ポスター発表 (Poster Sessions)

グリア、グリア-ニューロン相互作用 Glia and Glia-Neuron Interaction
P2-2-43 自閉症スペクトラム障害の神経病態に伴うミクログリアの関連性 Association of microglia with neuropathology of autism spectrum disorder ○古田島浩子¹, 中村泰子¹, 土屋明子¹, 鈴木恵里¹, 内野茂夫¹, 高坂新一¹ ○Hiroko Kotajima¹, Yasuko Nakamura¹, Akiko Tsuchiya¹, Eri Suzuki¹, Shigeo Uchino¹, Shinichi Kohsaka¹ 国立精神・神経医療研究センター神経研究所　代謝研究部¹ Department of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan¹ Autism spectrum disorder (ASD) is a developmental disorder characterized by deficits in social interaction and restricted and stereotyped patterns of interest and behaviors. Although ASD affects about 1-2% of children, the neuropathology and etiology of ASD remain unknown. Since recent neuroimaging studies have demonstrated abnormal microglia in the brains of ASD patients, the purpose of our study is to clarify the association between microglial function and neuropathology of ASD. Valproic acid (VPA) is used an anticonvulsant drug for the treatment of epilepsy. However, children exposed in utero to VPA demonstrate behavioral and neuroanatomical abnormalities similar to those seen in developmental disorders including ASD. In this study, we subcutaneously injected the pregnant mice on gestational day 13.5 with a dose of 800 mg/kg of VPA, and validated early life behavioral assessments as ASD model mice. The VPA-exposed pups less than one month after birth showed delayed physical development, poor motor performance and abnormal response to thermal stimulation. Furthermore, VPA-exposed pups at 10 weeks after birth showed the depression of sociability assayed by using three-chambered box social interaction test, while no differences were found in sociability between VPA-exposed pups and control pups at 4 weeks after birth. These results support that VPA-exposed pups are good model of ASD. To reveal the microglial function associated with the neuropathology of ASD using VPA-exposed mice, we examined the gene expression of several types of inflammation-related cytokines in the developing brain by quantitative RT-PCR using primer array, and found that the expression profiles of some chemokines, including CXCR4 and CCL5 (rantes), were different in between VPA-exposed pups and control pups. To clarify the role of microglia in the developing brain and the association of abnormal microglia with neuropathology of ASD, further study is now on going.	P2-2-44 Tenascin R陽性アストロサイトにおけるグルタミン酸取り込み能の調節 TenascinR modulates glutamate uptake activity by regulating astrocytic GLAST expression ○奥田洋明¹, 辰巳晃子¹, 森田晶子¹, 渋川幸直², 是金宏昭³, 堀井謹子⁴, 和田芳直², 谷口直之³, 和中明生¹ ○Hiroaki Okuda¹, Kouko Tatsumi¹, Shoko Morita¹, Yukinao Shibukawa², Hiroaki Korekane³, Noriko Horii-Hayashi⁴, Yoshinao Wada², Naoyuki Taniguchi³, Akio Wanaka¹ 奈良医大・第二解剖¹, 大阪府立母子保健総合医療センター研究所・代謝部門², 理研・基幹研究所・ケミカルバイオロジー研究領域・システム糖鎖生物学研究グループ³, 奈良医大・第一解剖⁴ Dept Anatomy & Neuroscience, Nara Medical Univ, Nara¹, Dept Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health², Systems Glycobiology Research Group, Chemical Biology Dept, Advanced Science Institute, RIKEN³, Dept Anatomy and Cell Biology, Nara Medical Univ, Nara⁴ TenascinR (TNR) is an extracellular matrix protein expressed primarily in the central nervous system, and is a multifunctional molecule with multiple domains that render various effects on cell functions, such as neuronal cell adhesion, neurite outgrowth, modulation of sodium channels. Our previous in situ hybridization analysis showed that TNR mRNA was selectively localized at a subpopulation of cortical astrocytes, but not at the WFA-positive GABAergic interneurons. Chondroitin sulfate chains of TNR, which were recognized by the CS56 antibody, constituted a patchy extracellular matrix in the adult mouse cortex. The TNR-immunoreactive structures uncover a subpopulation of cortical astrocytes and delineate astrocytic territories. In this study, we examined the molecular functions of TNR in cultured astrocytes. Astrocytes can modulate synaptic activity by releasing gliotransmitters and uptaking glutamate. We first examined whether gliotransmitters secretion was influenced by siRNA-mediated knock-down of TNR gene expression, however, knock-down of TNR expression did not influence gliotransmitters secretion. Next, we examined whether glutamate uptake and expression of glutamate transporters were influenced by TNR-knocked-down. Glutamate uptake activity was decreased and the expression level of GLAST but not GLT-1 was significantly lower in the TNR-knocked-down astrocytes than in the control astrocytes. TNR-knocked-down astrocytes further showed a decrease in Src activation, reduced phosphorylation on a stimulatory site (Tyr-416) and increased phosphorylation on an inhibitory site (Tyr-546). These results suggest that TNR regulates astrocytic GLAST expression possibly through Src signal pathways and thereby modulates extracellular glutamate concentration.
P2-2-45 アストロサイトへの変異SOD1遺伝子導入はノルアドレナリンによるアストロサイトを介した神経保護作用を阻害する The expression of mutant SOD1 in astrocytes abolishes the noradrenaline-induced astrocyte-mediated neuroprotection ○吉岡靖啓¹, 阿久根真人¹, 吉田貴秀¹, 山室晶子¹, 石丸侑希¹, 前田定秋¹ ○Yasuhiro Yoshioka¹, Masato Akune¹, Takahide Yoshida¹, Akiko Yamamuro¹, Yuki Ishimaru¹, Sadaaki Maeda¹ 摂南大学　薬学部　薬物治療学研究室¹ Dept. Pharmacotherap., Faculty Pharmaceut. Sci., Setsunan Univ., Osaka 573-0101, Japan¹ It has been reported that dysfunction of astrocyte plays an important role in motor neuronal death in the transgenic SOD1 (G93A) mouse model of amyotrophic lateral sclerosis. We have previously demonstrated that noradrenaline (NA) protects neurons from H₂O₂-induced death by increasing the supply of glutathione (GSH) from astrocytes by using the co-culture system of human neuroblastoma SH-SY5Y cells and human astrocytoma U-251 MG cells. In this study, we investigated the effect of mutant SOD1 (G93A) expression in U-251 MG cells on the neuroprotective effect of NA by using the co-culture system. We established U-251 MG cells that constitutively expressed wild-type or mutant (G93A) human SOD1. In co-culture, we used SH-SY5Y cells that constitutively express green fluorescence protein (GFP), and the cell viability was determined based on the morphology of GFP-positive cells under a fluorescence microscope. To investigate the intracellular GSH level in SH-SY5Y cells in co-culture, the cells were stained with reduced GSH-reactive probe monochlorobimane, and were analyzed by Cellomics ArrayScan. NA (10 μM) increased intracellular GSH levels in U-251 MG cells expressing wild-type SOD1, but not in the cells expressing mutant SOD1. In co-culture of SH-SY5Y cells and U-251 MG cells expressing wild-type SOD1, NA (10 μM) increased intracellular GSH levels of SH-SY5Y cells and protected the cells from H₂O₂-induced death. On the other hand, in co-culture using U-251 MG cells expressing mutant SOD1, NA (10 μM) neither affected intracellular GSH levels of SH-SY5Y cells nor protected the cells from H₂O₂-induced death. These results suggest that mutant SOD1 negated NA-induced astrocyte-mediated neuroprotection by inhibiting the increase of GSH in astrocytes.	P2-2-46 高細胞外カリウム濃度下でのアストロサイトによるカリウム輸送と細胞膨張の機序 Mechanisms of astrocytic K⁺ clearance and swelling under high extracellular K⁺ concentration ○村上慎吾¹, 倉智嘉久¹ ○Shingo Murakami¹, Yoshihisa Kurachi¹ 大阪大学大学院医学系研究科薬理学講座（分子・細胞薬理学）¹ Dept of Pharmacol, Graduate School of Medicine, Osaka Univ, Osaka¹ In response to elevation of extracellular [K⁺], astrocytic K⁺ clearance contributes to maintenance of a proper environment for neural activity. K⁺ clearance in astrocytes takes two forms: K⁺ uptake and K⁺ spatial buffering. On the other side high extracellular [K⁺] induces swelling of astrocyte, which leads to edema and cell death. Despite importance of the K⁺ clearance and swelling, their mechanisms are not fully understood. Here we report simulation analysis on mechanisms of astrocytic K⁺ clearance and swelling. An astrocyte model was constructed by incorporating into a compartment model elements that are related to astrocytic K⁺ clearance and swelling, such as intra/extracellular ion concentrations of Na⁺, K⁺ and Cl^-, cell volume, and models of NaKATPase, Na⁺-K⁺-Cl^- cotransporter (NKCC), K⁺- Cl^- cotransporters (KCC), inwardly rectifying K⁺ channel (KIR) channel, passive Cl^- channel, and Aquaporin (AQP) channels. Simulated response to high extracellular [K⁺] revealed significant contributions of NKCC and NaKATPase to increases of intracellular [K⁺] and [Cl^-], and swelling. Moreover, we show that KIR channel localized at synaptic cleft absorbs excessive K⁺ by depolarizing equivalent potential for K⁺ (E_K) above membrane potential, while K⁺ release through KIR channel localized at perivascular is enhanced by hyperpolarizing E_K and depolarizing membrane potential. Analysis of simulated drug effect under high extracellular [K⁺] shows that a drug may have distinct effects on K⁺ absorb, release and swelling, separately. Most notably, swelling of astrocyte under high extracellular [K⁺] can be suppressed by blockade of NKCC and AQP channels, although K⁺ clearance capability was slightly lowered. Thus, we identify here distinct roles of ion channels and transporters in astrocytic K⁺ clearance and swelling.
P2-2-47 神経細胞の生存に対するHPC-1/シンタキシン1Aとシンタキシン1Bの異なる役割 HPC-1/syntaxin1A and syntaxin1B have distinct roles in neuronal survival ○小藤剛史¹, 藤原智徳², 真田ますみ², 三嶋竜弥², 赤川公朗² ○Takefumi Kofuji¹, Tomonori Fujiwara², Masumi Sanada², Tatsuya Mishima², Kimio Akagawa² 杏林大学医学部共同研究施設RI部門¹, 杏林大学医学部細胞生理学教室² Radioisotope lab., Kyorin Univ. Sch. Med., Tokyo¹, Dept. Cell Physiol., Kyorin Univ. Sch. Med., Tokyo² In neurons, two types of syntaxin1 isoforms, HPC-1/syntaxin1A (STX1A) and syntaxin1B (STX1B), both of which are transcribed from distinct genes and thought to have similar functions in synaptic vesicle exocytosis, are predominantly expressed. Previously, we generated the gene knockout mice (KO) for STX1A or STX1B. Though STX1A-KO normally developed (Fujiwara et al, J Neurosci, 2006), STX1B-KO died within 2 weeks after birth. Structural abnormality was observed in CNS of STX1B-KO mice unlike STX1A-KO mice, suggesting that STX1A and STX1B might have distinct roles in CNS development. In this study, we examined the dissociated culture neurons from neonatal STX1A-KO or STX1B-KO. On poly-L-lysine-coated coverglass (PLL), STX1A-KO neurons normally survived compared with WT neurons, but STX1B-KO neurons died rapidly after 7-8 days in vitro (DIV). Interestingly, on WT or STX1A-KO glial feeder layers, STX1B-KO neurons survived as in WT neurons, however, STX1B-KO glial cells were less effective. Furthermore, conditioned medium from WT and STX1A-KO glial cells had similar survival effect, but not that from STX1B-KO. Since neurotrophic factors and some growth factors (NTs) from neurons and glial cells support neuronal survival, we examined effect of NTs on STX1B-KO neurons. We found that BDNF, one of NTs, supported neuronal survival of STX1B-KO neurons, but not NGF, bFGF nor insulin. Furthermore, co-culture of STX1B-KO neurons with WT neurons was less effective. We further generated the mutant mice lacking both STX1A and STX1B (DKO). DKO was embryonic lethal, and DKO neurons died within 7DIV on PLL. DKO neuronal survival was not supported by WT glial feeder layers, BDNF nor bFGF. These data suggest that STX1A and STX1B might have distinct roles in neuronal survival which might be supported by glia function lacking in STX1B-KO. Implication of these different roles in STX1A and STX1B will be discussed.	P2-2-48 ジクロフェナックナトリウムがサイトカイン存在下でのミクログリア貪食能に及ぼす影響－インフルエンザ感染に伴う脳症悪化のメカニズムの解明－ Diclofenac enhances proinflammatory cytokine-induced phagocytosis of cultured microglia via nitric oxide production ○青山峰芳¹, 垣田博樹^1,2, 長屋嘉顕^1,2, 鈴木美恵子^1,2, 浅井清文¹ ○Mineyoshi Aoyama¹, Hiroki Kakita^1,2, Yoshiaki Nagaya^1,2, Mieko Suzuki^1,2, Kiyofumi Asai¹ 名古屋市立大学大学院医学研究科　分子神経生物学分野¹, 名古屋市立大学大学院医学研究科　新生児・小児医学分野² Department of Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya¹, Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya² Influenza-associated encephalopathy (IAE) is a central nervous system complication with a high mortality rate, which is increased significantly by the non-steroidal anti-inflammatory drug diclofenac sodium (DCF). In the present study, we investigated the effects of DCF on brain immune cells (i.e. microglia) stimulated with three proinflammatory cytokines, namely interleukin-1β, tumor necrosis factor-α, and interferon-γ. Similar to previous findings in astrocytes, all three cytokines induced the expression of inducible NO synthase (iNOS), as well as NO production, in microglia. The addition of DCF to the culture system augmented iNOS expression and NO production. Immunocytochemical analysis and the phagocytosis assay revealed that cytokine treatment induced morphological changes to and phagocytosis by the microglia. The addition of DCF to the culture system enhanced microglial activation, as well as the phagocytic activity of cytokine-stimulated microglia. Inhibitors of nuclear factor (NF)-κB inhibited iNOS gene expression in cytokine-stimulated microglia with or without DCF, suggesting that the NF-κB pathway is one of the main signaling pathways involved. The iNOS inhibitor N-monomethyl-L-arginine (L-NMMA) reduced both cytokine-induced phagocytosis and phagocytosis induced by the combination of cytokines plus DCF. Furthermore, the NO donor sodium nitroprusside induced phagocytosis, indicating that NO production is a key regulator of microglial phagocytosis. In conclusion, DCF acts synergistically with proinflammatory cytokines to increase the production of NO in microglia, leading to phagocytic activity of the activated microglia. These findings, together with previous observations regarding astrocytes, may explain the significant increase in mortality of IAE patients treated with DCF.
P2-2-49 シスタミンによるトランスグルタミナーゼ阻害はミクログリアにおけるLPS誘導性のエンドサイトーシスを抑制する Blockade of transglutaminase activity by cystamine suppressed lipopolysaccharide induced endocytosis in microglia ○河辺憲司¹, 高野桂¹, 森山光章¹, 中村洋一¹ ○Kenji Kawabe¹, Katsura Takano¹, Mitsuaki Moriyama¹, Yoichi Nakamura¹ 大阪府立大学　生命環境科学部　獣医学科　統合生理学教室¹ Lab. Integrative Physiology, Vet. Sci., Osaka Pref. Univ.¹ Engulfment of neurons by activated microglia may play roles in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). Transglutaminase 2 (TG2) is a cross-linking enzyme, which is activated in AD, PD and Huntington's diseases. Coagulation factor XIII-A (FXIII-A), another TG, is reported to be expressed mainly in microglia in AD patient's postmortem brain. In monocyte/macrophage, TG2 and FXIII-A are reported to involve in phagocytosis of apoptotic cells; however, in microglia, it has not been investigated whether these TGs are associated with the mechanism of phagocytosis. We previously demonstrated that the expression of TG2 mRNA in mouse microglial cell line BV-2 was increased by lipopolysaccharide (LPS)-stimulation, and contrarily that the expression of FXIII-A mRNA was markedly decreased. In the present study, we examined the effects of cystamine, an inhibitor of TG activity, on phagocytosis and pinocytosis of BV-2 cells that are stimulated with LPS. As objects of phagocytosis of dead-cells, we treated in advance neuroblastoma cell line SH-SY5Y with 200 μM H2O2 for 24 h and stained with propidium iodide. When BV-2 cells were pre-stimulated with LPS (300 ng/mL) for 24 h, the phagocytosis of dead-cells for 30 min was significantly increased, and the pinocytosis of fluorescent-labeled 1 μm-microbeads for 1 h was also increased. When the cells were stimulated with LPS in the presence of 1 mM cystamine, the LPS-induced phagocytosis of dead-cells and pinocytosis were significantly suppressed. In addition, LPS increased TG2 protein expression, and it was inhibited by cystamine. On the other hand, cystamine had no effect on the LPS-induced down-regulation of FXIII-A mRNA. These results suggest that the increased expression of TG2 in activated microglia might be involved in the mechanism of their endocytosis that assumes important roles in various neurodegenerative diseases.	P2-2-50 間歇的な周期活動をするアストロサイト Intermittent oscillations in spontaneously active astrocytes ○宇治田早紀子¹, 松木則夫¹, 池谷裕二¹ ○Sakiko Ujita¹, Norio Matsuki¹, Yuji Ikegaya¹ 東京大院・薬・薬品作用学¹ Lab Chem Pharmacol, Grad Sch Pharm Sci, Univ Tokyo¹ Astrocytes exhibit various patterns of intracellular calcium elevations, possibly involved in gliotransmission. Many researches focus on this calcium activity from functional or mechanistic points of view. However reports on the precise activity patterns of calcium activities at single-cell level are limited and the temporal activity patterns of individual cells are not characterized at a long-time scale. Here, we utilized a large-scale calcium imaging technique to simultaneously visualize and analyze the activity patterns of hundreds of astrocytes in CA1 of acute hippocampal slices prepared from juvenile mice. We found that among the observed astrocytes, there are cells that show unique oscillation patterns, which we named "intermittent oscillations". Intermittently oscillating cells contributed 24.9 ± 17.1%, ranging from 4.4% to 50.6%, of all 1559 cells recorded from 10 slices. These cells repeatedly showed clustered oscillation events at an interval of 14.3 ± 11.0 min (mean ± SD), with only few sporadic activities between the events. Neither the frequency of the events nor the number of intermittently oscillating cells was affected by tetrodotoxin, but was dependent on intracellular calcium store. The population dynamics of IO fluctuated gradually but randomly over the recording period without any extracellular stimulation, and no intercellular propagation or synchronization was observed, indicating the single-cell property of this phenomenon. Observation of intracellular propagation of IO revealed a complex interaction of processes and the soma; the oscillation pattern consisted of propagation both to and from the soma, and the involvement of processes were variable between each cycles in one oscillatory event. This study suggests the existence of various activity states of astrocytes which fluctuates dynamically over time, and the possibility that the physiological function or sensitivity of individual astrocytes may not be homogeneous at all times.
P2-2-51 OPCの増殖を促すヘスペリジン/ナリルチン（チンピの有効成分）の分子機構にはプレニル化が関与する Hesperidin and Narirutin enhance the proliferation of OPCs through prenylation-mediated mechanism ○清和千佳¹, 山本雅浩², 植木俊之², 阿相皓晃¹ ○Chika Seiwa¹, Masahiro Yamamoto², Toshiyuki Ueki², Hiroaki Asou¹ 慶應大・医・漢方医学¹, （株）ツムラ　ツムラ研² Center for Kampo Medicine, Keio Univ School of Medicine, Tokyo¹, Tsumura Res Labs, Tsumura&CO., Ami² Proliferation of oligodendrocyte precursor cells (OPCs) is important for initial myelination as well as for remyelination in demyelinating diseases. Previously, we demonstrated that hesperidin (Hes), narirutin (Nari) (which are the active constitutions of Chinpi) play a role in proliferation and differentiation of OPCs recruited to areas of age-induced demyelination. Hesperidin and narirutin are citrus flavonoids that are known as have a wide variety of pharmacological activities. Recently, many prenylated flavonoids have been identified as active components in medical plant, however, the mechanisms responsible for their actions have not yet been elucidated. To emphasize the importance of the prenylation of Hes/Nari and its potential effect on proliferation, firstly we have performed BrdU-incorporation assay using OPC cultures. The cells were treated with BrdU for 24 hr or 48 hr and the proliferating cells were detected by immunostaining using a mouse monoclonal antibody. Evident difference was shown in the degree of BrdU incorporation with or without both farnesyltransferase inhibitor (FTI-277), and/or geranylgeranyltransferase inhibitor (GGTI-298). The efficacy of the Hes/Nari on the proliferation of OPCs was only inhibited treatment with FTI-277. These results suggest that prenylation of flavonone (Hes/Nari) is a pivotal step to process biological activities and may have therapeutic and physically potent effects on various neurological disorders in which demyelination plays a crucial role such as multiple sclerosis (MS).	P2-2-52 アストロサイトのギャップジャンクションによるネットワークは虚血ダメージを軽減する Astrocytic gap junctional networks reduce ischemic damages in vitro ○篠塚崇徳¹, 塗谷睦生¹, 安井正人¹ ○Takanori Shinotsuka¹, Mutsuo Nuriya¹, Masato Yasui¹ 慶大・医・薬理¹ Dept. Pharmacol., Keio Univ. Sch. Med. Tokyo¹ Astrocytes play pivotal roles in both physiology and pathophysiology of the brain. They communicate with each other via extracellular messengers as well as gap junctions, by which they may exacerbate or protect against the neuronal damage. However, their roles in the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged intracellular calcium concentration ([Ca2+]i) changes in astrocytes in mice cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy, combined with a whole cell patch clamp recordings in neurons. Under OGD condition, astrocytes showed [Ca2+]i oscillations followed by higher and sustained [Ca2+]i increases, which was synchronized with a neuronal anoxic depolarization. While pharmacological manipulations of extracellular transmissions by glutamate or ATP had no effect, blockade of gap junctional intercellular coupling between astrocytes significantly advanced the onset of sustained [Ca2+]i increase after OGD exposure. Furthermore, this selective blockade of astrocytic gap junctional coupling simultaneously affected the timing of a neuronal anoxic depolarization in the same manner, causing faster depolarization that was synchronized with the sustained [Ca2+]i increase in astrocytes. Finally, the effect of pharmacological inhibition of gap junctional coupling was abolished by a reduction of extracellular potassium concentration in the bath. These results indicate that astrocytes delay the appearance of pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network, which effectively handles local potassium fluctuations under ischemic conditions. Thus, astrocytic gap junctional networks work as a protection against tissue damage under pathological conditions.
P2-2-53 アストロサイトのP2Y₁受容体を介したCa²⁺シグナルとその生理的役割 Properties and its physiological role of P2Y₁-mediated calcium signals in astrocytes ○繁冨英治¹, 小松龍平¹, 小泉修一¹ ○Eiji Shigetomi¹, Ryohei Komatsu¹, Schuichi Koizumi¹ 山梨大学大学院医学工学総合研究部　薬理学¹ Dept Neuropharmacol, Univ Yamanashi, Yamanashi¹ Astrocytes are not merely supportive but regulate synaptic transmission actively by releasing gliotransmitters such as ATP, glutamate and D-serine via elevations of intracellular Ca²⁺. Ca²⁺ elevations occur spontaneously and/or via activation of G-protein coupled receptors (GPCR). Among various GPCRs known to express in astrocytes, P2Y₁ receptor play a central role in Ca²⁺ signals in astrocytes in vitro, especially in culture systems. We started to ask whether P2Y₁ receptor is also crucial for Ca²⁺ signals in astrocytes in situ. We imaged Ca²⁺ in astrocytes from acute brain slices using Fluo-4 and found that spontaneous Ca²⁺ signals were not reduced by pharmacological blockade and were not different from those in conventional P2Y₁ receptor knockout mice compared with the wild type mice. What makes the difference in Ca²⁺ signals between in vitro and in situ? If P2Y₁ receptor-mediated Ca²⁺ signals in astrocytes are relevant to synaptic transmission as past studies suggest, how and what type of Ca²⁺ signals are mediated by P2Y₁ receptor? We think that the past studies have not addressed two major issues. First, P2Y₁ receptors expression in other types of cells was not considered. Second, Ca²⁺ imaging methods used report signals from soma but not peri-synapse astrocytes (PSA) which are probably most interesting parts in astrocytes in term of the interaction with neurons. To resolve these issues, we developed two novel genetic approaches which allow to selectively 1) observe Ca²⁺ in PSA using membrane-targeted genetically encoded Ca²⁺ indicator , Lck-GCaMP3, and 2) manipulate gene expression of P2Y₁ receptor in genetic specified cell population using doxycycline controlled Tet systems. The combination of those two novel techniques will allow unique opportunities to investigate the role(s) of P2Y₁ receptor-mediated Ca²⁺ signals of PSA and its interaction with synapses.	P2-2-54 顔面神経傷害におけるアストロサイトの糖代謝関与 Involvement of astrocytes in energy metabolism in axotomized rat facial nucleus ○竹澤洋亮¹, 高坂新一², 中嶋一行^1,2 ○Yousuke Takezawa¹, Shinichi Kohsaka², Kazuyuki Nakajima^1,2 創価大学工学部生命情報工学科¹, 国立精神・神経医療研究センター神経研究所² Department of Bioinformatics, Faculty of Engineering, Soka University, Tokyo¹, Department of Neurochemistry, National Institute of Neuroscience, Tokyo² Glucose is used as a major energy source in the nervous system. It has been generally accepted that the glucose is taken into parenchyma from blood, and consumed directly or stored as glycogen in an emergency. Although glial cells are regarded to function in the regulation of energy metabolism in the normal nervous system, little is known about the role of glial cells in injured nervous system. Thus, in this study we investigated the process of glucose uptake and glycogen degradation/synthesis in axotomized rat facial nucleus from standpoint of glial function. We initially analyzed the glucose transporters in injured facial nucleus. There was no significant change in the levels of glucose transporters between contralateral and ipsilateral nucleus during 1-14 days after insult. Similarly, the levels of glycogen phosphorylase in the lesioned nucleus were found to be unchanged. These results suggested that the processes of glucose uptake and glycogen degradation are essentially unaffected in injured nucleus. However, Western blotting revealed that the levels of glycogen synthase in injured site decrease to below 75% during 5-14 days after injury. Immunohistochemical study indicated that astrocytes reduce the levels of glycogen synthase in injured nucleus. That astrocytes express glycogen synthase protein and contain glycogen in the cells was confirmed in in vitro system. Collectively, these results suggest that astrocytes in injured/regenerating facial nucleus function in energy supply by quitting glycogen synthesis.

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