg/s, and can perceive motion with a velocity at least higher than 1.5deg/s. [Jpn J Physiol 55 Suppl:S157 (2005)]

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In order to clarify the mechanism underlying the integration of multiple sensory stimuli in the neuronal network, the chemotactic responses of the nematode Caenorhabditis elegans to simultaneous presentation of water-soluble sodium acetate and an odorant diacetyl, which were sensed by different sensory neurons and integrated by common interneurons, were examined. The fraction of animals that gathered at the 0.7 M sodium acetate location was greater than that at the 0.1% diacetyl location in the presence of both attractants (p<0.05), although the chemotaxis indexes for 0.7 M sodium acetate and 0.1% diacetyl were similar in the presence of a single attractant. On the other hand, the fraction of animals that gathered at the 0.02% diacetyl location was greater than that at the 0.1 M sodium acetate location in the presence of both attractants (p<0.05), although the chemotaxis indexes for 0.02% diacetyl and 0.1 M sodium acetate were similar in the presence of a single attractant. These results suggest the existence of excitatory and/or inhibitory connections in the neuronal circuit for attractant selection, and that the efficacy of these connections may change according to the concentrations of both attractants. [Jpn J Physiol 55 Suppl:S157 (2005)]

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The aim of the present study was to examine whether the LC/SC modulates visceromotor function. An electromyogram (EMG) of the external abdominal oblique muscle evoked by colorectal distention was measured as a visceromotor response, and inhibitory effects of LC/SC stimulation were estimated by the decrease of EMG activity. Under halothane anesthesia (1% in air), graded colorectal distentions (30, 60, or 80 mmHg) were produced by inflating a balloon inside the descending colon and rectum. Colorectal distention at a pressure of 30 mmHg did not evoke any EMG activity in the external abdominal oblique muscle in all rats tested. Electrical stimulation of the LC/SC (30, 50 and 70 μA, 100 Hz, 0.1 ms pulses) reduced EMG responses evoked by colorectal distention to 60 and 80 mmHg. LC/SC stimulation was effective both ipsilaterally and contralaterally indicating a bilateral effect. EMG responses decreased with an increase of LC/SC stimulation intensity. Following recordings of the inhibitory effects of LC/SC stimulation, lesions of the LC/SC ipsilateral to the EMG recording site were induced. LC/SC stimulation did not reduce the EMG responses when stimulation was applied to the lesiond LC/SC, whereas EMG responses were observed by stimulation of the intact LC/SC contralateral to the EMG recording site. From lesion experiments, it could be considered that suppression of the visceromotor response to colorectal distention is due to activation of the LC/SC. The results suggest that the visceromotor function is under the control of the centrifugal pathways from the LC/SC. [Jpn J Physiol 55 Suppl:S157 (2005)]

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We examined the effect of the Ca²⁺ concentration in the endolymph ([Ca]_e) or in the endolymphatic surface cells on the endocochlear potential (EP) by using Ca²⁺-selective and conventional microelectrodes. 1) The abrupt increase in [Ca]_e up to ∼10⁻³ M with a fall in the EP was induced by transient asphyxia (∼1.5 min) or intravenous administration of furosemide (60 mg/kg), and significant correlation was obtained between the EP and p[Ca] _e (= –log [Ca] _e). 2) The perfusion of endolymph with 10 mM EGTA-containing solution for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP, suggesting that Ca²⁺ concentration across the stria vascularis did not contributed directly to the generation of EP. 3) The administration of 300 µM EGTA-tetraacetoxymethyl ester (EGTA-AM) with 10 mM EGTA-containing solution to the endolymph produced a gradual increase in EP and suppressed the asphyxia or furosemide-induced decrease in EP, although perilymphatic administration of 1 mM EGTA-AM caused no significant suppression of the asphyxia-induced change in EP. 4) The application of 30 μM nifedipine to perilymph also suppressed the fall in EP induced by transient asphyxia. These findings indicated that Ca²⁺ homeostasis in the stria vascularis cells plays an important role in generating/maintaining a large positive EP. [Jpn J Physiol 55 Suppl:S157 (2005)]

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Pendred syndrome, characterized by deafness and goiter, is caused by the mutation of the PDS gene SLC26A4, which codes for the pendrin. This protein is an anion exchenger, which can transport Cl⁻ and HCO₃⁻, has been found in the endolymphatic surface cells. Recent study demonstrated that positive endocochlear potential (EP) was disappeared in Slc26a4^−/− mice. In the present study, therefore, we examined the effect of CO₂/HCO₃⁻ on EP by using perilymphatic or endolymphatic perfusion technique in guinea pigs. The gradual decrease in EP from 70 to 25 mV was induced by a perilymphatic perfusion of CO₂/HCO₃⁻-free (Hepes buffer) solution. In this condition a transient asphyxia for ∼2 min produced further decrease in EP to -10 mV, whereas it produced the decrease in EP from 80 to 20 mV with the perilymphatic perfusion of 5% CO₂/25 mM HCO₃⁻ solution. The perfusion of perilymph or endolymph with 30 μM nifedipine suppressed the both Hepes buffer-induced decrease (HBID) or transient asphyxia-induced decrease (TAID) in EP. By contrast, perilymphatic administration of La³⁺ produced a decrease in EP and no significant suppression of HBID or TAID in EP. Perilymphatic administration of 1 mM EGTA-tetraacetoxymethyl ester (EGTA-AM) produced no significant suppression of HBID or TAID in EP. These findings suggest that effect of CO₂/HCO₃⁻ on EP is mediated by Ca²⁺ homeostasis in the cell of stria vascularis, especially by the nifedipine-sensitive Ca²⁺ channel. [Jpn J Physiol 55 Suppl:S158 (2005)]

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G-protein gated potassium channels contribute to the modulation of synaptic transmission in the CNS. Recent studies have implicated that an inactivation of spinal G-protein gated potassium channels elicits thermal hyperalgesia. In this study, we have shown that G-protein gated potassium channels are activated by an endogenous neurotrasmitter in the spinal dorsal horn using whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons of adult rat spinal cord slices. Although repetitive stimuli applied to the dorsal root did not induce any slow responses, repetitive stimuli focally-applied to the deep spinal dorsal horn produced slow inhibitory postsynaptic currents (IPSCs) at a holding potential of –50 mV in 32 of 101 SG neurons recorded. The slow IPSCs increased in amplitude and duration with increasing number of stimuli and significantly decreased by the removal of Ca²⁺ from external Krebs solution. The slow IPSC was associated with an increase in membrane conductance and reversed its polarity at a potential close to the equilibrium potential for K⁺, calculated from the Nernst equation. The slow IPSC was blocked by the addition of GDP-β-S into patch-pipette solution, and was reduced in amplitude in the presence of Ba²⁺. These results indicate that an endogenous neurotrasmitter released from interneurons or descending fibers induces slow IPSCs through the activation of G-protein gated potassium channels in SG neurons. This finding may serve to understand pathological pain sensations such as thermal hyperalgesia. [Jpn J Physiol 55 Suppl:S158 (2005)]

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Our eyes are in constant motion, but we are usually unaware of retinal image motions resulting from our own eye movements. This perceptual stability is assumed by an internal estimation of the external visual world derived from two signals, the retinal and extra-retinal signals. While we are tracking a moving object, this internal estimation of visual motion in space is known to work fairly well although not perfectly. Here we show the neuronal correlates of the estimated visual motion in space in the cortical area MST of macaque monkeys. We studied responses of MST and MT neurons to a visual stimulus moving at a series of constant speeds during smooth pursuit and stationary fixation. Most of the MST neurons responded in relation to the stimulus speed in space, although MT neurons responded in relation to the stimulus speed on the retina. The results are consistent with the idea of the internal estimation of the external visual world and suggest that the visual motion in space is served by the MST neurons through the information processing from the MT to MST. [Jpn J Physiol 55 Suppl:S158 (2005)]

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In oscine passerine birds (songbirds), auditory information which is conveyed from the higher auditory regions such as NCM (caudal medial nidopallium) in the posteritor telencephalon to HVC in "song control system" is thought to be necessary for "song perception" and/or "song learning". NCM in female songbirds and parrots is associated with perception of male song. In the budgerigar (Melopsittacus undulatus), a small parrot species, NCM is reported to have an indirect connection with NLC (central nucleus of the lateral nidopallium; corresponding to HVC of songbirds). On the other hand, another higher auditory regions such as NF (frontal nidopallium) in the anterior telencephalon have direct connection with NLC, suggesting that those regions have more of role for song perception. We, therefore, used immunocytochemistry for the protein product of the immediate early gene ZENK (Zenk protein; ZENK is also known as zif/268, egr-1, NGFI-A,krox-24, TIS 8) to investigate the role of NF for song perception in female budgerigars. We observed that there was no significant difference between song stimulus and control groups in Zenk immunoreactivity in NF. Thus, it seems plausible that the NCM (the posterior higher auditory region) in the posterior higher auditory regions is more important for the perception of song in female budgerigars. [Jpn J Physiol 55 Suppl:S158 (2005)]

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The primary source of salty taste is Na⁺ ions, not negatively charged anions paired with them. Experiments on gustatory nerves in animals have shown that anions modulate neural responses to Na⁺ ions. However, the mechanism of anion modulation remains unclear. The aim of the present study was to determine how anions modulate Na⁺-induced responses of the bullfrog (Rana catesbeiana) glossopharyngeal nerve (GL). We recorded responses of single fibers of the GL to various Na salts applied to the tongue surface. Since 1 mM NiCl₂ enhances the responses of the frog GL to Na salts, 1 mM NiCl₂ was always added to 0.01-0.5 M NaCl stimulating solutions. Large anions, such as GMP²⁻, glutamate⁻ and gluconate⁻, are unable to pass through the tight junctions between taste cells. Na salts with large anions elicited much smaller responses than did NaCl. They differently and strongly inhibited the responses to NaCl when they were mixed in the NaCl stimulating solutions. The large anions affected the dose-response curve for NaCl in a non-competitive manner. The present findings indicate that the Na⁺ receptor sites (X_Na) responsible for Na⁺-induced responses reside on the apical membrane of taste cells, not on the basolateral membrane. We hypothesize that anions can differently inhibit the efficacy of Na⁺-X_Na complexes by interacting with anion-binding elements that interact with X_Na. [Jpn J Physiol 55 Suppl:S159 (2005)]

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Frog taste discs contain three types of cells (type Ib, type II and type III cells). These three types of cells have voltage-gated Na⁺ inward currents. Merkel-like basal cells of the taste disc contain serotonin (5-hydroxytryptamin, 5-HT). Therefore, 5-HT has been thought to play an important role in gustatory transduction. In the present study, the effects of 5-HT on membrane properties of three types of cells of the bullfrog (Rana catesbeiana) taste disc were investigated. The patch clamp technique was used to make recordings from taste cells in vertical slices of taste discs. Cell types were identified by staining with Luciffer yellow in a pipette. The amplitudes of peak I_Na in type Ib, II and III cells obtained in the present study were –2153.1 ± 227.7 pA (mean ± SD, n = 5), –1423.1 ± 155.5 pA (n = 13) and –754.1 ± 59.0 pA (n = 6), respectively. External application of a 5-HT_1A receptor agonist, (+/-)-8-OH-2-(D1-n-propyl-amino)tetralin (8-OH DPAT) (20 μM), inhibited 14-22% of the peak I_Na in three types of cells, and the effect of 8-OH DPAT was reversible. These inhibitory effects were statistically significant (P < 0.05). The present findings indicate that the effects of 5-HT on the three types of cells were similar, suggesting that the release of 5-HT from Merkel-like basal cells affects electrical membrane properties of adjacent cells rather than a specific type of cell. [Jpn J Physiol 55 Suppl:S159 (2005)]

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In humans, Shingai et al. (1989) demonstrated that distilled water (DW) and NaCl solutions applied to the posterior tongue are effective for elicitation of swallowing reflex. They showed that excitation of water-sensitive receptors (WS-Rs) and salt taste receptors (S-Rs) elicits the swallowing reflex, but the precise sites of these receptors remain unclear. In the present study, the properties of the WS-Rs and S-Rs responsible for the swallowing reflex in humans were investigated. Each subject was instructed to repeat swallowing as fast as possible. DW or 0.05-0.3 M NaCl solution was delivered to the posterior tongue through a fine tube at a slow rate of 0.2 ml/min. The tip of the tube was located in the pharyngolaryngeal region (PL-R), where 0.3 M NaCl did not give rise to salty taste. The intervals between two consecutive swallowings in a test were measured. Swallowing interval was shortest when DW was used, and the interval increased with increase in NaCl concentration. The mean interval when 0.3 M NaCl was used was the same as that in the case of olive oil, which does not activate chemoreceptors, suggesting that 0.3 M NaCl inhibits excitation of WS-Rs. The present results suggest that WS-Rs for swallowing reflex reside in the PL-R, whereas S-Rs do not reside in this region. The tip of the infusion tube was moved to the dorsal tongue, where NaCl give rise to salty taste. Infusion of 0.15 M NaCl led to much shorter intervals than did DW. This suggests that S-Rs for swallowing reflex reside in the tongue, whereas WS-Rs do not reside in this region. [Jpn J Physiol 55 Suppl:S159 (2005)]

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We have previously showed desensitization of TRPV1 activity by successive applications of 100 nM capsaicin and its reversal by protein kinase C (PKC) in DRG neurons and CHO cells using Ca²⁺-imaging. We have performed whole-cell patch-clamp recordings in HEK293 cells and Ca²⁺-imagings in HEK293 and HeLa cells to clarify the more detailed mechanism. Capsaicin-activated currents were desensitized upon repetitive capsaicin application and re-sensitized upon activation of PKC by PMA in HEK293 cells expressing wild type TRPV1 but not S502A/S800A mutant insensitive to PKC. Furthermore, the re-sensitization was inhibited by PKCε translocation inhibitor. PKCε expression was significantly higher in HEK293 cells or CHO cells than in HeLa cells where re-sensitization was not observed using Ca²⁺-imaging method. The re-sensitization was restored by induction of PKCε into HeLa cells. The re-sensitization was accompanied by the increase in phosphorylated TRPV1 detected using anti-phospho-TRPV1 antibody made against phosphorylated S800. These results suggest that PMA-induced re-sensitization of TRPV1 activity occurs through PKCε-mediated phosphorylation of TRPV1 at S800. [Jpn J Physiol 55 Suppl:S159 (2005)]

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The processing of temporal information in the left and right auditory cortices of guinea pigs was investigated using click trains and optical imaging with a voltage-sensitive dye (RH795). Guinea pigs were anesthetized with ketamine (80mg/kg) and xylazine (40mg/kg). Click trains were presented at different repetition rates (4, 6, 8, 10, 12, 14, 20 Hz) and at 75 dBSPL ( equivalent to tone ). Neural activity was recorded from multiple auditory fields (primary: AI, dorsocaudal: AII, ventroanterior: VA) of both hemispheres. At the repetition rates of 4, 6 and 10 Hz, the neural activity followed well to each click and sometimes oscillated at the rate of the click even after the session of the stimulus. At 12 and 14 Hz, the neural activity showed one large peak followed by small peaks but at 20 Hz, it showed one large peak only. Repetition rate transfer functions (RRTF) in field AI were low-pass showing a sharp drop-off in evoked activity per click above 10 Hz but RRTFs in field AII were band-pass with the peak of 8 or 10 Hz. Synchronization to click trains was better in field AI than in other fields. In the same animals, the cut-off frequencies of RRTF in the left cortex were same as those in the right cortex but the slopes of the RRTF in the left cortex were sharper. We discuss the temporal processing in the left and right auditory cortices of guinea pigs. [Jpn J Physiol 55 Suppl:S160 (2005)]

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Cochlear hair cells receive efferent signals from neurons located in the superior olivary nuclear complex (SONs). The efferent signals are considered to alter sound sensitivity of inner hair cells by changing the length of outer hair cells. Little is known, however, about the mechanisms how the inhibitory efferent fibers form synapses with hair cells during the development. To address these questions, we cultured explants from SONs together with organ of Corti in the same collagen gel matrix, and examined how the efferent fibers were guided to the organ of Corti and formed synapses with their hair cells in the co-culture system. Small sections (approximately (0.5mm)3) dissected from SONs of embryonic or newborn rats were embedded in collagen matrix and were incubated in a serum-free medium supplemented with neurotrophins. These explants of SONs extended thousands of fine neurites in a BDNF-dependent manner. Immunostain against GAD revealed that these neurites contained many GABA-ergic ones. Co-culture of explants of SONs with sections of organ of Corti showed that many neurites from SONs made convergence to several regions on the organ of Corti at which outer hair cells were located. Many GABA-ergic fibers were identified among such fibers. These observations indicated that hair cells of the late embryonic and newborn stages were able to attract efferent fibers from the SONs neurons. [Jpn J Physiol 55 Suppl:S160 (2005)]

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The primary gustatory cortex (GC) is defined as the cortical region that receives fiber projection from the parvicellular part of the ventral posteromedial nucleus in the thalamus. The primary olfactory cortex is defined as the region that receives direct fiber projections from the olfactory bulb. The piriform cortex (PC) is the largest structure in the olfactory cortex and further the piriform region includes the endopiriform nucleus (EPN). In this study, we examined the possibility that the EPN is the region where olfactory and gustatory information are centrally integrated. Electrical and optical recordings were made from slice preparations including PC and GC. Electrical stimulation of the GC evoked a characteristic field potential in the EPN. A field potential was induced in the EPN by stimulation of the PC. Voltage-sensitive dye studies showed that stimulation of the PC induced signal propagation from the PC to EPN, whereas stimulation of the GC did the same from the GC to EPN via the agranular division of the insular cortex (AI). After stimulation of the EPN, optical signals propagated not only to the PC but also to the GC via AI. The olfactory and gustatory pathways appeared to be reciprocally connected. Unit recordings indicated that olfactory and gustatory activity converged onto a single neuron of the EPN. It is suggested that the cortical integration of olfactory and gustatory information could modulate mechanisms involved in food selection and emotional reactions relating to the chemical senses. [Jpn J Physiol 55 Suppl:S160 (2005)]

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Detail mechanism of the anesthetic effect of Dexmedetomidine (DX) is still unknown. The EEG activity and Fos expression were studied in the rats with different anesthetic levels. Two anesthetic levels were defined as light and deep anesthetic stages from the frequency analysis of the EEG activity during i.v. infusion of sodium pentobarbital (Nembutal), whereas those were defined as the occurrence of body movement during DX infusion. During Nembutal infusion, about 30% of δ and θ waves were observed at the light stage and about 50% of δ and 15% of θ waves were observed at the deep stage. On the other hand, EEG activity was not affected by DX infusion. A large number of Fos protein-LI cells were expressed in the MDH, area postrema, parabrachial nucleus, PAG, raphe, lateral hypothalamus and basolateral Amygdala after noxious heat stimulation of the face during Nembutal infusion, but no Fos protein-LI cells were expressed in the Amygdala during DX infusion. The strong depression of Fos protein-LI cells in the all areas of the CNS was observed at deep stage during Nembutal infusion. On the other hand, significant decrease in Fos expression was observed especially in the PAG and raphe nuclei following large amount of DX infusion. These data suggest that the DX may have a strong antinociceptive function as well as sedative effect. [Jpn J Physiol 55 Suppl:S160 (2005)]

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It is well known that the phosphorylation of extracellular signal-regulated protein kinase (pERK) in dorsal horn neurons is induced by noxious stimulation of the peripheral structures. The pERK is thought to be a good marker of the excitability of nociceptive neurons. This study was designed to elucidate the central mechanism of tooth pulp pain by use of pERK immunohistochemistry. The right upper maxillary tooth pulp was stimulated by 10 mM capsaicin (CAP) or vehcle (Veh) solution in the anesthetized rat. Rats were perfused at 2, 5, 10, 30 and 120 min after the stimulation. The number of pERK-like immunoreactive (LI) cells in the medulla and the upper cervical cord was counted in CAP or Veh groups. In addition, electromiographic (EMG) activities of digastric and masseter muscles were analyzed following the stimulation. Five min after CAP, a number of pERK-LI cells were expressed bilaterally in the trigeminal subnuclei interpolaris/caudalis (Vi/Vc) zone and paratrigeminal nucleus (Pa5). In the ipsilateral caudal subnucleus caudalis/upper cervical dorsal horn (C1/C2) zone, the pERK-LI cells were expressed significantly larger 5 min after CAP. EMG activities of the digastric and masseter muscles were significantly increased immediately after CAP.These results suggest that C1/C2 zone contribute directly to the tooth pulp pain, and the Pa5 and Vi/Vc zone would be involved in the other functions instead of tooth pain. [Jpn J Physiol 55 Suppl:S161 (2005)]

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We have shown that conditioning stimulation of the amygdaloid nucleus has an inhibitory effect on nociceptive responses of the somatosensory cortex. However, the site of inhibitory action in the nociceptive ascending system is not clear. The aim of this study was to determine whether amygdaloid conditioning stimulation exerts an influence on nociceptive neurons in the medullary dorsal horn of the rat. The animals were anesthetized with N₂O-O₂ and 0.5% halothane and were immobilized with pancuronium bromide. A peripheral test stimulus (a single rectangular pulse of 2.0 msec in duration) was applied to the receptive field of nociceptive neurons, and ipsilateral amygdaloid conditioning stimuli to the recording site were trains of 33 pulses (100∼300 μA) delivered at 330 Hz. The conditioning stimulation had almost no effect on the discharges of non-nociceptive neurons. In contrast, the conditioning stimulation markedly inhibited the activities in 32 of 51 nociceptive neurons (24/40 WDR neurons, 8/11 NS neurons). The amygdaloid inhibitory effect was 68.3±14.4% (n=32) at maximum and was not influenced by naloxone (2 mg/kg, i.v.). The present results suggest that the amygdaloid nucleus inhibits ascending nociceptive information at the 2nd-order neurons via a non-opioid system. The amygdaloid antinociceptive effect may provide a neurophysiological basis for the stress-induced analgesia (SIA) observed in the presence of stress and fear. [Jpn J Physiol 55 Suppl:S161 (2005)]

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