Ca²⁺ plays an important role in cell function. There are two mechanisms to regulate Ca²⁺ concentration inside the cell. One is through Ca² influx through Ca²⁺ channels on the plasma membrane. Another is Ca²⁺ release from internal store in side the cell. IP₃ is a second messenger to release Ca²⁺ from endoplasmic reticulum (ER) which is an intracellular store. We found that P400 protein deficient in Purkinje-neuron-degenerating mutant is IP₃ receptor (IP₃R). Studies on the role of IP₃R during development show that IP₃R is involved in fertilization and is essential for determination of dorsoventral axis formation. IP₃R is involved in neuronal plasticity. Double homozygous mutant of IP₃R2 and IP₃R3 shows deficit of saliva and pancreatic juice secretion. ERp44 works as a redox sensor in the ER and regulates IP₃R1 activity. We discovered that IP₃ not only releases Ca²⁺, but also releases IRBIT ( IP₃ receptor binding protein released with inositol trisphosphate). Since IRBIT binds to the IP₃ binding core in a phosphorylation dependent manner, IRBIT regulates IP3 induced Ca²⁺ release. In addition, IRBIT works as a third messenger to enchance pancreas type Na, Bicarbonate co-transporter 1 which regulates acid base balance of cells. And signaling pathway may be modified to be as follows: [signal →IP₃ →Ca²⁺ release] and [signal→IP₃ →IRBIT release →Na, Bicarbonate cotransporter 1 activation]. IP₃R is considered to work as a signaling center inside the cell by interacting with many molecules as “Calcio-signalsome”. [J Physiol Sci. 2007;57 Suppl:S2]

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Cell death occurs under physiological and pathophysiological conditions in response to a number of signals and stresses. Our recent studies have revealed that a number of mechanisms of cell death induction are based on physiological processes. Apoptotic cells exhibit whole-cell shrinkage, termed apoptotic volume decrease (AVD). Activation of volume-sensitive outwardly rectifying anion channel (VSOR) is coupled to the AVD induced by a mitochondrion- or death receptor-mediated apoptosis inducer. The regulatory volume increase (RVI) is impaired in apoptotic cells. The AVD event precedes cytochrome c release, caspase activation and DNA laddering. Even without stimulation by any specific apoptotic inducer, apoptotic cell death is induced under the conditions where persistent cell shrinkage is compelled by physical or physiological maneuvers. Prevention of AVD rescues cell death caused by an apoptotic inducer or ischemia-reperfusion. On the other hand, excitotoxicity, lactacidosis or acidotoxicity causes persistent cell swelling, termed necrotic volume increase (NVI), and necrotic cell death in neuronal, glial and epithelial cells. Impairment of the regulatory volume decrease (RVD) due to disordered VSOR activities is associated with NVI. Acidotoxicity leads to activation of a novel type of anion channel which serves as an anion influx pathway. Prevention of NVI or restoration of RVD rescues necrotic cell death. Thus, it is concluded that dysfunction of cell volume regulation and disordered activities of anion channels play indispensable roles in apoptotic, necrotic and ischemic cell death. [J Physiol Sci. 2007;57 Suppl:S2]

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Apoptosis is mediated by a cascade of caspases, which eventually activates a specific DNase (CAD). CAD^−/− cells do not undergo DNA fragmentation during apoptosis, but their DNA is degraded by DNase II in macrophages after they are engulfed. DNase II^−/− mice are embryonic lethal due to IFNΒ produced by macrophages carrying undigested DNA, indicating that mammalian DNA can activate innate immunity. Bacterial DNA activates innate immunity via a TLR-dependent system, but mammalian DNA does not use TLR to activate IFNΒ gene. When DNase II gene is inducibly inactivated after birth, they develop polyarthritis in an age-dependent manner. The mice carry numerous activated macrophages containing undigested DNA and produce TNFΑ. Administration of anti-TNFΑ blocks the development of polyarthritis, suggesting that TNFΑ produced by the macrophages is responsible to trigger the arthritis. Using the knowledge that DNA of apoptotic cells can be digested by macrophages, we established an assay for engulfment, and identified MFG-E8 that passes apoptotic cells to phagocytes. MFG-E8^−/− macrophages in germinal center show a defect in engulfment. MFG-E8^−/− mice produce antinuclear antibodies and suffer glomerulanephritis, confirming that if apoptotic cells are not efficiently engulfed, it will cause autoimmune diseases. Apoptotic cells expose phosphatidylserine (PS) on their surface in a caspase-dependent manner, and it is recognized by macrophages as an “eat me” signal. During erythropoiesis, nuclei are protruded from erythroid precursors, and engulfed by macrophages. We recently showed that nuclei protruded from erythroid precursors also expose PS on their surface. [J Physiol Sci. 2007;57 Suppl:S2]

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The discovery of leptin has led to the elucidation of a robust physiologic system that maintains fat stores at a relatively constant level. Leptin is a peptide hormone secreted by adipose tissue in proportion to its mass. This hormone circulates in blood and acts on the hypothalamus to regulate food intake and energy expenditure. When fat mass falls, plasma leptin levels fall stimulating appetite and suppressing energy expenditure until fat mass is restored. When fat mass increases, leptin levels increase, suppressing appetite until weight is lost. By such a mechanism total energy stores are stably maintained within a relatively narrow range. Recessive mutations in the leptin gene are associated with massive obesity in mice and some humans. Treatment with recombinant leptin markedly reduces food intake and body weight. The low leptin levels in patients with leptin mutations are also associated with multiple abnormalities including infertility, diabetes and immune abnormalities all of which are corrected by leptin treatment. These findings have established important links between energy stores and many other physiologic systems and led to the use of leptin as a treatment for an increasing number of other human conditions including a subset of obesity, some forms of diabetes including lipodystrophy and hypothalamic amennorhea, the cessation of menstruation seen in extremely thin women. Identification of a physiologic system that controls energy balance establishes a biologic basis for obesity and further establishes links between this system numerous other physiologic responses. [J Physiol Sci. 2007;57 Suppl:S2]

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Higher brain functions rely on functional units such as laminated structures and nuclei. To construct these structures, postmitotic neurons migrate from their sites of origin to their final destinations. Most CNS neurons are generated in the ventricular zone of the neural tube and then migrate radially towards the pial surface. Birthdate-dependent settlement followed by radial migration leads to the establishment of laminated structures such as the cerebral cortex. Neuronal migration also occurs in tangential directions. Although occurrence of tangential migration (TM) has been known for a long time, significance of TM has recently begun to be elucidated. A good example can be found in the cerebral cortex: here the excitatory neurons are born in the ventricular zone, and then migrate to the cortical plate (CP). On the other hand, inhibitory interneurons are generated in the basal forebrain and migrate tangentially to the CP. As a result, a structure with mixed excitatory and inhibitory neurons is established. TM of neurons also contributes to nucleogenesis. For example, neurons that are destined to form precerebellar nuclei originate from the lower rhombic lip, migrate circumferentially and then, they change the direction of migration from tangential to radial. These neurons eventually terminate radial migration at a distance from the pial surface to form nuclei (Kawauchi et al., 2006). Thus, nucleogenesis is a consequence of successive occurrence of TM and radial migration. Real time imaging further elucidates additional interesting features of TM. This will be shown with the aid of movies. [J Physiol Sci. 2007;57 Suppl:S3]

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In the central neuronal system, the synaptic strength changes dynamically, thereby playing pivotal roles in switching neuronal circuits. Despite a wealth of information accumulating on postsynaptic regulatory mechanisms, much less is known on the presynaptic mechanism, primarily because of small nerve terminal structures preventing direct electrophysiological approaches. The calyx of Held is a giant glutamatergic nerve terminal, which is visible in auditory brainstem slices, and allows one to address various questions on mechanisms operating in the presynaptic nerve terminal. In this talk, I'd like to present our recent results obtained using this preparation, with a particular focus upon presynaptic voltage-gated calcium channels (VGCCs). Facilitation and inactivation of presynaptic VGCCs contribute to dynamic changes in synaptic strength during high-frequency transmission, via modulating the probability of transmitter release. In addition, our results have revealed an unexpected role of the presynaptic VGCC, anchoring synaptic proteins involved in clathrin-coated synaptic vesicle endocytosis. Disruption of this interaction blocked endocytosis, as deduced from capacitance measurements. Thus, presynaptic VGCCs seem to play critical roles in both exocytosis and endocytosis of synaptic vesicles at the mammalian central synapse. [J Physiol Sci. 2007;57 Suppl:S3]

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It is well known that the systemic blood pressure(BP)is regulated by the baroreflexes of negative feedback mechanism, and its feedback loops consist of receptor regions, afferent pathways, coordinating center, efferent pathway and effectors (ie, heart and vessels). General understanding is that baroreceptor afferents firings increase in response to the increases in BP to mediate the short-term regulation of BP, and the receptors are mainly stretch sensitive. The aortic nerve fibers (ANs), which are located in the aortic arch, sense hemodynamic changes and send this information to the brain. In other words, the aortic baroreflex works in accordance with the alterations of several factors (eg, heart rate, stroke volume, intramural pressures), to maintain adequate BP and blood distribution. The hydrostatic pressure gradient due to the gravitational force in vessels disappear under weightlessness. We hypothesized that the range of alterations of BP caused by individual behavior narrows under microgravity in space, and it reduces the proportion of high-threshold unmyelinated ANs, which discharge at the higher BP level, and leads the lower baroreflex sensitivities. We studied the baroreflex mechanisms and its development in the young rats with dam, which flew on space shuttle for 16 days (Neurolab, 98, Shimizu et al.), and verified that these hypotheses were collect and reported the several findings. I would like to summarize these findings including the results from ground-based studies and discuss the future development of research for the regulation of cardiovascular system from the gravitational point of view. [J Physiol Sci. 2007;57 Suppl:S6]

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Gravity acts on the circulatory system to alter hydrostatic pressure gradient and then causes fluid shift; this could alter venous return and cardiac output, followed by an alteration of arterial pressure (AP). These hemodynamic changes have been thought to be mainly corrected by the baroreflex. However, recently we have demonstrated that the vestibular system has a significant role in maintaining AP during hypergravity. In conscious rats, AP decreases during hypergravity, if both the vestibular system and baroreflex are absent, probably due to blood redistribution. If the vestibular system but not the baroreflex operates, AP increases markedly, suggesting that the hypergravity is detected by the vestibular organ, which reflexively increases AP. This increase is suppressed, if the baroreflex operates. Thus, the vestibular system acts as a feedforward AP controller against hypergravity. The AP increase observed in the intact rats indicates that AP is overcompensated rather than compensated by the vestibular system. While feedforward control has the advantage of a short response delay, the major disadvantage is the instability of the response and the large correct error. Comparison of the AP responses between intact and baroreflex lesioned rats suggests that the overcompensated AP is compensated by the negative feedback control system, the baroreflex. The vestibular system is known to be highly plastic, i.e., the sensitivity of the vestibular system alters if subjects are maintained in a different gravitational environment. The plasticity of this system and the role of this system upon posture change in human will be discussed. [J Physiol Sci. 2007;57 Suppl:S6]

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The lymphatic system is a quite important part of microcirculation. The main function of the system is a drainage system of body fluid that leaks out of blood capillaries into the tissues, especially plasma protein, in the physiological condition. Also, the lymphatic system is a route of lymphocytes recirculation and metastasis of carcinoma cells. Thus to understand regulatory mechanisms of lymphatic circulation helps us investigate the function of microcirculation, immunology and oncology. In humans and animals, smooth muscles of the lymph vessels exhibit spontaneous contractile activity. Therefore the lymph vessels contribute to the active lymph transport mechanisms by the spontaneous lymphatic pump. We have demonstrated that ATP-sensitive K+ channels play crucial roles in mechanical activity of lymphatic smooth muscles and that endothelium-derived substances such as nitric oxide and prostanoids participate in the regulation of lymphatic pump activity. In addition, we have recently reported that lymphatic endothelium is a barrier against small sized-hydrophilic substances. In this symposium, we will demonstrate our current studies of the lymphatic functions indicating pump activity and permeability of lymph vessels, and then discuss the meaning and significance of lymphatic microcirculation in the cardiovascular physiology and pathophysiology. [J Physiol Sci. 2007;57 Suppl:S6]

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Besides secreting vasoactive substances such as nitric oxide and endothelin, vascular endothelial cells control the vascular tone through affecting the membrane potential of smooth muscle cells. Actually the vessel diameter is sensitive to the membrane potential. The smooth muscle and endothelial cells are electrically coupled with the myoendothelial gap junctions and they share a membrane potential and an intracellular ionic environment. In fact, the resting membrane potentials of both cells are quite similar, being around -50 mV, with the smooth muscle cells slightly more negative than the endothelial cells. Once separated from the smooth muscle cells, the endothelial cells cannot maintain their membrane polarization. So, two kinds of cells are functioning as a unit in terms of the membrane potential, and irrespective of which kind of cells is stimulated, the membrane potentials are changed in both kinds of cells. For example, ACh increases endothelial [Ca²⁺]_i, which in turn activates charybdotoxin-sensitive IK_Ca, apamin-sensitive SK_Ca and Cl_Ca channels in the endothelium. Resulting membrane responses vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization, and a variability in [Cl⁻]_i may be one of the reasons for the variations. [J Physiol Sci. 2007;57 Suppl:S7]

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The avascularity of cardiac valves is abrogated in several valvular heart diseases. This study investigated the molecular mechanisms underlying valvular avascularity, and its correlation with valvular heart diseases induction. Chondromodulin-I, an anti-angiogenic factor isolated from the cartilage, is abundantly expressed in normal murine, rat and human cardiac valves. It is first detected at developmental stage E9.5 in the left ventricle, outflow tract and valvular primordium, but is restricted to cardiac valves from late embryogenesis to the adult. Gene targeting of chondromodulin-1 resulted in enhanced VEGF-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography revealed aortic valve thickening, calcification and turbulent flow signifying early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation, mobilized endothelial cells and induced their apoptosis, and these effects were partially inhibited by chondromodulin-1 siRNA. In both ApoE^–/– mice and human valvular heart diseases, including infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in the areas of chondromodulin-1 down-regulation. These findings provide evidence that chondromodulin-I plays a pivotal role in maintaining valvular normal function by preventing angiogenesis, thickening and calcification that may lead to valvular heart diseases. [J Physiol Sci. 2007;57 Suppl:S7]

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The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals, which were immunopositive for vesicular glutamate transporter (VGluT) 2, were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. In contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both the compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both the compartment. Since axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment. We further demonstrate the output pattern originating from patch and matrix compartments, using single cell tracing with the recombinant Sindbis virus. [J Physiol Sci. 2007;57 Suppl:S7]

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It is known that μ opioid receptor (MOR) is located on the dendrite of striosomal medium-spiny (MS) projection neurons, and glutamatergic and non-glutamatergic presynaptic terminals innervating the MS neurons. The objective of this study was to investigate the MOR-mediated effects on the excitatory and inhibitory synaptic transmission in the striatal striosome/matrix compartment. To identify the striosomes, we used a transgenic mouse strain (TH-GFP mouse) harbouring an eGFP reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. Because dopaminergic neurons densely innervate the striosomal cells in early postnatal stage, a striosome is identified as a bright area under fluorescence microscope. Using corticostriatal slices obtained from TH-GFP mice (P14-P28), we made whole-cell recordings from MS neurons. DAMGO, an agonist of MOR, significantly suppressed GABAergic IPSCs in the striosomes (-18.4%±3.6%), whereas DAMGO had no effects in the matrix (+3.1%±5.5%). On the contrary, the effect of DAMGO on EPSCs in the striosomes was identical to that in the matrix. The suppression of IPSCs was also observed in cholinergic interneurons located near the striosomes, therefore MOR-mediated suppression of IPSCs might affect the release of endogenous acetylcholine. The MOR-mediated effects on synaptic transmission may control the activity of local neural circuits in striosome/matrix compartment. [J Physiol Sci. 2007;57 Suppl:S8]

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Recent studies suggested involvement of the striatum in decision-making and action selection. Striatal projection neurons receive action-related inputs from the cerebral cortices, reward prediction error signal from dopamine neurons and signals from cholinergic interneurons in the striatum. To study the signals conveyed by the striatal cholinergic neurons, projection neurons and midbrain dopamine neurons during decision-making and action selection, we recorded 265 tonically active neurons (TANs; presumed cholinergic interneuron) and 239 phasically active neurons (PANs; presumed projection neuron) from 2 macaque monkeys. After depressing a start button, the monkeys chose 1 of 3 target buttons with correct rates at 1st, 2nd, 3rd and repetition trials of 33, 50, 85 and 95%, respectively. Correct choices were followed by beep sound and reward water. 175 TANs responded to the reinforcer beeps after 1st, 2nd, 3rd choices with similar magnitudes, but showed almost no responses at the repetition trial. 18 of 147 PANs responding to the beeps showed the discharges varied with the reward probability. Other 18 PANs discriminated trials during repetition epoch from those during trial and error epoch. The reinforcer beeps evoked reward prediction error signals in dopamine neurons in other 3 monkeys. Our results suggested unique participations of cholinergic and dopamine signals in the striatal mechanisms for decision and action selection. [J Physiol Sci. 2007;57 Suppl:S8]

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The membrane phospholipid phosphatidylinositol phosphates (PIs) are critical signal transducer in eukaryotic cells. Deficient mice in leucin-rich phosphoiositide phosphatase (L-PIPase) showed involuntary movements that were ameliorated by an NMDA-receptor antagonist, MK-801. Enhancement of gliosis and apoptosis were observed in the striatum of the L-PIPase deficient mice. Whole-cell recordings were carried out in the slice preparations obtained from 15-20 day-old PIPase deficient and wild type mice. The resting membrane potential of both medium spiny neurons and cholinergic interneurones in the striatum of L-PIP deficient mice was less hyperpolarized than that of wild type mice. In the cholinergic interneurones of L-PIPase deficient mice, spontaneous firing rate was larger. In addition, NMDA receptor-mediated components of the synaptic currents were enhanced in the medium spiny neurons of L-PIPase deficient mice. These findings suggest that abnormalities in NMDA receptor-mediated synaptic transmission in the striatum could result in the involuntary movements observed in L-PIP deficient mice. [J Physiol Sci. 2007;57 Suppl:S8]

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Endocannabinoids (eCBs) are released from postsynaptic neurons, act retrogradely onto presynaptic cannabinoid CB1 receptors and cause transient suppression of transmitter release in various regions of the brain. The striatum contains a moderate level of CB1, and its function is regulated by the activity of cholinergic interneurons. In the present study, we examined how eCBs signaling interacted with the cholinergic system and modulated synaptic transmission in the striatum. We made whole-cell recording from medium spiny (MS) neurons and recorded inhibitory postsynaptic currents (IPSCs) in striatal slices from C57BL/6 mice (P15-21). We found that mAChR agonist triggered eCB-mediated suppression of IPSCs, and potently enhanced depolarization-induced, eCB-mediated suppression of IPSCs (DSI). Both effects were blocked by an M₁-preferring antagonist, pirenzepine, and by postsynaptic infusion of GDP-β-S, and were absent in M₁ knockout mice, indicating that postsynaptic M₁ receptor is required. Magnitude of DSI was significantly reduced by the suppression of cholinergic interneuron activity, whereas it was enhanced by inhibiting choline esterase. These results indicate that depolarization-induced eCB release from MS neurons is persistently upregulated by ambient ACh through M₁ receptors. [J Physiol Sci. 2007;57 Suppl:S8]

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Neuropathic pain, a highly debilitating chronic pain following nerve damage, is a reflection of the aberrant functioning of a pathologically altered nervous system. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. While the dominant theme in research on neuropathic pain has been to understand the roles of neurons in the peripheral nervous system and the dorsal horn, there is a rapidly growing body of evidence indicating that glia, especially microglia, in the spinal cord are activated following nerve injury. Here we will present recent evidence that activated spinal microglia are the cause and key cellular intermediaries in the pathogenesis of neuropathic pain. In activated microglia, P2X₄ receptor (P2X₄Rs), a ligand-gated cation channel activated by ATP, is a required molecular mediator. P2X₄Rs in the spinal cord are upregulated exclusively in activated microglia after nerve injury. Blocking P2X₄Rs leads to a reversal of neuropathic pain. We will also show recent findings that fibronectin, an extracellular matrix protein, causes an increase in the expression of P2X₄Rs in microglia and that P2X₄-stimulated microglia cause a release of brain-derived neurotrophic factor (BDNF) that mediates signaling between activated microglia and pain-transmission dorsal horn neurons. Understanding of how P2X₄Rs are expressed and activated in spinal microglia following nerve injury may lead to new strategies that may aid in the management of neuropathic pain. [J Physiol Sci. 2007;57 Suppl:S9]

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Using organotypic slice cultures, mechanisms for the glial cell activation during neuronal or tissue injury were investigated. Cortico-striatal slice cultures were prepared from postnatal day 2-3 Wistar/ST rats and cultured for 10-11 days. NMDA treatment (50 μM, 3h), which led to severe neuronal damages, induced MCP-1 production in astrocytes. MEK inhibitor U0126 and JNK inhibitor SP600125 significantly inhibited NMDA-induced MCP-1 production without affecting the NMDA-induced neuronal injury. On the other hand, p38 MAP kinase inhibitor SB203580 did not have any significant effects on NMDA-induced MCP-1 production. Immunostaining revealed that transient ERK phosphorylation was observed in neurons at early phase of the NMDA treatment, followed by the long-lasting ERK phosphorylation in astrocytes starting from 30 min after the administration of NMDA treatment. The delayed application of U0126 even at 3h after the end of NMDA treatment significantly suppressed MCP-1 production. These results suggest that NMDA-evoked neuronal injury induces MCP-1 production via the long-lasting activation of a MEK-ERK cascade in astrocytes. Next we examined the mechanism for the activation of microglial cells by acute tissue injury. Hippocampal slice cultures were prepared from Iba I-EGFP mice (postnatal day 6-7) and cultured for 10-11 days. Following tissue injury, microglia extended their processes toward the sites of tissue damage. This extension was significantly suppressed by apyrase, a hydrolyzing enzyme for ATP/ADP, suggesting the involvement of purinergic receptors. [J Physiol Sci. 2007;57 Suppl:S9]

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Glial cells are now recognized as important neuronal partners to regulate various brain functions. They release so-called "gliotransmitter" ATP or other nucleotides that control functions of neighboring cells including neurons, glial cells and vascular cells. In the present study, we show that astrocytes release nucleotides, by which they show both dynamic and static control of neuronal functions. Astrocytes release ATP in response to various stimuli or even spontaneously. Thus, astrocytes or adjacent cells including neurons receive ATP/P2 receptor-mediated signals in a tonic fashion. When incubated with P2 receptor antagonists or the ATP degrading enzyme apyrase, they alone dynamically affect neuronal activities. In addition, when incubated with these antagonist or apyrase for longer periods, expression of neurotransmitter receptors such as M1 muscarinic acetylcholine receptors in neurons were affected, which was totally dependent upon astrocytic nucleotides. Thus, astorocytes cause both dynamic and sustained change in neuronal activities by releasing gliotransmitter nucleotides. Astrcotyes also release uridine nucleotides that regulate vasculatures and microglial functions. We also discuss to differences between adenine and pyrimidine nucleotides for regulation of brain functions. Such wide-ranging regulation by astrocytic nucleotides may explain fine-tuning of complex brain functions. [J Physiol Sci. 2007;57 Suppl:S9]

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Most of the pre- and postsynaptic structures in the central nervous system are in close apposition to astrocytic processes, making it likely that the molecules expressed on the perisynaptic membranes mediate communication between neurons and astrocytes. In the nucleus of the solitary tract (NTS), extracellular ATP activates presynaptic P2X receptor channels (P2X), the Ca²⁺ entry through which facilitates glutamate release and initiates synaptic transmission in the absence of action potentials (Kato & Shigetomi, 2001; Shigetomi & Kato, 2004). Our hypothesis is that these presynaptic P2X receptors mediate the presynaptic action of the gliotransmitter ATP released from "synaptic processes" of the astrocytes. Immunohistochemical and electron microscopic analyses revealed a large number of GFAP-positive astrocyte processes surrounding NTS neurons and in close appositions of astrocyte processes and the presynaptic structures. Application of ATP in the vicinity of dendritic synapses in time- (–hundreds milliseconds) and space- (3 micrometers) delimited manners using laser-based photolysis of caged ATP in the brainstem slices resulted in an immediate release of glutamate. In a subset of small NTS neurons showing high-frequency spontaneous miniature EPSCs (mEPSCs), perturbation of glial ATP production with fluoroacetate immediately resulted in a sustained decrease in the mEPSC frequency. These data argue for a potential role of presynaptic P2X-Rs as an interface between astrocyte-derived ATP and initiation of the synaptic transmission without presynaptic excitation. Supported by MEXT, Japan 17300123 & 18053022. [J Physiol Sci. 2007;57 Suppl:S10]

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A wide variety of evidence has indicated that a high level of D-serine occurs in the mammalian brain. D-serine is predominantly concentrated in the forebrain, where the NMDA receptors are enriched. Because D-serine potentiates the NMDA receptor-mediated transmission by selective stimulation of the glycine site of the NMDA receptor, D-serine has been proposed as an endogenous coagonist for the NMDA receptor-associated glycine site in the mammalian brain. Serine racemase (SRR) that catalyzes the direct formation of D-serine from L-serine and D-amino acid oxidase (DAO) that catalyzes the oxidative deamination of neutral D-amino acids have been cloned from the mammalian brain. Several lines of evidence have demonstrated that the distribution of SRR and those of the D-serine and NMDA receptors share a similar regional pattern, whereas a regional distribution of DAO inversely correlates with both those of D-serine and SRR. Immunohistochemical studies at a cellular level have demonstrated that SRR and DAO were localized exclusively in GFAP-positive astrocytes. Recently, Wolosker's group and we revealed, however, the presence of D-serine and SRR in not only astrocytes but also neurons of rat brain. Because D-serine has been shown to improve the negative, positive and cognitive symptoms of schizophrenic subjects treated with conventional neuroleptics and because SRR and DAO have recently shown to be associated with the susceptibility to schizophrenia, SRR and DAO could play an important role in the regulation of the NMDA receptors via the D-serine metabolism and in the pathogenesis of schizophrenia. [J Physiol Sci. 2007;57 Suppl:S10]

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Prominent electron dense projections such as synaptic ribbons are observed at presynaptic terminals of sensory neurons that release transmitter tonically. These characteristic structures are thought to tether synaptic vesicles at the sites adjacent to active zones and to increase the size of the readily releasable pool. However, functional significance of prominent electron dense projections is not yet known. Applying total internal reflection fluorescence (TIRF) microscopy to goldfish retinal bipolar cell terminals, we visualized Ca²⁺ entry sites, synaptic ribbons, and vesicle fusion sites. Ca²⁺ entry sites were colocalized with ribbons. Depolarization-induced vesicle fusion occurred immediately around ribbons and then sustainedly at ribbon-free sites. Activation of protein kinase C (PKC), which is known to potentiate the delayed sustained component of transmitter release, specifically increased the number of vesicle fusion events at ribbon-free sites. Electron microscopy revealed that PKC activation selectively increased the number of docked vesicles at ribbon-free sites, which faced the neuronal processes with the postsynaptic density. Ribbon-associated and ribbon-free active zones were nearly equal in number in the Mb1 bipolar cell terminal. Our results suggest that the ribbon and ribbon-free synapses may transmit transient and sustained signals, respectively. [J Physiol Sci. 2007;57 Suppl:S10]

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In the mammalian retina, rod signals from rod bipolar cells are transferred to the cone circuit via AII amcrine cells. The AII amacrine cells make sign-conserving synapses, via gap junctions, with ON-cone bipolar cells at the arboreal dendrites and glycinergic sign-inverting synapses with OFF-cone bipolar cells at the proximal dendrites. It has been reported that AII amacrine cells generate TTX-sensitive repetitive spikes of small amplitude. Using in situ hybridization, we found the expression of Na_v1.1 (TTX-sensitive voltage-gated Na⁺ channel α-subunit) mRNA in AII amacrine cells, while the localization of Na_v1.1 protein remains unclear. We found that the spike frequency increased in proportion to the concentration of puffer-applied glutamate to the arboreal dendrite and was suppressed by the puffer-applied L-AP-4, a glutamate analogue that hyperpolarizes rod bipolar cells, to the outer plexiform layer. Therefore, it is most likely that the spike frequency generated by the AII amacrine cells is dependent on the glutamatergic input from rod bipolar cells. Puffer-application of TTX to the proximal dendrite of AII amacrine cells blocked the Na⁺ current significantly compared with the application to other regions, indicating that the voltage-gated Na⁺ channels were mainly localized around the proximal dendrites. It is possible that the spikes contribute to the OFF-pathway by promoting release of glycine from the proximal dendrites in response to light intensity in scotopic vision. [J Physiol Sci. 2007;57 Suppl:S11]

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The retina divides visual information into ON- and OFF signals, and processes each signal with independent subsets of neurons in parallel. So far no difference between the ON pathway and the OFF pathway has been found for neurotransmitter functions. We have previously reported that the immunoreactivity for P2X2-purinoceptors of cholinergic amacrine cells is OFF-pathway-specific in the mouse retina. We therefore examined if ATP has different functions between the ON pathway and the OFF pathway using the patch clamp technique and the multi-electrode recording system. Only the OFF-type, but not the ON-type, cholinergic amacrine cells responded to ATP vigorously. This response was mediated by the P2X2-purinoceptors. Furthermore, PPADS, a P2X-purinergic receptor antagonist, activated the tonic phase firing of the OFF-type ganglion cells, but not of the ON-type ganglion cells. Thus P2X-purinergic receptors selectively modulate OFF-pathway in the mouse retina. [J Physiol Sci. 2007;57 Suppl:S11]

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Neurons are often assumed to encode only one time-varying quantity, e.g. similarity between visual input and receptive fields, concentration of specific odorants, distance from specific locations, etc. However, when a neuron generates N spikes, there are N-1 interspike intervals. If these interspike intervals are on time scale much shorter than that of input modulation, they could, theoretically, encode up to N-1 quantities characterizing the input waveform. Here we show that the vertebrate retina performs such coding. In response to various visual inputs, retinal ganglion cells generate highly reproducible spike bursts, characterized by clusters of spikes separated by hundreds of milliseconds of silence. We asked if spike patterns, i.e., combinations of interspike intervals within these bursts, carry information about visual input. With multielectrode recoding from the retinas of salamanders and mice, we found that a subset of ganglion cells generate bursts with various spike patterns, which are unique to the preceding visual inputs. Surprisingly, when single bursts contain three spikes, the two interspike intervals nonlinearly encode two independent components of the preceding light intensity waveforms. Although these spike patterns are on time scales of a few milliseconds, they encode visual input as long as 400 milliseconds. Recordings from LGN slices indicated that the retinogeniculate synapses are reliable enough to transmit the information. We conclude that single ganglion cells encode multiple quantities in the form of burst spike patterns, and reliably transmit them to the brain. [J Physiol Sci. 2007;57 Suppl:S11]

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Retina receives centrifugal projection from the brain, and it is suggested that retinal function is somehow modulated, depending on the internal state of the brain. In birds, it is known that (1) most neurons in the isthmo-optic nucleus (ION) send their axons to the contralateral retina, (2) the retinopetal projection is organized topographically and (3) activity of isthmo-optic (IO) neurons enhances visual responses of the retinal ganglion cells. To clarify the functional significance of this pathway, we recorded spikes of IO neurons in awake Japanese quails with unrestrained heads using an implanted electrode assembly, while recording head movements with a high-speed video camera. The IO neurons fire passively by applying visual stimuli to their receptive fields and voluntarily without visual stimuli or eye-head movements. Voluntary activity was observed not only in the middle of eye-head fixation but also during about 200 ms before the onset of head saccades. The intensity of the activity before the onset of head saccades depended predominantly on the direction of motion in which the head saccades is initiated. That is, the IO neurons showed a large phasic elevation of activity before the onset of head saccades toward their receptive field, but substantially less response before the onset of head saccades in other directions. The IO neurons may convey signals reflecting covert spatial attention, which precedes overt attention shifts, to the retina, and that retinal local output may be enhanced by centrifugal signals before gaze shifts in a topographical manner specific to the direction of motion. [J Physiol Sci. 2007;57 Suppl:S11]

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1. Kyozo Koketsu (1922-): Dr. K. Koketsu first suggested the role of Ca ions in the cell membrane excitation and founded the general concepts of slow synaptic transmission and modulation in the nervous system. He guided and influenced many scholars all over the world and was the first among many students of Sir J.C. Eccles, who later established neurophysiology in Japan. 2. Hiroshi Irisawa (1922-1991):The outstanding achievement of Dr Irisawa is that he opened up the way to elucidating the mechanism of cardiac automaticity by applying the voltage clamp technique to sinoatrial node cells. Since then, through his tireless effort, he provided not only a vast amount of information on S-A node automaticity but also on that of other ion channels and transporters in the cardiac cells. He devoted himself until the last day of his life to cardiac cell physiology by carrying out experiments as well as encouraging young people and setting up places for the international exchange of scientific opinions. [J Physiol Sci. 2007;57 Suppl:S12]

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The aim of this talk is to discuss the reflex control of the human masticatory system and put forward a method for standardized investigation. To standardize the stimulation protocol, we need make sure that we stimulate peripheral receptors one receptor system at a time with minimal activation of other receptors. This stimulation should also be done in an efficient and reproducible way. It is also necessary to standardize the method for recording and analysing the response of the motoneurons to the stimulation of each receptor system. For that, a new technique is introduced and its advantages over the currently used methods are discussed. The new technique uses the changes in discharge rate of motor units in response to a stimulus to illustrate the synaptic potential that is induced in the motoneuron. With the delivery exact stimuli to individual receptor systems during various stages of mastication, and the use of the new frequency-based method to analyse synaptic connections between stimulated afferents and motoneurons, it is now possible to provide important new knowledge on the control of human mastication by the peripheral feedback. This knowledge is necessary in order to understand the functional connectivity of the jaw muscles and it will form the basis for improving current methods in diagnosing and treating painful disorders of the trigeminal region. Supported by the National Health and Medical Research Council of Australia. [J Physiol Sci. 2007;57 Suppl:S12]

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It has been known that masticatory movement, especially during chewing, is controlled by the brain stem. However, as mastication is a sequential action from food intake to swallowing, the higher brain, such as the cerebral cortex and basal ganglia, is thought to be needed for the control of mastication. In the cerebral cortex, the cortical masticatory area (CMA), to which repetitive electrical stimulation induces rhythmic jaw movements like masticatory movements, may be involved in the control of mastication. In the rabbit, the CMA is divided into two parts, based on induced jaw movement patterns. One is the part of CMA in which a jaw movement pattern (T-pattern) similar to food-transporting movement in natural mastication is evoked by electrical stimulation. The other part of the CMA is more dorso-medially located, and another jaw movement pattern (C-pattern) similar to chewing movement can be induced. What is the functional difference between two parts of the CMA? When we investigated the cortico-striatal pathway from two parts of the CMA in the rabbit, while the ventral part of the putamen receives the input from both parts, the input from the T-pattern inducing CMA was broader than that from the C-pattern inducing CMA. Recently, we also found that there were two parts of the CMA in the guinea pig as in the rabbit, and the distribution of the terminals of the cortico-cortical projections from these parts was different. From these findings, it is suggested that two parts in the CMA have different effects not only on the brain stem but also on the neuronal network in the higher brain. [J Physiol Sci. 2007;57 Suppl:S13]

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Swallowing is known to be one of the most complex reflexes in the body. It takes within a second for the food bolus to be propelled from the oral cavity through the oropharynx to the esophagus. The complexity may be partially caused by the fact that it can be evoked not only reflexively, but also voluntarily under the higher center control. In the past, it was believed that the swallowing event was critically divided into three, i.e., oral, pharyngeal and esophageal stages depending on where the bolus flew. Recently, Dr. Palmer and his colleagues showed that food passed into the oropharynx before swallowing during natural mastication. A videofluorographic study of feeding in humans showed that triturated food was routinely transported through the pillars of the fauces and the bolus was formed on the posterior surface of the tongue. What still remains unclear is the mechanism that coordinates the chewing and swallowing movements to complete the deglutition and that prevents aspiration of the forming oropharyngeal bolus. On the other hand, the swallowing can be modulated by the sensory feedback, such as the consistency or volume of the bolus, to handle the food bolus. In this symposium, kinematographic and electromyographic evaluation of swallowing function in humans and animals will be discussed. [J Physiol Sci. 2007;57 Suppl:S13]

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L-type Ca²⁺ channel serves as a primary step in the regulation of Ca²⁺ signaling in cardiac E-C coupling. We have shown in cardiac myocytes that L-type Ca²⁺ channels serve as a sensor and regulator of the SR Ca²⁺ content via CICR-dependent CDI (Ca²⁺-dependent inactivation) of L-type Ca²⁺ channels to ensure the efficacy of I_Ca to trigger CICR during APs. In atria, the down-regulation of L-type Ca²⁺ channels, in response to excess mechanical and electrical stimuli, is involved in arrhythmia such as atrial fibrillation. Two types of L-type Ca²⁺ channel pore-forming α subunits, Ca_V1.2 and Ca_V1.3, are expressed in atria. Aiming at elucidating the molecular mechanism of the regulation of atrial L-type Ca²⁺ channels, we studied the gating properties and their regulatory mechanisms of Ca_V1.2 and Ca_V1.3. The voltage-dependence of activation and inactivation of Ca_V1.3, were shifted to lower voltages, compared to those of Ca_V1.2, by approximately -15 mV. The voltage-dependent inactivation kinetics of Ca_V1.3 was slower than that of Ca_V1.2. Such voltage-dependence and kinetics of inactivation of Ca_V1.3 may contribute to maintain the threshold and duration of pacemaker action potential in atrial myocytes. The critical molecular region and the molecular complex involved in the unique gating properties and the regulation of atrial L-type Ca²⁺ channels, with respect to their roles in the fine-tuning of Ca²⁺ signaling, will be further discussed. [J Physiol Sci. 2007;57 Suppl:S13]

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L-type Cav1.2 Ca²⁺ channel is regulated by Ca²⁺-dependent facilitation (CDF) and inactivation (CDI). Although calmodulin (CaM) has been suggested to mediate both CDI and CDF, CaM-dependent protein kinase II (CaMKII) is also suggested to play an important role in CDF. In this study, we examined the roles of Ca²⁺, CaM and CaMKII in CDF and CDI using patch-clamp method in guinea-pig cardiomyocytes, in which run-down of the channel was controlled. Application of CaM (0.1-14 μM) + ATP ( [Ca²⁺]i <10 nM) to the intracellular side of the channels, within 1 min after patch excision, dose-dependently produced channel activity up to 250% of control. The relationship between [CaM] and channel activity was bell-shaped with a peak at ∼[CaM] 3 μM. Increasing [Ca²⁺]i (–500 nM) shifted the curve toward lower [CaM]. Thus, at a fixed [CaM] (e.g. 0.5μM), Ca²⁺ showed biphasic effects: facilitation at [Ca²⁺]i <500 nM, and inactivation at [Ca²⁺]i >500 nM. This Ca²⁺-dependent effect of CaM was considered to represent CDF and CDI and was not affected by protein kinase inhibitors KN-62 and K252a. The effects of CaM were attenuated, however, when the run-down time of the channels was >5 min. Application of CaMKII together with CaM + ATP, during the run-down period, restored the susceptibility of the channel to the modulation by CaM. These results suggested that CaM plays a key role in CDF and CDI, and that CaMKII and [Ca²⁺]i modulate the effect of CaM. [J Physiol Sci. 2007;57 Suppl:S14]

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Ca²⁺ dependent inactivation of L-type Ca²⁺ is well known phenomena. In this study, we would like to see the effect of NCX on ICaL kinetics by the change of subsarcolemmal Ca²⁺. We used the isolated cardiomyocytes in main pulmonary vein of rabbit and applied the whole cell voltage clamp techniques. In the presence of 0.1 mM EGTA in pipette solution, we applied a step depolarization prepulse from -40 mV to 10 mV for 2.5 msec to induce Ca²⁺ release from SR. And then, the test pulse of the same amplitude was applied. As the interval between the prepulse and the test pulse was increased, the inactivation time constants (taus) were decreased. In the absence of extracellular Na⁺, the taus were decreased further. The peak current amplitude was decreased at the first test pulse and became increased as the interval was increased to the control level. In the absence of extracellular Na⁺, the peak current decreased much larger and it increased much slowly. In the presence of BAPTA, ryanodine or thapsigargin, these phenomena were disappeared. SEA0400, a blocker of NCX, did not inhibit Ca²⁺ currents in the presence of BAPTA 10 mM but EGTA 0.1 mM. From these results, the release of SR Ca²⁺ could induce the rapid inactivation of L-type Ca²⁺ channels. Na⁺-Ca²⁺ exchange seems to be important to modulate L-type Ca²⁺ channel amplitude and inactivation kinetics by reducing subsarcolemmal Ca²⁺. SEA0400 was donated from Taisho Pharmaceutical Co. Ltd. This work was supported by the grant (No. R01-2004-000-10374-0) from KOSEF. [J Physiol Sci. 2007;57 Suppl:S14]

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Ca²⁺-dependent facilitation and inactivation of Cav2.1 channels modulate presynaptic P/Q-type Ca²⁺ currents. This dual feedback regulation by Ca²⁺ involves calmodulin (CaM) binding to the α1 subunit (α12.1). The molecular determinants for Ca²⁺-dependent modulation of Cav2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of α12.1, the CaM binding domain (CBD) and the IQ-like domain. In SCG neurons synapses transfected with brain-derived Cav2.1 channel and its mutants, Ca²⁺-dependent short-term synaptic plasticity, paired-pulse facilitation (PPF) and depression (PPD), augmentation and potentiation, was examined in the presence of Cav2.1 channels blocker, ω-conotoxin GVIA. PPF was completely prevented by alanine substitution of the first two residues of the IQ-like domain (IM-AA). In contrast, PPD was prevented by depletion of CBD (δCBD). Augmentation was reduced by IM-AA but not by δCBD. Potentiation was affected by neither IM-AA nor δCBD. These findings support a model in which Ca²⁺ binding to the C-terminal EF-hands of pre-associated CaM initiates facilitation of Cav2.1 channels via interaction with the IQ-like domain and initiates inactivation of Cav2.1 channels via interaction with CBD. This multifaceted mechanism allows positive and negative regulation of Cav2.1 channels in response to local Ca²⁺ increases. The Cav2.1 channel activity regulation, instead the residual Ca²⁺ at the active zone, is underlining the PPF, PPD and augmentation phenomena. [J Physiol Sci. 2007;57 Suppl:S14]

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Calmodulin (CaM) demonstrates biologically critical regulation of Ca_V1-2 Ca²⁺ channels. This talk explores three aspects of the powerful Ca²⁺ decoding that CaM exhibits in this context. (1) We summarize the current model of CaM/channel modulation, wherein Ca²⁺-free CaM (apoCaM) preassociates with channels, and subsequent Ca²⁺ influx drives conformational changes that induce regulation. Ca²⁺ binding to the individual lobes of CaM autonomously trigger different forms of modulation. The N-lobe usually responds to Ca²⁺ entry through sources other than the channel being regulated (global Ca²⁺ preference); the C-lobe favors Ca²⁺ entering via the regulated channel itself (local Ca²⁺ preference). (2) We describe a novel module that transforms the local/global Ca²⁺ preference of regulation. This module furnishes a vital clue (or 'Rosetta stone') for Ca²⁺ decoding. To start, Ca_V2.2 channels exhibit N-lobe Ca²⁺-dependent inactivation (CDI) with a global Ca²⁺ preference. Switching a small segment of Ca_V1.2 or Ca_V1.3 into Ca_V2.2 produces chimeric channels where CDI now has a local preference. Conversely, Ca_V1.3 channels exhibit an N-lobe CDI with a local preference, and deletion of this same portion within Ca_V1. 3 switches the Ca²⁺ preference from local to global. The key element of this Rosetta module is a Ca²⁺/CaM binding site. (3) This Rosetta module implicates a simple decoding mechanism wherein Ca²⁺ preference is specified by the ratio of channel affinities for Ca²⁺/CaM versus apoCaM. Indeed, the transforming actions of the CaM-binding Rosetta module satisfy stringent predictions of this new mechanism. [J Physiol Sci. 2007;57 Suppl:S14]

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Netwrok-forming interstitial cells with c-Kit-immunoreactivity are present in the myenteric plexus of the gut. From the histological resemblance these cells are referred to as interstitial cells of Cajal (ICC), and are now believed to act as pacemaker cells (1,2). Pacemaker potentials generated from ICC are synchronized with phase shifts over several hundred micrometers (3). Ca²⁺-dependent plasmalemmal ion channels periodically activated in ICC are responsible for pacemaker potentials (4,5). It is therefore deduced that intracellular Ca²⁺ oscillations in these cells are the primary mechanism. Suzuki et al. (6) have demonstrated that InsP₃ receptors play an essential role in ICC pacemaking. Furthermore, ICC pacemaker Ca²⁺ activity appears to also require ryanodine receptors and plasmalemmal Ca²⁺ permeable channels, such as TRP homologues (7,8). ICC-like interstitial cells are distributed throughout the body, e.g. urinary tract, uterus, vasculature and ES-gut. It is likely that these cells act as pacemaker cells and/or modulate smooth muscle spontaneous rhythmicity (8-10). (1) Huizinga JD et al. (1995) Nature 373, 347-349. (2) Dickens EJ et al. (1999) J Physiol 514, 515-531. (3) Nakayama S et al. (2006) J Physiol 576, 727-738. (4) Huizinga JD et al. (2002) Gastroenterology 123, 1627-1636 (5) Goto et al. (2005) J Physiol 559, 411-422. (6) Suzuki H et al. (2000) J Physiol 525, 105-111. (7) Aoyama M et al. (2004). J Cell Sci 117, 2813-2825. (8) Hashitani H (2006) J Physiol 576, 707-714. (9) Burdyga T & Wray S (2005) Nature 436, 559-562. (10) Takaki M et al. (2006) Stem Cells 24, 1414-1422. [J Physiol Sci. 2007;57 Suppl:S15]

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Interstitial cells of Cajal were discovered by Ramon y Cajal and thought to be part of the enteric nervous system of the gut. It has now been proven that ICC are the pacemaker cells of the stomach, small intestine and colon, generating unique electrical activities that instruct the musculature to perform peristaltic motor functions (1-3). ICC are innervated through synapse-like structures unlike smooth muscle cells that rely on non-synaptic neurotransmission. This special relationship to the nervous system gives ICC the possibility to modify neuronal signals and/or transmit information about muscle activity to the enteric nervous system. Rhythmic motor activity of smooth muscle is not restricted to the gut and ICC are now being characterized in the portal vein and the urinary tract. On the one hand, the potential widespread presence of ICC demands a rigorous definition and characterization. On the other hand, flexibility is needed because there is plasticity in ICC morphology, characteristics change dependent on circumstances such as the presence of inflammation or obstruction. ICC are implicated in the pathophysiology of many motor disorders in the gastrointestinal tract in the pediatric as well as adult population. Determining the exact role in pathophysiology is still difficult because of the intimate association with the enteric nervous system. New exciting avenues for research are aimed at a better understanding of motility control systems throughout the body. (1) Der-Silaphet et al. Gastroenterology 114: (2) 724-736, 1998; Huizinga et al. Nature 373: 347-349, 1995; (3) Thomsen. Huizinga et al. Nature Med 4: 848-851, 1998. [J Physiol Sci. 2007;57 Suppl:S15]

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Smooth muscles in the urinary tract and corporal tissue exhibit spontaneous contractile activity. Although this activity was assumed to be generated within smooth muscle cells (SMCs), it is now known that ICs, which share characteristics with ICC, the primary pacemaker cells in the gastrointestinal (GI) tract, are involved. In the urethra, isolated ICs generate spontaneous electrical activity, which are very similar to those of the intact urethra, suggesting that they may act as electrical pacemakers. Spontaneous Ca transients in urethral ICs in situ had a lower frequency (3 min⁻¹) and a longer half-width (4 s) than those in SMCs (10 min⁻¹ and less than 1 s, respectively). IC Ca transients depend on the release of Ca from intracellular stores and require the influx of Ca through non L-type Ca channel pathways. However ICs, did not form an extensive network nor consistently triggered Ca transients in adjacent SMCs, suggesting that signal transmission from ICs to SMCs in the urethra may be much less extensive than between ICC and SMCs. Interestingly, low frequency (1 min⁻¹),long duration (5-15s) Ca transients are recorded in ICs of the bladder, where SMCs generate spontaneous action potentials, and in ICs of the renal pelvis where atypical SMCs appear to provide the main pacemaker drive. Therefore, ICs in the urinary tact may be divisible into subpopulations in which ICs with a slower time course play a non-pacemaking role. Indeed, ICs in corporal tissue release prostaglandins via cyclooxygenase-2 activity to reinforce spontaneous activity initiated by SMCs. [J Physiol Sci. 2007;57 Suppl:S15]

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Mechanism of the pacemaker activity in the upper urinary tract, especially how single pacemaker cells form a pace maker region, has not been fully documented. To approach this problem, we developed in situ Ca²⁺ imaging technique of the rat utero-pelvic preparation using a macro zoom microscope (Olympus MVX 10). The bright clear view and smooth zooming operation with this technique enabled us to search upstream of Ca²⁺ transient and to identify the pacemaker region easily. Using the same technique we could also successfully record image of di-4-ANEPPS, a voltage-sensitive dye. The initial depolarization occurred at the exactly same place as the Ca²⁺ transient began. With higher magnification , we could observe that not only one cell but several cells increased their intracellular Ca²⁺ concentration simultaneously. Their propagating pathway were slightly different every time. Interestingly spontaneous Ca²⁺ rises in single cells were observed in the other part of renal pelvis and even in the ureter. They are not synchronous to the pristaltic movement and rarely propagated to neighboring cells. In the presence of low concentration of heptanol, a gap junction blocker, the cells forming the pacemaker region lost their synchronism and their function as the pacemaker. These results might suggest that summation of electric activity via gap junctions played an important role in the formation of the pacemaker region. [J Physiol Sci. 2007;57 Suppl:S16]

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One of the most intriguing, unanswered questions in uterine physiology is; what mechanism brings about spontaneous activity? As control of uterine activity is so very important for reproductive success, an answer to this question is vital. Despite an increased understanding of the mechanism of contractions and the underlying signalling pathways and changes in [Ca], our ability to prevent pre-term deliveries or dysfunctional labours has improved very little. Over the last few years, specialised cells with features similar to the classical gut pacemakers, the interstitial cells of Cajal (ICC), have been reported in a variety of rhythmic smooth muscles, and the cells referred to as ICC-like cells or modified smooth muscle cells. These cells have been attributed a pacemaker role in some but not all these smooth muscles. In the light of these reports we have examined pregnant rat myometrium for ICC-like cells, using intact preparations and single isolated cells and a variety of techniques (1). Both preparations revealed numerous ICC-like cells, with spider-like projections and enlarged central regions, making them readily distinguishable from myocytes; they were also non-contractile. In current and voltage-clamp experiments we studied their electrical properties. Membrane potential was -58 mV and outward K currents readily recordable. However we were not able to demonstrate any inward currents. We therefore conclude that at least in the rat, ICC-like cells are present but not acting as pacemakers, and suggest that the pacemaker ability resides within myocytes. A role for them remains to be determined. Reference Duquette, RA et al 2005, Biol Repro 72, 276-283 [J Physiol Sci. 2007;57 Suppl:S16]

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Recently, embryonic stem (ES) cells were shown to spontaneously (without LIF) give rise to a functional organ-like unit, the "ES gut", which undergoes rhythmic contractions and is comprised of enteric derivatives of all three embryonic germ layers: epithelial cells (endoderm), smooth muscle cells and interstitial cells of Cajal (ICCs) (mesoderm), and a small number of enteric neurons (ectoderm), but no ganglia. The ICC network within the GI tract is responsible for the generation of electrical pacemaker activity. Enteric neurons present within the GI tract innervate the smooth muscle and are essential for peristalsis. By adding brain-derived neurotrophic factor (BDNF)(10⁻⁸ g/ml) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (ENS) from enteric neural crest-derived cells in the ES gut. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) at single or multiple sites that were attenuated by nicotinic or muscarinic receptor blockade, or abolished by a neurotoxin, TTX. A 5-HT₄ receptor agonist elicited increases in [Ca²⁺]_i in the "ES gut" with enteric nervous system. This work was supported by Grants-in-aid for Scientific Research (14657311,16650090,17300130 and 18630007 for MT and 15300134 for SN) from the Ministry of Education, Science, Sports and Culture of Japan. [J Physiol Sci. 2007;57 Suppl:S16]

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Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the molecular properties of rhodopsin responsible for rod function, we have generated knock-in mice in which rhodopsin was replaced with mouse green-sensitive cone visual pigment (mouse green), whose molecular properties are considerably different from those of rhodopsin. Single cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were 3-4 times lower than those from rhodopsin. Noise analysis indicated that the rate of thermal activation of mouse green was about 900 times higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin would not be expected to affect the reduction of sensitivity to bright light, but would instead be expected to affect visual threshold under dim-light conditions, where the true light signals would be overwhelmed by dark noise due to the higher rate of thermal activation. Therefore, the ability of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision. [J Physiol Sci. 2007;57 Suppl:S17]

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In vertebrate retinas, cone cells exhibit photoresponses with a more restrained sensitivity and rapid shutoff kinetics than those of rods. For these characteristics, we have paid special attention to termination of the phototransduction through opsin phosphorylation by G protein-coupled receptor kinase (GRK) 1 and 7 in zebrafish. We identified GRK isoform, GRK1A expressed in the rods whereas GRK1B and GRK7-1 in the cones. Kinetics analysis revealed that V_max of a major subtype of the cone kinase, GRK7-1, was considerably higher than that of the rod kinase, GRK1A. The results suggest the activity of GRK being an important factor for photoresponse properties and also for the difference in photosensitivity. In the activation process, G protein transducin plays a central role in both cells. The γ-subunits of rod and cone transducin (Gγ1 = Tγ, Gγ8, respectively) are farnesylated, whereas the other subtypes of Gγ are geranylgeranylated. We investigated physiological significance of selective farnesylation of rod Tγ by using knock-in mice, in which farnesylation of Tγ was switched to geranylgeranylation. The isoprenyl replacement markedly impaired light-adaptation without affecting the phototransduction properties. The reduced light-adaptation was associated with slowdown of light-dependent translocation of Tβγ in rod cells, implying that the regulation of visual sensitivity requires the selective farnesylation of Tγ in order to facilitate its intracellular movement. The proper localization of transducin also required isoprenylation of Tγ, suggesting a more critical role of farnesylation of transducin for global organization of the visual cell. [J Physiol Sci. 2007;57 Suppl:S17]

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Ca²⁺ has various cellular functions. In olfactory cells, Ca²⁺ concentrations increase after odorant stimulation, which leads to desensitization of cAMP-gated channels and activation of Ca²⁺-activated chloride channel. In addition to these, we recently found a novel Ca²⁺-sensitive pathway mediated by a Ca²⁺-binding protein, dicalcin (formerly named p26olf), of which function is still unknown. In the present study we tried to find out the role of this protein. Dicalcin was originally found in frog olfactory epithelium and is now known to be present also in frog respiratory epithelium. Dicalcin is composed of two S100 domains aligned sequentially. S100 proteins are Ca²⁺-binding proteins, and functional in a dimer form. So far, dicalcin is found only in frogs, and therefore, this protein would be a functional form of S100 proteins in frogs. To investigate the role of this protein, we tried to identify target proteins of dicalcin, and found several candidate proteins. Among them, 35-38 kDa proteins were most abundant. We found that these proteins are annexin A1, A2 and A5, Ca²⁺-binding proteins found in many tissues. Because annexins are suggested to regulate membrane organization, we measured the effect of dicalcin on the membrane aggregation activity of annexins. Dicalcin enhanced the annexin activity in a Ca²⁺-dependent manner. We currently postulate that dicalcin facilitates reseal of the cilia membrane which could be broken by damages induced mechanically or chemically. [J Physiol Sci. 2007;57 Suppl:S17]

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Olfactory transduction starts at the sensory cilia that have 100 nm diameters. This signal transduction is now fairly well known to be conducted with cAMP metabolisms. Up to this point, however, knowledge about the spatial distribution of the system is very limited, mainly because of technical limitations accompanying the fine ciliary structure. To overcome these difficulties, we employed a combined technique of the patch clamp and photolysis of caged compound under fine visualization of sub-micron structure with the laser confocal microscope. Cilia were loaded with both caged cAMP for photolysis and Lucifer yellow for luminescent visualization. The laser light spot for photolysis was collected at the focal plane with the objective lens having a large NA(1.4) and with the short wavelength light (364 nm). When the intensity of the UV spot was assumed to express spatially a two-dimensional Gaussian distribution, σ was estimated to be < 300 nm. The On-Off and position of the UV spot were regulated by the AOTF device and Galvano-mirror. When local area (ca. 1 μm length) of cilium was illuminated (therefore, when local cAMP was jumped), the cell showed an inward current response of–10 pA at -50 mV. The current was observed at any part of cilia, but the amplitude gradually became smaller when the position was moved to the apical part. This may be consistent with the fact that the apical part becomes thinner than the proximal part. We conclude that olfactory transduction channels are present along entire cilium, presumably depending on the membrane area. [J Physiol Sci. 2007;57 Suppl:S18]

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It is widely accepted that horizontal cells (HCs) contribute to the formation of the center-surround receptive field, which is important to enhance the contrast of the image by lateral inhibition. HCs have a large receptive field due to electrical coupling. HCs are depolarized in the dark by a tonic glutamate release from photoreceptors (PRs). During surround illumination HCs are hyperpolarized. Three mechanisms have been proposed as responsible to the formation of receptive field surround. (1) GABA-mediated feedback from HC to PRs, (2) the electric field produced by the current flowing into HCs, and (3) proton extruded from HC into the invaginating cleft of the cone terminal. Tatsukawa et al (2005) have shown that GABAergic inputs from horizontal cells to cone is certainly present, but their contribution is very limited. Both the ephaptic hypothesis and proton hypothesis propose that shifts in I_Ca activation are responsible. In the ephaptic hypothesis current flowing into the invaginating cone synapse during changes in HC membrane potential through connexin 26 hemigap junctions at the tips of HC dendrites produce the requisite current sink (Fahrenfort et al. 2004; Kamermans et al. 2001). The proton hypothesis suggests that the modulation of I_Ca in cone PR is related to the membrane voltage of HCs (Hirasawa & Kaneko, 2003). It is kept acidic in the dark and is alkalinized by surround illumination. Recent studies by Vessey et al (2005) and by Cadetti and Thoreson (2006) favor the proton hypothesis, and our group succeeded to demonstrate that polarization of HC causes pH changes of the immediate surrounding extracellular surface of HCs. [J Physiol Sci. 2007;57 Suppl:S18]

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Analysis of miniature inhibitory postsynaptic currents (IPSCs) suggested the presence of non-NMDA and NMDA receptors at the presynaptic terminals of inhibitory connections in layer 2/3 pyramidal neurons of mouse visual cortex. We investigated the role of these presynaptic receptors in long-term potentiation (LTP) of IPSCs recorded from layer 2/3 pyramidal neurons. High-frequency-stimulation (HFS) of presynaptic fibers, which was applied while membrane potential was held at -70 mV to avoid Ca²⁺ entry into postsynaptic neurons, produced LTP of IPSCs recorded at the reversal potential of excitatory synaptic transmission (0 mV) in a normal solution. In the presence of the non-NMDA receptor antagonist NBQX, HFS produced LTP of IPSCs recorded at -70 mV, at which postsynaptic NMDA receptors are not activated, although the magnitude of LTP was smaller, compared with the control solution. In the presence of the NMDA receptor antagonist APV or the NR2B-selective NMDA receptor antagonist Ro 25-6981 together with NBQX, HFS failed to produce LTP. Even in the presence of NBQX and APV, HFS produced LTP in the solution containing a high concentration (4 mM) of Ca²⁺. Our previous study demonstrated the requirement of action potential-associated Ca²⁺ entry through voltage-gated Ca²⁺ channels into presynaptic terminals for the maintenance of LTP. Taken together, it is likely that presynaptic Ca²⁺ entry through NMDA receptors, in addition to the entry through voltage-gated Ca²⁺ channels, contributes to the maintenance of LTP. [J Physiol Sci. 2007;57 Suppl:S18]

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The amygdala is a crucial brain structure for the acquisition and expression of fear memory.The N-methyl-D-aspartate (NMDA)-type glutamate receptor channel, composed of the NR1 and NR2 subunits, plays a key role in synaptic plasticity in the CNS. NR2 subunits (NR2A-NR2D) are differentially expressed, depending on developmental stages and brain regions, but their functional roles are still unclear. In this study, we investigated the properties of synaptic NMDA receptors in the lateral nucleus of the amygdala (LA), comparing them with those of synaptic NMDA receptors in the CA1. We show that the electrophysiological properties of NMDA receptors and NR2A/NR2B ratio in the LA are distict from those of NMDA receptors in the CA1 and that the NR2B subunit contributes to synaptic transmission and long-term potentiation (LTP) induction more significantly in the LA than in the CA1. Our data suggest that these properties of NMDA receptors and the NR2B subunit in the LA are responsible for amygdala synaptic function and plasticity. [J Physiol Sci. 2007;57 Suppl:S19]

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Plasticity at the parallel fiber (PF)-Purkinje cell synapses is a putative cellular model of the cerebellar information storage. Predominantly expressed in Purkinje cells, the δ2 glutamate receptor (GluRδ2) plays a crucial role in regulating synaptic plasticity at the PF-Purkinje cell synapses and cerebellar functions; mutant mice deficient in GluRδ2 (GluRδ2^−/−) display impaired synapse formation and long-term depression (LTD) in the cerebellum. Nevertheless, the mechanism by which GluRδ2 functions in vivo has remained elusive. Previously, by using Sindbis virus-mediated expression system, we demonstrated that the most carboxy-terminal (C-terminal) domain of GluRδ2, which interacts with several PDZ proteins, is required for LTD induction (Kohda et al., J. Physiol. Sci. 56 Suppl., S166, 2006). To further examine the importance of the PDZ binding motif of GluRδ2 in cerebellar functions in vivo, we introduced a mutant GluRδ2 transgene lacking seven amino acids of the most C-terminal domain (GluRδ2^DCT7), into GluRδ2^−/− mice. Although GluRδ2^DCT7 proteins were expressed at PF-Purkinje cell synapses to a sufficient level required for rescuing all phenotypes of GluRδ2^−/− mice, LTD was still severely impaired in these mice, consistent with our previous study using a Sindbis virus. Furthermore, the mice showed poor performance in a rotor-rod test and an eye-blink conditioning task, reflecting impaired cerebellum-dependent motor learning. Taken together, the PDZ binding motif at the C-terminus of GluRδ2 plays an indispensable role for LTD induction and motor learning in the cerebellum. [J Physiol Sci. 2007;57 Suppl:S19]

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It is generally assumed that long-term memory is based on the long-lasting synaptic plasticity accompanied by the formation of new synapses. There is a debate whether LTP per se represents that process. Using stable cultures of rat hippocampal slices, we found recently that a long-lasting (lasting ≥3w) synaptic enhancement coupled with an increase in the number of synapses was established after repeated inductions of, but not a single induction of, LTP. We call this new plastic phenomenon RISE (Repetitive-LTP Induced Synaptic Enhancement) to discriminate it from conventional single LTP. RISE was established after ≥3 inductions of late-phase LTP spaced by proper intervals (3-24h). RISE required the activation of PKA, CaMK and MAPKK. In the developing phase of RISE, Ca²⁺-permeable AMPA receptors were transiently expressed and their activity was necessary for the establishment of RISE. DNA microarray analysis revealed that the genes up- and down-regulated after the 3rd LTP induction included those of regulatory proteins for actin-cytoskeleton, synaptic scaffold proteins and cell adhesion molecules, confirming that a dynamic structural reorganization leading to synaptogenesis occurred during the development of RISE. We propose that RISE should serve as a useful model system for an in vitro analysis of long-term memory. [J Physiol Sci. 2007;57 Suppl:S19]

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