In visual selection, stimulus uniqueness (oddity) is a key factor; an object with a feature contrast to the neighbor objects pops out and draws attention automatically. Although a natural scene may contain multiple objects odd in different stimulus dimensions, if we have knowledge about in what dimension the relevant objects are odd, we more easily detect them by attending to that dimension. Here, we investigated how visual responses were modulated by attention to the feature contrast in a particular dimension. In the present experiment, two odd stimuli, each unique in color and shape dimension, were presented with other identical stimuli. Monkeys were trained to search for the target stimulus that was odd in the relevant search dimension, while the search dimension was switched between shape and color dimension. Neuronal recording was made from area V4 and the frontal eye field (FEF). We found that, on average, V4 neurons showed the response enhancement when the relevant odd (target) stimulus fell in the receptive filed. However, the significant response enhancement occurred only when one particular odd stimulus became the target, suggesting that this neural modulation was exerted as the result of an interaction between the types of stimulus oddity and the search dimension. In contrast, FEF neurons showed the significant response enhancement for the target, irrespective of the types of stimulus oddity. [Jpn J Physiol 54 Suppl:S35 (2004)]

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Although even fish can convert large-field visual motion into eye velocity, the ability to track a small object moving across rich visual environment is unique to the primates. This indicates that the generation of smooth pursuit eye movements requires a number of neural processes beyond simply transforming visual motion into eye motion. Those processes differentiate sensory-guided voluntary movements from reflexes.
Recent studies of smooth pursuit have shown that there are at least two different signal flows in this system; one converts visual motion into command of eye velocity, while the other regulates the strength of this conversion. The former process is likely shared with the visually-guided reflexive eye movements such as the optokinetic response and the ocular-following response. The latter may play roles in choosing a response to given visual inputs, maximizing the sensorimotor conversion for visual motion derived from a selected object or location. I will review recent stimulation experiments showing that the frontal eye fields (FEF) have access to both pursuit pathways, and that the FEF can regulate the gains of both the initiation and the maintenance of pursuit. Taken together with the results of other behavioral and recording studies, I will discuss possible signal flows through the FEF that regulate smooth pursuit eye movements. [Jpn J Physiol 54 Suppl:S35 (2004)]

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The cholinergic pedunculopontine tegmental nucleus (PPTN) is one of the major ascending arousal systems in the brainstem, and is linked to motor, limbic and sensory systems. We hypothesize that PPTN is related to integrative control of motor behavior, and present a new model of the PPTN's involvement in the control of arousal, attention and reinforcement aspects of motor behavior, with a focus on the control of saccades. We investigated the relationship between the task performance and activity of PPTN neurons during visually-guided saccade tasks and the effects of reward schedule on them. We found that when the reaction time to the fixation point (RTFP) elongated, the animal tended to fail to complete the task. Activities of a group of PPTN neurons at the FP onset were larger in the success trials than in the error trials irrespective of the current eye position. These results suggested that the activity of the PPTN neurons is closely linked to the RTFP and encodes the animal's vigilance level. To investigate the correlation of PPTN neuron activity and its drive by motivation, we changed the volume of reward systematically and compared the reward volume with the RTFP, task performance and the PPTN neuronal activity in the following trial. Increase in the reward volume enhanced the PPTN neuron activity, decreased the RTFP and decreased the error rate in the next trials. [Jpn J Physiol 54 Suppl:S35 (2004)]

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The molecular characterization of the genes encoding the transient receptor potential (TRP) cation channels found in Drosophila photoreceptors gave rise to a systematic cloning strategy for mammalian isoforms. Using expressed sequence tag (EST) and genomic database searches, at least 20 new mammalian TRP-related genes have been cloned and the resulting channels characterized. The focus of this presentation will be on TRPM7 (LTRPC7, ChaK1, TRP-PLIK), a member of the TRPM channel subfamily. TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg²⁺ and Mg·ATP. Most interestingly, TRPM7 is a dual-function plasma membrane protein that acts both as an ion channel and as a protein kinase, whereby the protein's C-terminal alpha kinase domain proves not to be essential for the gating of the channel. Electrophysiological characterization of TRPM7 shows that the ion channel functions as a Ca²⁺- and Mg²⁺-permeable cation channel. Thus, endogenous TRPM7-like currents have been named MagNuM, for Magnesium-Nucleotide-regulated Metal ion currents. TRPM7 plays an essential role in cell proliferation and viability, which is implicated by the fact that cells made deficient in TRPM7 experience grow arrest within 24 hours and eventually die. Furthermore, most recent data provide evidence that TRPM7 regulates Mg²⁺ homeostasis in vertebrates. [Jpn J Physiol 54 Suppl:S36 (2004)]

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Magnesium must be actively extruded from the cell interior to keep cytoplasmic free Mg²⁺ concentration of about 1 mM or lower. One candidate for such active Mg²⁺ extrusion mechanisms is the Na⁺-Mg²⁺ exchange that has been postulated in many types of cells, including cardiac myocytes. However, the genes encoding the Mg²⁺ transporters have not yet been identified, and the Mg²⁺ transport is only poorly characterized. In this presentation, we will focus on modulation of the Mg²⁺ transport by membrane potential and intracellular/extracellular concentrations of Mg (Mg²⁺_i/Mg²⁺_o) and Na (Na⁺_i/Na⁺_o) ions in cardiac myocytes. We measured Mg²⁺_i of single ventricular myocytes of rat using the fluorescent Mg²⁺ indicator furaptra (mag-fura-2). After the cells were loaded with Mg²⁺ by incubation in the solution containing high Mg²⁺ concentration, and the rate of fall in Mg²⁺_i upon reduction of Mg²⁺_o was analyzed as an index of the Mg²⁺ efflux rate. Cell membrane depolarization slightly but significantly facilitated the Mg²⁺ efflux in the cells patch-clamped with the pipette containing amphotericin B. Activity of the Mg²⁺ transport was found to require extracellular Na⁺ with a half maximum Na⁺_o of ~50 mM, but was inhibited by intracellular Na⁺ with a half inhibitory Na⁺_i of ~40 mM. The transport was half-maximally activated by ~1.5 mM Mg²⁺_i, and was half-inhibited by ~10 mM Mg²⁺_o. In the light of these and other findings, contribution of the Na⁺-Mg²⁺ exchange as a major Mg²⁺ efflux pathway and intracellular Mg²⁺ buffering/sequestration to cellular Mg²⁺ regulation will be reviewed and discussed. [Jpn J Physiol 54 Suppl:S36 (2004)]

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Intracellular Mg²⁺ is known to affect many intracellular enzymes and other important cellular processes. In ³¹P-NMR spectrum, the observed chemical shifts of the ATP peaks indicate the ratio of metal-free to total ATP. Thereby, the intracellular Mg²⁺ concentration ([Mg²⁺]_i) can be estimated from the chemical shifts of ATP peaks assuming the dissociation constant of MgATP (K_D^"MgATP"). pH is known to affect the K_D^"MgATP". McGuigan's group have shown that K_D^"MgATP" at 25 and 37°C are described as quadratic functions of pH (Zhang et al. Anal Biochem 251, 246-250, 1997). In this presentation, we estimate [Mg²⁺]_i from the chemical shifts of intracellular ATP, using the formula modified for our experimental conditions. We will communicate the re-evaluated resting [Mg²⁺]_i in smooth and cardiac muscles, and also show various modulations of [Mg²⁺]_i caused by changing ionic substitutions and applying important hormones and drugs. [Jpn J Physiol 54 Suppl:S36 (2004)]

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Mg ions have been recognized as the important divalent cations for various physiological functions including the regulation of enzymatic activity, energy charge and signal transduction in cells. Recently, we have developed several types of novel Mg indicators, and we found that KMG-104 is a promising candidate for investigating intracellular Mg mobilization because of its high-sensitivity for Mg but not for Ca. The simultaneous measurement of Ca and Mg concentration with the combination of laser-photolysis of several caged compounds and dual-view imaging technique allows us to systematic investigation of divalent cation mobilization in cells. Transient increase of intracellular Ca and IP3 by photolysis induced no intracellular Mg concentration change in PC12 cells. This result indicates that Mg mobilization mechanism has no concern with Ca mobilization. Transient increase of intracellular Mg and Ca concentration was observed when FCCP, an uncoupler of oxidative phosphorylation at the inner membrane of mitochondria, was bath-applied. However, oligomycin, an inhibitor of ATP synthase, did not induce Mg and Ca concentration. These observations could be explained that intracellular Mg is not released from ATP during short-term exposure to oligomycin, and mitochondria are a plausible source of intracellular Mg release. We also observed that FCCP-induced intracellular Mg increase gradually returns to the basal level because of a Na/Mg ion exchanger on the cell membrane (Kubota et al. 2003). The simultaneous imaging of Ca and Mg ions will be revealed the cross talk of these important cations in single cells. [Jpn J Physiol 54 Suppl:S37 (2004)]

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Magnesium has an important influence on the excitability and viability of central neurons. Removal of extracellular magnesium results in activation of the NMDA subtype of glutamate receptor followed by excitotoxic neuronal death. We have investigated the role of magnesium using cultured neurons from embryonic rat brain along with fluorescent dyes to report intracellular magnesium concentrations. Our studies showed that magnesium can enter neurons through NMDA receptors under the appropriate ionic conditions, and that elevation of intracellular magnesium results in neuronal death. The mechanism by which this injury occurs is unknown, although we are currently exploring the impact of elevated intracellular magnesium on the function of neuronal mitochondria. These studies also suggest the presence of a sodium-magnesium exchange mechanism responsible for removal of magnesium from the cytoplasm. Interestingly, several studies have reported that magnesium is lost from intracellular compartments that include both mitochondria and the cytoplasm in conditions associated with neuronal injury. The transport mechanisms responsible for magnesium loss are not known. Collectively, these studies demonstrate that intracellular magnesium is an important variable controlling neuronal viability, but it remains to be seen whether magnesium overload or magnesium loss is the critical parameter. [Jpn J Physiol 54 Suppl:S37 (2004)]

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Physiological functions of transient receptor potential (TRP) Ca²⁺-permeable cation channels must be studied in "contexts" of dominant signaling pathways. In non-excitable cells, receptor-activated Ca²⁺ signalling comprises initial transient responses followed by a Ca²⁺ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP₃) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca²⁺ entry greatly enhanced IP₃ production, which terminated in phospholipase Cγ2 (PLCγ2)-deficient cells. Association between receptor-activated TRPC3 Ca²⁺ channels and PLCγ2, which cooperate in potentiating Ca²⁺ responses, was demonstrated by coimmunoprecipitation. PLCγ2-deficient cells displayed diminished Ca²⁺ entry-induced Ca²⁺ responses. However, this defect was canceled by suppressing IP₃-induced Ca²⁺ release, implying that IP₃ and IP₃ receptors mediate the second Ca²⁺ phase. Furthermore, confocal visualization of PLCγ2 mutants demonstrated that Ca²⁺ entry evoked a C2 domain-mediated PLCγ2 translocation toward the plasma membrane in a lipase-independent manner to activate PLCγ2. Strikingly, Ca²⁺ entry-activated PLCγ2 maintained Ca²⁺ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca²⁺ entry with PLCγ2 translocation and activation controls the amplification and coordination of receptor signalling. [Jpn J Physiol 54 Suppl:S37 (2004)]

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Intracellular Ca²⁺ forms spatial and temporal signals as a propagating Ca²⁺ wave and repetitive Ca²⁺ spikes, respectively. The mammalian egg is a good experimental system, since it has a large size and exhibits Ca²⁺ oscillations associated with a Ca²⁺ wave in each Ca²⁺ spike during fertilization as a pivotal signal for egg activation. Each Ca²⁺ spike is due to Ca²⁺ release from the ER mainly via the IP₃ receptor type 1. Ca²⁺ oscillations are induced by a cytosolic sperm factor that is introduced into the egg cytoplasm upon sperm-egg fusion. Recently, Saunders et al. have found a novel sperm-specific phospholipase C isoform, PLCζ which induces Ca²⁺ oscillations and early embryonic development when expressed in a mouse egg by injection of RNA encoding PLCζ. We expressed PLCζ fused with a fluorescent protein Venus and estimated PLCζ concentration for initiation of Ca²⁺ oscillations. Interestingly, expressed PLCζ-Venus translocated into the pronucleus formed after egg activation. We also synthesized recombinant PLCζ using baculovirus/Sf9-cell expression system. Ca²⁺ oscillations were produced by injection of PLCζ protein. PIP₂ hydrolysis assay in vitro showed that PLCζ has 70% maximal activity at 100 nM [Ca²⁺] that is usually the basal [Ca²⁺]_i level of cells. These characteristics not only qualify PLCζ for a strong candidate of the sperm factor but also provide a model of induction and regulation of Ca²⁺ oscillations. [Jpn J Physiol 54 Suppl:S38 (2004)]

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IP3 receptor (IP3R) is a Ca2+ channel localized on smooth endoplasmic reticulum and is involved in Ca2+ signaling and it plays essential role in development and brain function. We have already found by using IP3R type 1 (IP3R1) deficient mice that IP3R1 is essential for brain development and neural plasticity and other various cell functions. To understand fully the property of IP3R, it is extremely necessary to characterize the biochemical property of IP3R. We purified IP3R1 from the cerebellum and found that the dynamic conformation change occurs in the presence and absence of Ca2+. When it is trypsized into several pieces, they assemble together to function as intact IP3 induced Ca2+ release machinery. These unique biochemical properties may be related in producing Ca2+ wave and oscillation. Recently, we succeeded in screening and cloning many molecules which associate with IP3R1. 4.1N which binds to C-Terminus of IP3R1 is found to be important for translocation of IP3R1 to plasma membrane.IRBIT (IP3 receptor binding protein released with inositol 1,4,5-trisphosphate) binds to the IP3 binding core of IP3R in a phosphorylated form and is released with IP3. IRBIT may regulate IP3 induced Ca2+ release and may work as a third messenger. In addition, new molecules that regulate Ca2+ signaling will be discussed. [Jpn J Physiol 54 Suppl:S38 (2004)]

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Ca²⁺ signal is an extremely versatile intracellular regulator controlling various important cell functions such as contraction, secretion, immune responses and synaptic plasticity. The basis for the versatility has been attributed to the capability of Ca²⁺ signals to generate huge variations of spatiotemporal signalling patterns including Ca²⁺ oscillations. However, it remains elusive how Ca²⁺ oscillations are generated and how they are interpreted. We made two approaches to gain an insight into this problem. First, we studied the role of feedback regulation of the inositol 1,4,5-trisphosphate receptor (IP₃R) in Ca²⁺ oscillations. IP₃R is sensitive not only to IP₃ but also to the cytosolic Ca²⁺ concentration. We identified the Ca²⁺ sensor region of the IP₃R through mutagenesis experiments. Ca²⁺ oscillations was inhibited in cells expressing a mutant IP₃R with a decreased Ca²⁺ sensitivity. Therefore, Ca²⁺-mediated feedback regulation of IP₃R is essential for the generation of Ca²⁺ oscillations. We next studied the molecular mechanism that decodes the Ca²⁺ oscillations. For this purpose we used the nuclear factor of active T cells (NFAT), which is known to drive Ca²⁺ oscillation frequency-dependent transcription. Our results showed that NFAT functions as a working memory of Ca²⁺ signals thereby decoding the Ca²⁺ oscillation frequency and regulating its nuclear localization. We also showed that Ca²⁺ oscillation is a cost-effective way to control Ca²⁺-dependent cell functions. These studies provide an insight into the molecular mechanisms for generation and interpretation of Ca²⁺ oscillations. [Jpn J Physiol 54 Suppl:S38 (2004)]

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The PAS domain is a versatile protein fold that is found in many archaeal, bacterial, and plant proteins. It acts as a ligand-binding environmental sensor for detecting environmental changes in light intensity, oxygen concentration, and redox potentials. In mammals, several PAS domain-containing factors function as sensor molecules, including HIF, NPAS2, and AhR. In the mammalian circadian system, seven Clock gene products are PAS factors: PER1, PER2, PER3, CLOCK, NPAS2, BMAL1, and BMAL2. These clock genes are involved in the generation of circadian oscillations, the entrainment of animals to 24-hour light-dark cycles, and the output of signals from the central clock to the periphery. To investigate the system regulating the circadian clock with input from the environment, we established a system to monitor clock oscillations using a Bmal1 promoter construct as a reporter. Utilization of this system should facilitate the identification of factors that affect the circadian phase or amplitude. [Jpn J Physiol 54 Suppl:S39 (2004)]

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Physiology is an integrative science. The study on circadian clocks, those having period of approximately 24 hours, is a representative field of this integrative analysis from genes to behavior or physiology. Circadian rhythms are generated in pacemaker cells, the hypothalamic suprachiasmatic nuclei (SCN) in mammals, and entrained by environmental cues such as light and temperature. The output of a circadian oscillation appears as locomotive activity, hormonal secretion, the sleep-wake cycle, and other physiological processes. Recent molecular dissection for last several years has revealed that circadian rhythms are based on transcriptional regulation of clock and clock-controlled genes, and the interlocked feedback- and feedforward-loop of transcription is the basic concept of the circadian oscillator, conserved across the species. For the well-known example, the transcription of Per1 is activated by the binding of the CLOCK/BMAL1 hetero-complex, both of which are bHLH-PAS proteins, to the E-boxes in the promoter region of Per1. The translated PER1, together with other clock proteins, is returned into the nucleus to suppress its own transactivation, resulting in closing of a PER1 loop. On the other hand, BMAL1, which is a positive element to PER1, has itself another loop. New transcriptional regulation and oscillation by other molecules including orphan nuclear receptors, Rev-erb and ROR, in the BMAL1 loop will be described and discussed. [Jpn J Physiol 54 Suppl:S39 (2004)]

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New gene expressions play an essential role in consolidation of conditioned fear memory; a form of Pavlovian conditioning between a training context (CS) and an aversive stimulus such as footshock (US). To understand the molecular mechanisms of gene expression regulation during memory consolidation, we have investigated roles of transcription factor CREB using conditional mutation. We examined the both effects of lost and gain of CREB function. Indeed, we derived transgenic mice expressing inducible CREB repressor in forebrain. Dominant negative CREB (CREBS133A) fused with tamoxifen-dependent mutant of ER ligand binding domain is expressed under the control of alpha CaMKII promoter. These transgenic mice show that activation of CREB repressor by tamoxifen leads to severe impairment of memory consolidation. In contrast, forebrain specific expression of active CREB mutant having high affinity with CBP leads to the enhancement of memory consolidation. Together, these data indicates that CREB plays essential roles in memory consolidation, and suggests that CREB functions as molecular switch to determine the strength of memory consolidation. To further understand the regulation of CREB activity during memory consolidation in vivo, we have developed the method for monitor and visualization of CREB activity using fluorescence resonance energy transfer (FRET) from CFP to YFP. Indeed, we are trying to monitor the interaction of CREB with CBP, that is essential for activation of CREB mediated transcription, and investigating the mechanism of CREB activation by various signal transduction pathways in vivo. [Jpn J Physiol 54 Suppl:S39 (2004)]

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Bipolar disorder is a severe mental illness characterized by recurrent episodes of mania and depression affecting about 1% of the population. The pathophysiology of this illness is unclear. Strong effects of genetic factors have been evidenced by family, twin, and linkage studies. The mood stabilizers such as valproate, lithium and carbamazepine, are effective for this illness. We have recently reported that XBP1, a pivotal gene in endoplasmic reticulum stress response, is one of key genes associated with this illness (Kakiuchi et al, Nature Genetics, 2003). Using DNA microarray analysis of lymphoblastoid cells derived from two pairs of monozygotic twins discordant for the disorder, we found down-regulated expression of ER stress response-related genes in both the affected twins. A polymorphism (Rs2269577, C to G) in the promoter region of XBP1, losing the putative XBP1 binding site, was associated with this illness by genetic analyses. XBP1-dependent transcription activity of the G allele, the risk allele for bipolar disorder, was lower than that of the C allele, and in the cells having G allele, induction of XBP1 expression upon ER stress with thapsigargin was remarkably reduced. Valproate rescued the impaired ER stress response by inducing ATF6, the upstream gene of XBP1. Our results suggest a pathophysiological role of ER stress response pathway, especially the signaling involving XBP1 gene, in bipolar disorder. Even the function of XBP1 and ER stress response pathway in normal brain has not been clarified yet and it would be a good theme for understanding the pathophysiology of this illness from new aspect. [Jpn J Physiol 54 Suppl:S40 (2004)]

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Endocrine disruptors are usually considered to express their biologically adverse effects through taking over the nuclear receptors which have their cognate ligands, resulting in untimely activation or suppression of their transcriptional activities. 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), one of most toxic manmade chemicals, binds arylhydrocarbon receptor (AhR or dioxin receptor) with an extremely high affinity and expresses pleiotropic biological activities.
From their primary structures, AhR belongs to a distinct group of a super-gene family with the bHLH-PAS structural motif from that of nuclear receptors. PCB, 3-methylcholanthrene and benzo(a)pyrene are also ligands to AhR and these polycyclic aromatic chemicals are considered to display pleiotropic biological effects such as induction of drug-metabolizing enzymes, teratogenesis, tumor promotion, immuno-deficiency and estrogenic action.
Generation of the AhR-deficient mice revealed these toxic effects are mediated by AhR, because Ah(-/-) mice lost their susceptibility to these effects by TCDD and benzo(a) pyrene. It has recently been revealed that AhR is also involved in reproduction of female mice by controlling estrus cycle. In this presentation, I will talk about molecular mechanisms of expressing the transcriptional activity of AhR in response to exogenous chemicals and how AhR is involved in reproduction of female mice. [Jpn J Physiol 54 Suppl:S40 (2004)]

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In a semi-natural habitat, female mice were much more aggressive than males in both wild type (WT) and oxytocin gene knockout (OTKO). Parallel single-unit recordings show that the proportion and responses of OT-responsive neurons in ventromedial hypothalamus (VMH) are sexually different in both WT and OTKO mice. Similarly, single-cell RT-PCR study revealed sexual difference in the expression pattern of a battery of PKC isozyme genes in rat VMH neurons co-expressing OT and estrogen receptors (ERs). Such corresponding sexual differences are also observed for reproductive behaviors. Adult male rats are much less responsive to E than females in the induction of lordosis and proceptive behaviors, even after castration and high doses of E in VMH. This sex difference can be seen in pups as young as immediately after weaning. Using single-cell RT-PCR, VMH neurons of such young pups had already shown sexual differences in the expression of genes for ERs and PKC isozymes. Even earlier, during the critical period of sexual differentiation, sexual differences in gene expression in preoptic area and VMH were found with our microarray study. Responses of genes may also differ; certain mRNAs are induced by E in the VMH of females but not males. Even a same gene, ERα, can mediate different types of reproductive behavior and can have opposite effects on aggressive behaviors in different sexes. We now apply patch clamp techniques to determine whether E can both promote inward and inhibit outward currents on male rat VMH neurons as it did on female^,s. [Jpn J Physiol 54 Suppl:S40 (2004)]

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The choice of sexual partners is sexually dimorphic: estrous female rats access males, while sexually mature males pursue receptive females to copulate. In order to investigate the neuroendocrine mechanism of sexual orientation, we examined their preference of conspecific odors, showing that sexually active males preferred estrous female odors than those of males or ovariectomized females (masculine pattern), and estrous females preferred male odors than those of females or orchidectomized males (feminine pattern). Gonadectomy results in no preference at all, although males show transiently the feminine preference (2 weeks) after castration. Estrogen treatment in males induces the feminine preference, whereas testosterone in females activates their own, feminine pattern. Medial amygdala lesions in females eliminate the preference for sexually active males, but the same lesions in males failed to affect the preference for estrous females. These facts suggest that the masculine and feminine neural circuits are different and independent. Males may have potentially neural circuits for both masculine and feminine preferences, whereas females have only feminine one because of a lack of neonatal androgen. [Jpn J Physiol 54 Suppl:S41 (2004)]

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Lordosis, a typical feminine sexual behavior in rats, is seldom observed in the males even if large doses of estrogen are administered to them. The lateral septum (LS) plays an inhibitory role in lordosis expression and is involved in the sex difference in sexual behavioral patterns. In an attempt to clarify the lordosis-inhibiting neurons in the LS and the projecting site of those neurons, a retrograde tracer (Fluoro-Gold, FG) was injected into the midbrain central gray (MCG) in male rats with or without a transection of the ventral output fibers of the LS. Also, males were treated with estrogen, and then were tests for lordosis. In males without the transection, many FG-labeled neurons were located in the intermediate part of the LS (LSi), and lordotic activity was very low. Males with the transection had few FG-labeled neuron, and showed considerable levels of lordosis. Compared with males, females had greater numbers of FG-labeled neurons in the LSi after FG injection into the MCG. These results suggest that the sexually dimorphic LSi-MCG connection is involved in the inhibition of lordosis and responsible for the sex difference in the regulation of lordosis. The LSi-MCG connection is sexually differentiated during the neonatal period, because neonatal estrogen treatment to females decreased both lordotic activity and the density of the LSi-MCG connection to the level of males. During the postnatal development period, the number of apoptotic cells was greater in the LSi of males than females. Apoptotic cell death may contribute to the organization of the sexually dimorphic LSi-MCG connection. [Jpn J Physiol 54 Suppl:S41 (2004)]

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The central part of the bed nucleus of the stria terminalis was shown to be sexually dimorphic, i.e., smaller in women, with a female volume and neuron number in male-to-female transsexuals. The interstitial nuclei of the anterior hypothalamus (INAH) 3 was also sexually dimorphic, i.e., more than twice as large in heterosexual men than in homosexual men. These findings indicate that each sexual identity and sexual orientation has a different biological substrate. We studied the gonadotropin secretory pattern in 4 21-27 year-old female-to-male transsexual women (FTMs). In 2 FTMs, the surge secretion of LH was atypical, showing extremely low amplitudes, in accord with the clinical diagnosis of anovulatory menstrual cycles based on the basal body temperature (BBT), whereas pulsatile secretions of LH in both follicular and luteal phases were typical. One of these FTMs was bisexual and another homosexual. Two other FTMs showed almost typical LH secretory patterns and BBT during the cycle, being homosexual. Although it is controversial whether the control mechanism for gonadotropin secretion in the human hypothalamus is sexually dimorphic, we assume that FTMs are atypical not only in sexual identity, but also in sexual orientation and the control of gonadotropin secretion, suggesting that they have a general hypothalamic sexual differentiation disorder (GHSDD). [Jpn J Physiol 54 Suppl:S41 (2004)]

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Human gender/sexual differentiation is very complicated combination of following five factors: 1) true biological or genetic sex, that is, the gonad to be testis or ovary; 2) body sex in appearance which is judged mainly with form of the external genitalia; 3) sex type of the brain; 4) gender identity; and 5) sexual orientation. The sex type of the brain does not seem to determine gender identity, since some persons suffering from intense female-to-male (FTM) gender identity disorder have menstrual cycles, suggesting their brain possibly not to be male type. The gender identity, in turn, may not determine sexual orientation; there are persons who are living as male with completely male gender identity but are seduced sexually only by males (true homosexual). It is still mysterious what determines gender identity or sexual orientation. Any combination of the above five factors can be developed, and the situation is complicated more by the presence of hermaphrodites and persons with bisexual orientation. Besides, gender identity disorder, especially MTF, varies in intensity from transvestism to a core type disorder requiring sex reassignment surgery. By the way, why eating disorders, for example, are mostly seen in women? Here I try to discuss gender difference of such addicted behaviors, which develop after adolescence in very hard psychological conditions without any control by consciousness. Is a type of these behaviors induced by biological, environmental or cultural factors? [Jpn J Physiol 54 Suppl:S41 (2004)]

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We assessed the protective effects of s-nitroso (SNO)-polyethylene glycol-conjugated (PEG) hemoglobin (Hb), which was developed as an artificial oxygen carrier, after cerebral ischemia/reperfusion (I/R). Long-term potentiation (LTP) in the perforant path-dentate gyrus synapses of the rat hippocampus was evaluated as an index of functional damage after 2-vessel occlusion (2VO, 10 min). SNO-PEG-Hb, PEG-Hb, at a dose of 250 mg/kg or vehicle, was administered via tail vein. In vehicle-treated controls, 2VO produced impairment of LTP formation 4 days after ischemic insult. Marked pathological changes were not detected by light microscopy. SNO-PEG-Hb administered immediately or 24 hours after reperfusion, ameliorated the changes in LTP formation observed in vehicle-treated controls. In contrast, PEG-Hb did not exert any significant effects. Moreover, expressions of VEGF, bFGF, eNOS, iNOS and nNOS were up-regulated in the hippocampus in 2VO compared to controls. These expressions were differentially altered by SNO-Hb derivatives. Thus, it is suggested that an SNO derivative, SNO-PEG-Hb, has a beneficial effect to protect the brain from cerebral I/R damages via translocation of NO as well as NO quenching, and provides a new strategy for a pharmacological intervention against cerebral I/R. [Jpn J Physiol 54 Suppl:S42 (2004)]

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S1P is a platelet-derived angiogenic sphingolipid that activates a novel family of G-protein coupled receptors; eNOS has been implicated in angiogenic responses of vascular endothelial cells (EC). We explored whether and how S1P might regulate eNOS. We found that S1P induces robust eNOS activation to a degree comparable to those induced by classical eNOS agonists such as bradykinin or VEGF in cultured bovine aortic EC. eNOS activation by S1P is mediated via a signaling cascade comprising S1P₁ (EDG-1) receptors; PTx-sensitive G-proteins; PI3-kinase β; and a protein kinase Akt, which phosphorylates eNOS at serine 1179 to activate the enzyme. VEGF was known to activate eNOS and modulate angiogenesis independently of S1P. We discovered that VEGF induces marked up-regulation of S1P₁ protein/mRNA expression by 4 fold within 90 min, and thereby enhances subsequent S1P-dependent eNOS activation/S¹¹⁷⁹ phosphorylation in cultured EC as well as in intact rat mesenteric arterioles. These data indicate that VEGF specifically induces expression of S1P receptors, associated with enhanced eNOS responses to S1P. We propose that VEGF acts to sensitize the vascular endothelium to the effects of lipid mediators by promoting the induction of S1P₁ receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways. These studies establish that an angiogenic lipid mediator S1P is a novel and potent eNOS regulator in the vasculature, which modulates its specific receptors synergistically with VEGF. [Jpn J Physiol 54 Suppl:S42 (2004)]

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The discovery of endothelium-derived vasodilator factor (EDRF) have stimulated widespread interest in investigations of endothelial function in humans. EDRF has been identified as nitric oxide (NO). In the vasculature, either decreased synthesis or increased oxidative inactivation of NO leads to the impaired endothelium-derived vasodilatation, i.e. endothelial dysfunction.Atherogenic risk factors, including dyslipidemia, hypertension, diabetes mellitus, cigarette smoking, aging, and menopause can cause endothelial dysfunction in the early stage of atherosclerosis. After it has been proven that endothelial function can be assessed by measuring the vasomotor responses of coronary arteries undergoing cardiac catheterization, less or much less invasive methods have been developed, using peripheral arteries. The endothelial function test will be useful as a marker of treatment because it has been reported that assessment of endothelial function may predict future cardiovascular events.It has been postulated that oxidative stress increases blood pressure and may be responsible for the maintenance of high blood state as well as impaired endothelial function. In patients with renovascular hypertension, activation of the renin-angiotensin system enhances the vascular production of reactive oxygen species, resulting in endothelial dysfunction. Renal-artery angioplasty improved endothelial dysfunction through a reduction in oxidative stress. [Jpn J Physiol 54 Suppl:S42 (2004)]

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The blood-flow regulation by coronary resistance microvessels is performed by integrating various vascular-relaxing factors, among which nitric oxide (NO) and EDHF are key factors. Hierarchically, NO mainly controls small arteries (>100 μm), while EDHF functions predominantly in arterioles (<100 μm). Recently, we demonstrated that an oxygen radical H₂O₂ operates as a EDHF in the autoregulation of coronary circulation, although the natures of EDHFs may differ among species.As for the sensing mechanicsof flow, i.e., a key issue of controlling blood flow, the bush-like structure of glycocalyx on endothelial surface with an each distance of 20 nm amplifies a small shearing force acting on the cell, providing sufficient leverage to deform the underlining cortical cytoskeleton. In fact, NO production inboth in vivo arteries and cultured aortic endothelial cells by an application of shear stress became negligibly small following degradation of glycocalyx layer by hyaluronidase or heparinase. A distinguishing characteristics ofintramyocardial coronary arteriolar flow is forward and backward movement of blood (oscillating flow) due to cardiac contraction/relaxation. However, oscillatory flow causes O₂⁻ production of intramyocardial coronary arterioles which quenches NO, suppressing vasodilation. Thus, integrating effects of reactive oxygen species are very important to understand physiology and pathophysiology of coronary microcirculation. [Jpn J Physiol 54 Suppl:S43 (2004)]

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Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a newly identified endothelial cell surface major receptor for oxidatively modified low-density lipoprotein. Progression of artherosclerosis in the donor organ after organ transplantation is a major problem. We hypothesized that ischemia-reperfusion induces LOX-1. After 1 h ischemia of bilateral kidneys plus 3, 6, or 12 h reperfusion, we firstly revealed that LOX-1 mRNA expression was increased in renal cortex and medulla at 6 h after reperfusion, which was decreased by L-arginine supplement. Plasma nitric oxide (NO) end product nitrite plus nitrate and inducible nitric oxide synthase (NOS) expression were increased after reperfusion of 6 h. However, NOS substrate L-arginine did not augment but markedly decreased plasma NO end product, because L-arginine supplement suppressed inducible NOS expression in kidney. We hypothesized that available L-arginine is depleted by ischemia-reperfusion, leading to inducible NOS induction. Ischemia decreased L-arginine levels in kidney and L-arginine supplement increased NO end products in renal cortex in the earliest phase of reperfusion. These results disclosed for the first time that a deficiency in L-arginine by ischemia reperfusion causes uncoupling of constitutive NOS, which induces inducible NOS and LOX-1, implying why L-arginine is effective for stroke or transplantation in preventing atherosclerotic progress. [Jpn J Physiol 54 Suppl:S43 (2004)]

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Neuropeptides, nocistatin (NST) and nociceptin/orpahnin FQ (N/OFQ), are produced from the same precursor, prepronociceptin/orphanin FQ (ppN/OFQ). Although all opioid precursors contain several bioactive peptides with similar functions, NST and N/OFQ have opposite central functions including pain transmission, learning and memory. Protein processing has been measured biochemically in the lysate of destroyed cells and a cell-free system. To investigate the regulation of NST and N/OFQ production from ppN/OFQ in living cells and in vivo, we developed a novel intra-molecular BRET system between a luciferase and fluorescent protein. This BRET system demonstrated high efficiency of BRET and the real-time and continuous BRET assay, and the change in BRET signals quantified the processing of the protein. This BRET had the desired profile for monitoring the protein processing of NST and N/OFQ in intact cells, and this BRET system showed that proprotein convertases, PC 1 and furin, were involved in the production of these peptides. Since our novel intra-molecular BRET system is particularly useful for studies on protein processing in living cells, it can be applicable to the elucidation of regulation of biological functions of NST and N/OFQ in vivo. [Jpn J Physiol 54 Suppl:S43 (2004)]

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Roles of afferents activity on the development of neural circuits has been intensely studied on the sensory system. In contrast to a number of studies regarding the influence of peripheral inputs on excitatory circuits development, less is known on its action on inhibitory circuits development. Recent studies demonstrates that the main inhibitory transmitters in the CNS, GABA and glycine, play as excitatory substances and promote various neuronal development. Actions of GABA/glycine change from a depolarization to a hyperpolarization with age, resulted from developmental upregulation of KCC2 in expression and function, a main molecule extruding intracellular chloride out of neurons, and a decrease of intracellular chloride concentration. This switch was regulated by afferent activity during development. Even after maturation, neuronal insults, e.g. injury, induced a down-regulation of KCC2 in expression, resulting in an increase intracellular chloride concentration and GABA-induced excitation. Re-acquisition of immature characteristics following injury was also well demonstrated in the excitatory transmitter. For example, in injured neurons the subunits making up NMDA receptor changed from predominantly NR2A to other type subunits, resulting in less sensitivity to extracellular magnesium ion and prolonged decay time of NMDA currents, which are characterized in immature neurons. If developmental strategy were the case in the regeneration, sensory afferent activity might largely regulates the remodeling of the neurotransmitter function in regeneration. [Jpn J Physiol 54 Suppl:S44 (2004)]

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We have been studying the role of ATP receptors in pain, and already reported that activation of P2X2/3 heteromeric channel/receptor in primary sensory neurons causes acutely tactile allodynia. We report here that tactile allodynia under chronic pain state requires an activation of P2X4 ionotropic ATP receptor and p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. Two weeks after L5 spinal nerve injury, rats displayed a marked mechanical allodynia. In the rats, activated microglia were detected in the injury side of the dorsal horn where the level of the phosphorylated p38MAPK (phospho-p38MAPK) was increased. We performed the double-immunostaining analysis using cell-type specific markers and found that phospho-p38MAPK-positive cells were microglia. Moreover, intraspinal administration of p38MAPK inhibitor, SB203580, suppressed the allodynia. We also found that the expression level of P2X4 was increased strikingly in spinal cord microgila after nerve injury and that pharmacological blockade of P2X4 reversed the allodynia. Intraspinal administration of P2X4 antisense oligodeoxynucleotide (ODN) reduced the expression of P2X4 and suppressed tactile allodynia. Taken together our results demonstrate that activation of P2X4 or p38 MAPK in spinal cord microglia is necessary for tactile allodynia following nerve injury. [Jpn J Physiol 54 Suppl:S44 (2004)]

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Prostaglandin E₂ (PGE₂) is one of the inflammatory mediators that play important roles in nociceoption and hyperalgesia in inflamed tissues by exciting or sensitizing nociceptors. The capsaicin receptor TRPV1 is a non-selective cation channel expressed predominantly in unmyelinated C-fibers and activated not only by capsaicin, but also by protons or heat both of which cause pain in vivo. The functional interaction between TRPV1 and PGE₂ was examined using electrophysiological and behavioral analyses. PGE₂-induced thermal hyperalgesia was almost completely abolished in TRPV1-deficient mice. In HEK293 cells expressing cloned TRPV1 and EP₁ receptors, PGE₂ increased capsaicin-evoked TRPV1 currents through EP₁ receptors in a PKC-dependent pathway. In addition, in the presence of PGE₂, temperature threshold for TRPV1 activation was reduced from 40.8 to 30.9^oC. Similar EP₁-mediated increase in TRPV1 activity could be observed in mouse dorsal root ganglion neurons. In behavioral analyses, both PGE₂-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in EP₁-dificient mice. Thus, the potentiation of TRPV1 activity through EP₁ activation might be one important mechanism underlying the nociceptive actions of PGE₂. [Jpn J Physiol 54 Suppl:S44 (2004)]

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The origin of the most space deconditioning arises from lack of gravity in the environment. Because the gravity is transparent and not escapable as long as all animals stay on the ground, we usually do not recognize the gravity itself, only feel as weight. Many of the fundamental physiological systems such as equilibrium control, motor function, cardiovascular system with autonomic reflexes as well as the brain functions including sensation and recognition strongly depend on the gravitational factors. The most of the acute space deconditioning is initiated immediately after the gravitational unloading affecting both gravity dependent and co-working gravity independent functions. The subsequent chronic changes are the adaptive responses to compensate the mismatches between the gravity dependent and independent feedbacks. Major issues of the space deconditioning are the chronic changes induced by the compensative responses, such as bone demineralization, reduction of muscle innervation and changes in autonomic nerve responses, and even higher-order brain functions which may undergo a self-reprogrammed state. The present paper summarizes the role of gravity in the major issues of the space deconditioning including some recent findings from the long-term space flights and ground based simulation of weightlessness. [Jpn J Physiol 54 Suppl:S45 (2004)]

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The use of short-arm centrifugal system (SACS) could be one of the best means for the prevention of several physiological problems including cardiovascular deconditioning (CVD) caused by a prolonged exposure to microgravity. The CVD consists of: 1) hypovolemia, 2) a decrease of diastolic blood pressure, 3) a decrease of stroke volume, 4) a decrease of left ventricular mass, and 5) resetting of the carotid baroreceptors. The CDV appears as: 1) dizziness, 2) increased heart rate, 3) orthostatic intolerance, 4) presyncopal feeling, and 5) reduced exercise capacity. First, we should establish a prescription (the strength, length, and frequency) of artificial gravity loads using the SACS. We studied the effect of the SACS as a countermeasure on the prevention of the CVD during 4 days of 6° head-down bed rest (HDBR). Half of 20 young healthy subjects was loaded total gravity of +2Gz twice a day (up to 30min×2) during the HDBR period. R-R intervals and continuous blood pressures were simultaneously measured at supine position prior to the HDBR and immediately after the HDBR. Previous reports have indicated that prolonged weightlessness impaired the baroreceptor reflex responses and vagal modulation to heart rate. Our result suggests that daily loading to 2Gz-1hr can lessen the changes in autonomic cardiovascular control during the exposure to microgravity, and consequently might be useful for the prevention of CVD. The periodic loads couldn't prevent a decrease in exercise capacity. Therefore, exercises during the centrifugal loading are needed. [Jpn J Physiol 54 Suppl:S45 (2004)]

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The purpose of this study was to investigate effect of high intensity bicycle training during 6 deg head-down tilt bed rest on muscle function and volume. Ten healthy men were divided into either two groups: countermeasure group (CM, n = 5) and control group (Cont, n = 5). The CM group performed two sessions centrifuge-induced artificial gravity with bicycle exercise training consisted of two sessions (20 min per session, 40 min total) during bed rest period. First session was 0.8-1.4 Gz in supine position with a constant exercise intensity (60 W). Second session was 0.3 Gz in supine position with an interval exercise protocol (40% to 80% of heart rate maximum). Before and after the bed rest period, maximum voluntary contraction (MVC) during knee extension, muscle volume of the thigh, muscle functional MRI (mfMRI) and EMG of the quadriceps femoris during fatiguing isotonic knee extension. CM subjects showed no change in MVC (525 to 485 N, P = 0.31), however the Cont group demonstrated significant change in MVC after bed rest (593 to 457 N, P = 0.008). Muscle volume of the knee extensors, knee flexors, and adductors of the CM group was unchanged, whereas muscle volume of the knee extensors of the Cont group decreased by 9% (P = 0.02). There was no significant change in exercise-induced mfMRI signal change and EMG activity of the knee extensors for both the CM and Cont groups. These results suggested that short-arm centrifuge with high intensity bicycle training would be effective for maintain of function and volume in skeletal muscle. [Jpn J Physiol 54 Suppl:S45 (2004)]

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Introduction: We have developed a bone loss countermeasure program for Japanese astronauts with the aid of the Bone Loss Countermeasure Advisory Working Group. Pharmacological intervention (Bisphoshonate administration) is planned for astronauts with osteoporosis BMD (T-score <2.5) as treatment, and for astronauts with osteopenia BMD (T-score -2.5 to-1.0) as preventive therapy for long duration space flight. To validite this program, JAXA participated in the long duration bed rest study at MEDES, co-sponsored by European and French Space Agencies.
Methods: Twenty-five male volunteers were split into three groups: control (9 subjects), drug (7), and exercise (9) groups. Bisphosphonate was infused intravenously 14 days before the bed rest. Data collection including BMD, urinary Ca excretion, abdominal X-P for renal stone was performed 15 days before, during 90-day head-down tilt bed rest, and up to one year after bed rest.
Results: DXA results showed the BMD of total femoral bone decreased by 6.2% in the control group and by 3.6% in the exercise group, while pharmacological group showed no change at the end of 3-month bed rest period. Urinary Ca excretion significantly increased in the control group and in the exercise group during bed rest. In the pharmacological group, urinary Ca excretion decreased immediately after infusion, and stayed low for one month. Moreover, existence of renal calculi was detected on the plain abdominal X-ray after bed rest, in two from the control group and four from the exercise group, but none from the pharmacological group. [Jpn J Physiol 54 Suppl:S46 (2004)]

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Treadmill exercise in lower body negative pressure to counteract deconditioning from spaceflight. D.E. Watenpaugh, S.M.C. Lee, W.L. Boda, D.D. O'Leary, S.M. Schneider, and A.R. Hargens. Research performed at the Department of Orthopaedics, University of California, San Diego, USA. Over the last 15 years, we developed treadmill exercise in lower body negative pressure (LBNP) as a countermeasure. We hypothesize that LBNP exercise during bed rest (simulated spaceflight) maintains muscle mass and strength, bone metabolism, gastrointestinal (GI) motility, balance, sprint speed, exercise capacity, and orthostatic tolerance. We currently study identical twins in 6 degree head-down bed rest for 30 days. One twin from each pair exercises supine in LBNP at 1.0-1.2 bodyweight and 40-80% VO2 peak 6 days per week; 5 min of resting LBNP follows exercise. The siblings serve as non-exercising controls. Preliminary findings indicate control subjects experience: reduced leg muscle strength and endurance; reduced paraspinal muscle cross-sectional area and spinal bone mineral density; increased biochemical evidence of bone catabolism; reduced GI motility; reduced balance, sprint speed, and VO2 peak; and orthostatic intolerance. The countermeasure attenuates or prevents all of these effects except GI dysmotility. Full protection of orthostatic tolerance may require longer post-exercise resting LBNP (10 min?), and resistance exercise may be needed for full maintenance of lower body musculoskeletal tissues. NASA, NIH, and the Canadian Space Agency support this work. [Jpn J Physiol 54 Suppl:S46 (2004)]

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We manufactured the loading system of artificial gravity and ergometric exercise by centrifuge. To test the effectiveness of centrifuge-induced artificial gravity with ergometric exercise, 12 healthy young men (20.7 ± 1.9 yrs) were exposed to simulated microgravity for 14 days of -6° head-down bedrest. Half the subjects were randomly selected and loaded 1.2 G artificial gravity with 60 W (four out of six subjects) or 40 W (two out of six subjects) of ergometric workload on days 1, 2, 3, 5, 7, 9, 11. 12, 13, 14 (CM group). The rest of the subjects served as the control. Anti-G score, defined as the G-load × running time to the endpoint, was significantly elongated by the load of the centrifuge-ergometer. Plasma volume loss was suppressed -5.0 ± 2.4 vs -16.4 ± 1.9%), and fluid volume shift was prevented by the countermeasure load. Elevated heart rate and muscle sympathetic nerve activity after bedrest were counteracted, and exaggerated response to head-up tilt was also suppressed. Centrifuge-induced artificial gravity with exercise is effective in preventing cardiovascular deconditioning due to microgravity exposure, however, an effective and appropriate regimen (magnitude of G-load and exercise workload) should be determined in future studies. [Jpn J Physiol 54 Suppl:S46 (2004)]

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In eukaryotic cells, activation of receptors that couple to the phosphoinositide pathway evoke a biphasic increase in cytosolic free Ca²⁺ concentration: an initial transient release of Ca²⁺ from intracellular stores is followed by a sustained phase of Ca²⁺influx. This influx is generally store-dependent and is required for controlling distinct Ca²⁺-dependent processes like exocytosis and cell growth. Store-operated Ca²⁺ entry is often manifest as a non-voltage-gated Ca²⁺current called I_CRAC. CRAC channels are activated by emptying intracellular Ca²⁺ stores but how stores open the channels is unclear. Three models have been proposed: 1) the vesicular fusion model in which CRAC channels are inserted into the plasma membrane upon store depletion via an exocytotic mechanism; 2) conformational-coupling and secretion-like coupling where InsP₃ receptors on the stores physically attach to CRAC channels in the plasma membrane; 3) the diffusible messenger model in which a factor is generated from empty stores and which then opens CRAC channels. We have systematically tested the predictions of each model. Our results are hard to reconcile with the vesicular fusion and coupling-type models. On the other hand, we find that inhibition of lipoxygenase enzymes impairs the activation of I_CRAC, raising the possibility that the enzyme might be involved in regulating Ca²⁺ influx. We are now measuring lipoxygenase activity to see whether it is modulated by store depletion. Our recent results will be described. [Jpn J Physiol 54 Suppl:S47 (2004)]

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Antigen receptors on B lymphocytes (BCRs) play a central role in immune regulation by transmitting signals that regulate B lymphocyte survival, growth, and differentiation. Among a number of signaling pathways, the importance of phospholipase C (PLC)-γ2 pathway has been recently underscored by gene targeting experiments in mice. PLC-γ2 activation leads to hydrolysis of phospholipids, yielding IP3 and diacylglycerol (DAG). IP3 binds IP3 receptors located in the endoplasmic reticulum (ER), leading to calcium release from internal stores. Triple knock-out of three IP3 receptor isoforms in DT40 B cells abolishes the BCR-induced calcium mobilization both from internal stores and from extracellular stores, therefore supporting the capacitative calcium entry (CCE) model in which calcium influx channels in the plasma membrane is opened upon depletion of intracellular calcium stores.TRPC1 could be one component of these calcium influx channels in that genetic disruption of TRPC1 significantly attenuates calcium-release-activated calcium currents in DT40 B cells. Moreover, IP3 receptor function is suppressed by loss of TRPC1, suggesting the existence of a functional feed-forward regulatory loop between IP3 receptors and TRPC1 in calcium signaling in B lymphocytes. [Jpn J Physiol 54 Suppl:S47 (2004)]

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The major Ca²⁺ influx pathways through the cytoplasmic membrane in cardiac and skeletal muscles are believed to be dihydropyridine receptors (DHPR). However, we recently showed the presence of store-operated Ca²⁺ influx (SOC) pathway in skeletal muscle (Kurebayashi & Ogawa, J Physiol;2001;533;185). In this study, we intend to further characterize the properties of the SOC channel in mouse skeletal muscle and extend the study to rat cardiac muscle. In skeletal muscle, Ca²⁺ influx through the SOC channel was inward rectifying and the magnitude at the resting membrane potential was greater than that through the DHPR upon depolarization. Although severe store depletion was necessary for enhancement of Mn²⁺ influx, the Ca²⁺ entry through SOC was observed at a lesser extent of Ca²⁺ depletion, suggesting its important role in Ca²⁺ homeostasis. Among putative inhibitors of SOC, 2-aminoethoxydiphenyl borate (2-APB) was most effective. With cardiac muscle, nifedipine-insensitive Ca²⁺ entry also was detected at the resting state when the Ca²⁺ store was partially decreased. Its influx rate was lower than that through DHPR. Econazole, a putative inhibitor to TRPV5, moderately inhibited the influx whereas 2-APB did not, indicating that the Ca²⁺ influx pathway in cardiac muscle is pharmacologically different from that in skeletal muscle. Based on the above results and RT-PCR studies, candidates for these pathways will be discussed. [Jpn J Physiol 54 Suppl:S47 (2004)]

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Circumstantial evidence of store operated channels (SOCs) has been accumulated in exocrine acinar cells. After depletion of internal Ca storage pools with strong agonist stimulations or with a SERCA pump inhibitor, thapsigargin, Ca-influx from the external milieu induces a substantial part of cytosolic Ca-increases. However, no direct measurement of SOCs has been carried out in the acinar cells. The foreground channel currents of the acinar cells, sensitive to an increase in cytosilic Ca, make the assessment of SOC current not easy. Eliminating the foreground currents with large size cation and anion, we successfully detected divalent cation currents after the store depletion in whole-cell patch-clamp recordings from rat pancreatic acinar cells. The peak magnitude of the divalent cation currents reached the order of tens pico-amperes and seemingly inactivated at negative membrane potentials during the time course. The divalent currents may play a role as a route of capacitative Ca entries in pancreatic Ca signaling. [Jpn J Physiol 54 Suppl:S48 (2004)]

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Neurons with similar response specificity cluster in columns in cerebral cortices. This structure is considered to be a universal characteristic of cortical areas. However, modular organizations such as columns are not fully characterized in higher association cortices, including monkey inferior temporal (IT) cortex. In the present study, we used intrinsic signal imaging and voltage sensitive dye imaging to explore modular organizations in IT, and extracellular recordings to understand functional structures at cellular levels in IT cortex. Intrinsic signal imaging revealed that object images activate multiple spots in IT cortex. Each spot (diameter, 0.5+/-0.13 mm) was specific to a particular visual feature included in the object images. On the other hand, voltage sensitive dye imaging revealed that a region 5-10 times larger than the spots was specifically activated by a set of the object images. Taking into account that intrinsic signals emphasize spiking activity and that voltage sensitive dye signals emphasize synaptic potentials, these results suggest that there are modules in different scales that are distinguished by measures of neural activity. In addition to these results, extracellular recordings from individual cells showed that object selectivity of nearby cells within a spot was similar enough to distinguish cells in one spots from the other. Among the cells in a spot, however, object selectivity was not exactly the same. Based on these results, we proposed that there is a hierarchical modular organization where levels of the hierarchy are different in scales and probably in stimulus selectivity. [Jpn J Physiol 54 Suppl:S48 (2004)]

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In an attempt to study the cortical control of motor systems, intra- or juxta-columnar connections of pyramidal neurons to corticospinal neurons of layer V in the rat motorsensory cortices were examined in brain slices by combining intracellular staining with Golgi-like retrograde labeling of corticospinal neurons. The pyramidal neurons were electrophysiologically classified into several types by the firing patterns in response to long depolarizing current pulses. Interestingly, all layer IV neurons were star-pyramidal neurons exhibiting phasic responses, whereas the vast majority of pyramidal neurons in layer II/III displayed tonic discharges in response to depolarizing current pulses. Morphologic analysis revealed that a large number (110-558) of axon varicosities of a pyramidal neuron located in layers II-VI were apposed to the dendrites of corticospinal neurons. In particular, the varicosities of a layer IV star-pyramidal neuron made 2- to 3-fold more appositions to the dendrites of corticospinal neurons than those of a pyramidal neuron in the other layers. The present results suggest that thalamic inputs are conveyed to corticospinal neurons preferentially via layer IV excitatory neurons, which have phasic response properties and might contribute to the initiation or switching of motor activity. In contrast, excitatory inputs from layer II/III seem to be integrated in corticospinal neurons with from layers IV-VI, and might be useful in maintaining the activity of corticospinal neurons by the tonic response properties of layer II/III pyramidal neurons. [Jpn J Physiol 54 Suppl:S48 (2004)]

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Synchronized neuronal activity plays an important role in cortical information processing. The synchrony of spiking in pyramidal and non-pyramidal cells may be caused by synchronous excitatory synaptic inputs. We previously demonstrated that GABA_B receptor-mediated IPSPs evoked by stimulation of layer II spread vertically throughout all layers in a columnar pattern, but not horizontally. Our study also indicates that paired pulse depression of excitatory synaptic currents is mediated by the activity of GABA_B receptors. Then, it can be assumed that GABA_B receptor-mediated presynaptic inhibition is suitable for modulating the coherent activity of excitatory synaptic inputs in each column. To test the hypothesis, we have performed dual whole-cell patch clamp recording in slice preparations of the barrel cortex of rats and GAD67-GFP knock-in mice. By stimulation of layers II/III, EPSCs were evoked in a pair of pyramidal or non-pyramidal cells located in neighboring two columns. Paired pulse depression of evoked EPSCs (inter-stimulus interval = 200 ms) were observed in one pyramidal or non-pyramidal cell located in the same column as the stimulating electrode was placed, but not in the other cell located in the adjacent column. Correlation coefficient was calculated between the first EPSCs and between the second EPSCs evoked in the proximal and distal cells. Coherent activity of excitatory synaptic inputs was modulated independently in each column by the activity of GABA_B receptors. [Jpn J Physiol 54 Suppl:S49 (2004)]

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Activities of cortical pyramidal cells are regulated by GABAergic nonpyramidal cells. Recently several dynamic properties of cortical circuitry have been revealed, but the precise intracortical wiring by each nonpyramidal cell subtype remains largely unknown. We investigated synaptic connections made by double bouquet cells, a subtype of nonpyramidal cells, in rat frontal cortex. Non-pyramidal cells were identified in isolated slices of frontal cortex from 18-22 days postnatal rats by whole cell, current-clamp recording, followed by intracellular injection of biocytin. After overnight fixation, the slices were histochemically stained with DAB and embedded in Epon. The postsynaptic structures were reconstructed three-dimensionally from serial ultra-thin sections of the intracellulary-stained axon terminals. Double bouquet cells innervated dendritic shafts, spine heads, spine necks, and rarely somata. About half of the dendrites contacted by double bouquet cell axons received numerous asymmetrical synapses on their dendritic shafts, which indicated these postsynaptic targets were nonpyramidal cells. The rest of the target dendrites were proximal dendrites and distal dendrites of pyramidal and nonpyramidal cells. The target spines usually had an asymmetrical input, which may indicate that double bouquet cell specifically inhibits the excitatory input. These observations indicate the double bouquet cells have a several specific synaptic targets and may have a specific role in a regulation of cortical activity. [Jpn J Physiol 54 Suppl:S49 (2004)]

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At present, investigating the neural correlates of conscious percept (Crick and Koch 2003) is one of the promising scientific approach to the problem of subjective experience. The neural correlates paradigm is a direct extension of the standard model of neural information representation based on the response selectivity paradigm (Hubel and Wiesel 1962). With the advent of various imaging techniques such as fMRI and MEG, the neural correlate approach is an obvious and useful way of approaching the physical origin of consciousness. Here I argue that in order to come to a full understanding of the neural correlates of conscious percept, we need to "reclaim homunculus" in a nontrivial way. The old idea of a single central area monitoring the neural activities in various areas of the cortex is gone. However, in order to account for the "integrated parallelism" of information processing in the brain, as is evident from observations in active vision (e.g. binocular rivalry) and sensori-motor coordination (e.g. body image, mirror neurons), we do need to come to understand a neural basis of subjectivity in which all information representation in the brain should become relevant.Citing several neurophysiological evidences from my own laboratory and elsewhere, I put forward several working hypotheses regarding the neural basis of subjectivity. In particular, I discuss how the time parameters relevant in the neural coding of information across the multiple cortical regions are coordinated in a systematic way to give rise to a coherent sense of agency. I also propose several specific experiments to be conducted in order to test the suggested hypotheses. [Jpn J Physiol 54 Suppl:S49 (2004)]

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One of the main topics of science in this century will be the mind-brain problem: how does the brain generate the human mind? Today various kinds of scientists and scholars join the discussion on this issue. Physiology has played a vital role to investigate the functions of the central nervous system, so physiologists are expected to contribute to the solution of the mind-brain problem, too. I will suggest a physiological framework to deal the mind-brain problem. The mechanism that generates the human mind seems to be beyond our comprehension probably because we lack some crucial understanding about the mind or brain, which could be brought by an unexpected finding during the experiments. However, it would be possible that we might fail to pick up such a finding because we do not known what kind of facts seem to be important for the mind-brain problem. Indeed, we cannot deny the possibility that we actually lost such a fortune in the past history. The loss might be prevented by the 'theoretical physiology.' This new kind of physiology should be specialized to focus on reviewing the meaning of experimental findings, developing innovative ideas to understand the nature of the mind and brain, and giving predictions to gain a new experimental finding in the near future. I will show some topics that could be good targets of 'theoretical physiology' for the mind-brain problem.
I appreciate the comments from Prof. Masahiro Sokabe. [Jpn J Physiol 54 Suppl:S50 (2004)]

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The seminal paper by Crick and Koch (2003) opened a gate to the bland new realm of research in science. A decade ago, the problem was still called "mind-body" problem and was thought of as a somewhat obsolete topic in philosophy. I would like to discuss the problem of 'self-awareness.' In particular, I will focus my attention on "network science" and "quantum mechanics" and the possible role they might play in brain science. First of all, does self-awareness somehow emerge out of classical, i.e. non-quantum, networks? If so, is it limited to biological networks such as human brain, or is it possible for the inorganic networks, Internet for example, to have self-awareness? I will argue that Alexa, a web search program launched in 1996, had changed the whole landscape of Internet. Before Alexa, the flow of information on the Internet was 'one-way,' due to the original design, i.e. HTML. After Alexa, truly two-way or feedback flow emerged. I will discuss some implications of this (seemingly innocuous) 'add-on.' Next, does quantum mechanics have anything to do with self-awareness, and with the function of human brain system in general? Extravagant as it may seem, the claim that quantum mechanics is vital when we try to understand the origin of self-awareness, is far from dismissible. (I will cite some examples from the standard text books.) Finally, and most importantly, I must utter the following question: do all the above argument have anything to do with physiology? [Jpn J Physiol 54 Suppl:S50 (2004)]

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The cerebro-cerebellar communication system proposed by Allen and Tsukahara (1974) was a major landmark for behavioral neuroscience. Their insights on the two cerebro-cerebellar loops, a closed sensorimotor-cerebellar-sensorimotor loop through the intermediate zone and a more open association/sensorimotor-cerebellar-sensorimotor loop, have contributed tremendously to our understanding how motor commands are programmed and generated in this system. But, new developments of our studies go beyond the notion, and are opening a gate to new functional aspects of the system. Namely, the use of viral tracing and fMRI revealed that not only the cerebral motor areas, but also the parietal and prefrontal cortex, closely link with the cerebellum, thus contributing to various higher brain functions. Among various possible roles of the cerebro-cerebellar communication system, however, the central issue is still its role in motor control and learning. In the light of recent neurophysiological findings, the ventral premotor cortex (PMv) will be focused, and a tight linkage between the PMv and the cerebellum in prism adaptation that requires dynamic changes in frames of reference for visually-guided reaching movements will be discussed in this symposium. [Jpn J Physiol 54 Suppl:S50 (2004)]

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An internal model is a neural mechanism that can mimic the input-output properties of a controlled object such as a tool. Recent interests have moved on to how multiple internal models are learned and switched under a given context. Two representative computational models for switching propose distinct neural mechanisms, thus predicting different brain-activity patterns in the switching of internal models. In one model, called the mixture-of-experts architecture, switching is commanded by a single executive called a "gating network," which is different from the internal models. In the other model, called the MOSAIC, the internal models themselves play crucial roles in switching. Consequently, the mixture-of-experts model predicts that neural activities related to switching and internal models can be temporally and spatially segregated, while the MOSAIC predicts that they are closely intermingled. Here, we examined the two predictions by analyzing fMRI activities during the switching of one common tool and two novel tools. The switching and internal-model activities temporally and spatially overlapped each other in the cerebellum and in the parietal cortex whereas the overlap was very small in the frontal cortex. These results suggest that 1) switching mechanisms in the frontal cortex can be explained by the mixture-of-experts model, while those in the cerebellum and the parietal cortex are explained by the MOSAIC, or 2) the prefrontal regions estimates contribution of each internal model, and gives the cerebellum feedback within the MOSAIC. Supported by the TAO. [Jpn J Physiol 54 Suppl:S51 (2004)]

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