Endocannabinoids mediate short- and long-term suppression of synaptic transmission. Nature of presynaptic cannabinoid receptor has been investigated for both excitatory and inhibitory synapses in various regions of the brain. For inhibitory synapses, importance of type 1 cannabinoid receptor (CB1) is generally accepted. For excitatory synapses, however, results are controversial. In the present study, we used electrophysiological and immunohistochemical techniques, and examined the type of cannabinoid receptors functioning at hippocampal and cerebellar excitatory synapses. Using CB1-knockout mice, we demonstrate predominant contribution of CB1 to excitatory synaptic transmission on CA1 pyramidal neurons in the hippocampus and that on cerebellar Purkinje cells from climbing fibers and parallel fibers. The presence of CB1 at presynaptic terminal was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy of the hippocampus and the cerebellar cortex, densities of CB1-positive signal in excitatory terminals were much lower than in inhibitory terminals, but clearly higher than the background level. These results clearly indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum. [J Physiol Sci. 2006;56 Suppl:S84]

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Drebrin A is a neuron-specific actin-binding protein, which is localized in mature dendritic spines. During synapse formation, embryonic isoform, drebrin E is converted into neuron-specific isoform, drebrin A. We have demonstrated that suppression of the upregulation of drebrin A attenuated spine formation in vitro. To investigate physiological differences of drebrins E and A, we generated mice, in which isoform conversion of drebrin did not occur, by targeted disruption of drebrin A specific exon (DAKO). In these mice total amount of drebrin was not changed since drebrin E was overexpressed instead of drebrin A. We first analyzed their dendritic spine morphology on apical dendrites of layer V pyramidal cells in somatosensory cortex using rapid Golgi staining. The number and length of dendritic spine in adult DAKO mice (16-18 week old) were comparable to that in wild-type mice. We next analyzed behavioral phenotypes in DAKO mice. These mice showed impaired context-dependent fear conditioning, a hippocampal NMDA receptor-dependent learning task. Our findings indicate that isoform conversion of drebrin is required for regulation of synaptic function. In contrast, total amount of drebrin is important for regulation of spine morphology. [J Physiol Sci. 2006;56 Suppl:S84]

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Dopamine is thought to play important roles in various brain functions including learning and memory. In the hippocampus, dopamine has been shown to modulate synaptic transmission and plasticity in the CA1 region. However, it remains largely unknown how dopamine affects synaptic transmission in the CA3 region where forms of synaptic plasticity distinct from those in the CA1 region have been demonstrated. In the present study, we show that application of dopamine reversibly potentiates synaptic transmission between the mossy fibers (MFs) and CA3 pyramidal cells. Pharmacological experiments showed that D1-like receptors mediate the potentiation induced by dopamine. This potentiation was accompanied by a decrease in the magnitude of synaptic facilitation and occluded by application of forskolin, an adenylate cyclase activator, suggesting the involvement of presynaptic cAMP-dependent mechanisms. The MF synaptic transmission is kept inhibited by tonic activation of presynaptic adenosine A1 receptors and GABA_B receptors. It is known that dopamine and adenosine counteract each other in some brain regions and that adenosine receptor antagonists can enhance effects of dopamine receptor agonists. At the MF synapse, application of an adenosine receptor antagonist alone, or together with a GABA_B receptor antagonist, enhanced the synaptic transmission, but suppressed the dopamine-induced potentiation. These results suggest that dopamine-induced presynaptic potentiation of the MF synaptic transmission is unmasked by the tonic presynaptic inhibition mainly mediated by adenosine. [J Physiol Sci. 2006;56 Suppl:S84]

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Associative LTP by two independent inputs is known to be an important feature of synaptic plasticity observed in the CA1 of rat hippocampus. However, it is still unclear what kind of physiological role, especially in relation with memory and learning mechanisms, it plays and how it is inducted by two pathways. In order to investigate these questions, the specio-temporal pattern of neuronal activities during LTP induction followed by long lasting later period were studied by using both voltage-sensitive dye optical and extracellular electrical recording. Moreover, the possible contribution of back-propagating action potentials invaded into dendritic trees to the induction mechanisms of the present LTP was investigated under the assumption of STDP (spike timing dependent plasticity) mechanisms. In order to induce associative LTP rat acute slice preparations stained with Di-4-anepps were stimulated by two independent Schaffer collateral pathways with the stimulation protocol reported previously; one pathway is stimulated weakly under threshold (single pulse) while another pathways is strong over the threshold (multiple pulses). Optical signals were recorded and analyzed by using the photo-diode array system (Neuro-plexVI; Redshirt Imaging, USA). The obtained results from the present study are discussed from the view point of non-linear summation of two independent inputs and its distribution along the dendrite, together with the possible induction mechanisms obtained from low TTX experiment. [J Physiol Sci. 2006;56 Suppl:S84]

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NEURON AND SYNAPTIC FUNCTIONS Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo.ResultsFluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ∼1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. [J Physiol Sci. 2006;56 Suppl:S85]

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It has been hypothesized that hippocampal mossy fiber terminals of young rodents release GABA in addition to glutamate. Using whole cell-clamp recordings in mouse slice preparations, I re-examined this possibility with paying attentions about the conditions to evoke monosynaptic GABAergic responses by stimulation of mossy fibers. Strong stimulus to the stratum granulosum of dentate gyrus or stimulus to the stratum lucidum of CA3 region elicited IPSCs in CA3 neurons in the presence of glutamate receptor antagonists 10 μM CNQX and 25 μM D-AP5, and these putative "monosynaptic IPSCs" were abolished by addition of GABA_A receptor antagonist 100 μM picrotoxin. In contrast, weak stimulus to the stratum granulosum never elicited IPSCs in the presence of CNQX and D-AP5. The responses to weak and strong stimuli also displayed differential sensitivity to group II mGluR agonist DCG-IV; application of 1 μM DCG-IV almost abolished the responses to weak stimulus and left substantial responses to strong stimulus which were inhibited by further application of picrotoxin. These results suggested that strong stimulus to stratum granulosum causes monosynaptic IPSCs by stimulating inhibitory interneurons in addition to mossy fibers, and mossy fiber terminals themselves may not release GABA. [J Physiol Sci. 2006;56 Suppl:S85]

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Regulation of neurotransmitter release by tomosyn phosphorylation. Sumiko Mochida¹, Takeshi Baba², Toshiaki Sakisaka², and Yoshimi Takai². ¹Depatment of Physiology, Tokyo Medical University, Shinjuku 160-8402, Japan, and ²Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.PKA phosphorylation of tomosyn, a SNARE regulatory protein, significantly decreased its binding to syntaxin-1 in vitro, and cAMP stimulation increased the phosphorylation of tomosyn and decreased tomosyn binding to syntaxin-1, resulting in enhanced SNARE complex formation. Overexpression of tomosyn in cultured superior cervical ganglion neurons inhibited neurotransmitter release which could be rescued by the introduction of cAMP into the presynaptic neuron. Expression of tomosyn S724A, a PKA unphosphorylated mutant, or knock-down of tomosyn by siRNA introduction also decreased neurotransmitter release, but was not rescued by cAMP. Under high frequency stimulation, expression of tomosyn S724A and S724D, a PKA phosphorylated mimic mutant, increased the EPSP failure rate and asynchronous EPSPs. These results indicate that tomosyn is a physiologically significant PKA target that controls neurotransmitter release through the regulation of SNARE complex formation. [J Physiol Sci. 2006;56 Suppl:S85]

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Tetraethylammonium (TEA), a K⁺ channel blocker, reportedly induces long-term potentiation (LTP) of hippocampal CA1 synaptic responses in an NMDA receptor-independent manner. However, the characteristics of TEA-induced plasticity and modulation by inhibitory interneurons at CA3 and the dentate gyrus (DG) remain unclear. This study recorded field EPSPs from CA1, CA3 and DG to examine the involvement of GABAergic modulation in TEA-induced synaptic plasticity at each region. In Schaffer collateral-CA1 synapses, bath application of 25 mM TEA for 12 min induced LTP in the presence and absence of 100 μM picrotoxin (PTX), a GABA_A receptor blocker, suggesting little modulation by interneurons in the CA1 region. In CA3, associational fiber (AF)-CA3 synapses showed TEA-induced LTP regardless of PTX, but mossy fiber (MF)-CA3 synaptic plasticity were influenced by PTX application; TEA-induced LTP was detected only in the absence of PTX. In DG, synaptic plasticity was modulated by GABAergic inputs, but characteristics differed between lateral perforant path (LPP) and medial perforant path (MPP). LPP-DG synapses showed TEA-induced LTP in the presence of PTX, but no changes in the absence of PTX. At MPP-DG synapses, TEA-induced long-term depression (LTD) was observed in the absence of PTX, but no changes were seen in the presence of PTX. This series of results demonstrated that TEA-induced plasticity at perforant path-DG synapses and MF-CA3 synapses are modulated by GABAergic inputs, and that the effects of GABAergic modulation on plasticity are inhibitory (LPP- and MPP-DG) or excitatory (MF-CA3). [J Physiol Sci. 2006;56 Suppl:S85]

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Considering the anticonvulsant effects of adenosine and the anatomical connections between piriform cortex (PC) and amygdala, in this study, the effect of kindling implementation on adenosine A1 receptor mediated neuromodulation in PC pyramidal neurons was tested at 24 h and 1 month after amygdala kindling. Field potentials were recorded from layer II of PC following stimulation of the lateral olfactory tract. Obtained results showed that N6-cyclohexyleadenosine (CHA; i.c.v.; 1, 10 and 100 μM, as a selective A1 agonist) reduced A1 slope (as an index of EPSP slope) and B1 amplitude (as an index of spike amplitude) of field potentials in both kindled and non-kindled rats. However, its effects were more potent at 24 h, but not 1 month after kindling. Pretreatment of 1,3-dimethyl-8-cyclohexylexantine (CPT; i.c.v.; 50 μM, as a selective A1 antagonist), eliminated effects of CHA (i.c.v.; 10 μM). These results indicate that A1 receptors of PC have anticonvulsant effects on amygdala kindled seizures and the efficiency of the A1 receptor neuromodulation is increased at short- (24 h), but not long-term (1 month) after kindling implementation. [J Physiol Sci. 2006;56 Suppl:S86]

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The genome project elucidates that the whole genome possesses approximately 25,000 genes. Although it is thought that single cells express approximately 10,000 genes in various copy numbers in a cell-type specific manner, differences of gene expressions inside and among defined cell-types are not examined at a single cell level. Here, we examined mRNA expressions in acute rat hippocampal slices where the classification of cell types was most extensively examined. CA1 and CA3 pyramidal cells, dentate granule (DG) cells and interneurons were identified from their morphologies and the locations and cellular contents were harvested into patch pipettes. We performed RT-multiplex PCR of 31 genes, including biochemical markers of calcium binding proteins, neuropeptides and neurotransmitter receptors. Detection rates of each mRNA were examined. Except for correlations linked to glutamatergic and GABAergic neuronal markers, strong correlations were scarcely found. When cells were grouped according cell types, however, large differences of detection rates in many genes were observed. χ-square analysis revealed that 37% of tested genes were differently expressed in interneurons and principal neurons and that 30% were differently expressed in pyramidal cells and DG cells. Genes differentially expressed between CA1 and CA3 pyramidal neurons were not found. Dendrogram showing the distances of cell types was obtained by cumulative differences of detection rates. [J Physiol Sci. 2006;56 Suppl:S86]

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Small molecule G-protein Arf1 in combination with phospholipase D (PLD) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to Golgi apparatus. However, it is recently reported that it also regulate ionic channel activity presumably though recycling of the receptors and ionic channels at the cytoplasmic membrane. To examine possible involvement of Arf and subsequent PLD in regulation of receptor-induced responses in neuron, we recorded K⁺-current response induced by dopamine (DA) in the ganglion cells of Aplysia under conventional two-electrode voltage clamp. Intracellular application of brefeldin A, a specific blocker of Arf GEF, significantly depressed the K⁺-current response to DA. The DA-induced response was also inhibited by injection of 2-(4-fluorobenzoilamino)- benzoic methyl ester (Exo1), an activator of GAP for Arf1. Intracellular injection of N-terminal peptide of Arf1 markedly suppressed the DA-induced response. In contrast, application of those of Arf6 did not affect the response to DA. Furthermore, intracellular application of α-synuclein, a specific blocker of PLD, significantly depressed the K⁺-current response to DA. In contrast, all these reagents had no significant effect on the Na⁺-current response induced by acetylcholine in the same type of cells. These results suggest that Arf1 and subsequent PLD may regulate the K⁺-current response induced by DA. [J Physiol Sci. 2006;56 Suppl:S86]

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5-hydroxytryptamine (5-HT; serotonin) inhibits transmitter release via activating GTP binding (G) proteins, but the target of this effect in the nerve terminal is not determined. We addressed this question at the calyx of Held synapse in brainstem slices of developing rats. In 5-day-old rats bath-application of 5-HT (10 μM) attenuated the amplitude of evoked EPSCs and facilitated paired-pulse ratio, whereas 5-HT had no effect on the amplitude of spontaneous miniature EPSCs. The 5-HT_1B receptor agonist CP93129 mimicked the inhibitory effect of 5-HT, but the 5-HT_1A agonist 8-OHDPAT had no effect. 5-HT_1B receptor antagonist NAS-181 blocked the inhibitory effect of 5-HT. These results suggest that 5-HT activate 5-HT_1B receptors in the nerve terminal, thereby inhibiting transmitter release. In whole-cell recordings from calyceal nereve terminals, 5-HT attenuated voltage-gated Ca²⁺ currents, but had no effect on voltage-gated K⁺ currents. Upon repetitive application 5-HT showed tachyphylaxis with its effect on both EPSCs and presynaptic Ca²⁺ currents becoming weaker in the second application. Surprisingly 10 mM BAPTA loaded into the nerve terminal abolished the tachyphylaxis. The presynaptic inhibitory effect of 5-HT was robust at postnatal day 5, but became weaker as animals matured. We conclude that 5-HT_1B receptors can mediate presynaptic inhibition of transmitter release in immature calyceal terminals via inhibiting voltage-gated Ca²⁺ channels. Upon repetitive activation 5-HT_1B receptors may be internalized or desensitized by a Ca²⁺-dependent mechanism. [J Physiol Sci. 2006;56 Suppl:S86]

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At the excitatory synapse neurotransmitter glutamate is released from synaptic vesicles. After exocytosis vesicles are retrieved by endocytosis and recycled for reuse, thereby maintaining synaptic transmission. During recycling vesicles are refilled with glutamate by vesicular glutamate transporters (VGLUTs), using electrochemical gradient produced by vacuolar-type H⁺-ATPase (V-ATPase). Multiple recycling mechanisms of different speeds are thought to operate in the nerve terminal. However, the speed of vesicle refilling with glutamate is not known. Here we manipulated vesicular glutamate directly in the nerve terminal, depleting it by whole-cell dialysis and refilling it by the photolysis of caged glutamate. Vesicle refilling, monitored by postsynaptic currents, was abolished by blocking VGLUT or V-ATPase, and attenuated by increasing cytosolic Cl⁻ concentrations. The refilling time constant ranged 2-13 s depending upon the magnitude of refilling, with 100% refilling time constant being 18 s. This rate is faster than the "full-fusion"-type slow recycling time (1 min), but slower than "kiss-and-run"-type fast recycling time (<1s). We conclude that transmitter glutamate can fully refill vesicles recycled via slow, but not fast, pathways. [J Physiol Sci. 2006;56 Suppl:S87]

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In hippocampal CA1 neurons, delivery of low-frequency afferent stimuli <LFS;80 pulses at 1 Hz> induced LTP in both the field EPSP and population spike <PS>. In the same cells, reversal of LTP in the EPSP and PS was achieved by the same LFS given 20 min after the first LFS <second LFS>. We investigated the effects of metabotropic glutamate receptor <mGluR> antagonists on LTP or LTD induced by the second LFS. When the first LFS was given in the standard solution, both the field EPSP and PS were attenuated by the second LFS. In contrast, when the first LFS was delivered in the presence of MCPG, a broad spectrum mGluR antagonist or 4CPG, a type 1 mGluR antagonist, the field EPSP and PS were enhanced by the second LFS. Thus, activation of mGluRs during preconditioning LFS stimulation determines the direction of synaptic plasticity at CA1 neurons. Then, we investigated the effects of an NMDA receptor antagonist, AP5 on synaptic plasticity induced by the second LFS. When the first LFS was given in the standard solution or in 4CPG but the second LFS was given in the presence of AP5, both the EPSP and PS were enhanced after the second LFS. These results indicate that the synaptic plasticity induced by the second LFS depends on NMDA receptor activation at CA1 synapses. Thus, it is possible that activation of mGluRs during prior synaptic activation resulted in enhancement of NMDA receptor activation, leading to reversal of LTP. [J Physiol Sci. 2006;56 Suppl:S87]

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The present study was aimed to elucidate the possible mechanism of glucocorticoids on the conditioned fear extinction by using fear-potentiated startle paradigm. We found that (1) Systemic administration of dexamethasone (DEX, 1.0 mg/kg, i.p.) accelerated extinction of conditioned fear. (2) Administration of glutamate NMDA receptor antagonists (±)-HA966 (6.0 mg/kg, i.p.) and intra-amygdala infusion of MK801 (0.5 ng/side, bilaterally) or D,L-2-amino-5- phosphonovaleric acid (AP5, 2.0 ng/side, bilaterally) blocked the DEX facilitation effect. (3) Blockade of corticosteroid synthesis inhibitor metyrapone (25 mg/kg. s.c.) on extinction was removed by co-administration of NMDA receptor agonist D-cycloserine (DCS, 5.0 mg/kg, i.p.). (4) Co-administration of DEX and DCS in a sub-threshold dose provided a synergistic facilitation effect on extinction (0.2 mg/kg and 5 mg/kg, respectively). However, DEX and DCS co-administration did not alter the expression of conditioned fear. (5) The facilitation effect of DEX was blocked by intra-amygdala infusion of mitogen activated protein kinase (MAPKs) inhibitors PD98059 (500 ng/side, bilaterally) or U0-126 (20 μM/side. bilaterally). DEX significantly enhanced the phosphorylation of MAPKs which induced by the extinction training. These results suggested that glutamate NMDA receptors and MAPKs within the amygdala participated in the modulation effect of glucocorticoids on extinction. [J Physiol Sci. 2006;56 Suppl:S87]

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Drebrin A (DA), an F-actin binding protein, is involved in spine morphogenesis. We have recently demonstrated that knock down (KD) of DA expression attenuates synaptic clustering of PSD-95, and decrease the number of dendritic protrusions in developing hippocampal neurons. Furthermore, in-vivo experiments indicate that DA-KD in the rat hippocampus impairs pre-pulse inhibition. These data suggest that DA plays a role in the regulatory mechanism of glutamate receptor activity in addition to that of spine shapes. To investigate the effect of DA-KD on glutamate receptor activities in vitro, 12-DIV hippocampal neurons were treated with antisense oligonucleotide specific to DA (AOD) and reverse AOD (ROD). Then at 14 DIV, NMDA and AMPA currents in mock-treated, AOD-treated, and ROD-treated neurons were measured using whole cell patch-clamp technique. We applied glutamate (1, 10, 30, 100 and 300 μM) in the presence of 50 μM AP5, and NMDA (1, 10, 30, 100 and 1000 μM) in the presence of 20 μM CNQX. The AMPA currents seemed larger in AOD-treated neurons than mock-treated and ROD-treated neurons. There were no clear differences in the NMDA currents between AOD-treated and ROD-treated neurons. The data suggest that drebrin A is involved in the regulatory mechanism of glutamate receptor activities. [J Physiol Sci. 2006;56 Suppl:S87]

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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, which induces the morphological changes of dendritic spines into filopodia-like protrusion. Timelapse imaging of GFP-drebrin expressing neuron revealed that translocation occurred within 60 sec. Since myosin II B is involved in actin-drebrin complex in the brain, contribution of actin-myosin system to the translocation was investigated in this study. In the present study, we analyzed the effect of blebbistatin, a specific inhibitor of myosin II ATPase, on the glutamate-induced translocation of drebrin and F-actin in cultured hippocampal neurons. We prepared the low density cultures of rat hippocampal neurons from 18-day embryo. After pretreatment of 21 DIV neurons with 100 μM (±) blebbistatin for 60 min, we applied 100 μM glutamate for 10 min and analyzed the location of drebrin and F-actin immunocytochemically. 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. [J Physiol Sci. 2006;56 Suppl:S88]

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Dense expression of cocaine- and amphetamine-regulated transcript (CART) mRNA and peptide in the nucleus accumbens (NA) and striatum (ST) are found. NA and ST are the main projection sites of mesolimbic (ML) and nigrostriatal (NS) dopaminergic (DA) systems which are involved in the extrapyramidal motor system and ewarding and emotional behaviors, respectively. Whether gonadal hormones, estradiol (E) and testosterone (T), play the regulatory roles on the stimulation of CART peptide in the MLDA and NSDA systems were examined in Sprague-Dawley rats in this study. DA neuronal activities were determined by measuring the concentration of DOPAC (3,4-dihydroxyphenylacetic acid), the major metabolite of DA, in the NA and ST by HPLC-ECD. Intracerebroventricular administration of CART peptide increased the DOPAC content of NA and ST in ovariectomized (OVX) priming E, but not in OVX only female rats. The stimulation by CART peptide on the DOPAC contents of NA and ST was found only in intact, castrated (CAS) with E or T priming, but not in CAS only male rats. Finally, E and T antagonists blocked T effects, but only E antagonist could block E effects. All these findings indicate that gonadal hormones play the regulatory roles on the stimulation of CART peptide in MLDA and NSDA systems, and suggest that E is through intracellular genomic rather than extracellular non-genomic action. [J Physiol Sci. 2006;56 Suppl:S88]

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Extracellular recordings in inferotemporal (IT) cortex suggest that there are columnar organizations in terms of the optimal stimuli for individual neurons (Fujita, et al., 1992). This finding is consistent with optical imaging experiments showing that a visual stimulus elicits a localized activation of the spots in IT cortex (Wang, et al., 1996, 1998). However, it is also known that object selectivity of nearby cells is not necessarily the same. Thus, our understanding of columnar organizations in IT cortex is still partial. Here, we identified active spots revealed by optical imaging, and then recorded single unit activities and multiple unit activities (MUAs) from these spots. To quantify the similarity of object selectivity, we calculated correlation coefficients of responses to 100 object stimuli. We found that only 30% of pairs of nearby single units showed significant correlation in object selectivity. However, many pairs of MUAs (63%) showed significant correlation in object selectivity. The difference in number of significant pairs suggests (1) that there is a common property among single cells, and (2) that averaging of responses of individual cells, such as MUA, decreases cell-to-cell variability in object selectivity and disclose common property among cells within a spot. In fact, when all MUAs in a spot were averaged, single unit activity and the averaged MUA showed significant correlation in object selectivity in 63% of single cells within the spot. However, we could not find such significant correlation between the averaged MUA of a spot and single unit activity recorded from outside of the spot. [J Physiol Sci. 2006;56 Suppl:S88]

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The inhibitory effect of pain on tolerance development to analgesic effect of opioids is reported to be mediated by stress aspect of pain and activation of HPA axis. We tried to investigate whether the chronic pain and co-administration of dexamethasone (Dex) is able to reverse the tolerance and to evaluate expression of G_αi/o and G_β subunits of G proteins following chronic pain, chronic Dex, tolerance and their combination.Tolerance was induced by chronic intraperitoneal (i.p.) administration of morphine to male Wistar rats and analgesia was assessed using tail flick test. Lumbar spinal tissues were assayed for expression of G proteins using "semi-quantitative PCR" normalized to beta-actin.Both chronic pain and chronic Dex could reduce and reverse the tolerance. Chronic morphine did not change G_αi/o gene expression, while chronic pain and Dex both increased its expression. Expression of Gβ was increased following chronic morphine, but not following chronic pain and Dex. None of these increases were observed when morphine was co-administered with pain or Dex. It seems that the development of tolerance to analgesic effect of morphine is partially mediated by increased G_β gene expression. The increase in G_αi/o genes expression produced by chronic pain and chronic Dex can facilitate opioid signaling pathway and compensate for morphine-induced tolerance. [J Physiol Sci. 2006;56 Suppl:S89]

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Recent brain imaging studies have been revealing central processing of muscle pain. However, there is hardly a consensus on a brain region specifically responsible for muscle pain, not for skin pain. To seek out this specific region, we used event-related functional magnetic resonance imaging (MRI) and the 3-T MRI scanner. Twelve healthy male subjects participated in this study. Electric stimulation with 1-ms duration was applied to two sites: the left anterior tibial muscle and the skin just above it. The stimuli consist of three levels in strength both for skin and muscle stimulation, i.e. nonpainful, painful (5/10 in visual analog scale, VAS) and more painful (7/10 in VAS). Group analysis revealed that brain regions activated by muscle stimulation included ipsilateral superior frontal gyrus, postcentral gyrus (primary somatosensory cortex, SI), posterior cingulate; contralateral precentral gyrus, medium dorsal nucleus (thalamus), superior temporal gyrus; bilateral middle frontal gyrus, cingulate gyrus and lenticular nuclei. These regions are similar to those reported by other researchers and some of them are known to be activated during pain. Brain regions specifically activated by muscle stimulation, not by skin stimulation, were ipsilateral culmen, cingulate gyrus; contralateral lenticular nucleus, pons, paracentral lobule (SI), substantia negra, medial frontal cortex and bilateral middle temporal gyrus. These regions may play an essential role to distinguish muscle pain from skin pain in the brain. [J Physiol Sci. 2006;56 Suppl:S89]

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To date, eight subtypes of metabotropic P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃ and P2Y₁₄ receptors, have been identified in mammals. Although numerous studies have demonstrated that ionotropic P2X receptors play a crucial role in facilitating pain transmission in the spinal cord, possible roles for P2Y receptors in nociceptive signaling have received limited attention. In this study, we examined whether the activation of P2Y receptors can modulate synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices using whole-cell patch-clamp recordings. Bath application of UTP (100 μM), an agonist for P2Y₂ and P2Y₄ receptors, and UDP (100 μM), an agonist for P2Y₆ receptors, affected neither excitatory (glutamatergic) nor inhibitory (glycinergic and/or GABAergic) synaptic transmission in all 11 SG neurons recorded. 2-Methylthio ADP (30 μM), an agonist for P2Y₁, P2Y₁₂ and P2Y₁₃ receptors, also did not change excitatory transmission in all 11 SG neurons examined, but significantly increased the frequency and amplitude of spontaneous inhibitory (glycinergic and/or GABAergic) postsynaptic currents in 12 of 52 SG neurons recorded. These results indicate that the activation of 2-methylthio ADP-sensitive P2Y receptors enhances inhibitory but not excitatory synaptic transmission, probably through a direct action on spinal inhibitory interneurons. Thus, in contrast to P2X receptors, P2Y receptors can be involved in an inhibitory effect on pain transmission in the spinal dorsal horn. [J Physiol Sci. 2006;56 Suppl:S89]

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Previous studies show that the spinal cord and subcortical components of the somatosensory evoked potentials are not attenuated during movement in man. The objective of the present study was to investigate the effect of simultaneous fractionated finger movement on cortically generated somatosensory evoked potentials. Left median nerve somatosensory evoked potentials were recorded from a scalp array of 21 electrodes over the right scalp in 9 normal subjects aged 18-31 years. They reclined, eyes closed as recordings were made at rest and during fractionated finger movements of the left hand. Latency and peak-to-peak amplitude of the parietal N20 and frontal P20 waves did not show statistically significant difference between rest and movement conditions. This represents activity in the tangentially arranged pyramidal neurons in area 3b. Amplitude of the parietal P25 wave, representing activity in radially arranged area 1 pyramidal neurons was attenuated during finger movement by 70% (P<,0.01; Wilcoxon's test). Fractionated finger movements utilise the corticospinal pathway from the motor cortex. In animal studies it has been shown that there is evidence for an inhibitory cortico-cortical pathway from area 4 (motor) to area 1 (sensory) cortex but not to area 3b. We propose that activity in this pathway is responsible for our findings. This can be considered as a physiological evidence for the function of a cortico-cortical pathway in man. [J Physiol Sci. 2006;56 Suppl:S89]

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Interhemispheric neural interactions between bilateral cortical regions are critically dependent on interhemispheric time lag of cortical activation. In somatosensory systems the time lag information is processed for integrating bilateral stimulus inputs. Here we investigated interhemispheric interactions between bilateral somatosensory cortices by simultaneous recording of neural and hemodynamic signals and by analyzing their dependence on stimulus time lag between conditioning stimuli (CS) and test stimuli (TS). We measured electrophysiological signals (local field potentials, 1<LFPs<100 Hz; multiunit spiking activity, 300<MUA<5k Hz) and optical intrinsic signals (586 nm, cerebral blood volume, CBV; 605 nm, oxygenation) in rat somatosensory cortex evoked by electrical pulses to the contralateral hindpaw (TS) while delivering electrical pulses to the ipsilateral hindpaw (CS) under α-chloralose anesthesia. Both responses to CS-TS were normalized by the responses to TS without CS. The results showed that both electrophysiological (LFPs and MUA) and optical (586 nm monophasic and 605 nm biphasic activity) responses were significantly suppressed around 40-60 ms time lag and slightly augmented at 0 ms. Average and trial-by-trial correlation analyses revealed that CBV-related optical signals have high fidelity to integrated MUA and LFP negative components. Activity-related microcirculatory responses may more faithfully reflect neural interactions through brain network than we imagined. [J Physiol Sci. 2006;56 Suppl:S90]

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TRPV4 is first reported to be a "hypoosmolality-sensing" cation channel. On the following studies with knockout mice (Trpv4-/-), we have reported that response of vasopressin to hypertonicity was exaggerated but another group has reported that it was abolished in Trpv4-/-. Although controversial in response, both reports suggest that TRPV4 can be responsible to hypertonic stimuli. To elucidate "hyperosmolality-sensing" in TRPV4, we designed to re-examine the response in vivo and investigate whether TRPV4 was sensitive to hyperosmolality in cultured neuronal cells. Trpv4-/- and Trpv4+/+ mice were subjected to dehydration from 24 to 96 hrs. Then serum osmolality and water intake were measured. There was not remarkable difference in serum osmolality at any period of dehydration but a significant decrease in serum osmolality of Trpv4-/- at 72 hrs dehydration. Water-crave behavior and amount of water intakes after the dehydrations were not changed. Thus TRPV4 channel may respond to hyperosmolality. Neuronal cell lines with and without TRPV4 were established from a cell line. Hyperosmoliality (500 mOsm) induced robust Ca influx in TRPV4 (+) cells by the method of fluorescence quenching, while not in TRPV4 (-) cells. The influx was partially blocked with genistine, a blocker of tyrosine kinase, and blunted with pBPB, a blocker of PLA2. Therefore, TRPV4 is hyperosmolality-sensng channel through several biochemical cascades. [J Physiol Sci. 2006;56 Suppl:S90]

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Molecular mechanisms underlying olfactory signal amplification were investigated by monitoring cAMP dynamics in the intact sensory cilia. [cAMP]i increased superlinearly with time during odorant stimuli for over a second. This time course was remarkably different from that obtained with the rapid quench method previously applied to the in vitro preparation, in which [cAMP]i change has been reported to be transient. The superlinear increase of [cAMP]i was due to a gradual increase of cAMP production rate that was consistent with the thermo-dynamical interaction model between elemental molecules, as has been revealed on the rod photoreceptor cell. It thus seems likely that the fundamental mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (–200,000 molecules/s/cell at the maximum), in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique mechanism would be essential to reduce the loss of ATP. Transduction by a smaller number of second messenger formations would be achieved by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired high amplification at the final stage of transduction channels, utilizing Ca²⁺ as a third messenger. [J Physiol Sci. 2006;56 Suppl:S90]

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The effect of unsaturated fatty acids on taste sensitivity was explored by human psychophysical study, molecular biological study and mouse neuroethological study. Inhibitory effects of fatty acids on bitter taste including QHCl and denatonium were demonstrated in perceived taste intensity test in human, the mouse behavioral experiment using a short -term lick test (10 sec) and responses in the chorda tympani (CT) and the glossopharyngeal (GL) nerve in wild type mice. In contrast, fatty acids have no effect on bitter tasting L-amino acids, NaCl, HCl, sucrose and MSG in all study performed. To investigate involvement of Ggustducin in this inhibitory effect of fatty acids, nerve recording from gustducin KO mice and in vitro G-protein activation assay using bovine taste membrane were employed. Results from gustducin KO mice showed no suppression in bitter taste responses both in the CT and the GL. In vitro G-protein activation assay using bovine taste membrane showed that the activation of both gustducin and transducin by denatonium was significantly inhibited by DHA and oleic acid, and that the activation of transducin by rhodopsin was not inhibited. These results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of T2R receptors which coupled with Ggustducin and Gtransducin. [J Physiol Sci. 2006;56 Suppl:S90]

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It has been suggested that pH of the invaginating synaptic cleft of the cone terminal is related to the membrane voltage of horizontal cells (HCs), low in the dark and high during surround illumination (1). High pH enhances the release of L-glutamate from the cone terminal, resulting in the formation of the centre-surround receptive field and the colour opponency in the visual system. We measured the pH of the immediate external surface (pH_o) of an HC isolated from carp or goldfish retina to study the mechanisms producing the pH change by a ratio imaging technique, using a pH-sensitive lipophilic dye, 5-hexadecanoylaminofluorescein. When HCs were depolarized by application of 20 μM kainate or by high extracellular K⁺, pH_o acidified. The amount of pH acidification was monotonically dependent on the depolarization, as much as 0.21±0.05 pH unit by 100 mM K⁺ (approx. 94 mV depolarization). Acidification of the HC surface was suppressed by 0.4 μM bafilomycin A1, a specific inhibitor of vacuolar type H⁺-ATPase (V-ATPase), suggesting the existence of an outward electrogenic H⁺ pump enhanced by the HC depolarization. These are consistent with the hypothesis that proton released from the depolarized HCs can act as the inhibitory feedback transmitters onto cone synaptic terminals. (1):Hirasawa, H. and Kaneko, A. (2003) J.Gen.Physiol.,122:657-71. [J Physiol Sci. 2006;56 Suppl:S91]

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Retinal amacrine cells (ACs) regulate activities of retinal ganglion cells, the output neurons to higher visual centers, through cellular mechanism of lateral inhibition in the inner plexiform layer (IPL). Electrical properties of gap junction networks between ACs in the IPL were investigated using combined techniques of intracellular recordings, Lucifer yellow and Neurobiotin injection, dual patch-clamp recordings and high voltage electron microscopy in isolated retinas of cyprinid fish. Six types of gap-junctionally connected ACs were classified after recordings of their light-evoked responses to light flashes. Among them gap junction networks of three types of ACs were studied with structure-function correlation analysis. Cellular morphology of intercellular connections between three homologous cell classes was characterized. High voltage electron microscopy (Hitachi 1250M, NIPS, Okazaki, co-operative program 2005-HVEM02) revealed localization of gap junctions between the dendritic tips of Neurobiotin-coupled cells. Receptive field size, space length constant, response latency and conduction velocity were measured. Simultaneous dual patch-clamp recordings revealed that the lateral gap junction connections between homologous ACs expressed bidirectional electrical synapses passing Na⁺ spikes. Lateral inhibition regulated by ACs in the IPL appears to be associated with directional extension of the dendrites and orientation of dendrodendritic gap junctions (J. Integr. Neurosci. 4(3): pp 313-340, 2005). [J Physiol Sci. 2006;56 Suppl:S91]

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Recent studies showed that the cholinergic amacrine cells have a unique membrane properties but the membrane properties of the cholinergic amacrine cells have not been characterized systematically. Here, we studied the voltage-gated ionic channels of transgenic mouse line whose cholinergic amacrine cells were selectively labeled with GFP electrophysiologically and immunohistochemically. Voltage-gated K currents were inhibited by 4-aminopyridine (A current) and tetraethylammonium (delayed rectifier). Voltage-gated Ca currents had &omega-conotoxin GVIA-sensitive component (N-type) and &omega-AgaIVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na current and dihydropyridine-sensitive Ca current (L-type) were not observed. The immunoreactivity for K channels subunits (Kv. 3.1 (delayed rectifier) and Kv. 3.3 (A-current)) and Ca channel subunits (&alpha1A (P/Q-type) and &alpha1B (N-type)) was colocalized with GFP signals. Immunoreactivity for Ca channel subunits (&alpha1C (L-type)) did not colocalize with GFP signals. Immunoreactivity for Na channel subunit existed in the nuclear region but not in the cell surface of the GFP positive cells. Our findings indicate that the signal propagation of the cholinergic amacrine cells are mediated by the combination of the voltage-gated K channels (A-type K current and delayed rectifier K current) and the voltage-gated Ca channels (P/Q-type and N-type) in the mouse retina. [J Physiol Sci. 2006;56 Suppl:S91]

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We examined whether Y39983, a novel Rho/ROCK inhibitor, can make injured RGC axons regenerate into the crushed optic nerve (OpN) of adult cats. Methods: In vitro: retinal pieces were cultured with Y39983 for 14 days to count number of TUJ-1-positive neurites. In vivo: the left OpN of anesthetized cats was crushed with 0.2 N tension for 60 s. On day 12, 0.5 mg of WGA-HRP was injected into the vitreous to label regenerated axons. On day 14, the cats were perfused with fixative, the OpN was dissected, embedded in gelatin. Frozen sections were reacted for HRP with TMB. Results: Retinal culture: To obtain the optimum concentration for axonal regeneration, we examined effect of Y39983 on neurite outgrowth of cultured retinal pieces. The number of TUJ-1 positive processes was greatest at the concentrations of 3 and 10 μM in central to peripheral retinal areas. Similarly, length of neurites in retinal pieces was longest at 3 and 10 μM. Axonal regeneration in crushed OpN: We injected Y39983 at 10 and 100 μM. An injection of 10 μM Y39983 increased regenerated axons longer than 0.5 to 2 mm from the crush site. The second injection of 10 μM Y39983 at day 7 increased the number 2 to 3.5 fold than the number in one injection. Single or double injections of 100 μM 39983 increased the number of regenerated axons. Conclusion: A Rho/ROCK inhibitor, Y39983, enabled injured axons of RGCs of adult cats to regenerate into the crushed OpN. [J Physiol Sci. 2006;56 Suppl:S91]

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Previous studies have shown that sustained-response neurons in the primary auditory cortex (A1) of awake animals have sensitivities to the spectral-edge and fundamental frequency (F0) and that the sensitivity originates from the specific inhibition pattern of frequency receptive field (FRF). This study investigated cell-type correspondence between the different experiments by estimating the excitatory and inhibitory summation patterns for harmonic components with a given F0. The findings show that there are, at least, four types of sustained-response neurons sensitive to specific spectral features of the complex tone: energy-integrator cells integrate sound energy on the excitatory FRF; high-edge-sensitive cells are sensitive to the spectral high edge on the best frequency (BF); low-edge sensitive cells detect the spectral low edge on BF; F0-sensitive cells are sensitive to two F0s of harmonics corresponding to BF and an octave below, but not to noise with a similar spectral location. The spectral-cue sensitivity originates from specific inhibitory FRF: less dominant inhibition for energy-integrator cells, asymmetric inhibition for high- and low-edge-sensitive cells, and selective inhibition of non-preferred harmonics for F0-sensitive cells. A1 operates as filters with pass bands and reject bands, which correspond to the peak and trough in FRF. The filter specification is well organized for decoding three acoustic features: sound energy in a given spectral region, spectral edges, and F0 of harmonics. [J Physiol Sci. 2006;56 Suppl:S92]

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We have analyzed traces of vergence eye movements while subjects watch three-dimensional (3D) and two dimensional (2D) movies, and found that 2D images without binocular disparity sometimes evoked convergence similar to those found in the 3D condition. In this study, we investigated factors that drove convergence other than binocular disparity. As previously presented, 3D/2D movies were projected on an 80 inch-screen in a dark room (10 lux) by two liquid crystalline projectors with/without polarized filters to provide binocular disparity, field-sequentially. To monitor the gaze of the subject, we measured binocular eye movements and head movements with a new binocular video oculography (Newopto) and a magnetic motion sensor (Polhemus). A 2D movie representing driver's view of a go-cart (160 sec), which included forward scenes and expanding optic flows, was shown to fifteen subjects. The motion vectors localized to their gaze points were calculated, and the optic flow was estimated based on flame-by-flame analyses of images. The traces of vergence eye movements consisted with the changes in the optic flow. It is suggested that most of factors that drove convergence in 2D movies are closely related to optic flow. [J Physiol Sci. 2006;56 Suppl:S92]

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It was reported that after the lesion of the corticospinal tract (CST) at C5, recovery of the finger dexterity was completed in 1-3 months (Sasaki et al. 2004, Nishimura et al. 2005). In this study, to clarify the neuronal mechanism of the functional recovery, we performed following two experiments; 1) In 3 monkeys, we performed functional imaging by positron emission tomography (PET) during precision grip. At postoperative 1 month (during early recovery), increased activity was observed in bilateral primary motor cortex (M1). At postoperative 3 months (after complete recovery), in addition to bilateral M1, activities in bilateral ventral premotor cortex (PMv) were increased. 2) In 2 monkeys we investigated the effect of reversible inactivation of areas where we found increased activity in the PET study. Inactivation of M1 contralateral (c) to the lesion caused deficit in control of independent finger movements both at postoperative 1 and 3 months. Inactivation of M1 ipsilateral (i) to the lesion caused deficit in precision grip at postoperative 1 month. Inactivation of iPMv also caused deficit in precision grip at postoperative 3 months. These results indicate that bilateral M1 and iPMv play an important role in the functional recovery. [J Physiol Sci. 2006;56 Suppl:S92]

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The midbrain locomotor region (MLR) is located in the lateral part of the mesopontine tegmentum. Either electrical or chemical stimulation of the MLR first increases the level of postural muscle tone and then initiates locomotion. The MLR receives GABAergic basal ganglia output from the substantia nigra pars reticulata (SNr). The present study was designed to understand synaptic mechanisms acting on motoneurons involved in the basal ganglia control of locomotion. Intracellular recording was made from hindlimb motoneurons (n=47) in two types of decerebrate cats (n=11). One was mesencephalic cats decerebrate at precollicular-postmammilarly level, and the other was hypothalamic cats decerebrated at precollicular-premammilarly level. In mesencephalic cats, short trains of stimuli applied to the MLR (3 pulses, 5 ms intervals, 30-50 μA) induced a sequence of EPSPs and IPSPs. Although stimulation of the SNr (20-60 μA, 50-100 Hz) alone neither changed membrane potentials nor input resistance of motoneurons, it greatly reduced the amplitude of the MLR-induced IPSPs, resulting in an enhancement of the EPSPs. In hypothalamic cats, fictive locomotion, sequences of membrane oscillations with depolarizing and hyperpolarizing phases, was induced in motoneurons. SNr stimuli reduced the hyperpolarizing phases and finally stopped the oscillations of both extensor and flexor motoneurons. These results suggest that enhancement of the basal ganglia output to the brainstem may stop locomotion by suppression of the postsynaptic inhibitory drive acting on motoneurons and initiate locomotion by removal of the inhibition. [J Physiol Sci. 2006;56 Suppl:S93]

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For pursuit of small objects moving slowly and smoothly in space close to the observer, two independent eye movement systems are used: frontal smooth pursuit and vergence-tracking. Signals for both systems must be synthesized for pursuit of a target moving in three dimensions. Recent studies in our laboratory have demonstrated that among the cerebral cortical pursuit areas, three dimensional (3D) pursuit signals are generated primarily in the frontal eye fields (e.g. Akao et al. 2005). To drive ocular motoneurons, 3D pursuit signals must be sorted into signals for each eye movement system and finally into oculomotor signals. Studies in our laboratory indicate that such conversion was not detected in the cerebeller floccular region (Tsubuku et al. Soc Neurosci Abstr 2004). The cerebellar dorsal vermis is well known as another pathway for frontal pursuit. To examine whether vergence signals are present in this area, we examined simple-spike discharge of vermal pursuit Purkinje (P-) cells in 2 monkeys. Of a total of 64 P-cells that were examined during both frontal pursuit and vergence-tracking, 50% discharged for both, 37.5% only for vergence-tracking, and 12.5% only for frontal pursuit. These results indicate that about 90% of vermal pursuit P-cells discharged for vergence-tracking and that half of them still had 3D pursuit signals. Majority (71%) of these P-cells discharged before onset of vergence eye movements with the typical lead time of 50 ms, suggesting their involvement in the initiation of vergence eye movements. [J Physiol Sci. 2006;56 Suppl:S93]

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Initial tracking responses were larger when smooth pursuit eye movements were executed after a steady fixation and the initial fixation target disappeared before the onset of the pursuit target motion (Miura et al. JJP, 55 (Suppl), 2005). To study whether this phenomenon is common in the genesis of visually-guided reflexive ocular behaviors, we observed human ocular following response, a reflexive eye movement elicited by the motion of a wide-field visual stimulus. At the beginning of each trial, a stationary fixation point (placed at the center of the screen) and a stationary random-dot pattern (covering the wide visual field) were presented. The subjects fixated the fixation point and immediately (no-gap condition) or 200 ms after the extinction of the fixation point (gap condition), the random-dot pattern moved briefly at 20 deg/s rightward or leftward for 0.2s, and then turned off. The latency of the ocular following responses elicited by the random-dot pattern motion was not affected by the presence of the gap. In all 3 subjects, the change in eye position during the open-loop period of the ocular following responses was significantly larger under the gap condition than under the no-gap condition. The effect of the gap on the ocular following responses started about 15 ms after the onset of the ocular following responses. This result suggests that the efficacy of visuomotor transmission for ocular following responses was facilitated by the release from the fixation before the onset of the random-dot pattern motion, as was seen in the smooth pursuit initiation. [J Physiol Sci. 2006;56 Suppl:S93]

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During food intake 2-3 mM glucose concentration in CSF become twice. When 6 mM glucose is injected into the hippocampus spatial learning and memory are facilitated. We analized this mechanism. In vitro experiments, glucose concentration in Krebs Ringer solution was changed from 3 to 6 mM for15 min and returned to the original 3mM glucose. By the change the amplitudes of CA1 synaptic potentials were augmented more than 3 times and continued for more than 40 min. Furthermore pre- synaptic transmitter release and postsynaptic responses to NMDA applied at the apical dendrites were also significantly facilitated. Neurochemically phosphorylations of presynaptic synapsin 1-3, postsynaptic PKC&alpha, ERK, CaMK II of CA1 neurons were all significantly facilitated by the glucose change.LTP of CA1 neurons produced by a tetanic stimulation of the Schaffer collateral / commissure whichwas applied just before returning from 6 to 3 mM glucose was significantly facilitated, while only STPwas produced in 3 mM glucose. During LTP meintenance only phosphorylations of MARCKS (related to plasticity) and PKC&alpha were significantly facilitated. We are now studying the effect of blockers of ATP sensitive K channels on CA1 neurons.These evidences indicate the importance of food intake for reinforcement of the higher brain function.. [J Physiol Sci. 2006;56 Suppl:S94]

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We have previously shown that the defense response against stressor was attenuated in prepro-orexin gene knockout mice and orexin neuron-ablated mice (Jpn J Physiol 55: S87, 2005). We have proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. It is still open question, however, how information of stressor activates the orexinergic neurons. In this experiment, we examined possible contribution of the amygdala as one of the afferent nuclei to activate orexinergic neurons. In urethane-anesthetized mice, a GABA-A receptor antagonist, bicuculline, was microinjected into the amygdala, of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiratory frequency. Bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated mice and wild-type controls. However, dose-response curve was rightward shifted in the orexin neuron-ablated mice. We conclude that the amygdala constitutes one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. [J Physiol Sci. 2006;56 Suppl:S94]

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The cholinergic neurons in the brainstem (laterodorsal tegmental nucleus: LDT) have a role in the regulation of REM sleep. M. Schmidt have suggested that the cholinergic input to the preoptic area is crucial for induction of penile erection during REM sleep. In the present study, we examined the possible involvement of the cholinergic LDT neurons in the regulation of penile erection during REM sleep. Unanesthetized, head-restrained rats were used. Single neuronal activity was recorded through a glass pipette electrode. Penile erection is composed of two components; slow increase of the corpus spongiosum penis (CSP) pressure and sharp peaks riding on the slow increase. Several types of the LDT neurons showed firing patterns in close relation with penile erection including 1) the cholinergic neurons which showed tonic firing increase 20-30 sec before the erection. The firing increase of this type of neurons was well correlated with the slow increase of CSP pressure. 2) the cholinergic neurons which showed phasic firing in synchronous with the sharp CSP peaks. 3) the non-cholinergic neurons which decreased or stopped firing during erection. These results suggest that the cholinergic neurons in the LDT are involved in induction and excursion of penile erection during REM sleep, while the non-cholinergic neurons have inhibitory influences on penile erection. [J Physiol Sci. 2006;56 Suppl:S95]

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This report deals with effects of five basic taste stimuli on the activity of sympathetic and vagal nerves innervating visceral organs in anesthetized rats. Under urethane anesthesia, basic taste stimuli such as sweet (10% sucrose or 5% glucose), or salty (5% NaCl), sour (0.6% acetic acid), bitter (0.2% quinine sulfate)and Umami (2.8% MSG) were applied into the oral cavity for 10 minutes. Efferent nerve activity was recorded from central cut end of sympthetic branches (adrenal and WAT) and vagal branches (gastric and celiac). Results: Sensory stimulations with four basic taste stimuli (salty, sour, bitter and umami) evoked an activation of sympathetic nerve activity to adrenal gland and WAT, however,taste stilulation with sweet taste caused a suppresseion in sympathetic efferents as well as vagalgastric efferents. On the contrary, vagal celiac efferents were activated by sweet taste and suppressed by other stimuli (salty, sour, bitter. and UMami). These observtions suggest that the visceral functions are regulated by taste stimulithrough autonomic outflows. [J Physiol Sci. 2006;56 Suppl:S95]

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The spinal and supraspinal components of both A- and C- reflexes were studied in the somato-ovarian sympathetic reflex discharges elicited by a single shock either to a spinal (T9-11) afferent nerve or to a limb (tibial) afferent nerve in urethane anesthetized rats. In central nervous system (CNS) intact rats, a single shock to a T9-11 spinal afferent nerve produced early and late A-reflex discharges with latencies of about 51 ms and 117 ms, respectively, and a C-reflex with a latency of about 200 ms in a ovarian sympathetic efferent nerve. After spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an A-reflex with the same latency as the early A-reflex in CNS-intact rats and a C-reflex discharge with a latency of about 112 ms. On the other hand, a single shock to a tibial afferent nerve evoked an A-reflex discharge with latency of about 91 ms, and a C-reflex discharge with a latency of about 228 ms in CNS-intact rats. In most cases, the A-reflex could be divided into two subcomponents of different latencies. These A- and C- reflex discharges elicited by stimulation of a tibial afferent nerve were not observed after spinalization. It was concluded that ovarian sympathetic A- and C- reflex discharges evoked by stimulation of a segmental spinal afferent nerve in CNS-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. [J Physiol Sci. 2006;56 Suppl:S95]

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We have demonstrated the distribution of nNOS neurons in the brainstem and diencephalon, which was upregulated in hypertensive Dahl-salt sensitive (DSS) rats compared with normotensive DSS rats. In this study, we directly compared nNOS activity in the brainstem and diencephalon between hypertensive DSS and normotensive Sprague-Dawley (SD) rats. The DSS and Dahl-salt resistant (DSR) rats were fed on 8% NaCl food (DSS8% and DSR8%) or 0.4% NaCl (DSS0.4% and DSR0.4%). SD rats were fed only on 0.4% NaCl food (SD0.4%). The level of nNOS activity in the brainstem of SD0.4% was almost the same as that of DSS8%, which had been significantly higher than those of DSS0.4%, DSR0.4% and DSR8% (normotensive Dahl rat strain). Although the level of nNOS activity in the diencephalon of SD0.4% was significantly higher than that of DSS8%, which had been almost the same as those of the normotensive Dahl rat strain. All these results indicated that at normal blood pressure, the nNOS neuronal system in both the brainstem and diencephalon of Dahl rat strain might be downregulated compared with SD rat strain. In hypertensive DSS rats, the nNOS neuronal system specifically in the brainstem seems to be reversed to the level in SD rats, although the nNOS neuronal system in the diencephalon stays to be downregulated compared with SD rats. [J Physiol Sci. 2006;56 Suppl:S95]

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Feedforward control of human thermoregulation by skin sympathetic nerve activitySatoshi IWASE1, Naoki SAWASAKI2, Daisaku MICHIKAMI3, Tadaaki MANO4, Junichi SUGENOYA1, Jian CUI51. Department of Physiology, Aichi Medical University, 2. Department of Surgery, Tokai Hospital3. Ohtsuka Pharmaceutical Co. Ltd.4. Director, Tokai Central Hospital5. Pennsylvania State UniversityTwo ways of thermoregulation has been recognized, feedforward and feedback mechanisms. Feedforward mechanism employs neural afferent pathway, whereas feedback uses convection of the blood stream from the peripheral to the core. We investigated the relation between the microneurographically recorded skin sympathetic nerve activity (SSNA) and the tympanic temperature (Tty) measured as the core. Four exposure conditions, 1) local cold, 2) generalized cold, 3) local heat, 4) generalized warming, were loaded to the subjects and the response of SSNA and Tty were analyzed in time series analysis. The abilities to activate and to suppress SSNA were correlated to the changes in Tty, indicating that the individuals who are excellent in activating/suppressing SSNA are excellent in thermoregulation. The time lag of neural activation in Tty was within 1 min, whereas that of convectional Tty change was approx. 10 min. We concluded that skin sympathetic regulation is critical in rapid thermoregulation in humans. [J Physiol Sci. 2006;56 Suppl:S96]

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CO₂ water immersion at 1000 ppm affects thermoregulation through the increased cutaneous blood flow on the immersed skin area. However, the effect of CO₂ water immersion on cardiovascular function remains to be clarified in humans. To examine whether CO₂ bathing affects cardiac autonomic function, we analyzed heart rate variability and measured the cardiac output by Echo (Apio XV, Toshiba, Japan) during CO₂ and fresh water immersion. Tympanic temperature (thermistor thermometry), cutaneous blood flow (laser-Doppler flowmetry) and electrocardiogram (ECG) were monitored continuously. The subjective thermal and comfort sensations were asked every 10-min during experiments. After a rest for 10 min, the subject immersed up to the breast level to CO₂-rich water at 1000ppm or fresh water at thermoneutral water. The results were shown as follows: 1) HF was significantly higher in CO₂ water immersion than in fresh water immersion. 2) LF/HF ratio was significantly lower in CO₂ water immersion than in fresh water immersion. 3) Heart rate was not significantly different between CO₂ water and fresh water immersion. 4) Tympanic temperature was significantly lower during CO₂ water immersion than during fresh water immersion. 5) Cutaneous blood flow in immersed forearm was significantly higher during CO₂ water immersion than during fresh water immersion. [J Physiol Sci. 2006;56 Suppl:S96]

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Although dramatic changes of lighting environments are occurring in modern society, the influence of these changes has not been fully elucidated. It is demonstrated that, early life experiences (i.e. maternal care) induce various changes in the function of circadian rhythm, emotionality and memory. Here, we examined whether ELC affected circadian rhythm and maternal care of dam, and the offspring's circadian rhythm, anxiety and memory. Prolonged dark phase (PDP) was used to study the effects of ELC. Dams and their litters were kept in a PDP cycle (L/D=6:18h; postnatal days 2-14) or under a normal condition (L/D=12:12h). Throughout this period, locomotor activity of dams was measured and all cages were video recorded for behavioral scoring. At age of 10-week, behavioral observations of the offsprings were undertaken. Circadian rhythm of locomotor, avoidant behavior, social interaction and object recognition memory were examined. Under PDP, the morning offset of dams' motor activity was delayed and amounts of maternal care decreased for the first 2-7 days of lactation. In the adult offspring, circadian rhythm of locomotor was not affected. Whereas PDP increased avoidant behavior, PDP decreased social interaction and memory. In conclusion, the PDP mothers showed impaired circadian rhythm and maternal care, and the offsprings exhibited depressive-like behaviors. Therefore, it is conceivable that ELC may alter mother-infant interaction and subsequently change the offsprings' emotionality. [J Physiol Sci. 2006;56 Suppl:S96]

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We examined the neural mechanism of discriminative taste preference learning, using the procedure with some modifications of flavor-postingestive consequence learning paradigm in C57BL/6 male mice. Wild mice were allowed to drink water for 10 min daily with the two-bottle method after 16 h water- and food-deprivation. When mice were alternately exposed to sucrose (Suc, 0.5 M) or saccharin (Sacch, 5 mM) instead of water during 2 weeks, the amount of 0.5 M Suc consumption markedly increased but that of 5 mM Sacch did not. Because naive mice prefer to 0.5 M Suc much more than 5 mM Sacch, we employed 0.15 M Suc, to which mice showed the same preference as 5 mM Sacch, instead of 0.5 M Suc. The amount of 0.15 M Suc tended to increase with decreasing of the Sacch-consumption only when the intragastric injection of 0.5 M Suc was done. These results suggest that mice can discriminate between subtle difference cues of Suc from Sacch, mediating the association with intragastric sensory feedback in the brain. [J Physiol Sci. 2006;56 Suppl:S97]

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Major components of energy homeostasis are subjected to circadian regulation that synchronizes energy intake and expenditure. Recently, relationship of circadian clock and lipid metabolism is highlighted. The CLOCK transcription factor is a key component of the molecular circadian clock. Adipocytes play essential metabolic roles not only serving as energy reserves but also secreting hormones and cytokines that regulate metabolic activities. Clock mutant mice were fed with high fat diet for 13 weeks, and lipid metabolism was investigated. Both wild type and Clock mutant mice gained body weight. But, in Clock mutant mice, increases of body weight and of adipocyte tissue were significantly attenuated. In Clock mutant mice, total cholesterol of plasma and liver, and triglyceride of liver were significantly lowered. Again, we examined clock controlled gene mRNA in the adipocyte by real-time RT-PCR. Plasminogen activator inhibitor type 1 (Pai-1) which is related to cardiac infarction was significantly down-regulated in Clock mutant mice. As a summary, we showed that Clock mutant mice may have abnormal lipid metabolism. [J Physiol Sci. 2006;56 Suppl:S97]

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It is suggested that the GABAergic system in the ventral pallidum (VP) plays a role in taste palatability. Taste palatability shift occurs as a function of conditioned taste aversion (CTA). To elucidate the role of VP GABAergic system on CTA, we examined the effects of microinjections of a GABA_A receptor antagonist, bicuculline, on the retrieval of CTA memory. We measured consumption of conditioned stimulus (CS) using one-bottle test (Experiment 1) and observed the ingestive or aversive behavior to CS using taste reactivity test (Experiment 2). Rats received 5 mM saccharin or 0.3 mM quinine hydrochloride as CS, immediately followed by an i.p. injection of 0.15 M lithium chloride (20 ml/kg). After this conditioning, vehicle or bicuculline (12.5–200 ng) was bilaterally infused into the VP immediately before re-exposure to the CS. In Experiment 1, the bicuculline microinjections significantly increased the intake of the saccharin CS, but not the QHCl CS. In Experiment 2, while the control rats infused with vehicle showed a variety of aversive responses (e.g. gaping, chin rubbing, head shaking, forelimb flails), the rats infused bicuculline failed to show these aversive responses. These results indicate that the blockade of GABA_A receptors in the VP attenuates aversion to saccharin CS, and this may be due to elimination of aversive responses. Thus, it is suggested that the GABAergic system in the VP plays a critical role in the expression of CTA. [J Physiol Sci. 2006;56 Suppl:S97]

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The experiment aimed at knowing whether exercise stress on acute phase proteins may be influenced differently, depending on bright (5,000 lx) or dim (50 lx) light exposure during a day.Eight healthy women volunteered as the subjects. The subject entered a bioclimatic chamber at 08:00 h on first day.The light intensity measured at eye level from 08:00 to 18:00 hours was either 50 lx in the dim light condition (first day) or 5,000 lx in the bright light condition (second day), 10 lx from 18:00 to 23:00 hours.Subjects exercised moderately for 20 min by a bicycle ergometer with 60 W intensity.Blood samples were drawn 30 minutes later after the end of exercise on first, second and third day. Concentrations of&alpha1-antichymotrypsin (ACT), transferin (Tf), &alpha2-macroglobulin (&alpha2-M) and haptoglobulin (Hp) were analyzed by the usage of immunoelectrophoresis. Interleukin-6 and TNF&alpha concentrations were analyzed by ELISA kits. There did not exist any significant differences for acute phase proteins on first day between exercise and no exercise. ACT was significantly higher (p<0.05) on 3rd day morning than on 2nd day morning, suggesting that an increase of ACT due to the exercise stress was amplified by bright light exposure during one diurnal day before the exercise stress was applied to the subjects. [J Physiol Sci. 2006;56 Suppl:S97]

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