Fatigue can be the primary symptom of a disease itself–this is the case in chronic fatigue syndrome (CFS), which might therefore prove to be a good model for studying the mechanisms underlying fatigue. CFS is an illness characterized by profound disabling and unexplained fatigue sensation lasting at least 6 months resulting in severe impairment in daily functioning and a combination of nonspecific accompanying symptoms including impaired neurocognitive functions such as concentration, attention, and short-time memory. From neuroimaging studies, various neural deteriorations have been shown: for example, patients with CFS had reduced gray-matter volume in the bilateral prefrontal cortex, and within these areas, the volume reduction paralleled the severity of fatigue; the density of serotonin transporters in the brain was significantly reduced in the rostral subdivision of the anterior cingulate; during the fatigue-inducing period, although the functional magnetic resonance imaging (fMRI) responsiveness of task-unrelated brain regions was not changed in healthy subjects, it was attenuated in the CFS patients, and the attenuation speed was correlated with the subjective fatigue sensation. Although the neural deterioration in fatigue has mostly been studied for this specific disorder, prolonged or accumulated fatigue may introduce both functional and organic deteriorations of central nervous system among healthy population. [J Physiol Sci. 2008;58 Suppl:S19]

抄録全体を表示

Although the vestibular system plays a significant role in the arterial pressure (AP) response to gravitational changes, the physiological significance of this response remains unclear. Hypergravity causes orthostatic fluid shift from the intrathoracic compartment to the legs; this could result in reduced venous return and cardiac output, followed by decreased AP. Thus, the vestibular system-mediated pressor response may counteract the hypergravity-induced hypotension. In this regard, the vestibular system acts as a regulator of AP in order to prevent hypotension. On the other hand, microgravity causes a headward fluid shift that could result in increased venous return and cardiac output, followed by increased AP. Thus, if the vestibular system has a physiological significance in the control of AP during gravitational changes, it should induce a depressor response under microgravity conditions. Conversely, however, it induces a pressor response; i.e., the vestibular system induces a pressor response irrespective of the direction of the changes in gravity and the AP. These results suggest that the vestibular system-mediated pressor response is a type of stress response but not a purposeful response. This pressor response is, however, effective in preventing hypotension under hypergravity conditions and on postural change from recumbency to upright standing. In this presentation, I will review our resent data that supports a physiological significance of the vestibulo-cardiovascular reflex for preventing orthostatic hypotension. [J Physiol Sci. 2008;58 Suppl:S19]

抄録全体を表示

We succeeded in attemptting targeted, direct in vivo protein transduction by microinjecting β-galactosidase (β-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS) (Neurosci Letts, 378; 18-21, 2005). In this study, we investigated changes in blood pressure (BP) by direct in vivo nNOS (neuronal nitric oxide synthase) protein transduction into the rat NTS in order to examine whether the direct in vivo transducted protein in the brain has a function. The BP at 45 min to 3 hr after nNOS microinjection was significantly lower than that of control. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas may have a function. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system. [J Physiol Sci. 2008;58 Suppl:S20]

抄録全体を表示

Sympathetic nerve activity has been thought to play a significant role in long-term regulation of physiological functions, including haemodynamics and body fluid metabolism. However, very few attempts have been made to perform long-term recording of sympathetic nerve activity in conscious animals. We have recently succeeded in recording renal sympathetic nerve activity (RSNA) over a month in rats. In this symposium, we would like to describe a technique of long-term recording of RSNA in freely moving rats, and then introduce our recent experiments in which RSNA and sodium balance were measured during 3 days changes in salt intake in rats. Rats were anesthetized with pentobarbital sodium and then renal nerve was exposed; the exposed nerve and the electrode were embedded in a two-component silicone gel. At least 7 days after the implantation of the electrodes, RSNA and sodium balance were measured continuously over three weeks. Animals were allowed to drink four different concentration of NaCl solutions, that was 0, 50, 154, 308 mEq/L NaCl. Sodium excretion changed significantly in proportion to the sodium loading levels of 0, 154 and 308 mEq/L NaCl. However, we found that changes in RSNA were not always correlated with the changes in sodium excretion during changes in dietary sodium intake. It is therefore that long-term recording of sympathetic nerve activity would provide new aspects on roles of sympathetic nerve activity in prolonged adaptive responses of renal and other physiological functions. [J Physiol Sci. 2008;58 Suppl:S20]

抄録全体を表示

Neurohypophyseal homones, vasopressin and oxytocin, are known to be involved in body fluid homeostasis as anti-diuretic hormone, milk-ejection reflex and maternal behavior. The gene targeting techniques have already generated knockout mice of vasopressin receptors and oxytocin receptor. Recently, we have generated vasopressin-green fluorescent protein (GFP) transgenic and oxytocin-cyan fluorescent protein (CFP) transgenic rats to visualize vasopressin- and oxytocin-secreting neurons in the hypothalamus and their terminals in the posterior pituitary. In vasopressin-GFP transgenic rats GFP fluorescence was observed in the magnocellular division of the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the suprachiasmatic nucleus and axon terminals in the posterior pituitary. In the magnocellular division of the PVN and SON GFP fluorescence was marked increased after dehydration and chronic salt loading. Acute and chronic stresses caused a significant increase of GFP fluorescence in the parvocellular division of the PVN. In oxytocin-CFP transgenic rats CFP fluorescence was also observed in the magnocellular division of the PVN, SON and axon terminals in the posterior pituitary. Strong CFP fluorescence in the PVN and SON were observed in lactating transgenic rats. These rats give us unique new tool to study vasopressin- and oxytocin-secreting neurons in in vivo experiments, using electrophysiology and monitering system of these fluorescences. [J Physiol Sci. 2008;58 Suppl:S20]

抄録全体を表示

The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes including autonomic nervous and neuroendocrine system. Although we know much about of molecular clock mechanism, we still do not fully understand how the molecular clock is coupled to the output pathways which controls circadian phenotypes. The neural output of the suprachiasmatic nucleus (SCN) in the mammalian hypothalamus is essential in the circadian regulation of behavioral activity. We have examined the downstream pathway of the central clock by means of real-time monitoring of multi-unit neural activity in feely moving mice. The results demonstrate that SCN neural activity is tightly coupled to environmental photic input that is relayed to the subparaventricular zone (SPZ), the neural activity rhythm in the SPZ being reversed in phase compared in the SCN. In Clock mutant mice exhibiting attenuated circadian locomotor rhythmicity, neural rhythmicity in the SCN and SPZ is affected in a similar manner. These results suggest that the SPZ plays a functional role in relaying circadian and photic signals from the SCN to centers involved in generating behavioral activity. [J Physiol Sci. 2008;58 Suppl:S20]

抄録全体を表示

To study the mechanisms and consequences of dendritic peptide release in vivo, we have combined microdialysis with single cell recording. A purpose-designed small microdialysis loop is placed on the exposed ventral surface of the supraoptic nucleus (SON) in anaesthetised rats to allow sampling of extracellular fluid which enables the in vivo measurement of dendritic peptide release and the direct application of substances via the dialysate to influence dendritic activity. Through the centre of the loop we insert a microelectrode into the SON to monitor the electrical activity of identified magnocellular neurons, classified as oxytocin and vasopressin cells by their firing patterns and physiological responses. By placing an additional stimulating electrode in a brain area of interest we can electrically-stimulate afferent inputs to the SON (e.g. organum vasculosum of the lamina terminalis, nucleus tractus solitarius, olfactory bulb). Post-stimulus histograms reveal the effects of transmitters released from these inputs. We can thus monitor physiological regulation of dendritic release in vivo during pharmacological control of dendritic mechanisms, and with concurrent monitoring of somatic action potential activity. We have used this approach to determine the actions of neuroactive substances such classical transmitters, endogenous opioids, adenosine, nitric oxide, steroids, and glial-derived factors, which affects both neuronal activity and dendritic peptide release. These studies provided insights in many local neuronal autoregulatory mechanisms. [J Physiol Sci. 2008;58 Suppl:S21]

抄録全体を表示

Macula densa (MD) modulates tubuloglomerular feedback system by generating nitric oxide (NO) in response to changes of luminal NaCl concentration. A newly established mouse cell line of MD (NE-MD) shows up-regulation of nNOS protein when NE-MD cells were incubated with either low [NaCl] or 12 μM furosemide for 5 hrs. Molecular weights of furosemide-induced nNOS proteins were 65 kDa and 150 kDa (Western blotting), although a faint single band of 150 kDa was only recognized under the control condition. This suggests that truncated nNOS protein may play a key role for regulation of NO generation. To further investigate nNOS, we examined the control and furosemide-induced proteomes of NE-MD by two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MALDI-TOF-MS) as well as a study of nNOS mRNA by RT-PCR technique. We found that nNOS mRNA was about 4.0 kb corresponding to a nearly full length of nNOS protein (150 kDa). More over, we found that 6 protein spots were increased by 1.5 times in NE-MD cells treated with furosemide (2-DE), and that only one unique protein spot, determined as nNOS, increased by more than 5 times. By using MALDI-TOF-MS, although N-terminal heme binding domain was conserved, a C-terminal reductase domain was missing. In conclusion, N-terminal oxidative domain of nNOS protein may explain a unique regulation of NO generation in the mouse kidney macula densa. [J Physiol Sci. 2008;58 Suppl:S21]

抄録全体を表示

Arginine-vasopressin (AVP) is well-known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported that the plasma volume and blood pressure are decreased in V1a receptor-deficient (V1aR-KO) mice. In this study, we investigated the role of V1aR in renin-angiotensin system (RAS) using V1aR-KO mice and found that RAS was suppressed in V1aR-KO mice. The V1a receptor is detected at macula densa and granule cells of kidney in control wild mice by the analysis with in situ hybridization and found to be co-localized with the expression of neuronal nitric oxide synthase (nNOS) and cyclooxygenase (COX)-2 in the macula densa (MD) cells. The expression of renin in the granule cell was decreased in V1aR-KO mice, which led to decreased plasma renin level. In addition to the decreased renin expression, the expression of renin stimulators such as neuronal nitric oxide synthase (nNOS) and cyclooxygenase (COX)-2 in the macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-KO mice. These data indicate that AVP regulates body fluid homeostasis via the V1aR in the MD cell by activating RAS. The disruption of V1aR led to decreased plasma volume and hypotension. [J Physiol Sci. 2008;58 Suppl:S21]

抄録全体を表示

The electrogenic Na-HCO₃ cotransporter NBC1, encoded by SLC4A4, plays important roles in whole-body acid/base homeostasis and intracellular/extracellular pH regulation. NBC1 has two major spliced variants, kidney-type cotransporter kNBC1 and pancreas-type cotransporter pNBC1. kNBC1 is predominantly expressed in renal proximal tubules, where it mediates a majority of bicarbonate absorption. On the other hand, pNBC1 is more widely distributed in several tissues such as pancreas, eye, and brain, and is involved in other biological processes. Unlike kNBC1, pNBC1 is fully activated only after an IP3 receptor binding protein IRBIT binds to the N-terminal specific region of pNBC1. Furthermore, the binding of IRBIT to pNBC1 is dependent on phosphorylation status of IRBIT. Such a unique regulation of pNBC1 by IRBIT might at least partially explain diverse physiological roles of NBC1 variants in vivo. Homozygous mutations in NBC1 cause proximal renal tubular acidosis associated with ocular abnormalities (OMIM 604278). At least 50% reduction of NBC1 activity is required to induce severe acidemia. In addition to functional defects, some NBC1 mutants showed abnormal trafficking in both polarized and non-polarized cells. Interestingly, several lines of evidence suggest that defective membrane expression of NBC1 mutants in astrocytes is associated with migraine, supporting an indispensable role of NBC1 in controlling neuronal excitability. Physiological and pathophysiological roles of NBC1 in renal and extrarenal tissues will be discussed in this symposium. [J Physiol Sci. 2008;58 Suppl:S22]

抄録全体を表示

In many epithelial cells, intracellular Cl⁻ concentration ([Cl⁻]_i) is maintained by Na⁺/K⁺/2Cl⁻ cotransport (NKCC, influx) and Cl⁻ channels (efflux). Inhibition of NKCC (bumetanide) decreased [Cl⁻]_i and inhibition of Cl⁻ channels increased it. Our recent studies demonstrated that bumetanide (20 μM) or a Cl⁻-free (NO₃⁻) solution enhanced ACh-stimulated exocytosis in guinea pig antral mucous cells, in contrast, NPPB (a Cl⁻ channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO₃⁻ solution. An [Cl⁻]_i decrease modulates ATP-dependent priming in Ca²⁺-regulated exocytosis. And, [Ca²⁺]_i measurements showed that bumetanide and NO₃⁻ solution enhanced the ACh-stimulated [Ca²⁺]_i increase. Measurements of [Cl⁻]_i revealed that ACh decreases [Cl⁻]_i, and that bumetanide and NO₃⁻ solution decreased [Cl⁻]_i and enhanced the ACh-evoked [Cl⁻]_i decrease; in contrast, NPPB increased [Cl⁻]_i and inhibited the [Cl⁻]_i decrease induced by ACh, bumetanide or NO₃⁻ solution. Thus, an [Cl⁻]_i decrease accelerates ATP-dependent priming and [Ca²⁺]_i increase, which enhance Ca²⁺-regulated exocytosis in ACh-stimulated antral mucous cells. Moreover, in bronchiolar ciliary cells, an [Cl⁻]_i decrease also enhanced ciliary beat frequency by modulating cAMP actions. These observations indicate that the Cl⁻ transport plays an important role in the regulation of cellular functions in epithelia. [J Physiol Sci. 2008;58 Suppl:S22]

抄録全体を表示

Cellular responses to the hypotonic media have been extensively investigated in various tissues. However, the response to osmotic changes in the renal collecting duct (CCD) is not well known yet, although the luminal osmolarity of CCD is occasionally lowered in some diuretic states. In this study, we examined effects of hypotonic solution on the apical ionic conductance and the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the principal cells of the freshly isolated rat CCD. Exposure of the cells to hypotonic solution activated the apical Ca²⁺-dependent BK channel in the cell-attached mode of the patch-clamp technique and evoked a transient increase in [Ca²⁺]_i measured by fura-2. These responses were both markedly attenuated by removal of extracellular Ca²⁺. The hypotonicity-induced [Ca²⁺]_i elevation was not influenced by nifedipine, rutheniumred, La³⁺, Gd³⁺ and a prinergic receptor antagonist, suramin. On the other hand, 2-APB, which is widely used as an IP₃ receptor antagonist and as an inhibitor of several types of TRP channel, significantly suppressed the hypotonicity-induced [Ca²⁺]_i elevation. These results suggest that extracellular hypotonicity induces [Ca²⁺]_i elevation, which elicits the activation of BK channel. Moreover, our data provide a possibility that extracellular Ca²⁺ and 2-APB sensitive components would play a crucial role in the mechanism underlying the hypyonocity-induced [Ca²⁺]_i elevation in the principal cell of CCD. [J Physiol Sci. 2008;58 Suppl:S22]

抄録全体を表示

The multispecific organic anion transporters in the liver play an important role in the elimination of toxic substances and in the turnover of endogenous biomolecules. In this study, we functionally characterized a novel organic anion transporter OAT7 (SLC22A9) exclusively expressed in human liver. When expressed in Xenopus oocytes, OAT7 mediated high affinity transport of sulfate-conjugated steroid hormones, estrone sulfate (ES) (Km = 8.7 μM) and DHEA sulfate (Km = 2.2 μM), in a sodium independent manner. ES transport mediated by OAT7 was inhibited by negatively charged BSP, ICG and several sulfate-conjugated xenobiotics. In contrast, glucuronide and glutathione conjugates exhibited no inhibitory effects on the OAT7-mediated transport. Immunohistochemical analysis revealed that OAT7 protein was located in the sinusoidal membrane of hepatocytes. We further examined the trans-stimulatory effects of various endogenous organic anions to investigate the driving force for OAT7-mediated transport. Short chain fatty acids trans-stimulated the OAT7-mediated ES uptake by the injection of those cold compounds into the oocytes. Furthermore, OAT7 mediated [¹⁴C]butyrate uptake and [³H]ES efflux in exchange for extracellular butyrate both in Xenopus oocytes and OAT7-stably expressing cells. These results suggest that OAT7 is the liver-specific organic anion transporter using butyrate as a counterion for its transport of steroid sulfates. [J Physiol Sci. 2008;58 Suppl:S22]

抄録全体を表示

In the adult mammalian CNS, it is well known that injured axons exhibit very limited regeneration ability. Due to this lack of appropriate axonal regeneration, a traumatic damage to the adult brain and spinal cord frequently causes permanent neuronal deficits. Several axon growth inhibitors in the CNS have been identified in the myelin. Downstream of these inhibitors, activation of RhoA and its effector Rho-kinase has been shown to be a key element for neurite growth inhibition and growth cone collapse elicited by these inhibitors. Consistent with these findings in vitro, inhibition of RhoA or Rho-kinase in vivo promotes axon growth and functional recovery after spinal cord injury. Recently, several developmental guidance proteins, including repulsive guidance molecules, semaphorin, and ephrin are suggested to be involved in axon growth inhibition after injury to the CNS. Thus, multiple axon growth inhibitors seem to contribute to inability of the injured axons to regenerate, and therapeutic strategy to block the multiple axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the CNS. In addition, it is noted that synaptic plasticity in pre-existing pathways and the formation of new circuits through collateral sprouting of lesioned and unlesioned fibers are important components of the spontaneous recovery process. The molecular mechanism of this phenomenon is poorly understood, and elucidation of this will contribute to enhancement of functional recovery after incomplete injury to the CNS. [J Physiol Sci. 2008;58 Suppl:S23]

抄録全体を表示

Although it has been long believed that the damaged central nervous system does not regenerate upon injury, there is an emerging hope for regeneration-based therapy of the damaged central nervous system (CNS) due to the progress of developmental biology and regenerative medicine including stem cell biology. In this talk, I will summarize recent studies aimed at the development of regeneration-based therapeutic approaches for spinal cord injuries, including therapy with anti-inflammatory cytokines, transplantation of neural stem/precursor cells and induction of axonal regeneration. [J Physiol Sci. 2008;58 Suppl:S23]

抄録全体を表示

Transection of the direct cortico-motoneuronal (CM) pathway at the mid-cervical segment of the spinal cord in the macaque monkey results in a transient impairment of finger movements. Finger dexterity recovers within a week to a few months. Combination of brain imaging with positron emission tomography (PET) and reversible pharmacological inactivation of motor cortical regions with focal injection of muscimol showed that the recovery involves the bilateral primary motor cortex during the early recovery stage, and more extensive regions of the contralesional primary motor cortex and bilateral premotor cortex during the late recovery stage. These changes in the activation pattern of frontal motor related areas represent an adaptive strategy for functional compensation after spinal-cord injury. Furthermore, we recorded the focal field potentials in the motor cortices and EMG activities of hand/arm muscles in 2 monkeys. Cortico-muscular coherence at beta band (peak at 17Hz), which was observed in one of the monkeys disappeared and never recovered even after the functional recovery (3 months postoperatively). On the other hand, musculo-muscular at gamma band (peak at around 30 Hz), which was never observed preoperatively, became evident and prevailed widely among the hand/arm muscles and increased as the recovery progresses. These results suggest that some unknown low level common oscillator takes over the function to compensate for the dexterous finger movements after the lesion of the direct cortico-motoneuronal pathway. [J Physiol Sci. 2008;58 Suppl:S23]

抄録全体を表示

We have investigated the effects of post-lesion training on motor recovery after a lesion of the primary motor cortex (M1). In the monkeys that had undergone intensive daily training after lesion, behavioral indexes used to evaluate manual dexterity recovered to the same level as in the pre-lesion period 1 or 2 months after the lesion Relatively independent digit movements, including precision grip, were restored in the trained monkeys. While the behavioral indexes of manual dexterity recovered to some extent in the monkeys without the post-lesion training, they remained lower than those in the pre-lesion period until several months after M1 lesion. The untrained monkeys frequently used alternate grip strategies to grasp a small object with the affected hand instead of using precision grip. These results suggest that the recovery of precision grip after M1 lesion requires intensive post-lesion training. To investigate the reorganization of neural circuits underlying the training-induced recovery of precision grip, both histochemical and brain imaging studies are in progress. [J Physiol Sci. 2008;58 Suppl:S24]

抄録全体を表示

In mammalian central nervous system, upon arrival of an action potential, glutamate is released from presynaptic terminals by exocytosis. Direct imaging of neurotransmission should greatly contribute to analyze an exocytosis dynamics at synapses and improve our understanding of the mechanisms in synaptic transmission. Aiming at imaging glutamate, we developed a novel optical glutamate probe named EOS, which consists of a ligand binding domain of AMPA receptor GluR2 subunit and a small molecule fluorescent dye. Glutamate binding to EOS can be reported as a change of fluorescence intensity. By immobilizing EOS on the cell surface of the hippocampal neuronal culture, we successfully visualized released glutamate following presynaptic firing with a single synapse resolution. Furthermore, we succeeded in continuous monitoring of the changes in presynaptic activity induced by phorbol ester. These results indicate that EOS can be used in evaluation of presynaptic modulation and plasticity. To clarify the mechanism of exocytosis in an excitatory synapse, we tried to quantitatively analyze released glutamate at individual synapses. We found that released glutamate varied in amount among synapses. Also, changes in release probability by manipulating extracellular calcium concentration were associated with changes in the amount of glutamate released at individual synapses. Our results suggest that single hippocampal synapses contain several release units. In summary, EOS-based glutamate imaging method is useful to address numerous fundamental issues in exocytosis process in the central nervous system. [J Physiol Sci. 2008;58 Suppl:S24]

抄録全体を表示

Two-photon microscopy is a less-invasive cross-sectional imaging technique for long-term visualization of living cells within deeper layers of organs. This microscopy is based on the multi-photon excitation process and has been used widely in medical and biological sciences. An attractive property of two-photon microscopy, multicolor excitation capability has enabled quantification of spatiotemporal patterns of [Ca²⁺]_i, ion transport and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocyotosis" for the first time. Sequential compound exocytosis has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Further exploration has revealed dynamics and physiological roles of actin cytoskeleton, and soluble NSF attachment receptor (SNARE) proteins. In addition, our newly developed method (TEPIQ method) can be used to determine fusion pores and the diameters of exo- and endocytotic vesicles smaller than the diffraction-limited resolution. Recently, we have successfully observed neurons deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. We have also improved the spatial resolution needed to visualize fine structures of basal dendrites in layer V in vivo. These results suggest that we can follow long-term changes of neural or glial cells in vivo. Two-photon microscopy will thus be important in advancing the study of the molecular basis of secretory and neural activities in the intact human body. [J Physiol Sci. 2008;58 Suppl:S24]

抄録全体を表示

Arl8 is the first Arf-like GTPase found to be localized to lysosomes and a highly conserved GTPase in multi-cellular organisms. In the present study, we analyzed possible role(s) of Arl8 in lysosome biogenesis and functions using C. elegans. We isolated loss-of-function mutants for arl-8 and found that the individual late endosomes and/or lysosomes got smaller with the increasing number of these vesicles in the coelomocytes (macrophage-like scavenger cells in C. elegans) of arl-8 mutants. arl-8 mutants also showed that endocytosed macromolecules, which are efficiently degraded in lysosomes of wild-type coelomocytes, are abnormally accumulated in late endosomes of arl-8 mutants. These results suggest that arl-8 is required for trafficking from late endosomes to lysosomes. Recent research has suggested that lysosomes can fuse with late endosomes, leading to the formation of an endosome-lysosome hybrid organelle; however, the molecular mechanisms underlying the hybrid-organelle formation remain unresolved. cup-5, the C. elegans mucolipin-1 homolog, functions in the reformation of lysosomes from endosome-lysosome hybrid organelles. We found that the enlarged hybrid-organelle formation in cup-5 mutants is strongly suppressed by null mutations in arl-8, indicating that arl-8 acts genetically upstream of cup-5. Collectively, these results suggest that arl-8 positively regulates the hybrid-organelle formation between late endosomes and lysosomes. [J Physiol Sci. 2008;58 Suppl:S25]

抄録全体を表示

The Sec1/Munc-18 (SM) family members are essential factors that regulate docking/fusion of membrane-bound organelles in various eukaryotic cells, but their precise roles are still unclear. To elucidate the physiological roles of SM proteins in membrane traffic and organelle biogenesis in a multicellular organism, we have systematically isolated deletion mutations of all the SM genes (unc-18, vps-33.1, vps-33.2, vps-45, T07A9.10 and F43D9.3) on the C. elegans genome, using a TMP (trimethylpsolaren)/UV method. We found that the SM mutations result in various phenotypes, such as embryonic lethality, larval lethality, small brood size, and reduction of motility. To investigate which trafficking pathways are impaired in these mutants, transgenic analyses using fluorescent reporters were performed. vps-33.1 and vps-45 mutations caused the defects on both the fluid-phase and the receptor-mediated endocytosis pathways, and these endocytic defects were linked to abnormal morphologies of endosomes or lysosomes in the mutant cells. The mutation of the neuron-specific gene, unc-18, resulted in the defect of secretion of synaptic vesicles. These functional analyses of SM mutants revealed that SM proteins are key molecules regulating membrane traffic on both the exo-and the endocytic pathways in a multicellular organism. [J Physiol Sci. 2008;58 Suppl:S25]

抄録全体を表示

The crystal structure analyses of ion channels had been awaited for a long time to elucidate the structure-function relationship, but the application was limited by the difficulty of crystallization of membrane proteins. Significant progresses were made in the last ten years by MacKinnon's and other groups, and many important findings such as the structural basis of K⁺ selectivity in K⁺ channels have been reported. These facts prove the undoubted effectiveness of the structural biological approach to the ion channel research. It is also true, however, that there are many important questions remaining unanswered even after the initial crystal structural analysis. These include dynamic aspects of structural rearrangements, environment dependent changes, and formation and function of channel complexes. Our group aims to know the dynamic aspect of membrane proteins in function towards the elucidation of functioning mechanisms. In this presentation, I would like to first introduce very briefly three topics from our previous works: (1) FRET approach to the structural rearrangements of metabotropic glutamate receptor1; (2) Density dependent changes of pore properties of ATP receptor channel P2X₂; (3) Voltage and [ATP] dependent gating of P2X₂ which lacks voltage sensor. I will then introduce in detail our recent work using cys accessibility analysis focusing on (4) the effect of accessory subunit KCNE1 and KCNE3 on the movement of voltage sensor of KCNQ1 channel, which gives insight into how the assembly of KCNE1 and 3 modifies the activation of KCNQ1 channel. [J Physiol Sci. 2008;58 Suppl:S25]

抄録全体を表示

Memories are thought to be formed and stored as changes in fast neurotransmission mediated by AMPA-type glutamate receptors in the mammalian CNS. Several lines of evidence indicate that changes in the number of AMPA receptors at the postsynaptic sites are one of the major mechanisms underlying such synaptic plasticity. Therefore, a better understanding of molecular mechanisms regulating a life of AMPA receptors in neurons is crucial. An odyssey of AMPA receptors begins when they are synthesized in the endoplasmic reticulum (ER) mostly located in somata of neurons. Interestingly, mutant AMPA receptors that cannot bind to ligands, form channels, or improperly assembled complexes, are actively retained and degraded at the ER by an unknown mechanism. AMPA receptors are then selectively trafficked to dendrites by an adaptor protein complex-4 at the trans-Golgi network (TGN). For the ER exit and TGN sorting processes, transmembrane AMPA receptor regulatory proteins (TARPs) play an indispensable role. AMPA receptors are exocytosed by the vesicle fusion apparatus, such as the exocyst complex, at yet unknown extrasynaptic sites. AMPA receptors then laterally diffuse into synapses, where they are anchored by several proteins including TARPs. AMPA receptors are removed from the synapses by lateral diffusion followed by clathrin-mediated endocytosis at the perisynaptic endocytosis zones. Some AMPA receptors recycle back to the synapses, but others are degraded by a ubiquitin-proteasome system. In this symposium, I would like to introduce recent progresses at each step of the life of AMPA receptors, focusing on our own findings. [J Physiol Sci. 2008;58 Suppl:S26]

抄録全体を表示

Production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) by phospholipase C is critical in lymphocyte activation. In contrast to store-operated Ca²⁺ entry activated by IP₃-induced Ca²⁺ release from ER Ca²⁺ stores, importance of DAG-activated Ca²⁺ entry pathways remains elusive. Here, we describe the physiological role of DAG-activated Ca²⁺ entry in B cell receptor (BCR) signaling using TRPC3-deficient avian DT40 B lymphocytes. They showed significant impairment of DAG-activated cation currents but an intact SOC activity. TRPC3 deficiency suppressed the sustained translocation of protein kinase C (PKC)β to the plasma membrane (PM) and PKC-dependent ERK activation upon BCR stimulation. Since TRPC3 directly associated with PKCβ in a Ca²⁺ entry-dependent manner, TRPC3 functions as both a Ca²⁺-entry channel and a molecular anchor for PM translocation of PKCβ. [J Physiol Sci. 2008;58 Suppl:S26]

抄録全体を表示

Neuropathic pain is a severely disabling state that affects more than 15 millions of people in the world. This type of pain may be experienced after nerve injury. In the neuropathic pain state, touch stimulation frequently evokes strong pain sensation. We have previously shown that P2X4 receptors (P2X4Rs), which are upregulated in activated microglia in the spinal cord after nerve injury (Nature, 2003), and the stimulation of P2X4 causes release of brain-derived neurotrophic factor (BDNF) (Nature, 2005). BDNF causes a collapse of transmembrane anion gradient of lamina I neurons, and this results in changing the inhibitory action of GABA to excitatory one. Thus, we postulate that BDNF released by the stimulation of P2X4 in microglia is a crucial signalling molecule evoking the modal sift. We have also examined the mechanisms of the up-regulation of P2X4Rs in microglial cells. We found that fibronectin (FN), an extracellular matrix protein, increased P2X4R expression in microglia via an interaction with integrins (Glia, 2006) through activation of PI3K and MEK/ERK. Lyn tyrosine kinase is also required for this upregulation (Glia, 2008). These results indicate that FN may be regulators acting directly and indirectly on the P2X4R gene in microglia. Understanding of mechanisms underlying P2X4R expression in spinal microglia may lead to new strategies for the management of neuropathic pain. [J Physiol Sci. 2008;58 Suppl:S26]

抄録全体を表示

Taste responses to glutamate are initiated by G-protein coupled receptors and transduced via G-protein signaling cascade, which ultimately activates the taste-transduction channel (TRPM5). Molecular genetics and heterologous expression implicate T1r1/T1r3 heterodimer as umami taste receptor. T1r3-KO mice, however, showed diminished but not abolished taste responses to glutamate, suggesting the existence of umami taste receptors other than T1r1/T1r3. Recordings from mouse single fibers and taste cells revealed that both glutamate sensitive fibers and taste cells were classified into sucrose-best (S-type) and mono potassium glutamate (MPG)-best (M-type). Each type was further classified into 2 subgroups: one type showing synergistic effect between MPG and IMP (S1, M1) and the other type showing no synergism (S2, M2). In T1R3- or TRPM5-KO mice, S1-type was absent, but S2, M1 and M2 types still remained, supporting the existence of multiple receptors, transduction pathways and fiber types for umami taste. Metabotropic glutamate receptors are possible umami taste receptors other than T1r1/T1r3. Group I and III antagonists, AIDA and CPPG, showed inhibitory effects on taste responses to glutamate, suggesting that mGluR1 and mGluR4 may be involved in glutamate signaling. Thus, taste of glutamate is mediated by multiple receptors and transduction mechanisms in the taste bud. [J Physiol Sci. 2008;58 Suppl:S27]

抄録全体を表示

This report deals with responses of vagal afferents to amino acidsin the gut. To observe the effects of infusion of amino acids solutions into thestomach or intestine, afferent nerve activities were recorded fromperipheral cut end of gastric or celiac branch of the vagus nerve in anesthetized rats. Isotonic (150 mM) solution of 8 essential and 10 non-essential aminoacids (1 ml) were slowly infused.Reults: Infusion of MSG solution into the stomach evoked an activationof vagal gastric afferents, however, infusions of other 17 amino acidsolutons and saline evoked no siginificant response. Infusion of most of amino acid solutions including essential and non-essential aminoacidsinto the intestinal canal evoked the excitatory afferent response in celiac branch of the vagus nerve, and four amino acids, methionine,threonine, glycine and histidine evoked inhibitory response. These observations suggest that gastric MSG sensor works as an proteinmarker and intestinal amino acid sensors send their infomation tothe brain through vagal afferents which may play a role in appetiteand proein metabolism. [J Physiol Sci. 2008;58 Suppl:S27]

抄録全体を表示

It has been reported that application of glutamate into the rat stomach induces excitation of the gastric branch of the vagus nerve and activates specific brain loci. Since a specific blocker for serotonin receptor type 3 (SR3) inhibited the above excitation, and no report for the distribution of SR3-containing cells in the gastric mucosa was available, we examined the distribution of SR3 and serotonin (5-hydroxytryptamine, 5-HT) within the gastric and intestine mucosae. For SR3, prominent immunoreactivity was localized in the neck region of gastric glands. SR3-immunoreactive cells were irregular in shape, which were different population from mucous neck cells. A scattered population of 5-HT-immunoreactive cells was localized within the mucous epithelium and the basal region of the gastric glands. By contrast, no-SR3-immunoreactive cells were localized within the mucous epithelium and glandular components of the duodenum, whereas 5-HT-immunoreactive cells that were probably typical intestinal endocrine cells were frequently seen within its mucous membrane. Thus, it is very likely that the novel distributional pattern of 5-HT- and SR3-producing cells demonstrated in this study plays a crucial role for glutamate signaling in the gastric mucosa. The further characterization of these cells, together with other cell types related to the glutamate signaling, will be presented, and their physiological significance will be discussed. [J Physiol Sci. 2008;58 Suppl:S27]

抄録全体を表示

Postingestive consequences (nutrition, satiation, etc.), as well as oro-nasal sensory stimuli (taste, smell, and texture), are the key factors that determine preference and appetite for foods and fluids. Recent studies have demonstrated expression of taste receptors and their taste transduction elements in the gastrointestinal (GI) epithelium, suggesting an existence of chemical sensing systems both in the oral cavity and GI tract. Oral stimulation with monosodium L-glutamate (MSG), a representative umami-taste substance, elicits neuronal activation along with ascending gustatory pathways (dorsal "taste" and ventral "hedonic" pathways). For example, some neurons in the lateral hypothalamic area (LHA), a feeding center, respond to MSG during ingestion in rats. In addition to taste-related responses, we have demonstrated spatio-temporal activation of rat forebrain regions, including the cortex, hypothalamus, basal ganglia, and limbic system following intragastric delivery of taste substances (glucose, MSG, NaCl) by using functional magnetic resonance imaging (fMRI). Subdiaphragmatic total vagotomy (TVX) substantially eliminated brain activation induced by intragastric administration with MSG and NaCl. Consumption of MSG solution was greatly reduced by TVX. These results suggest that taste substances can activate higher brain centers via neural signaling pathway (gustatory and vagus nerves), which is involved in regulation of preference and appetite for foods and fluids. [J Physiol Sci. 2008;58 Suppl:S28]

抄録全体を表示

Recent concept of brain science began to propose that formation of emotion initially depends on the interoception, in which visceral perception is one of the demonstrable phenomena. Visceral hypersensitivity and dysregulation of brain-gut interactions are the key features of pathophysiology of irritable bowel syndrome (IBS). Therefore, clarification of pathophysiology and pathogenesis of IBS may contribute to not only gastroenterology but also research on physiology of perception and emotion. Determining substance that plays a key role in brain-gut interactions is a crucial step for clarifying pathophysiology of IBS. Peripheral administration of CRH antagonist, α-helical CRH (αhCRH), improves visceral stimulation-induced increase in colonic motility, abdominal pain, and anxiety in IBS patients. Peripheral administration of CRH receptor-1 antagonist selectively induces similar phenomena in IBS model rats. Studies using positron emission tomography (PET) demonstrate that IBS patients show greater activation in the brain stem to the mild and intense distention than controls. Significantly more activated brain regions with intense distention between placebo and αhCRH treatments in IBS patients than that in controls are right anterior insula, right PFC (BA11), and left parahippocampal gyrus. Functionally crucial role of limbic and prefrontal cortices and related substances during normal and abnormal visceral perception are suggested. Further research of visceral perception and emotional awareness of the body is warranted. [J Physiol Sci. 2008;58 Suppl:S28]

抄録全体を表示

Vagal afferents monitor sensory events originating in the internal organs and convey the signals to the brain; this is an essential process of homeostatic regulation. Accordingly, vagal afferents and related brainstem nuclei have been studied with respect to autonomic regulation. By contrast, while vagal afferent activity diverges in the central nervous system and exerts a variety of influence on the brain functions, involvement of forebrain areas other than limbic or basal forebrain remains less understood. In this talk, cortical, thalamic and striatal representation of vagal afferents will be described. 1. Visceral information transmitted via vagal afferents ascends through a thalamocortical pathway parallel to the gustatory system. Input from the solitary tract nucleus (NTS) and / or the parabrachial nucleus (PB) arrive at the basal ventromedial thalamic nucleus, which in turn projects to both the insular and ventral sensorimotor cortex. These areas may qualify as the viscerosensory cortex. 2. Vagal afferents project to the thalamic parafascicular nucleus (Pf) via both NTS and PB. Since Pf vagal-responsive region massively projects to the striatum, this pathway would provide vagal input to the basal ganglia. Functional significance of these central projections can only be speculated at present. Vagal afferent activity contributes little to our conscious experience. These forebrain areas may work within the extended framework of autonomic regulation that includes behavioral adjustment of homeostatic needs, in part by affecting basic emotions. [J Physiol Sci. 2008;58 Suppl:S28]

抄録全体を表示

The ontogenetic approach to physiological events is a useful strategy for understanding the functional organization of the CNS. However, conventional electrophysiological techniques are difficult or impossible to employ with early embryonic neurons. Optical imaging with voltage-sensitive dyes has made it possible to monitor electrical events in embryonic neurons that are inaccessible to microelectrodes. Using this technique, we have investigated the developmental organization of neural circuits related to the cranial and spinal nerves in the embryonic CNS. As easily accessible models, we focused on the brainstem neural circuits related to some cranial nerves. From developmental pursuits of the brainstem network formation, we found that (1) in the 1st-ordered nucleus, postsynaptic responses are observed from early stages of development, before differentiation of pre- and postsynaptic neurons has been completed and the morphological structure of conventional synapses has appeared; (2) neural excitability and synaptic function in a higher-ordered nucleus have already been generated by the time synaptic function in the first-ordered nucleus is initially expressed; and (3) connections between the first- and higher-ordered nuclei are established in a manner similar to the adult pattern from the beginning of nuclear organization. We also found a widely spreading depolarization wave that was triggered by multi-sensory inputs and spontaneous activities and that was mediated by a dual network of chemical synapses and gap junctions. It was accompanied by a Ca-wave, indicating its nurturing effects on CNS development. [J Physiol Sci. 2008;58 Suppl:S29]

抄録全体を表示

The visceral afferent fibers arising from various cervico-abdominal receptors form the first intracerebral synapse in the dorsal vagal complex composed of the nucleus of the solitary tract (NTS), area postrema and the dorsal motor nucleus of the vagus nerve. Recent identification of molecular and functional characteristics of the purinoceptors gave rise to an emerging view of the synaptic function of the extracellular purines such as ATP and its metabolite adenosine. Here I discuss three lines of evidence pointing to an essential role of ATP and adenosine receptors expressed on the synaptic terminal of the vagal primary afferents in the NTS: (1) target cell-dependent modulation of the frequency-filtering properties by adenosine of the primary afferent to second-order neuron transmission, (2) frequency-dependent involvement of P2X receptors in the pulmonary stretch receptor-mediated modulation of the central respiratory rhythm, and (3) suppression of adenosine-mediated presynaptic inhibition by in vivo gene silencing in the nodose ganglion. Supported by MEXT, Japan, 2005-2007. [J Physiol Sci. 2008;58 Suppl:S29]

抄録全体を表示

Phylogenetical and ontogenetical evidence suggests that visual self-recognition requires cognitive mechanisms that are partially distinct from those for recognition of other person, but closely related to a higher social cognition ability, such as empathy. Recent functional imaging studies demonstrated a pattern of cortical activation observed specifically during the recognition of one's own face. A characteristic of the pattern includes an absence of activation in the temporoparietal regions, which is observed during other-face recognition, and has been implicated in social perception. Given that this pattern has been replicated in the self-name recognition, the lack of the involvement of the social perception network seems to be a domain-general characteristic of self-recognition. On the other hand, several cortical regions, predominantly in the right parietal and frontal cortices, exhibit activation specifically during the self-face but not self-name recognition. While these regions have been typically associated with the sensorimotor integration, results of a functional connectivity analysis on intersubject variability in activation suggested that the right parietal and frontal networks play distinct roles in self-face recognition. Taking these findings together, cortical activation specific to the self-face appears to reflect self-other distinction at multiple levels, at least including physical and social levels. [J Physiol Sci. 2008;58 Suppl:S29]

抄録全体を表示

Individuals can experience negative emotions (for example, embarrassment) accompanying self-evaluation immediately after recognizing their own facial image, especially if it deviates strongly from their mental representation of ideals or standards. In this study, we conducted an fMRI study to identify the cortical regions involved in self-recognition and self-evaluation along with self-conscious emotions. We used facial feedback images selected from a video recording, some of which deviated significantly from normal images. In total, 19 participants were asked to rate images of their own face (SELF) and those of others (OTHERS) according to how photogenic they appeared to be. After scanning the images, the participants rated how embarrassed they felt upon viewing each face. As the photogenic scores decreased, the embarrassment ratings dramatically increased for the participant's own face compared with those of others. The SELF versus OTHERS contrast significantly increased the activation of the right prefrontal cortex, bilateral insular cortex, anterior cingulate cortex, and bilateral occipital cortex. Within the right prefrontal cortex, the anterior region, which is located in the right middle inferior frontal gyrus, was modulated by the extent of embarrassment. This finding suggests that the right middle inferior frontal gyrus is engaged in self-evaluation preceded by self-face recognition based on the relevance to a standard self. [J Physiol Sci. 2008;58 Suppl:S30]

抄録全体を表示

Alexithymia, proposed by Sifneos (1972) as a common personal trait in psychosomatic patients, is characterized by difficulty in identifying and describing feeling of the self. On the other hand, alexithymia has been also found in a broad spectrum of psychiatric disorders featured by a dominant disability of recognition of other's mental states or intentions. Furthermore, recent evidences from neuroscience indicate that common neural coding between perception and action occurs in various parts of the monkey brain (Rizzolatti, 2001 for review), and that, in humans, similar brain areas and computational processing are involved during mental representation of both one's own and others' actions and mental-state reasoning (c.f., 'shared representation' by Decety and Sommerville, 2003). These notions inspires us to suppose common (neural) components of representing self and other, and alexithymia could be proposed as a good model of its fault simulation. Using functional MRI, we evaluated whether individuals with alexithymia have impairment in recognizing other's mental states, movements, and in empathizing others etc. Our studies would address a general interesting issue: can a person who can not understand oneself understand others? It also suggests a common neural component for representing self and other. [J Physiol Sci. 2008;58 Suppl:S30]

抄録全体を表示

Our sense of self is greatly influenced by how, we believe, others view us, and a drive to seek a good reputation has a profound impact on our everyday social decision-making and behaviors. Theoretical research on the evolution of human cooperation also highlights the importance of establishing a good reputation. To depict the neural basis of this enhanced motivation, we conducted functional magnetic-resonance imaging while subjects disclosed their own behavioral tendencies with reference to social norms. The mere presence of observers enhanced the task-related activities in the dorsal striatum and medial prefrontal cortex (mPFC). Moreover, these activities positively correlated with the individual's tendency to behave in a socially desirable manner. As the striatum is part of the brain's reward system, our findings suggest that the motivation for a good reputation, which is an important social reward for humans, shares the same neural basis as that for basic material rewards (e.g., food). Furthermore, considering that the mPFC functions in the formation of reflected self-knowledge, the present study suggests that the dorsal striatum and mPFC constitute a social reward system that motivates us to seek a good reputation when our public aspect of the self is salient and thus play a pivotal role in regulating our social behaviors. [J Physiol Sci. 2008;58 Suppl:S30]

抄録全体を表示

The purpose of this study was to identify the neural correlates of psychosocial maturity during understanding others from the viewpoint of self. In psychology, ego identity is one of the most famous theories for psychosocial maturity. Those who have established ego identity have a definite purpose for one's life and an ability of goal directed behavior. We assumed that such a psychosocial maturity is related to the ability to distinguish others from self when one understands the others. We used an fMRI technique to identify cortical regions where activity during understanding others' decision-making is correlated with personality maturity. We measured subjects' personality maturity using the Rasmussen's Ego Identity Scale. There were three conditions, S, F and G which were perspective taking conditions for inferring self, best friend and people in general's decision-making, respectively. Differential activation of both the contrasts F-S and G-S was significantly correlated with the ego identity scale in the left lateral prefrontal cortex (LPFC). The results indicate that the activity of the left LPFC is related to psychosocial maturity when understanding others from the viewpoint of self. We suggest that psychosocial maturity is related to the development of the executive function which is supported by the left LPFC. [J Physiol Sci. 2008;58 Suppl:S30]

抄録全体を表示

In intestinal smooth muscle (SM), gastrointestinal (GI) inflammation induced motility disorder. However, possible mechanisms to induce GI dysmotility are not well understood. We here propound that inflammatory cytokines, IL-1β and TNF-α induce motility disorder of intestinal SM by altering contractile machinery in vitro and in vivo. In the organ cultured ileal SM tissue, IL-1β or TNF-α significantly inhibited carbachol-induced contraction in accompanied with decrease in MLC-phosphorylation. In the western blot analysis for detecting contractile proteins, only CPI-17 expression was significantly decreased by those cytokines treatment. In the series of experiments using IL-1αβ or TNF-α deficient mice, IL-1β did not induce down-regulation of CPI-17 expression in the organ cultured ileal tissue isolated from TNF-α deficient mice, indicating that IL-1β down-regulates CPI-17 expression via TNF-α production. In ileitis model induced by hapten, carbachol-induced contraction in the inflamed intestine was gradually inhibited with decrease in CPI-17 protein content within 2-3 days after the inflammation. This phenomenon could not detect in the ileitis model by using TNF-α deficient mice. In conclusion, we found that TNF-α directly affects on SM to induce impairment of Ca sensitization via down-regulation of CPI-17 expression. We concluded that CPI-17 is one of key molecules to induce motility disorder during GI inflammation. [J Physiol Sci. 2008;58 Suppl:S31]

抄録全体を表示

Myosin light chain kinase (MLCK) of smooth muscle consists of the actin-binding domain at the N-terminal, the catalytic domain in the central portion, and the myosin-binding domain at the C-terminal. The kinase activity is mediated by the catalytic domain that phosphorylates the myosin light-chain of 20 kDa (MLC20), activating smooth muscle myosin to interact with actin. Although the regulatory role of the kinase activity is well-established, the role of the non-kinase activity derived from actin-binding and myosin-binding domains remains unknown. We expressed full length MLCK (WT MLCK) as a recombinant protein to examine their effects on the actin-myosin interaction in vitro. We mutated it so as to lose the kinase activity (ΔATP MLCK), followed by the expression. We found that both MT MLCK in the absence of calcium calmodulin and ΔATP MLCK were able to activate the ATPase activity of unphosphorylated smooth muscle myosin. The author will also refer to the effect of arachidonic acid that enhanced the ATPase activity of smooth muscle myosin without phosphorylating myosin ATPase activity and will discuss about the myosin heavy chain consisting of myosin heads as the site of arachidonic action. [J Physiol Sci. 2008;58 Suppl:S31]

抄録全体を表示

It is known that, in solution, smooth muscle myosin filaments are less stable than the myosin filaments of striated muscles, and assembly of the smooth muscle myosin filaments is strongly correlated with phosphorylation level of myosin regulatory light chain (MLC₂₀) at physiological concentrations of MgATP. Supporting the results in solution, increment of the thick filament (myosin filaments with the accessory proteins) contents during contraction have been reported at least in some smooth muscle cells, although a number of the thick filaments exits even in the resting states. Role of structural dynamics of the thick filaments in excitation-contraction coupling of the smooth muscle cells is, however, still unclear. Recently, using electron microscopes, X-ray diffraction technique and florescent microscopes, we found that blebbistatin, a potent inhibitor of myosin II, irreversibly suppressed Ca²⁺-induced contraction and also disrupted the thick filament structure of skinned (cell membrane permeabilizsed) smooth muscle cells without affecting MLC₂₀ phosphorylation level. Our results indicated that myosin ATPase activity regulates lability of the thick filaments as well as cross bridge cycling in the smooth muscle cells. Contribution of thick filament lability in excitation-contraction coupling of the smooth muscle cells will be discussed. [J Physiol Sci. 2008;58 Suppl:S31]

抄録全体を表示

Rho-kinase (ROK)-mediated Ca²⁺-sensitization plays an important role in abnormal contractions of vascular smooth muscle (VSM) such as vasospasm. As an upstream mediator for this pathway, we previously identified sphingosylphosphorylcholine (SPC), which indeed induced vasospasm in vivo. The SPC-induced contraction of human VSM depended on serum total and LDL-cholesterol. Cholesterol depletion by β-cyclodextrin removed caveolin-1, a membrane raft marker, from VSM membrane and abolished the SPC-induced contraction and translocation of ROK and Fyn, a member of Src family tyrosine kinase (Src-TK). Src-TK inhibitors blocked the SPC-induced contraction and activation of ROK. Furthermore, siRNA-mediated knockdown of Fyn tyrosine kinase inhibited the SPC-induced contraction. In β-escin-permeabilized VSM, constitutively active and dominant negative Fyn induced and abolished the Ca²⁺-sensitization, respectively. The similar results were obtained with overexpression of these mutated Fyn. A functional proteomics approach identified candidate molecule and its phosphorylation site as a possible target of Fyn. These findings suggest that membrane raft and its associated Fyn tyrosine kinase plays an essential role in ROK-mediated Ca²⁺-sensitization of VSM contraction. [J Physiol Sci. 2008;58 Suppl:S32]

抄録全体を表示

The logic of complex and dynamic biological networks such as circadian clocks is difficult to elucidate without (1) comprehensive identification of network structure, (2) prediction and validation based on quantitative measurement and perturbation of network behavior.First, we comprehensively determined the transcriptional regulatory circuits composed of 20 transcription factors, and three type of DNA elements including "morning" element (E-box), "day-time" element (D-box) and "night-time" element (RevErbA/ROR binding element, RRE)^1,2. The following quantitative measurement and static perturbation of clock circuits revealed that E-box/E'-box regulation represents a topological and functional vulnerability in mammalian clocks^2,3. Second, we synthetically implemented photo-responsiveness within mammalian cells by introducing a photoreceptor, melanopsin, and continuously monitored the effect of photo-perturbation on the state of cellular clocks. We report that the phase and amplitude of cellular clocks can be regulated by changing the timing and duration of light pulses. We reveal that a critical light pulse drives cellular clocks into a singularity behavior where robust circadian rhythmicity ce abolished after a certain stimulus. Theoretical analysis and subsequent single-cell-level observation predicts consistently and proves directly that desynchronization of individual cellular clocks underlies this singularity behavior⁴.Reference 1. Ueda, H.R. et al, Nature 418, 534-539 (2002). 2. Ueda, H.R. et al, Nat. Genet. 37, 187-192 (2005). 3. Sato T K, et al, Nat Genet. 38:312-9 (2006). 4. Ukai H, et al, Nat Cell Biol. 9, 1327–1334 (2007). [J Physiol Sci. 2008;58 Suppl:S32]

抄録全体を表示

Spike-timing-dependent synaptic plasticity (STDP), which depends on the relative timing of pre- and postsynaptic spiking, plays an important role in neural development and information storage. However, the mechanisms by which spike-timing information is encoded into STDP remains unclear. Here we show that a novel allosteric kinetics of N-methyl-D-aspartate receptors (NMDARs) codes spike-timing information into STDP (Figure). We developed a biophysical model of STDP, and found a requirement of slow and rapid suppression of NMDARs by Ca²⁺·calmodulin with pre- -> post- and post- -> pre-spiking, respectively, which led us to predict an allosteric kinetics of NMDARs during induction of synaptic plasticity. We experimentally validated the allosteric kinetics by examining peak-amplitudes and time of NMDAR-mediated EPSPs. The allosteric kinetics of NMDARs was also experimentally valid for synaptic plasticity induced by more complex spike trains. Simplification of the model revealed that intracellular Ca²⁺ concentration, at the time when glutamate binds to NMDAR, is the dominant spike-timing information carrier. These findings demonstrate that the simple allosteric kinetics of NMDARs governs the coding of complex spike-timing information into long-term changes in synaptic strength, which may restructure neural circuits and embed experiences into the brain. In this symposium, we discuss the possible interaction between experiments and simulation in terms of synaptic plasticity. [J Physiol Sci. 2008;58 Suppl:S32]

抄録全体を表示

Physiome inherently investigates hierarchical layers of biological system. In the post genome era, the number of layers under study should increase because possible mechanisms at molecular level are always referred to. However, biological modeling has to face unavoidable uncertainties in biological measurements and explosion of degree of freedom. An exhaustive modeling seems to be necessary to understand possible mechanisms underlying biological phenomena at every hierarchical level. However, simultaneously it looks like an impossible or reckless trial. In order to get rid of these difficulties, we propose a novel modeling strategy that integrates the top-down retrospection and bottom-up reduction modeling. Here, our strategy is applied to modeling of biological rhythms which is an appropriate system to study because of the penetration of rhythmic dynamics through all of the hierarchical layers. [J Physiol Sci. 2008;58 Suppl:S33]

抄録全体を表示

The integration of knowledge from many disciplines and vast amount of biological data in the post-genome era together with mathematical and information sciences is moving the world towards a new generation of life science where physiological and pathological information from the living human body can be quantitatively described in silico across multiple scales of time and size and through diverse hierarchies of organization. The Physiome Project represents such emerging sciences. The challenge is to understand and quantitatively integrate not only structure and function of biological entities such as ion channel proteins and enzymes on a single spatio-temporal scale, but also functional relationships between entities across multiple scales. This integrative approach is in stark contrast to the linear approach of reductionist life science, and it will allow us to understand the mechanisms underlying biological functions that will emerge through the dynamics of each element and large aggregations of the elements. Here we discuss several points of the challenge that are expected to be resolved through the Physiome Project We then introduce our recent efforts towards realization of a physiome database and biological simulation platform. [J Physiol Sci. 2008;58 Suppl:S33]

抄録全体を表示

Estrogen binds the estrogen receptor (ER), and the subsequent regulation of gene transcription is called the slow genomic mode of estrogen action. In contrast, rapid, non-genomic actions of estrogen initiated at the membrane also exist. Though the genomic actions of estrogen have been more prominent, both rapid and slow modes of action are important in reproduction. Using a two-pulse paradigm, we have shown that a first pulse of a membrane-limited estrogen conjugate (E2-BSA) rapidly initiates non-genomic actions and potentiates transcription induced by a subsequent pulse of 17β-estradiol from a consensus estrogen-response element (ERE) driven reporter gene in neuroblastoma cells, transfected with ERα. In these cells, we have shown that rapid effects that are important to this transcriptional potentiation by E2-BSA include kinase activation and calcium flux. In addition, we show that these actions are initiated via a Gα_q-coupled receptor at the membrane and may involve βγ signaling to the protein kinase B pathway. Behaviorally, pulsatile estradiol benzoate (EB) administration to ovariectomized female rats induces lordosis behavior comparable to a single continuous administration of EB. Infusion of E2-BSA into the ventromedial hypothalamus could potentiate lordosis behavior induced by EB, demonstrating the applicability of this paradigm in vivo. We are currently searching for endogenous estrogen-induced genes, relevant to reproduction, whose transcription is affected by E2-BSA. [J Physiol Sci. 2008;58 Suppl:S33]

抄録全体を表示

In the rat, receptive females are attracted by sexually active males, whereas matured males approach estrous females. In order to explore the neuroendocrine mechanisms of such odor-based preferences, we established a 3-compartment apparatus and demonstrated clear sexual dimorphism. Furthermore, castrated male rats implanted with Silastic capsules containing testosterone (T), dihydrotestosterone (DHT) or estradiol benzoate (EB), and gonadally intact males with aromatase inhibitor all showed normal, male-typical preference pattern. Removal of the T and EB capsules induced transient reversal of their sexual orientation, that is, those males spent longer time to explore odor of sexually active males to that of receptive females or castrated males. This phenomenon occurs for 2–3 weeks after orchidectormy, and female rats never show the reversal after removal of sex steroids. The results suggest that in male rats, a high level of estrogen provokes male-type preference, and a residual low level of estrogen 2 weeks after castration elicits female-type preference. We also investigated the effect of lesions in the medial amygdala and the preoptic area on odor preference of male rats. Medial amygdala lesions disrupted preference for castrated male odor to sexual active male odor, but did not affect preference for receptive female odor, while medial preoptic lesions eliminated all of the preferences. In a point of view of the chemosensory processing system, the MeA is located in the upstream of the POA. Nevertheless, POA lesions had broader influences on preference, suggesting the circuit processing preference for estrous odor that bypassed the MeA. [J Physiol Sci. 2008;58 Suppl:S34]

抄録全体を表示

Conventional wisdom holds that gonadal steroid hormones sexually differentiate (organize) neural circuits mediating behavior during a perinatal critical period of neural development, and at puberty, gonadal hormones act on those circuits to facilitate (activate) adult- and sex-typical social behaviors. Using the Syrian hamster as a model for understanding mechanisms of behavioral maturation during adolescence, my laboratory has demonstrated that pubertal hormones both organize and activate the adolescent brain. Initial experiments showed that the presence or absence of gonadal hormones during adolescent development programs the degree to which steroid hormones activate adult male and female reproductive behavior. More recent work indicates that puberty and adolescence mark the end of an extended postnatal period of sensitivity to testosterone remodeling of neural circuits mediating reproductive behavior. Remodeling of the adolescent brain appears to involve changes in dopaminergic systems and in synaptic organization of the medial amygdala, a brain region involved in the integration of sensory and steroidal stimuli that regulate reproductive behavior. Our work demonstrates that interactions between gonadal hormones and the adolescent brain play a pivotal role in the maturation of adult social behaviors. We propose that variation in the timing of interactions between steroid hormones and the human adolescent brain, such as those that occur with precocious or delayed puberty, contribute to individual differences in adult behavior. [J Physiol Sci. 2008;58 Suppl:S34]

抄録全体を表示

The molecular and cellular mechanisms underlying experience-dependent plasticity of brain function are poorly understood. Recent in vitro studies have identified the regulated trafficking of AMPA receptors (-Rs) into synapses as a major molecular component of neural plasticity. Here we ask if experience-driven plasticity in the developing rat barrel cortex is accompanied by and/or requires AMPA-R delivery to synapses. By combining in vivo gene delivery with in vitro recordings, we show that experience drives recombinant GluR1, an AMPA-R subunit, into synapses formed between layer 4 and layer 2/3 neurons. Furthermore, expression of the GluR1 cytoplasmic tail in layer 2/3 neurons, a construct that inhibits synaptic delivery of endogenous AMPA receptors during long-term potentiation, blocks experience-driven synaptic potentiation. These studies show that synaptic delivery of AMPA-Rs contributes to plasticity driven by natural stimuli in the mammalian brain.Although social isolation early in life has been shown to alter behaviors including learning and emotion, how neonatal isolation affects synaptic connection is poorly understood. Since whisker-barrel system is involved in social interaction, we examined if social isolation changes experience-dependent synaptic delivery of AMPA receptors in the developing rat barrel cortex. We found that neonatal social isolation disrupted in vivo trafficking of GluR1 and GluR4 into synapses formed from layer 4 to layer 2/3 of developing rat barrel cortex. These data suggested that neonatal social isolation alters neuronal reorganization by disruption of experience-driven synaptic delivery of AMPA receptors. [J Physiol Sci. 2008;58 Suppl:S34]

抄録全体を表示