Among glutamatergic synapses, contribution of AMPA receptor (AMPAR) desensitization to short-term synaptic depression is variable. We addressed the mechanism by which AMPAR desensitization is involved in synaptic depression at the calyx of Held synapse of developing rats. AMPA-EPSCs showed a strong paired-pulse depression (PPD) at postnatal day (P) 7, but PPD became weaker as rats matured. The recovery time from AMPAR desensitization, assessed by paired-pulse glutamate applications to outside-out patches from postsynaptic neurons, became shorter with development. Cyclothiazide (CTZ), which completely blocked AMPAR desensitization, attenuated PPD at P7, but not later than P14, suggesting that AMPAR desensitization contributes to synaptic depression only at immature synapses before the hearing onset (P10). Matching Pr at P14 synapses to that at P7, by increasing external Ca²⁺ and adding tetraethylammonium, recruited a CTZ-sensitive component in PPD. Quantitative single-cell RT-PCR analyses indicated that the R/G-edited GluRs increase with development, and that the recovery AMPARs from desensitization was faster in patches from cells having R/G-edited GluRs more abundantly. We conclude that a developmental decrease in Pr, together with a developmental increase in the R/G-edited GluRs, reduces contribution of AMPAR desensitization to synaptic depression. [J Physiol Sci. 2007;57 Suppl:S19]

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Cisplatin, a platinum-based drug widely used in cancer therapy, is thought to kill cancer cells by causing the induction of apoptosis. We have found that in the KB-3-1 human epidermoid cancer cell line, cisplatin treatment causes a potentiation of activity of the volume-sensitive, outwardly rectifying (VSOR) Cl⁻ channel, which has previously been shown to play an important role in apoptosis. Caspase-3 activation and cell death induced by cisplatin treatment were reduced by inhibition of the VSOR Cl⁻ channel with the blocker DIDS, suggesting that activity of the channel contributes to cisplatin sensitivity. Furthermore, we have found that in the KCP-4 cell line, a cisplatin-resistant derivative of the KB-3-1 cell line, VSOR Cl⁻ channel activity is virtually absent. We hypothesized that the lack of the channel activity contributes to the cisplatin resistance of these cells, and to test this, we attempted to restore the channel activity. Treatment with trichostatin A (TSA) or apicidin, a histone deacetylase inhibitor, caused VSOR Cl⁻ channel function to be partially restored. The TSA-induced restoration reduced the cisplatin resistance of KCP-4 cells, as measured by caspase-3 activity and cell viability assays. DIDS inhibited the reduction in cisplatin resistance. We conclude that activity of the VSOR Cl⁻ channel is involved in the response of KB-3-1 and KCP-4 cells to cisplatin treatment. [J Physiol Sci. 2007;57 Suppl:S20]

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Previous studies have shown that ion channels and transporters are indispensable for cell function including normal cell growth and proliferation. Particularly, modulation of Cl⁻ transport via Cl⁻ channel, K⁺/Cl⁻ cotransporter (KCC) and Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) occurs during cell proliferation. However, it is unknown whether proliferation of cancer cells has correlation to NKCC expression and activity. We determined mRNA and functional expression levels of the NKCC and investigated an inhibitory effect of NKCC blocker, furosemide (a loop diuretic), on proliferation in two human cancer cell lines, MKN28 and MKN45, which were respectively established from moderately and poorly differentiated adenocarcinoma. We found that both mRNA and functional expression levels of NKCC were higher in MKN45 than MKN28 cells. Furthermore, furosemide inhibited the cell proliferation delaying the G₁/S phase progression in MKN45 cells. Since furosemide may diminish the intracellular Cl⁻ concentration ([Cl⁻]_i) by blocking NKCC, we studied the effect of [Cl⁻]_i reduction on the proliferation of MKN cells. The cells cultured in low Cl⁻ media showed the elevation of the G₀/G₁ population associated with increased expression of cyclin-dependent kinase inhibitor, p21, one of the critical molecules for G₁/S checkpoint. These observations suggest that the NKCC plays important roles in cell cycles and cell proliferation of human gastric cancer cells via regulation of [Cl⁻]_i. [J Physiol Sci. 2007;57 Suppl:S20]

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The inhibitory neurotransmitter GABA evokes excitation in immature brain instead of inhibition in adult brain. Since GABA_A receptor is a Cl⁻ channel, such a developmental switch of GABA action between excitation (Cl⁻ efflux) and inhibition (Cl⁻ influx) would be induced by a shift of Cl⁻ homeostasis generated by a dynamic balance shift of cation-Cl⁻ cotransporters (Cl⁻ importer, NKCC1, and exporter, KCC2) during development. The excitatory GABA actions may be involved in neural circuitry development, since reduction of [Cl⁻]_i by inhibiting NKCC1 resulted in less formation of inhibitory synapse formation. High [Cl⁻]_i and excitatory GABA actions may also contribute to neuronal cell migration, since migrating cells had the higher [Cl⁻]_i, the higher expression of NKCC1 and the lower expression of KCC2 than already-settled cells, which were depolarized instead of hyperpolarized by GABA. Indeed, a blockade of GABA_A receptors resulted in migration disorder. A conversion of the GABA response from inhibition to excitation was induced by injuries. In cortical freeze-lesion model, cells adjacent to the lesion site regained immature status of Cl⁻ homeostasis and GABA actions, so that they anomalously migrated. In axotomized motoneurons, the increases in [Cl⁻]_i induced by KCC2 downregulation turned GABAergic inhibition to excitation. The induced spontaneous [Ca²⁺]_i oscillation might be related with re-wiring of afferent inputs. Such a downregulation of KCC2 also induced in postsynaptic neurons by the injury of primary afferents, suggesting functional dynamics of inhibitory pathway regulated by active changes in Cl⁻ homeostasis. [J Physiol Sci. 2007;57 Suppl:S20]

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Intracellular chloride concentrations ([Cl⁻]i) in CNS neurons are regulated by several transporters to be lower than that expected from passive distribution. Among these transporters, ATP-dependent chloride pump acts as an outwardly directed and primarily active chloride transporter requiring phosphatidylinositol 4-monophosphate (PI4P) for its maximal activity. A decrease in plasma membrane PI4P level reduces the pump activity resulting in an increase in neuronal [Cl⁻]i. Such cases can be observed when PI turnover was accelerated with poor supplies of PI substrates, or plasma membrane type II PI 4-kinase (PI4KII) was inhibited by pathophysiological concentrations of amyloid-beta proteins of Alzheimer disease. Neuronal [Cl⁻]i increases from 6 mM to around 30 mM, and this enhances glutamate toxicity at its physiological concentration resulting in neuronal cell death. Mechanisms of this enhancement are partially explained by inhibition of chloride-sensitive activity of PDK2 by higher [Cl⁻]i, and by resulting decreases in cell survival molecule phospho-Akt Ser473 level. Regulation of pathological changes in neuronal [Cl⁻]i is protective against the enhancement of glutamate toxicity. Supplement of substrates for PI or PI4P production, cyclic GMP as PI4K activator, GABAc receptor agonists and short peptides blocking amyloid-beta-induced inhibition of PI4K, reversed the inhibition of chloride pump activity as well as the increases in neuronal [Cl⁻]i, and finally recover the cell viability. Regulation of [Cl⁻]i thus seems to be essential for neuronal cell survival. [J Physiol Sci. 2007;57 Suppl:S21]

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In vivo molecular imaging has become a key technology for pathophysiological science and drug development. We are mostly utilizing PET (Positron Emission Tomography) as a first-choice modality, because of its ultra-high sensitivity for molecules, adequate temporal and spatial resolution, and especially broad spectrum of target molecules. The present status for development of PET molecular probes, instrumentations including microPET, and the methods for quantitative analyses will be introduced with some examples. In vivo molecular imaging could bring the high-quality information about:1. Molecular diagnosis for living patients with symptoms2. Closer approach for etiology and differential diagnosis3. Direct follow-up of key molecules as disease markers4. Pharmacokinetics/Pharmacodynamics in primates/human5. Dose finding information for individuals, corresponding to SNP6. Direct evidence for accumulation in non-target organs: Related to adverse effects7. Drug effects with surrogate markers8. Early decision of dropout substances (drug candidates) In 2005, RIKEN and National Institute of Radiological Science were selected as the key centers for development of All-Japan research network to further promote mutual international and multi-disciplinary collaboration on in vivo molecular imaging. On this occasion, the concept and project themes will also be introduced. [J Physiol Sci. 2007;57 Suppl:S21]

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Positron emission tomography(PET) is an imaging of interaction between radio-labeled molecular probes and bioactive proteins/genes. Targets of PET are, mRNA, protein or reporter gene products, and appropriate radio-labeled molecular probes should be designed for the corresponding targets.For the detection of gene expression at mRNA level, antisense-oligo concept is applicable. However, radio-labeled antisense-oligo for in-vivo hybridization imaging is quite demanding. There are two approaches for the detection of gene expression at protein level, namely detection of target protein itself, or use of reporter gene system. As an example, FDG as a molecular probe for radiation therapy monitoring will be demonstrated using molecular biological findings in radiation therapy monitoring. The latter approach uses a general radio-ligand–reporter gene system, which requires an introduction of a reporter gene in addition to a target gene. As an example, estrogen receptor ligand binding domain and F-18-estradiol are demonstrated to be a candidate for gene-reporter system.These tactics in nuclear medicine will bring new information and approaches in the field of molecular and cellular medicine. [J Physiol Sci. 2007;57 Suppl:S21]

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The genome project for various biological species succeeded in sequencing species-specific genomes and derived genome information, which gave rise to a new era in biology. Biological species comprise cells in which genomic products work on each physiological system. Therefore, cells are considered as the fundamental working unit of each living system, and it is important to characterize the cells in a living system. For this purpose, we are developing a non-invasive technique to visualize cells of interest in living systems. Among various methods to analyze cells, MRI is remarkable because subjects are not disturbed by medium or drugs, and is non-invasive to biological species. However, MR cannot detect signals from small amounts of cells. Therefore, it is necessary to introduce magnetic tags to the cells of interest. To overcome the limitations of MR sensitivity, we developed an efficient method to transport MR tags (superparamagentic iron oxide; SPIO) into cells by using non-virus vehicles. MRI can detect the incorporated MR marker inside the cells non-invasively. Our aim is the in vivo development of more efficient probes for cell tracking with better sensitivity to detect targeted cells. We would show a case study about the application of this cell labeling technique to detect transplanted microglia cells in animal model of Alzheimer disease. [J Physiol Sci. 2007;57 Suppl:S22]

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In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue heterogeneities in oxygen concentration in health and disease. Here, I report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP expressing COS7 cells were briefly photo activated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after a photo activation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knocked-in mouse (green mouse) heart. Then, we tried molecular imaging of hypoxia in in vivo organs. Using the macroscopic optics, the red shift could be imaged in the ischemic liver and kidney in the green mouse provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. It was concluded that present technique using GFP as an oxygen indicator may allow molecular imaging of oxygen in in vivo organs. [J Physiol Sci. 2007;57 Suppl:S22]

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Two respiration-related rhythm generators have been identified in the medulla of rodents to produce intrinsic periodic bursts: One is the pre-Bötzinger complex (PBC), producing inspiratory (Insp) neuron bursts, and the other is the para-facial respiratory group (pFRG), predominantly producing pre-inspiratory (Pre-I) neuron bursts. Although both generators can be independently active under specific conditions, the pFRG-Pre-I rhythm generator interacts with PBC-Insp rhythm generator as a coupled oscillator system to regulate the rhythm of the intact system. Previous studies reported existence of excitatory and inhibitory synaptic connections from Pre-I to Insp neurons and inhibitory synaptic connection from Insp to Pre-I neurons. Several lines of evidence support that the pFRG-Pre-I rhythm generator functions as a primary respiratory rhythm generator to determine frequency of inspiratory burst at least in the brainstem-spinal cord preparation of newborn rats. Since these two rhythm generators possess different sensitivity to various neuromodulators, we hypothesized that the dominance of these rhythm generators to determine basic respiratory rhythm depends on the background stimulation level of the system. These rhythm generators may function also in vivo in which the background stimulation level may differ depending on conditions such as arousal level, anesthesia and afferent inputs. Thus, consideration of rhythm generator dominance in the determination of output frequency may be important in understanding the neuronal mechanisms of respiratory rhythm generation under various in vivo as well as in vitro conditions. [J Physiol Sci. 2007;57 Suppl:S22]

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Recently the rat has become the most popular animal model for in vivo and in vitro studies of central ventilatory control. We examined the pattern of the abdominal expiratory activity in the adult and neonatal rat. In the anesthetized or decerebrate vagotomized paralyzed and artificially ventilated adult rats, the abdominal expiratory nerve showed two discharge patterns, low amplitude expiratory discharge that persisted throughout the expiratory phase (E-all activity) and late expiratory, high-amplitude bursts (E2 activity) superimposed on this steady activity. The E2 activity occurred only at the higher levels of respiratory drive (i.e. hypoxia or hypercapnia). In the decerebrate vagotomized paralyzed and artificially ventilated neonatal rats, the E-all activity was frequently observed in the abdominal nerve under control conditions. Noxious stimuli such as tail pinch increased the respiratory frequency, and enhance the inspiratory and expiratory motor activity. When FICO₂ was increased, the abdominal expiratory activity was enhanced and showed biphasic discharge pattern that consisted from the pre- and post-inspiratory discharges. The pre-inspiratory discharge had larger amplitude and shorter duration than the post-inspiratory discharge in most of cases. Since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity should change during the postnatal development. The importance of the change in the expiratory motor pattern will be discussed. [J Physiol Sci. 2007;57 Suppl:S23]

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Limb movements during walking in mammals are highly coordinated. The basic spatial and temporal activity patterns of each muscle during such movements are generated in a neuronal circuit in the spinal cord. In this circuit also called the central pattern generator (CPG), synaptic inhibition mediated by inhibitory neurons is considered to be an important element. In vitro isolated spinal cord preparations from neonatal rats and mice have been widely used to study the neuronal mechanism underlying locomotion in mammals. Utilizing this preparation, we have previously shown that GABA and glycine, two major neurotransmitters in the spinal cord, each play important roles in the coordination of the left and right limb activity and its maturation in the prenatal period. Recent studies utilizing genetically engineered mice have facilitated the investigation of the activity and roles of identified interneurons during locomotion. We have recently recorded the activity of Renshaw cells, a population of inhibitory neurons that form recurrent inhibitory connections to motor neurons in isolated spinal cord preparations taken from GAD67EGFP-knock-in mouse neonates. Their synaptic inputs and firing patterns during locomotor-like motor activity suggested that Renshaw cells play a role in regulating the firing of motor neurons during locomotion in rodents. Furthermore, these recordings revealed that Renshaw cells are modulated by the CPG while their firing is not essential for the generation of locomotor activity. [J Physiol Sci. 2007;57 Suppl:S23]

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Feeding behavior in mammals changes from suckling to mastication during postnatal development. In order to investigate the postnatal development in the neuronal circuits controlling feeding movements of orofacial structures, we have examined the location of excitatory premotor neurons for trigeminal motoneurons (TMNs), and postnatal changes of inputs from these premotor neurons to TMNs in brain stem slice preparations and anesthetized adult rats. Electrical stimulation of the supratrigeminal region (SupV) and the reticular formation dorsal to the facial nucleus (RdVII) in the brainstem slices evoked excitatory postsynaptic currents (EPSCs) in jaw-closing (JCMNs) and jaw-opening (JOMNs) motoneurons. Electrical and chemical stimulation of RdVII also induced excitatory responses in JCMNs of anesthetized rats. Intracellular staining of SupV neurons by biocytin revealed synaptic contacts with TMNs. Application of CNQX and APV inhibited EPSCs induced by stimulation of SupV or RdVII in both JCMNs and JOMNs of neonatal and juvenile slices. Strychnine and GABA also inhibited EPSCs in neonatal JCMNs and JOMNs, whereas strychnine enhanced EPSCs in juvenile JCMNs and JOMNs. We conclude that neurons located in SupV and RdVII most likely excite TMNs. It is also suggested that excitatory inputs from SupV to TMNs are mediated by activation of glutamate, GABA and glycine receptors in neonatal rats, whereas glycine receptor activation in TMNs becomes inhibitory in juveniles. Such postnatal change of synaptic transmission from SupV to TMNs might be involved in the transition from suckling to mastication. [J Physiol Sci. 2007;57 Suppl:S23]

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Rhythmic masticatory muscle activity (RMMA) of jaw-closing muscles can occur during sleep in healthy human subjects although its physiological significance remains unknown. If RMMA is exaggerated, however, the condition is regarded as sleep bruxism, which can be associated with clinical problems. Until recently, mechanisms generating sleep bruxism had not been well understood. Recent studies have reported that episodes with RMMA mostly occur in light (non-rapid eye movement) NREM sleep during the ascending phase of NREM sleep cycles, and that the genesis of episodes is secondary to a sequence of arousal-oromotor events; cardiac and cortical activation precedes jaw-opening suprahyoid muscle contraction, and is followed by rhythmic jaw-closing muscles activation. Since neurophysiological mechanisms generating RMMA have yet to be studied in animals, masseter muscle activity in guinea pig was recorded during sleep and analyzed. Among various muscle bursts occurring during sleep, some episodes with repetitive masseter muscle activity in NREM sleep were associated with a brief increase in heart rate and a transient decrease in delta EEG activity. This presentation provides an overview of current knowledge of RMMA during sleep in humans and introduces a challenge in developing a putative animal model for sleep bruxism. [J Physiol Sci. 2007;57 Suppl:S23]

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The aim of this symposium is to present recent results of studies of medial frontal lobe including the supplementary motor area, the pre-supplementary motor area, cingulate cortex and medial prefrontal areas. The symposium especially focused on the cognitive role of the medial frontal lobe. Each speaker will bring recent data and discuss possible role of the medial frontal lobe from his/her point of view. As one of co-organizers of this symposium, I will introduce recent study of anterior cingulate cortex in my lab. We recorded neuronal activity from the anterior cingulate area, while animals performed a path-planning task. I will discuss a possible role of this area for monitoring and updating cognitive rules of actions. [J Physiol Sci. 2007;57 Suppl:S24]

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To achieve a certain behavioral goal, we have to arrange multiple actions with different effectors in correct temporal order. It is not known how the supplementary motor area (SMA) and presupplementary motor area (pre-SMA) are involved in bimanual sequential movements. To address this question, we trained two monkeys to perform bimanual two sequential movements, consisting of either pronation or supination of the right or left arm, in sixteen different temporal orders. Correct sequences were initially instructed with visual signals but had to be memorized while performing a particular sequence. Thereafter, the sequential movements were performed based on memory. In this report, we focus on neuronal activity during the period just before the onset of the first movement. We examined how well the information about the arm use (right or left arm) and action (pronation or supination) for the first or second movement were represented in the neuronal activity in these areas. A group of neurons changed their activity depending on the arm use. In contrast, another group of neurons predominantly reflected the action to be performed. In addition, activity of a majority of neurons was strongly influenced by the action of the second movement, even before the initiation of the first movement. Herein, we consider the functional implication of neuronal activity in these areas concerning preparation and execution of bimanual sequential movements. [J Physiol Sci. 2007;57 Suppl:S24]

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We studied neuronal activity in medial prefrontal cortex in monkeys performing a conflict solving task in which a visual cue appeared either on the left or the right side of the panel placed in front of them. The cue's color instructed the monkeys to reach either to the left or right target, regardless of its location. The location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. We found a focus of reaching-related neurons in the medial prefrontal cortex, rostrally to the presupplementary motor area (pre-SMA), where previous studies had not found neurons related to arm movements. The activity of some of the neurons in this region was modulated by the response conflict, but the majority of them showed task-related activity even in the absence of response conflict (i.e. congruent trials). Interestingly, neurons in this region lost task-related activity modulation when these monkeys were re-trained on congruent trials alone. In contrast, in the adjacent pre-SMA and SMA, neurons still showed task-related activity after a prolonged absence of response conflict. These results suggest that the functional map in this region is dynamically reorganized by the task demand. [J Physiol Sci. 2007;57 Suppl:S24]

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We recorded single-cell activity from lateral frontal cortex of a rhesus monkey discriminating relative durations and distances. In the duration task, two different visual stimuli appeared sequentially at the monkey's fixation point (screen center), each followed by a delay period (D1 after the first stimulus and D2 after the second). Stimulus durations ranged from 50 ms to 3.2 s. After D2, the two stimuli appeared simultaneously,one to the left and one to the right of screen center. To receive a reward, the monkey had to choose the stimulus that had persisted longer in its initial presentation. In the distance task, the two stimuli appeared sequentially at different distances from screen center, one above and one below it. Matching the duration task, the stimuli later appeared simultaneously, and the monkey had to choose the stimulus that had been located farther from the center. We used one-way ANOVA (p<0.05) on neuronal activity during the first 400 ms of D2, with relative duration or distance as the factor. In the duration task, 21% of 182 neurons encoded relative duration: shorter and longer in roughly equal numbers. In the distance task, 21% of 312 neurons encoded relative distance from screen center: nearer and farther in roughly equal numbers. For 37 significant neurons studied in both tasks, 12 encoded only relative duration, 19 only relative distance, and 6 both. These findings indicate that during comparative judgments, frontal neurons can contribute to decisions about relative magnitude in both the spatial and temporal domains. [J Physiol Sci. 2007;57 Suppl:S25]

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To adapt behavior to changing environment, one must monitor outcomes of executed actions and adjust subsequent actions accordingly. However, little is known about neural processes underlining this link from performance monitoring to performance adjustment. To reveal neural mechanisms of the link, we trained two monkeys in a task that required frequent learning of action based on visual feedback stimuli. While the monkey was performing the task, we recorded the activity of single cells from the medial and lateral prefrontal cortex (PFC). About one-fourth of cells in both medial and lateral PFC responded to the feedback stimuli. However, the proportion of cells that differentially responded to positive and negative feedback stimuli was significantly larger in the medial PFC than in the lateral PFC (p<0.05). The visual features or novelty of the stimuli, or stay/shift for the next action did not account for the selectivity of responses, and therefore the responses represented evaluation of the executed action. Moreover, the response to the positive-feedback stimuli decreased along the course of action learning, in proportion to the amount of prediction error of action value calculated based on the behavior. Therefore, we suggest that the medial PFC cells signal the direction and amount of errors in the predicted value of executed action to specify the adjustment for subsequent action selection. [J Physiol Sci. 2007;57 Suppl:S25]

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The prefrontal cortex can be divided into two parts, the lateral and medial, traditionally held to have dissociable functions. In the present talk, we demonstrate the functional dissociation of medial and lateral prefrontal regions that contribute to behavioral flexibility along cognitive-emotional axis. Decomposition of brain activity measured by fMRI revealed dissociation of three groups of prefrontal regions and related mental components involved in set shifting required during performance of the Wisconsin Card Sorting Test (WCST): the left lateral region associated with update of behavioral set, the right lateral region associated with inference of the meaning of negative feedback, and the medial region associated with emotion evoked by the presentation of negative feedback. The medial region was segregated from the pre-supplementary motor area activated during set shifting and from the anterior cingulate regions known to be associated with conflict monitoring and error detection. The medial region was also reproducibly activated during presentation of negative feedback in a motion prediction task, a situation simpler than the WCST. Furthermore, the medial prefrontal activity in the prediction task correlated with emotional aspects of personality traits of individual subjects as indexed by psychological assessments. These results suggest that traditional cognitive-emotional dichotomy of the lateral and medial prefrontal functions holds during set shifting, and that the medial prefrontal cortex is functionally heterogeneous, as has been widely accepted regarding the lateral prefrontal cortex. [J Physiol Sci. 2007;57 Suppl:S25]

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Sound, light, tactile, taste and odor stimuli are all translated into action potentials in the brain. At the same time, even in the absence of sensory stimuli, neurons continue to emit spikes. Such evoked and spontaneous activity is based on the intrinsic properties of neurons as well as circuit mechanisms. Thus, understanding the brain requires comprehensive observation of the function and structure of circuits woven by numerous neurons. This is attainable, al least in part, with large-scale recordings of neuronal activity that allow reconstructing the temporal and spatial patterns of firing activity in a large population of neurons. We developed functional multineuronal calcium imaging (fMCI) to analyze the net structure of spontaneous CA3 network activity in hippocampal slice cultures loaded with Oregon green 488 BAPTA-1 using a spinning disk confocal microscope (up to 1000 frames/s). Principal component analysis revealed that network states, defined by active cell ensembles, were stable, but heterogenous and discrete. These states were stabilized through synaptic activity and maintained against external perturbations. Networks tended to stay in a single state for tens of seconds and then suddenly jump to a new state. After a state transition, the old state was rarely, if ever, revisited by the network during our observation period. Within each state, the pattern of network activity tended to stabilize in a specific configuration. These findings suggest that the network states are metastable, rather than multistable, and might be governed by local attractor-like dynamics. [J Physiol Sci. 2007;57 Suppl:S26]

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Until recently, neural-glial communication has been assumed to be mediated by low concentration transients of transmitter that result from spillover from the synaptic cleft. Therefore such communication has been thought to be a secondary effect of synaptic transmission between neurons. In the cerebellum, Bergmann glia cell (BG) processes encase synapses between presynaptic climbing fiber and parallel fiber elements and postsynaptic Purkinje cell (PC) spines and glutamate released from these fibers can activate Ca²⁺-permeable AMPA receptors on BGs. We have found evidences that support the idea that this rapid neural signaling to BGs is mediated not by spillover but by ectopic vesicular release of glutamate from presynaptic elements directly facing the BG membrane. It has been suggested that such neural-glial communication is responsible for the encasement and isolation of excitatory synapses on PCs by BGs. Therefore we have started to employ two-photon microscopy to study the impact of neural-glial communication on BG morphology. Rapid remodeling of the extracellular space caused by spontaneous motility of BG protrusions was observed and synaptic activation lead to enhanced motility of these protrusions. Synaptic activation also lead to Ca²⁺ influx at the tip of the protrusions via Ca²⁺-permeable AMPA receptors followed by a broader propagation of Ca²⁺ increase throughout the entire process possibly via activation of ATP receptors. We are currently trying to understand the mechanism of BG motility and how neural-glial communication leads to the refinement of synaptic and glial complex environment. [J Physiol Sci. 2007;57 Suppl:S26]

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Evidence for astrocytic participation of neuronal signal processing has cumulated in recent years. In particular, large and long-lasting cytosolic calcium surges in astrocytes, which may result in neurotransmitter release from the astrocytes, have been described in in vitro preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain have just started to be addressed. We demonstrate that a topical application of Fluo-4 AM to the neocortical surface of a juvenile rat results in specific uptake of the dye in astrocytes. Using this method, we have imaged cytosolic calcium fluctuation in astrocytes of superficial cortical layers in vivo with two-photon laser scanning microscopy. Surface application of AM-ester dyes becomes increasingly difficult as the brain matures, possibly due to increased brain density. We have employed a bolus loading method [1] to apply Oregon Green 1 BAPTA AM to layer 2/3 neurons and glia of the somatosensory cortex. Co-application of sulforhodamine 101 [2] made it possible to separate astrocytes from neurons among the dye loaded cell populations. The bulk-loaded cells with the fluorescent calcium indicator were imaged simultaneously with the local field potential and the associated multi-unit activity. Spontaneously calcium surges in astrocytes were also seen in adult (> P25) rodents. Correlation of neural activity and astrocytic calcium surges in mature animal is currently being investigated.[1] Stosiek C. et al. PNAS, 100 (2003) 7319-24[2] Nimmerjahn A. et al. Nat. Methods, 31 (2004) 1: 31-37 [J Physiol Sci. 2007;57 Suppl:S26]

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Making patch-clamp recordings in vivo under direct visual control is highly desirable since it allows specific cell types to be targeted reliably. Previous methods for targeted patching (Margrie et al., 2003) require neurons to be fluorescently labeled, either by use of transgenic animals or viral transfection.We have developed a new approach for making targeted patch-clamp recordings from unlabeled neurons in vivo visualized using two-photon microscopy. The method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. The same electrode can then be placed on the neuron under visual control to allow gigaseal formation. This approach, which resembles that used by Euler et al. (2002) to target amacrine cells in whole-mount retina, thus offers the prospect of targeted recording and labeling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations.We demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in barrel cortex and cerebellum in adult mice and rats anaesthetized with ketamine/xylazine or urethane. We also show that the method can be used for targeted electroporation in single identified neurons in vivo. [J Physiol Sci. 2007;57 Suppl:S27]

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Today we have a nice technique to look at deeper tissue or cells, called two-photon laser scanning microscope (2PLSM). We are taking up some topics here related to this sexy technique. I will give a talk about in vivo two-photon targeted patch (TPTP) method which is an example of combination 2PLSM and electrophysiological technique.First we made a shRNA against the voltage gated sodium channel subunits, Na_v1.1, 1.2, 1.3, which are reported to be mainly expressing in the dendrites. We packed it into a self-inactivated lentiviral vector with GFP as an indicator. Then we injected it in the barrel cortex before critical period of it at P8 old. Ten days after injection, we patched the cell which expressed the shRNA and GFP under the 2PLSM to investigate the property of somatosensory receptive field. We could reviel that the reduction of somatosensory responses of neurons in layer 2/3 of the barrel cortex with TPTP. This result suggest that the dendritic sodium channel played a critical role for maturation of sensory receptive field during its critical period, which could be accomplished via a form of neural plasticity, such as spike timing dependent plasticity.Now we are standing at the starting point with this result, because we have many molecules, brain areas and speices which have to be done with this method, TPTP. [J Physiol Sci. 2007;57 Suppl:S27]

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We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43°C and below about 15°C evoke not only a thermal sensation, but also a feeling of pain. The first found molecule detecting ambient temperatures is capsaicin receptor TRPV1 which belongs to a large TRP ion channel super family and can be activated by elevated temperatures with a discrete threshold near 43°C. Three other TRPV channels, TRPV2, TRPV3 and TRPV4 have been cloned and characterized as heat or warm thermosensors. In addition, three of TRPM channels (TRPM2, TRPM4 and TRPM5) have been reported to be thermosensitive. The threshold temperatures for activation of these channels range from relatively warm (TRPV3, TRPV4, TRPM2, TRPM4 and TRPM5) to extremely hot (TRPV2). In contrast to these warmth- or heat-activated TRP channels, two other TRP channels, TRPM8 and TRPA1 have been found to be activated by cold stimuli although cold sensitivity of TRPA1 is controversial. Thus, there are nine TRP channels to be thermosensitive, and the fact that five TRP channels can be activated by warm stimuli suggests that many cells in our body are sensing ambient temperatures. I would like to present our recent works about warmth-sensitive TRPV3, TRPV4 and TRPM2 channels. [J Physiol Sci. 2007;57 Suppl:S27]

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Voltage sensing nature has long been thought as a trait unique to ion channels. Voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels. We have recently identified two novel voltage-sensing proteins that have VSD but lack pore domain. Ci-VSP consists of VSD and phosphatase domain similar to a tumor suppressor enzyme, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Ci-VSP shows robust gating current and voltage-regulated phosphoinositide phosphatase activity. We have also isolated vertebrate orthologs to Ci-VSP and found that they showed similar molecular properties as Ci-VSP including voltage-dependent charge movements and phosphoinositide phosphatase activities. Among vertebrate orthologs, teleost VSP shows robust gating currents in mammalian cells, serving an ideal system to study coupling between VSD and phosphatase domain. Many examples are known in which interaction between distinct modules in a single protein is bidirectional. We found that this was true for VSP, since phosphatase activity modified movement of gating current. In this talk, further findings on the mechanisms of this protein and biological functions will be discussed. [J Physiol Sci. 2007;57 Suppl:S28]

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How physical force is sensed by cells and transduced into cellular signaling is poorly understood. When we found that phosphorylation of p130Cas (Cas) was involved in a physiological force transduction, we postulated four possible mechanisms of stretch-dependent tyrosine phosphorylation of Cas: 1) Kinase (Src family kinase) is activated by stretching, 2) Phosphatase of Cas is inactivated by stretching, 3) Kinase and substrate (Cas) spatially interact upon stretching, 4) Susceptibility of Cas to phosphorylation is enhanced by stretching. Because our experimental data suggested that mechanisms 1)–3) were not primarily responsible for the stretch-dependent phosphorylation of Cas, we tested the possibility 4). To eliminate any extraneous biochemical interactions or signaling pathways, we constructed an in vitro protein extension system in which bacterially expressed Cas substrate domain proteins (CasSD) were extended and analyzed biochemically. We found that mechanical extension of CasSD enhanced its tyrosine phosphorylation by c-Src kinase with no apparent change in Src kinase activity in vitro. Physiological relevance of in vitro CasSD extension was confirmed by immunostaining intact cells fixed in the late phase of spreading using an extension-specific anti-Cas antibody. Thus, Cas converts force into a biochemical signal through mechanical extension of its substrate domain, causing priming to phosphorylation. We propose such "substrate priming" is a general mechanism regulating intracellular signal transduction. [J Physiol Sci. 2007;57 Suppl:S28]

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Vascular functions are regulated not only by chemical mediators,such as hormones, cytokines, and neurotransmitters, but by shear stress, a mechanical force generated by blood flow. The endothelial cells that line blood vessels recognize shear stress, and transmit the signal into the interior of the cell, where it triggers cell responses that involve changes in cell morphology, cell function, and gene expression. Ca²⁺signaling plays an important role in shear-stress signal transduction. Our previous studies demonstrated that a shear-stress-dependent Ca²⁺ influx occurs in endothelial cells when exposed to flow, and that an ATP-operated cation channel, P2X4, is the major contributor to flow-induced Ca²⁺ influx. The flow-induced activation of P2X4 requires ATP, which is supplied by the endogenous ATP released by endothelial cells. Cell surface ATP synthase localized in lipid rafts in the endothelial cell membrane is involved in ATP release in response to shear stress. To gain insight into the physiological significance of this shear-stress signal transduction via P2X4, we generated a P2X4-deficient mouse, which does not exhibit normal endothelial cell responses to flow, such as Ca²⁺ influx and subsequent production of nitric oxide (NO), a potent vasodilator. The vasodilation induced by an acute increase in blood flow in situ is markedly suppressed in P2X4--deficient mice, and they have higher blood pressure values and excrete smaller amounts of NO products in their urine than wild-type mice. Thus, endothelial P2X4 channels are critical to the flow (shear-stress)-sensitive mechanism that regulates vascular functions. [J Physiol Sci. 2007;57 Suppl:S28]

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The retinal ganglion cells (RGCs) in mammals cannot regrow their axons and become apoptotic after optic nerve lesion. In contrast, fish optic nerve can spontaneously regenerate and finally their visual function can also restore even after axotomy. We determined a correct time schedule of goldfish optic nerve regeneration process using morphological and behavioral parameters. The process was divided into four periods 1) preparation period (0-6 days), ii) axonal elongation and synaptic formation period (1-6 weeks), iii) synaptic reinforcement and refinement period (1.5-4 months) and iv) recovery of visual function period (4-6 months). For screening of interest molecules at early stage, retinal cDNA library was prepared from axotomized goldfish retinas 5 days previously. We obtained several cDNA clones with differential hybridization of normal and injured retinas. Among of them, purpurin and IGF-I mRNAs rapidly increased in goldfish retina 1 or 2 days after axotomy, whereas Na,K-ATPase and transglutaminase (TG) mRNAs increased in the RGCs 10-20 days after axotomy. We conclude that the former involves in the preparation of optic nerve regeneration and the latter involves in the axonal elongation. Actually, IGF-I and TG identified from goldfish regenerating retinas could induce a striking neurite outgrowth from adult rat retina in culture. Thus the fish visual system offers a useful clue for searching for reliable and effective molecules of mammalian CNS regeneration. [J Physiol Sci. 2007;57 Suppl:S29]

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We examined whether Y-39983, a novel ROCK inhibitor, and Y-27632 can make injured RGC axons regenerate into the crushed optic nerve (OpN) of adult cats. Small pieces of the cat retina were cultured in medium containing Y-27632 or Y-39983 for 14 days. After fixation, the neurites were stained with anti-TUJ-1 antibody. Number and length of TUJ-1 positive processes were obtained. Microcrush of optic nerve (OpN): The left OpN of anesthetized cats was tied up and crushed. Y-39983 or PBS was injected into the vitreous and the crush site. Anterograde labeling of regenerated axons: 2 days after a WGA-HRP injection into the vitreous, the cats were deeply anesthetized, perfused with fixation on day 14. The OpN was sectioned in a cryostat, reacted for HRP with TMB. Retinal culture: To obtain the optimum concentration for axonal regeneration, we examined effect of Y-27632 and Y-39983 on neurite outgrowth of cultured retinal pieces. On day 14 the number of TUJ-1 positive processes was maximum in the culture containing 3 and 10 μM Y-39983: the number was greater than that in 10-100 μM Y-27632. No neurites protruded in culture containing 30 μM Y-39983 or 1 mM Y-27632. Axonal regeneration in crushed OpN:From the culture results we injected Y-39983 at 10 and 100 μM. An injection of 10 μM Y-39983 increased regenerated axons longer than 0.5 mm from the crush site. Second injection of 10 μM Y-39983 at day 7 increased the number 2 fold at 0.5 mm, 3.5 fold at 2 mm than in one injection of 10 μM Y-39983. ROCK inhibitor, Y39983, enabled injured axons of RGCs of adult cats to regenerate into the crushed OpN. [J Physiol Sci. 2007;57 Suppl:S29]

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Optic nerve injury induces a loss of vision which proceeds rapidly within several hours, together with retinal ganglion cell death in much slower time course. Electrical stimulation has previously been shown to rescue the injured retinal ganglion cells from cell death. It is not clear whether electrical stimulation is effective for acute impairment of visual function. We tested whether transcorneal electrical stimulation could preserve visual function after an optic nerve crush. Transcorneal electrical stimulation was given immediately after a calibrated optic nerve crush. We measured visually evoked potential (VEP) in the visual cortex of rats before, immediately after the optic nerve crush, and after the transcorneal stimulation to estimate an extent of damage and effects of stimulation in individual animals. In addition, we labeled the retinal axons with a fluorescent anterograde tracer to determine whether the transcorneal electrical stimulation can protect the retinal axons from degeneration. The optic nerve crush was made at an intensity that does not allow a spontaneous recovery of VEP for 1 week. The transcorneal stimulation immediately increased VEP amplitude impaired by the optic nerve crush and this augmentation was often preserved after one week. In the stimulated animals, a larger amount of retinal axons projected centrally beyond the crushed region in comparison to the unstimulated animals. Transcorneal electrical stimulation would restore the functional impairment of optic nerve by traumatic injury at an early stage and protect retinal axons from the following degeneration. [J Physiol Sci. 2007;57 Suppl:S29]

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Retinal prosthesis has been investigated as an innovative treatment to outer retinal degeneration such as retinitis pigmentosa. Epiretinal stimulation and subretinal stimulation were developed as the methods to stimulate the residual retinal neurons. Both systems, however, have some difficulties in safety of electrode implantation because of direct attachment of the implanted electrodes to the retina. We proposed the novel retinal prosthesis method, suprachoroidal transretinal stimulation (STS), the electrodes of which are implanted in the sclera and the vitreous body. This approach of retinal stimulation can reduce surgical insult on the implantation. Electrophysiological studies with normal and RCS rats revealed that STS was able to evoke focal excitation on superior colliculus and that its threshold was comparably low to epiretinal or subretinal stimulation (Kanda et al., 2004). To evaluate the area activated by STS quantitatively, the response probability elicited by STS and the distance from the scleral electrode to the receptive field of LGN relay cells of cats were measured for each unit and each stimulus intensity. The units with high response probability were limited near the scleral electrode, and the radius of the activated area at half maximum response was 1.8 degrees on 100 and 150 uA of STS. These physiological evaluations indicated that STS is a good candidate as well as other retinal prosthesis. The integrated efforts of neuroscientists, ophthalmologists and engineers should be required to realize the retinal prosthesis. [J Physiol Sci. 2007;57 Suppl:S30]

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Visual prostheses are electric devices that provide visual signal to the nervous system of blind patients. To design the visual prostheses, effects of electrical stimuli on the neuronal circuits have to be elucidated. We investigated the spatio-temporal properties of neuronal responses elicited by externally applied current and photolysis of caged-glutamate in the rodent visual cortex slice, using a multi-electrode array and Ca-imaging. Bipolar current pulses were applied to layer 4, which receives inputs from the lateral geniculate nucleus. The two-dimensional current source-density analysis revealed that current sink induced by the stimulus pulse higher than 40 μA propagated vertically to layer 2/3 and then spread horizontally along layer 2/3. This response was confined within 50 μm around the stimulation site when excitatory transmissions were blocked by CNQX and AP-5. The response was completely abolichsed by TTX. In the Ca-imaging experiment, high [Ca²⁺]i region spread vertically from layer 4 to layer 2/3 and to the upper part of layer 5. The region was confined within several tens of micrometer around the stimulus under the administration of CNQX and AP-5 and was completely abolishied by TTX. In response to the photolysis of caged-glutamate applied to layer 4, on the other hand, high [Ca²⁺]i region propagated to layer 5. The vertical spread to layer 2/3 was also observed but not as prominent as those induced by electrical stimuli. In conclusion, the electrical stimuli applied to layer 4 induces similar but not the same response as that evoked by the photolysis of caged-glutamate, which is thought to mimic natual inputs to the visual cortex. [J Physiol Sci. 2007;57 Suppl:S30]

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Retinal regenerative medicine is expected as one of the future treatments of obstinate retinal diseases. There are two strategies for the recovery of lost retinal function especially in the outer retina. One is retinal regeneration from intrinsic progenitor cells and another is retinal cell transplantation. As for retinal regeneration, we revealed that the Muller glia acquired the progenitor like property, proliferated and differentiated into retinal neurons including photoreceptor cells after retinal damage even in the adult mammalian retina. We could promote the proliferation with Wnt. Notably this photoreceptor regeneration was observed also in the retinal degeneration model mice. For cell transplantation, we need sufficient amount of specific types of cells. One of the solutions is the embryonic stem (ES) cell. We have established a feeder-free and serum-independent culture method that induces directed differentiation of primate ES cells (human and non-human) into mature retinal cells. The next step is to select the required cells out of undifferentiated cells that may cause tumors. Furthermore, modification of transplantation condition is needed, although recently the recovery of retinal function has been reported by transplantation of immature retinal cells in mice and a retinitis pigmentosa patient. Research of retinal cell transplantation entered the stage of confirmation of functional recovery. [J Physiol Sci. 2007;57 Suppl:S30]

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In this symposium, I would like to introduce present status of cooperative participation between men and women in Nagoya University. I also overview an ideal environment to work together with bright prospects, and will discuss the point at issue to settle the problems. [J Physiol Sci. 2007;57 Suppl:S31]

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Tokyo Women's Medical University (TWMU) is a medical university with over one hundred years of history which now has a modern and sophisticated educational, clinical and research environments. Traditionally all of our undergraduate schools are devoted to develop women's professionalism. Our graduate schools, faculties and hospitals are open to both genders. The uniqueness of TWMU derives from the founder's strong volition to establish women's professionalism for women patient. Her conviction, faith and compassion, enlightens is our commitment for medical services and care provided to our clients. However, under the present condition, there are few women's doctors who continue the research and become a leader in the field because of their child care and employment conditions. In this program, we established a day nursery center for sick children and introduced a work share and flex system to support their child care, research activity and life. We also established Support Center for Female Medical Scientists (SCFMS) to give them counsel by senior scientists and psychologists, and show role models as an objective and a goal of their research life. In this symposium, I would like to introduce an outline and present status of this program, and discuss the point at issue to settle the problems. [J Physiol Sci. 2007;57 Suppl:S31]

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The brain is deeply involved in individual- and species-conservation via regulating visceral functions, behaviors, and higher cognitive functions. The energy monitoring network in the brain cooperatively controls not only energy metabolism and feeding behavior but also other physiological functions such as reproduction, thermoregulation and learning and memory processes. Glucose monitoring cells such as glucose-sensitive neurons and glucoreceptor neurons play pivotal roles in this energy monitoring neural network. The energy-monitoring system in the brain is highly sensitive to not only appetite-regulating substances but also many other chemicals related to reproductive functions, thermo-regulation and immune functions. Even environmental chemicals influence this system. These multi-sensing systems provide a model of sophisticated system which accelerate the energy saving and environmental preservation in the individuals, species and environment. Although the brain has such a sophisticated system to maintain energy homeostasis to maximize the adaptive function, recent drastic changes in chemical environments affect this process and disturb the homeostatic functions conduced by the brain. These unexpected changes may concern our increasing problems of obesity, eating disorders, reproductive problems, immune dysfunction and emotional disorders. Physiological and pathophysiological significance of cooperative mechanisms of feeding and other homeostatic functions in various environments will be discussed. [J Physiol Sci. 2007;57 Suppl:S32]

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Introduction Body temperature is finely regulated, although feeding condition is a factor modulating the regulation. Fasting is a strong stimulus reducing metabolism (i.e. heat production). However, body temperature is well maintained in the active phase, indicating change in thermoregulation. I discuss about neural and hormonal mechanisms involved in the change in thermal regulation during fasting and the possible role of clock genes. Experiments and Results 1) Fasting induced greater reduction of metabolism and its circadian amplitude. Although the reduction of heat production, body temperature rhythm was well maintained except for greater decrease of body temperature around the lights onset. During the fasting, body surface temperature was greatly reduced, meaning that heat loss mechanisms were suppressed. 2) During fasting, heat producing response to the cold was attenuated in rats. However, this response is attenuated in the presence of i.c.v. leptin. 3) Feed-fast cycle generated daily oscillation of body temperature in clock mutant and cry knockout mice, both mice could not modulate heat loss responses to the fasting. 4) Fasting affects neural activities in the suprachiasmatic nucleus, estimated by c-Fos expression. Conclusion Fasting is a strong signal modulating thermal regulation. Leptin level in the central may be a factor to change metabolic activity, deactivating the sympathetic nerves to the brown adipose tissue. Moreover, circadian clock receive fasting signals, which may alter heat loss responses and maintain body temperature rhythm. [J Physiol Sci. 2007;57 Suppl:S32]

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To investigate homeostatic characteristics of the glucose-monitoring (GM) neuronal network in the rodent and macaque forebrain, complex series of electrophysiological and behavioral experiments have been performed. Extracellular single neuron recording studies in anesthetized Wistar rats and alert rhesus monkeys revealed specific and differential responsiveness of GM cells of the nucleus accumbens, globus pallidus, mediodorsal and ventrolateral (or orbitofrontal) prefrontal cortex during: 1) microelectrophoretic administration of the diabetes inducing streptozotocin (STZ) and the primary cytokine interleukin 1beta (IL-1); 2) intraoral taste stimulation, as well as 3) performing a conditioned bar press feeding task. Bilateral microinjection of STZ into the above structures of male Wistar rats caused type 2 diabetes like acute and chronic metabolic alterations, whereas that of IL-1 elicited hypophagia, hypodipsia, hyperthermia, and metabolic deficits as well. Data substantiate that the forebrain GM neuronal network is indispensable in the maintenance of an adaptive homeostatic balance for the well being of the organism.Supported by: National Research Fund of Hungary (T042721, M036687), Health Care Science Council (ETT 315/2006), NKTH MEDIPOLIS RET-008/2005, and the H.A.S. [J Physiol Sci. 2007;57 Suppl:S33]

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Orexigenic and anorexigenic peptides in the hypothalamus are involved in feeding behavior and also related to the stress responses. The noradrenergic pathway from the brainstem to the hypothalamus is also known to mediate stress responses. Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of an orphan G-protein coupled receptor and colocalized with the noradrenergic neurons in the brainstem. Intracerebroventricular administration of PrRP activated CRH neurons in the parvocellular division of the PVN and caused increase of plasma corticosterone levels in rats. We found that restraint stress, nociceptive stimulus and acute inflammatory stress upregulated the PrRP gene expression in the brainstem in rats. In streptozotocin-induced diabetic rats and fa/fa Zucker diabetic rats, PrRP mRNA levels were significantly reduced in comparison with controls. In both diabetic rats PrRP mRNA levels were significantly increased after restraint stress. These data suggest that PrRP may be an important mediator of the stress response from the brainstem to the hypothalamus under normal and diabetic conditions. [J Physiol Sci. 2007;57 Suppl:S33]

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Lowering glucose concentrations from 5-10 mM to 1-3 mM increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in 20% of isolated neurons from the rat hypothalamic arcuate nucleus (ARC), exhibiting the property of glucose-sensitive (GS) neurons. More than 90% of these GS neurons were neuropeptide Y (NPY)-containing neurons. In ARC GS neurons, low glucose-induced ([Ca²⁺]_i) increases were mimicked by a non-metabolizable glucose analogue 2-deoxy-glucose, a glucokinase inhibitor mannoheptulose, and a mitochondrial inhibitor KCN. Furthermore, low glucose-induced ([Ca²⁺]_i) increases were inhibited by blockers of L- and N-type Ca²⁺ channel blockers. These results indicate that changes in extracellular glucose concentrations are transduced to intracellular changes in glucose metabolism and energy production, which eventually stimulate Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The coupling mechanisms between reduced energy state and Ca²⁺ channel activation remain to be elucidated. Intracerebroventricular injection of 2-deoxy-glucose and a glucokinase inhibitor, as well as NPY, enhanced food intake in rats. Fasting increased NPY mRNA level in ARC. Overexpression of NPY mRNA in ARC accounted for hyperphasia in the young-adult Goto-Kakizaki rats, a type 2 diabetic model. These results support physiological relevance of GS-NPY neurons in the regulation of feeding. [J Physiol Sci. 2007;57 Suppl:S33]

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A striated muscle fiber is composed of a bundle of myofibrils in which sarcomeres run in series from one end of a myofibril to the other end. The sarcomere is composed of contractile proteins (actin and myosin), and structural proteins (Z-disc proteins, connectin/titin) together with many other proteins. The contractile force produced in each sarcomere is transmitted to the neighboring sarcomeres via Z-discs. Recently by applying nano-manipulation techniques (an atomic force microscope; AFM, optical tweezers, etc.), detailed mechanical properties of myofibrils of striated muscles have been studied. It was found that, by rupturing specific sarcomere components of myofibrils, the mechanical strength of myofibrils is marginal to sustain the force they produce. This suggests that various protein components are delicately involved in maintaining the molecular architecture of myofibrils intact against the force they produce. It is known that cardiac muscle is substantially stiff compared with skeletal muscle, possibly due to the difference in the content of connective tissues. In our recent study, the transverse stiffness of myofibrils of the two muscles was examined. Interestingly it was found that the transverse stiffness of cardiac myofibrils was significantly greater than that of skeletal myofibrils. This suggests that the transverse stiffness difference between skeletal and cardiac myofibrils may come from the difference in the physiological role of the two types of striated muscles. [J Physiol Sci. 2007;57 Suppl:S34]

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Connectin (also called titin) is a giant fibrous elastic protein of vertebrate striated muscle with a molecular mass exceeding 3 MDa. A single molecule, more than 1 μm in length, connects the Z-line and the M-line of the striated muscle sarcomere. Because of its elasticity, connectin plays a role in maintaining the thick filament at the center of the sarcomere during contraction and relaxation of the muscle. Observations with fluorescent antibodies and immunoelectron microscopy using antibodies against connectin have demonstrated that this function occurs only in the I-band region, not in the entire sarcomere. It has been reported in human muscle that cardiac muscle connectin had fewer amino acids in the middle immunoglobulin domain and PEVK segments than psoas and soleus muscle connectins. Both segments reside in the I-band region, which is related to extension and contraction between the Z-line and the A-I junction in the muscle sarcomere. We measured passive tension generation with the stretching of mechanically skinned myofibril bundles and revealed that tension development in rabbit cardiac muscle began at shorter sarcomere length than in psoas and soleus muscles of rabbit. We infer that the cardiac muscle connectin remains in a slightly stretched state even at rest. This would explain why the cardiac sarcomere does not extend to the same degree as that of the psoas and soleus muscles under force. [J Physiol Sci. 2007;57 Suppl:S34]

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Muscle contractile apparatus (myofibrils) is a highly ordered structure, where actin and myosin filaments are organized in a hexagonal lattice with correct polarity and precise spatial position to generate cross-striated sarcomeric structures. They are anchored to Z- or M-lines, and supported by connectin/titin and nebulin filaments. Recently, numbers of new proteins have been reported to be involved in filament organization Therefore, regulatory mechanisms that control formation and maintenance of myofibrillar structure are complex. Here, we report our observations regarding how cofilin, C-protein and myosin-actin interaction are involved. Cofilin is an actin-binding protein that controls actin filament dynamics in a variety of cells. In muscle cells, we found that both over-expression and knockdown of cofilin with cDNA manipulation leads to disorganized actin assembly during myofibrillogenesis and disruption of actin filaments in sarcomeric structures. Thus, cofilin plays a critical role for the regulated assembly of actin in the myofibrils. C-protein is a myosin- and connectin-binding protein characteristic of striated muscles. We found that it also binds to actin filaments at the N-terminal side. Thus, C-protein is very likely involved in myofibril organization by interacting with multiple proteins. On the other hand, we verified that myosin-actin interaction is indispensable for myofibrillogenesis. Inhibitors of myosin or actin-myosin interaction such as BTS drastically suppressed myofibril assembly and caused disruption of sarcomeric structure when applied to muscle cells in culture. [J Physiol Sci. 2007;57 Suppl:S34]

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The transverse stiffness of single myofibrils of skeletal and cardiac muscles was examined by applying an atomic force microscope (AFM). The contribution of various sarcomere components to the transverse stiffness of myofibrils was estimated by treating myofibrils with proteolytic enzymes and examining resulting changes in the transverse stiffness of the myofibrils. Single myofibrils were prepared by homogenizing glycerinated muscle fibers of the left ventricle of rat heart and the psoas muscle of rabbit. The transverse stiffness of myofibrils was determined as previously based on indentation of myofibrils by pressing a cantilever of AFM to the surface of myofibrils. The overall transverse stiffness of cardiac myofibrils was 3-4 times greater than that of skeletal myofibrils. The calpain treatments selectively digested α-actinin, and clarified that Z-bands of cardiac myofibrils were 3-4 times stiffer than those in skeletal myofibrils. The trypsin treatments selectively digested connectin/titin, and suggested that the connectin/titin filaments contribute to the transverse stiffness more in cardiac myofibrils than in skeletal myofibrils. The results obtained suggest that the transverse stiffness difference between skeletal and cardiac myofibrils is due to the difference in the content and the arrangement of specific protein components in their sarcomere structures. [J Physiol Sci. 2007;57 Suppl:S35]

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Biological systems structurally and functionally constitute a hierarchy from single molecules to supra-molecular assemblies, and further up to organelles, cells, and tissues. Here we focus on the hierarchical organization of biological motile systems: 1) "Bio-nanomuscle", in which a single actin filament is pulled into an A-band, a bundle of thick (myosin) filaments. This model system was invented to bridge the gap between a single-molecule system and a muscle fiber, which is considered to be a minimum unit that mimics a single (half) sarcomere of striated muscle. 2) "Single myofibril", in which mechanical properties are studied under an optical microscope. The contractile system takes either one of three states, i.e., contraction, relaxation, and self-oscillatory (SPOC) state. Here we focus on the dynamic properties of SPOC. 3) "Mitotic spindle" extracted from Xenopus eggs, which is, as we consider it, a complex system located at the highest level in the hierarchy of biomotile systems. In the experiments we used several optical microscopy techniques, such as optical tweezers and a glass microneedle. We stress that the force production due to the structural transitions in motor proteins and the molecular strain induced by the applied force are the key factors driving the self-organization in the higher-ordered biological systems. [J Physiol Sci. 2007;57 Suppl:S35]

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Highly ordered lattice structure is an outstanding feature of striated muscle. It realizes condensed packing of myoproteins to a concentration unattainable with the ultracentrifugation of myoproteins. It is of interest how sarcomere stabilizes such condensed lattice structure. Skinned muscle specimen is suitable to study the intrinsic lattice stability, since it is free from the isovolumic constraint across the cell membrane. The force balance between electrostatic repulsive force and van der Waals attractive force (DLVO-theory) fails to account for the effect of low ionic strength on the lattice. The mechanical force born by sarcomeric structures such as connectin/titin and acto-myosin affinity would be significant. Actually, strong acto-myosin affinity at rigor and contracting states is considered to shrink the lattice. At resting state, the significance of acto-myosin affinity on the lattice is unclear. We prepared gelsolin treated skinned muscle specimens, from which actin filaments were successfully removed. Their lattice was observed with x-ray diffraction at BL45XU of SPring8. The results indicated that actin filament little contributes to the lattice of resting skeletal and cardiac muscles. Since lattice spacing is expected to be a significant modulator of the activation process in both muscles, reconsideration on the activation process would be necessary. [J Physiol Sci. 2007;57 Suppl:S35]

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TRPV1 is a ligand-gated cation channel activated by capsaicin, acid and heat. Because TRPV1 is activated by pain producing agent, capsaicin, thus, it is presumed to mediate an inflammatory pain. Indeed, some types of thermal pain are reduced in TRPV1-deficient mice. Previously, we showed that metabolic products of lipoxygenase (LO) such as 12-HPETE activate TRPV1. Because metabolites of LO mediate inflammatory processes, activation of capsaicin receptors by metabolites of LO further suggests a role of the receptor in mediating inflammatory pain. Bradykinin, a potent algogenic substance released in response to injury, initiates multiple inflammatory responses and excites sensory neurons. In this symposium, lines of evidence that suggest that bradykinin excite sensory neuron by opening the capsaicin receptor via the PLA2/LO pathway will be discussed. In addition, we also present unequivocal evidence that histamine, another inflammatory mediator, also uses the PLA₂/LO/TRPV1 pathway for excitation of sensory neurons. Application of histamine caused robust scratching behaviors in mice in capsazepine, quinacrine, and NDGA dependent pathways. Furhtermore, the scratching induced by histmamin was much reduced in TRPV1 deficient mice. Because histamine is a major pruritogenic (itch causing) substance, identification of the histamine signaling pathway is much helpful to developing anti-pruritogenic substance to cure itch sensation in atopic dermatitis patients. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain or itch. [J Physiol Sci. 2007;57 Suppl:S36]

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Dental primary afferent neurons and odontoblasts are cellular components by which noxious stimuli are perceived as painful by teeth. Our lab tries to elucidate molecular mechanisms underlying these transmission processes. Tooth pain is commonly induced by hot or cold substances. Temperature signaling can be initiated by members of transient receptor potential family (thermo-TRP) channels. We hypothesized that thermo-TRP channels expressed by dental primary afferent neurons and/or odontoblasts mediate tooth pain evoked by noxious thermal stimuli. Single-cell RT-PCR and immunohistochemistry revealed expression of TRPV1, TRPM8 and TRPA1 in subsets of such neurons. Capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist) and icilin (a TRPM8 and TRPA1 agonist) increased intracellular calcium and evoked cationic currents in subsets of neurons, as did the appropriate temperature changes (>42°C, <25°C and <17°C respectively). Individual neurons sometimes expressed two or three channels, and responded to two or three corresponding stimuli. Subfamilies of thermo-TRP channels were not only expressed but also functionally working in odontoblasts. The results suggest that activation of thermo-TRP channels expressed by dental afferent neurons and/or odontoblasts contributes to tooth pain evoked by temperature stimuli. Accordingly, blockade of thermo-TRP channels will provide a novel therapeutic intervention for the treatment of tooth pain [J Physiol Sci. 2007;57 Suppl:S36]

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