Dysfunctions of vesicular transport are linked to neurodegenerative disease, including Alzheimer disease (AD). Amyloid β-prrotein precursor (APP) has been implicated in the development and progression of AD. Recent reports suggest that APP functions as cargo receptor for kinesin I. APP interacts with kinesin light chain (KLC) indirectly via JNK-interacting protein 1b (JIP1b). We have reported that APP associates with Alcadein, a novel type I membrane protein, in neuron through their cytoplasmic interaction with X11-like (X11L) protein. We also found that Alcadein associates with KLC directly, thus Alcadein and APP/JIP1b competed for KLC. Alteration in APP- and Alcadein-transport system in neuron impairs the vesicle trafficking in axon, suggesting that inappropriate assignment of these cargos leads to neuronal malfunction and the degeneration in future. [J Physiol Sci. 2006;56 Suppl:S34]

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Alzheimer's disease (AD), the most common form of senile dementia, is characterized by excessive production and accumulation of neurotoxic β-amyloid (Aβ) peptides which are proteolytically derived from β-amyloid precursor protein (APP) via β- and γ-secretase cleavages. Experimental evidence from several groups including our own has demonstrated that the production of Aβ occurs largely in the trans-Golgi network (TGN) where APP molecules predominantly reside. Mutations in presenilins genes are associated with the majority of familial AD likely through a mechanism of increase Aβ42 production. Presenilins (PS, PS1 and PS2) along with their associated proteins including nicastrin (Nct), PEN2 and APH1 are essential for the γ-secretase activity. The precise functions of Nct, APH-1 and PEN-2 have not been fully elucidated. Recent studies including ours suggest that PEN-2 mediates endoproteolysis of PS1, while APH-1 and Nct play regulatory roles in maintaining the stability of PS1 and the complex. PS1 knockout mice exhibit pre-neonatal lethality and PS1 has also been shown to affect numerous physiological functions including calcium homeostasis, skeletal development, neurite outgrowth, apoptosis, synaptic plasticity, tumorigenesis. These data strongly indicate critical physiological roles of PS1 addition to its essential role in γ-secretase activity. We and others have reported that PS1 plays an important role in intracellular trafficking (especially from the TGN to the plasma membrane) of select membrane proteins including APP, PEN2 and nicastrin. The detailed cell biological mechanism for PS-mediated protein trafficking will be discussed. [J Physiol Sci. 2006;56 Suppl:S35]

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Alzheimer's disease is a slowly progressive neurodegenerative disorder which causes severe dementia. Amyloid-beta peptide (Aβ) deposition in senile plaques is one of the pathological hallmarks in Alzheimer's disease. Aβ peptide is derived from the amyloid precursor protein (APP) by proteolytic processing by beta-secretase which cleaves APP at the N-terminus of Ab, and by gamma-secretase which cleaves at the C-terminus of Aβ. In spite of extensive research, the precise subcellular localization of Ab generation has not been identified yet. Using the recently developed fluorescence resonance energy transfer (FRET) approach and pulse-chase ELISA, we examined the subcellular localization of interactions between APP and beta-secretase. Our data showed a close APP-BACE interaction in early endosomes, and highlight the cell surface as an additional potential site of APP-BACE interaction. Furthermore, we identified a novel interaction between LRP, an endocytic receptor for APP, and beta-secretase, in the early endosomes and on the cell surface. The interaction between LRP and beta-secretase was not detected when cholesterol was depeleted, suggesting that LRP encounters beta-secretase in the lipid raft of the membranes. Taken together, we propose that APP interacts with beta-secretase in the lipid rafts of the cell membrane and in early endosomes, and that LRP may be a scaffold protein which links APP and BACE upon endocytosis. We believe that investigation of the interaction between APP and its secretases helps us understand the mechanisms of Aβ generation and pathogenesis of Alzheimer's disease. [J Physiol Sci. 2006;56 Suppl:S35]

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Cyclin-dependent kinase 5 (Cdk5) is a multifunctional Ser/Thr protein kinase activated by binding to its activator p35 or p39, which is expressed predominantly in neurons. Cdk5 is shown to be involved in neuronal migration during brain development, synaptic activity in matured neurons, and neuronal cell death in aged brains. However, exact roles of Cdk5 in those neuronal activities have not been answered yet. Cellular localization would be critical to understand the detailed functions of Cdk5/p35 or Cdk5/p39. p35 or p39 activator controls the cellular localization as well as the kinase activity. The Cdk5/p35 and Cdk5/p39 complexes bind to plasma membranes and Golgi apparatus via myristoylation at the N-terminal Gly of p35 or p39 that may compartmentalize the active Cdk5 complexes in the cytoplasm. When Gly is mutated to Ala, Cdk5/p35 and Cdk5/p39 become soluble in the cytoplasm and then is translocated into nucleus. This mislocalization is observed at the time of neuronal cell death. For example, endoplasmic reticulum (ER) stress deregulates the Cdk5 activity by cleavage of p35 to p25 with calpain. The cleavage of p35 changes the cellular distribution of active Cdk5, stabilizes the p25/Cdk5 complex, and stimulates the kinase activity of Cdk5, thereby allowing potentially aberrant phosphorylation of neuronal proteins, which would adversely affect the survival of neurons. We would also like to discuss on its localization in relation to membrane trafficking in living neurons. [J Physiol Sci. 2006;56 Suppl:S35]

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Clathrin-mediated endocytosis plays a key role in the recycling of synaptic vesicles in nerve terminals and amphiphysin I is one of the components of the molecular machinery involved in this process. Amphiphysin I mediates invagination and fission of synaptic vesicles in cooperation with dynamin. We found that amphiphysin I was cleaved to three fragments by treatment with high KCl (80 mM) and by high-frequency electrical stimulation in the mouse hippocampal slices. The cleavage sites were localized in the CLAP domain. The cleaved amphiphysin I was unable to interact with dynamin and disrupted the co-polymerization into a ring formation with dynamin and liposome in a cell-free system. The calpain-dependent cleavages inhibited clathrin-mediated endocytosis. Finally the amphiphysin I cleavages were found in the hippocampus of kainate-treated FVB/N mice and the cleavages inhibited the neural hyperexcitation of the mice. I will review these findings and discuss the role of calpain-dependent cleavages of amphiphysin I in protecting neurons against excitotoxicity and hyperexcitation. [J Physiol Sci. 2006;56 Suppl:S35]

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The Na-Ca exchangers family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding calcium from the cell. Two basic properties characterize them: 1-Their activity is not predicted by thermodynamic parameters of classical electrogenic counter-transporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na and Ca) and non-transported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra, intra and trans-membrane protein domains. 2- Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This presentation emphasizes the interrelations between ionic and metabolic modulations of Na-Ca exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP related pathways in these two systems is that although they are different (PIP2 in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: sodium-calcium-proton interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger will be discussed. [J Physiol Sci. 2006;56 Suppl:S36]

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Anion channels play a stabilizing role in excitability in muscle and neuronal cells and a Cl⁻ transporting role in epithelial cells. Recent investigations have revealed their more general functions including cell volume regulation and cell proliferation. Here, we present additional roles that have been found in our laboratory. First, the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel, which is ordinary activated by cell swelling, plays an inductive role of apoptotic cell death. An apoptotic inducer rapidly activated the VSOR current without cell swelling and thereby induced apoptotic volume decrease (AVD) in epithelial and cardiac cells. Second, the same channel is involved in excitotoxic neuronal cell death. Stimulation with NMDA induced activation of the VSOR Cl⁻ channel, varicosity formation, somatic swelling and eventually necrotic death in cortical neurons. Third, the maxi-anion channel with a single-channel conductance of around 400 pS serves as the release pathway of ATP, which is an extracellular signal for cell-to-cell communication, in mammary cells, kidney macula densa cells, cardiomyocytes and astrocytes activated by a variety of stimuli. Forth, the maxi-anion channel also mediates glutamate release from cortical astrocytes under ischemic conditions. Molecular understanding of physiological or pathoplysiogical functions of these anion channels will progress after identification of their molecules. [J Physiol Sci. 2006;56 Suppl:S36]

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The cerebellum is involved in the control of not only motor but also autonomic functions. I will summarize roles of the cerebellum in cardiovascular control. I propose that the cerebellum contains five distinct modules (cerebellar corticonuclear microcomplexes) dedicated to cardiovascular control. First, a discrete rostral portion of the fastigial nucleus and the overlying medial portion of the anterior vermis (Lobules I, II and III) conjointly form a module that controls the baroreflex. Second, anterior vermis also forms a microcomplex with the parabrachial nucleus. Third, a discrete caudal portion of the fastigial nucleus and the overlying medail portion of the posterior vermis (lobules VII and VIII) form another module controlling the vestibulosympathetic reflex. Forth, the medial portion of the uvula may form a module with the nucleus tractus solitarius and parabrachial nucleus. Fifth, the lateral edge of the nodulus and the uvula, together with the parabrachial nucleus and vestibular nuclei, forms a cardiovascular microcomplex that control the magnitude and /or timing of sympathetic nerve responses and stability of the mean arterial blood pressure during changes of head position and body posture. Another region of the flocculus, which has recently been found to be related to cardiovascular control, will be also discussed (Nisimaru and Ito, 2005). [J Physiol Sci. 2006;56 Suppl:S36]

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Troponin, one of the sarcomeric proteins, plays a central role in the Ca²⁺ regulation of contraction in vertebrate skeletal and cardiac muscles. More than two hundred of mutations in the cardiac sarcomeric proteins, including myosin heavy/light chains, actin, troponin, tropomyosin, myosin-binding protein-C, and titin/connectin, have been found to cause various types of cardiomyopathy in human since 1990, and over sixty mutations in cardiac troponin subunits have been identified in the hypertrophic (HCM), dilated (DCM) and restrictive (RCM) cardiomyopathies. To explore molecular mechanisms for the pathogenesis of these cardiomyopathies, recombinant mutants of human cardiac troponin subunits were exchanged into permeabilized rabbit cardiac muscle fibers and their effects on the Ca²⁺-dependent force generation in cardiac muscle were examined. Most mutations in cardiac troponin subunits associated with HCM had Ca²⁺-sensitizing effects. In contrast, DCM-linked mutations in cardiac troponin T decreased the Ca²⁺ concentrations required for force generation, strongly suggesting that changes in the Ca²⁺ sensitivity of force generation in cardiac muscle in opposite directions, i.e. Ca²⁺-sensitization and desensitization, play important roles in the pathogenesis of these two distinct forms of cardiomyopathy. RCM-linked mutations in cardiac troponin I had much greater Ca²⁺-sensitizing effects on force generation than HCM-linked mutations, suggesting that HCM and RCM-linked mutations in troponin subunits share a common feature of increased Ca²⁺ sensitivity of cardiac myofilament, but more severe change in Ca²⁺ sensitivity is associated with the clinical phenotype of RCM. [J Physiol Sci. 2006;56 Suppl:S37]

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One of the first functionally identified groups of inhibitory neurons in the mammalian central nervous system are Renshaw cells (RCs, Renshaw 1946). RCs are excited by axon collaterals from motor neurons (MNs), and provide recurrent inhibition of MNs (Eccles et al. 1954). It has been shown by studies using cat spinal cord since then that, 1) excitatory synaptic inputs from MNs are mediated by acetylcholine (Curtis and Ryall 1966) and 2) these inputs are the main driving force for RC activity during locomotion (Noga et al 1987). We examined the physiological nature of RCs in detail using visually guided whole cell recording in isolated spinal cord preparations from glutamic acid decarboxylase (GAD) 67-EGFP knock-in mouse neonates. Among the GFP-positive cells in the lumbar ventral horn, RCs were uniquely identified by electrically stimulating the adjacent ventral root to evoke a short latency EPSC and by filling the cell with alexa-dyes to confirm its expression of calbindin-28k after recording. The short latency EPSCs that were reduced to 20-40% of control in amplitude by nicotinic receptor blockers and further reduced by blocking ionotropic glutamate receptors indicating that glutamate is also mediating synaptic inputs to RCs from MNs. During the locomotor-like rhythmic activity evoked by bath-application of 5-HT and NMDA, RCs fired rhythmically and modulated not only by excitatory synaptic inputs but also inhibitory ones. These results show that such technique is a powerful tool to reveal the neuronal mechanisms of motor control. [J Physiol Sci. 2006;56 Suppl:S37]

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Orexin A and B are a pair of neuropeptides implicated in the regulation of sleep/wakefulness and energy homeostasis. The regulatory mechanism of orexin neurons is poorly understood since the small number of orexin neurons is sparsely distributed in the lateral hypothalamus. We made the following transgenic (Tg) mice to study the physiological role in the regulation of sleep/wakefulness.Orexin/cameleon Tg mice, in which orexin neurons specifically express calcium sensing protein, were used for calcium imaging to screen what kind of neurotransmitter affects the activity of orexin neurons.Orexin/EGFP Tg mice, in which orexin neurons express EGFP, were used for electrophysiological studies to reveal intracellular mechanisms involved in the activation or inhibition response. Orexin/GFP::TTC Tg mice, in which orexin neurons express a retrograde tracer, were used for immunohistochemical studies to reveal which neurons directly innervate orexin neurons. These studies using Tg mice revealed how orexin neurons are regulated by afferent neurons. [J Physiol Sci. 2006;56 Suppl:S37]

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Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluid. For Na homeostasis of the body, controls of Na/water-intake and -excretion are of prime importance. However, the system for sensing the Na level within the brain that is responsible for the control of Na/water-intake behavior remains to be elucidated. Using physiological and behavioral techniques in combination with genetic manipulations, we have demonstrated that a sodium channel Na_x is indispensable for the Na-level-sensing in the brain. We previously showed that Na_x channel is preferentially expressed in glial cells in the circumventricular organs (CVOs) and that Na_x-knockout mice ingest saline in excess under dehydrated conditions. Subsequently, we demonstrated that Na_x is a Na-level-sensitive Na channel. Recently, we confirmed the physiological role of the Na_x in vivo by infusion of hypertonic Na solution to the cerebral ventricle. The infusion induced prompt intake of water and aversion to salt in wild-type mice. In contrast, such aversive behavior was not observed in the knockout mice. When Na_x cDNA was introduced into the brain of the knockout mice with an adenoviral expression vector, only animals with a transduction of the Na_x gene into the subfornical organ (SFO) among the CVOs recovered salt-avoiding behavior under dehydrated conditions. These results clearly indicate that the SFO is the center of the control of salt-intake behavior in the brain, where the Na-level-sensitive Na_x channel is involved in sensing the physiological increase in the Na level of body fluids. [J Physiol Sci. 2006;56 Suppl:S38]

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The fate of grafted neuroepithelial stem cells in the mature brain environment was assessed to confirm their feasibility in the functional repair of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of enhanced-GFP-carrying rat embryos, and implanted into the striatum of normal adult rat or Parkinson's disease model rat. The differentiation pattern of donor-derived cells was monitored immunohistochemically. The functional abilities of the donor-derived cells and communication between them and the host were investigated using host-rat brain slices incorporating the graft with whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were following their genetically programmed fate. In the long-term, the large number of donor-derived neurons was sustained, but the staining pattern showed appearance of medium spiny or cholinergic neurons, suggesting that some neurons were following environmental cues. Some donor-derived astrocytes were also seen in the graft. Firing pattern and membrane properties suggest the presence of both dopaminergic and non-dopaminergic neurons in the donor-derived neurons. Glutamatergic and GABAergic post-synaptic currents could be evoked by electrical stimulation applied in the host region. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor material for intracerebral grafting in functional repair. [J Physiol Sci. 2006;56 Suppl:S38]

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In order to clarify the implications of morphological brain changes in development of schizophrenia, we have made extensive comparisons of brain morphology using MRI between established schizophrenia and schizotypal disorder, a schizophrenia-spectrum disorder without overt and sustained psychotic episode. Compared with controls, bilateral volumes of the amygdala, hippocampus and posterior superior temporal gyrus were reduced comparably in both schizotypal and schizophrenia patients. Total prefrontal grey matter was smaller bilaterally in schizophrenia patients than in controls, whereas schizotypal patients had larger right prefrontal grey matter than controls. In schizophrenia patients, the bilateral superior frontal, inferior frontal and straight gyri, and the left middle frontal gyrus were smaller than those in controls, while schizotypal patients had larger bilateral middle frontal gyri and smaller right straight gyrus. In white matter, decreased volume of the anterior limb of the internal capsule, a fiber bundle connecting the frontal cortex and thalamus, was found bilaterally in schizophrenia but only on the right in schizotypal disorder. These findings suggest that volume reductions in the medial and postero-lateral temporal regions are the common morphological substrates for the schizophrenia-spectrum which presumably represent the vulnerability. Additional widespread involvement of the prefrontal cortex might lead to the loss of inhibitory control in other brain regions and play a critical role in the manifestation of overt psychosis. [J Physiol Sci. 2006;56 Suppl:S38]

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The previous neuropsychological studies demonstrated that the human amygdala (AM) increased its response to emotional facial expressions and gaze direction toward to the subject, and suggested that the AM is essential in social cognition. In the present study, the monkey AM neuronal activity was recorded during performance of a delayed non-matching to sample task using human photos with various facial expressions and gaze direction. Some neurons were further tested with various human actions such as approaching toward the monkey. Autonomic activity (pupil radius) of the monkey, which reflected emotional expression, was simultaneously recorded. The results indicated that the AM neurons differentially responded to various emotional expressions and/or gaze directions. These facial expression-differential neurons were most sensitive to those of the familiar persons to the monkeys. These results suggest that social cognition might develop based on learning through social interaction, and the AM is involved in such learning. Activity of other AM neurons increased when the experimenter approached toward the monkey, or when the experimenter moved its arm or leg. Pupil radius also increased during this approaching. These results suggest that the AM is essential in primate social cognition as well as emotional expression. [J Physiol Sci. 2006;56 Suppl:S39]

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It is a continuing question how emotions enhance memory formation. To answer the question, we study the connection between the hippocampus and the hypothalamus, which is involved in Papez circuit. We have hypothesized that the hippocampal activity is enhanced by direct inputs from the supramammillary nucleus (SuM) of the hypothalamus to the dentate gyrus (DG) and the CA2 region. Immunocytochemistry of Fos positive neurons (FN) demonstrated that the SuM-hippocampal pathway was activated when animals were exploring a novel environment. Number of FN in SuM and the hippocampus increased when rats were placed in an open field. SuM lesions significantly suppressed the increase of FN in the entire hippocampus. Small lesions in the lateral SuM significantly suppressed the increase of FN in the ipsilateral CA2 compared with the control side, although there was no difference in the number of FN between both sides of DG. These data suggest that CA2 neurons is specifically activated by ipsilateral inputs from SuM, while DG is activated by bilateral inputs from SuM. Since SuM is related to anxiety, anxiety enhances the neuronal activity of the dentate granule cell and CA2 neurons and results in the enhancement of memory formation. [J Physiol Sci. 2006;56 Suppl:S39]

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The primate amygdala plays an important role in differentiating between facial expressions, yet the neural properties underlying this process are largely unknown. We recorded from the monkey amygdala neural responses to images of monkey faces, human faces, and objects. Most neurons differentiated between these image categories, yet monkey faces, human faces, and objects were equally likely to elicit stimulus-selective responses. In certain animals threatening faces appeared to elicit increased firing rates compared to neutral or appeasing faces suggesting a processing bias in favor of stimuli that signals potential danger. Neural responses to monkey faces were further examined to determine whether the observed changes in firing rate can be best accounted for by face identity or facial expression. The majority of neurons responded to unique combinations of identity and expression suggesting that in the amygdala identity and facial expressions are merged into a single representation. This representation might carry information about the emotional and social significance of facial expressions encountered during social interactions. The amygdala is also involved in orchestrating overt behavioral and autonomic responses to images with emotional value. We recorded skin conductance response, heart rate, and facial muscular activity in conjunction with neural responses in the amygdala and found correlations between stimulus-selective neural activity and peripheral autonomic responses. [J Physiol Sci. 2006;56 Suppl:S39]

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Scientific institution in Japan established an institutional animal care and use committee for reviewing animal experimentation protocols and advise the institutional director to improve the welfare of laboratory animals according to the administrative guidance "Notification Concerning Animal Experimentation Conducted by Universities etc."(1987). In 2004, Science Council of Japan proposed a new regulation rule of animal experimentation to promote the public understanding of ethical and scientific animal experimentation. Major points of revision are as follows:1. Establishment of a guideline for animal experimentation commonly applicable to the all scientific institutions in Japan.2. Establishment of an objective evaluation system on the institutional self regulation for animal experimentation. Establishment of new regulation system is now on going. On the other hand, Amended Law for the Humane Treatment and Management of Animals (2005) stipulated the 3Rs principle in animal experimentation.For the development of health research for humans as well as animals, balance between science and animal welfare is indispensable.(Former chair of Committee on Laboratory Animal Science, Science Council of Japan) [J Physiol Sci. 2006;56 Suppl:S40]

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The Ministry of Health, Labour, and Welfare introduced the Notification System for the Importation of Animals, which is effective from 1st September 2005, to prevent the outbreaks of human infectious diseases derived from imported animals. Any importers of animals, including experimental animals or transgenic animals, are required to submit a written declaration giving the specified information on the animals, such as their species name and quantity, to the quarantine station of the Ministry of Health, Labour, and Welfare. This declaration has to be accompanied by a health certificate issued by the government authorities of the exporting country certifying that the animals are free from the infectious diseases specified for each species. Procedure to issue the health certificate by the government authorities varies among the exporting countries. For example, in the United States the animal facility that stores the exporting animals publishes the heath certificate corresponding to the regulation proposed by the Ministry of Health, Labour, and Welfare, and sends the certificate to the USDA office located in each state, where the official veterinarians endorse the certificate. In addition, the Ministry of Health, Labour, and Welfare is asking to register the health certificate form for the government authorities in other countries that have not yet responded to the notifying system for importation of animals. Researchers who are planning to import the experimental animals need to understand the current situation of the importation and correspond to this new system to ensure the smooth importation of animals into Japan. [J Physiol Sci. 2006;56 Suppl:S40]

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Introduction Reduced gene expression of SERCA2 impairs calcium handling and represents a hallmark of heart failure. Unlike adenovirus- or adenoassociated vectors, lentivirus can stably integrate into host genome of terminally differentiated cardiac myoctes and induces permanent expression. We developed lentivirus-based SERCA2 gene transfer system and examined its feasibility as a therapy for heart failure. Results The therapeutic effect of Lenti-SERCA2 vector (1x10¹¹ IU/300g BW) was compared with the Lenti-β-Gal control vector in the failing heart induced by myocardial infarction (MI) in rats. Echocardiography revealed that Lenti-SERCA2 introduction prevented an increase in left ventricular diameter and a decrease of fractional shortening by 10-15% compared with Lenti-β-Gal group rats from days 30 to 180. Pressure-volume analysis demonstrated that Lenti-SERCA2 introduction improved systolic (dP/dt max, 7677 vs 3028 mmHg/sec; Emax, 0.68 vs 0.37) and diastolic function (tau, 18.4 vs 22.6). Northern and Western blot analyses revealed that SERCA2 mRNA and protein were elevated and the BNP mRNA was significantly decreased in Lenti-SERCA2 group. Finally, SERCA2 gene transfer prevented the expansion of MI region and decreased the mortality rate. Conclusion Our study showed that the SERCA2 gene was successfully integrated into hearts and supports the premise that a lentivirus-based SERCA2 gene therapy improves heart failure. [J Physiol Sci. 2006;56 Suppl:S41]

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Rho-kinase (ROK)-mediated Ca²⁺ sensitization plays a pivotal role in abnormal vascular smooth muscle (VSM) contraction such as vasospasm. Previously we identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as an upstream signaling molecule of ROK-mediated Ca²⁺ sensitization. Since VSM contains both Fyn and c-Src among Src-TK, we analyzed which Src-TK was truly important for the Ca²⁺ sensitization mediated by SPC/ROK pathway. Immunofluorescent study showed that SPC induced the translocation of Fyn, but not c-Src, to plasma membrane in cultured VSM cells and eicosapentaenoic acid, a specific inhibitor of SPC-induced VSM contraction, blocked the translocation of Fyn. The siRNA which specifically knockdown Fyn diminished SPC-induced contraction remarkably in cultured VSM cells. In β-escin permeabilized VSM strips, constitutively-active Fyn, which was expressed by baculovirus system, induced Ca²⁺ sensitization and dominant-negative Fyn blocked Ca²⁺ sensitization. In confocal study, SPC induced the translocation of Fyn to plasma membrane where it colocalized with caveolin-1, a membrane-raft-associated protein. A functional proteomics approach identified p160 and its phosphorylation site as a possible target of Fyn. Those findings suggested that membrane rafts and its associated Fyn played an essential role in ROK-mediated Ca²⁺-sensitization of VSM contraction. [J Physiol Sci. 2006;56 Suppl:S41]

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Excitatory receptor agonists such as noradrenaline stimulate the activity of the small G protein Rho and inhibit myosin phosphatase (MP) through mechanisms involving Rho kinase-dependent phosphorylation of the MP regulatory subunit MYPT1 in VSM. We have recently demonstrated that a novel, Ca²⁺-dependent mechanism for Rho activation and myosin phosphatase (MP) inhibition is operating in receptor agonist- and membrane depolarization-induced vascular smooth muscle (VSM) contraction. We found that phosphoinositide 3-kinase (PI3K) was required for Ca²⁺-dependent Rho activation; the PI3K inhibitors wortmannin and LY294002 inhibited all of the Ca²⁺-dependent Rho activation, MYPT1 phosphorylation, MP inhibition, MLC phosphorylation and contraction. We tried to identify a PI3K isoform by adopting RNA interference and cultured VSM cells. The selective down-regulation of the expression of class II alfa isoform (PI3K-C2&alpha), but not the class I p110&alpha, by a specific siRNA markedly inhibited Rho kinase-dependent MYPT1 phosphorylation, MLC phosphorylation and contraction in differentiated VSM cultures. Noradrenaline as well as membrane depolarization stimulated the activity of PI3K-C2&alpha, but not p110&alpha, in a Ca²⁺-dependent manner. Thus, these observations unveiled a novel role of the PI3K-C2&alpha as an upstream regulator of Rho and consequently MP and contraction. [J Physiol Sci. 2006;56 Suppl:S41]

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Recent evidence suggests that stent-associated inflammation is a prominent feature in animals and humans, and thus can be a promising next-generation target for prevention of restenosis. We have shown great benefit of anti-monocyte chemoattractant protein-1 (MCP-1) therapy by systemic transfer of an N-terminus deletion mutants of human MCP-1 (called 7ND) gene for prevention of restenotic changes in animals. Therefore, to translate our achievement on MCP-1 pathobiology to clinic, we tested the hypothesis that stent-based local delivery of 7ND gene reduces in-stent neointimal formation. Bare, polymer-coated, and 7ND plasmid-coated stents were implanted in iliac arteries of hypercholesterolemic rabbits (n=8-10 each) and cynomolgus monkeys (n=7-10 each). 7ND gene-eluting stents attenuated stent-associated monocyte infiltration/activation and neointimal formation (about 30% reduction) in rabbits. In monkeys, significant reduction of neointimal formation was noted 1, 3, and 6 month after stenting, indicating long-term benefits of 7ND gene ES in monkeys. No evidence of incomplete healing process was noted in 7ND-eluting stent sites. In conclusion, anti-MCP-1 strategy with 7ND gene-eluting stents was strikingly effective in reducing experimental restenosis in rabbits and monkeys. Our finding in nonhuman primates has significant clinical significance, implying that this anti-inflammation strategy targeting MCP-1 might be a promising therapy against human restenosis. [J Physiol Sci. 2006;56 Suppl:S42]

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The role of Angiotensin II (Ang II) in the regulation of the cardiovascular system under normal and pathologic conditions have been well documented. A variety of angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) are selected as first choice medicine for hypertension or heart failure. Although two major subtypes Ang II receptors, exist type1 (AT₁) and type2 (AT₂), most studies and treatments have focused on AT₁ coupled events. Previous reports indicated that AT₂ plays a role in essentially growth suppression such as through the tyrosine phoaphatase SHP-1 and MKP-1 activation. However, a detailed signaling mechanisms of these responses still remain unclear. Interestingly, an increasing number of recent reports indicate that AT₂ plays a role in growth promoting similar to the AT₁ function. We reported that AT₂ gene-deleted mice lose the ability to develop cardiac hypertrophy in response to pressure overload or to chronic Ang II stimulation, and also found a novel signaling mechanism of AT₂ mediated by the transcription factor promyelocytic leukemia zinc finger (PLZF) leading to cardiac hypertrophy. PLZF is selectively expressed in the heart, but not in the kidney or aorta. Upon Ang II stimulation, AT₂ and PLZF are internalized, and PLZF translocates into the nucleus, whereby nuclear PLZF activates phosphoinositide 3-kinase (PI3K) regulatory subunit 85α leading to cardiac hypertrophy. However, in the absence of PLZF, Ang II evoked SHP-1 activation leading to growth suppression via AT₂. These results suggest that AT₂ may have dual switching functions mediated by PLZF. [J Physiol Sci. 2006;56 Suppl:S42]

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Contraction of vascular smooth muscles is under the regulation of sympathetic activity and vasoactive hormones. It is known that release of Ca²⁺ from inositol 1,4,5-trisphosphate (IP₃)-sensitive stores causes the initial phase of agonist-induced vasocontraction. In the present study, phenylephrine (PE)-induced contraction was measured in thoracic aortas isolated from the wild-type (WT) and IP₃ receptor type 1 knockout (IP3R1-KO) mice, in order to specify the IP₃ receptor subtype responsible for the agonist-induced contraction. PE (10⁻⁸–10⁻⁶ M)-induced aortic contraction in the IP3R1-KO mice was greatly diminished, compared to that in WT mice, and lacked the steep contraction which was invariably seen in WT aortas immediately after PE application at 10⁻⁶ M. But, high K⁺-induced contraction was indistinguishable between WT and IP3R1-KO aortas. Immunoblotting analysis demonstrated the presence of three IP₃ receptor subtypes (IP3R1, IP3R2 and IP3R3) in WT mouse thoracic aorta; however, abundance of each subtype was in the order of IP3R1 > IP3R3 >> IP3R2. These results indicate that IP3R1 constitutes the Ca²⁺ release channels critical to vasocontraction regulated by sympathetic activity and vasoactive hormones. [J Physiol Sci. 2006;56 Suppl:S42]

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The vestibuloocular reflex (VOR) stabilizes vision during head turn by counter-rotating the eyes in the orbit. Its gain (eye velocity/head velocity) can be modified by visual-vestibular mismatch, but following cerebellar inactivation, the gain cannot be further modified. Thus, the VOR has been a model system to study potential cerebellar roles in motor learning. The cerebellum may have different roles in acute versus chronic VOR motor learning, because cerebellar inactivation entirely eliminates any acutely learned component, but it only partially eliminates the memory of long-term gain change, suggesting multiple loci for the chronic memory. To pinpoint these, a series of experiments in which activities of cerebellar Purkinje cells (PCs) and their target neurons in dorsal Y group (YNs) were recorded before and after chronic VOR motor learning. The sensitivities of PCs to both vestibular (V) and efference copy (E) signals changed with learning, YNs changed their sensitivities to V modalities, and these changes are asymmetric for gain increase and decrease. Computational modeling revealed significant changes in 1) V pathway to cerebellar flocculus (FL), 2) direct V pathway to YNs after gain increase, and in 3) E pathway to FL, 4) direct V pathway to YNs, 5) pathway from PCs to YNs, and 6) V pathway excluding those through FL and YNs after gain decrease. The results suggest involvement of several loci in chronic learning and different neuronal mechanisms for gain increase and decrease. [J Physiol Sci. 2006;56 Suppl:S43]

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Adaptation of ocular reflexes is a prototype of cerebellum-dependent motor learning. Two different views are proposed for its neural mechanisms: one that the memory of adaptation is formed within the cerebellar flocculus through the cerebellar long-term depression (LTD) of parallel fiber-Purkinje cell synapses, and the other that the memory is formed within the vestibular nuclear neurons using signals mediating through the flocculus. We developed a long-term adaptation paradigm adaptation of mouse eye movements. We revealed that the memory trace of motor learning induced by short-term (day-long) training is located within the cerebellar cortex, while that induced by long-term (week-long) training in the cerebellar or vestibular nuclei, by evaluating the effects of reversible pharmacological shutdown of the cerebellar cortex. These results suggest that the memory trace of motor learning is initially formed in the cerebellar cortex, and later shifts transsynaptically to cerebellar/vestibular nuclei for consolidation. We further revealed that LTD plays a critical role in both the acquisition and consolidation of memory by pharmacological and gene-knockout mouse experiments. [J Physiol Sci. 2006;56 Suppl:S43]

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There are some unresolved problems in motor learning theory. One is determining the source of a learning signal, sometimes called a motor error signal. Another one is the credit assignment problem of the motor error, since the erroneous performance perceived by a subject is due to the actions of many elementary motor units. The feed-forward associative learning theory attributes the source to the movement system itself. When a subject performs a corrective movement after his primary movement, the proposed neural learning device learns to associate the primary motor command with the corrective motor command by using a place-coding system. In the subsequent trials, the primary movement will involve a correction due to the participation of this mechanism, thus resulting in better performance. The device consists of many adaptive units each of which is specialized for a particular elementary motor unit, and naturally resolves the assignment problem. The theory assumes three conditions, namely, that a motor center and the learning device share the same place-encoded motor information; the motor center issues a command and a learning signal simultaneously from the same unit; and a learning signal issued with a corrective command has a heterosynaptic interaction with the previous primary command. The cerebellum is a reasonable candidate for the device satisfying these conditions. The reaction time of a corrective movement, usually 100-300 ms, almost satisfies the coincidence condition for long-term depression of the granule-to-Purkinje synapses. As an application, this theory is demonstrated to account for behavioral results regarding saccadic adaptation. [J Physiol Sci. 2006;56 Suppl:S43]

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The cerebellum plays critical roles in motor control and learning. Relative simplicity of the cerebellar cortical circuit has prompted the study on how it works, which has made the cerebellum one of the best characterized structure of central nervous system. However, respective role of each component or its function such as a particular type of synapse, neuron or synaptic regulation has been elusive. We have been addressing these issues using several types of mutant mice, and here I present our recent data on the GluRδ2 (glutamate receptor δ2 subunit) knockout mice (δ2-/-). GluRδ2 is a molecule related to ionotropic glutamate receptor, which is specifically expressed at parallel fiber (PF)–Purkinje neuron (PN) synapses. The δ2-/- show impairment in the long-term depression, synaptic stabilization of PF-PN synapses and elimination of surplus climbing fiber (CF) inputs resulting in the multiple innervation to a PN, and also show motor discoordination and motor learning failure. We studied the eye movements and found that δ2-/- show involuntary spontaneous eye movements and large phase delay in the optokinetic response (OKR). We have been analyzing the mechanism of these abnormal motor regulations by simultaneous recording of eye movements and PN activity. Our results suggest that the enhanced CF activity in δ2-/- largely disturbed the normal pattern of PN activity regulating eye movement, that highlights the importance of synaptic inputs balance on a PN in motor regulation. [J Physiol Sci. 2006;56 Suppl:S44]

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The concept for glial cells as non-excitable- supporting elements has been accepted until almost the end of 20th century without doubt. However, total number of glial cells in human brain was found to be far larger than that of neuronal cells, and the ratio of glial cells to neurons in the brain was found to be higher in the highly developed animals than that in primitive animals. Those evidences suggested that the cells may be required for establishing higher order brain function. The dynamic feature of the cells has been revealed by the Ca²⁺ imaging techniques. Since then astrocytes have been recognized as dynamic cells and are regarded as intimate collaborators with neuronal cells. The concept of"tri-partite synapse" has been put forward to explain the possible participation of astrocytes into the synaptic transmission and information processing in the brain. However, the interaction among neuronal cells and astrocytes may not be such small scale. The transmission of the Ca²⁺ waves from an astrocyte to the other has been found to be performed through gap-junctions and also specific transmitters and receptors system. Thus astrocytes themselves form a wide network among them, which may be woven into the neuronal networks and construct large and highly organized information processing system. Neuronal networks as main system in the brain information processing may be controlled slowly and widely by astrocytes networks. This "glia-neuron functional coupling" will establish a higher order brain function and its deficit will cause brain dysfunctions. [J Physiol Sci. 2006;56 Suppl:S44]

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Recent studies have demonstrated the existence of direct interactions between neurons and glial cells. To evaluate these interactions, we focused on interneuron/peri-interneuronal glial cell pairs in the hippocampal CA1 region, because of the close proximity of these two cells. Based on the electrophysiological, morphological and immunohistochemical studies, the peri-interneuronal glial cells were classified into astrocytes and oligodendrocytes, and we worked with the peri-interneuronal astrocytes (PNAC) in this study. Excitatory postsynaptic currents (EPSCs) recorded in an adjacent interneuron were suppressed by the depolarizing current injection into the PNAC. These suppression of EPSCs accompanied the increase of paired-pulse ratio and were blocked by the application of adenosine A₁ receptor antagonist, indicating the involvement of presynaptic adenosine A₁ receptors. Moreover, PNAC depolarization modified the directly induced firing of the interneuron. These results demonstrate directly modulatory effects of the PNAC on neuronal activities. [J Physiol Sci. 2006;56 Suppl:S44]

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Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed 6-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). The prolongation of the decay kinetics of CF-EPSCs in GLAST(-/-) mice is found only in the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors. We attempted to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporters, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of CTZ, DHK prolonged the decay time constant (τ_w) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by ∼80%, caused no change in τ_w in WT in the absence of CTZ, whereas it prolonged τ_w in the presence of CTZ. This prolonged value of τ_w was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion (∼20%) of functional transporters is preserved, and that GLT-1 alone in GLAST(-/-) mice is sufficient to keep the fast kinetics of EPSCs in the absence of CTZ. [J Physiol Sci. 2006;56 Suppl:S45]

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Neural-glial communication has been assumed to be mediated by spillover of transmitter from the synaptic cleft. In the cerebellum, Bergmann glia cell (BG) processes encase synapses between presynaptic climbing fiber (CF) and parallel fiber elements and postsynaptic Purkinje cell (PC) spines and glutamate released from these fibers can activate Ca²⁺-permeable AMPA receptors on BGs. Quantal responses recorded from BGs were not coincident with quantal responses recorded in adjacent PCs sharing the same CF input. By combining electrophysiological recordings and quantitative immunogold electron microscopic analysis, high-concentration (1.5 mM) rapid-transients (0.5 ms) of glutamate were estimated to underlie BG quantal events. We propose that exocytosis can occur from ectopic release sites located directly across from BG membranes. Ectopic release may be necessary to activate low affinity AMPA receptors on BGs, which may provide a geographical cue to guide BG membrane to surround active synapses and ensure efficient glutamate uptake. We have recently started to employ two-photon microscopy to study the result of such neural-glial communication. Morphological refinement of BG processes occurs within a few days in early development and rapid motility and spontaneous remodeling of extracellular space by BG protrusions were observed in minutes. Synaptic activation leads to Ca²⁺ influx at the tip of the protrusions via Ca²⁺-permeable AMPA receptors. We are currently probing the mechanisms that manipulate the motility and refinement of BG processes and their effect on synaptic transmission. [J Physiol Sci. 2006;56 Suppl:S45]

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Alexander disease is caused by heterozygous mutation in glial fibrillary acidic protein (GFAP). The pathological hallmark is the presence of astrocytic GFAP aggregation called Rosenthal fibers (RF). To understand the pathophysiology of Alexander disease and utilize those results to the understanding of normal function of astrocytes in vivo, we have established transgenic mice that express hunan GFAP R239H mutant under the control of mouse GFAP promoter. Immunohistochemistry using human GFAP specific antibody, SMI21, showed that this immunoreactivity was present only in S100 beta positive astrocytes and ependymal cells. Some astrocytes possessed the aggregations with SMI immunoreactivity that was co-labeled by small heat shock protein, alpha B crystalline and HSP25. Contrary to the case of human, transgenic mice showed no abnormality of myelin formation and structure. To examine whether the presence of human GFAP aggregation altered brain functions, we challenged kainic acid administration to transgenic and wild type mice and studied the susceptibility of convulsion and the vulnerability of hippocampal cell damage. Transgenic mice were more susceptible to systemic administration of kainic acid (20 mg/kg) than wild type mice as revealed by the number of mice showing tonic-clonic convultion: 8 out of 11 for transgenic mice and 2 out of 14 for wild type mice. Transgenic mice were more vulnerable to excitotoxicity of kainic acid as revealed by the appearance of Fluoro Jade positive dying hippocampal neurons after 24 hours administration. We concluded that Alexander disease model mice compromised the protective function against kainic acid excitotoxicity. [J Physiol Sci. 2006;56 Suppl:S45]

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During CNS development, neural stem cells (NSCs) give rise first to various kinds of specified precursor cells, which proliferate extensively before terminally differentiating into either neurons or glial cells. It is still not clear, however, whether the precursor cells are irreversibly determined to differentiate into their particular cell types. Neither is it clear how proliferation of the precursor cells are terminated, although it is widely accepted that control of the proliferation plays a crucial role in determining the number of neurons or glial cells. We have been addressing these issues using the developing mouse cerebellum as a model system. We found that cerebellar granule cell precursors (GCPs) can differentiate into astroglial cells when exposed to Shh and BMP. This indicates that GCPs are not irreversibly committed to neuronal development, but can be induced to differentiate into astroglial cells by appropriate extracellular signals. We also examined the role of cyclin-dependent kinase inhibitors in the control of proliferation of GCPs. Among the inhibitors we examined, only p27 was expressed at significant levels in cells of the granule cell lineage in the developing cerebellum. We found that there was an inverse correlation between BrdU uptake and p27 expression by GCPs. Even in the presence of saturating amounts of Shh, a potent mitogen, the cells eventually stopped dividing and differentiated, expressing p27 strongly. These results suggest that there is an intracellular mechanism that stops GCP division and causes GCPs to differentiate and that p27 is part of this mechanism. [J Physiol Sci. 2006;56 Suppl:S46]

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We report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1 and Zipro1). After purification by FACS and co-culture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system. [J Physiol Sci. 2006;56 Suppl:S46]

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It has become apparent that epigenetic modification plays a critical role in the regulation of lineage-specific gene expression. We have previously reported that the change in DNA methylation at the promoter of astrocytic genes, such as glial fibrillary acidic protein (GFAP), controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. The methylated promoter at midgestation undergoes demethylation as gestation proceed, corresponding to the onset of astrocytogenesis. However, the exon1 of the gene remains hypermethylated even in the adult neural progenitors and in cells differentiated from the progenitors, i.e. neurons, astrocytes and oligodendrocytes. The methyl-CpG binding proteins (MBDs) bind to methylated DNA and suppress the target gene expression. They are strongly expressed only in neurons in the nervous system and the cells do not respond to astrocyte-inducing signals to express GFAP. In contrast, by using Cre-recombinase fate tracing, we show here that oligodendrocytes, in which MBDs are not expressed, expressed GFAP upon stimulation with the astrocyte-inducing cytokines. Overexpression of MeCP2, one of the MBD family proteins, in oligodendrocytes inhibited the GFAP expression by the cytokines, implicating MBDs as key molecules to restrict the transdifferentiation of neural cells. It is well known that astrocytes increase dramtically in number after insult to the nervous systems. Taking the above results into consideration, oligodendrocytes could be a source of the newly generated astrocytes in damaged nervous systems in vivo. [J Physiol Sci. 2006;56 Suppl:S46]

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There is increasing evidence that some kinds of glial cells in central nervous system (CNS) can behave as multipotent neural stem cells (NSCs) and generate neurons, astrocytes, and oligodendrocytes in vivo and in vitro. However it is still unknown how such glial cells acquire multipotentiality. Oligodendrocyte precursor cells, which exist in many area in CNS, can also behave as multipotent NSC when the cells are exposed to specific conditions. Recently we have shown that sox2, which is an essential transcription factor in NSCs, is reactivated in the OPC reversion. We have also shown that in the reversion SWI/SNF chromatin remodeling complex is recruited to an enhancer in the sox2 promoter and lysine 4 and 9 of histone H3 in the enhancer are methylated and acethylated, respectively. We propose that the reversion of OPCs to NSCs depends on extensive chromatin remodeling, which is in part mediated by SWI/SNF. [J Physiol Sci. 2006;56 Suppl:S47]

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Cortical neural precursor cells (NPCs) sequentially undergo expansion, neurogenic and gliogenic phases during development, although the underlying mechanisms are poorly understood. We have recently shown that Wnt signaling instructively induces neuronal differentiation of NPCs. Importantly, Wnt signaling does so only in midgestation stage (neurogenic phase) of NPCs but not in early embryonic stage (expansion phase) or in perinatal stage (gliogenic phase) of NPCs. In early embryonic stage, Wnt signaling rather promotes proliferation of NPCs. Here I will discuss possible mechanisms that might account for these stage-dependent responses. Likewise, STAT3-activating ligands induce astrocytic differentiation in late (gliogenic phase) but not in early (expansion and neurogenic phases) NPCs. These stage-dependent responses of NPCs might play a central role in determining the timing of differentiation and the size of final population of each differentiated cell type. [J Physiol Sci. 2006;56 Suppl:S47]

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Neuroepithelial cells are first generated from the ectoderm, forming the neural plate. These cells undergo symmetric cell divisions to produce more neuroepithelial cells. After neural tube formation, they become radial glial cells, which undergo asymmetric cell divisions, forming one radial glial cell and one neuron (or a neuronal precursor) from each cell division. After production of neurons, radial glial cells finally give rise to glial cells such as astrocytes. Thus, neural stem cells change their characteristics of morphology and competency over time during development. We found that inactivation of the bHLH genes Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. We also found that the boundary structures such as the isthmus and the zona limitans intrathalamica are not maintained and that the boundary cells are differentiated into neurons and lose the organizer activity. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of neural stem cell differentiation and by maintaining the boundaries with the organizer activity. [J Physiol Sci. 2006;56 Suppl:S47]

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Vascular endothelial cells (ECs) alter their morphology, function, and gene expression in response to shear stress generated by blood flow. However, the molecular mechanism of shear stress sensing by ECs has not been clarified. We investigated the mechanism from the aspect of calcium (Ca) signaling. Human pulmonary artery ECs (HPAECs) loaded with the Ca indicator Indo-1/AM were exposed to laminar flow and changes in intracellular Ca concentrations were monitored. A stepwise increase in flow rate elicited a corresponding stepwise-increase in Ca concentrations. Apyrase or EGTA completely abolished the flow-induced increase in Ca concentrations, indicating that ATP and influx of extracellular Ca are essential for the Ca responses. Flow increased the release of ATP from HPAECs in a shear stress-dependent manner. HPAECs predominantly express a subtype of ATP-operated cation channel P2X4, and antisense oligonucleotides targeted to P2X4 abolished the flow-induced Ca influx. Pulmonary microvascular ECs cultured from P2X4-deficient mice showed no flow-induced Ca influx and nitric oxide production. Human embryonic kidney 293 cells became sensitive to flow and show flow-induced Ca influx when transfected with P2X4 cDNA. Flow-induced dilation of skeletal muscle arterioles was markedly suppressed in P2X4-deficient mice. P2X4-deficient mice had higher systolic blood pressure values than wild-type mice. Thus, ECs transduce the signal of shear stress into Ca influx via P2X4, and that the purinoceptor-mediated blood flow-sensing plays important roles in endothelial NO production and vascular tone control. [J Physiol Sci. 2006;56 Suppl:S48]

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Human umbilical endothelial cells (HUVECs) show various responses including morphological changes and protein expressions in response to mechanical stretch. Our previous studies revealed that intracellular Ca increase in response to mechanical stretch via Ca permeable stretch-activated (SA) channel activation is critical in HUVECs cultured on an elastic PDMS (polydimethylsiloxane) membrane. Since recent reports suggest that the transient receptor potential (TRP) channels may form the SA channel, we investigated the involvement of TRPV2 in the stretch-induced Ca increase in HUVECs. Human TRPV2 was isolate from a HUVEC cDNA library. Heterologous expression of the human TRPV2 in COS7 cells resulted in the stretch-induced Ca increase and injection of a TRPV2-specific siRNA in HUVECs abolished the stretch-induced Ca increase.These observations indicate that TRPV2 plays a critical role in the stretch-induced Ca increase in HUVECs. [J Physiol Sci. 2006;56 Suppl:S48]

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Hemostasis is a physiologic mechanism that maintains blood in a fluid state within the circulation. The blood-coagulation cascade has the ability to transduce a small initiating stimulus into a large fibrin clot, which is mediated by cellular components and soluble plasma proteins. The potentially explosive nature of this cascade is counterbalanced by natural anticoagulant mechanisms. The maintenance of adequate blood flow and the regulation of cell-surface activity control the local accumulation of activated blood-clotting enzymes and complexes. Antithrombin is a plasma protein that inhibits the blood serine proteases of the intrinsic and common coagulation pathways. Heparin-like molecules, heparan sulfate proteoglycans, are closely associated with endothelial cells and enhance the action of circulating antithrombin. In this session, the endothelial heparan sulfate and antithrombin system, which plays an important role in the natural hemostatic balance to maintain the blood fluidity, will be discussed through the data from the congenital deficient mouse-models, i.e. KO mice. [J Physiol Sci. 2006;56 Suppl:S48]

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Tissue plasminogen activator (t-PA), the primary PA in vasculature, is synthesized and secreted from vascular endothelial cells (VECs). Besides other serine proteases involved in both coagulation and fibrinolysis, t-PA has unique characteristics of possessing physiological activity as a single chain form and being secreted as an active form. In blood there also exist plasminogen activator inhibitor type 1 (PAI-1), a member of serine protease inhibitor superfamily (SERPINS), which inhibits the activity of both single chain- and two chain- forms of t-PA by forming an equimolar high molecular weight complex. We have reported that total fibrinolytic activity in plasma is regulated by the balance between these two molecules, showing that increase in PAI-1 level under either physiological or pathological conditions suppresses fibrinolytic activity, whereas the enhanced t-PA secretion accelerates fibrinolysis. Recently, we have studied the dynamics of t-PA secretion from its containing granules in VECs using total internal reflection fluorescence microscopy (TIRFM). We obtained results suggesting that secreted t-PA by regulatory exocytosis stays on the membrane of VECs for certain period of time, and expresses its specific activity on VECs. PAI-1 appeared to modify the dynamics of t-PA secretion, and thus fibrinolytic activity on VECs.Showing these resuls, we discuss how fibrinolytic activity is regulated by t-PA and PAI-1 both in plasma and on VECs. We also want to discuss the physiological relevance of this regulatory mechanism which is naturally modified by many physiological stimuli [J Physiol Sci. 2006;56 Suppl:S49]

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Introduction. Atherothrombosis, including myocardial infarction and ischemic stroke, is a leading cause of death in the modern world. Platelet, forming thrombi at site of ruptured or disrupted atherosclerotic plaque, play crucial role in the onset of atherothrombosis. Mechanism of platelet thrombus formation under blood flow condition may not be the same as platelet aggregation under static conditions. Method. Whole blood, containing platelets rendered fluorescent by addition of mepacrine or calcium sensitive dye of Fluo-3, was perfused on the immobilized collagen fibrils at various shear rate conditions. Two-dimensional and three-dimensional growth of platelet thrombi on the collagen fibrils were detected by epi-fluorescent video-microscpy and ultra-fast laser confocal miscroscope equipped with piezo-motor control unit, respectively, Real time visualization of intra-cytoplasmic calcium ion concentration was also achieved with the use of laser confocal microscopy. Results. Unlike platelet aggregation under static condition, platelet thrombus growth on the collagen fibrils under blood flow conditions was markedly inhibited by blocking von Willebrand factor binding with glycoprotein Ibα, ADP binding with P2Y₁₂ ADP receptor, and so on. Our results also revealed that cyclic increase in intracytoplsmic calcium ion concentration was abolished when P2Y₁₂ was blocked. Collagen and thrombin also play important roles in the growth of platelet thrombi. Conclusion. Mechanism of platelet thrombus formation under blood flow conditions are different from that of platelet aggregation under static condition. [J Physiol Sci. 2006;56 Suppl:S49]

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Microglia play an important role as immune cells in the central nervous system. Recently, accumulating evidences indicate the important role of ATP receptors of activated microglia in the neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, cancer, diabetes or infection. The expression of P2X4 receptor is enhanced in spinal microglia after peripheral nerve injury model, and blocking pharmacologically and suppressing molecularly P2X4 receptors produce a reduction of the neuropathic pain (Tsuda et al. Nature 424, 778-783, 2003). Several cytokines such as interleukin-6 and tumor necrosis factor in the dorsal horn are also increased after nerve lesion and have been implicated in contributing to nerve-injury pain. ATP can activate MAPK leading to the release of bioactive substances including cytokines from microglia (Shigemoto-Mogami et al., J Neurochem 78, 1339-1349, 2001; Suzuki et al., J Neurosci 24, 1-7, 2004). Thus, diffusible factors released from activated microglia by the stimulation of purinergic receptors may have an important role in the development of neuropathic pain (Tsuda, M., Inoue, K., & Salter, M.W. Trend Neurosci 28, 101-107, 2005). I will discuss the mechanism of P2X4-evoked allodynia with an effect of a neurotrophic factor from activated microglia based on the latest our findings (Nature, in press). [J Physiol Sci. 2006;56 Suppl:S49]

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Brain-derived neurotrophic factor (BDNF) is known to involved in the development of spinal plasticity underlying inflammation-induced hyperalgesia. An injection of complete Freund adjuvant (CFA) into rat plantar surface produced hyperalgesia, which was significantly attenuated by intraperitoneal administration of anti-BDNF antiserum performed a day before and just after CFA. In vivo patch-clamp recordings from the spinal substantia gelatinosa (SG) neurons of the inflamed rats demonstrated a marked enhancement of excitatory synaptic responses to noxious and non-noxious stimuli, suggesting an increase in the activity-dependent synthesis and release of BDNF in the SG. In the spinal slice preparations, BDNF, but not nerve growth factor (NGF) or neurotrophin-3 (NT-3), acted presynaptically to increase frequency of miniature EPSCs in SG neurons of the inflamed, but not naive rats, through an activation of lidocaine-sensitive, TTX-resistant sodium channels. This effect was observed in slices of the inflamed rat only 2-4 days after CFA injection. On the other hand, the number of monosynaptic A-beta afferent inputs to the SG significantly increased a week after the onset of the inflammation, and this increase was significantly suppressed by treatment with anti-BDNF antiserum. These findings, taken together, suggest that BDNF, which is considered to be released from the sensitized primary afferents, increases the excitability of SG neurons through its action on the presynaptic terminals, and may thereafter trigger plastic changes in the spinal sensory transmission to develop hyperalgesia/allodynia during inflammation. [J Physiol Sci. 2006;56 Suppl:S50]

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Here I report the initiation mechanisms underlying neuropathic pain (NP) following nerve damages. In this experiment, we used partial sciatic nerve ligation (PSNL) to induce NP in mice. The abnormal chronic pain including hyperalgesia and tactile allodynia was observed 3 days after the PSNL and lasted for more than 2 weeks. We found many nociceptive fiber-specific changes in protein and/or gene expression of molecules involved in pain transmission. They include the novel expression of Ca channel alpha2-delta1 subunit in myelinated fiber DRG neurons, which may account for hyperalgesia. At the same time, we found the loss of function in unmyelinated fiber-mediated pain transmission. They include the down-regulation of substance P in dorsal horn of spinal cord. In addition to these phenotypic changes in the expression, we observed marked demyelination and remyelination in dorsal root, a phenomenon which is closely related to allodynia. All these changes were abolished in lysophosphatidic acid (LPA) receptor (LPA1) knock-out mice, and mimicked by the intrathecal single injection of LPA. In ex vivo culture system of dorsal root, the addition of LPA caused demyelination and sprouting/remyelination, which were accompanied with down-regulation of myelin basic protein expression. We also found that LPA causes microglia activation in the spinal cord. I will discuss how LPA plays the initiation role in many molecular events observed in PSNL-induced NP. [J Physiol Sci. 2006;56 Suppl:S50]

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It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. Here, we found that chronic pain caused a dramatic down-regulation of μ-opioid receptor function to activate its coupling with G-proteins of ventral tegmental area (VTA), and produced of the suppression of morphine-induced rewarding effect. Using the fluoro-gold (FG) microinjection into the VTA, numerous FG-labeled cells were detected in the lateral preoptic nucleus (LPO) and dorsolateral hypothalamus (DMH) of nerve-ligated rats. Subpopulations of β-endorphin-positive fibers in the LPO and DMH were co-labeled by FG. Furthermore, we found that chronic pain caused a dramatic down-regulation of cortical δ-opioid receptor function to activate its coupling with G-proteins, which is associated with the increased δ-opioid receptor phospholylation, and produced anxiety-like behaviors in mice, as characterized by both the light-dark and elevated plus-maze tests. These data provide direct evidence that the endogenous opioid-containing neuron projecting from the pain processing regions may be continuously activated by nerve ligation, resulting in the long-lasting down-regulation of μ- or δ-opioid receptors. This phenomenon may lead to the suppression of the morphine-induced rewarding effect and emotional disorders including aggravated anxiety under chronic pain-like state. [J Physiol Sci. 2006;56 Suppl:S50]

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To understand molecular mechanisms of learning and memory is one of major topics in neuroscience research. To identify genetic components underlying synaptic plasticity and memory processes, I have been so far focusing on analyzing behavioral phenotypes, especially learned fear, of several strains of genetically manipulated mice for plasticity-related genes. One example is Fyn-overexpressing mice exhibiting hyper-tyrosine-phosphorylation of the NMDA receptor (NR). These mice showed NR activity-dependent impairment of fear conditioning, suggesting that this tyrosine kinase is a key molecule that controls conditioned fear through NR phosphorylation. Another is knockout (KO) mice for drebrin A, a F-actin binding protein in the dendritic spines. Context-dependent fear conditioning and MK-801-induced hyperlocomotion were changed in these KO mice. These results suggest that drebrin A has a pivotal role in the regulatory mechanism of NR function. The mutant mice for ICER, a CRE-binding transcriptional repressor, also showed the phenotype on fear conditioning. In ICER-overexpressing mice, long-term retention of fear memory was impaired, while the short-term memory remained intact. ICER-KO mice conversely showed a better performance in the retention of conditioned fear. These results suggest that ICER acts as a negative regulator for memory consolidation. Thus, these three examples demonstrate that the learned fear is a good target of behavioral analysis of mutant mice for genes being critical for synaptic plasticity and it should be included in the behavioral test battery. [J Physiol Sci. 2006;56 Suppl:S51]

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