Liver plays a central role in energy metabolism depending upon nutritional states and hormones. Since long-term regulation of carbohydrate and lipid metabolisms is controlled at the transcriptional level, hepatic nutritional transcription factors play a pivotal role in energy metabolism. SREBP-1c has been established as a transcription factor that controls synthesis of fatty acids and triglycerides based upon our data from SREBP-1c transgenic and knockout mice. SREBP-1c also regulates insulin sensitivity via direct regulation of IRS-2, a key insulin-signaling molecule in the liver. Nutritional induction of hepatic SREBP-1c by dietary carbohydrates and saturated fatty acids well explains how liver shifts metabolism from glycogen synthesis to lipogenesis in the feeding cycle. To excess, SREBP-1c activation contributes to components of metabolic syndrome such as dyslipidemia, diabetes, fatty liver, and insulin resistance, and finally leading to atherosclerosis as observed in our murine models. Meanwhile, we have identified TFE3 as a strong activator of insulin signaling. TFE3 transcriptionally activates IRS2 and diversely participates insulin signaling and markedly ameliorates diabetes in different models. TFE3 and FOXOs synergistically activate, and SREBP-1c competitively suppresses IRS-2 promoter. Collectively, these energy transcripton factors regulate carbohydrate-lipid metabolism, insulin signaling, and might be involved in metabolic syndrome and diabetes. Thus, these factors could be future therapeutic targets. [J Physiol Sci. 2006;56 Suppl:S18]

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The release of neurotransmitters at synapses requires the temporally-ordered trafficking of synaptic vesicles. More than 20 proteins are involved in this process, including the SNARE proteins that participate in membrane fusion during exocytosis. To discern the specific role that each protein plays, it is necessary to sort out the temporal order in which they interact. Here we consider the timing of action of synaphin (also named as complexin), a SNARE-binding protein that plays an important role in calcium-triggered neurotransmitter release. A light-activated binding-site peptide was used to perturb the interaction of synaphin with SNARE proteins at precise time intervals before neurotransmitter release. We find that this peptide inhibits neurotransmitter release within 180 milliseconds before synaptic vesicles fuse with the presynaptic plasma membrane. These results indicate that synaphin binds to SNARE complex after synaptic vesicles dock but well before the fraction of a millisecond required for calcium ions to trigger synaptic vesicle fusion. These results are compatible with a role for synaphin in preparing trans-SNARE complexes for membrane fusion. [J Physiol Sci. 2006;56 Suppl:S19]

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After exocytosis the synaptic vesicle (SV) membrane is recycled by endocytosis. Exocytosis requires external Ca and so does endocytosis, which is supplied through voltage-gated Ca channels in the presynaptic membrane. Multiple types of calcium channels in the presynaptic terminal are participating in these processes. Ca channels designated for exocytosis are highly localized at the release site. Other types of Ca channels are probably diffusely distributed and may contribute to endocytosis. Two types of endocytosis have been demonstrated in EM at the Drosophila neuromuscular junction, namely, active-zone endocytosis that occurs at the presynaptic active zone and non-active-zone endocytosis that operates at the area away from the active zone. Recently we found that two separate types of Ca channels support these two types of endocytosis. Non-active-zone endocytosis is blocked by low concentrations of La, while active-zone endocytosis is inhibited by a spider toxin, PLTXII. Yet another type of Ca channel encoded by the gene, cacophony, is specifically designated for exocytosis. This type of channel, cac-Ca channel, is highly localized at the presynaptic active zone. The distribution of other types of Ca channels is difficult to demonstrate, but physiological findings indicate that PLTXII-sensitive channels are located close to or within the active zone, while the La-sensitive channels reside away from it. Ca is ubiquitously used a messenger. Its temporal and spatial characteristics mold its specific role. [J Physiol Sci. 2006;56 Suppl:S19]

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Regulation of synaptic expression of AMPA type glutamate receptors (AMPA-Rs) through a receptor-trafficking is one of the underlying mechanism for synaptic plasticity in hippocampal and cerebellar neurons. In cerebellar Purkinje cell, induction of the long-term depression (LTD) of parallel fiber (PF)-EPSC requires clathrin-mediated endocytosis, however, relation between the constitutive rapid trafficking and LTD-induction was unclear. Suppressing exocytosis or endocytosis, we addressed whether regulation of the rapid constitutive trafficking of AMPA-Rs was underlying synaptic plasticity in cerebellar Purkinje cell. Effects of intracellular infusion of tetanus toxin (TeTx), a blocker of VAMP2, on PF-EPSC were analyzed in cerebellar slice using whole-cell patch-methods. Infusion of TeTx reduced amplitude of PF-EPSCs to 60% within 20 min. Since infusion of botulinum neurotoxin C (a blocker of syntaxin) reduced PF-EPSC amplitude to similar extent, contribution of TeTx-insensitive receptor-trafficking was suggested to be negligible. As a counterpart of constitutive elimination of synaptic AMPA-R, constitutive insertion of AMPA-Rs into PF-synapse was demonstrated by blocking of dynamin. As for relation between constitutive trafficking of AMPA-Rs and synaptic plasticity, LTD did not occlude with constitutive elimination of AMPA-Rs at PF-synapse, suggesting that internalization of synaptic AMPA-Rs during LTD did not belong to constitutively recycling pool of AMPA-Rs. [J Physiol Sci. 2006;56 Suppl:S19]

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The synaptic transmission is potentiated by the activation of adenylyl cyclase (AC) and protein kinase A (PKA) at hippocampal mossy fiber-CA3 synapses. Although the AC/PKA activation was suggested to facilitate the transmitter release from MF presynaptic terminals, the most of these classical evidences were indirect. In this study the presynaptic exocytosis was directly investigated in the hippocampal slice of a synaptopHluorin (SpH) transgenic mouse (TV-42 line) which expresses SpH specifically at the mossy fiber terminals of hippocampus (Araki et al. 2005). The repetitive stimulation (10 Hz for 1 s) of mossy fiber bundle transiently increased the SpH fluorescence in the presynaptic terminal. The SpH fluorescence was sampled before and after application of forskolin (50 μM) and IBMX (100 μM), a combination which activates AC/PKA. The AC/PKA activation increased the activity-dependent increment of SpH (ΔSpH) by 2.42 ± 0.49 (mean ± SEM, n = 29) on average (p<0.001). However, both the rising and falling time course of ΔSpH was not changed. The ΔSpH was also largely facilitated in the presynaptic terminal of which it was null at baseline. It is suggested that the AC/PKA activation facilitates the presynaptic exocytosis and that it turns some presynaptically silent synapses into active. Ref: Araki R. et al. (2005) genesis 42, 53-60. [J Physiol Sci. 2006;56 Suppl:S20]

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The vestibular nucleus (VN) is a center for stabilizing gaze and posture in response to the head rotation and tilt. VN neurons are physiologically classified into regularly and irregularly discharging neurons on the basis of the regularity of spacing of action potentials. The segregation of VN neurons is involved in different response properties to head movements. Although the discharge regularity has been considered to be attributed to afterhyperpolarization (AHP), the relationships between the discharge regularity and profiles of AHP are still unclear. In this study, we investigated discharge patterns of VN neurons using whole-cell patch clamp technique not only in vitro slice preparations but in vivo preparations obtained from young rats. Previously, AHPs were classified into AHP without a slow component [AHP(s-)], AHP with a slow component [AHP(s+)], and AHP with a slow component and an afterdepolarization [AHP(s+) with ADP]. Both in vitro and in vivo, neurons exhibiting AHP(s+) fired more regularly than the other types of neurons. Application of 100 μM apamin to block Ca²⁺-dependent K⁺ channels abolished the slow component of AHP(s+) and made regular discharges of neurons exhibiting AHP(s+) to be irregular. These suggest that neurons exhibiting AHP(s+) are regularly discharging neurons, whereas neurons exhibiting AHP(s-) and AHP(s+) with ADP are irregularly discharging neurons. The regular firings of neurons exhibiting AHP(s+) are attributed to activation of apamin-sensitive Ca²⁺-dependent K⁺ channels. [J Physiol Sci. 2006;56 Suppl:S20]

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Localizing sound sources requires discriminating differences of sound arrival time of a microsecond order between the two ears (interaural time difference, ITD). In nucleus laminaris (NL) of birds, neurons calculate ITDs by detecting the coincidence of binaural synaptic inputs. We utilized slice-patch recordings, immunohistochemistry and computer simulations to explore the acuity and cellular mechanisms of coincidence detection in NL neurons of the chick. At 40 °C, the avian body temperature, the acuity of coincidence detection was high enough to account for the animal behavior. This acuity was achieved by the acceleration of EPSP time course due to the activation of Kv1.2-mediated low-threshold K⁺ conductance. In NL, neurons are tuned to a specific frequency of sound (characteristic frequency, CF), and are arranged so that the CF decreases from rostro-medial (high-CF) to caudo-lateral (low-CF) direction. Along this tonotopic axis, NL neurons were specialized morphologically and functionally depending on their CF. In the high- and middle-CF neurons, dendrites were short and expression of Kv1.2 channels was strong, which made the EPSP time course rapid and improved the coincidence detection. In the mid-high CF neurons, the process of generating spikes was also specialized; the axon initial segment was myelinated and Nav channels were clustered at some distance from the soma (20-50 μm) in the axon. Theoretical model predicted that this unique distribution of Nav channels in the axon is essential for making the high-frequency generation of action potentials and enhancing the ITD detection. [J Physiol Sci. 2006;56 Suppl:S20]

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Disinhibition such as a loss of inhibitory interneurons or a shift in the transmembrane anion gradient especially in the substantia gelatinosa (SG) of the spinal dorsal horn is thought to be a crucial etiology for chronic pain syndromes. However, there is little direct evidence to elucidate the natural inhibitory mechanism for nociceptive transmission because of difficulties in recording inhibitory synaptic responses from small size SG neurons in vivo. In this study, whole-cell recordings were obtained from SG neurons in vivo and in slice preparations to analyze how inhibitory synaptic inputs modulate noxious transmission and the underlying neuronal circuits. SG neurons in vivo responded to cutaneous pinch accompanied with a barrage of EPSCs. On the other hand, touch evoked a barrage of IPSCs during the stimulation and the receptive fields were larger than those of pinch-evoked EPSCs. After cessation of a brief touch, a burst of IPSCs lasted for about 10 s in some cells. The number of action potentials generated by pinch was decreased by the simultaneous stimulation of touch applied to the surrounding area. In slice experiments, activation of C fiber was required to elicit the burst of inhibitory response and large islet cells known as an inhibitory interneuron received C fiber inputs. The results suggest that innocuous stimulation even brief touch sufficiently suppresses noxious sensation in the SG through activation of C fibers. [J Physiol Sci. 2006;56 Suppl:S21]

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In the cerebellar circuit, Golgi cells receive inputs from granule cells and in turn terminate their axons on granule cell dendrites. Since Golgi cells are the only element that controls the activity of granule cells, Golgi cells are thought to play an important role in information processing via feedback mechanisms.First we investigated the role of Golgi cells by selective ablation using the immunotoxin-mediated cell targeting technique. The elimination of Golgi cells caused severe acute motor disorders. These mice gradually recovered but retained a continuing difficulty in performing fine movements. Electrophysiological analyses indicated that disruption of Golgi cells not only eliminates GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells. These results demonstrate that synaptic integration involving GABA inhibition and NMDA receptor activation is essential for motor coordination. Next we investigated the synaptic mechanisms of postsynaptic metabotropic glutamate receptor subtype 2 (mGluR2) on Golgi cell dendrites, using whole-cell patch-clamp recording of green fluorescent protein-positive Golgi cells of wild-type and mGluR2-deficient mice. Postsynaptic mGluR2 was activated by glutamate released from granule cells and hyperpolarized Golgi cells via G protein-coupled inwardly rectifying K channels. This hyperpolarization induced long-lasting silencing of Golgi cells, the duration and extents of which were dependent on stimulus strengths. Postsynaptic mGluR2 thus senses inputs from granule cells and plays a pivotal role in spatiotemporal modulation of mossy fiber-granule cell transmission. [J Physiol Sci. 2006;56 Suppl:S21]

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Sick house syndrome is a disorder of nerve function, mainly affecting the central nervous system/autonomic nervous system, caused by a sensitivity reaction induced by exposure to trace amount of deleterious chemical substances present in the living environment. Diagnosis is not easy because pathophysiological understanding of the syndrome is not sufficiently complete. In this syndrome, functional assessment of nerve function is especially relevant. For example, the electronic iriscorder is useful as one of the tests of autonomic nerve functioning in this syndrome. There are many cases of this syndrome in which some abnormality and/or instability of pupillary light reaction, that is primarily caused by functional abnormality of autonomic nerve function, has been observed. Furthermore, evaluation of eye movement by Electro-Oculograph is also very useful, as many patients have some disorder of smooth pursuit movement. Modulation Transfer Function, which evaluates the higher optical center (visual cortex) is also useful, and a decrease in Visual Contrast Sensitivity is often observed. Genetic polymorphism testing of drug metabolizing enzymes, such as CYP, GST, NST, and PON1 are useful in evaluating an inherited sensitivity to chemical substances in the patient. From our latest investigation, differences such as absence, decrease in concentration, and delay in induction of these enzymes are observed in some of the patient groups which are obviously different from healthy people. This is important knowledge leading to possible specification of the genes active in expression of this syndrome. [J Physiol Sci. 2006;56 Suppl:S21]

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Polychlorinated biphenyl (PCB) is an environmental chemical that may cause adverse health effects. Previous studies have shown that developing central nervous system is one of the most vulnerable organs against its exposure. However, the molecular mechanisms of PCB action have not yet been fully understood. Since PCB exposure induces abnormal brain development similar to those seen in perinatal hypothyroid animal, we have been studied the effect of PCB/dioxin on thyroid hormone (TH) receptor (TR)-mediated transcription. We have previously identified that PCB may not competitively bind to TR ligand binding domain. Instead, it partially dissociated TR from TH-response element located on the promoter region of target gene. The mechanisms of such dissociation in not well known. However, our recent findings have indicated that PCB may bind to DNA binding domain of TR, which may alter the structural conformation of TR protein.In addition to PCB action on TR, we have also studies the effects of PCB on several other nuclear receptors. It may affect to estrogen receptor (ER)-, and steroid and xenobiotic receptor (SXR)-mediated transcription, but not to glucocorticoid or progesterone receptor action. Further, PCB may also act to neuronal membrane to induce an increase in intracellular calcium concentration, which then stimulate the expression of calcium-induced transcription factors such as c-Jun.These results indicate that PCB may act at multiple systems to alter the gene expression profile of neuronal cells that may affect the normal brain development. [J Physiol Sci. 2006;56 Suppl:S22]

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1-Bromopropane (CH₃CH₂CH₂Br 1-BP) is a newly introduced substitute for specific chlorofluorocarbons whose production was prohibited because of depletion of ozone layers, and is mainly used for degreasing agents and spray adhesives. Although case studies in the USA and China have demonstrated that 1-BP could adversely affect the human nervous system, the underlying mechanism for the effects of 1-BP inhalation exposure on the CNS has not been understood. We investigated the effects of 1-BP exposure on the CNS using different models of exposure. 1-BP potentiated GABA but inhibited ACh responses in Xenopus oocytes expressing GABA_A and nicotinic ACh receptors, respectively, and enhanced recurrent inhibition in the rat hippocampus. On the other hand, hippocampal disinhibition was observed in a concentration-dependent manner (200–1500 ppm) after chronic inhalation of 1-BP in the rats and, at the highest concentration of 1-BP inhalation, epileptic potentials were evoked in the dentate gyrus. Moreover, prenatal exposure to 1-BP vapor resulted in enhanced stimulation/response (S/R) curve of population spikes in the CA1 area of PND 11-15 rats and reduced S/R curve of field excitatory postsynaptic potentials in the CA1 area in adults (6-8w). These results suggest that 1-BP inhalation exposure disrupts neuronal excitability of the hippocampal formation. [J Physiol Sci. 2006;56 Suppl:S22]

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The effects of environmental chemicals on sexual differentiation of exploratory behavior and emotional behaviors and brain were investigated. We exposed bisphenol A (BPA, 0.05-5 ppm) to mother rats pre-, peri- or postnatal period or 1-bromopropane (1-BP, 700 ppm) for 6 h/day during prenatal period. In the open field test, control females explored more frequently than males. This sex difference was not affected by neonatal BPA treatment but was abolished by pre- and perinatal treatment or 1-BP. The time spent in open arms in the elevated plus maze test decreased by neonatal or prenatal BPA treatment although sex difference was not clearly affected. In the forced swimming test, both prenatal and neonatal BPA exposures increased immobility time, an index of depressive behavior, in male rats and reduced immobility latency in both sexes. The duration of immobility decreased in the forced swimming test and the sex difference were disappeared by 1-BP. These findings suggest that prenatal BPA or 1-BP exposure is more effective to impair sexual differentiation of exploratory behavior than neonatal BPA exposure but neonatal period is also important for development of emotional behavior [J Physiol Sci. 2006;56 Suppl:S22]

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Homer is a postsynaptic scaffold protein with the N-terminal target binding and C-terminal self-assembly domains. Homer multimers likely link their targets, including proteins related to the Glu receptor and Ca²⁺ signaling (mGluR1a/5, Shank, IP3R) and to the actin cytoskeleton (Drebrin and Cdc42), at postsynaptic density (PSD). The Homer family consists of three long-form Homers H1b/c, Cupidin/H2, and H3. A natural dominant-negative, short-form H1a with only the N-terminal domain is also activity-dependently expressed. In hippocampus, H1b/c and Cupidin/H2 predominate in CA1 region, whereas H3 is largely localized in CA2-CA3 region. In cultured hippocampal cells, dendritic clustering and synaptic targeting of long Homers coincide with those of NMDAR and PSD-95 throughout development. Overexpression of long Homers increases mature-shape spines, whereas that of H1a alters PSD target contents and spine morphology. In cerebellum, H1b/c and Cupidin/H2 are concentrated in PSDs of granule cells. In cultured granule cells, their clustered distribution is changed by NMDAR-mediated Ca²⁺ influx, and H1a has a neuroprotective action against excess Glu exposure probably by interfering a target linkage via long Homers, which inhibits NMDAR activity. On the other hand, H3 is exclusively localized in Purkinje cells. mGluR1a binding and dendritic localization of H3 is controlled by its Ca²⁺-dependent phosphorylation states. Thus, Homer is involved in synapse formation and function by regulating molecular organization of PSD and spine morphology. [J Physiol Sci. 2006;56 Suppl:S23]

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Drebrin is a major F-actin binding protein in the brain. We have recently demonstrated that the expression of drebrin A (neuron specific isoform) is rapidly upregulated in parallel with synapse formation, and that it governs the targeting of postsynaptic density (PSD) protein PSD-95 to synapses. Immunoelectron microscopy demonstrated that drebrin A is first appeared at the submembranous regions of developing excitatory postsynaptic sites at the initial stage ofsynapse formation. To determine the role of drebrin A on excitatory synapse formation, we analyzed whether the suppression of drebrin A expression affects filopodia-spine morphology and synaptic targeting of NMDA receptors in cultured hippocampal neurons. Suppression of developmentally programmed upregulation of drebrin A by antisense treatment significantly decreased the density and width of filopodia-spines. Immunocytochemistry showed that the antisense treatment did not attenuate synaptic clustering of NMDA receptors under condition that permitted spontaneous activities, but inhibited the accelerated targeting of NMDA receptors into synapses by its antagonist AP5. These results indicate that drebrin A upregulation play a pivotal role in spine morphogenesis and activity-dependent synaptic targeting of NMDA receptors. [J Physiol Sci. 2006;56 Suppl:S23]

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NMDA receptors play a key role in synaptic plasticity in the hippocampal CA1 pyramidal cells. Two subunits of NMDA receptors NR2A and NR2B have distinct expression patterns in development and may contribute differently to induction of long-term potentiation and depression. We discovered input-dependent left-right asymmetry of NR2B subunit allocation in Schaffer collateral (Sch)- and commissural fiber- pyramidal cell synapses. However, it has not been known if NR2B has such asymmetrical distribution in Sch-interneuron synapses, and if NR2A has also asymmetry to neutralize the NR2B asymmetry in pyramidal cell synapses. Here, we investigated distribution of NR2A and NR2B in single synapses by postembedding immunogold and SDS-digested freeze-fracture replica labeling methods. To facilitate the detection of NR2B density difference, we utilized NR2A knockout mice, which have a simplified NMDA receptor subunit composition.The labeling density for NR2B but not NR1 in Sch-CA1 pyramidal cell synapses was significantly different between the left and right hippocampus with opposite directions in strata oriens and radiatum. No significant difference in NR2B density, however, was detected in CA1 stratum radiatum between the left and right Sch-interneuron synapses. Immunoblot analysis of PSD fractions from CA1 radiatum confirmed significant difference in protein amount for NR2B but not for NR1 and NR2A between left and right hippocampus. These results indicate that the asymmetry of NR2B distribution is target-cell specific and unique to this subunit. [J Physiol Sci. 2006;56 Suppl:S23]

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While NMDA-type ionotropic glutamate receptor (NMDAR) is widely accepted as a key regulator for certain forms of memory and learning and LTP induction, not much is known about how NMDAR function is regulated at physiological circumstances. The NR2B subunit of the NMDAR is tyrosine phosphorylated in brain, with Tyr-1472 as a major phosphorylation site. Phosphorylation of neural proteins is one of the main mechanisms underlying dynamic changes in neural functions, so we investigated the physiological significance of NR2B phosphorylation in neuronal plasticity and learning behavior. Mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) showed impaired induction of amygdaloid long-term potentiation and fear-related learning. Basic properties of synaptic transmission were normal in YF/YF mice, suggesting that impaired LTP in YF/YF mice is not caused by direct modification of NMDAR current properties but is associated with some intracellular signaling downstream from NMDAR activation. In fact, CaM kinase II, a key regulator for synaptic plasticity was undetectable in NMDAR complex of YF/YF mice. Electron microscopic analyses revealed that NMDAR localization at synapses was impaired in YF/YF mice, presumably resulting in altered NMDAR complex of YF/YF mice. These results strongly argue that phosphorylation of Tyr-1472 regulates NMDAR localization at synapses leading to modulating synaptic plasticity and fear-related learning. [J Physiol Sci. 2006;56 Suppl:S24]

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Human is a social animal and unique to communicate with language. We want to understand the neurobiological basis of this feature in the light of comparative neuroethology. Social animals, like primates and birds, could communicate with conspecific mates by vocalization. We have established the developmental model of social communication with domestic chick which is a precocial bird and can grow itself without parent care. This allows us to investigate the development of peer relationship without considering the effect of parent-infant relationship. We reared chicks under grouped or individual condition, and compared socialization between two conditions. We measured the association and calling behavior as indicating socialization. The chicks reared as a group for 8-14 days after hatching showed socialization. In contrast, the chicks reared under socially deprived condition showed strong fear response in the novel environment and could not develop socialization. We, next, tested uni-modal social deprivation, that is, either visual or vocal cue was presented during individually rearing. Under these conditions, the fear response was suppressed when the behavior was tested on 8-14th day. Socialization, however, was different between the sensory social cues presented. Only vocally communicated chick could develop socialization. These results suggest that social interaction during infant is critical to develop peer relationship and mutual vocalization is important. Now, we are localizing the neuronal substrate for the development of peer relationship [J Physiol Sci. 2006;56 Suppl:S24]

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Young rats prior to eye opening depend on somatosensory and olfactory function for survival, as they can learn their dam's odor and approach her without visual information. In order to establish olfactory learning, the pairing of odor and tactile stimulation is crucial. Noradrenergic activation through the locus coeruleus by a somatosensory stimulus is implicated in olfactory learning. Within the olfactory bulb (OB), the noradrenergic innervation modulates the efficacy of dendrodendritic synapses between the mitral and granule cells. At the dendrodendritic reciprocal synapses, mitral cell activity is inhibited by GABA released from the granule cells. It is noteworthy that disinhibition of the mitral cells is a crucial step in the formation of an olfactory memory. We previously showed that intrabulbar infusion of the GABA receptor antagonist, bicuculline facilitated olfactory learning. These results implicate the OB as a critical site for olfactory learning. Since the transcription factor, CREB is well known to be involved in plasticity, we examined whether CREB is involved in olfactory learning. Behavioral pharmacology shows that only long-term olfactory memory was prevented by CREB antisense infusion, but short-term memory was intact. Western blot analyses reveal that P-MAPK/ERK was increased for 1 hour after odor exposure paired with shock, followed by increase of P-CREB lasting for 6 hours. These may be evidence suggesting that synaptic plasticity in the OB underlies aversive olfactory learning. [J Physiol Sci. 2006;56 Suppl:S24]

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The life of newborn rats totally depends on their maternal care. Although the first several days of their life are characterized by stress-hyporesponsiveness, absence of mother acts as a strong stressor which overrides it and results in long-lasting stress-vulnerability. Periodic maternal deprivation (MD) for the first few days act as a strong non-photic time cue and entrains pups' circadian clock. However, little is known as to the mechanisms how the MD affects on the pups' circadian clock and stress-responsiveness in the adulthood. We imposed newborn rats to MD of various durations at different time in the light phase, at different period in the postnatal life, with or without keeping pups warm. In addition, by restricting food access of mother rats to 2h (RF), behavioral rhythms of mothers were modified without depriving them from pups. We measured clock gene expression rhythms in the suprachiasmatic nucleus of the fetes and neonates, and behavioral rhythms after weaning. At 8 weeks of age, stress responsiveness was examined by measuring plasma corticosterone levels after exposing to mild stress of cage exchange. 12 h MD during the light phase in day1-6 completely reversed the circadian rhythms of clock gene expression. The behavioral rhythms after weaning were also shifted depending on the phase of MD. Rats exposed to MD exhibited hyper responsiveness to the novelty stimuli, which was abolished by MD with warming. These results suggest that the maternal care is important to entrain pups' clock, while heat loss due to isolation is critical for the stress hyper-responsiveness in adulthood. [J Physiol Sci. 2006;56 Suppl:S25]

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Although an early adversity is a major risk factor for the vulnerability to stress later in life, the mechanism of the stress vulnerability remains to be unknown. It is known that while neonatal isolation (NI) induces stress vulnerability in adult rats, environmental enrichment (EE) following NI leads to resilience. We examined whether NI induced the susceptibility to learned helplessness (LH) (animal model of depression) and EE ameliorated this susceptibility. Pups were individually isolated from postnatal day 2 to 9. After weaning, EE was administrated until the beginning of LH session. In adulthood, we measured the number of escape failures and escape latency 24 hours after exposure to inescapable shock session. Behavioral analyses revealed that whereas the population of LH in NI rats was significantly higher than that in sham rat, EE markedly decreased the population of LH in NI rats. We tried to identify genes involved in the molecular mechanism underlying the susceptibility to LH using a cDNA array, and real-time PCR. The comparison of hippocampal gene expression between NI-LH and sham-nonLH rats revealed the significant decrease in LIMK1 mRNA in NI-LH. EE prevented the decrease in the expression of LIMK mRNA in the hippocampus of NI rats. These findings suggest that LIMK may play an important role in stress vulnerability developed by an early environment. [J Physiol Sci. 2006;56 Suppl:S25]

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It is well established that blood pressure (BP) is controlled by feedback mechanisms, e.g. via baroreceptor reflex. The feedback mechanism supports blood pressure homeostasis to save life at sudden orthostasis or massive hemorrhage. We have shown that sinoaortic denervation abolished BP resetting to a higher level at daily physical activity. This indicates that the baroafferent signal supports active resetting of BP during exercise, suggesting that BP should be controlled not only homeostatically by feedback but also homeodynamically by feedforward mechanisms. The above mechanisms are involved in the short-term control of BP. Resting level of BP is thought to be relatively constant for life-long in health subjects. Lesion of the area postrema, which is a site for neurohumoral interaction with vasopressin and angiotensin II, did not show any abnormality in day-to-day control of BP. In animal-models with salt-sensitive hypertension, vascular endothelial functions or renal functions are impaired but central sympathetic control is preserved, although the central nNOS systems are upregulated to inhibit sympathetic activation. These data lead to the possibility that main causes for abnormality in long-term control of BP might be present in the effector organs but not in the central control system, rather that the central sympathetic system functions to compensate hypertension. These data suggests that BP should be regulated by feedback and local mechanisms homeostatically for saving life and by feedforward mechanisms homeodynamically for helping organ-functions. [J Physiol Sci. 2006;56 Suppl:S25]

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The word "homeostasis" implies a regulatory mechanism that stabilizes the biological parameters around an operating point during the resting condition. During exercise or stressful condition, however, a new operating point will be set. This operating point is different from that under the resting condition and should be suitable for the bodily demand during the new condition. To explore neural mechanisms of such resetting of the operating point, we have recently focused on the stress-induced defense response because stressor induces not only cognitive, emotional and behavioral changes but also autonomic changes. These changes include increases in blood pressure, heart rate, muscular blood flow, respiratory frequency, and tidal volume and suppression of the baroreceptor reflex and pain sensitivity. Although research on the neural circuits underlying such autonomic changes has implicated the hypothalamus in the defense response against stressors, neurotransmitters in this multifacet and coordinated response have not been revealed. In my talk, I will summarize our recent discovery of possible contribution of orexin as a master switch to elicit multiple efferent pathways in the defense response and discuss future directions. [J Physiol Sci. 2006;56 Suppl:S26]

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A major source of excitatory drive to the pre-ganglionic sympathetic neurons originates from the rostral ventrolateral medulla (RVLM). RVLM sympathetic premotor neurons (RVLM neurons) have spontaneous activity in vivo. For the generation of RVLM neuron activity, "Network theory" and "Pacemaker theory" have been suggested by in vivo and in vitro studies. However, it is still unclear which one is right. We studied how RVLM sympathetic premotor neurons generate their activity using an in situ arterially perfused preparation (the working heart-brainstem preparation). We recorded activities of the thoracic sympathetic chain and RVLM neurons. The firing response of RVLM neurons during hypoxic-induced gasping is heterogeneous with some inhibited and others persisting. The finding that some RVLM neurons continue to fire after blockade of fast synaptic transmission is consistent with the hypothesis that they may have intrinsic pacemaker activity. Since some RVLM neurons remained responsive to hypoxia after blockade of fast excitatory and inhibitory synaptic transmission, they may be directly sensitive to hypoxia. Some RVLM neurons may be central oxygen sensors and exhibit pacemaker activity during hypoxia to ensure high levels of sympathetic activity and hence maintenance of arterial pressure. [J Physiol Sci. 2006;56 Suppl:S26]

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Cerebral blood flow is vital for the survival and function of the brain. In particular, the hippocampus and cerebral cortex are very sensitive to transient ischemia. The hippocampus and cerebral cortex receive cholinergic vasodilative fibers that originate in the medial septum and the nucleus basalis of Meynert (NBM), respectively, of the basal forebrain (see a review by Sato et al., 1995, Alzheimer Dis. Assoc. Disord. 9: 28). Recently, we showed that increases in blood flow in the hippocampus and cerebral cortex in rats during activation of the vasodilative system, either by pharmacological (i.v. nicotine) or physiological (electrical stimulation of the NBM) methods, can prevent delayed death of hippocampal and cortical neurons following transient ischemia. Stimulation of the NBM increased the diameter of cortical parenchymal microvessels during stimulation. In addition, after the end of stimulation, an increase in the concentration of brain-derived neurotrophic factor (BDNF) in the cortical extracellular fluid was observed. From these findings, we suggest that activation of the intracranial vasodilative system provides protection against ischemia-induced delayed neuronal death by inducing increases in both the diameter of parenchymal microvessels and the release of an endogenous neuroprotective factor, BDNF. We also showed that activation of the vasodilative system occurred during passive somatosensory stimuli and active movements such as walking. Thus, this intracranial vasodilative system may contribute to the beneficial effect of physical activity on cognitive brain functions. [J Physiol Sci. 2006;56 Suppl:S26]

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Catecholamine signal is a major mechanism of regulating cardiac function. Norepinephrine released from the synaptic terminal binds to beta adrenergic receptors, leading to the activation of the stimulatory G protein and thus adenylyl cyclase. Cyclic AMP generated by adenylyl cyclase activates protein kinase A, which initiates multiple phosphorylation reactions within cardiac myocytes. A major impact of catecholamine stimulation is the enhancement of Ca cycling within myocytes. In the past decade, multiple molecules have been identified that are involved in Ca cycling. Transgenic studies using mouse models have elucidated the function of such molecules. Indeed, a growing body of evidence has shown that Ca cycling and Ca-dependent signaling pathways play a pivotal role in cardiac hypertrophy and heart failure. In addition, recent studies identified that mutations of the genes encoding sarcoplasmic reticulum proteins cause human cardiomyopathies and lethal ventricular arrhythmias. The regulation of Ca homeostasis via the SR proteins may have potential therapeutic value for heart diseases such as cardiomyopathy, heart failure and arrhythmias. Similarly, molecular mechanisms of catecholamine signal have been elucidated and the diversity of cAMP signal within the heart has been demonstrated. For example, it is now well known that the heart expresses multiple isoforms of adenylyl cyclase. The role of each adenylyl cyclase isoform is different in regulating cardiac function and the viability of cardiac myocytes under normal and pathological conditions. We will summarize our recent progresses in the study of this pathway in the heart. [J Physiol Sci. 2006;56 Suppl:S26]

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Leptin is an adipocyte-secreted hormone that regulates body energy metabolism. We have recently shown that leptin stimulates fatty acid oxidation in skeletal muscle by activating α2 AMP-activated protein kinase (AMPK). Leptin exerts this effect directly at the level of muscle and through the hypothalamic-sympathetic nervous system. In contrast, hypothalamic α2 AMPK activity is inhibited by anorexigenic hormones (leptin and insulin), a melanocortin (MC) receptor agonist (anorexigen), high glucose and refeeding. AGRP (orexigenic neuropeptide), fasting and MC4 receptor-KO obese mouse increase hypothalamic AMPK activity. Expression of dominant-negative (DN) and constitutively active (CA) AMPK in the hypothalamus is sufficient to change food intake, body weight and expression of orexigenic neuropeptides such as NPY, AGRP and MCH. CA-AMPK blocks leptin-induced suppression of food intake. We recently examined the signaling pathway of leptin's effects on AMPK, using muscle and neuronal cell lines that express leptin receptor Ob-Rb. Leptin activates α2 but not α1 AMPK in muscle cells through activation of ataxia telangiectasia mutated and calcium/calmodulin-dependent protein kinase kinase β. Furthermore, cellular localization of α2 AMPK is changed in response to leptin. In contrast, leptin suppresses α2 AMPK activity and NPY expression in neuronal cells. Thus, leptin reciprocally regulates AMPK activity in neuronal and muscle cells. Our data indicate that leptin-AMPK system plays a critical role in peripheral and central regulation of body energy metabolism. [J Physiol Sci. 2006;56 Suppl:S27]

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Adiponectin/Acrp30 is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. We reported that AdipoR1/R2 serve as receptors for adiponectin and mediate increased fatty-acid oxidation and glucose uptake by adiponectin. Moreover, obesity was associated with decreased plasma adiponectin levels as well as decreased expression levels of AdipoR1/R2, the latter reduced adiponectin sensitivity, both of which finally lead to insulin resistance. In this study, to clarify the physiological and pathophysiological roles of AdipoRs in vivo, we studied the effects of adenovirus-mediated upregualtion of AdipoRs in the mice liver. Here we show that adenovirus-mediated expression of AdipoR1 in the liver of db/db mice increased adiponectin effect such as increased activation of AMP kinase by adiponectin, decreased molecules involved in gluconeogenesis and increased fatty-acid oxidation, thereby ameliorating diabetes. Moreover, adenovirus-mediated expression of AdipoR2 in the liver of db/db mice increased adiponectin effect such as increased PPARalpha target genes including molecules involved in fatty acid oxidation and energy dissipation, thereby ameliorating diabetes. These data raised the possibility that AdipoR1 may be more tightly linked to activation of AMP kinase pathway, while AdipoR2 may be more tightly linked to activation of PPARalpha pathway. Adiponectin receptor agonists and adiponectin sensitizers should serve as versatile treatment strategies for obesity-linked diseases such as diabetes and metabolic syndrome. [J Physiol Sci. 2006;56 Suppl:S27]

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Feeding is a basic behavior that is necessary for life. Long-term lack of food results in death. It is well accepted that appetite is controlled by the brain and that feeding behavior is regulated by complex mechanisms in the central nervous system, in particular the hypothalamus. However, recent identifications of novel neuropeptides and peptide hormones develop a paradigm in appetite regulatory mechanisms in the central nervous system. In this presentation, I will discuss the two appetite-regulating peptides, ghrelin and neuromedin U. Ghrelin is a growth-hormone releasing and appetite-stimulating hormone secreted mainly from stomach. On the other hand, neuromedin U (NMU) is a potent appetite-suppressing peptide. Moreover, we recently revealed that ghrelin directly acts on osteoblast cells to regulate bone formation, and NMU is involved in the regulation of peripheral inflammation. Thus, both ghrelin and NMU are more than appetite regulators, but have multifaceted roles in, for example bone formation and inflammation. [J Physiol Sci. 2006;56 Suppl:S27]

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Skeletal muscle and fat tissue are involved in the homeostasis of glucose metabolism. Here, we introduce new bioactive factors derived from skeletal muscle and fat tissue. Musclin was identified via signal sequence trap of mouse skeletal muscle cDNAs. Musclin protein contained a region homologous to nariuretic peptide family. Its mRNA was expressed almost exclusively in skeletal muscle of mice, and regulated by nutritional changes. Recombinant musclin protein significantly attenuated insulin-stimulated glucose uptake and glycogen synthesis in myocytes. A newly identified adipocytokine, visfatin, is highly enriched in the visceral fat of both human and mice and whose expression level in plasma increases during the development of obesity. Visfatin exerted insulin- mimetic effects in cultured cells and lowered plasma glucose levels in mice. Heterozygous knockout mice of visfatin had modestly higher levels of plasma glucose relative to wild type littermates. Surprisingly, visfatin binds to and activates the insulin receptor. Production of ROS increased selectively in fat tissue of obese mice. In cultured fat cells, oxidative stress caused dysregulated production of adipocytokines, including adiponectin, PAI-1, IL-6, and MCP-1. In obese mice, treatment with NADPH oxidase inhibitor reduced ROS production in fat tissue, attenuated the dysregulation of adipocytokines, and improved diabetes. Further study on the physiological role of these factors may lead to new insights into glucose homeostasis. [J Physiol Sci. 2006;56 Suppl:S28]

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To be filled in... [J Physiol Sci. 2006;56 Suppl:S28]

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It is thought that gap junctions may be closely associated with tight junctions. However, the mechanisms are still undefined. We found that Cx32 but not Cx26 was closely related to tight junctional proteins in primary cultured rat hepatocytes (Exp. Cell Res. 263, 193-201, 2001) and that Cx32 formation and/or Cx32-mediated intercellular communication could induce expression and function of tight junctions in a mouse hepatic cell line (Exp. Cell Res. 276, 40-51, 2002). When we performed cDNA microarray analysis of Cx32-transfectants, compared to parental cells derived from Cx32-deficient hepatocytes, an increase in expression of membrane-associated guanylate kinase with inverted orientation -1 (MAGI-1), which is known to be localized at adherens and tight junction regions, was observed (Cell Tissue Res. 319, 341-347, 2005). More recently, we performed to express short interfering RNA (siRNA) for Cx32 in primary cultured rat hepatocytes which highly expressed Cx32 and tight junction proteins and examined changes in expression of tight junction proteins and activated MAP-kinase. Down-regulation of Cx32 was associated with a decrease of claudin-1 and an increase of claudin-2. Furthermore, up-regulation of phosphorylated MAP-kinase was observed by the siRNA. Cx32 expression may in part regulate expression of tight junctions through the signal transduction pathway such as MAP-kinase. [J Physiol Sci. 2006;56 Suppl:S28]

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It has been known that intracellular acidification leads to gap junction channel closure. This phenomenon is called "chemical gating", which may be one of the causes of lethal arrhythmia during cardiac ischemia. Chemical regulation of Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. However, the location of the "receptor" for the CT has been unknown.Electrophysiological analysis shows that Cx43 channels reside in three states; closed (C), open (O) or residual (R). Since the R state is eliminated by truncation of the CT, it is hypothesized that the R state results from the interaction of the CT with the receptor. Recently, we showed in vitro that there is an intramolecular interaction of the CT with a region in the cytoplasmic loop of Cx43 (amino acids 119-144; dubbed "L2"). To determine the function of the L2, Cx43 channels were recorded in the presence of a peptide corresponding to the L2 region, delivered via the patch pipette. This manipulation eliminated the R state in a manner similar to that observed after truncation of the CT, indicating that L2 peptide competitively inhibits the interaction between the CT and the native L2 region. Thus, we propose that the L2 acts as a "receptor" that interacts with the CT during channel gating. [J Physiol Sci. 2006;56 Suppl:S29]

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In cardiac muscle, the gap junction greatly contributes to intercellular impulse propagation. The remodeling of the gap junction is induced in pathological myocardium and influences the cardiac function. We investigated remodeling of connexin 43 (Cx43) which is dominant in the ventricular muscle cells, in reference to phosphorylation and dephosphorylation of the protein, using methods of electrophysiology, immuno blot and immunohistochemistry in adult guinea-pig and rat hearts.An activation of PKA promoted the PKA-mediated phosphorylation of Cx43 in association with an increase in the electrical intercelluar coupling and in expression of Cx43. In hypoxia, intracellular Ca-overload or acidosis, the PKA-mediated phosphorylation of Cx43 was inhibited in association with a suppression of the intercellular coupling and of expression of Cx43. These deteriorated changes of Cx43 were alleviated by PKA-activators. In the diabetic or PMA-treated heart, the PKC-mediated phosphorylation of Cx43 was augmented in association with an inhibition of the intercellular coupling and of expression of Cx43. These effects of an activation of PKC were ameliorated by a treatment of PKC-inhibitors, proteasome inhibitors or lysosome inhibitors. These results indicate that Cx43 hyperphosphorylated by PKC is highly susceptible to proteolytic degradation. It is concluded that Cx43 is up-regulated by PKA and down-regulated by PKC, and the remodeling of Cx43 is essentially induced by an excess activation of PKC. [J Physiol Sci. 2006;56 Suppl:S29]

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Gap junctions are considered to play an important role in moderating cell death including apoptosis. However, the basic phenomena underlying when and where the alterations of gap junctions occur during apoptosis have not been well documented. In this study, To answer these questions, we analyzed the spatiotemporal changes of Cx during UV light-induced apoptosis using Cx43-EGFP-expressing HeLa cells, and compared them with those of mitochondrial membrane potential (MMP) using tetramethylrhodamine ethyl ester (TMRE) and nuclear morphological observation using Hoechst 33342. At 2 hr post-UV-irradiation, a third of the cells became TMRE-negative, i.e., they showed the loss of MMP, but with slight nuclear fragmentation, and high percentages of linear Cx43-EGFP plaques were found among both TMRE-positive and TMRE-negative cells. At 4 hr post-UV-irradiation, the percentage of these linear plaques was decreased, and both punctate and diffuse localization of Cx43-EGFP were noted in the cytoplasm of TMRE-negative cells without nuclear fragmentation. At 8 hr post-irradiation, punctate cytoplasmic localization of Cx43-EGFP was noted in TMRE-negative cells with nuclear fragmentation. Treatment with the caspase inhibitor Z-VAD-FMK blocked nuclear fragmentation and partially preserved both gap junctional plaques and MMP. These results indicate that, during apoptosis, Cx mobilization into the cytoplasm occurs after MMP depolarization but before nuclear fragmentation and that this alteration partly depends on caspase. [J Physiol Sci. 2006;56 Suppl:S29]

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Connexins are expressed in vascular endothelial and smooth muscle cells. However, the roles of connexins in the regulation of arterial tone are unclear. In this symposium, we would like to introduce recent evidence that connexins mediate endothelium dependent arterial relaxation caused by endothelium-derived hyperpolarizing factor (EDHF). The molecular identity of EDHF is not convincing. We assessed that whether NO, PGI₂, K⁺, anandamide, H₂O₂ or EET act as EDHF. However, none of that act as EDHF in rat mesenteric artery. Recent studies suggest that gap junctional communication between endothelium and smooth muscle may account for EDHF responses. In rat mesenteric artery, endothelium-dependent relaxation and hyperpolarization by EDHF were inhibited by gap junction inhibitors. RT-PCR experiment showed that connexin 37, 40, 43 & 45 were expressed in the artery. In immunohistochemistory, connexin37, 40 & 43 were expressed in endothelium and connexin 43 was expressed in smooth muscle cells. EDHF-mediate hyperpolarization and relaxation were correlated with serum estrogen level. The expressional levels of connexin40 & 43 were also dependent on estrogen level. These results suggest that EDHF is not a molecule and its responses are mediated by gap junctional communications. Connexin may play a pivotal role in the regulation arterial tone in physiological and pathophysiological states in especially small arteries. [J Physiol Sci. 2006;56 Suppl:S29]

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There are several lines of evidence suggesting that connexin expression is suppressed and/or aberrantly localized in pre-cancerous lesions in several organs and many, if not all, tumor-promoting agents have been shown to inhibit gap junctional intercellular communication (GJIC) of cultured cells as well as those in vivo, suggesting that the loss of GJIC enhances clonal dispersion, causing loss of the growth-suppression signals from the surrounding cells. For endometrial carcinogenesis, it may be concluded that the loss of GJIC caused by the suppressed expression and the aberrant localization of connexin support the clonal evolution of endometrial cancer cells originating in the hyperplasia cells. In the present study, GJIC of IK-ER1, which overexpresses ER-alpha was markedly reduced in the estradiol-containing medium and the reduction was found to be inhibited by ICI182.780, a pure anti-estrogen substrate, as demonstrated by Lucifer-Yellow dye-transfer assay. Western blot analysis indicated that the expression of both Cx26 and Cx32 also decreased in E(+) and the reduction was inhibited by adding ICI182.780. These results supported the result of the dye-transfer assay. Thus, estrogen, which suppresses connexin expression of endometrial epithelium and causes cell proliferation, may act as a tumor-promoting agent for endometrium. [J Physiol Sci. 2006;56 Suppl:S30]

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Dendritic spines are highly motile structures, but how spines move while keeping their contacts with presynaptic varicosities is not clear. To analyze movements of these synaptic structures simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. We obtained stable expression of GFP in a limited population of CA1 pyramidal neurons by using transgenic mice with a Cre-loxP recombination system. CA3 pyramidal neurons of the slice cultures were loaded with rhodamine-dextran by electroporation. Labeled varicosities of CA3 pyramidal cells and spines of CA1 pyramidal cells were visualized using two-photon microscopy to detect close association of the two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall structural changes between varicosities and spines was correlated, while the direction of short-term volume changes was regulated independently. Furthermore, dendritic morphological changes induced by electrical stimulation had little effect on their association. These results suggest that synaptic junctions provide stable attachment sites functioning to correlate presynaptic and postsynaptic motility. [J Physiol Sci. 2006;56 Suppl:S30]

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Actin cytoskeletal organization in dendrites and dendritic spines are believed to form a molecular basis for the morphological plasticity at brain synapses. We here demonstrate that actin cytoskeleton of hippocampal neurons is rapidly rearranged by N-methyl-D-aspartate (NMDA) receptor activation in both neuronal phenotype and developmental activity-dependent manner. In rat hippocampus primary cultures, a certain population (20-30%) of neurons showed drastic redistribution of its dendritic filamentous (F-) actin after a stressful NMDA stimulation (50μM for 30 s). The NMDA-induced actin rearrangement correlated with changes of spine morphology and disruption of several postsynaptic components like Homer1b/c, GluR1 and NMDAR2A, even though the synaptic contacts seemed to stay preserved. Immunohistochemical characterization showed that NMDA-susceptible cells did not express calbindin-D₂₈k. Reduction of network activity by chronic tetrodotoxin application resulted in an increased number of calbindin-D₂₈k -negative and NMDA susceptible cells. Exogeneous calbindin expression in these neurons could recover their resistance to NMDA induced F-actin redistribution. These data indicate that F-actin organization is diverse among different populations of neurons, which are selectively sensitive to hyper excitatory input. Such neuronal type-specific heterogeneity also points toward specific molecular mechanisms that contribute to cytoskeletal regulation in dendrites. [J Physiol Sci. 2006;56 Suppl:S30]

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Dendritic spines represent the developmentally-regulated and activity-dependent pleomorphism based on actin cytoskeleton. However, molecular mechanisms governing the pleomorphism are unclear. First, we find that during development, synaptic drebrin clustering in dendritic filopodia is required for spine morphogenesis. Drebrin clustering with actin filaments occurs at postsynaptic sites of axon-filopodia contact. The drebrin clustering precedes and governs synaptic PSD95 clustering and spine morphogenesis. Second, using fluorescence recovery after photobleaching (FRAP) technique and immunocytochemistry, we find that activities of AMPA receptors (AMPA-Rs) and NMDA-Rs orchestrate drebrin dynamics for synaptic clustering of drebrin and PSD95. AMPA-R blockade reduces binding capacity of drebrin within spines, observed as a reduction of unrecoverable fraction. Consequently, Chronic AMPA-R blockade inhibits synaptic clustering of drebrin and PSD95. NMDA-R blockade facilitates transport of drebrin into spines, observed as a reduction of time constant. Further, chronic NMDAR blockade promotes synaptic targeting of NMDA-Rs but inhibits that of PSD95. Finally, we find that drebrin is involved in activity-dependent synaptic NMDA-R targeting. Drebrin-A knockdown inhibits the accelerated targeting of NMDA-Rs into synapses by NMDA-R blockade despite no effect on NMDA-R localization under conditions of spontaneous activities. In conclusion, activity-regulated actin-cytoskeletal system based on drebrin is critical for the diversity of spine structure. [J Physiol Sci. 2006;56 Suppl:S31]

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Endocannabinoids (eCB) mediate retrograde signal at various brain regions. Postsynaptic release of eCB can suppress neurotransmitter release through activating presynaptic CB1 receptor and cause short-term or long-term synaptic plasticity. The eCB release is induced by strong increase in postsynaptic [Ca²⁺]_i or activation of G_q/11-coupled receptors. Furthermore, coincidence of [Ca²⁺]_i elevation and receptor activation markedly enhances eCB release. Phospholipase C (PLC) is involved in biosynthesis of the major eCB 2-arachidonoylglycerol. To determine the role of PLC in eCB release, we used cultured hippocampal neurons and monitored the eCB release by measuring CB-sensitive synaptic currents. We found that the receptor-driven eCB release was absent in PLCβ1-knockout mice. This PLCβ1-mediated eCB release was dependent on physiological levels of [Ca²⁺]_i. We measured PLCβ1 activity in intact neurons by using exogenous TRPC6 channel as a biosensor for the PLC product diacylglycerol. The receptor-driven TRPC6 currents were absent in PLCβ1-knockout mice and showed a similar [Ca²⁺]_i dependence to that of receptor-driven eCB release. These results indicate that PLCβ1 serves as a coincidence detector for triggering eCB release in the hippocampus. PLCβ contributes to various neuronal signaling. Therefore, Ca²⁺ dependency of PLCβ may play an important role in various synaptic modulations and plasticity. [J Physiol Sci. 2006;56 Suppl:S31]

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Learning and memory formation requires continuous synaptic plasticity at both the functional and structural level. The stability of synapse is maintained by bidirectional signals between pre- and post-synaptic molecules in response to synaptic activity. However, very little is known about molecules that are involved in such a transsynaptic action. Synaptotrophins (Sptn1-Sptn4) structurally belong to C1q/tumor necrosis factor (TNF) family. Recently, Sptn1 has been revealed to be glycoprotein secreted from cerebellar granule cell, and to regulate synaptic plasticity and synaptic integrity between parallel fiber-Purkinje cell (PF-PC) synapse. sptn1-null mice are ataxic, and exhibit molphological abnormalities of PF-PC synapse [e.g., naked spine, mismatched Postsynaptic density (PSD)]. Whereas sptn1 mRNA is predominantly expressed in the cerebellum, other members of synaptotrophin family, of which structure are highly similar to that of Sptn1, are expressed in not only cerebellum but also other brain regions. We hypothesized that the transsynaptic action of the secreted synaptotrophins plays a critical role in structural remodeling of synapses in various central nervous systems. Here we present the expression and the biochemical characteristic of Synaptotrophin family. [J Physiol Sci. 2006;56 Suppl:S31]

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DANCE AND SCIENCE Observation, invention, experiment, control, speculation, discussion and presentation...these are processes that a dance work is born. Movements come from nature both inside and outside me. To pick up and compose such movements, what I need to do everyday is sharpen my senses and hone my skill. It is same for doing science, isn't it? PHYSIOLOGY FOR ART Sharpen senses, make imagination from what we sense and express the internal image as a work...these are the processes of artistic expression. These processes are composed of every physiological functions beginning from the sensory inputs. When I do dance, I need to act on natural physiological limits such as joint angle, muscle mobility and so on. In other words, these limitations are the origins of the characteristic dance movement. Such limitation is the very source of beauty and confidence for movements. Audible sound, visible color, actable movement...these physiological limitations have important meanings for both artistic expression and impression. With these physiological limitations, we can converge and decide the artwork and can share impressions over the cultures and age universally. Many people believe that art is too subjective to be a scientific object. Indeed,individual experience of art is hard to be described in objective way. However, I believe it still should have a great importance to study about art in physiological paradigm, because art is the work of the human beings limited physiologically. [J Physiol Sci. 2006;56 Suppl:S32]

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Rave is the outside dance music party, started in 1988 in Britain, and has spread all over the world. Now it's one of the biggest youth culture. And before all, it's a strong and personal experience of sound, dance and ecstasy. I was in the scene from 80's, and wrote some books about the rave culture, dance music and trance, dancing high and ecstasy, the party as a temporary autonomous zone etc. What Rave gave the youth is the shout & feeling of "We are alive." But what's that? And for me, it's a "big somewhere" related to the writing. Where is there? Is it the place religion calls "a holy", or Timothy Leary's LSD revolution, or alcoholic junkie's blue devil? Nobody explain yet, but Rave clearly gives us. "We are alive." [J Physiol Sci. 2006;56 Suppl:S32]

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In the Western modern framework, which had Cartesian dualism as one of its vital origins, music, which induces beauty and pleasure in human mind, and physiology, which illuminates physical mechanism of human body, were considered to belong to exclusively independent domains. Physiological approaches to music, therefore, are basically challenges against the paradigm dating back to Descartes and inevitably involve essential difficulties in practice. As a successful instance of physiological approach to music, I will examine the discovery of the "hypersonic effect" in this presentation. The hypersonic effect is the phenomenon that imperceptible high-frequency component of air vibration above human audible range activates neural circuit of beauty and pleasure, and makes the sound more comfortable to hear. Regarding the phenomenon, there had long been a serious disagreement between artists and researchers. It may be easy to recognize that the critical factor for the discovery of the hypersonic effect, beyond this historical conflict, was the fact that the discoverer, Tsutomu OOHASHI, was a distinguished artist, Shoji YAMASHIRO, at the same time. Sharing scientific ability and artistic sensibility in one single personality, however, was just a necessary condition but not a sufficient condition for this discovery. This paper will introduce "Eiffel-Tower, Pyramid, and volcanic-islands models of human activity" that realized the physiological approach to music. [J Physiol Sci. 2006;56 Suppl:S32]

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Activities of cortical pyramidal cells are regulated by GABAergic nonpyramidal cells with temporally and spatially differentiated inhibitotry wiring. Cortical inhibitory synapses were believed to make synaptic contacts mainly on soma and/or proximal dendrites of pyramidal cells, however the latest our finding shows approximately 1/3 of axon terminals of cortical nonpyramidal cells, such as double bouquet cell, Martinotti cell and neurogliaform cell, make synaptic contact on spine head, which also receive an asymmetrical input, called double innervated (DI) spine. From morphological point of view, the inhibitory synapse on DI spine probably has a vetoed function to the asymmetrical excitatory input. We studied morphological properties of the double innervated spines using vesicular glutamate transporters (VGLUTs) positive terminals, which show complementary distribution in cortex, VGLUT1 positive terminals were mostly originated from cortical pyramidal cells and VGLUT2 positive terminals mostly from thalamic projection neurons. This complementary localizations permitted to study an origin of the excitatory terminals on the DI spines. We observed 291 VGLUT1 innervated and 442 VGLUT2 innervated spine heads and found the target of these inhibitory synapses were almost exclusively DI spines received VGLUT2 positive excitatory synapse. Forty four (9.6%) out of 442 spine heads innervated by VGLUT2 positive synapses received symmetrical synaptic input and only 2 (0.7%) out of 291 VGLUT1 innervated DI spines were found. These results indicated that part of thalamo-cortical efferent fibers were vetoed by inhibitory synapse selectively at spine head. [J Physiol Sci. 2006;56 Suppl:S33]

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We studied the roles of presynaptic glutamate receptors in synaptic transmission and plasticity in cortical inhibitory connections. Whole-cell recording was conducted from layer 2/3 pyramidal cells in visual cortical slices of young mice. Inhibitory postsynaptic currents (IPSCs) were recorded at the reversal potential of excitatory synaptic transmission. The frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin was increased by bath application of glutamate, AMPA and NMDA, while it was decreased by the AMPA receptor antagonist NBQX and the NMDA receptor antagonist APV. These compounds did not change mIPSC amplitude, suggesting that AMPA and NMDA receptors are present at the presynaptic terminal of inhibitory synapses and their activation facilitates inhibitory synaptic transmission. Indeed, application of either APV or NBQX decreased the amplitude of evoked IPSCs considerably with accompanying increases in both paired-pulse ratio and coefficient of variation of IPSC, consistent with presynaptic action of these antagonists. High-frequency stimulation produced long-term potentiation (LTP) of IPSCs in normal solution. The magnitude of LTP decreased in the presence of NBQX and LTP did not occur in the presence of NBQX and APV, suggesting that AMPA and NMDA receptors both contribute to the facilitation of LTP production. These results indicate that both AMPA and NMDA receptors are present at the presynaptic terminal of inhibitory synapses in layer 2/3 pyramidal cells and that their activation facilitates inhibitory synaptic transmission and LTP. [J Physiol Sci. 2006;56 Suppl:S33]

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Mammalian cortex receives dense cholinergic innervation from basal forebrain cholinergic neurons, but how acetylcholine (ACh) regulates cortical circuits is still unclear. Recent experiments favor the hypothesis that instead of producing simple facilitation or inhibition, ACh serves to shift the cortical circuits into a condition where cortical neurons are influenced predominantly by afferent inputs from thalamus rather than other cortical inputs. This is achieved by muscarinic suppression of intracortical connections and nicotinic facilitation of thalamocortical inputs, both presynaptically. Indeed, excitatory postsynaptic potentials (EPSPs) in layer 4 neurons elicited by thalamic stimulation were enhanced or in some cases "unsilenced" in the presence of a nicotinic agonist. Optical recordings further supported facilitatory effect of ACh, but it also revealed that this facilitation was followed by suppression only in layer 4. By comparing the sensitivity to a nicotinic agonist between excitatory and inhibitory cells, we found that inhibitory neurons were more susceptible to nicotine. Incidentally, we also found that thalamic activation of GABAergic neurons preceded that of excitatory neurons in a given barrel, which effectively works to produce a feedforward inhibition on excitatory relay cells. Thus, exploiting such intrinsic network property, ACh not only facilitate thalamic input to cortex, but also restrict the excitation of postsynaptic cells to a narrow window of time by selectively enhancing thalamic innervation to inhibitory neurons in the cortex. [J Physiol Sci. 2006;56 Suppl:S33]

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Strengthening GABA-A receptor α1 subunit-mediated inhibition with diazepam triggers ocular dominance (OD) plasticity prematurely (Fagiolini et al., 2004). Yet, the endogenous determinant of critical period (CP) induction remains unknown. Several biochemical analyses intriguingly reveal an elevated GABA uptake with a peak before the CP onset. The main GABA transporter (GAT1) localized to inhibitory axon terminals may be responsible for this transient activity based on its developmental expression profile. Here, we tested directly whether loss of GAT1 function regulates CP onset. In GAT1 knockout (KO) mice, neuronal response strength in visual cortex was normal, but prolonged discharge reflecting weak intracortical inhibition was lost earlier, suggesting an accelerated maturation of GABA function in vivo. Brief monocular deprivation (MD) revealed that OD plasticity of GAT1 KO mice was prematurely activated and rapidly eliminated. When CP onset is delayed by genetic disruption of GABA synthesis, infusion of a GAT1 inhibitor into visual cortex concomitant with MD restored robust OD shifts. Similarly, natural CP delay by dark-rearing is counteracted by diazepam treatment (Iwai et al., 2003) and was also prevented by loss of GAT1. Interestingly, western blot analysis showed that 2d diazepam treatment in the dark triggers a reduction of GAT1 expression, similar to the normal developmental decline in the light. Indeed, GAT1 heterozygous mice mimic such a downregulation and also exhibited an accelerated CP. These findings indicate that GAT1 expression acts as a "brake" to delay CP onset. [J Physiol Sci. 2006;56 Suppl:S34]

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Neurons in area TE of the monkey inferior temporal cortex respond selectively to images of particular objects and are considered to be a neural basis for visual object recognition. The mechanism of generation of the stimulus selectivity, however, is largely unknown. We addressed the role of inhibitory TE neurons in this process by examining their visual response properties and interactions with adjacent target neurons. We applied cross-correlation analysis to spike trains simultaneously recorded from pairs of adjacent neurons in anesthetized macaques (Macaca fuscata). Neurons whose activity preceded a decrease in activity from their partner were presumed to be inhibitory neurons. Most inhibitory neurons responded to a variety of visual stimuli in our stimulus set, which consisted of several dozen geometrical figures and photographs of objects, with a clear stimulus preference. On average, 10% of the stimuli increased firing rates of the inhibitory neurons. Degree of stimulus selectivity of inhibitory neurons was similar to that of excitatory neurons. Although inhibitory neurons occasionally shared the most preferred stimuli with their target neurons, overall stimulus preferences were less similar between adjacent neurons with inhibitory linkages than adjacent neurons with common inputs and/or excitatory linkages. These results suggest that inhibitory neurons in area TE are activated selectively and exert stimulus-specific inhibition on adjacent neurons, contributing to shaping of stimulus selectivity of TE neurons. [J Physiol Sci. 2006;56 Suppl:S34]

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