Clinical observations imply that vascular compression and demyelination of the trigeminal nerve root have been major etiological factors. LPA, released after nerve injury, also induces demyelination, which may cause neuropathic pain and may increase nociceptive activity. Surgical procedures were performed under pentobarbital sodium (40 mg/kg, ip). Under anesthesia, LPA (1 nmol/ 3 µl) was injected into the left trigeminal ganglion. We examined free behavior (spontaneous pain), air-puff test (mechanical allodynia), pinprick response (mechanical hyperalgesia), and acetone test (cold allodynia) at -3, 3, 7, 10, 14, 17, 21, 24, 30, 40, 55, 70, 85, 100 and 130 days after surgery. The most obvious behavioral changes that occurred following microinjection of LPA into the trigeminal ganglion were a dramatic increase in the responses to mechanical stimulation of the face. The thresholds of air-puff significantly decreased after microinjection of LPA. In the almost LPA-treated animals, mechanical allodynia was established within 3-7 days bilaterally and strongly remained over 40 days following LPA injection. Microinjection of LPA produced mirror-image mechanical allodynia. Microinjection of LPA produced demyelination of axon in the trigeminal ganglion. These results suggest that demyelination plays a major etiological role in trigeminal neuralgia and that LPA-induced trigeminal neuralgia animal model help us study pathogenesis of trigeminal neuralgia. Supported by: grant (No. R01-2006-000-10488-0) from KSEF. [J Physiol Sci. 2007;57 Suppl:S36]

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Peripheral nerve injury produces increments of the primary afferent activity with an abnormal pattern at the early period after injury, and a reorganization of the large diameter nerve fiber terminals that occurs at later period, which is manifested as allodynia. This primary afferent discharge and reorganization of large diameter nerve fiber terminals in the central nervous system following peripheral nerve injury may occur in a wide area of the spinal cord, beyond the area innervated by the injured nerve. We established a trigeminal nerve injury model and analyzed the neuronal activity in order to clarify the response properties of nociceptive neurons in the trigeminal spinal nucleus caudalis (Vc) of rats with inferior alveolar nerve (IAN) transaction or infraorbital nerve (ION) ligation. The escape threshold from mechanical stimulation applied to the whisker pad area ipsilateral to the transection or ION ligation were significantly lower than that for the contralateral and sham-operated whisker pad. Background activity and mechanically evoked responses of WDR neurons on the ipsilateral side relative to the injured nerve were significantly increased after the operation. We did not observe any modulation of the thermal responses of WDR and NS neurons following IAN transection or ION ligation. The present findings suggest that the increment of neuronal activity of WDR neurons in the Vc following IAN transection or ION ligation may play an important role in the development of the mechano-allodynia in the trigeminal region. [J Physiol Sci. 2007;57 Suppl:S37]

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The sensation of pain is carried by A-delta and C afferents to the superficial spinal dorsal horn, while the non-nociceptive information is carried by A-beta fiber to the deep dorsal horn. Inflammation induced by CFA injection to the rat hind paw triggered sprouting of subpopulation of A-beta afferents into the superficial laminae, in particular lamina II, so called substantia gelatinosa (SG). The sprouted A-beta afferents retracted back to deep laminae in 3 to 4 weeks after inflammation that was consistent with a disappearance of pain behavior. On the other hand, the majority of SG neurons received A-beta afferent inputs in an immature condition with lacking of C afferent inputs, because of significant delay of innervation of dorsal horn neurons with small afferents in the spinal cord during development. In analogy with the fact that a change of multiple innervaion in immature to single innervation of skeletal muscle by motor fibers in mature state switches to multiple in pathological conditions, the sprouting of A-beta afferents following inflammation and retraction of the afferents to deeper laminae would be a part of regenerative processes of the sensory pathway. [J Physiol Sci. 2007;57 Suppl:S37]

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The cortical areas of primates are structurally and functionally highly specialized compared with their rodent counterpart. To study the molecular basis of cortical differentiation in primates, we searched for genes that are specifically expressed in restricted areas of macaque monkeys and found the striking enrichment of serotonin (5-HT)1B receptor mRNA in the primary visual area (V1). In situ hybridization (ISH) analyses revealed that 5-HT2A but no other 5-HT receptors are also enriched in V1. 5-HT1B and 5-HT2A receptor mRNA expression in V1 was upregulated in geniculorecipient layers 4A and 4C during early postnatal periods to achieve their area and lamina specificity in adults. In adults, the expression of these mRNAs were dependent on retinal activity. These observations strongly suggest the central role of visual activity in sculpting the cortical expression of these 5-HT receptors. We also show evidence that 5-HT1B and 2A receptors are functional in monkey V1. The pharmacological study suggested that these receptors have opposite modulatory effects on the firing of V1 neurons. Interestingly, the modulatory effects were dependent on the firing rate of the recorded neurons. So far, the enriched expression of 5-HT1B and 2A receptor mRNAs in V1 is observed only in primates. The potential role of these serotonin receptors in visual function of primates will be discussed. [J Physiol Sci. 2007;57 Suppl:S37]

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Fast-spiking (FS) interneurons are one of the strong sources of cortical inhibition. In this study, we examined developmental changes in the connections between pyramidal and FS cells in rat primary visual cortex. We simultaneously recorded FS and pyramidal neurons from layer 2/3 with the whole-cell recording method. At postnatal 14-17 days (P14-17), FS cells provided weak inhibitory inputs to many neighboring pyramidal cells, while excitatory connections from the latter to the former neurons were found much less frequently. Although the proportion of pairs with inhibitory connections was considerably high, the proportion of pairs reciprocally connected was quite low. The inhibitory connections were weak irrespective of the presence of the reciprocal connections. At P20-25, the proportion of pairs with inhibitory connections decreased, but the proportion of reciprocally connected pairs increased. In addition, the inhibitory connections in the reciprocally connected pairs were much stronger than those in non-reciprocally connected pairs. The specificity of the connections and the strength of the inhibition at P30-35 were indistinguishable from those at P20-25. Taken together, these results indicate that FS cells provide neighboring pyramidal neurons with weak inputs at an early stage and then inhibitory connections in reciprocally connected pairs survive and strengthen. This age-dependent change in synaptic connections may contribute to the maturation of visual responsiveness of cortical neurons. [J Physiol Sci. 2007;57 Suppl:S38]

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In many cells of the primary visual cortex (V1), stimuli in the receptive field surround suppress the responses to stimuli presented in the classical receptive field. We have studied surround suppression in membrane potential and conductance changes of simple cells in anesthetized cats. Like surround suppression in spike responses, suppression in membrane potential was selective for surround orientation. Suppression was amplified by spike threshold, being stronger for firing rate than for membrane potential. Surprisingly, surround stimulation reduced both the excitation and inhibition received by a cell. This reduction is not caused by a reduction of the input from geniculate relay cells: Suppression in relay cells was not orientation selective. These results suggest that V1 operates as an inhibition-stabilized network, in which recurrent excitation is by itself unstable, but is stabilized by feedback inhibition. We assume that surround stimuli activate excitatory input onto inhibitory cells. The sequence of events when a surround stimulus is added to a center stimulus is as follows. The activity of inhibitory cells is transiently increased. The activity of excitatory cells is decreased by the increase in inhibition. The activity of excitatory cells is further decreased by the reduction of recurrent excitation. The activity of inhibitory cells is also decreased by the reduction of total excitation in the network. As a result, both the excitation and inhibition onto cells are reduced. [J Physiol Sci. 2007;57 Suppl:S38]

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In the primary visual cortex (V1), a neuronal response to stimulation of the classical receptive field (CRF) is suppressively modulated by the stimulus presented at the receptive field surround (SRF). This surround suppression is considered as a neuronal basis of perceptual figure-ground segregation. Using stationary flashes of sinusoidal grating as stimuli, we examined the dependency of surround suppression on the stimulus size (Exp. 1) and that on the orientation-contrast between CRF and SRF stimuli (Exp.2) in V1 neurons of anesthetized cats. In Exp.1, CRF was stimulated with a flash (500 ms) of the grating patch with optimal parameters and varying diameters. Temporal analysis of SRF effect demonstrated followings; 1) there were two groups of cells exhibiting different types of suppression in early component (0-80 ms) of response (fast suppression), that is, SRF diameter-independent (type I) and SRF diameter-dependent (type II) suppression, and 2) late component (100 ms <) of response was progressively suppressed with an increment of stimulus size (late suppression) in both type I and II cells. In Exp.2, CRF was stimulated with a flash (500 ms) of circular grating and SRF was stimulated with a flash (50 ms) of annular grating that was either iso- or cross-oriented to the CRF orientation. In type I cells, fast suppression is less orientation-specific, but both fast and late suppressions were orientation-specific in type II cells. There seems to be an interaction of functionally distinct pathways with different spatiotemporal properties for underlying mechanism of surround suppression. [J Physiol Sci. 2007;57 Suppl:S38]

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The detection of chemicals in the external environment -so called, chemosensation- is essential for the survival in many organisms. Odorants (volatile odorous chemicals) and pheromones (species and gender-specific chemicals) are two major olfactory cues by which information about food and suitable mating partners is transmitted. I herein describe how odorant or pheromone signal is detected and transduced via olfactory sensory systems in vertebrate and invertebrate. I will first present our data demonstrating molecular mechanisms underlying odorant and pheromone detection in the insect olfactory system. It turns out that there is a unique and previously-unappreciated receptor signal transduction mechanism, which appears to be the basis for the remarkable sensitivity and selectivity in insect chemosensation. Unlike insect, social and reproductive behaviors in mammals are modulated by not only volatile pheromones but also non-volatile cues that are likely detected by the vomeronasal organ located at the base of the nasal septum. We identified a male-specific peptide that was encoded by a gene from a previously-unrecognized large family in mice. This peptide, named ESP1, is secreted from male mice and transferred to the female vomeronasal organ wherein it elicits an electrical response. I will summarize current knowledge on structure and function of the ESP family and also describe most recent data on ESP1 receptor and its neural signaling pathway in the vomeronasal system. Supported in part by PROBRAIN, JSPS, and MEXT. [J Physiol Sci. 2007;57 Suppl:S39]

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ATP, an intracellular high energy phosphate compound, extracellularly functions as a neurotransmitter or neuromodulator. Receptors for ATP, P2 receptors, consist of seven ionotropic (P2X) and eight G-protein-coupled (P2Y) receptor subtypes. Although the involvement of two P2X and four P2Y subtypes were shown in taste responses, that of other subtypes remained to be investigated. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 μM ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca²⁺-imaging showed that similarly applied 1 μM ATP, 30 μM BzATP (a P2X7 agonist), or 1 μM 2MeSATP (a P2Y1 and P2Y11 agonist) increased intracellular Ca²⁺ concentration, but 100 μM UTP (a P2Y2 and P2Y4 agonist) and α,β-meATP (a P2X agonist except for P2X2, P2X4, and P2X7) did not. RT-PCR suggested the expression of P2X2, P2X4, P2X7, P2Y1, P2Y13, and P2Y14 among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X2 and P2X7. The exposure of the basolateral membranes to 3 mM ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 μM PPADS (a non-selective P2 blocker) and 1 μM KN-62 (a P2X7 blocker). These results showed for the first time the functional expression of P2X2 and P2X7 on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed. [J Physiol Sci. 2007;57 Suppl:S39]

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Taste is the sensory system primarily responsible for selection of food to be ingested. In this sense, the ability to taste the sweetness is important for animals to detect carbohydrate source of calories and has a critical role in the nutritional status of animals. Our recent studies suggested that sweet-sensitive cells may act as sensors not only for external calorie sources but also for internal energy store, represented by plasma leptin level, and external temperature (15-35^oC). Decrease in plasma leptin level and increase in temperature enhance sweet sensitivity of taste cells. Such modulation of perceived sweetness transmitted to the brain through afferent fibers may help maintaining animal's energy homeostasis. Recently, it is reported that taste cells expressing sweet or bitter receptors, such as T1rs and T2rs, have no conventional synapses with taste axons, providing a new taste mystery on peripheral information transmission. To challenge this mystery, we compared response characteristics of mouse fungiform taste receptor cells with action potentials and chorda tympani nerve fibers. We found that taste cells and fibers share similar characteristics in (1) response selectively to the four basic taste stimuli, (2) the grouping based on hierarchical cluster analysis, and (3) the occurrence of each class of taste cells with different taste responsiveness. These results suggest that information derived from receptor cells generating action potentials may be transmitted to taste nerve fibers with no major modifications by some unknown system. [J Physiol Sci. 2007;57 Suppl:S39]

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Although neurons operate more slowly than transistors, the brain can process information with impressive speeds. Our psychophysical studies in rats showed a single sniff of 150 ms can provide precise information about an odor (Uchida and Mainen, 2003). These findings suggested that the sniff cycle may be a fundamental unit of odor information coding and processing, and raised the question of how olfactory information is encoded on such a time scale. In an attempt to identify underlying mechanisms of rapid odor coding, we performed multi-electrode recordings in the olfactory cortex while rats performed an odor discrimination task. The rats were trained to perform a two-alternative olfactory discrimination task using 2-3 odor pairs. Sniffing was simultaneously recorded using a temperature sensor implanted in the nostril. Olfactory cortical responses were robust and widely distributed. For neurons tested with >= 6 odors, over half showed a significant response to at least one odor. For responsive neurons, spikes were tightly locked to the sniff cycle. Typical responses constituted of a fast transient (–50 ms duration) with a fixed latency (<100 ms) from the inhalation onset. To quantify the time course of the development of piriform odor representations, the ability of single neurons to discriminate between odor pairs was quantified. These analyses showed discriminability developed rapidly, usually peaking or saturating within 100 ms of the onset of the first odor inhalation. [J Physiol Sci. 2007;57 Suppl:S40]

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The sensation of taste has both cognitive and hedonic aspects of reward and aversion. Recognition of quality and quantity of food is necessary to compare it to the previous experiences and to remember it for future identification. Hedonic evaluation, on the other hand, plays a critical role in the decision of acceptance or rejection of the food. For example, the sweet taste of sugar is innately associated with palatability, or positive hedonics, and its ingestion is facilitated. However, if the ingestion of sugar is followed by illness, the hedonic value of the sugar changes from positive to negative and its ingestion is rejected even if its sweetness is essentially the same. Taste information is conveyed through the central gustatory pathways to the cortical gustatory area and is processed in terms of qualitative and quantitative aspects. Taste information is also sent to the reward system and feeding center via several brain sites including the prefrontal cortex, insular cortex and amygdala. The reward system contains the ventral tegmental area, nucleus accumbens and ventral pallidum and finally sends information to the lateral hypothalamic area, the feeding center. The dopamine system originating from the ventral tegmental area mediates the motivation to consume palatable food. The actual ingestive behavior is promoted by the orexigenic neuropeptides from the hypothalamus. Finally, the neural substrate of learning and memory of taste is introduced and the biological significance will be discussed in terms of survival of life. [J Physiol Sci. 2007;57 Suppl:S40]

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Fatigue is a very important biological alarm that signals the need for rest. For many years, we have studied the molecular and neural mechanisms of fatigue. In parallel with these studies, we have developed quantification methods, treatments for fatigue, and even proposals for citizens. Motivation is correlated with creativity, new planning, concentration, excitation, positive thinking, curiosity, and interest. Additionally, motivation is associated with academic successes. Therefore, we are working on the development of quantification methods for motivation and fatigue. Specialists in the fields of neuroscience, neuroimaging, genetics, childhood type chronic fatigue syndrome (CCFS), and education, are participating in the cohort study on school-age children's learning motivation with functional neuroimaging toward the goal of developing an efficient method for learning. Therefore, we had started our studies to develop methods to evaluate motivation in tasks; to perform follow-up studies on development of learning in elementary and junior high school students; to perform follow-up studies on CCFS; to examine the correlation between the imaging data and the results from the cognitive function battery, personality tests, gene expression related to the functional development of the brain, and environmental factors. [J Physiol Sci. 2007;57 Suppl:S40]

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Motivation and working memory are essential for learning progresses. Although neural substrates of motivation by monetary reward during working memory processing have been clarified to some extent, those by academic reward are still unclear. We here studied effects of academic reward on neural activities during working memory processing by using a 3.0 T functional magnetic resonance imaging in college students. Although the brain regions activated during working memory processing in the academic reward condition were totally overlapped with those in the monetary reward or control condition (condition without reward), addition or enlargement of activated brain regions were not found. Correlation analyses showed that neural activities of bilateral putamen during the academic reward condition were positively correlated with the baseline academic achievement motivation score and the task performance. Although the extents of the neural activities of bilateral putamen in the monetary reward condition were greater than those in the academic reward condition, the neural activities in that condition were not correlated with the motivation score or the task performance. Hence, putamen is a common brain region that is activated by monetary or academic reward and the brain region plays a crucial role in driving individual academic motivation and task performance of college students during working memory processing. [J Physiol Sci. 2007;57 Suppl:S41]

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We examined the effects of one-night sleep deprivation (SD) on the brain GABA_A receptor binding using PET with macaque monkeys(macaca mulatta). Monkeys were trained in a continuous simple reaction task (CSRT), which is required to repeat a visual-guided lever pressing, until they had been skillful in performing the task by a long-term training. The reaction time (RT) which is required for responding to the visual cue and for repeating trials was measured to estimate the behavioral performance. To measure the binding activity of the GABA_A receptor, two ¹¹C-labeled benzodiazepine analogues, [¹¹C]Ro15-4513 and [¹¹C]Ro15-1788, were used. Parametric images of the binding potential (BP) which were generated by a simplified reference tissue model (SRTM) based on pixel-wise kinetic modeling using a time activity curve of the pons as a reference were statistically analyzed using SPM99 software. RT for CSRT was significantly prolonged by the SD. BPs of [¹¹C]Ro15-4513, but not of [¹¹C]Ro15-1788, in the limbic structures such as the anterior cingulate and amygdala were significantly increased by SD. In addition, BPs of [¹¹C]Ro15-4513 in the mediodorsal nucleus of thalamus was changed dependent on the performance state that was estimated by the reaction time of CSRT. These results strongly indicate that changes of behavioral performance state caused by SD might be associated with changes in GABAergic neurotransmission in the limbic structure and the mediodorsal nucleus of thalamus. [J Physiol Sci. 2007;57 Suppl:S41]

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In Japan, the majority of school children in previously healthy children and adolescents are suffering from chronic fatigue, according to the report by the research group of the Ministry of Health and Welfare (Okuno et al, 1999). These symptoms in child and adolescent cases greatly interfere with normal function at school. We have encountered many such patients with associated indefinite or definite complaints; their major symptoms are general fatigue, a low-grade fever, central nervous system symptoms, such as moderate or severe depression, memory disturbance, confusion, and poor concentration, and/or sleep disturbance. Although the diagnostic criteria for chronic fatigue syndrome (CFS), such as the Center for Disease Control (CDC) are useful for adult cases, each of which has a distinct view of childhood CFS cases so that not all pediatric patients meet diagnostic criteria for CFS. Our major goal in this study is to determine the diagnostic criteria for childhood type chronic fatigue in Japan to carry out its definitions. Putting the different views together in pediatric cases is valuable to get a more complete picture. To better understand the pediatric cases of CFS in Japan we conducted a detailed analysis of our patients and proposed new diagnostic criteria for CCFS. [J Physiol Sci. 2007;57 Suppl:S41]

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The Na⁺/H⁺ exchanger (NHE, SLC9 family) is an ion transporter that regulates the ionic homeostasis such as intracellular pH (pH_i), Na⁺ concentration and cell volume. Such physiological function is intimately linked to the remarkable propertie of NHE, i.e., it is rapidly activated in response to a variety of extracellular stimuli such as hormones, growth factors and mechanical stressors. In recent years, we have focused on several essential factors for function of the ubiquitous isoform NHE1. We obtained evidence that at least three processes are necessary: 1) interaction of multiple protons with the regulatory "pH-sensor" sites in the cytoplasmic side of NHE1, 2) homo-dimerization of NHE1 and 3) interaction with calcineurin B-homologous protein CHP at the juxtamembrane cytoplasmic region of NHE1. CHP is one of EF-hand Ca²⁺ binding proteins and now at least three isoforms are known. We recently suggested that CHP2 is almost exclusively expressed in cancer cells and may be involved in maintenance of high pH_i in these cells, while CHP1 is ubiquitously expressed in virtually all tissues. More recently, we determined the crystal structure of CHP2 complexed with the cytosolic region (aa 503-545) of NHE1 at 2.7 Å and clarified the molecular basis for producing the target specificity of related Ca²⁺ binding proteins at atomic level. In this symposium, we will particularly focus on the crystal structure of the CHP/NHE1-peptide complex and the mechanism of pH_i regulation by NHE1/CHP complex. [J Physiol Sci. 2007;57 Suppl:S42]

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Kidney is the most important organ for water and electrolytes metabolism within body. This process is mostly attained by numerous water and electrolytes transport systems automatically regulated in response to ever changing external conditions. Recently, some of the disturbances of water and electrolytes metabolism were identified to be caused by the mutations of specific renal transporters and channels. However, identification of a responsible gene does not always guarantee that molecular pathogenesis of the disease is fully understood. In addition, naturally occurring mutations often tell us important functional domains of the protein of our interest and its regulatory mechanism. Accordingly, functional analysis of disease-causing mutants is important for understanding pathophysiological roles of channels and transporters. However, it has been hampered by the lack of good cell lines endogenously expressing these proteins. To overcome this situation, we generated AQP2 water channel and WNK4 kinase knock-in mice to study molecular pathogenesis of autosomal dominant type nephrogenic diabetes insipidus (AD-NDI) and pseudohypoaldoseronism type II (PHAII), respectively. Analysis of these mice revealed novel molecular mechanisms of the diseases. In addition, these mice were good resources for further studies using proteomics techniques and also for testing possible therapeutic strategies for the diseases. [J Physiol Sci. 2007;57 Suppl:S42]

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Septins are ubiquitous GTP-binding proteins which form filamentous heteropolymer complexes. Although they are required for cortical organization in cell division and cellular morphogenesis, postmitotic roles of septins beneath neuronal and glial membrane are unknown. To explore their physiological functions, we examined mice lacking a brain-specific septin subunit, Sept4. By comprehensive behavioral screening, we pinpointed a hypo-dopaminergic defect in the nigrostriatal transmission. Although the nigral dopamine neurons without Sept4 were morphologically normal, the dopamine transporter (DAT) and a few related molecules were reduced from their axons projecting to the striatum. These molecules and Sept4 were colocalized in dopaminergic presynaptic terminals and co-immunoprecipitated from the striatal homogenate. Transgenic mice overexpressing Sept4 exhibited a slightly higher DAT level, and transgenic supplementation of Sept4 normalized the DAT level in Sept4^−/− striatum. These data concordantly demonstrate that association of Sept4 has a positive effect on the DAT level. We hypothesize that the presynaptic septin scaffolds help organize and/or stabilize the macromolecular complex containing DAT. We previously reported that Sept4 is sequestered into α-synuclein aggregates under pathological conditions such as Parkinson disease. Thus, Sept4 insufficiency may contribute to the pathophysiology by attenuating dopaminergic neurotransmission. [J Physiol Sci. 2007;57 Suppl:S42]

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The small G proteins including Rab5 and Rheb, which cycle between active (GTP-bound) and inactive (GDP-bound) states, play essential roles for membrane budding and trafficking in cells. Here we show novel Rab5-binding proteins (RIN family), which contain many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer for GTP-Rab5. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes. Rheb appears to be involved not only in cell growth but also in nutrient uptake. We identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. [J Physiol Sci. 2007;57 Suppl:S43]

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After the completion of human genome project, about 350 transporters have been identified in human, including those identified from the genomic sequences. The recent progresses in transporter research have revealed numbers of transporter function, although many orphan transporters still remain. Now the transporter research moves the interests to physiological and pathological roles of transporters. For such analyses, the quantitative information is essential in addition to the qualitative information, e.g. functional changing of transporters in disease conditions, and functional regulation of interacting proteins including scaffold proteins. To accelerate this movement, we have developed two fundamental techniques by means of HPLC-linked tandem mass-spectrometry (LC-MS/MS). Identification of substrates and evaluate the transport activity is one of limiting steps in transporter research, since transport activity was examined using single radio-labeled compound. We have developed the technique to analyze transport function using mixture of non-radio-labeled compounds. The expression was usually analyzed in mRNA levels and there is no method to quantify the transporter protein comprehensively. We have succeeded to quantify the absolute protein amount of multiple transporters simultaneously with high-sensitivity. This method can produce the transporter map, which is necessary to evaluate the physiological and pathological contribution of transporters. The new techniques will open the new field in transporter research. [J Physiol Sci. 2007;57 Suppl:S43]

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Thyroid hormone plays a critical role in cerebellar development. Its deficiency results in various abnormalities, including reduced dendritic arborization of Purkinje cells, prolonged proliferation and delayed migration of external granule cells, and decreased synaptogenesis between Purkinje and granule cells. Such abnormal development cannot be rescued unless thyroid hormone is replaced within first two weeks of postnatal life in rodents. Thyroid hormone acts by binding to nuclear thyroid hormone receptor (TR) that is a ligand dependent transcription factor, which binds to thyroid hormone response element (TRE) located at the promoter region of its target gene. TR further recruits cofactors such as SRC-1, which is also strongly expressed in cerebellum. It regulates transcription of target genes only during first two weeks of postnatal life in rodents. Target gene includes RORα, neurotrophic factors and myelin basic proteins. Among such genes, RORα may play a critical role in TR-mediated cerebellar development. Cerebellar phenotype and alteration of neurotrophin expression of mutant mouse (staggerer) harboring RORα mutation is similar to that of hypothyroid mouse. Furthermore, although RORα augments TR-mediated transcription, staggerer type mutant RORα does not have such action. These results indicate that RORα may play a major role in TR-mediated cerebellar development. [J Physiol Sci. 2007;57 Suppl:S43]

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Normal cerebellar development requires several hormones. Recently, it has been a concern that environmental endocrine disrupters may interfere with hormonal functions in brain development. Polychlorinated biphenyls (PCBs) and hydroxylated PCB (OH-PCB) metabolites are widely distributed bioaccumulative environmental chemicals and have similar chemical structures to those of thyroid hormones (THs). Previously, we reported that THs are essential for the normal dendritic development of Purkinje cells, and low doses of two OH-PCBs, namely, 4-OH-2',3,3',4',5'-pentachlorobiphenyl (4'OH-PCB106) and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl (4'OH-PCB159), inhibited their TH-dependent development in mouse cerebellar cultures (Develop. Brain Res., 154: 259, 2005). Koibuchi et al. clarified in reporter gene assay that they interfere with TH-dependent gene expressions (JBC, 279:18195, 2004). On the other hand, 4-OH-2,2',3,4',5,5',6-heptachlorobiphenyl (4OH-PCB187), which was found to have the highest concentration among OH-PCB congeners in human cerebrospinal fluid (CSF) (Takasuga et al. Organo Halog Compounds 66:2529, 2004), did not inhibit TH activity in cerebellar cultures. However, it promoted an abnormal dendritic development of Purkinje cells and affects their survival rate. Furthermore, 4OH-PCB187 showed an inhibitory effect on synapse formation, which is essential for normal brain development. These results indicate that OH-PCB congeners may interfere with hormonal functions in brain development via several mechanisms depending on their chemical structure. [J Physiol Sci. 2007;57 Suppl:S44]

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ERα and ERβ, are expressed in many brain regions of rodents, but the expression pattern is different between the two receptors. In previous studies, the expression of ERβ but not ERα was detected in the adult cerebellum, by in situ hybridization and immunohistochemistry. In the present study, quantitative real-time RT-PCR demonstrated that levels of cerebellar ERα mRNA in neonatal pups were significantly higher than in adults. In contrast, cerebellar ERβ mRNA remained at similar levels during postnatal development. Using in situ hybridization and immunohistochemistry, ERα mRNA and protein were detected in Purkinje cells of the developing rat cerebellum. However, ERα immunoreactivity was detected only in a few Purkinje cells in the adult cerebellum. Thus, ERα expression was transiently increased during the time when Purkinje cell dendritic growth and synapse formation proceed, suggesting that a role for ERα in Purkinje cell differentiation. ERβ expression occurred in Golgi type neurons in the granular layer at P7, Purkinje cells at P14, and basket cells in the molecular layer at P21, and was detected in all the cell types in the adult cerebellum, suggesting a role for ERβ associated with neuronal differentiation and maintenance. Furthermore, double-label immunofluorescence for ERα and ERβ demonstrated their co-localization in Purkinje cells at P14, suggesting a possible interaction. between the two receptors. The discrete expression profiles for ERα and ERβ in the developing cerebellum suggest the two ERs play distinct roles in cerebellar development. [J Physiol Sci. 2007;57 Suppl:S44]

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The brain has traditionally been considered to be a target site of peripheral steroid hormones. By contrast, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo from cholesterol, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain is a conserved property of vertebrates. Recently the Purkinje cell, a cerebellar neuron, has been identified as a major site for neurosteroid formation in a variety of vertebrates. This is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. Since this discovery, diverse actions of neurosteroids are becoming clear by the studies on mammals using the Purkinje cell as an excellent cellular model. In mammals, the Purkinje cell possesses several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase (3β-HSD) etc., and actively synthesizes progesterone and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. The Purkinje cell also expresses the key enzyme of estrogen formation, cytochrome P450 aromatase (P450arom), and may actively produce estradiol in the neonate. Both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in Purkinje cells. 3α,5α-THP is also involved in Purkinje and granule cell survival. [J Physiol Sci. 2007;57 Suppl:S44]

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Chloride ions subserve many physiological functions, including regulation of cell volume, intracellular pH, fluid secretion, and stabilization of the resting membrane potential. Cl⁻ is absorbed along the length of the gastrointestinal tract via region-specific pathways. The major route is an electroneutral NaCl absorptive pathway involving parallel functioning of the Na⁺/H⁺ exchanger and Cl⁻ /HCO₃⁻ exchanger. SLC26A3, Cl⁻ /HCO₃⁻ exchanger, is highly expressed in colonic epithelial cells, and mutations in the SLC26A3 cause congenital chloride diarrhea. These suggest that SLC26A3 play the role in NaCl absorption in the colon. However, the transport characteristics of SLC26A3 have not been fully studied. Since multiple isoforms of the Cl⁻ /HCO₃⁻ exchanger are co-expressed in an intact intestinal cell, complicating the functional analysis of an individual isoform, we generated an N-terminal hemagglutinin epitope-tagged human SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Using this system, we have characterized SLC26A3 by measuring of its activity with fluorescent pH-sensitive dye, BCECF and chloride-sensitive dye, MEQ. We measured substrates specificity, inhibitors sensitivity and regulation of pH. We also measure Cl⁻ /HCO₃⁻ exchanger activity in the isolated colonocytes to determine the functional expression of SLC26A3 in native tissue. We will discuss the role of SLC26A3 in NaCl absorption in the colon. [J Physiol Sci. 2007;57 Suppl:S45]

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Patients with cystic fibrosis (CF;cystic fibrosis transmembrane conductance regulator) caused by CFTR gene mutations often fail to produce alkaline pancreatic juice, in which Na⁺-H⁺ exchangers (NHE) may be involved. We examined the activity of apical NHE in interlobular pancreatic ducts isolated from δF mice, a CF mouse model. The ducts (diameter:100 μm) were isolated by microdissection and superfused with HCO₃⁻-free Hepes-buffered solutions at 37°C and the lumen was microperfused separately. Intracellular pH (pHi) was measured by microfluorometry using BCECF. Duct cells were acid-loaded with a pulse of 20 mM NH₄⁺, which was followed by a Na⁺-free solution in both the bath and lumen. The rate of pHi recovery after re-addition of Na⁺ to the luminal solution was calculated as a measure of the activity of the apical NHE. The rate of pHi recovery was 0.12 ± 0.01 pH unit/min (mean ± SD, n = 8) in wild-type ducts which was completely inhibited by 100 μM HOE642, an inhibitor of NHE. Forskolin (1 μM) reduced the apical NHE activity to 0.05 ± 0.01 (n = 9, p <0.01). The apical NHE activity in CF (δF/δF) ducts was 0.20 ± 0.01 (n = 6), which was significantly (p < 0.01) higher than that in wild-type ducts and was further accelerated to 0.66 ± 0.11 (n = 6, p < 0.01) by forskolin. Under normal conditions, the apical NHE activity in mouse pancreatic duct cells is inhibited by cAMP via CFTR-dependent mechanisms. In the absence of functional CFTR, the apical NHE activity is stimulated by cAMP, which may be responsible for reduced HCO₃⁻ secretion in CF. [J Physiol Sci. 2007;57 Suppl:S45]

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The FXYD family proteins are small proteins which contain a single transmembrane domain, and the conserved FXYD (Phe-Xaa-Tyr-Asp) motif in the ectodomain. They associate with the Na⁺,K⁺-ATPase to modulate its function. They also modulate epithelial membrane transport of small solutes and ions. Among seven members of human FXYD proteins, the FXYD3 (Mat-8) associates with the Na⁺,K⁺-ATPase , and slightly decreases its affinity for Na⁺ and K⁺. The FXYD3 was also involved in cell proliferation and tumorigenesis. Its mRNA was highly expressed in human and murine breast tumors. However, there has been no report about the relationship between cell proliferation and modulation of Na⁺,K⁺-ATPase function by the FXYD3. Here, we studied the mRNA and protein expression of FXYD3 in human cultured cell lines and normal tissues. The mRNA expression was high in a breast cancer cell line, MCF-7 and a pancreatic cancer cell line, T3M-4. Among normal tissues, the mRNA expression of FXYD3 was highest in colons. By using a monoclonal antibody raised against human FXYD3, the protein was observed as a single band with a molecular mass of 13 kDa in the membrane fraction of human colon mucosae as well as MCF-7 and T3M4 cells. It should be noted that the FXYD3 protein was down-regulated in human colorectal cancers compared with the surrounding normal mucosae. [J Physiol Sci. 2007;57 Suppl:S45]

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Arachidonic acid (AA, 10 nM–2 μM), enhanced Ca²⁺-regulated exocytosis in acetylcholine (ACh) stimulated antral mucous cells of guinea pig. The AA actions were inhibited by MK886 (50 μM, an inhibitor of peroxisome proliferation activation receptor α (PPARα)), and were mimicked by PPARα agonists (Eicosatetraynoic acid (ETYA) and WY14643). The enhancement of ACh-stimulated exocytosis induced by AA, ETYA and WY14643 was eliminated by a PKG inhibitor (Rp8BrPET-cGMPS) and was mimicked by 8Br-cGMP. Nitro-L-arginine methyl ester (L-NAME) also inhibited the enhancement induced by AA, ETYA and WY14643, and NOC-12 (an NO donor) enhanced ACh-stimulated exocytosis similar to AA, ETYA and WY14643, which was inhibited by Rp8BrPET-cGMPS. ACh-stimulated exocytosis was partially inhibited by MK886, L-NAME and Rp8BrPET-cGMPS. AA, ETYA or WY14643 did not increase intracellular Ca²⁺ concentration ([Ca²⁺]_i) and did not enhance an ACh-stimulated [Ca²⁺]_i increase in antral mucous cells. Measurements of cGMP contents in antral mucosa demonstrated that stimulation with ACh, AA and PPARα agonists stimulate cGMP accumulation, which is inhibited by MK886 and L-NAME. In conclusion, in ACh-stimulated antral mucous cells, AA accumulated via phospholipase A2 stimulates PPARα, which activates the NO/cGMP cascade, and PKG activated by cGMP enhances Ca²⁺-regulated exocytosis in antral mucous cells. [J Physiol Sci. 2007;57 Suppl:S46]

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A short-chain fatty acid butyrate is produced in the colonic lumen and taken up by epithelial cells, playing an essential role in the maintenance of homeostasis of the colonic epithelium. Previous studies have demonstrated that butyrate suppresses cell proliferation and induces cellular differentiation and apoptosis in a wide variety of cell types. To identify the detailed mechanisms by which sodium butyrate (SB) induces the differentiation of colonic epithelial cells, GeneChip microarray and computational gene network analyses were performed. A differentiated phenotype accompanying elevations of alkaline phosphatase activity and histone acetylation was observed in the mouse colonic epithelial MCE301 cells treated with 2 mM SB. Of the 22,690 probe sets analyzed, 2,604 differentially expressed probe sets were identified in the differentiated cells and were classified into 4 groups. Of these, the gradually increased group and the gradually decreased group contained the genetic networks for cellular development and cell cycles, respectively. Moreover, the expression levels of transcripts for ion channels and transporters were changed remarkably by the differentiation of the cells. The present results provide a basis for understanding the detailed molecular mechanisms of the cell differentiation induced by SB in colonic epithelial cells. [J Physiol Sci. 2007;57 Suppl:S46]

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Depth with unambiguous sign is created when one fuses dichoptic pairs of discs with one member of each pair black and the other white. This is referred to as the sieve effect. The stimulus contains no positional disparities. Howard (1995) noted qualitatively that the sieve effect occurs when the rivalrous regions are within the range of sizes, contrasts, and relative sizes where exclusive rivalry occurs, rather than binocular lustre, stimulus combination, or dominant rivalry. This suggests that perceived depth in the sieve effect should be at a maximum when exclusive rivalry is most prominent. We used a disparity depth probe to measure the magnitude of perceived depth in the sieve effect as a function of the sizes, contrasts, and relative sizes of the rivalrous regions. We also measured the rate of exclusive rivalry of the same stimuli under the same conditions. Perceived depth and the rate of exclusive rivalry were affected in the same way by each of the three variables. Furthermore, perceived depth and the rate of exclusive rivalry were affected in the same way by changes in vergence angle, although the configuration of the stimulus surface was held constant. These findings confirm the hypothesis that the sieve effect is correlated with the incidence of binocular rivalry. [J Physiol Sci. 2007;57 Suppl:S46]

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How are three-dimensional surface orientations represented in the visual cortex? We have examined an idea that these surface orientations are encoded by neurons with a variety of tilts in their binocular receptive field (RF) structure. To examine if neurons in the early visual areas are capable of encoding surface orientations, we have recorded from single neurons extracellularly in areas 17 and 18 of the cat using standard electrophysiological methods. Binocular RF structures are obtained using a binocular version of the reverse correlation technique. About 30% of binocularly-responsive neurons have RFs with statistically significant tilts from the frontoparallel plane. The degree of tilts is sufficient for representing the range of surface slants found in typical visual environments.We also investigated how tilted binocular receptive fields may be constructed. We proposed two models, an interocular spatial frequency difference model (dif-frequency model) and a pooling model. To test the dif-frequency model, we compared the actual slant of the receptive field with the tilt predicted from the spatial frequency tuning of the neuron in the left and right eyes. A subset of neurons exhibited responses consistent with the dif-frequency model. The pooling model was also partially consistent with some neurons, but inconsistent with others. Our results indicate that encoding of 3D surface orientation begins with binocular neurons in early visual cortex, and this selectivity has contributions from both spatial frequency differences and pooling. [J Physiol Sci. 2007;57 Suppl:S47]

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It has revealed that neurons in the caudal part of intraparietal sulcus (caudal intraparietal, CIP) selectivity respond to 3D axis and surface orientation, suggesting that this region might be important for processing of visual information of 3D structure. To elucidate the importance of CIP in 3D vision further, we investigated response of CIP neurons to more integrated structure–3D curvature. A set of 3D curvatures, such as ellipsoids, cylinders and saddles, was presented to a monkey performing fixation task. Single unit activity was recorded from CIP of the animal during the task. Some CIP neurons showed significant response to one of curvatures. We defined these neurons as curvature selective neurons (CSN). Notably, some CSN were highly selective to a particular shape. The preferred shapes of CSN were biased toward convex and concave ellipsoid, whereas they were distributed over all shapes tested. These results suggest that 3D curvature might be represented in CIP as well as axis and surface orientation. We also investigated architectonic structure of CIP using immunohistochemical staining with SMI-32 antibody. We observed a thick immunoreactive band of SMI-32 in layer 3 and 5 of the lateral bank of intraparietal sulcus (IPS) near the fundus, and found that most of recording sites of CSN fell in that region. These results suggest that CIP might be an architectonically distinct visual area in IPS and might be functionally specialized for 3D vision. [J Physiol Sci. 2007;57 Suppl:S47]

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Most of the patient with visual impairment show category specific visual deficit such as letter (alexia), object (visual object agnosia), landmark (topographical agnosia), or faces (prosopagnosia). However, in rare cases, some patients show element specific visual impairment. In this session, I introduce such a patient. His main characteristic was visual impairment for two-dimensional object. The patient was a 62 year-old right handed man. Due to a progressive visual problem, he visited Showa University Hospital. The magnetic resonance imaging (MRI) showed a right dominant mild atrophy of the parieto-occipital lobe. In the neuropsychological examination, his feature was as follows. When the patient was asked to name the objects of photograph or line drawing, he could not identify them; however, he could identify the real object immediately after presentation. In order to define the effect of three-dimensional information, we examined the patient using two tasks: Binocular vs. Monocular (BM) task and Single Viewpoint (SV) task. In the BM task, the patient was asked to identify real objects using monocular or binocular vision. In the SV task, he was asked to name the real objects from single viewpoint using chin rest. As a result he showed no significant differences between monocular and binocular vision. On the other hand, his identification time was significantly delayed when he was asked to name the object from single view point rather than free view condition. Considering these results, we suspected that his disorder of recognition of two-dimensional object was due to disturbance of recognition from a single viewpoint. [J Physiol Sci. 2007;57 Suppl:S47]

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Brain is one of the most lipid-rich tissues containing large variety of lipid molecules. Recent advances in lipid research have been attracting great interests in lipid metabolism in the brain and its physiological and/or pathophysiological significance. Membrane phospholipids, when hydrolyzed by phospholipases, can cause two classes of lipid mediators: fatty acid derivatives such as prostaglandins (PGs) and leukotrienes (LTs), and lysophospholipid-related mediators such as platelet-activating factor (PAF). Possible involvement of specific lipid mediators in the brain physiology and/or pathophysiology has been suggested; however, the information has been rather limited because of lack of a strategy for comprehensive understanding of lipid mediator production profiles. In the present symposium, we introduce our recent development of a multiplex quantitation system by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) that covers various lipid mediators and its application to a comprehensive profiling analysis of lipid mediator production in the rodent brain, especially in the hippocampus during kainic acid-induced seizure, an animal model of human temporal lobe epilepsy. [J Physiol Sci. 2007;57 Suppl:S48]

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Marijuana affects neural functions through the binding of its active component to CB1 cannabinoid receptors. The CB1 receptor is widely distributed in the brain, and primarily localized on axons and presynaptic terminals. Endogenous ligands for cannabinoid receptors (endocannabinoids), such as anandamide and 2-arachidonoylglycerol, are lipid in nature, and produced on demand from membrane lipids through two enzymatic reactions. Recent studies have revealed that endocannabinoids play important roles in activity-dependent modulations of synaptic transmission as retrograde messengers. Endocannabinoids are released from postsynaptic neurons, retrogradely act on presynaptic CB1 receptors, and cause transient or long-lasting suppression of transmitter release. In this symposium, we summarize our studies with cultured hippocampal neurons, and show how the endocannabinoid signal is generated and terminated. We demonstrate the roles of voltage-gated Ca²⁺ channels, NMDA-type glutamate receptors, G_q/11-coupled receptors, and phospholipase Cβ in the generation, and the roles of monoacylglycerol lipase and cyclooxygenase-2 in the termination of the endocannabinoid signal. Wide distributions of these molecules as well as CB1 in the brain suggest importance of the endocannabinoid signal in various aspects of brain function. [J Physiol Sci. 2007;57 Suppl:S48]

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Upon depolarization and Gq protein-coupled receptor activation, endocannabinoids are synthesized in postsynaptic neurons and act on presynaptic CB1 receptor, causing endocannabinoid-mediated retrograde suppression (ERS) of transmitter release at activated excitatory synapses and nearby inhibitory synapses. Through immunohistochemical studies on distribution and localization of CB1, endocannabinoid synthetic enzyme diacylglycerol lipase (DAGL), and relevant receptors, the following facts are disclosed so far. In Purkinje cells, CB1 is highly expressed at both excitatory and inhibitory synapses, and DAGLa accumulates at the neck of dendritic spines. Notably, DAGLa is selectively excluded from the spine head, at which mGluR1 is exclusively concentrated. In hippocampal pyramidal cells, CB1 is expressed at much higher levels in CCK interneuron synapses than excitatory synapses, and, reciprocally, DAGLa is confined to dendritic spines forming excitatory synapses. In the striatum, CB1 is highly expressed at medium spiny neuron (MSN)-MSN neuron synapse and parvalbumin interneuron-MSN synapse, with lower levels at corticostriatal synapses. In MSNs, DAGLa and mGluR5 exhibit similar gradient-like distribution with the highest level in dendritic spines and gradual decrease toward the soma, while muscarinic acetylcholine receptor M1 is more evenly distributed. Therefore, the occurrence of physiological ERS at excitatory and inhibitory synapses are regulated, at least partly, through these different fine synaptic localization of endocannabinoid signaling molecules in given neurons forming distinct synaptic organization. [J Physiol Sci. 2007;57 Suppl:S48]

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Prostaglandins (PGs) are thought to regulate brain functions. We have reported that prostaglandins including PGD₂, PGE₂, and PGF_2α are produced by COX-2 expression in neurons in the cerebral cortex following cortical spreading depression, the propagation of neuronal membrane depolarization throughout the cortical hemisphere, in rats. In such a model for neuronal excitation in the cortex, the amount of non-REM sleep, but not of REM sleep, increased subsequently for several hours in the animals, and the increase was completely attenuated by application of NS-398, a COX-2 inhibitor. In the same model, furthermore, perineuronal satellite cells which exist ubiquitously all over the cortex and were recently identified as cortical progenitor cells, started to proliferate in the hemisphere and differentiated into oligodendrocytes, astrocytes, and immature neurons in weeks. The proliferation activity depended on numbers of induced spreading depression, and was also attenuated by NS-398. The number of cells involved in the cell cycle increased in the cortical slices by application of prostaglandins including PGF_2α. These observations indicate that prostaglandins regulate cell renewal in the cortical tissue. Prostaglandins are thought to be key molecules which relieve excessive brain activity by inducing non-REM sleep and bring about brain tissue remodeling for building up tolerance to the excessive activity. [J Physiol Sci. 2007;57 Suppl:S49]

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Intracerebroventricular (i.c.v.) injection of arachidonic acid (AA) evokes fever. This response has been thought to occur simply because AA is converted to prostaglandin E₂ (PGE₂), the final mediator of fever, by constitutively expressed enzymes of the AA cascade. However, our recent study suggested that AA might not only be the precursor of PGE₂ but also induce an enzyme cyclooxygenase-2 (COX-2) that catalyses AA to form prostaglandins. We here examined in rats whether AA-induced fever is dependent on COX-2, and, if so, where COX-2 is induced by AA. I.c.v. injection of AA evoked biphasic fever. The first phase started within 15 min of injection and peaked at 30 min. The second phase started at 60 min and peaked at 2 h after AA injection. COX-2 specific inhibitor completely suppressed the second phase of fever while COX-1 specific inhibitor suppressed the first phase. Two hours after AA injection, PGE₂ level in the cerebrospinal fluid was elevated. This elevation was also suppressed by the COX-2 inhibitor. Immunohistochemistry and immunoblotting revealed enhanced COX-2 expression in subarachnoidal tissues. Double immunohistochemistry revealed that the major cell type that expressed COX-2 was endothelial cell. These results indicate that AA itself or its metabolite induces COX-2 expression that accelerates the formation of PGE₂ from AA, and, hence, develops the second phase of fever. [J Physiol Sci. 2007;57 Suppl:S49]

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Osteoclasts resolve calcified tissue by secreting a massive amount of protons and play significant roles in bone remodeling and bodily Ca²⁺ metabolism. Osteoclasts express two distinct types of acid-secreting pathways, the vacuolar type H⁺-ATPase (V-ATPase) and the voltage-gated proton channel (H⁺ channel). Both could acidify the extracellular space, but would work differently in cellular functions. The V-ATPase is rich at the plasma membrane faced to bone surface (the ruffled membrane) and serves as the primary mechanism responsible for H⁺ secretion into the resorption pit. Inadequate control of the H⁺ pump may impair bone remodeling and cause pathological states like osteoporosis. We for the first time recorded the proton currents mediated by plasmalemmal V-ATPases in murine osteoclast-like cells. The H⁺ pump current was tiny as compared to that of the H⁺ channel, but continued to carry protons uphill against large negative electrochemical gradients. The V-ATPase activity was also regulated by the concentration of intracellular ATP, interaction with the actin cytoskeleton, recruitment from the internal vesicles, the membrane potential and pH gradients across the membrane. In addition, we found an elevation of extracellular calcium reduced the V-ATPase activity. Accumulation of acids and calcium in the resorption pit might serve as a negative feedback mechanism for the V-ATPase in osteoclasts. H⁺ releasing ability of the plasmalemmal V-ATPase could change over a wide range in response to variable cellular conditions. [J Physiol Sci. 2007;57 Suppl:S49]

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Inhibitory action of extracellular H⁺ on neural activity occurs through pH-sensitive channels, such as voltage-gated Ca ²⁺ channels and NMDA receptors. In order to interpret the formation of receptive field surrounds in the retinal neurons, it has been suggested that GABA mediates the feedback signal from horizontal cells (HCs) to cones. Hirasawa and Kaneko (2003) proposed an H⁺-mediated feedback hypothesis to substitute for the GABA hypothesis since picrotoxin, a GABA-receptor antagonist, could not suppress the feedback signal. To verify the idea that the depolarized HCs release H⁺ we measured, by a fluorescence ratio imaging technique, the pH of the immediate external surface (pH_o) of HCs isolated from carp or goldfish retina. When HCs stained by 5-hexadecanoylaminofluorescein, a pH-sensitive lipophilic dye, were depolarized by application of kainate or by high extracellular K⁺, pH_o decreased. The amount of pH_o reduction was monotonically dependent on the amount of depolarization, as much as 0.21 ± 0.05 pH unit by 100 mM K⁺. Reduction of pH_o was suppressed by 0.4 μM bafilomycin A1, a specific inhibitor of V-ATPase (proton pump), suggesting that the HC depolarization enhanced an outward H⁺ flux by voltage-sensitive H⁺ pump. Immunohistochemical analysis using an anti V-ATPase antibody revealed the existence of V-ATPase on the HC cytoplasmic membrane. Double labelling of V-ATPase and GAD revealed that the V-ATPase was essentially localized at the surface of HC which would affect the cone terminals. [J Physiol Sci. 2007;57 Suppl:S50]

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Substantial evidence indicates that voltage-gated proton channels are activated during the phagocyte respiratory burst, when NADPH oxidase produces superoxide, a precursor to many reactive oxygen species (ROS). Originally, Henderson and colleagues (Biochem. J. 246:325-329) proposed that the function of proton extrusion was to compensate the charge translocated by the electrogenic extrusion of electrons by NADPH oxidase. (1) We believe that charge compensation remains an important function of proton current. However, proton current during the respiratory burst has additional useful consequences that could not be accomplished if the charge were compensated by other ions. (2) Proton movement from cytoplasm into the phagosome prevents large pH changes in either compartment. (3) Proton flux into the phagosome prevents excessive phagosome swelling that would occur if charge compensation were mediated by other ions. (4) Protons are required in the phagosome as substrates to produce enormous quantities of H₂O₂ (the immediate dismutation product of superoxide anion) as well as HOCl (believed to be a key bactericidal ROS). Quantitative estimates and modeling of events during the respiratory burst in human neutrophils and eosinophils suggest that proton current compensates at least 90-95% of the charge translocated by NADPH oxidase. Only proton current can perform all four required tasks. Supported by the Heart, Lung and Blood Institute of the NIH (HL61437). [J Physiol Sci. 2007;57 Suppl:S50]

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In phagocytes such as neutrophil, macrophage and microglia, regulation of pH of phagosomes and cytoplasm is critical for pathogen killing, antigen presentation and elimination of abnormal cells such as apoptotic cells and cancer cells. NADPH oxidase that mediates ROS generation during phagocytosis, called respiratory burst, leaves proton in the cytoplasm while it releases superoxide anions. This causes charge imbalance in the cytoplasm leading to membrane depolarization and acidification. Proton also needs to be supplied to phagosome for production of hydrogen peroxide and HOCl. Voltage-gated proton channel (Hv channel) has been believed to be the most likely candidate for these roles in phagocytosis. However, its molecular basis has long been elusive. We have reported a protein called VSOP (voltage-sensor only protein, also called as Hv1) that in heterologous expression recapitulates most properties of endogenous Hv currents (Sasaki et al, Science, 2006). To understand roles of VSOP in phagocytosis, immunohistochemistry and western blot analysis were performed with VSOP-specific antibody. Prominent expression was detected in many types of cells in spleen. Induction of VSOP protein expression was also studied in an injured tissue: cryoinjury was induced in adult mouse brain. Up to one month, population of VSOP-positive cells including neutrophils, lymphocytes and monocytes were observed around the injured site. We are also studying detailed pattern of subcellular localization of VSOP proteins in these cells. [J Physiol Sci. 2007;57 Suppl:S50]

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During exercise, oxygen must be taken out of the air and sent to the exercising muscles, and carbon dioxide derived from metabolism must be discharged quickly from our bodies. Otherwise, acid-base and/or blood gases homeostasis will be lost and exercise cannot be continued. Accordingly, ventilation should increase quickly and precisely in accordance with metabolic demand during exercise (Exercise Hyperpnea). It is recognized that the chemoreceptor reflex should keep the tension of blood gases fixed thus maintaining homeostasis. However, during moderate constant load exercise blood gases are kept at nearly the same levels as during rest although ventilation markedly increases. The alternating mechanisms for exercise hyperpnea are assumed to be two neural pathways, central command and peripheral neural reflex. These neural mechanisms should operate from the onset of exercise so that ventilation increase rapidly and roughly before the metabolic byproducts reach the carotid body. Thus, the neural mechanisms are indispensable in the maintenance of homeostasis during exercise.The initial ventilatory response to exercise within 20s at the start of exercise, named Phase I, is derived solely from neural origin suggesting that to investigate Phase I response is very valuable for better understanding of exercise hyperpnea and its contribution to homeostasis. Our laboratory has been focused on Phase I and in this symposium I will introduce the accumulated data concerning the characteristics of Phase I in various subject groups (e.g. elderly, athletes etc.) and conditions (e.g. bedrest, muscle soreness etc.). [J Physiol Sci. 2007;57 Suppl:S51]

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Exercise causes an increase in blood flow to active muscle and a decrease in vascular conductance in non-active muscle to meet a metabolic demand in the exercising muscle. Several neural mechanisms, such as arterial and cardiopulmonary baroreflex, central command and a feedback from skeletal muscle, are involved in maintenance of circulatory homeostasis. We now focus on the role of feedback signal from skeletal muscle via afferents arising from muscle mechanoreceptor and metaboreceptor. In single limb, post-exercise muscle ischemia which is conducted by circulatory cuff occlusion on the exercised arm decreased vascular conductance in non-active muscle via arm metaboreceptor activation. Simultaneous post-exercise muscle ischemia in the lower limb attenuated the decreased vascular conductance in non-active muscle (Tokizawa et al., Exp Physiol, 2006). Further passive muscle stretch or venous occlusion assumed to activate mechanoreceptor in the lower limb suppressed the decreased vascular conductance in non-active muscle (Tokizawa et al., J Appl Physiol, 2004a,b). These inhibitory effects on vasoconstriction in non-active muscle might play an important role to maintain circulatory homeostasis against excessive cardiovascular response during exercise when the feedback signal from multiple limbs occurred. [J Physiol Sci. 2007;57 Suppl:S51]

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Lactate had been considered to be a waste which is produced due to lack of oxygen supply during exercise. However, it is now recognized that lactate is not a waste but an intermediate of carbohydrate and therefore an oxidizable substrate. Production of lactate does not necessarily mean lack of oxygen but rather increase in glycolysis and glycogenolysis. Therefore, lactate is an intermediate which balances glycolytic flow with mitochondrial oxidation. The transport of lactate into and out of tissues are done via transporter called MCT (Monocarboxylate Transporter). There are several isoforms in MCTs. MCT1 is abundant in slow type muscle fibers and is related to oxidation of lactate. MCT4 is rich in fast type muscle fibers and is related to extrusion of lactate out of tissues. These characteristics of distribution of MCTs in muscle fibers reflect characteristics of lactate metabolism particularly during exercise when a great deal of lactate is produced. Because lactate is an oxidizable substrate, ingestion of exogenous lactate can prevent hypoglycemia during endurance exercise. Fatigue during exercise is not necessarily caused by accumulation of lactate. For example, in marathon running, runners feel fatigue toward the end of race while there is less accumulation of lactate because of depletion of glycogen. There are some reports showing beneficial effect of production of lactate against fatigue. These suggest that oxidation is the major fate of lactate during and after exercise. Further studies are needed to clarify what is the major cause of fatigue other than lactate during exercise. [J Physiol Sci. 2007;57 Suppl:S51]

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The heat loss responses (sweating and skin blood flow responses) during exercise are very important for maintaining internal temperature. This study focuses on the characteristics of heat loss responses during exercise from a viewpoint of investigating non-thermal factors. In the mild hyperthermic condition, short-term dynamic exercise induces to show an intensity-dependent increase in sweating rate and decrease cutaneous vascular conductance (skin blood flow) without changes in thermal factors. These responses are caused by mainly non-thermal factors include central command, peripheral mechanisms activating the mechanosensitive and metabosensitive receptors in exercising muscle, baroreflex, mental stress, and so on. In case of activating mostly central command and metaboreflex from exercising muscle, these factors affect the heat loss responses during exercise by facilitating the sweating response, and inhibiting heat loss by altering cutaneous vascular conductance. On the other hand, an activation of mechanoreflex from exercising muscle only seems to influence the increase in the sweating response but not cutaneous vascular conductance. On the bases of these results, non-thermal factors associated mainly with central command, peripheral mechanisms activating the mechanosensitive and metabosensitive receptors in exercising muscle enhances the sweating response which may compensate the inhibition of heat loss by the altered skin blood flow which might play a role in maintenance of cardiovascular system during exercise. [J Physiol Sci. 2007;57 Suppl:S52]

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With the advent of green fluorescent protein (GFP), the subcellular distribution of sex steroid hormone receptors has been found to be more dynamic than previously thought. Upon estradiol treatment ERa and b in the same cell were colocalized to show discrete pattern, suggesting that both subtypes of ERs were bound to the same nuclear sites. In fact, FRET (fluorescence resonance energy transfer) clearly showed the interaction of ERa and ERb. In the presence of the estradiol, however, the discrete staining pattern of ERa and b were mostly overlapped with Brg-1, indicating that most of the ERs clusters are involved in the chromatin remodeling machinery. FRAP (fluorescence recovery after photobleaching) analysis showed that nuclear ERa and b are most dynamic and mobile in the absence of the ligand, but its mobility was decreased after the ligand treatment. When ATP was deleted from the culture medium, even liganded ERa significantly lost its free mobility in the nucleus, indicating that ERa dynamism is ATP-dependent. Transgenic mice in which GFP was expressed under the ERa promoter activity were generated. Ovariectomy caused significant reduction of cell bodies of GFP neurons containing ERa in the medial preoptic area, but not in the ventromedial nucleus. These results suggest that estrogen affects ERa containing cells at the region-specific manner. Sexually dimorphic nucleus of the preoptic area (SDN-POA) showed distinct male-female difference and contained estrogen receptor a (ERa). Relationship between the intrauterine position of fetuses and the volume of the SDN-POA is discussed. [J Physiol Sci. 2007;57 Suppl:S52]

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Because there are sex differences in feeding behavior in rats, we looked for a possible sex difference in the activity of melanin-concentrating hormone (MCH) and orexin neurons in the lateral hypothalamic area using phosphorylated cyclic AMP response element-binding protein (pCREB) as a marker of neural activity. Under free feeding condition, the activity of MCH neurons was increased and decreased in association with a meal initiation and meal termination, respectively. The activity of orexin neurons did not correlate with feeding states. Though fasting activated MCH neurons in both male and female rats, while the activation of orexin neurons was only observed in female rats. In fasted rats, glucose injection decreased the activity of MCH neurons more promptly in females than in males. The magnitude of decrease caused by glucose was greater at proestrus and estrus than at diestrus 2. Gonadectomy in males enhanced and in females attenuated the response of MCH neurons to glucose. Testosterone and estrogen replacement in males and females, respectively, restored the response of MCH neurons to glucose. Sex differences in the response of MCH neurons to glucose correlated well with the gonadal steroid milieu; thus, MCH neurons may play an important role in sex differences in feeding behavior. On the other hand, orexin neurons may responsed to stress induced by fasting only in female rats. Sex differences in MCH and orexin neurons may be the neural basis for the fact that females are more suspectable to eating disorder. [J Physiol Sci. 2007;57 Suppl:S52]

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We examined the effect of estrogen (E2) replacement in ovariectomized rats on body fluid regulatory responses to plasma hyperosmolality and central angiotensin II (ANGII), and on the systemic and central ANGII-induced pressor responses. E2 replacement attenuated fluid intake and c-Fos expression of arginine vasopressin (AVP) neurons at the PVN and SON in response to both iv hypertonic saline infusion and icv ANGII injection. E2 replacemnet, however, did not influence the c-Fos expression at the osmosensitive regions, such as OVLT, SFO or MnPO during systemic osmotic challenge, suggesting that E2 does not modify osmoreceptor sensitivity. The c-Fos expression at the ANGII sensitive sites such as SFO and MnPO induced by central ANGII injection were attenuated by E2 replacement, indicating that the central sensitivity to ANGII is attenuated by E2 replacement. Thus, the attenuated osmoregulatory responses by E2 replacement might be due to the reduced central sensitivity to ANGII, because it has been reported that central ANGII is involved in the osmoregulatory thirst and AVP release. The pressor response to iv and icv ANGII was not affected by E2 replacement, while the decrease in heart rate induced by iv ANGII was attenuated by E2 replacement, suggesting that vasoconstrictor response and baroreflex response controlling heart rate are attenuated by E2 replacement. The central ANGII-induced pressor response was not affected by E2, suggesting that E2 differentially affect the central ANGII-induced body fluid and blood pressure regulations. [J Physiol Sci. 2007;57 Suppl:S53]

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