Neuroendocrinology, during the last several decades, has elucidated brain effects on hormones and hormone effects on brain. Estrogenic effects on a specific reproductive behavior, lordosis, were analyzed in three steps: discovery of hormone receptors in brain, neuronal circuitry for this behavior, and estrogenic effects on gene expression (Estrogens and Brain Function, Springer-Verlag, 1980; Drive, MIT Press, 1999). This work proved that specific chemical reactions in specific parts of the brain regulate a specific mammalian behavior. Now we are studying the most primitive, fundamental function of the brain: generalized CNS arousal (Brain Arousal, Harvard Univ. Press, 2006). So far, we have defined four chemical routes by which generalized arousal influences sexual arousal and lordosis mechanisms: histamine, norepinephrine, opioid peptides and prostaglandin D. [J Physiol Sci. 2008;58 Suppl:S2]

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People who suffer from anxiety tend to interpret ambiguous situations, situations that could potentially be dangerous but not necessarily so, as threatening. It is also recognized that a person's propensity for anxiety can be influenced by early life experiences. We have shown that signaling by the neurotransmitter serotonin during early postnatal development in the mouse is about to moderate life-long anxiety behavior. Specifically, genetic or pharmacological blockade of the serotonin 1A receptor (Htr1aKO) during this period leads to increased anxiety both to innate anxiety cues (exposure novel arena) as well as enhanced fear conditioning to ambiguous, but not non-ambiguous conditioned stimuli. To examine the involvement of specific forebrain circuits in this phenotype, we developed a pharmacogenetic technique for the rapid tissue and cell-type specific silencing of neural activity in vivo. Inhibition of neurons in the central nucleus of the amygdala suppressed conditioned responses to both ambiguous and non-ambiguous cues. In contrast, inhibition of hippocampal dentate gyrus granule cells selectively suppressed conditioned responses to ambiguous cues and reversed the Htr1aKO phenotype. These data demonstrate that Htr1aKO mice have a bias in the processing of threatening cues that is moderated by hippocampal mossy fiber circuits and suggest that the hippocampus plays an important role in the response to ambiguous aversive stimuli. [J Physiol Sci. 2008;58 Suppl:S2]

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Male prairie voles (Microtus ochrogaster) display mating-induced pair bonding, demonstrated by a preference for a familiar partner versus a conspecific stranger (partner preference) and aggression towards a stranger (selective aggression). Therefore, this species provides an excellent opportunity to study the neurobiology of social attachment. In a series of experiments, we systematically examined the role of dopamine (DA) in the nucleus accumbens (NAcc) in the regulation of pair bonding in male prairie voles. The vole NAcc contained DA terminals and receptors, and mating increased DA release/turnover. Intra-NAcc activation of D2-type, but not D1-type, DA receptors facilitated partner preference formation whereas blockade of D2-type, but not D1-type, receptors inhibited this behavior induced by mating or by D2 receptor activation. This receptor-specific effect of DA on partner preference formation was further confirmed by manipulations of the cAMP signaling transduction pathway in the NAcc. In addition, two weeks of pair bonding resulted in an increase in the density of D1-type receptors in the NAcc in males that displayed selective aggression. Blockade of these receptors blocked selective aggression. Together, these data demonstrate that NAcc DA regulates pair bond formation and maintenance in a region- and receptor-specific manner. We also examined the interaction of DA with other neurotransmitter systems, including vasopressin and oxytocin, and our data illustrate a neural circuit important in pair bonding. (supported by NIH grants from NIMH and NIDA) [J Physiol Sci. 2008;58 Suppl:S2]

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Biomolecules assemble to form molecular machines such as molecular motors, cell signal processors, DNA transcription processors and protein synthesizers to fulfill their functions. Their collaboration allows the activity of biological systems. The reactions andbehaviors of molecular machines vary flexibly while responding to their surroundings. This flexibility is essential for biological organisms. The underlying mechanism of molecular machines is not as simple as that expected from analogy with man-made machines. Since molecular machines are only nanometers in size and has a flexiblestructure, it is very prone to thermal agitation. Furthermore, the input energy level is not much difference from average thermal energy, kBT. Molecular machines can thus operate under the strong influence of this thermal noise, with a high efficiency of energyconversion. They would not overcome thermal noise but effectively use it for their functions. This is in sharp contrast to man-made machines that operate at energies much higher than the thermal noise. In recent years, the single molecule detection (SMD) and nano- technologies have rapidly been expanding to include a wide range of life science. The dynamic properties of biomolecules and the unique operations of molecular machines, which were previously hidden in averaged ensemble measurements, have now been unveiled. The aim of our research is to approach the engineering principle of adaptive biological system by uncovering the unique operations involved in utilizing fluctuations by biosystems from molecular machines to brain. [J Physiol Sci. 2008;58 Suppl:S2]

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Puberty/adolescence is a transition between childhood and adulthood, after which reproductive, cognitive, emotional, and social maturations are attained. My talk will focus on the recent progress on the mechanism of the onset of puberty and further review current knowledge on brain development during adolescence. Puberty is a consequence of hypothalamic maturation. An increase in pulsatile LHRH release from the hypothalamus triggers the onset of puberty. Tonic GABA inhibition suppresses LHRH neuronal activity throughout juvenile period. Its reduction shortly before puberty leads to an increase in glutamatergic activity resulting in the pubertal increase in LHRH release. In addition, the role of GPR54 and kisspeptin (metastin54) have been introduced into this scheme. Nonetheless, it is unclear what decreases tonic GABA inhibition shortly before puberty and how the kisspeptin/GPR54 neuronal system fits into the pubertal changes in GABA and glutamate neuronal activity. Brain imaging data in humans show that maturation of neocortical and limbic circuits occurs throughout puberty completing in the early twenties. The pubertal increase in gonadal steroids would greatly influence the maturation of their circuits, although the degree to which gonadal steroids are involved in adolescent brain development is still unknown. Collectively, despite the recent progress in understanding the puberty brain and adolescent brain, further research with collaboration between reproductive neuroendocrinologists, behavioral scientists, and brain imaging specialists is required to illustrate brain development during the pubertal/adolescent period. [J Physiol Sci. 2008;58 Suppl:S3]

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Ascidian embryonic development is known to be phylogenetic prototype of the vertebrate embryogenesis. The cleavage-arrested large embryonic cells derived from the early 2 to 8 cell ascidian embryos revealed the multi-directional differentiation under the condition of cell-cell interaction like vertebrate stem cells. This differentiation without cell-cleavage gives us a rare chance to examine the fate selection mechanism under the condition of mixed cell-fate determinants in single cells, which will not be possible in mammalian systems. Especially either cell in an ascidian 2-cell embryo (2C cell) has multiple fates and develops into any cell-types in a tadpole. To find the condition for controlled induction of a specific cell-type, cleavage-arrested cell-triplets were prepared in various combinations, in which a 2C cell was in contact with any one of four types of cell-pairs from 8-cell embryos. The differentiation resulted from the fate selected in the 2C cell was quantitatively identified in terms of cell-type specific ion channel expressions on the cell membrane. Differentiation of the 2C cell into major cell-types, such as neural, muscular and epidermal, was reproducibly induced by selecting the type of contacting cell-pair and the developmental time difference between the cell-pair and 2C cell. Similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells will be discussed and a proposal that the ascidian 2C cell is a simple model for toti-potent stem cells will be presented. [J Physiol Sci. 2008;58 Suppl:S3]

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The analysis of biological function using mathematics has been rather limited to simple reactions. Recent progress of computer science has made it possible to integrate these simple model schemes into cell models or even tissue and organ models. The most advanced example is in the field of membrane excitation. The biophysical behavior of ion channels has been analyzed at both the single channel and whole cell levels. The large variety of membrane excitation in cardiac cells has been explained by combining different types of ion channels. Further integration of individual cell excitation into the whole organ model well explained the modulation of the electrocardiogram under various pathological conditions. Extending this integration of molecular events to metabolism and signal transduction potentially makes it possible to clarify mechanisms underlying homeostatic regulation of the biological functions mediated by negative and positive feedback reactions among numerous molecular pathways. The integration of the experimental knowledge is not an easy job to achieve. To overcome the complexity of the biological system, the integration platform for developing the comprehensive cell model should be thoroughly designed, and also knowledge of basic sciences is required in addition to the medical biology. Here, obviously collaboration of scientists from different field of study is necessary. This vast effort in interdisciplinary collaboration should be encouraged by realizing that development of biosimulation will introduce innovation in the medical sciences in near future. [J Physiol Sci. 2008;58 Suppl:S3]

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Long-term potentiation (LTP) at glutamatergic synapses is considered to underlie learning and memory, and is associated with enlargement of dendritic spines. Since consolidation of memory and LTP requires protein synthesis, it is important to clarify how protein synthesis affects spine enlargement. We here report that repetitive pairing of postsynaptic spikes and two-photon uncaging of glutamate at single spines (spike-timing protocol) displayed both immediate and gradual phases of spine enlargement in CA1 pyramidal neurons. The gradual enlargement was strongly dependent on protein-synthesis and BDNF action, often associated with spine twitching, and was induced specifically at the spines which were immediately enlarged by the synaptic stimulation. Thus, spike-timing protocol is an efficient trigger for BDNF secretion, and induces protein-synthesis dependent long-term enlargement at the level of single spines. [J Physiol Sci. 2008;58 Suppl:S6]

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It has become a consensus that the AMPA type glutamate receptor (AMPA-R) is transported to the synapse by LTP induction, thereby contributing to the enhanced synaptic transmission. However, AMPA-R itself does not exist by itself. It binds with many other synaptic proteins which support the AMPA-R both from inside and outside of the synapse through direct or indirect interaction. Then, when AMPA-R is transported to the synapse, other proteins should be transported as well. To test this, we chose several representative postsynaptic proteins and investigated their transport after LTP induction. They were fused with GFP and coexpressed RFP, as a volume filler, in hippocampal CA1 pyramidal neurons. Upon LTP induction with two-photon-uncaging of glutamate on the single dendritic spine, these proteins showed different kinetics of delivery. While some proteins are immediately transported to the synapse, others were significantly slower. We then tested whether these slow proteins are moving at all by using FRAP assay. As opposed to our expectation, FRAP assay indicates that those molecules are constantly undergoing turnover between dendritic spine and shaft. Our results indicate that a synapse that has undergone synaptic plasticity is different in the composition compared with naive synapes. This may explain phenomenon known as metaplasticity, where a prior plasticity induction changes the properties synapse to further induction of synaptic plasticity. [J Physiol Sci. 2008;58 Suppl:S6]

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Despite a plethora of information available on the synaptogenesis during development, molecular mechanisms underlying synapse formation and maintenance in adult CNS remain largely unknown. The C1q family is an expanding family consisting of 32 members, such as C1q of the classical complement pathway and adiponectin. We recently demonstrated that Cbln1, a brain-specific C1q family protein, was secreted from cerebellar granule cells and played two crucial roles at granule cell-Purkinje cell synapses: the matching and maintenance of pre- and postsynaptic structures; and the induction of long-term depression, a synaptic plasticity model underlying cerebellar motor learning. Here, we show that Cbln1 is also present outside the cerebellum and other Cbln family proteins Cbln2 and Cbln4 are expressed throughout the CNS with distinct spatial and temporal patterns. In mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N-linked glycoproteins like Cbln1. We found that purified Cbln1, 2, and 4 proteins could bind to distinct postsynaptic regions in the cerebellum and hippocampus. Interestingly, application of exogenous Cbln1 to mature Purkinje cells rapidly increased excitatory synapse formation not only in vitro but also in vivo. These results indicate that Cbln family proteins likely serve as transneuronal regulators of synapse formation and maintenance in distinct brain regions in adult. [J Physiol Sci. 2008;58 Suppl:S6]

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Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, an E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating thatSCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed alteredelectrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning. [J Physiol Sci. 2008;58 Suppl:S7]

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The synaptic transmission is regulated by the vesicular exocytosis and the subsequent recycling in the presynaptic terminal. These vesicular dynamics were quantified by measuring the synaptopHluorin fluorescence from the individual large mossy fiber bouton in the hippocampus. Slices were made from the hippocampus of a TV-42 transgenic mouse in which synaptopHluorin is specifically expressed in the mossy fiber boutons. We found that there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool. The initially docked vesicles are easily depleted and the readily releasable pool (RRP) is replenished by the reserve subpopulation of releasable pool ("reserve" releasable pool). Phorbol esters, the C1-domain receptor agonists, modified the vesicular dynamics in one of three modes: (1) recruiting a presynaptically silent synapse to be releasable (OFF-ON type), (2) increasing the size of reserve releasable pool (ON-ON₁ type), and (3) facilitating the replenishment of RRP (ON-ON₂ type). These effects were almost completely blocked by a PKC inhibitor, staurosporine. However, there remained the phorbol ester-dependent potentiation of synaptic transmission even in the presence of staurosporine. It is possible that phobol esters increase the size of RRP, which is hardly detectable by our synaptopHluorin method, through activating non-PKC C1-domain receptors. Ref: Suyama et al. Neurosci Res 59:481-490 (2007). [J Physiol Sci. 2008;58 Suppl:S7]

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To understand physiological mechanism of acupuncture effectively, it is required to study in bi-directional way, translational approach. As a "from bedside to bench study", we developed an experimental ankle sprain model for persistent pain showing clear acupuncture analgesia based on the widespread clinical usage. The analgesic effect of electroacupuncture (EA) was specific to the acupoint and the effect could not be blocked by systemic injection of opioid antagonists. However, the α-adrenoceptor antagonist phentolamine completely blocked the EA-induced analgesia by intrathecal injection. These data suggest that this analgesic effect is produced by applying EA to a site remote from the painful area in a stimulus point-specific way and the effect of EA in ankle sprain pain is, at least in part, mediated by spinal α-adrenoceptor mechanisms. As a "from bench to bedside study", we examined the preemptive analgesia of acupuncture in inflammatory pain. EA was applied prior to the induction of inflammation. Pretreatment of EA applied to ST-36 significantly prevented hyperalgesia. However, EA applied to control point was not effective. Furthermore, the effect of EA was blocked by intraperitoneal injection of naloxone. These data suggest that EA could be used as preemptive analgesic treatment on inflammatory pain in human. Complementing these studies are laboratory investigations of clinical use that contribute to a fuller understanding of the acupuncture. [J Physiol Sci. 2008;58 Suppl:S7]

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Peripheral nerve discharges, one of the physiological bases around acupoints, evoked by manual acupuncture (MA) and EA at acupoints ST-36, SP-6 and ST-25 were investigated in rats. MA at ST-36 but not at SP-6 and ST-25 evoked significant discharges from afferents innervating ST-36, and such discharges could be simulated by EA with strengths of 10-15 V. The results suggest that different effects of acupoints might be due to the different nerves innervating particular acupoints.In somatic pain treatment, EA was applied to ST-36 and BL-60 acupoints bilaterally in the hind limbs of mice with inflammatory pain in the right hind paw induced by CFA injection. The time of paw-withdrawal latency (PWL) responded to a heat source was used for judging pain intensity. It was found that both single (30 min) and repeated (14 days) EA treatments significantly shortened PWL in the CFA injected paw. There was no treatment effect applying EA at acupoints LI-10 and PL-6 in the fore limbs.In treatment of visceral pain induced by colorectal distention (CRD) stimuli in rats with irritable bowel syndrome (IBS), both single and repeated EA treatments at ST-36 and ST-37 acupoints significantly inhibited abnormally increased abdominal withdrawal reflex (AWR) scores and the magnitude of electromyogram (EMG) recorded from the abdomen responded to CRD stimulation at strengths of 20, 40, 60 and 80 mmHg. The above results suggest that both somatic and visceral pain can be effectively treated by EA. (Financial support: FSMCST: 06DZ19732; 973 Program: 2005CB523306) [J Physiol Sci. 2008;58 Suppl:S8]

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Acupuncture has been used to alleviate cardiac dysfunction (e.g., tachycardia). There have been several reports that acupuncture produces a decrease in heart rate in healthy human subjects. However, the neural mechanism of this response is still controversial. I would like to introduce our recent study on the neural mechanism of acupuncture on heart rate in anesthetized rats. Acupuncture stimulation of various areas (forepaw, forelimb, chest, abdomen, hindlimb, hindpaw) produced decrease in heart rate. The acupuncture-induced decrease in heart rate was abolished by severance of the somatic nerves ipsilateral to the site of stimulation. Heart rate was decreased by acupuncture stimulation of the muscle alone, but not of the skin alone. The acupuncture-induced decrease in heart rate was not significantly influenced by severance of both vagus nerves at the cervical level but was abolished by bilateral stellectomy. Acupuncture stimulation decreased cardiac sympathetic efferent nerve activity as well as heart rate. After spinal transection at the C1 level, the bradycardiac responses to stimulation of hindpaw and hidlimb were abolished, while those of the forepaw, forelimb, chest and abdomen reversed to tachycardiac responses. These results suggest that the decrease in heart rate produced by acupuncture stimulation is a reflex response. The afferent pathway is composed of muscle afferents while the efferent pathway is composed of cardiac sympathetic nerves. The reflex center is located in the brain. The spinally-mediated reflex increases in heart rate seem to be inhibited by inhibitory descending pathways originating in the brain. [J Physiol Sci. 2008;58 Suppl:S8]

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Our previous study demonstrated that splenic NK cell activities were significantly higher in the EA-treated rats than in the non-acupunctured rats, and that lesion of the lateral hypothalamic area (LHA) abolished the effects of EA on NK cell activity. We also examined EA-induced alterations of gene expression in splenic NK cells using microarray technique and real time RT-PCR. Our data shows that EA treatment increased CD94, PTK and VCAM-1 expressions while decreased PTP and SHP-1. In additional sets of experiments, we showed that successive EA treatment reduces IgE production in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH) by suppression of the Th2 cell lineage development. Furthermore, pretreatment of phentolamine (α-adrenergic receptor antagonist) or Y-25130 (5-HT3 receptor antagonist), but not naloxone (general opioid receptor antagonist), completely blocks the EA-induced suppression of antigen-specific and total IgE levels in serum, and IL-4 production in anti-CD3 mAb-activated splenocytes in DNP-KLH immunized mice. Therefore, it is suggested that EA modulate NK cell activities and Th1/Th2 cell responses through the neural-immune interaction.This work was supported by the SRC program of KOSEF (R11-2005-014), Republic of Korea. [J Physiol Sci. 2008;58 Suppl:S8]

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Tadashi Hisamitsu, Department of Physiology, School of Medicine, Showa University, Tokyo, Japan The effect and possible mechanism of acupuncture and moxibustion on the collagen induced arthritis in rats Acupuncture and moxibusution is known to have beneficial effects on the human rheumatoid arthritis. To evaluate the effects and to explore possible mechanism of these treatments on the arthritic disease, the influence of electroacupuncture (EA) and moxibustion on collagen-induced arthritis (CIA) animal was examined in vivo. DBA/1J mice were immunized intradermally twice at the 3-week interval with bovine type II collagen (CII). The main incidence of arthritis started about on day 30 and lasted to day 60 after the first immunization. EA stimulation or moxibustion, begun on day 21 simultaneously with the second immunization, was applied three times a week for 3 weeks at the acupoint equivalent to GV4 (governor vessel 4, Ming Men, Meimon). The results showed that EA and moxibustion delayed the onset, attenuated the severity of arthritis, and reduced the anti-collagen antibody level. Furthermore, EA stimulation significantly inhibited the concentrations of splenic endogenous interleukin-1β (IL-1β) and serum prostaglandin E₂ (PGE₂). The expression of IL-1β mRNA in spleen cells was obviously down-regulated and that in synovial tissues was modestly affected by EA. These data suggest that EA has an inhibitory effect on murine CIA, and the partial mechanism of its therapeutic result may be attributed to inhibiting the productions of IL-1β and PGE₂. [J Physiol Sci. 2008;58 Suppl:S8]

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Stretch-activated cation (SAC) channels play an essential role in sensing and transducing external mechanical stresses in living cells. However, its molecular identity in mammalian cells is not as yet firmly established. In human epithelial HeLa cells, we found the activity of non-selective cation channel which is delicately sensitive to membrane stretch with a negative pressure for half-maximum activation of around 3 cm-H₂O. Also, the whole-cell cation current was augmented by osmotic cell swelling. The Mg²⁺-sensitive, Ca²⁺-conducting, stretch-activated cation channel current exhibited the hallmark biophysical and pharmacological features of TRPM7 at both single-channel and whole-cell levels. The endogenous expression of TRPM7 in HeLa was confirmed by RT-PCR and western blotting. Treatment with siRNA targeted against TRPM7 led to abolition of stretch-activated single-channel cation currents and of swelling-activated whole-cell cation currents. When the TRPM7 cDNA was transfected into HEK-293T cells, the robust stretch-activated cation channel current exhibited similar biophysical and pharmacological features of endogenous TRPM7 at both single-channel and whole-cell levels. Suppression of the regulatory volume decrease (RVD) upon a hypotonic challenge was observed by application of siRNA for TRPM7, by elimination of extracellular Ca²⁺ or by addition of a TRPM7 channel blocker. Thus, it is concluded that TRPM7 is the SAC channel endogenously expressed in human epithelial cells and is involved in volume regulation of the cells by serving as a swelling-induced Ca²⁺ influx pathway. [J Physiol Sci. 2008;58 Suppl:S9]

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TRPC6 is ubiquitously expressed in vascular smooth muscle cells (VSMCs) and implicated in the regulation of vascular tone and remodeling. In several different types of VSMCs, stimulation of phospholipase C-liked receptors can activate this channel via generation of diacylglycerol. In other VSMCs showing prominent blood flow autoregulation, elevated intravascular pressure itself can activate TRPC6 channel eliciting a reflex vasoconstriction (myogenic response). However, our recent investigations demonstrate that TRPC6 channel can be activated synergistically by receptor stimulation and mechanical forces in both heterologous expression system and native VSMCs. Application of mechanical forces (e.g. hypotonic or shear stresses), albeit not effective by themselves, induced large cationic currents, when subthreshold receptor activation or weak direct activation of G-protein by GTPγS preceded, and inclusion of agonist in the patch pipette greatly lowered the threshold of negative pressure to elicit single channel activities. Importantly, this synergism was not affected by a mechanosensitive cation channel blocker GsMTx but was strongly attenuated by specific inhibition of ω-hydroxylation which generates 20-hydroxyeicosatetraenoic acid, a reportedly potent vasoconstrictive lipid messenger. Consistent with these results, video-microscopic diameter measurement of cannulated mesenteric artery revealed that this mechanism may operate in sensitized myogenic response to intravascular pressure increase under weak receptor stimulation. [J Physiol Sci. 2008;58 Suppl:S9]

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Transient receptor potential (TRP) non-selective cation channel is involved in various sensory systems. TRP channels that respond to low or high temperatures have been identified in cell bodies of sensory neurons in dorsal root ganlia. Some of these thermo TRP channels have been reported to induce autonomic thermoregulatory responses, and sensations such as cold and hot, leading to behavioral thermoregulation. In the present paper, we state TRP melastatin 8 (TRPM8) channel that is sensitive to low temperature less than 28 ^oC and menthol (ingredient of mint). In TRPM8 knockout (KO) mice, when cooling stimuli is applied to plantar skin, cold-escape behavior is inhibited, indicating that TRPM8 at plantar skin mediates cooling-induced cold-escape behavior. We showed previously that when menthol is applied to whole body skin of wild type (WT) mice, core temperature is increased by 1-2 ^oC through activation of heat-gains responses such as shivering and non-shivering thermorgenesis. This indicates that there are pathways from TRPM8-expressing sensory neurons to thermoregulatory effectors for autonomic and behavioral thermoregulation. However, the structure of TRPM8-exressing nerves in the skin is not known. In the present paper, we investigated nerve endings of TRPM8-expressing neurons of mice using anti-TRPM8 antibody, which was produced in out laboratory. We report that TRPM8 is positively stained at free nerve endings in various areas of skin. [J Physiol Sci. 2008;58 Suppl:S9]

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Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells at inflamed sites in injury or infection. Here, we demonstrate that the plasma membrane Ca²⁺-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, H₂O₂ evokes Ca²⁺ influx through TRPM2 to activate Ca²⁺-dependent tyrosine kinase Pyk2 and amplify Erk signal via Ras. This elicits nuclear translocation of NF-kB essential for the production of the chemokine interleukine-8 (CXCL8). In monocytes from TRPM2-defecient mice, H₂O₂-induced Ca²⁺ influx and production of the macrophage inflammatory protein 2 (MIP-2/CXCL2), the murine CXCL8 functional homologue, were significantly impaired. In the inflammation model dextran sulfate sodium-induced colitis, CXCL2 expression, neutrophil infiltration, and ulceration were significantly attenuated by TRPM2 disruption. Thus, TRPM2 Ca²⁺ influx controls the ROS-induced signal cascade responsible for chemokine production which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases. [J Physiol Sci. 2008;58 Suppl:S10]

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pH sensation is critical for nociception in sensory neurons or avoiding spoiled foods and harmful solutions. It has been reported that several TRP channels could be related to the pH sensation in mammals. We present here a recent progress in the molecular mechanisms of pH sensing through TRP channels. TRPV1 has been reported as a polymodal nociceptive receptor activated by noxious heat or acid pH. Acidic solution evoked ionic currents with a EC₅₀ value of about pH 5.4. Extracellular protons could activate TRPV1 by increase in P_O rather than by altering unitary conductance, although protons slightly reduce the amplitude of currents. Single channel currents of TRPV1 were observed by outside-out but not inside out configuration. These results suggest that protons act on amino acids in the extracellular domain of TRPV1. Recently, an acid-sensing channel complex, PKD1L3/PKD2L1, has been reported as a possible candidate for sour taste receptor. PKD2L1 (TRPP3) belongs to TRPP subfamily. In mouse tongue, PKD1L3 and PKD2L1 are co-expressed in a subset of taste receptor cells. Large currents were evoked by citric acid and HCl. PKD1L3/PKD2L1 channel complex showed a unique property named as an off-response, which implies that the channel is activated upon the removal of acid stimulus. Acid-induced responses were observed only after the removal of an acidic solution with a pH lower than 3.0. As described above, TRP channels play important roles for acid pH sensing in mammals. Furthermore, we will also mention about a recent study in which TRPA1 could be activated by alkaline pH. [J Physiol Sci. 2008;58 Suppl:S10]

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Glial cells outnumber neurons by about ten to one in the adult human brain but their physiological roles have been traditionally postulated to be restricted to nutritional support of neurons. Progress in glial cell biology in the last ten years revealed a variety of dynamic functions of glial cell, such as regulated exocytosis in astrocytes, coordination of motility and phagocytosis in microglial cells, and synaptic communication of NG2 glial cells with neurons. Furthermore, sophisticated optical approaches in combination with development of in vivo preparation for brain imaging illustrated intimate functional and morphological relationship between neurons and glial cells in situ. Neuron-glia interaction may also be a key component in pathophysiology of neurodegenerative diseases and psychiatric disorders. I will summarize the recent advancement in this field and briefly introduce our own findings on the role of astrocytes in excitatory synapse formation and maturation in the hippocampus. [J Physiol Sci. 2008;58 Suppl:S10]

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S100B is a brain specific calcium binding protein predominantly expressed in astrocytes. Previous studies using gene manipulated animals have suggested that the protein has a role in synaptic plasticity and learning. In order to assess the physiological roles of the protein in active neural circuitry, we have recorded spontaneous neural activities from the neocortex and hippocampus in urethane anesthetized S100B knockout (KO) and wildtype control (WT) mice. Typically occurring local field oscillation patterns including the slow (0.5-2 Hz) oscillations in the neocortex, theta (3-8 Hz) and sharp wave associated fast ripple (120-180 Hz) oscillations in the hippocampus were observed in both genotypes and appeared virtually indistinguishable. When seizure was induced by intraperitoneal injection of kainic acid, gamma (30-80 Hz) oscillation in hippocampal CA1 stratum radiatum was significantly smaller in S100B KO mice [1]. The results suggest that deficiency of S100B does not have a profound impact on spontaneous neural activity in normal conditions. However, when neural activity was sufficiently raised, activation of S100B related pathways may take effect, resulting in modulation of neural activities. As S100B is known to be secreted to the extracellular space by astrocytes, we have performed a series of S100B infusion and antibody blockade experiments. Our current results suggest that the attenuation of kainate induced gamma oscillation is likely to be an extracellular effect of S100B.[1] Sakatani et al., EJN 25:1144-1154 (2007) [J Physiol Sci. 2008;58 Suppl:S11]

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Abnormalities in L-glutamate signal transmission have been postulated to play a role in major mental illnesses. Recent studies suggest that glial glutamate transporters play critical roles in normal glutamatergic signal transmission. The glial disruption results in decreased uptake of glutamate and an elevation of extracellular glutamate levels. Elevated extracellular glutamate may cause cytotoxic damage to neurons and glia. Significant down-regulation of glial glutamate transporters, GLT1 and GLAST, in major depressive disorder has been reported. In the present study we examined the role of glial glutamate transporters in the pathogenesis of autism. Autism is a neurodevelopmental disorder characterized by impairments in reciprocal social interaction, communication deficits and repetitive and restricted patterns of behavior and interests. Yet, the etiology of autism is largely unknown. Aberrant glutamate function is often cited as an important element of risk for autism, but little is known about the underlying molecular determinants and neural mechanisms. In the present study, we generated animal models in which glutamate receptors are overstimulated by genetic down-regulation of glial glutamate transporters. Resulting mutant mice showed abnormal social interaction and increased anxiety-like behavior. We observed enlarged amygdala and hippocampus. These mutant mice replicate many aspects of the behavioral and neuroanatomical abnormalities seen in autism. Scanning the genomes of autism spectrum disorder families revealed that GLT1 falls close to one of linkage peaks. Thus, these mutants are new animal models of autism. [J Physiol Sci. 2008;58 Suppl:S11]

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Dominant mutations in the ubiquitously expressed Cu/Zn superoxide dismutase (SOD1) lead to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motor neurons. Although ubiquitous expression of mutant SOD1 provokes progressive, selective motor neuron degeneration in human and rodents due to an acquired toxic property(ies) of the mutant, the cell types that contribute to the onset and progression of the motor neuron disease are not known. To test whether mutant SOD1 toxicity within specific cell types contributes to motor neuron degeneration, we have generated a mouse ubiquitously expressing a "floxed" mutant SOD1 transgene (LoxSOD1^G37R) which can be removed within specific cell populations by the action of Cre recombinase. To eliminate mutant SOD1 within motor neurons or non-neuronal cells, Lox SOD1^G37R mice were mated to Islet1-Cre, CD11b-Cre, or GFAP-Cre that express Cre specifically in motor neurons, microglia, or astrocytes, respectively. Removing mutant SOD1 from motor neurons slowed the timing of disease onset and early disease progression, indicating mutant action in neurons as an initiating factor in triggering disease. More importantly, silencing of SOD1 mutant expression selectively within microglial cells or astrocytes has minimal effect on age of disease onset, but sharply slows disease progression. Thus, onset and progression represent distinct disease phases defined by mutant action within different cell types to generate non-cell-autonomous killing of motor neurons, findings that validate therapies, including cell replacement, targeted to the glial cells. [J Physiol Sci. 2008;58 Suppl:S11]

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Recent studies have revealed a novel role for astrocytes in the neuronal synaptic plasticity as they can release excitatory amino acids (EAAs) such as glutamate that activate neuronal NMDA receptors. Vesicular and non-vesicular mechanisms have been suggested for controlling astrocytic glutamate release, but exact molecular correlates are unclear. Here we report that mouse astrocytes express functional Ca²⁺-activated anion channels (CAACs), which are readily activated by increases in intracellular Ca²⁺ by various astrocytic G_q-coupled receptors, and encoded by mouse bestrophin 1 (mBest1) channel. mBest1 provides a molecular mechanism for Ca²⁺-dependent non-vesicular and channel-mediated glutamate release from astrocytes. Furthermore, glutamate release through mBest1 induces the potentiation of hippocampal CA3-CA1 synaptic activity by modulating the threshold of synaptic plasticity. Our results both provide a novel mechanism of gliotransmitter release by direct permeation through astrocytic CAACs and suggest a potential role in regulating hippocampal synaptic plasticity by modulation of neuronal NMDA receptors. [J Physiol Sci. 2008;58 Suppl:S11]

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ATP and glutamate are the principal gliotransmitters released from astrocytes through various mechanisms. Glutamate released from astrocytes activates neuronal receptors including NMDA receptors (Araque et al., 1998) and facilitates glutamate release (Perea & Araque, 2007). In contrast, ATP of astrocytic origin is rapidly hydrolysed into adenosine in the brain slices that are rich in ecto-nucleuotidase and activates presynaptic A₁ receptors, which in turn attenuates excitatory transmission (Zhang et al, 2003; Pascual et al., 2005; Serrano et al., 2006). In the nucleus of the solitary tract, activation of presynaptic P2X receptors, extracellular ATP-gated cation channels, facilitates glutamate release (Kato & Shigetomi, 2001; Shigatomi & Kato, 2004). Here I present lines of structural, pharmacological, and circumstantial evidence supporting that these presynaptic P2X receptors are the "interface" molecules directly transducing excitation of "syncytia network" of astrocytes into synaptic excitation of neurons. Supported by MEXT, Japan. [J Physiol Sci. 2008;58 Suppl:S12]

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The hypothalamic suprachiasmatic nucleus (SCN) is the master regulator of the systemic circadian organization. Many neurons in the SCN are capable to generate circadian output and are synchronized in a steady light:dark condition. However, light exposure during the night disrupts the synchrony. Previously, we demonstrated that the endogenous desynchrony in the SCN occurs after the rapid light:dark cycle shift in rats, which suggested that jet lag syndrome is caused by endogenous desynchrony in the SCN and that the aberrant peripheral circadian rhythm faithfully reflects the state of the central clock in the SCN. How does the SCN deliver its rhythm to the locomotor activity? SCN transplantation is an effective method to investigate the question. The transplanted SCN restored the circadian rhythms of locomotor in SCN-lesioned arrhythmic mice. Since the graft of the embryonic SCN in the third ventricle has a few number of neural connections to the surrounding brain tissues, it is assumed that the humoral factors from the SCN mainly deliver the rhythm of the master oscillator to the periphery in the SCN grafted animals. In our present analysis, some characteristics of the circadian rhythms in the locomotor activity and peripheral organs were not fully restored. The finding suggests that the humoral factor is able to evoke the peripheral circadian rhythm but neural projection from the SCN is also involved in the formation of the circadian peripheral rhythm. [J Physiol Sci. 2008;58 Suppl:S12]

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A major challenge in modern physiology is to integrate molecular mechanisms with tissue level organization. Studies of circadian clock gene function have relied heavily on behavioral phenotypes of knockout mice, with little attention to system-level interactions. We therefore studied clock function at the level of individual cells, using bioluminescence imaging of PER2::LUC expression in tissue explant or dissociated cell cultures from Per1-, Cry1-, Cry2-, Bmal1-, and Clock-deficient mice. Similar lengthening of circadian period was observed in Cry2-deficient cells, explants, and behavior. However, behavior did not predict the cellular and tissue level phenotypes of other genetic perturbations. In particular, Per1 and Cry1 were required for persistent circadian rhythmicity in single cells, but oscillator network interactions uniquely present in the suprachiasmatic nucleus, the master neuronal circadian pacemaker, were able to compensate for Per1 or Cry1 deficiency, preserving sustained rhythmicity in SCN slices and in behavior. We are currently analyzing data from Bmal1- and Clock-deficient cells. By examining effects of genetic perturbations at the level of single cells and tissues, we have demonstrated that cellular interactions are in fact essential to the operation of the circadian clock. [J Physiol Sci. 2008;58 Suppl:S12]

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The understanding of the mammalian circadian system profoundly progressed in recent years. The recent molecular dissection of the biological clock has revealed that circadian oscillations are generated by interacting positive and negative transcription/translation feedback loops of a set of clock genes. This molecular core oscillator is present in most cells in the body, and thus it is believed that mammalian circadian system is composed of multiple oscillatory systems with a hierarchical architecture. The master clock in the hypothalamic suprachiasmatic nucleus (SCN) drives slave oscillators in peripheral tissues. In this circadian system, the central and peripheral autonomic nervous system has an important role in carrying the circadian signals from the SCN clock to the peripheral organs. In all airway tissues, we investigated the role of vagal nerve activity on the respiratory clock. We will discuss the circadian role of airway mucin and muscarinic acetylcholine receptors. Vagal signaling will be essential gears in conferring circadian time information to airway glands. [J Physiol Sci. 2008;58 Suppl:S13]

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Daily restricted feeding (RF) causes a circadian rhythm even after SCN lesion, and the mechanism of this rhythm is poorly understood. Here, we examine the role of E-coupled breakfast oscillator in RF-entrained oscillation using Bmal1-luciferase transgenic mouse (gift from Honma, Hokkaido University). Circadian rhythm change of liver bioluminescence was recorded through Lumicycle after establishment RF oscillation. In SCN intact mice under LD cycle, daily RF applied during ZT0-4, ZT6-10, ZT12-16 or ZT18-22 for 1 week caused phase-advance of bioluminescence rhythm during ZT6-10 and phase-delay during ZT18-22. Interestingly, daily twice RF (ZT0-2 and ZT12-14) or (ZT6-8 and ZT18-20) did not caused phase change. This data suggests that daily twice RF is ineffective signal for entrainment under intact SCN. We did same experiment using SCN lesioned mice. These mice showed the clear bioluminescence rhythm in the liver, but its peak time was ranging from day to night. Daily RF for 4hrs entrained liver bioluminescence rhythm with a peak time (2 hrs after RF start time). Daily twice RF (2hrs with12-hr interval) also entrained liver bioluminescence rhythm with a peak time (2 hrs after former RF) when RF started at former clock time, and with a peak time (2 hrs after later RF) when RF started at later clock time. Thus, peak time was dependent on RF start clock time (breakfast time).In the present experiment, it is strongly suggested that fasting period is important for entrainment signal of RF-entrainment. [J Physiol Sci. 2008;58 Suppl:S13]

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The suprachiasmatic nucleus (SCN) works as the central circadian oscillator that controls phases of circadian oscillators in peripheral tissues, although the mechanism of the control is not well known. Current understanding is that the timing of ovulation in rats is determined by the luteinizing hormone (LH) surge from the pituitary, itself timed by the SCN through gonadotropin releasing hormone surge. In this study we tried to reveal how the phase of ovarian circadian oscillator is controlled. To monitor phases of circadian oscillator we used transgenic rats in which luciferase is rhythmically expressed under the control of the Period1 promoter. First, we asked whether ovarian clocks were influenced by signals from the pituitary. To determine whether the phase was set by neural or endocrine signals we surgically denervated or heterotopically transplanted ovaries with or without encapsulation in dialysis membrane. Our results indicated that endocrine signals are sufficient to transmit circadian phase information to the ovary. We next evaluated LH and follicle stimulating hormone as potential endocrine signals for phase control. Peak phase of Per1-luc rhythm was shifted by the pituitary hormones when applied to the cultured granulosa cells. These results raises the possibility that ovulation is timed by interaction between circadian clocks in the hypothalamus/pituitary and those in the ovary. [J Physiol Sci. 2008;58 Suppl:S13]

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Feeding behavior regulates body weight. Hyperphagia (increased food intake) causes obesity, diabetes and metabolic syndrome. Ghrelin is a potent orexigenic hormone of the stomach origin. Ghrelin increases [Ca²⁺]i in neuropeptide Y (NPY) neurons isolated from the hypothalamic arcuate nucleus (ARC) of adult rats, and this effect depends on cAMP-PKA pathway. Leptin suppresses ghrelin-induced [Ca²⁺]i increases via PI3 kinase- and phosphodiesterase 3 (PDE3)-mediated pathway. Stimulation of feeding by intracerebroventricular (icv) ghrelin injection is counteracted by leptin in a PDE3-dependent manner. Thus, leptin signaling via PI3 kinase-PDE3 counteracts cAMP-dependent ghrelin signaling, suppressing ARC NPY neurons and thereby feeding.We found that Goto-Kakizaki (GK) rats, a suitable model of human type-2 diabetes, display young-adult specific hyperphagia at 6-14 weeks, while body weight is unaltered, compared to control Wistar rats. In GK rats at 11 weeks, visceral fat is accumulated and plasma leptin level is elevated. Leptin suppresses food intake and phosphorylates STAT3 in the hypothalamic ARC less potently in GK than Wistar rats. In ARC, mRNA levels for NPY are elevated. Icv injection of NPY Y1 receptor antagonist corrects the hyperphagia. In conclusion, hyperphagia in GK rats is due to the ARC NPY neuron hyperactivity, which may result from and/or cause visceral fat accumulation and leptin resistance. Young-adult GK rats provide an excellent model for elucidating the mechanisms of hyperphagia. [J Physiol Sci. 2008;58 Suppl:S14]

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With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid hormone receptors has been found to be much more dynamic than previously thought. FRET (fluorescence resonance energy transfer) clearly showed the interaction of estrogen receptor (ER) a and ERb. In the presence of the estradiol, however, the discrete staining pattern of ERa and b were overlapped with Brg-1, indicating that most of the ER clusters are involved in the chromatin remodeling machinery. FRAP (fluorescence recovery after photobleaching) analysis showed that nuclear ERa and b_ are mobile in the absence of the ligand, but its mobility was slightly decreased after the ligand treatment. Glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are localized in the cytoplasm in the absence of the ligand and they translocate to the nucleus after ligand binding. FRET demonstrated that importin a is involved in GR/MR translocation from the cytoplasm to the nucleus and GR and MR dimerize within the nucleus. FRAP showed that the movement of ligand-binded GR/MR is restricted in the nucleus. Steroid hormone receptor is composed of a transactivation domain (NTD), a DNA binding domain (DBD) and a ligand binding domain (LBD). Androgen receptor (AR) was chosen to investigate the domain specific nuclear import. Nuclear localization signal (NLS) of DBD was Ran- and importin a/b-dependent, whereas the NLS signal of NTD and LBD were Ran-dependent but importin-a/b independent, suggesting that the nuclear import of AR is regulated by the interplay between each domain of receptor. [J Physiol Sci. 2008;58 Suppl:S14]

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Somatostatin is widely distributed in the central nervous system and the periphery, and is implicated in neuronal survival or neurogenesis. The number of somatostatin neurons is sexually dimorphic in the human or rat bed nucleus of the stria terminals. In the rat, one of the most prominent brain sex differences is found in the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA), and which is significantly larger in the male than in the female. The difference also depends on the gonadal-steroid milieu during the critical period. In the present study, a transient, sex-specific transcription of somatostatin gene was detected by in situ hybridization in the rat SDN-POA, during the establishment of sex difference. On postnatal day 1 (day of birth), somatostatin mRNA was detected in the SDN-POA of both sexes. On days 8 through 35, the area of somatostatin mRNA-positive cells was significantly larger in males than in females. In males, the area attained its maximum size on day 15 and diminished gradually thereafter. In females, the area did not change in size during this period. On day 60, expression of somatostatin mRNA was low and not different between sexes. As with Nissl staining and calbindin immunohistochemistry, somatostatin mRNA hybridization on day 15 revealed a reversal of the sexual dimorphism in the size of the SDN-POA in males that had been neonatally orchidectomized or females given estrogen as pups. Sex-specific, transient transcription of the somatostatin gene may causally relate to the estrogen-dependent organization of the SDN-POA. [J Physiol Sci. 2008;58 Suppl:S14]

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Novel neuropeptides of G-protein coupled receptor (GPCR) ligands are shown to be localized in brain and play a range of physiological functions including feeding regulation. This review concerns some novel GPCR ligands of feeding regulating neuropeptides such as orexin, ghrelin, galanin-like peptide (GALP) and neuropeptide W such as those studied by our research group, and neuronal interactions among these neuropeptides in the hypothalamus. Cross-talk among several these neuropeptides-containing neurons in the hypothalamus plays a key role in determining feeding states as well as feeding behavior. We will show some structural and functional characteristics of these novel GPCR ligands and summarize the interactions between these different kind of feeding regulating neurons and leptin-targeting neurons in brain. Moreover, we will present a new strategy for analyzing the neural circuit of these feeding-regulating GPCR ligands-containing neurons in brain by use of transgenic model mice. Finally, we will present our very recent results of GALP which are involved in regulation of feeding as well as energy homeostasis and body temperature. Research in this field will serve a very important role of clarifying neurologically-based causes for appetite dysfunctions and diseases and it may help in establishing and leading to new therapies for people who are suffering such conditions. [J Physiol Sci. 2008;58 Suppl:S15]

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The identification of endophenotypes is an essential step for investigating psychiatric illness, but represents a considerable challenge in human patients. Here we show that alpha-CaMKII heterozygous knockout mice have dysregulated behaviors, including a severe working memory deficit and an exaggerated infradian rhythm. The neurons of the dentate gyrus (DG) in the mutants resembled immature DG neurons in normal rodents. Biomarkers derived from the mutants classified individuals into two clusters, one of which had higher susceptibility to schizophrenia and differential expression of genes related to neural development. Based on these results, we propose the "immature DG" as a candidate endophenotype of psychiatric disorders. [J Physiol Sci. 2008;58 Suppl:S15]

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Bipolar disorder is one of major mental disorders affecting about 1% of the population. Lithium, a mood stabilizer, is known to neuroprotective effect. MRI studies showed increased incidence of white matter hyperintensity. Blood cells of patients with bipolar disorder. These findings suggest that bipolar disorder is characterized by vulnerability or impaired resilience at the cellular level. We proposed the mitochondrial dysfunction hypothesis of bipolar disorder based on several findings in patients with bipolar disorder such as abnormal energy metabolism in the brain and abnormal mitochondrial DNA in the postmortem brains. This hypothesis may explain the molecular basis of such abnormality at the cellular level in bipolar disorder. Other groups are also studying the possible role of mitochondrial dysfunction in bipolar disorder. To verify the mitochondrial dysfunction hypothesis, we produced transgenic mice that accumulate abnormal mitochondrial DNA in the brain by introducing a mutation in the gene that encodes an enzyme which synthesizes mitochondrial DNA, called POLG. The wheel running activity of the transgenic mice changed periodically following the estrous cycle. The periodic activity change in mutant mice was improved by lithium, a mood stabilizer. Mitochondrial dysfunction is not specific to bipolar disorder, but common to several diseases such as Parkinson's disease or diabetes mellitus. This model mouse may be useful to identify the neural systems responsible for bipolar disorder. [J Physiol Sci. 2008;58 Suppl:S15]

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Since the pathophysiology of schizophrenia have been discussed with a functional alteration of dopaminergic transmission in the brain, we have focused the dopaminergic components for the research target of schizophrenia using PET. Using high affinity ligand [11C]FLB 457, we found reduced D2 receptor binding in the anterior cingulate cortex of patients with schizophrenia, and a significant negative correlation was observed between D2 receptor binding and the positive symptom score. Subregions of interest were defined on the thalamus using individual magnetic resonance images. D2 receptor binding was also lower in the central medial and posterior subregions of the thalamus in patients with schizophrenia. Based on the recent findings, cortical D2 receptor may interact with GABA system working to modulate DA release, while thalamic D2 receptor is likely to participate in sensory gating function over the prefrontal cortex. We have found decreased D2 receptor in the anterior cingulate cortex and thalamic subregions of the patient with schizophrenia. These observations could lead to the view that alterations of extrastriatal D2 receptor is involved in dysregulation of DA release and sensory signals from the thalamus to the cortex. On the other hand D1 receptor binding was found to be lower in the prefrontal cortex and a significant negative correlation was observed between D1 receptor binding and the negative symptom score. Abnormality of D1 receptor function would be at the bottom of the negative symptoms and cognitive impairment of schizophrenia. [J Physiol Sci. 2008;58 Suppl:S16]

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We focused on investigating neural network deficits in schizophrenia using functional Magnetic Resonance Imaging (fMRI) while participants performed cognitive task. Patients with schizophrenia have a variety of cognitive deficits. The cognitive deficits associated with schizophrenia can be divided into (1) deficits in preparation for the task (task set) (2) deficits in execution of the task (executive control) (3) deficits in feedback of the task (evaluation). Though previous research of schizophrenia mainly focused on deficits of executive control, it is important to investigate the function of preparation for the task before the execution. For example, when the instructions of the task are given, the human brain established a task set before the task is actually performed. We investigated brain activity of patient with schizophrenia and normal volunteer related to both task set and task performance. In turn our study allows us to determine whether patients with schizophrenia set up different or similar task sets prior to the task performance and then use different or similar task strategies as normal participants. The result of this study showed that patients with schizophrenia form different task sets than normal participants due to attention control disturbances. [J Physiol Sci. 2008;58 Suppl:S16]

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The classical theory of neurotransmission is that a neurotransmitter is released from the nerve terminal to convey either excitatory or inhibitory signal in the postsynaptic target through anatomically defined synaptic connections. During the last decade, however, it has been becoming clear that neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to change the strength of neurotransmission within common neural pathways. Such heterosynaptic interactions underlie diverse brain functions. Our studies have provided examples of such interactions among different synaptic inputs in the cerebellar cortex, which include: (1) the excitatory neurotransmitter released from climbing fibers produces not only direct excitation of Purkinje cells but also presynaptic inhibition of GABAergic transmission to the same PC through activation of AMPA-type glutamate receptors localized on cerebellar interneuron terminals; (2) the inhibitory neurotransmitter GABA released by cerebellar interneurons acts on adjacent parallel fiber synapses to enhance slow synaptic excitation via cross-talk between perisynaptic GABAB and metabotropic glutamate receptors; and (3) GABAergic synapses are also controlled by monoamine-induced presynaptic and ATP-mediated postsynaptic modulation with short- and long-lasting time-courses. Detailed mechanisms underlying reciprocal interactions between excitatory and inhibitory inputs would provide not only basic knowledge to better understanding of the brain function but also clues to therapeutic targets for psycho-neurological disorders. [J Physiol Sci. 2008;58 Suppl:S16]

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At the calyx of Held, excitatory transmitter L-glutamate inhibits transmitter release. This effect is mediated by mGluRs (Takahashi et al, 1996) and iGluRs, such as AMPARs (Takago et al, 2005) and also possibly by NMDARs at the nerve terminal. We investigated the mechanisms underlying presynaptic inhibitions by AMPARs and NMDARs using whole-cell recordings from presynaptic terminals and postsynaptic cells. Activation of AMPARs inhibited presynaptic Ca²⁺ currents (I_pCa) and attenuated NMDA-EPSCs. The AMPA-induced I_pCa inhibition was disinhibited by a strong depolarization and occluded by GTPγS loaded into the terminal. Thus, activation of presynaptic AMPARs inhibits voltage-gated Ca²⁺ channels via interaction with Gβγ, thereby attenuating glutamate release. Our preliminary data also suggest that glutamate-induced I_pCa inhibition can be mediated by NMDARs. Bath application of NMDA inhibited I_pCa. Loading of the NMDAR channel blocker MK-801 into the calyx presynaptic terminal, or bath application of NMDAR antagonists abolished this effect. As a result bath-applied NMDA attenuated AMPA-EPSCs. Surprisingly, intraterminal GTPγS did not affect NMDA-induced I_pCa inhibition. Thus at the calyx of Held, the G protein-dependent I_pCa inhibition by glutamate is mediated by both mGluRs and AMPARs, but not by NMDARs. [J Physiol Sci. 2008;58 Suppl:S17]

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Hippocampal long-term potentiation (LTP) of excitatory synaptic transmission has been regarded as a cellular model of learning and memory. Its induction is regulated by many functional molecules at synapses, including the neuropeptide nociceptin identified as an endogenous ligand for the orphan opioid receptor. Mutant mice lacking the receptor exhibit enhanced LTP and hippocampus-dependent memory formation; however, the precise molecular and cellular mechanism is largely unknown. Here, we show that LTP in the hippocampal CA1 region is inhibited by nociceptin synaptically released from interneurons by tetanic stimulation. This endogenous nociceptin down-regulates the excitability of pyramidal cells by the hyperpolarization induced by the activation of K⁺ channels, which are the common target shared with γ-aminobutyric acid type B (GABA_B) receptors although the mode of action is considerably different. Interestingly, the modulation of LTP by endogenous nociceptin is not observed when theta-burst stimulation is used in stead of tetanic stimulation, suggesting that relatively longer high-frequency synaptic activation is required for the release of endogenous nociceptin. These results indicate that, in addition to GABA, nociceptin released from interneurons by their high-frequency activation is a novel endogenous neuromodulator that negatively regulates LTP induction in the hippocampus through direct modulation of pyramidal cells. [J Physiol Sci. 2008;58 Suppl:S17]

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Endocannabinoids (eCBs) are lipid mediators and serve as retrograde messengers at synapses in various brain regions. eCBs are produced and released on demand in activity-dependent manners, bind to presynaptic CB1 receptors, and cause short- or long-term suppression of transmitter release. Previous studies have revealed that eCB-mediated retrograde synaptic suppression is induced by Ca²⁺ influx into postsynaptic neurons through voltage-gated Ca²⁺ channels. We tested whether Ca²⁺ entry through NMDA receptors (NMDARs) can trigger eCB release. Using cultured hippocampal neurons, we demonstrated that NMDA transiently suppressed cannabinoid-sensitive IPSCs. This NMDA-induced suppression was eliminated by blocking NMDARs and CB1 receptors, and was significantly reduced by intracellular BAPTA. These results indicate that NMDAR activation caused Ca²⁺ influx into postsynaptic neurons and induced eCB release. We then examined how released eCBs are degraded and eCB signaling is consequently terminated. We found that inhibition of monoacylglycerol lipase (MGL), a hydrolyzing enzyme of the major eCB 2-arachidonoylglycerol, markedly prolonged the suppression of IPSCs induced by Ca²⁺ influx. Together with the anatomical evidence for the localization of MGL in presynaptic terminals, our data indicate that presynaptic MGL contributes to termination of retrograde eCB signaling. [J Physiol Sci. 2008;58 Suppl:S17]

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Neurotrophic factors are a family of diffusible intercellular messengers and indicated to be involved in differentiation, development and survival of neurons in nervous systems. In the present study, we demonstrate an involvement of brain-derived neurotrophic factor (BDNF) in functional maintenance of excitatory synapses in mature cerebellum.The original interest of the study is to examine functional roles of inositol 1,4,5-trisphosphate (IP3) signaling in glutamatergic synapses in the mature cerebellum. We first examined changes in synaptic functions after a chronic in vivo suppression of IP3 level in cerebellar Purkinje cells (PCs) by overexpressing exogenous IP3 5-phosphatase (5-Ppase), which selectively hydrolyzes IP3. The suppression of IP3 production in PCs for 1 days resulted in the decrease in the strength of parallel fiber (PF) -PC synapses, accompanied with the decrease in the probability of transmitter release from PFs. Because IP3 5-Ppase was specifically expressed in PCs, postsynaptic site of PF synapse, the observation indicates an involvement of retrograde signaling in the functional maintenance of PF synapse. Among many candidate factors involved in the retrograde signaling, application of exogenous BDNF rescued the attenuation of synaptic function in IP3 5-Ppase expressing PCs. Furthermore, chronic neutralization of endogenous BDNF with function-blocking antiserum not only inhibited transmitter release from PFs but also occluded the effect of IP3 5-Pase. These results indicate that IP3-BDNF signaling maintains function of PF-PC synapse in the mature cerebellum. [J Physiol Sci. 2008;58 Suppl:S17]

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What is fatigue? Why do we feel tired sometimes or seemingly all of the time? What is the physiological role or meaning of the sensation of fatigue ? How is chronic fatigue related to various diseases? How can we prevent chronic fatigue and exhaustion? In the past we really did not know very much about the mechanisms of fatigue. Fatigue is really an important bio-alarm, without which we might drop into an unrecoverable exhaustive state and in the most severe case even die, referred to in Japanese as Karoshi. As compared with the mechanisms of other bio-defense systems such as pain and fever, little is known regarding molecular/neuronal mechanisms of fatigue. Cytokine-prostaglandin systems are involved as the major factors in the induction and/or mediation of pain and fever. Although some pre-inflammatory cytokines may be the central mediator(s) in fatigue, Matsumura et al. will present their results indicating that the prostaglandin systems are probably not involved in the mechanisms of fatigue. Lactate, which was previously considered to be a candidate fatigue-inducing substance accumulating during severe exercise, is no longer thought to be a causative substance of fatigue. Although surprisingly a lot of people are suffering from chronic fatigue lasting longer than 6 months (more than one-third of the Japanese population), integrated research on fatigue had not been organized until our research projects under MEXT. Here, I would like to summarize the present status of fatigue research and am going to present the efforts to develop anti-fatigue foods, medicine, and daily life goods. [J Physiol Sci. 2008;58 Suppl:S18]

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Two experimental animal models for central fatigue have been developed by excessive activation of the central nervous system of rats; induction of cortical spreading depression, the propagation of neuronal membrane depolarization throughout the cerebral cortex, and long-term intracranial self-stimulation. We have reported that prostaglandins including PGD₂, PGE₂, and PGF_2α were produced by COX-2 expression in neurons in the cerebral cortex following cortical spreading depression. In such a model for neuronal excitation in the cortex, the amount of non-REM sleep, but not of REM sleep, increased subsequently for several hours in the animals, and the increase was completely attenuated by application of NS-398, a COX-2 inhibitor. In a long-term intracranial self-stimulation study, resting behavior and non-REM sleep appeared a few hours after the start of stimulation, and such behavioral changes were also inhibited by application of an inhibitor of COX-2. These observations indicate that arachidonic acid cascade plays an important role in a common molecular and neural system in such animal models for fatigue following neuronal activation in the central nervous system, and relieves excessive brain activity by inducing resting behavior and non-REM sleep. [J Physiol Sci. 2008;58 Suppl:S18]

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Accumulated fatigue disrupts homeostasis in various organs, which sometimes leads to chronic fatigue and work-overload induced death. We examined the cellular changes that occur with the accumulation of fatigue in a rat fatigue model in which locomotor activity and a partial sleep disturbance are continuously induced. Prolonged fatigue stress induced a marked development of the rough endoplasmic reticulum (rER), enlargement of Golgi apparatus in the cells of the intermediate lobe of the pituitary gland (IL), and elevation of alpha-melanocyte stimulating hormone (a-MSH) in the peripheral blood. Further continuous stress caused dilation of the ER. The cells became full of dilated ER and began to degenerate. The prolonged fatigue stress markedly decreased tyrosine hydroxylase (TH)-positive nerve fibers in the IL. Injection of the dopamine antagonist to non-fatigued rats induced the same morphological changes of the ER and Golgi structures. Conversely, injection of a dopamine agonist to fatigued rats inhibited these changes. These findings suggest that prolonged fatigue stress suppressed dopamine release in the IL, causing oversecretion of IL hormone from the melanotrophs and leading to melanotroph dysfunction and subsequent cell death. This characteristic response observed in melanotrophs in chronic stress provides a possible marker for diagnosis and a potential therapeutic target for chronic fatigue. [J Physiol Sci. 2008;58 Suppl:S18]

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Acute viral infection associates fatigue and fever. Using an animal model of viral infection, we studied molecules and neural substrates possibly involved in fever and fatigue. In the animal model, rats or mice were injected intraperitoneally with poly IC, a synthetic double-stranded RNA, as a viral mimic. Animals received poly IC developed fever and showed a decrease in locomotor activity during the dark period, which we used as an index of fatigue. Fever was completely suppressed by a prostaglandin (PG)-synthesis inhibitor. On the other hand, the inhibitor partially suppressed fatigue in the early phase (18:00-24:00) of the dark period, and failed to reduce fatigue in the late phase (24:00-6:00). These results indicate that poly IC-induced fatigue consists of PG-dependent and PG-independent mechanisms. We next examined neurons possibly involved in poly IC-induced fatigue using Fos as a neuronal activity marker. Three hours after poly IC injection, the numbers of Fos-positive neurons were increased in the ventromedial preoptic nucleus (VMPO), hypothalamic paraventricular nucleus (PVN), bed nucleus of stria terminalis (BNST), central nucleus of amygdala (CeA), lateral parabrachial nucleus (LPBN), and nucleus of solitary tract (NTS). The PG-synthesis inhibitor suppressed Fos expression in VMPO, PVN, LPBN and NTS, but did not suppress Fos expression in BNST and CeA. These brain loci may be involved in PG-dependent and PG-independent fatigue, respectively. [J Physiol Sci. 2008;58 Suppl:S19]

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