Poster Sessions

Poster Sessions イオンチャンネル、神経伝達物質

2P-01 IL-1β enhances H₂S-evoked Ca²⁺ signals via polysulfide production in RIN-14B cells Ken-ichi Otsuguro，Ayako Ujike，Tomoki Kuraishi，Soichiro Yamaguchi，Shigeo Ito Lab Pharmacol, Facult Vet Med, Hokkaido Univ Hydrogen sulfide (H₂S) is increasingly recognized as a gassotransmitter. Recently, it is proposed that the effects of H₂S are mediated via polysulfides, which are converted from H₂S by oxidation. We have previously reported that H₂S has excitatory and inhibitory effects on Ca²⁺ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K⁺ channels, respectively, suggesting that changes in the balance of inhibitory and excitatory effects of H₂S modulate H₂S signals. In this study, effects of interleukin-1β (IL-1β), a proinflammatory cytokine, on H₂S-induced Ca²⁺ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na₂S, a H₂S donor, evoked three types of Ca²⁺ signals, i.e., 1) the inhibition of spontaneous Ca²⁺ oscillation, 2) inhibition followed by increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and 3) [Ca²⁺]_i increase without inhibition. IL-1β treatment (3 ng/ml, 24 h) enhanced H₂S-evoked [Ca²⁺]i increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca²⁺ channel blockers. However, IL-1β did not affect [Ca²⁺]_i increases evoked by a TRPA1 agonist or high concentration of KCl. The expression level of TRPA1 mRNA was also unchanged by IL-1β. Na₂S increased intracellular polysulfide levels, which were enhanced by IL-1β. L-NMMA, a NOS inhibitor, suppressed polysulfide production and [Ca²⁺]i increase enhanced by IL-1β. These results indicate that IL-1β enhances the conversion of H₂S to polysulfides, resulting in [Ca²⁺]i increases. NO is suggested to contribute to polysulfide production from H₂S by oxidation. Under inflammatory conditions, polysulfides may play an important role in the effects of H₂S.		2P-02 Study on the function of MEGF10 in neurons Yu Fujita¹，Tomoji Maeda²，Koji Kamaishi¹，Rui Saito¹，Koyo Chiba¹，Xuefeng Shen¹，Kun Zou¹，Hiroto Komano¹ ¹Department of Neuroscience, School of Pharmacy, Iwate Medical University，²Department of Pharmacotherapy, Nihon Pharmaceutical University Multiple-EGF like domains 10 (MEGF10) is the mammalian homologue of Draper, a Drosophila phagocytosis receptor that plays an important role in synapse elimination and cell type-specific recognition. However, the expression and function of MEGF10 in the brain remain to be elucidated. Therefore, we aimed to clarify the regions and types of neurons that express MEGF10 in the brain, and to determine whether cells expressing MEGF10 possess phagocytic abilities. Our results indicated that MEGF10 is expressed in cholinergic and glutamatergic neurons of the cortex, hippocampus, and substantia nigra. Furthermore, the ratio of neurons expressing MEGF10 was higher in the cortex and hippocampus of hAPP transgenic mice than in those of wild-type mice. Phagocytic activity was also observed in neuronal cells expressing MEGF10. Thus, our results indicate that MEGF10 may be responsible for phagocytic activity targeted toward unwanted substances such as amyloid in cholinergic and glutamatergic neurons.

2P-03 Phosphorylation of serotonin 1A receptor (5HT1AR) by Cdk5 Miyuki Takahashi¹，Yuki Kobayashi²，Kanae Ando¹，Yumiko Saito²，Shin-ichi Hisanaga¹ ¹Dept. of Biol. Sci., Tokyo Met. Univ，²Grad Sch Integ Arts and Sci, Hiroshima Univ. Mental disorders including depression are one of urgent issues to be addressed. To prevent the onset and develop the treatment, it is important to understand a mechanism of the diseases at molecular level. Serotonin (5HT) is a neurotransmitter involved in releasing anxiety and depression. Serotonin 1A receptor (5HT1AR) is a subtype of 5-HT receptors expressed highly in central nervous system and is thought to be involved in psychiatric activity. 5HT1AR is a seven transmembrane G-protein-coupled receptor, which binds to Gi or Go of trimeric G proteins to inhibit adenylyl cyclase or open K+ channels in neurons. Dysfunction of the serotonin signal is considered as a cause of many mental diseases. It is not fully known, however, how expression or activity of 5HTlAR is regulated. We found three possible Cyclin-dependent kinase 5 (Cdk5)-phosphorylation sites in 5HT1AR. Cdk5 is a neuron-specific membrane-bound Ser/Thr kinase. We examined phosphorylation of 5HT1AR by Cdk5-p35. 5HT1AR was indeed phosphorylated by Cdk5-p35. We constructed non-phosphorylatable Ala mutants at T149A, S245A, or T314A, and examined their phosphorylation. Thr314 was identified as a phosphorylation site in 5HT1AR. Expression level of 5HT1AR was decreased by cotransfection with Cdk5-p35, but not with kinase negative Cdk5-p35, in COS-7 cells. We investigated the effect of decreasing 5HT1AR expression by Cdk5 activity on serotonin signaling by measuring cAMP. Co-expression of Cdk5-p35 suppressed 5HT1AR’s inhibitory action on cAMP synthesis. These results suggest that Cdk5-p35 modulates the serotonin signal through phosphorylation-dependent downregulation of 5HT1AR.		2P-04 Immunoanalysis of melanin-concentrating hormone receptor 1 located in neuronal primary cilia Tomoya Okada，Shogo Yamato，Daisuke Miki，Yuki Kobayashi，Yumiko Saito Grad Sch Integrated Arts Sci, Hiroshima Univ Non-motile primary cilia are sensory organelles that present in most vertebrate cell types. The importance of primary cilia is underscored by a group of disease, known as ciliopathies often presenting clinical manifestations such as obesity. Many neurons in the mammalian brain possess primary cilia that are enriched for certain G protein-coupled receptors (GPCRs), including melanin-concentrating hormone (MCH) receptor 1 (MCHR1). The MCH-MCHR1 system is known to mediate distinct aspects of energy balance and vital behavior. Recently, we reported that MCH causes an effective reduction in cilia length via ciliary MCHR1-expressing epithelial cells. Although short cilia have been observed in genetic obese mice, a possible correlation between MCHR1-positive neuronal cilia length and energy metabolism has not been characterized. To this end, first we established a novel protocol to detect MCHR1 colabeled with the neuronal ciliary marker (adenylyl cyclase 3) in frozen section of mice brain. Ciliary MCHR1 were located in the striatum, nucleus accumbens, hippocampus, cerebral cortex and hypothalamus. The features in each sub-region were not uniform, especially, the length of ciliary MCHR1 in striatum were significantly longer than that in the other regions. Next, brains were collected from mice subjected to a 48-h fast. Starved mice displayed shorter MCHR1-positive cilia than did normal diet-fed mice in their hippocampus. According to histogram analysis, fasted mice showed an increased frequency of shorter cilia and a decreased frequency of longer cilia. The present results provide the first evidence that MCHR1-positive cilia length in terminally differentiated neuron is actively reduced by metabolic alteration, and this appears to selectively occur in hippocampus neuron.

2P-05 Metabolic GPCR-mediated primary cilia shortening through dynamic cytoskeletal reorganization Sakura Tomoshige¹，Kousuke Hosoba²，Akie Hamamoto³，Shogo Yamato²，Tatsuo Miyamoto²，Yuki Kobayashi¹，Yumiko Saito¹ ¹Graduate School of Integrated Arts and Sciences, Hiroshima University，²Research Institute for Radiation Biology and Medicine, Hiroshima University，³Institute of Life Science, Kurume University Primary cilia are antenna-like sensory protrusion mediated sensory and neuroendocrine signaling. Defects in cilia formation and function are associated with a spectrum of human diseases including diabetes and obesity. The G-protein-coupled receptor (GPCR) melanin-concentrating hormone (MCH) receptor 1 (MCHR1), a key regulator of feeding, is selectively expressed in a subset of neuronal primary cilia in rodent brain. We have previously shown that the MCH-ciliary MCHR1 axis causes cilia shortening via the Gi/o-dependent Akt pathway. Many factors can participate in cilia length control. However, the mechanisms for how these molecules are coordinated to activate cilia shortening are poorly understood. In the current study utilizing our unique cell model, we investigated the role of cytoskeletal dynamics in regulating MCH-induced cilia shortening. Pharmacological and biochemical approaches showed that an increased level of soluble tubulin in the cell body was accompanied by MCHR1-mediated cilia shortening. We next observed an enhancement of F-actin fiber intensity in MCH-treated cell. Then, the actions of various pharmacological agents revealed that coordinated actin machinery, especially actin polymerization, was required for MCHR1-mediated cilia shortening. A recent report indicated the existence of actin-regulated mechanism for cilia shortening through GPCR agonist-dependent ectosome release. However, our live-cell imaging experiments showed that MCH progressively elicited cilia shortening without exclusion of fluorescence-positive material from the tip. Our findings may contribute to a better understanding of how the cytoskeleton is involved in the neuropeptide-triggered cilia shortening, which underlies clinical manifestations such as obesity.		2P-06 X-irradiation elicits decrease of drebrin accumulation within dendritic spines via activation of NMDA receptors Shuchuan Miao，Noriko Koganezawa，Kenji Hanamura，Tomoaki Shirao Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine Drebrin, an important synaptic protein, is localized in postsynaptic sites of excitatory synapses and it is known to decrease in early phase of cognitive impairments. We previously reported that drebrin intensity was transiently decreased in molecular layer of dentate gyrus (MLDG) of mice after X-irradiation in vivo. Intriguingly, this decrease was associated with fear memory impairment. We also found similar phenomenon using primary cultured neuron in vitro. In addition, our previous study shows that the activation of NMDA receptors induces the disappearance of drebrin from dendritic spines. To elucidate the molecular mechanism of the synaptic pathology, we tested whether NMDA receptors were involved in the decrease of drebrin after X-irradiation. For in vitro study, primary hippocampal cultured neurons were pretreated with APV (an NMDA receptor antagonist; 50 µM) 1 hour before X-irradiation. The cells were fixed 8 hours after irradiation. Then we quantitated the number of drebrin clusters. For in vivo study, whole brains of mice were irradiated with X-irradiation and fixed 8 hours after irradiation. Immunostaining intensity of drebrin and PSD-95 in MLDG was analyzed. We showed that number of drebrin clusters significantly decreased in X-irradiated group but this decrease was inhibited by the APV treatment. The intensity of drebrin in MLDG significantly decreased in irradiated mice while the intensity of PSD-95 was not changed. Interestingly, pretreatment with MK-801 (an NMDA receptor antagonist) could inhibit the decrease of drebrin intensity in vivo. Our results indicate that decrease in drebrin accumulation induced by X-irradiation is caused by activation of NMDA receptors, which may cause synaptic dysfunction and impair fear memory acutely after X-irradiation.

2P-07 H3 receptor antagonist ameliorated allodynia in chronic neuropathic pain model rat Takuro Matsuzawa¹，Takeo Yoshikawa¹，Tadaho Nakamura^1,2，Maria Mogilevskaya¹，Asuka Mogi¹，Kazuhiko Yanai¹ ¹Dept Pharmacol. Tohoku Univ Grad Sch Med，²Div of Pharmacol. Fac of Med. Tohoku Med and Pharm Univ Neuropathic pain is a chronic condition following nerve damage and degeneration induced by various pathologies such as diabetes, infection, autoimmune diseases and malignancies. Allodynia, a painful abnormal sensation caused by innocuous stimuli, is a major symptom of neuropathic pain, but currently there is no effective clinical treatments. Histamine functions as a neurotransmitter in the CNS. Previous studies indicated that elevation of histamine concentration in CNS alleviates a pain and a H3 antagonist attenuated allodynia in chronic neuropathic pain model rats. The site of action for H3 antagonist in CNS, however, is not totally understood. In this study, we used neuropathic pain model rat with L5 spinal code ligation to examine the central effects of H3 receptor antagonists, JNJ-101814572, (JNJ). First, we confirmed that H3 receptor was highly expressed in thalamus and spinal cord (SC) using quantitative RT-PCR. Next, we intraperitoneally injected JNJ to evaluate its effect on allodynia quantified by von Frey filament. JNJ injection decreased allodynia in a dose-dependent manner. JNJ increased histamine concentration in the ventral posterolateral nucleus of thalamus (VPL), periaqueductal grey matter (PAG), and locus ceruleus (LC). Finally, we directly injected JNJ to VPL, PAG, LC and SC to evaluate its effect on allodynia. Local injection of JNJ into these areas ameliorated allodynia, indicating that JNJ activates the descending inhibitory pathways. In conclusions, these data suggested that JNJ ameliorated allodynia in chronic neuropathic pain model rat via the descending inhibitory pathways.		2P-08 The involvement of histamine receptors with loss of conscious induced by inhaled anesthetics Toru Tamii^1,2，Tadaho Nakamura^1,3，Takeo Yoshikawa¹，Tomomitsu Iida¹，Aniko Karpati¹，Takuro Matsuzawa¹，Haruno Kitano¹，Fumito Naganuma³，Nobuyuki Okamura³，Masanori Yamauchi²，Kazuhiko Yanai¹ ¹Dept Pharmacol. Grad Sch Med, Tohoku Univ，²Dept Anesthesiol. Grad Sch Med, Tohoku Univ，³Div Pharmacol. Facul Med, Tohoku Med Pharm Univ Background: The mechanism of loss of conscious (LOC) induced by inhaled anesthetics is little understood. Recent studies suggested that inhaled anesthetics inhibit arousal pathway in the central nervous system to induce LOC. Histaminergic nervous system is originated from posterior hypothalamus and modulates evoking and maintaining arousal. However, the relation between histamine receptors in brain and inhaled anesthetics is unclear. The aim of this study is to examine the involvement of histamine receptors, H₁, H₂ and H₃ with LOC induced by desflurane and sevoflurane, which are inhaled anesthetics mainly used in clinical practice.Methods: We studied the LOC effect of desflurane and sevoflurane by behavioral experiments and electroencephalography (EEG) using wild type (WT), H₁ knockout (H₁KO), H₂KO and H₃KO male mice. We measured loss of righting reflex (LORR), immobilization time and LORR recovery time as substitutions for LOC in mice. We recorded EEG under general anesthesia and performed power spectrum analysis.Results: EC₅₀ for LORR of desflurane and sevoflurane were lower than WT (desflurane: WT 8.94% vs H₁KO 6.74% vs H₂KO 7.54% vs H₃KO 9.24%, sevoflurane: 2.81% vs 2.01% vs 2.53% vs 2.58%). H₁KO and H₂KO had shorter immobilization time than WT (p<0.0001). H₁KO had lower EEG power than WT in induction phase. These results indicated that H₁ and H₂ receptors might have antagonistic properties to LOC induced by general anesthetics. There were no significant differences of LORR recovery time between WT and each KO mice. H₁KO and H₂KO had equivalent power of EEG compared to WT in recovery phase. Conclusions: H₁KO and H₂KO had shorter time of induction LOC induced by inhaled anesthetics. However, H₁KO and H₂KO did not have significant difference in recovery from LOC.

2P-09 Alteration of GABA release in developing cerebellar cortex of VPA-administrated autistic model rat Kana Miyamoto¹，Tetsuri Mikami²，Yukiko Fueta³，Susumu Ueno³，Yuko Sekino⁴，Yasunari Kanda⁵，Naohiro Hozumi⁶，Sachiko Yoshida² ¹Dept Environ Life Sci, Toyohashi Univ of Tech, Toyohashi, Japan，²Dept Environ Life Sci, Toyohashi Univ of Tech, Toyohashi, Japan，³Univ Occupational Environmental Hlth, Kitakyushu, Japan，⁴University of Tokyo，⁵Natl Inst Health Sci, Tokyo, Japan，⁶Dept Electrical-Electronic-Information-Engineering Toyohashi Univ of Tech, Toyohashi, Japan Background: Neurotransmitters and neuronal releasing molecules are not only the regulator of neuronal function but also the indicator of neuronal conditions. γ-amino butyric acid (GABA) plays important roles in cerebellar differentiations and functions. Sodium valproate (VPA), the antiepileptic drug by promoting the release of GABA, increases the risk of neurodevelopmental disorder such as autism due to the intervention to the transition from excitatory GABA function to inhibitory one. In this report, we investigated the alteration of GABA transition in VPA- or other neurotoxic chemicals-administrated rat pups.Methods: Released neurotransmitter was detected using an oxidoreductive enzyme-linked photo assay system. GABase and glutamate dehydrogenase were available to detect GABA and glutamate, respectively. Enzymes oxidize substrates, and generate NAD(P)H stoichiometrically. Using this system, both spontaneous and AMPA-stimulated GABA release in the developing cerebellar slices were observed. Additionally, spontaneous GABA release was detected using the spectrophotometer.VPA, Tributyltin (TBT), or Chlorpyrifos (CPF) was once administrated to embryonic day (E) 16 p.o. with 600mg/kg, 20 mg/kg or 10 mg/kg of mother weight, respectively. The cerebellum in pups was observed from postnatal day (P) 5 to P14. Result: Spontaneous GABA release in the normal developing cerebellar cortex was transiently decreased and recovered after birth. On the other hand, Spontaneous GABA release in VPA treated cerebellum kept higher than control one. Additionally, AMPA-stimulated GABA release pattern was different between VPA-administrated pups and control.Conclusions: Spatio - temporal GABA release would reflect developing progress and neuronal network activities.		2P-10 Alteration of neurotransmitter release in cerebellar cortex of Valproate-administrated Autistic model rat Tetsuri Mikami¹，Yukiko Fueta²，Susumu Ueno²，Yuko Sekino³，Yasunari Kanda⁴，Naohiro Hozumi⁵，Sachiko Yoshida¹ ¹Dept Environ Life Sci, Toyohashi Univ of Tech, Toyohashi, Japan，²Univ Occupational Environmental Hlth, Kitakyushu, Japan，³University of Tokyo，⁴Natl Inst Health Sci, Tokyo, Japan，⁵Dept Electrical Electronic Information Engineering, Toyohashi Univ of Tech, Toyohashi, Japan Background: Valproate (VPA) is one of the candidates of inducer of autism. In VPA-administrated rat, we have observed facilitation of Purkinje cell development and inhibited granule cell migration in young animals. Neurotransmitter and neuronal releasing molecules are not only the regulator of neural cell proliferation and differentiation, but also the indicator of neuronal conditions. Observation the spatio - temporal distribution of neurotransmitter releasing is important to know that neural functions. Glutamate and γ-amino butyric acid (GABA) play important roles in cerebellar differentiations and functions. Methods: Released neurotransmitter was detected using an oxidoreductive enzyme-linked photo assay system. GABase and glutamate dehydrogenase were available to detect GABA and glutamate, respectively. Enzymes oxidize substrates, and generate NAD(P)H stoichiometrically. Using this system, both spontaneous and AMPA-stimulated GABA release in the developing cerebellar slices were observed.Embryonic day 16 rat pups were administrated 600mg/kg VPA maternally. Postnatal day 19 (P19) to P24, VPA-administrated or vehicle rat cerebellum were sliced sagittally at 400 µm thick and mounted on the enzyme-linked glass device for observation of fluorescence with 20 msec time resolution.Result: 0.25 µM AMPA-stimulated Glutamate or GABA release in the cerebellar slices were observed. In the vehicle rat cerebellum, Glutamate or GABA release were translated for about 60 msec, while VPA-administrated cerebellar slices showed shorter duration time of neurotransmitter release.Conclusions: We suggest that the difference of neural activities between VPA-administrated and vehicle cerebellum would be due to the development of inhibitory GABA response.

2P-11 Serotonin induced synchronization to both respiratory rhythm and body movement Ooka Hirotaka，Chiaki Uchida，Akiko Arata Dept. of Physiol. Hyogo Coll. of Med. Respiratory rhythm is belonging to the medullary rhythm generator and fetal movement is belonging to the spinal rhythm generator. Our previous result showed postnatal spinal activity induced by strychnine was thought to be same property as a fetal movement. Parabrachial Nucleus (PB) in the dorsolateral pons conjunctive system of medulla respiratory activity and spinal motor activity. It is known that serotonin strongly modulates the rhythm generator like respiratory or body movement. However, the relationship between respiratory rhythm and body movement at the PB modulated by serotonin has not been well investigated. In this study, we analyzed the relationship between respiratory rhythm and body movement using neonatal rat medulla-spinal cord preparation with/without pons; and we also examined the effect of serotonin on the relationship between respiratory rhythm and fetal movement in PB. In the case of the pons-medulla-spinal cord preparation, the respiratory rhythm was synchronized to body movement when serotonin was applied. On the other hand, even if serotonin was applied, the relationship between respiratory rhythm and body movement was not synchronized without pons. In addition, we examined the distribution of optical signals in the pons triggered by body movement. We found the optical signals which was induced by body movement in the dorsal pons. These results suggested that serotonin activated the synchronization between respiratory rhythm and body movement through the pontine structure.