The traditional view that the cerebellum is involved solely in the control of movement has been challenged in recent years. One of the pivotal reasons for this reappraisal has been new information about cerebellar connections with the cerebral cortex. The use of transneuronal transport of neurotropic viruses has enabled us to identify cerebello- thalamocortical pathways to selected motor (Hoover & Strick, ‘93,‘99; Dum & Strick, ‘03), prefrontal (Middleton & Strick, ‘94,‘00,‘01; Kelly & Strick, ‘03) and posterior parietal areas of cortex (Clower et al., ‘01, ‘04). Thus, our results indicate that cerebello- thalamocortical pathways project to a wider set of cortical areas than previously imagined. The output stage of cerebellar processing displays a surprising degree of topographic organization. We proposed that localized regions within the dentate form distinct output channels that are directed at specific cortical areas. As the data on these circuits has accumulated, a general rule has emerged- each cortical area that projects to the input stage of cerebellar processing receives (via the thalamus) efferents from the output stage of cerebellar processing. This rule implies that multiple closed loops represent a fundamental unit of cerebro-cerebellar circuitry. These circuits provide the cerebellum with the neural substrate to influence not only the generation and control of movement, but also aspects of cognition and visuo-spatial perception. Support: VA Medical Research Service, USPHS grants NS24328 & MH56661. [Jpn J Physiol 55 Suppl:S2 (2005)]

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In 1965, it was thought that acetylcholine (ACh) activates an electrogenic Cl⁻ pump in the basal acinar membrane, but in the late 1960s it was shown that ACh opens a K⁺ pathway¹. In the 1970s, Akinori Nishiyama² and Noriyuki Iwatsuki³, characterized properly the salivary and pancreatic acinar cell membranes and intracellular Ca²⁺ injection was shown to mimick the action of external ACh³. Yoshio Maruyama's pioneering patch clamp work in the early 1980s, resulted in a molecular characterization of the conductance pathways and quantification of the K⁺ channels^{4, 5}. In the late 1980s and early 1990s, Makoto Wakui and Yuri Osipchuk defined the principal mechanisms of the ACh- and inositol trisphosphate- elicited cytosolic Ca²⁺ spiking in the acinar apical pole⁶. The intracellular Ca²⁺ tunnel was discovered by Hideo Mogami⁷ and directly visualized by Myoung Kyu Park⁸. Hanna Tinel discovered the perigranular mitochondrial belt⁹ and Jose Cancela showed the action of new Ca²⁺ releasing messengers¹⁰. The nuclear Ca²⁺ pool was characterized by the Gerasimenkos¹¹ and their latest data show two intracellular pools. 1. Petersen OH J Physiol 208, 431-447, 1970 2. Nishiyama A & Petersen OH J Physiol 244, 431-465, 1975 3. Iwatsuki N & Petersen OH Nature 268, 147-149, 1977 4. Maruyama Y & Petersen OH Nature 300, 61-63, 1982 5. Petersen OH & Maruyama Y Nature 307, 693-696, 1984 6. Wakui M et al Cell 63, 1025-1032, 1990 7. Mogami H et al Cell 88, 49-55, 1997 8. Petersen et al TINS 24, 271-276, 2001 9. Tinel H et al EMBO J 18, 4999-5008, 1999 10. Cancela J et al EMBO J 21, 909-919, 2002 11. Gerasimenko J et al J Cell Biol 163, 271-282, 2003 [Jpn J Physiol 55 Suppl:S2 (2005)]

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Ca is involved in diverse cellular functions. In the brain Ca controls signal transmission at the synapse. Ca influx through voltage-gated Ca channels (Ca channels) times transmitter release. Late Professor Susumu Hagiwara and his associates are the pioneers who have characterized Ca channels in various cell types in a variety of animals, i.e., in barnacle muscles, mammalian endocrine cells, etc. The Ca channel has been shown to have distinct biophysical properties from the Na channel. Later it has become evident that there are multiple types of Ca channels, each specialized for specific functions. The synapse is considered to be the site of higher brain functions. There, Ca enters during an action potential and forms a microdomain within the presynaptic terminal. A Ca sensor, Synaptotagmin I (Syt I), on the synaptic vesicle detects a rise of Ca concentration in the microdomain. Synaptic vesicles are docked and primed at the release site and ready to fuse with the terminal membrane upon a cue through Syt I. Syt I Ca-dependently binds phospholipids resulting in vesicle fusion. Then the transmitter in the vesicle is released instantaneously into the synaptic cleft and binds to receptors in the postsynaptic membrane. Since this process of transmitter release and the properties of postsynaptic receptors are modulated by a variety of mechanisms the efficacy of synaptic transmission can be controlled by the past experience of the synapse. This is considered to be the basis of memory. Thus Ca channels that were initially characterized in barnacle muscles assume a central role for the brain function. [Jpn J Physiol 55 Suppl:S4 (2005)]

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The retina converts the image into the neural signal and, in this process, the individual photoreceptors work as pixels. But the conversion of the image is not simply pixel by pixel. An important function of the retina is to enhance the contrast of the image by lateral inhibition. As a result, a neuron in the early visual system has a concentric receptive field with a center-surround antagonism. Many vision scientists agree now that horizontal cells (HCs) contribute to the formation of the center-surround receptive field. HCs have a large receptive field due to electrical coupling. They have AMPA receptors, and a tonic glutamate release from photoreceptors keeps HCs depolarized in the dark. During surround illumination HCs are hyperpolarized. We recently found that pH of the invaginating synaptic cleft of the cone terminal is related to the membrane voltage of HCs (Hirasawa & Kaneko, 2003). It is kept acidic in the dark and is alkalinized by surround illumination. The pH change we found in the retinal slices of the newt disappeared when the slice was superfused with a solution with enriched pH buffering capacity. We concluded that the surround illumination enhances the amount of L-glutamate release from the alkalinized cone terminal (the effect opposite to spot illumination), resulting in the formation of the center-surround receptive field of the second- and higher-order neurons in the visual system. By an imaging technique, we measured the pH of the extracellular space of an HC isolated from the carp retina and found that depolarization of HC acidifies the immediate surrounding of the HC. The mechanisms of pH change are still under study. [Jpn J Physiol 55 Suppl:S4 (2005)]

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Blood glucose levels are tightly controlled in normal subjects: 70-110 mg/dl in the fasting state and below 140 mg/dl two hours after 75 g oral glucose challenge. Brain uses glucose at very high rate, 5 g/hr. Suppose the circulating blood volume being 5 l, the subject with 100 mg/dl glucose level has 5 g glucose in the blood. Therefore, glucose levels would decrease to zero in one hour if there were no supply of glucose to the circulation. 75 g oral glucose challenge would give rise to 1500 mg/dl blood glucose. However, these things never happen. I will focus on three issues in my talk. 1) Glucose transport requires conformational change of membrane protein termed glucose transporter and C-terminal deletion locks glucose transporter into an inward-facing form without transport activity. Similar mutations reportedly caused human disease. For insulin stimulation of glucose transport, insulin receptor, IRS, PI3 kinase, Akt and glucose transporter GLUT4 are sequentially stimulated. 2) Tight control also depends on glucose-regulated insulin secretion. A key molecule for recognition of blood glucose levels is glucokinase in beta cells and mutations of this gene cause diabetes. Clinical characteristics of these patients we found are consistent with the role of glucokinase in insulin secretion. 3) To understand the disease such as diabetes, not only a role of a specific molecule in cells but also cross-talk between cell and cell, tissue and tissue, and overall whole body metabolism are of much interest. I will discuss brain sensing and regulation of peripheral metabolism through autonomic nervous system. [Jpn J Physiol 55 Suppl:S4 (2005)]

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Cardiac function is controlled by autonomic nervous system. The released ACh decelerates the heart beat and delays the artrio-ventricular conduction. This is due to activation of K⁺ channels in nodal cells first identified by Prof. Sunao Tawara in 1906. The ACh-deceleration of heart beat is the first example of synaptic chemical transmission revealed by Prof. Otto Levi in 1920s. It is a great honor for me to talk about its ionic mechanism, i.e., muscarinic activation of cardiac K⁺ channel, on which I have been working for more than 20 years. In cardiac nodal and atrial myocytes, application of ACh elicits a K_ACh current. The K_ACh channel comprises Kir3.1 and Kir3.4. The activation time-course is sigmoidal and takes several hundred ms to reach a peak. Thereafter, the current decreases to a quasi-steady state level in 1 min in the presence of ACh. When I started studying activation mechanism of K_ACh channel by ACh, I observed three phenomena; 1) rapid run-down of the response, 2) the short term-desensitization, and 3) the agonist-dependent relaxation of the current. We found wash-out of intracellular GTP caused rapid rundown because the channel is activated by PTX-sensitive G proteins. We for the first time clarified that the βγ subunit of G proteins directly activates the channel. We have recently revealed the agonist-dependent relaxation of K_ACh current is caused by the voltage-dependent action of RGS proteins, which facilitates GTPase activity of G_α. On the basis of these results, we could have modeled physiological behavior of G protein-activation of K_ACh channel. [Jpn J Physiol 55 Suppl:S4 (2005)]

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Mammalian homologues of Drosophira transient receptor potential (trp) proteins (TRP) form Ca²⁺ permeable cation channels, which are activated in response to stimulation of G-protein-coupled receptors. We have previously demonstrated that TRPC5 permeates Ca²⁺ in response to stimulation through direct cysteine oxidation by reactive oxygen species (ROS) and reactive nitrogen species (RNS). We have examined activity of TRPC5 channel recombinantly expressed in HEK293 cells using patch-clamp technique and the fluorescent Ca²⁺ indicator fura-2 for measurements of intracellular free calcium concentration ([Ca²⁺]_i). In bovine aortic endothelial cells (BAEC) treated with all-trans-retinoic acid (RA) or recombinantly expressing TRPC5, ATP receptor stimulation caused significant production of nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) and further [Ca²⁺]_i rises. RNA interference targeting bovine TRPC5 attenuated ATP-induced [Ca²⁺]_i rises and NO production in BAEC treated with RA. Furthermore, physical association between TRPC5 and eNOS at the caveolin-rich plasma membrane area was revealed by immunocytochemistry and co-immuneoprecipitation experiments. These results suggest that receptor-activated TRPC5 Ca²⁺ influx activity by coexpressed eNOS leads to an idea that NO generated by nearby eNOS activates TRPC5 in caveolae. There is a positive feedback cycle between TRPC5 and eNOS that amplifies NO production and [Ca²⁺]_i rises through TRPC5 nitrosylation. [Jpn J Physiol 55 Suppl:S6 (2005)]

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Nitric oxide (NO) is a gaseous signaling molecule, having diverse biological actions. In addition to the well-known guanylate cyclase/cGMP/protein kinase G pathway, direct protein s-nitrosylation and s-glutathiolation provide a novel mechanism of NO-dependent signaling. In cardiac myocytes, the slowly activating delayed rectifier potassium current (I_Ks) channels appear to co-localize with NO synthase-3 (NOS3) in caveolae (ref 1), and is a target of s-nitrosylation by NO (ref 2). S-nitrosylation by NO underlies physiologically important regulatory mechanisms of I_Ks by cytosolic Ca²⁺ and by sex hormones. Rise in intracellular Ca²⁺ activates NOS3 and produces NO via a Ca²⁺/calmodulin-dependent pathway, resulting in activation of I_Ks channel (ref 3). Sex hormones, such as testosterone, estradiol, and progesterone, activate NOS3 via their non-genotropic pathway, which also enhances I_Ks (ref 4). It has been shown that scaffolding proteins belonging to striatin family (striatin, zinedin, and SG2NA) compose macromolecular complex involving caveolin, calmodulin, NOS, and sex hormone receptor. Dynamic changes in protein-protein interaction around this macromolecular complex play an important role in NO-dependent I_Ks regulation. [References] (1) J. Biol. Chem. 2004;279:40778-40787.(2) Br. J. Pharmacol. 2004;142:567-575.(3) Circ. Res. 2005;96:64-72.(4) Circulation (in revision). [Jpn J Physiol 55 Suppl:S6 (2005)]

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In adults, the extent of renal excretion of inorganic phosphate (Pi) is determined by the abundance of the Na-dependent Pi-cotransporter (NaPi-lla; SLC34A1) localized at the brush border membranes of proximal tubular cells. A number of hormonal and metabolic factors, PTH being a paradigm, define the abundance of NaPi-lla. PTH, via apically and basolaterally localized receptors, leads to an internalization and lysosomal degradation of NaPi-lla. In order to understand the mechanisms involved in the apical positioning and the regulation of the NaPi-lla cotransporter, yeast two-hybrid screens have been performed. In a screen, using the C-terminus of NaPi-lla as a bait, among others, two PZD proteins, NHERF1/2 and PDZK1, were identified to interact with the C-terminus of NaPi-lla. These interactions were assigned to specific PDZ domains and to the PDZ binding motif TRL. In addition, applying a membrane split-ubiquitin two-hybrid system, we recently demonstrated that these interactions occur also between full length NaPi-lla. Both, NHERF1 and PDZK1 are expressed in the brush border of proximal tubular cells. Studies performed with NHERF1 and PDZK1 deficient mice demonstrated that ablation of NHERF1 impairs apical localization and regulation of NaPi-lla whereas ablation of PDZK1 was without effect. Furthermore, studies performed with OK-cells indicated that NHERF1 directly or indirectly is involved in the apical positioning of NaPi-lla. As upon PTH stimulation NaPi-lla is internalized but NHERF1/PDZK1 not, the interactions of NaPi-lla with respective PDZ domains are suggested to be regulated. PTH dependent phosphorylation of NHERF1/PDZK1 was investigated in OK-cells and mouse kidney slices. [Jpn J Physiol 55 Suppl:S6 (2005)]

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Twelve-membrane-spanning amino acid transporters of SLC (solute carrier) 7 family require single membrane spanning proteins for their proper membrane targeting. Two single membrane spanning accessory units 4F2hc and rBAT are identified so far in SLC3 family. They are connected to the specific 12-membrane-spanning catalytic units via a disulfide bond and form heterodimeric transporters. In the epithelial cells of small intestine and renal proximal tubules, rBAT is localized in the apical membrane, whereas 4F2hc is found in the basolateral membrane. We performed extensive chimera analysis in which we expressed the chimeric proteins of rBAT and 4F2hc with BAT1, a partner of rBAT, or LAT1, a partner of 4F2hc and found that the single membrane-spanning accessory units recognize their partner catalytic units at the membrane-spanning domains. Furthermore, the accessory units were proved to possess signals responsible for membrane sorting. rBAT is connected with BAT1 and form a cystine transporter on the apical membrane of the epithelia. By analyzing cystinuria patients, we found a loss-of-function mutation in the C-terminus intracellular domain of BAT1. The motif-like sequence with similarity to the "targeting domain" of Ca_v1.2 turned out to be responsible for the transition from endoplasmic reticulum to Golgi complex and for the membrane targeting of heterodimeric transporters. Based on the deletion analysis and yeast two hybrid analysis, more than one proteins are proposed to interact with the C-terminus of BAT1 and promote the proper membrane targeting of the heterodimeic complex. [Jpn J Physiol 55 Suppl:S7 (2005)]

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Genetic defects in cardiac Na channel gene (SCN5A) result in multiple distinct arrhythmic syndromes such as long QT syndrome (LQTS) and Brugada syndrome. Brugada syndrome is a subgroup of idiopathic ventricular fibrillation characterized by the ST elevation in the right precordial leads. Mutant Na channels responsible for Brugada syndrome heterologously expressed in tsA-201 cells have shown several distinct biophysical abnormalities including 1) non-functional channels, 2) reduced Na current due to gating modulation and 3) defect of channel protein trafficking to the plasma membrane. Loss-of-function of Na current of the cardiac action potential leaves transient outward K current (Ito) unopposed in the phase 1, and results in the transmural voltage gradient predominantly observed in the right ventricle. Genetic variations and subclinical mutations in LQTS-related K channel genes have been suggested as risk factors for drug-induced LQTS. We have identified a silent SCN5A mutation (L1825P) in a case with cisapride-induced acquired LQTS. The L1825P channel showed a prominent persistent component most commonly observed for LQT3, and the loss-of-function features characteristic of Brugada syndrome. Despite severe channel dysfunctions, the patient exhibited normal QT-interval until exposure to the potent IKr blocker cisapride. Our study suggests that SCN5A mutations may underlie some cases of acquired LQTS and potentially predispose apparently stable individuals to life-threatening arrhythmias when treated with agents that inhibit cardiac K channels. [Jpn J Physiol 55 Suppl:S7 (2005)]

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We reported a dominantly inherited Japanese family with myotonia and cold-induced hypokalemic periodic paralysis. The proband showed hypokalemia and flaccid paralysis in cold atmospheric temperatures. This phenotype is associated with a novel mutation in the voltage dependent skeletal muscle sodium channel (Na_v1.4) alpha subunit gene (SCN4A). This P1158S mutation is localized between the fourth and fifth transmembrane segments of domain III in Na_v1.4. Using the amphotericin B-perforated-patch clamp method, sodium currents were recorded at 22°C and 32°C from the wild type (WT) and P1158S mutant Na_v1.4 expressed in tsA201 cells. Computer simulation was performed incorporating the gating-parameters of the P1158S mutant Na_v1.4. P1158S mutant Na_v1.4 exhibited hyperpolarizing shifts in voltage-dependence of both activation and inactivation curves at a cold temperature and a slower rate of inactivation than the WT. Computer simulation reproduced the abnormal skeletal muscle electrical activities of both paralysis at a low potassium concentration in the cold, and myotonia at a normal potassium concentration. For the first time we found that a single amino acid mutant sodium channel shifted the voltage dependency in the hyperpolarizing direction in a temperature dependent manner. Recently, it was reported that generalized epilepsy with febrile seizures plus (GEFS+) were caused by sodium channel mutations, and the heat-induced paralysis of the smellblind drosophila mutant was also found to be due to a sodium channel mutation. Thus, sodium channel abnormalities may be responsible for the temperature sensitive pathological states of various diseases. [Jpn J Physiol 55 Suppl:S7 (2005)]

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Chloride channels had not been paid much attention by many physiologists, partly because of the lack of drastic gating. Investigations of muscle disorders with myotonia (muscle stiffness due to continued firing of action potentials), however, has conveyed numerous information about the physiology of chloride channels. The study linking chloride channel to diseases goes back to an electrophysiolgical recording from myotonic goat in 80’s, which has showed the reduction in chloride conductance, which contributes two-third of normal resting conductance. After the cloning of the chloride channel in skeletal muscle, its mutations have been identified in two forms of inheritable myotonia; autosomal dominant myotonia congenita (Thomsen) and recessive generalized myotonia (Becker). Co-expression of dominantly inherited mutant and wild type channels has suggested the dominant-negative effect of the mutant, sufficient to reduce chloride conductance drastically. The cardinal symptoms of myotonic dystrophy, the most common muscular dystrophy in adult, are muscle atrophy and myotonia. The genetic defect is abnormal expansion of CTG repeat located at 3’ untranslated region of a putative kinase gene. Electrophysiological recording from the muscle fibers of the model mice expressing expanded repeats, revealed reduced chloride conductance, attributable for myotonia. Decreased expression of channel protein due to aberrant splicing of channel mRNA has successively been shown in patients. Therefore, the myotonic dystrophy is a new form of channelopathy, caused by disrupted splicing of the ion channel mRNA. [Jpn J Physiol 55 Suppl:S8 (2005)]

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The voltage-dependent Ca_V2.1 (P/Q-type) calcium channel is a predominant calcium channel in the central nervous system (CNS) and is an essential molecule for neurotransmitter release in presynaptic neurons and regulation of neuronal excitability and gene expression in postsynaptic neurons. Mutations in the gene encoding the Ca_V2.1 calcium channel α₁2.1 subunit are associated with neurological disorders in human and mice. In human, familial hemiplegic migraine, episodic ataxia type 2, spinocerebellar ataxia type 6 and others are associated with the α₁2.1 mutations. In mice, two major phenotypes of neurological disorders are cerebellar ataxia and absence epilepsy. Because several strains of the α₁2.1 subunit mutations are available, we have analyzed the impacts of the mutations on the channel properties, the synaptic transmission, the axonal extension, the network activity, and other factors, using the morphological and electrophysiological techniques. The analyses have provided insights into mechanisms how the symptoms of the genetic disorders become overt in some specific areas of CNS or in some developmental stages. The results will contribute to understanding the pathophysiology of human neurological disorders. An overview of the extensive results will be presented in the symposium. [Jpn J Physiol 55 Suppl:S8 (2005)]

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Until recently, all genes found to be mutated in hereditary idiopathic epilepsies encoded subunits of ion channels or functionally related proteins, leading to the view of this class of diseases as channelopathies. Exceptions to this rule are the Mass1, the LGI1, and the EFHC1 genes. Mutations of the CHRNA4 and the CHRNB2 have been detected in families with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To study pathogenesis of ADNFLE, a transgenic rat strains inserted a ADNFLE mutant CHRNA4 (S284L) has been developed. The KCNQ2 and KCNQ3 gene mutations were identified in families with benign familial neonatal convulsions (BFNC). The age-dependent development and spontaneous remission of BFNC reported to be associated with the functional switching of the GABAergic system from excitatory to inhibitory in neonatal CNS may be explained by the facts that NKCC1 (Cl-accumulating Na+, K+, -2C-, cotransporter) plays a pivotal role in the generation of GABA-mediated depolarization in immature cortical plate cells and KCC2 (Cl-extruding K+-Cl- cotransporter) promotes the later maturation of GABAergic inhibition in the rat neocortex. Mutations in the voltage-gated Na+-channel (VGSC:SCN1A, SCN2A, SCN1B) and GABAA receptor (GABRG2) genes have been identified in generalized epilepsy with febrile seizure plus (GEFS+), and those of SCN1A and GABRG2 in severe myoclonic epilepsy in infancy (SMEI), although GEFS+ and SMEI are clinically distinct from each other. The functional comsequences of these VGSC mutations remain controversial. Thus, functions should be analyzed not only in cells but in animal models such as S284L transgenic rat. [Jpn J Physiol 55 Suppl:S8 (2005)]

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Physiologically, it is believed that past, present, and pre-future memory is stored in the parietal and temporal lobes, hippocampus, and frontal lobes respectively. The hippocampal networks consist of three types of synapses that form the circuit. The spatial-signal which serves as the input to the hippocampus is transmitted through a synapse in the dentate girus (DG) to the CA3 then through another synapse to the CA1. The hippocampusCA3 is characterized by a distinct biological neural network which has a recurrent network. This circuitry compiles past memory into the present. On this subject, Dr. Tonegawa (Massachusetts Institute of Technology) et al have reported that when they used genetic techniques to knock out feedback in the hippocampal CA3 neural network, an extremely large number of cues were required to accomplish one action. According to this , it can be believed that the hippocampal CA3 forms a context of time sequence, and the CA1 maps the spatiotemporal context into its synaptic weight space. For the CA3 →CA1 network, Tsuda(1996 and 1998) proposed a computational model of chaos-driven contracting systems in which the unstable network(chaos-driven network, CA3) forms a context of events via chaotic itinerary and stable network(contracting dynamics, CA1) encodes its information as Cantor coding. In the CA1 Tsukada, et al. (1996,2005) proposed a spatiotemporal learning rule in mapping of spatiotemporal information onto CA1 synaptic weight space. Short-term memory is formed in this way. [Jpn J Physiol 55 Suppl:S9 (2005)]

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The transitory activity of neuron assemblies has been observed in various areas of animal and human brains. We here highlight some typical transitory dynamics observed in laboratory experiments and provide a dynamical systems interpretation of such behaviors. The transitory dynamics typically observed in the brain seems to appear in high-dimensional systems. A new dynamical systems interpretation for the cortical dynamics is reviewed, cast in terms of high-dimensional transitory dynamics. This interpretation differs from the conventional one, which is usually cast in terms of low-dimensional attractors. We focus our attention on, in particular, chaotic itinerancy, a dynamic concept describing transitory dynamics among “exotic attractors”, or “attractor ruins”. We also emphasize the functional significance of chaotic itinerancy. We further show that Cantor coding can be used to hierarchically embed temporal sequences produced by a chaotic or a random information source. We propose a hypothesis on the possibility of Cantor coding for the formation of episodic memory in the hippocampus and also for the category formation of episodic memory. [Jpn J Physiol 55 Suppl:S9 (2005)]

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A distinctive function of the hippocampus is to learn a sequence of places so that animals can recognize the places where they are. In this paper, we will show that theta burst stimulation organizes radial propagation of neuronal activity from the stimulus site in a CA3 network model that contains recurrent excitatory synapses having nature of spike-timing-dependent plasticity (STDP). We will also report that a neuronal assembly coding a temporal sequence of signals may be organized in CA1 through the radial propagation of neuronal activity in CA3 when Schaffer collateral synapses are subject to a STDP rule. When firing of the presynaptic neuron precedes firing of the postsynaptic neuron, the synaptic connection is potentiated by a STDP rule, while the synaptic connection is depressed when the neurons fire in reverse order. Radial propagation of neuronal actvity was therefore produed by theta burst stimulation fed to a local site in CA3. When theta burst signals were successively fed to several local sites in CA3 and CA1, radial propagations of neuronal activity were produced first, like ripples spreading on the surface of water. Since the radius of the ripples depends on the time when signals are fed, the distribution of ripples reflects the sequence of signals. The coincident firing of CA3 pyramidal cells on the ripples then potentiated Schaffer collateral synapses to specific CA1 neurons together with proper perforant path signals. These CA1 neurons may code a sequence of signals fed to the hippocampus. [Jpn J Physiol 55 Suppl:S9 (2005)]

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It is suggested that episodic memory is defined as a context-dependent sequence memory, and that the hippocampal formation (HF) is essential in such memory. To investigate HF involvement in a context-dependent sequence memory, multiple single unit activities were recorded from the monkey HF during performance of real translocation and virtual navigation tasks. The results indicated that place-related neuronal activity in the HF was task- or context-dependent, and cross-correlation data suggest that the context-dependent information may be encoded by interaction among pyramidal neurons based on asymmetrical connections. Rat CA1 HF neurons were also recorded during a conditional sequence memory task. Consistent with the computational studies, 2 types of the HF neurons were found; some neurons responded to single item regardless of sequences in which the item was presented, while other neurons displayed sustained firing during serial presentation of several items. In humans, event-related potentials (ERPs) were recorded during a sound-sequence memory task. The results suggest that the ERPs around 300-700 msec latency were specifically involved in sound sequence information processing. Furthermore, equivalent dipoles for the ERPs were localized in the medial temporal lobe including the HF and parahippocampal gyrus. These results suggest that the HF is crucial in context-dependent sequence information processing, which may be the neural basis of episodic memory. [Jpn J Physiol 55 Suppl:S10 (2005)]

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Animals sense various ranges of temperatures, pressure and irritants through skin-nerve pathway at the maximum level, pain sensation. Molecular mechanism under the response to physical factors is thus essential for understanding pain. Transient receptor potential vanilloid 4 (TRPV4) is a family of capsaicin receptor encoding a cation channel activated by cell swelling. We early cloned the gene and made its deficient mice, TRPV4-/-. Based on the findings from molecule to mouse, TRPV4 plays a key role in sensing of certain ranges of these stimuli and I here summarize putative molecular mechanisms. TRPV4 is located in DRG, nerve endings and deep keratinocytes. In vivo, TRPV4-/- becomes unable to respond to cutaneous pressure in a range of 80-300 mmHg. TRPV4 current or a rise in intracellular Ca²⁺(iCa) was observed in response to shear stress less than 10 mPa. The deformity of cell surface transmits the signal through a coiled-coil protein or lipid-based signal transduction. On the other hand, TRPV4 plays a cardinal role in warmth sensation. Neuronal activity in the femoral nerve revealed that number and activity of neurons decreased in response to a warm temperature in TRPV4-/- mice. TRPV4-/- mice displayed a significantly longer latency to escape from the hotplates at 35-45 ^oC when hyperalgesia was induced by carrageenan. Single channel analysis of TRPV4 suggested that warming did not directly activate TRPV4 in inside-out patches but that an intracellular heat-sensing protein may help for TRPV4 to cluster. Although the protein is not yet isolated, we could suggest in collaboration with Dr. Koide (National Institute of Genetics) that a mouse lacking this protein reveals a heat-insensitive phenotype. [Jpn J Physiol 55 Suppl:S10 (2005)]

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Acid sensing ion channel 3 (ASIC3) is highly expressed on the cardiac afferents and has been proposed to transduce the tissue acidification caused by myocardial ischemia into electrical signals leading to chest pain (angina). However, this proposal requires that ASIC3 has certain kinetic properties. Occlusion of a coronary artery for several minutes decreases myocardial tissue pH just to ∼7.0 or 6.7 at the lowest. Thus, the acid sensor must respond within this critical range of extracellular pH. Furthermore, ASIC3 channels desensitize, whereas the chest pain is persistent. Here we show that the small changes in pH do indeed trigger sustained ASIC3 current associated with persistent neuronal excitation expected during angina. Successive 20-sec step changes in pH between 7.4 and 6.7 produced the sustained Na⁺-selective current in ASIC3-transfected CHO cells. The peak magnitude of the sustained current occurred at pH 7.0. The bell-like shape of the I-pH relationship suggests that the sustained ASIC3 current is a 'window current', caused by overlap of activation and desensitization curves. Lactate, an anaerobic metabolite, enhanced the sustained current at pH 7.2 and 7.1, but not at pH 7.0-6.8. Thus, ASIC3 has appropriate properties to trigger lactic acid-induced nociception during cardiac ischemia. When ASIC3 is co-expressed with ASIC2a, the activation curve shifts to more acidic pH. Thus, the presence of homomers and heteromers of ASIC3 channels provides a cell the ability to generate prolonged current in response to a range of pH below 7.2. [Jpn J Physiol 55 Suppl:S10 (2005)]

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Recently, the molecular substitutes of thermoreceptors of the primary afferents have been clarified. These include six members of the transient receptor potential (TRP) family of nonselective cation channels; TRPV1 (renamed from VR1), TRPV2 (renamed from VRL1), TRPV3, TRPV4, TRPM8 (also known as CMR1), and TRPA1 (renamed from ANKTM1). Several mechanisms have been implicated in underlying the perception of cold, most notably the activation of TRPA1 and TRPM8. Between the two channels, TRPA1 is more likely to be involved in nociception because the temperature threshold for its activation is about 17°C, close to the reported threshold for cold nociceptors. We investigated the expression of TRPA1 and TRPM8 in dorsal root ganglion (DRG) neurons following peripheral inflammation induced by complete Freund's adjuvant (CFA). TRPA1 mRNA expression increased in the small- and medium-sized DRG neurons at 1 and 3 days after peripheral inflammation and the levels were back to normal by 7 days. This up-regulation corresponded well with the development and maintenance of inflammation-induced cold hyperalgesia of the hind paw. In contrast, there was no change in expression of TRPM8 mRNA, or in the percentage of TRPM8-immunoreactive neurons observed over 7 days after the CFA injection. Our data suggests that increased TRPA1 in primary afferent neurons may contribute to the exaggerated cold response observed in this peripheral inflammation model. Silencing of the TRPA1 gene in nociceptors may be potential targets for the development of novel analgesics. [Jpn J Physiol 55 Suppl:S11 (2005)]

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Activity-dependent long-term potentiation (LTP) in the spinal cord is believed to underlie hyperalgesia after inflammation or nerve injury. We report a presynaptic form of LTP in the rat spinal cord slices by visualizing pre- and postsynaptic excitation. To record presynaptic excitation, we stained primary afferent fibers anterogradely from the dorsal root. A single-pulse test stimulation of C fiber-activating strength to the dorsal root (TS) elicited action potential (AP)- or compound AP-like optical signals. After conditioning stimulation (CS, at 2 Hz for 2 min), the presynaptic excitation was augmented. Furthermore, new excitation was elicited in previously silent areas. For postsynaptic recording, projection neurons in spinal lamina I were stained retrogradely from the brain stem. TS elicited AP-like or EPSP-like optical signals in the stained neurons. After CS, the EPSP-like responses were augmented, and previously silent neurons were converted to active ones. The facilitation and generation of new excitation in pre- and postsynaptic excitation were inhibited by nitric oxide (NO) synthase inhibitors and a glial metabolism inhibitor. Application of a NO donor with shorter CS, that normally did not induce facilitation, augmented the excitation and elicited new excitation. Furthermore, the augmentation of presynaptic excitation was inhibited by metabotropic glutamate receptor (mGluR) antagonist, and mGluR agonist without CS augmented presynaptic excitation. This NO-, glia- and mGluR-mediated presynaptic LTP may contribute to the induction of hyperalgesia. [Jpn J Physiol 55 Suppl:S11 (2005)]

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Introduction: The goal of pain research is the establishment of new strategies for relieving pain in patients suffering from intolerable pain. Postoperative pain, that is, surgical incision-induced pain, is commonly encountered in a clinical setting, but little is known about its mechanisms. Methods and results: In this symposium, I will first present results showing that development of primary hyperalgesia and spread of secondary hyperalgesia following an experimental incision in human volunteers are caused by peripheral mechanisms, not by central mechanisms. I will then show that spinal mechanisms of postoperative pain in the adult rat are different from those of tissue injury-induced pain in conventionally used animal models. I will also show that spinal mechanisms of postoperative pain in neonatal rats are different from those in adult rats; one example is that postoperative pain is associated with activation of NMDA receptors of the spinal cord in neonatal rats but not in adult rats. Finally, I will show that different mouse strains have different sensitivities of postoperative pain, reflecting different characteristics of spinal dorsal horn neurons in the strains following surgical incision. Conclusions: The results suggest that pain intensity and pain mechanisms depend on the type of injury and on the age and genetic background of the individual. Since mechanisms of pain seen in a clinical setting may thus be different from those in animal models conventionally used, translational research should play more important roles in the field of pain research. [Jpn J Physiol 55 Suppl:S11 (2005)]

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The nicotinic acetylcholine (ACh) receptor is a ligand-gated ion channel involved in rapid signal transmission at the neuromuscular junction. The structure of the receptor in the tubular crystal has been investigated by cryo-electron microscopy. The mechanism of activation by ACh binding to the receptor is not known in detail, but a precise interpretation of the structural change in the activation is given by the comparison of the extracellular domain of the receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. Substitution in the 3-dimensional maps of the receptor by AChBP mimics the changes seen on activation. When ACh binds, it leads to an extended conformational change, involving 15 degrees rotations of polypeptide chains on the inner pore-facing parts of the extracellular domain of the α subunits. These conformational changes most likely act as the trigger that opens the gate in the membrane-spanning pore of the receptor. The ACh receptor has four predicted membrane-spanning segments, M1-M4, in each subunit. The pore shaped by an inner ring of five α-helices (the second membrane-spanning segment, M2), and an outer ring of fifteen α-helices (M1,M3, and M4), which shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the membrane, formed by weak interactions between neighboring inner helices. When ACh enters the ligand-binding domain, it triggers rotations of the protein chains on the opposite sides of the entrance to the pore. These rotations are communicated through the inner helices and open the pore by breaking the girdle apart. [Jpn J Physiol 55 Suppl:S12 (2005)]

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Mechanosensation is involved in various biological responses such as hearing, gravity perception, and osmoregulation. The membrane excitation on cell deformation is mainly brought about mechanosensitive channels. Two types of mechanosensitive channels have been identified: one that is directly activated by membrane tension and the other that detects stress through the linkage between extracellular and intracellular components. Bacterial mechanosensitive channels, MscL and MscS, belong the former type. In fact, incorporation of amphypath or lisophosphatidylcholine increases the mechanosensitivity of MscS and MscL. Random mutagenesis study indicates that hydrophobic interaction between the residues in the transmembrane domain and the lipid is essential for the mechanosensitivity. Scanning mutagenesis in which the hydrophobic interaction is interfered by asparagine substitution indicates that hydrophobic residues at the periplasmic ends of the transmembrane α helices of MscL are important for mechanosensing. Similar approach on MscS revealed that both ends of the transmembrane α helices have a interaction essential to the mechanosensitivity. These findings are consistent with the idea of the lateral pressure profile of the lipid bilayer, which suggests that membrane protein is subject to negative pressure only near the surface of the bilayer. Therefore, the hydrophobic residues identified at the ends of transmembrane α helices probably receive the force from the lipid and form the mechanosensor of MscS and MscL. [Jpn J Physiol 55 Suppl:S12 (2005)]

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CooA is a transcriptional activator found in a purple non-sulfur photosynthetic bacterium, Rhodospirillum rubrum and is responsible for the expression of the coo operons in response to CO. CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. CooA shows several unique features for the coordination structure of the heme compared with other heme proteins, as described below. First, a proline residue is coordinated to the ferric and ferrous hemes as an axial ligand. CooA is the first and only example of the coordination of a proline residue to a metal ion in metaloproteins. Second, a redox-controlled ligand exchange occurs between Cys⁷⁵ and His⁷⁷ during the change in the oxidation state of the heme in CooA. Cys⁷⁵that iscoordinated to the ferric heme in CooA is replaced by His⁷⁷ upon the reduction of the heme iron, andvice versa. Third, CO can easily react with the ferrous heme in CooA to form the CO-bound form under physiologicalconditions, though the ferrous heme is saturated coordinationally with two endogenous axial ligand. The ligand exchange proceedsbetween Pro² and CO during formation of the CO-bound CooA. I will also discuss some properties of a CooA homologue we have found recently. [Jpn J Physiol 55 Suppl:S12 (2005)]

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Heme enzymes conduct a broad range of functions such as mediators of electron transfer with cytochromes, O₂ storage / carriage with myoglobin and hemoglobin, and catalytic activators of oxygen species with P450s and peroxidases. A new class of heme enzymes involved in intramolecular signal transduction is emerging, known as heme-based sensors. Most of the heme-based sensors are composed of two domains, one is an N-terminal heme-bound domain and the other is a C-terminal catalytic domain. We characterized a heme-regulated phosphodiesterase (Ec DOS) from E. coli. Catalysis of Ec DOS is regulated by the heme redox state in that the enzyme is active when the heme iron is in the Fe (II) form, but not in the Fe (III) complex form, suggesting that the heme is a sensor that regulates catalysis. A change in the heme redox state causes profound protein structural changes of the heme-bound domain, resulting in transmission of signals to the catalytic domain and initiation / termination of the activity. We also identified several genes associated with Ec DOS genes by generating knockout E. coli. and showed that they are involved in cell differentiation / development. Heme-regulated kinase associated with globin translation will be discussed as well. Refs. H. Kurokawa et al. J. Biol. Chem. 279, 20186 (2004).; J. Igarashi et al. J. Biol. Chem. 279, 15752 (2004).; S. Taguchi et al. J. Biol. Chem. 279, 3340 (2004).; T. Yoshimura et al. J. Biol. Chem. 278, 53105 (2003).; A. Sato, et al. J. Biol. Chem. 277, 32650 (2002).; Y. Sasakura et al. J. Biol. Chem. 277, 23821 (2002). [Jpn J Physiol 55 Suppl:S13 (2005)]

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It has been well established that primary role of cellular electrical signal is to change ion fluxes via ion channels and transporters, leading to alteration of intracellular chemical conditions. However, other signaling pathways coupled with membrane potential have not been fully addressed. I will talk about a novel membrane protein, denoted as VSP (voltage sensor-containing phosphatase), which contains an ion channel-like voltage sensor and phosphatase domain similar to PTEN, a tumor suppressor protein. The C-terminal enzyme domain of this protein shows the robust activity of dephosphorylating phosphatidylinositol (3,4,5) trisphosphate, which is known to play a critical role in regulating cell morphology, chemotaxis, apoptosis and cell proliferation. Channel-like domain of VSP exhibits robust asymmetrical charge movements that resemble gating currents of voltage-gated channels. We provide evidence that the enzymatic activity of VSP changes with membrane potential through the conformational change of the voltage sensor. This protein provides a completely novel pathway of cell signaling by which an electrical signal is transduced into biochemical signals without requiring ionic flow. This also gives the first example in which enzymatic activity is regulated by membrane potential through the operation of channel-like voltage sensor, providing a new platform to understand structure-function basis for coupling between voltage sensor and its effecter. Expression in the sperm membrane, gut and human hepatic cancer also suggests its important involvement in reproduction, development and oncogenesis. [Jpn J Physiol 55 Suppl:S13 (2005)]

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Recent evidence indicates the existence of interaction between neurons and glial cells. In view of the bidirectional interaction between these cells, the spatial relation may be important because the close proximity is essential to influence each other. To investigate the mutual interaction, therefore, we chose and focused on the perineuronal glial cells (PGs) in the stratum radiatum of CA1 region of rat sliced hippocampus. As a first step, we attempted to make a classification of the PGs. For this purpose we performed whole-cell recording from the PGs and examined their morphological and immunohistochemical properties. Results indicated that PGs could be classified into two groups based on their membrane properties, one group with relatively low input resistance and relatively deep resting membrane potential, and the other with high input resistance and shallow resting membrane potential compared to the former. Combining the morphological and immunohistochemical studies, we confirmed cells in the former group belong astrocytes and those in the latter group oligodendrocytes. These results will provide cues to distinguish glial cell type during physiological experiment and may contribute to the study to understand the interaction between neurons and glial cells. We also performed dual whole-cell recording from neurons and PGs and revealed the direct evidence of the effect of PGs on the synaptic transmission. [Jpn J Physiol 55 Suppl:S13 (2005)]

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Noxious information from the skin, joints and muscles of the limbs and trunk is carried to the superficial dorsal horn, especially to the substantia gelatinosa (SG) through the fine myelinated Aδ- and unmyelinated C-afferents. SG neurons are interneurons that have different morphological and electrophysiological properties from those of the neurons in other laminae of the spinal dorsal horn and they play an important role in the modulation of the nociceptive transmission receiving inputs from primary afferent fibers. In the present study we investigated how the information from the primary afferents are processed in the spinal cord over several segmental levels, and what the SG neurons receiving excitatory and inhibitory inputs have morphological and electrophysiological characteristics by whole cell patch clamp recordings with patch pipettes filled with neurobiotin in a horizontal slice preparation of adult rat spinal cord. We found (1) that the C-afferent-mediated EPSC in the SG spreads more rostrocaudally than the Aδ-mediated EPSC, and (2) that IPSCs mediated by both Aδ- and C-afferents distribute more rostrocaudally than EPSCs, working as a feedforwad (lateral) inhibition. Furthermore, the SG neurons are classified into four cell types based on the dedritic arborization; islet cell, small islet cell, radial cell, and vertical cell types, which have characteristic inputs depending on Aδ- or C-mediated, and excitatory or inhibitory in nature. [Jpn J Physiol 55 Suppl:S14 (2005)]

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The area postrema (AP) located in the dorsal medulla is one of the circumventricular organs that are characterized by the lack of a blood brain barrier. The neuronal activity of AP neurons contributes to the control of several autonomic functions. The AP have been implicated to contain uniformly shaped small neurons and glia, however, AP cell bodies, which is visually identified in brain slices using Nomarski optics, are different sizes. We, therefore, hypothesized that there may be morphological correlates of the electrophysiological cell classes. Whole-cell recordings were performed to examine the morphological properties of electrophysiologically-classified area postrema (AP) neurons in rat brain slices. Using electrophysiological criteria, AP neurons were subdivided into three groups: 1) cells displaying both the hyperpolarization-activated cation current (I_h) and the fast transient outward current (fast I_to); 2) cells displaying only the fast I_to; and 3) cells displaying only the slow I_to. Majority of group 3 cells showed relatively larger values of the cell soma diameter and the total electrical capacitance. Interestingly, a number of cells from group 1 and 3 but not group 2 were found to extend their dendrites into the nucleus tractus solitarius (NTS) suggesting that AP neurons could receive vagal afferent inputs at their dendritic termini within the NTS. This study indicates the presence of significantly different subpopulations of AP neurons, which are characterized not only electrophysiologically but also morphologically. [Jpn J Physiol 55 Suppl:S14 (2005)]

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Our aim is to analyze the brainstem respiratory network, which involves a variety of respiratory neurons and generates the respiratory rhythm. First, we revealed detailed morphological features of individual respiratory neurons. In anesthetized, paralyzed and artificially ventilated rats, physiologically identified single neurons were labeled juxtamembranously with a mixture of Neurobiotin and HRP. Juxtamembranous injection was used because intracellular injection frequently results in killing of the injected neurons especially if they are small. The mixture of Neurobiotin and HRP improved visualization of myelinated fibers that Neurobiotin alone dose not stain well. Thus, we can reveal the details of dendritic, somal, and axonal morphology of a number of key respiratory neurons. Second, we determined which transmitter is used by inhibitory respiratory neurons: glycine or GABA. Since reliable identification of glycinergic or GABAergic cell bodies by immunohistochemical labeling is difficult, we employed in situ hybridization for mRNAs encoding glycine transporter 2 (GLYT2) as a marker for glycinergic neurons and glutamic acid decarboxylase isoform 67 (GAD67) as a marker for GABAergic neurons. We labeled single respiratory neurons juxtamembranously with Neurobiotin and then performed double-detection of GLYT2 and GAD67 mRNAs using DIG-labeled and FITC-labeled riboprobes. Thus, we determined which respiratory neurons were glycinergic and which were GABAergic. In addition, we found some respiratory neurons that expressed both mRNAs, suggesting they co-release both glycine and GABA. [Jpn J Physiol 55 Suppl:S14 (2005)]

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Cortical GABAergic nonpyramidal cells (NP) regulate activities of cortical pyramidal cells. We investigated morphological dendritic parameters, cross sectional areas of their dendrites, synapse density, in the NPs: basket cell, double bouquet cell, Martinotti cell and so on. The NPs were identified in isolated slices of young rat frontal cortex by whole cell, current-clamp recording, followed by intracellular injection of biocytin. The cells were stained with DAB and embedded in Epon. Many portions of the dendrites from each NP subtype were reconstructed three-dimensionally from serial ultra-thin sections. Each NP dendrites received many excitatory and GABAergic inhibitory synapses. The synaptic densities on their dendrites were almost uniform from proximal to distal dendrites. The dendrite was simply recognized as a pillar of a true circle by most scientists. However the identified NP dendrites were not a right pillar but an ellipse pillar. The distortion rate: a cross sectional area of a reconstructed dendrite which section was ellipse / an area of a right circle with the circumferential length of the reconstructed dendrite, is 0.2–0.9 and they were distorted more in thicker dendrites. The dendritic cross sectional area proximal to a branching point is almost equal to sum of cross sectional area of two daughter branches. Dendritic diameters could not be defined correctly under light microscope due to the distortion, but input conductance estimation using the circumference and cross sectional areas suggested impedance matching between parent and daughter branches at the node according to Rall's idea. [Jpn J Physiol 55 Suppl:S14 (2005)]

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Revealing spatiotemporal aspects of cellular signalling molecules is one of the major challenges to current biomedical sciences. We developed new fluorescent indicators to monitor signalling molecules upstream and downstream of intracellular Ca²⁺ signals within living cells. Using the indicators we have clarified important cellular mechanisms, and some of these are listed below. Imaging of inositol 1,4,5-trisphosphate (IP₃) concentration changes within fine dendrites of cerebellar Purkinje cells identified robust cross talk between metabotropic and ionotropic glutamate receptors for the generation of IP₃ signals. Nitric oxide (NO) generation at the parallel fiber-Purkinje cell synapse was imaged and a NO- and frequency-dependent synaptic plasticity was identified. Using organelle-targeted Ca²⁺ indicators, we clarified the involvement of mitochondrial Ca²⁺ handling in the generation of Ca²⁺ oscillations. We generated a fluorescence energy transfer-based indicator of myosin light chain phosphorylation, which can monitor the phosphorylation state of myosin light chain within intact cells. These results demonstrate the power of fluorescent indicators in physiological studies of a variety of cell functions. These new methods may also shed light on the regulatory mechanism of smooth muscle contraction. [Jpn J Physiol 55 Suppl:S15 (2005)]

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It is known for many years that arachidonic acid (AA) enhances the contraction of smooth muscle by elevating the phosphorylation level of regulatory light chain (RLC) of smooth muscle myosin (SmM). The elevation was reported to be due to enhancement of be myosin light chain (MLC) phosphatase and Rho kinase activities. In spite of the reports, we found that AA stimulated the ATPase activity even if these phosphatase and kinase were absent (J.Pharm.Sci. 94 Suppl. I, 140P, 2004). This stimulation holed true of SmMy that had been fully phosphorylated; AA stimulated the ATPase activity as well as myosin motor activity of the phosphorylated SmMy. We will show that the binding of AA to SmMy caused the stimulation.To relate of such an enhanced motor activity to the hyper-contraction such as the cerebrovascular spasm, the rabbit femoral artery was allowed to contract by K ⁺-induced depolarization in the presence of phosphatase inhibitor of calyculin A and was observed a long-lasting, tonic contraction of the muscle. The addition of 250 μM AA caused further contraction. The western-blot of the muscle showed that RLC remained fully phosphorylated throughout the period of this tonic contraction. We speculate that AA binds to the SmMy, causing further contraction of vascular muscle. [Jpn J Physiol 55 Suppl:S15 (2005)]

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Smooth muscle is generally resistant to the transduction of exogenous genes, which hampers gene manipulation-utilizing analysis of smooth muscle functions. Enzymatically dispersed rat aortic vascular smooth muscle (VSM) cells which are cultured under a serum-free condition on laminin-coated dishes can maintain differentiated phenotypes including high levels of contractile protein and receptor expression. The green fluorescent protein expression in these cells combined with observation using a fluorescent microscope allowed quantitative analysis of the contractile responses. We found that siRNA effectively knocks down gene expression in this differentiated cell system. We previously demonstrated that excitatory receptor agonists such as noradrenaline (NA) induce small GTPase Rho activation in a Ca²⁺-dependent manner, resulting in inhibition of myosin phosphatase (MP) through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the search of signaling molecules involved in the Ca²⁺-dependent Rho activation, we found that silencing a PI3-kinase subtype inhibited NA-induced phosphorylation of MYPT1 and consequent 20-kDa myosin light chain (MLC) phosphorylation and contraction. These findings implicate the novel lipid kinase as an essential regulator of Ca²⁺-dependent, Rho/Rho kinase mediated VSM contraction. By taking advantage of the differentiated VSM cells and gene manipulation techniques, we can dissect signaling pathways for contraction. [Jpn J Physiol 55 Suppl:S15 (2005)]

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While Ca²⁺-dependent contraction of vascular smooth muscle (VSM) regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction plays a pivotal role in abnormal VSM contraction such as vasospasm. As an upstream mediator for such abnormal Ca²⁺-sensitizing pathway, we previously identified sphingosylphosphorylcholine (SPC), which induces the Ca²⁺-sensitization through the activation of ROK, which was blocked by dominant negative ROK. Inhibitors of Src family tyrosine kinases (Src-TKs) blocked the SPC-induced contraction and activation of ROK and tyrosine phosphorylation of several proteins, suggesting the involvement of a Src-TK-mediated tyrosine phosphorylation. Furthermore, using the selective knockdown of the target molecule by siRNA, we determined that Fyn, a member of Src-TK, mediates the SPC-induced ROK-mediated Ca²⁺-sensitization of VSM contraction. Subsequently, in order to screen for the molecular target(s) downstream of Fyn, we performed mass spectrometry of possible signal molecules whose tyrosine was phosphorylated by a SPC/Fyn pathway, and identified p160 and p40. Further characterization and roles of these molecules are currently performed and some of the results will be presented. [Jpn J Physiol 55 Suppl:S16 (2005)]

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Noradrenergic (NA) neurons in the brain stem have been implicated in transmission of the stress and emotional inputs to the hypothalamus. However, it is not yet clear how the NA system functions in relationship with other neuronal systems. Nor is it clear how NA interacts with other neurotransmitters that are co-expressed in the NA neuron. First, rats were exposed to hypotensive hemorrhagic shock, then Fos expression was examined in the brain stem tyrosine-hydroxylase (TH)-immunoreactive neurons. The ratio of Fos-positive neurons increased not only in the A1, A2, and A6 regions following hemorrhagic shock, but also in the prolactin-releasing peptide (PrRP)-containing neurons in the A1 and A2. Thus, PrRP, together with NA, may mediate the visceral stress signals from the medulla to the hypothalamus. Second, a novel approach to examine the functions of the TH neurons is introduced. A transgenic mouse line was used that express green fluorescent protein (GFP) under the control of TH promoter. The embryonic brain of the transgenic mouse was digested with trypsin and the dispersed GFP-positive neurons were sorted by flow-cytometry (FACS). The sorted GFP-positive neurons can be used for many purposes, but preliminary results are presented focusing on the relationship between the TH-neurons and the proopiomelanocortin (POMC) neurons. Expression of melanocortin receptor mRNAs was demonstrated in the GFP-positive (TH) neurons by PCR, suggesting a functional role of POMC peptides in the regulation of TH neurons. [Jpn J Physiol 55 Suppl:S16 (2005)]

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Arginin vasopressin (AVP) as well as corticotropin-releasing hormone (CRH) is well known to modulate ACTH release from the anterior pituitary gland via V1b receptor. The parvocellular neurosecretory cells in the paraventricular nucleus (PVN) of the hypothalamus synthesize AVP and CRH, project their axons to the external layer of the median eminence (ME) and secrete AVP and CRH into the portal blood flow. The synthesis and secretion of AVP and CRH are modulated by various stressors. We examined the effects of adrenalectomy (ADX), endotoxin shock and chronic inflammatory stress on the expression of AVP and CRH in the PVN of rats. In AVP-enhanced green fluorescent protein (eGFP) transgenic rats eGFP was marked increased in the external layer of the ME and the parvocellular CRH-producing neurons of the PVN 5 days after bilateral ADX. After acute and chronic inflammatory stress one of novel G-protein coupled receptor ligands, galanin-like peptide (GALP), expressed in the pituicytes of the posterior pituitary gland. In in vitro preparation GALP stimulated AVP release from the posterior pituitary. During chronic inflammatory stress such as adjuvant arthritis the expression of the AVP gene in the PVN was upregulated, while the expression of the CRH gene in the PVN was downregulated. These results suggest that AVP and CRH in the PVN may have different regulatory mechanism under acute and chronic inflammatory stress. The monitoring the eGFP expression in the PVN and the ME in AVP-eGFP transgenic rats may give us new information about the physiological role of central AVP system under acute and chronic stress. [Jpn J Physiol 55 Suppl:S16 (2005)]

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Food intake affects neuroendocrine and behavioural responses to stressful stimuli. Peripheral administration of cholecystokinin, a satiety factor, activates prolactin-releasing peptide (PrRP) neurones. PrRP neurones have been suggested to play a facilitative role in ACTH and oxytocin release after stressful stimuli. We investigated whether PrRP also plays a role in behavioural stress responses. We examined effects of icv administration of PrRP and anti-PrRP antibody upon fear/anxiety behaviour in response to stressful stimuli in rats. Icv administration of PrRP reduced anxiety-related behaviour in an elevated plus maze test, while administration of anti-PrRP IgG increased it. On the other hand, PrRP administration increased plasma oxytocin concentrations and administration of anti-PrRP IgG attenuated plasma oxytocin increase after fear-related stimuli. All these data suggest that PrRP neurones have differential effects upon neuroendocrine and behavioural responses to stress. PrRP neurones may play a role in food intake-associated modulation of neuroendocrine and behavioural responses to emotional stress. [Jpn J Physiol 55 Suppl:S17 (2005)]

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Here we introduce the behavioral study of the mice lacking oxytocin (OXT) or OXT receptor (OXTR) genes. Oxtr-/- males showed elevation in their aggression, with normal plasma concentration of testosterone. In contrast, Oxt-/- males showed no elevated aggression, demonstrating discrepancy between the functions of oxt and oxtr genes. In detection of remanent receptor activity for OXT, no binding capacity to OXT in brain of Oxtr-/- mice was found. In the receptor mutants, concentration of OXT and AVP in pituitary, and of plasma OXT, and expression level of mRNAs of OXT and AVP in hypothalamus were all not altered. We suspected the influence of maternal OXT through placenta on the development of fetus brain to establish normal social behavior in Oxt-/- male. Oxt-null males from cross of Oxt-null parents, showed elevated aggression, according to our expectation. On the other hand, Oxtr-/- female showed abnormal maternal behavior in retrieving and crouching over the pups. Virgin Oxtr-/- females also displayed a similar defect, suggesting that OXTR is required for nurturing responses to pups outside the physiological context of pregnancy and parturition. In addition, decrease in isolation-induced ultrasonic vocalizations and increase in locomotor activity of infant males, and impairment of social discrimination as well as Oxt-/- were found in Oxtr-/- male. Our study demonstrates that OXTR may play a critical role in regulating several social behaviors, related to developmental psychiatric disorders. [Jpn J Physiol 55 Suppl:S17 (2005)]

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Emotional responses are thought to play a vital role in survival and in our ability to adapt to our environment. It has been suggested that the mechanism by which we process emotional information consists of evaluative, experiential, and expressive components. However little is known about human emotional responses in the brain. From these standpoints, we have done neuroimaging analyses to explore human emotional responses in the brain used by a functional magnetic resonance imaging (fMRI) in healthy volunteers and depressed patients. Brain activation was measured in healthy subjects while the subjects performed a warned reaction task using pictures which evoked emotionally pleasant or unpleasant content, because evaluation in the present of emotional events that are destined to happen in the future is a necessary form of adaptive behavior. These data suggest that left prefrontal cortex (PFC) activation is associated with the expectancy of pleasant stimuli and that right PFC activation is associated with the expectancy of unpleasant stimuli. We also investigated the brain activity associated with the expectancy of emotional stimuli in depressed patients. In contrast to healthy control, depressed subjects showed attenuated activation in the left PFC during pleasant stimuli and increased activation in the right PFC during unpleasant stimuli. These findings suggest that abnormal emotional responses in depressed patients are associated with altered brain activities within these regions during the warned reaction task. [Jpn J Physiol 55 Suppl:S17 (2005)]

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The endoplasmic reticulum (ER) appears as a three-dimensional network formed by an endomembrane. In neurones, the ER extends from the nucleus and the soma to the dendrites and, through the axon, to presynaptic terminals. The ER lumen is packed with enzymes that allow synthesis and post-translational folding of proteins. At the same time, the ER is involved in neuronal Ca²⁺ signalling via Ca²⁺-induced Ca²⁺ release or InsP₃-induced Ca²⁺ release, controlled by two subsets of Ca²⁺ release channels residing in the ER membrane, the ryanodine receptors (RyRs) and the InsP₃-receptors (InsP₃Rs). The ER Ca²⁺ store emerges as a single interconnected Ca²⁺ pool, although the RyRs and InsP₃Rs show heterogeneous localisation in distinct cellular sub-compartments, conferring thus specificity in local Ca²⁺ signals. The intimate mechanisms of ER integration remain largely undiscovered, yet a central role for Ca²⁺ is emerging. First, Ca²⁺ is a key input and output signal of the ER as cytosolic Ca²⁺ increases affect its concentration in the ER, and in turn, the ER Ca²⁺ release and uptake influence the cytosolic Ca²⁺ concentration. Second, the intra-ER chaperones are Ca²⁺ binding proteins, and changes in ER Ca²⁺ content affect their functional activity. Therefore, fluctuations in the ER Ca²⁺ concentration provide the link between rapid signalling and long-lasting adaptive responses. The disruption of intra-ER Ca²⁺ homeostasis can be involved in neurodegenerative disorders such as diabetic peripheral neuropathies and Alzheimer disease. [Jpn J Physiol 55 Suppl:S18 (2005)]

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In neurons of the central nervous system, the integrative function depends on the somato-dendritic distribution and properties of voltage-gated ion channels, pumps and transporters. These membrane-associated proteins are highly regulated by an intracellular second-messenger system consisted of a variety of functional molecules and divalent cations including Ca²⁺. In this talk, particular emphasis is placed on results concerning Ca²⁺ dependent modulation of excitability in CNS dendrites. We examined how Ca²⁺ signals play pivotal roles in 1) the hippocampal neuronal death after transient forebrain ischemia, and in 2) protein kinase C (PKC) signaling in cerebellar Purkinje cells. A combination of intracellular recording and fluorescence Ca²⁺ imaging was applied to each neuron in the brain slice obtained from rodents. In the former study, we found that excitatory synaptic inputs induced relatively larger calcium transients in the apical dendrites of post-ischemic CA1 neurons than in those of normal neurons, although somatic depolarization induced smaller increases in dendritic signals than those of the control. These results suggest that the Ca²⁺ homeostasis at the apical dendrite is impaired in CA1 pyramidal neurons following transient ischemia. In the later study, we found propagation of Ca²⁺ dependent γPKC translocation along Purkinje cell dendrites. The present results suggest that local intracellular signals activated by parallel fiber input could transmit to the other parts of cell through dendritic arbor. Our findings obtained from those studies may provide us a new insight for understanding molecular mechanisms of the integrative function in CNS dendrites. [Jpn J Physiol 55 Suppl:S18 (2005)]

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Recent studies have revealed that endocannabinoids (eCB) are released from postsynaptic neurons and suppress transmitter release in various regions of the CNS. In this presentation, we introduce our recent studies on the eCB-mediated retrograde suppression and its regulation by Ca²⁺ in hippocampal neurons. We used highly cannabinoid-sensitive inhibitory synapses as biosensor of released eCB. We found that eCB release is induced through two different pathways. One is dependent on phospholipase Cβ1 (PLCβ1), and triggered by activation of G_q-coupled receptors such as group I metabotropic glutamate receptors and M₁/M₃ muscarinic receptors. This receptor-driven eCB release is dependent on physiological levels of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and markedly enhanced by depolarization-induced [Ca²⁺]_i elevation. The other pathway is independent of PLCβ1, and triggered by [Ca²⁺]_i elevation without receptor activation. The [Ca²⁺]_i level required for this pathway is significantly higher than that required for the enhancement of PLCβ1-dependent pathway. The released eCB acts retrogradely onto presynaptic cannabinoid CB1 receptors and suppresses transmitter release. These results indicate that transmitter release is regulated by postsynaptic G_q-coupled receptors and [Ca²⁺]_i through the retrograde eCB signal, and that PLCβ1 serves as a coincidence detector through its Ca²⁺ dependency for triggering eCB release in hippocampal neurons. [Jpn J Physiol 55 Suppl:S18 (2005)]

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The hypothalamic suprachiasmatic nucleus (SCN) has a pivotal role in the mammalian circadian clock. SCN neurons generate circadian rhythms in action potential firing frequencies and neurotransmitter release, and the core oscillation is thought to be driven by “clock gene” transcription-translation feedback loops. Various neurotransmitters, receptors and second messenger systems have been proposed for the signal transduction mechanisms in SCN neurons. Among these, glutamate-induced Ca²⁺ flux is one of the most important pathways to regulate the circadian oscillations. Cytosolic Ca²⁺ mobilization followed by receptor stimulation thus has been studied extensively in SCN neurons, whereas contribution of steady-state cytosolic Ca²⁺ levels to the rhythm generation was unclear. Therefore, we challenged ultra long-term (up to 2 weeks) Ca²⁺ imaging experiment in cultured SCN neurons with a Ca²⁺ indicator protein, Yellow Cameleon 2.1, and found robust circadian rhythms in the cytoplasmic concentration of Ca²⁺ (Ikeda et al., Neuron 38:252-263, 2003). The circadian Ca²⁺ rhythms are driven by the release of Ca²⁺ from ryanodine-sensitive internal stores and resistant to the blockade of action potentials. These results raise the possibility that clock gene translation/transcription loops may interact with the autonomous Ca²⁺ oscillations in the production of circadian rhythms in SCN neurons (Ikeda, Trends Neurosci 26:654, 2003), and thus demonstrate that Ca²⁺ is a key intracellular messenger for the circadian clock system. [Jpn J Physiol 55 Suppl:S19 (2005)]

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Prolonged wakefulness or sleep deprivation induces fatigue effects, such as drowsiness and a decrease in learning and memory in humans. A person taking a nap after sleep deprivation shows deep sleep, i.e., non-rapid eye movement (non-REM) sleep, for recovery from fatigue. However, the mechanism involved in these responses remains unclear. Previously, we demonstrated that prostaglandin (PG) D₂-induced non-REM sleep was mediated by DP receptor (DPR) in mice. Sleep deprivation by gentle handling in the light period increased the PGD₂ content in the brain of wild-type (WT) mice and induced the rebound of both non-REM sleep and REM sleep in a sleep deprivation-time dependent manner. However, the gene-knockout (KO) mice for PGD synthase (PGDS) or DPR did not show any rebound of non-REM sleep after sleep deprivation, indicating PGD₂ to be crucial for induction of the non-REM sleep rebound to recover from sleep deprivation stress. Furthermore, we investigated the effect of sleep deprivation on spatial learning and memory for 5 days, as assessed by the Morris water maze test with the hidden platform. When we performed 6 h sleep deprivation before swimming from Day 2 to Day 5, WT mice increased drowsiness day by day to be inactive during the sleep deprivation treatment, whereas PGDS KO mice remained active even on Day 5. The sleep deprivation suppressed the decline of the escape latency as a function of training days for WT mice, but not at all for the KO mice, indicating that sleep deprivation induced PGD₂-mediated drowsiness and impaired spatial learning and memory. [Jpn J Physiol 55 Suppl:S19 (2005)]

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The orexinergic system has a crucial role in regulation of wakefulness and a deficiency of orexinegic system causes narcolepsy. The orexinergic neurons project to and excite the brainstem aminergic and cholinergic neurons. So, the mechanisms of wakefulness regulation are well understood by these excitatory projections. However, current knowledge that orexins excite the cholinergic neurons does not explain the mechanisms of the suppression of cataplexy by the orexinergic system, because activation of the brainstem cholinergic neurons induces sudden reduction of muscular tonus (muscular atonia), equivalent to cataplexy. In decebrated cats, injection of orexin A into the muscular atonia inducing area (pedunculopontine tegmental nucleus; PPN) suppressed the cataplexic state. The effect was reversed by additional injections of bicuculline (GABA_A antagonist) into the PPN. The similar effect of orexin was observed when injected into the substantia nigra pars reticulata (SNr), one of the origins of GABAergic input to the PPN, and was also reversed by bicuculline into the PPN. In freely moving rats, orexin injection into the PPN increased GABA release in the PPN which was measured by microdialysis-HPLC method. These findings suggest that the orexinergic system suppresses the atonia system by enhancement of GABAergic effects upon the cholinergic PPN neurons. In the absence of orexin, the attenuation of GABAergic excitability would result in cataplexy. [Jpn J Physiol 55 Suppl:S19 (2005)]

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In the hibernation-regulation system, we have shed light on the activation of the A1 receptor by adenosine for the generation of hypothermia in the entrance phase of hibernation, and the activation of the μ1-opioid receptor by opioid peptides for maintenance of hypothermia in the maintenance phase. Moreover, recent studies in our laboratories have demonstrated that tyrosine-releasing hormone (TRH) is a key endogenous substance involved in the elevation of body temperature in hamsters during the arousal phase. It is well known that central prostaglandin (PG) D2 and the adenosine systems play important roles in sleep induction. In addition, we have clarified that the central opioidergic systems also contribute substantially to sleep maintenance. However, PGE2 and the histaminergic systems play critical roles in awakening from sleep. Similar histaminergic systems are thought to contribute to awakening from hibernation as well. There are many similarities in the central control mechanisms of sleep and hibernation. Although the arousal system (TRH system) acts tonically during hibernation, hibernation is in fact maintained by inhibiting the arousal system in phases via activations of the adenosine and opioidergic systems. All these systems are believed to collaboratively modulate sleep regulation. [Jpn J Physiol 55 Suppl:S20 (2005)]

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