ポスター発表 (Poster Sessions)

突起伸展、回路形成 Axonal/Dendritic Growth and Circuit Formation
P3-1-71 神経成長円錐におけるカルシウム、cAMPおよびcGMPのクロストーク Crosstalk among calcium, cAMP, and cGMP in nerve growth cone ○小林孝彦¹, 永瀬史章¹, 堀田耕司¹, 岡浩太郎¹ ○Takahiko Kobayashi¹, Fumiaki Nagase¹, Kohji Hotta¹, Kotaro Oka¹ 慶應義塾大学大学院理工学研究科基礎理工学専攻生命システム情報専修生物物理・神経情報学研究室¹ Center for Biosciences and Informatics, School of Fundamental Science and Technology, Graduate School of Science and Technology, Keio University, Kohoku-ku, Yokohama, Kanagawa, Japan¹ During formation of specific neural connections, growth cones navigate axons toward their targets via physical and chemical interactions with environmental cues. Chemotactic substances, mechanical barriers, substratum adhesive substances are vital keys for neural circuit formation. Activation of receptors at axonal tips by these cues may generate signals that are transmitted back to the growth cone where they are transduced into changes in its motility. Calcium signal plays a crucial role in axon pathfinding. Asymmetric calcium signal induces activation of CaMKII and CaN; leading to asymmetric cytoskeletal protein reorganization and endocytosis/exocytosis, which steers the growth cone toward attractive guidance cue (Tojima et al, 2011). cAMP and cGMP are known as the key regulators of cytosolic calcium through IP₃R and RyR modulation. Previous reports have shown that the growth cone turning responses to attractive guidance cue netrin-1 depends on the ratio of cAMP and cGMP (Nishiyama et al, 2003). As such both cAMP and cGMP are key players in axon guidance, however these dynamics in the migrating growth cone are poorly understood. In this research, we report spatial and temporal dynamics of second messengers in freely extending growth cone with dual FRET microscopy: cAMP, cGMP, and calcium. We found that the reciprocal dynamics between cAMP and cGMP with delay. Spatial asymmetry of cyclic nucleotide signal was also found in freely turning growth cone. Our results suggest the crosstalk mechanism among the second messengers in the growth cone.	P3-1-72 AhRシグナルの活性化は発達期脳における海馬CA1錐体細胞の樹状突起成長を制御している Activation of AhR signaling pathway regulates dendritic growth of hippocampal CA1 pyramidal neurons in the developing brain ○木村栄輝¹, 松吉智瑛里¹, 宮崎航^1,2, 久保健一郎³, 遠藤俊裕¹仲嶋一範³, 掛山正心¹, 遠山千春¹ ○Eiki Kimura¹, Chieri Matsuyoshi¹, Wataru Miyazaki^1,2, Ken-ichiro Kubo³, Toshihiro Endo¹, Wenting Ling¹, Kazunori Nakajima³, Masaki Kakeyama¹, Chiharu Tohyama¹ 東京大学院・医・CDBIM・健康環境医工学部門¹, 熊本大学大学院生命科学研究部環境生命科学講座公衆衛生・医療科学分野², 慶應義塾大学医学部解剖学教室³ Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan¹, Department of Public Health, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan², Department of Anatomy, Keio University School of Medicine, Tokyo, Japan³ Dendritic arborization plays an important role for neural network formation, and the abnormality of higher brain function has been reported to be accompanied with abnormal dendritic arborization in humans and laboratory animals. In this study, we reported that perinatal exposure of mice to dioxin, an environmental pollutant, altered gene expression and dendritic arborization of the CA1 pyramidal neurons in the developing hippocampus. Pregnant C57BL/6 mice were orally administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxin, at a dose 0, 0.6 or 3.0 μg/kg (Control, TCDD-0.6 or TCDD-3.0 group, respectively) in gestation day (GD) 12.5, and brains of male progeny were collected on postnatal day (PND) 1. Gene expression in the brain was determined by real-time RT-PCR analysis. In TCDD-3.0 group, an increase in AhR-target genes such as Cyp1b1 and Ahrr mRNA abundance was observed. To analyze dendritic morphology of CA1 neurons, Thy1-GFP transgenic mice were used. Wild-type C57BL/6 female mice were mated with Thy1-GFP transgenic mice, and pregnant mice were orally administered TCDD using the same dosing scheme. Thy1-GFP heterozygous male progeny on PND 14 was euthanized, followed by collection of brains for dendritic morphology analysis. CA1 neurons were traced, and morphology of apical and basal trees was analyzed. The length of apical dendritic branching on 2nd order was shown to be significantly decreased in TCDD-3.0 group. TCDD-0.6 group was found to have a significant increase in the 3rd branch length of apical dendrite than both Control and TCDD-3.0 groups. No effects of TCDD exposure were observed on the basal tree. In conclusion, perinatal dioxin exposure induces the altered dendritic morphology of pyramidal neurons in the mouse hippocampal CA1.
P3-1-73 ドネペジルのシグマ1受容体を介した神経突起の再構築 Dendritic remodeling by donepezil through the sigma-1 receptor ○平田朗¹, 山本秀子², 山本敏文¹ ○Akira Hirata¹, Hideko Yamamoto², Toshifumi Yamamoto¹ 横浜市立大学生命ナノシステム科学研究科、分子精神薬理学研究室¹, 東京都医学総合研究所、依存性薬物プロジェクト² Lab of Mol Psychopharmacol, Grad School of Nanosci, Yokohama City University, Yokohama¹, Addictive Substance Project, Tokyo Metropolitan Inst of Medical Science² The potent and selective acetylcholinesterase inhibitor donepezil is the most widely prescribed drug for Alzheimer's disease. Donepezil has a high affinity for the sigma-1 receptor and exerts its neuroprotective effect by acting as an agonist of the sigma-1 receptor. However, the mechanisms underlying this neuroprotective effect are not clearly understood. In this study, we investigated the neuroprotective effect of donepezil by using a rat model of kainic acid-induced cellular injury. Juvenile male rats (4 weeks old) were administered kainic acid (10 mg/kg, i.p.). We then assessed the effect of donepezil on the dendritic structure of hippocampal and amygdaloid neurons, as well as behavioral changes in the rats. Dendritic morphology analysis using Golgi-Cox staining revealed that kainic acid administration induced dendritic changes in hippocampal CA3 and amygdaloid pyramidal neurons, resulting in decreased dendritic length and spine density in both regions. In addition, behavioral studies showed that kainic acid induced anxiety-like behavior in the rats during the elevated plus maze (EPM) test, that is, kainic acid-treated rats spent less time in the open arms of the maze. However, these kainic acid-induced changes in dendritic morphology and behavior in the EPM could be restored by repeated (2 weeks) treatment with donepezil (3 mg/kg/day) initiated 1 week after the kainic acid treatment. Moreover, the sigma-1 receptor antagonist NE100 attenuated the recovery effect of donepezil. These findings suggest that donepezil may improve kainic acid-induced anxiety via dendritic remodeling in hippocampal CA3 and/or amygdaloid spiny neurons.	P3-1-74 コンドロイチン硫酸合成酵素欠損マウスにおける神経発生・発達異常 Abnormal brain development and maturation in mice deficient for chondroitin sulfate-synthesizing enzymes ○吉岡望^1,4, 武内恒成^1,2,4, 岡田正康^1,3, 和田芳野¹, 川野仁⁴, 五十嵐道弘^1,2 ○Nozomu Yoshioka^1,4, Kosei Takeuchi^1,2,4, Masayasu Okada^1,3, Yoshino Wada¹, Hitoshi Kawano⁴, Michihiro Igarashi^1,2 新潟大・医・分子細胞機能学¹, 新潟大・超域学術院², 新潟大・脳神経外科³, 都医学研⁴ Div Mol Cell Biol, Niigata Univ Grad Sch Med Dent Sci¹, Transdisciplinary Res Ctr, Niigata Univ², Dept Neurosurg, Niigata Univ³, Tokyo Metropolitan Inst for Medical Science⁴ Chondroitin sulfate proteoglycans (CSPGs) are one of the major components of the extracellular matrix (ECM) in brain. CSPGs may have multiple functions, because highly sulfated glycosaminoglycans interact with various molecules, such as other ECM molecules, cell adhesion molecules and cytokines. In the postnatal brain, perineuronal nets (PNNs), high-density complexes of ECM molecules, appear around a subset of neurons temporally coincides with decrease in the neural plasticity. CSPGs are accumulated in PNNs and CS-degrading enzyme chondroitinase ABC (ChABC) increases the plasticity, suggesting that CSPGs are involved in the postnatal brain maturation. In the present study, we investigated the involvement of CSPGs in the brain development and maturation through the analysis of knockout (KO) mice lacking in a crucial CS-synthesizing enzymes CSGalNACT1. In the CSGalNAcT1-KO mice, the amount of CS was significantly decreased, and the cortical formation process was delayed. Moreover, we generated double knockout (DKO) mice for CSGalNAcT1/2, to expect that amount of CS in the brain will be reduced further. In CSGalNAcT1-KO mice, accumulation of CS there was diminished, as well as ChABC treatment, however, some differences in remaining extent of WFA-positive PNNs have been observed between the KO mice and ChABC treatment. Taken together, CSGalNAcT1 and CSPGs are thought to be closely related to the regulation of brain development and its functional maturation.
P3-1-75 シナプトフィジン陽性斑の低速・長距離の逆行性移動－皮質脊髄スライス共培養系における解析 Characterization of slow and long-distance retrograde movement of synaptophysin positive puncta along axons in corticospinal slice coculture ○吉岡昇¹, 磯尾紀子¹, 村部直之¹, 亀田浩司¹, 高橋一郎¹, 桜井正樹¹ ○Noboru Yoshioka¹, Noriko Isoo¹, Naoyuki Murabe¹, Hiroshi Kameda¹, Ichiro Takahashi¹, Masaki Sakurai¹ 帝京大学医学部生理学講座¹ Dept Physiol, Teikyo Univ Sch Med, Tokyo¹ One of the important processes in synaptogenesis is the transport of presynaptic components along axons to the synaptic sites. In corticospinal slice coculture system, we have been using synaptically enriched protein synaptophysin that is tagged with EGFP as a marker for presynaptic components. By way of in vitro electroporation, we labeled cortical cells with synaptophysin-EGFP and monitored the dynamic properties of the distribution of the protein. We also introduced DsRed2 to visualize axonal morphology. We developed a technique using a custom made stage top CO₂ incubator equipped to a non-inverted confocal microscope system to obtain time-lapse fluorescence images from the cortical axons in slice coculture at the interval of 5 - 60 min for more than 10 hours. We found that, during 3 - 7 days in vitro (DIV), synaptophysin-EGFP signal was accumulated to form puncta, or varicosities visualized with DsRed2. When monitored at an interval of 60 min, those puncta showed slow (10-50 μm/hour) and long-distance (sometimes exceeding 300 μm) movement. This movement was dominantly retrograde at all the culturing days examined. In cases movement lasted for more than 10 hours, we found these puncta moved at a constant rate interrupted by long-time (more than 1 hour) pauses. The movement was also monitored at 5 min interval and rate of constant movement comparable to that monitored at 60 min interval was observed, which further supported continuous and constant movement during the moving phase. The frequency of a punctum to show movement in 6 hours was 30 % at 3 DIV and it declined gradually until 7 DIV with a frequency of less than 10 %. As this period coincides with that of synaptogenesis, we hypothesized that those moving puncta are prepared package for presynaptic apparatus, searching for potential synaptic sites, and cease their movement when captured by those sites.	P3-1-76 軸索に存在するDINEはシュワン細胞と相互作用することにより適切な軸索ブランチングおよび神経筋接合部形成を促す Axonal DINE affects the axon-Schwann cell interactions to form appropriate nerve terminal arborization and neuromuscular junction ○桐生寿美子¹, 松本早紀子¹, 木山博資¹ ○Sumiko Kiryu-Seo¹, Sakiko Matsumoto¹, Hiroshi Kiyama¹ 名古屋大院・医・機能組織学¹ Dept Functional Anat & Neurosci, Nagoya Univ, Nagoya¹ Damage-induced neuronal endopeptidase (DINE) is a membrane-bound metalloprotease belonging to the neprilysin family, which we identified as a nerve regeneration-associated gene. DINE-deficient mice die of respiratory failure shortly after birth due to perturbed terminal arborization of motor neuron in the diaphragm, indicating the abundant expression of DINE in motor neurons is responsible for this phenotype. Detailed histochemical analysis demonstrated that the phrenic nerve in DINE-deficient embryo reached the diaphragm and extended their processes into the muscle, however it failed to find the proper direction for axonal branching in the muscle. At this time point, Schwann cell precursor cells transit to immature Schwann cells, which migrate and align to the axons in response to axonal cues. The immature Schwann cells along the axons of DINE-deficient mouse showed abnormal morphology and alignment, together with a decreased expression of Schwann cell differentiation markers such as Oct6. We hypothesize that DINE derived axonal signals allow immature Schwann cells to differentiate and assist axonal behavior. Time-lapse imaging of neurons and Schwann cells co-culture showed that Schwann cells were capable of making contact with axons, associating and aligning to the axons of the normal mouse. However, the Schwann cells failed in making tight alignment to the DINE-deficient axons. Furthermore, the supernatant application of cultured motor neurons from DINE-deficient mouse did not promote the expression of differentiation marker molecules in Schwann cells. Recent human genome-wide analyses have reported that mutations in DINE (ECEL1 in human) cause arthrogryposis which is a congenital joint contracture disorder, suggesting that DINE plays an important role in human as well as in rodent. These data suggest that DINE provides axonal cues to immature Schwann cells so that they assist axonal branching as well as axonal elongation to form neuromuscular junction properly.
P3-1-77 脊髄交連ニューロンの軸索分岐形成の時空間制御のin vivo解析 Temporal and spatial regulation of commissural axon branching in the developing mouse spinal cord ○金山武司¹, 池内彬¹, 稲又靖之¹, 白崎竜一¹ ○Takeshi Kaneyama¹, Akira Ikeuchi¹, Yasuyuki Inamata¹, Ryuichi Shirasaki¹ 大阪大院　生命機能　細胞分子神経生物学¹ Grad Sch Frontier Biosci., Osaka Univ., Japan¹ During development, growing axons navigate toward their targets in response to variety of guidance cues. Intermediate targets play a key role in regulating axon guidance programs. We have been focusing on the development of commissural axons in order to elucidate the molecular mechanisms of axon guidance. In the spinal cord, commissural neurons generated from Atoh1-expressing progenitors (i.e., DI1-type commissural neurons) initially extend axons ventrally toward the floor plate (FP) at the ventral midline, an intermediate target of these axons. After crossing the FP, these axons turn and grow rostrally to project to their final targets such as the cerebellum. Previously, we unraveled precise pathway choices made by DI1-type commissural axons until they reach around the cerebellum in mice. We found that rostrally growing axon branching occurs from the proximal axon segment on the ipsilateral side of the post-crossing commissural neurons. Intriguingly, these axon branching emerged only after their first-bearing axons had crossed the FP, raising the possibility that generation of these unique axon branching is causally linked to an interaction between commissural axons and FP cells. In the present study, to test this possibility, we examined the detail behavior of DI1-type commissural axons in vivo under a condition where the midline crossing by these axons is blocked. It has been shown that, for successful midline crossing, it is crucial for Robo3 to silence the function of Robo1, a receptor for FP-derived chemorepellent Slits. Here we employed RNAi strategy to knockdown Robo3 expressed in commissural neurons. This also enabled us to assess whether an earlier activation of Robo1 function prior to midline crossing is capable of temporally earlier induction of the ipsilateral axon branching. The results suggest that commissural neurons intrinsically possess molecular programs required for timely-generating ipsilateral axon branching independently of midline crossing.	P3-1-78 Dnmt3b依存的なDNAメチル化は単一神経細胞におけるクラスター型プロトカドヘリン遺伝子群の確率的発現と樹状突起の自己忌避を制御する Dnmt3b-dependent DNA methylation regulates stochastic expression of clustered Protocadherin genes and dendritic self-avoidance in single neurons ○豊田峻輔^1,2, 川口将史³, 小林俊寛⁴, 足澤悦子^2,5, 遠山知子¹, 岡野正樹⁶, 小田昌朗^1,7, 中内啓光⁴, 吉村由美子⁵, 三宝誠^2,5, 平林真澄^2,5, 平山晃斉^1,2, 平林敬浩^1,2, 八木健^1,2 ○Shunsuke Toyoda^1,2, Masahumi Kawaguchi³, Toshihiro Kobayashi⁴, Etsuko Tarusawa^2,5, Tomoko Toyama¹, Masaki Okano⁶, Masaaki Oda^1,7, Hiromitsu Nakauchi⁴, Yumiko Yoshimura⁵, Makoto Sanbo^2,5, Masumi Hirabayashi^2,5, Teruyoshi Hirayama^1,2^1,2, Takahiro Hirabayashi^1,2, Takeshi Yagi^1,2 大阪大院・生命機能¹, 国立精神・神経セ³, 東大医科研⁴, 生理研⁵, 理研CDB⁶, 大阪大院・医⁷ Grad Sch of Frontier BioSci, Osaka Univ, Japan¹, JST CREST, Japan², Natl Inst of Neurosci, NCNP, Japan³, Inst of Med Sci, Univ of Tokyo, Japan⁴, Natl Inst for Physiological Sci, Japan⁵, RIKEN CDB, Japan⁶, Grad Sch of Med, Osaka Univ, Japan⁷ The mammalian brain contains enormous numbers of neurons that have distinct physiological properties and circuit specificities. Protocadherin (Pcdh) -α, Pcdh-β and Pcdh-γ gene clusters encode diversified transmembrane proteins that are expressed stochastically and combinatorially in individual neurons. Previous study revealed that each Pcdh gene has its own promoters and their methylation patterns are correlated with its expression in neuroblastoma cell lines. Here we studied in vivo mechanisms and found that the Pcdh promoters are differentially methylated by de novo DNA methyltransferase Dnmt3b during early embryogenesis. Because Dnmt3b deficiency causes embryonic lethality, we produced chimeric mice with Dnmt3b-knockout (KO) induced pluripotent stem cells (iPSCs). These mice survived to adulthood, and Dnmt3b is dispensable for neural differentiation. However, Dnmt3b-KO Purkinje cells impaired dendritic self-avoidance and patterning. To quantify the expression of each Pcdh isoform in single cells, we developed a high-throughput expression analysis using a microfluidic real-time PCR system. We found that individual Dnmt3b-KO Purkinje cells expressed increased numbers of the Pcdh isoforms at single neuron level, whereas total expression level within the cluster was maintained. These results indicate that Dnmt3b-mediated DNA methylation during early embryogenesis regulates the probability of stochastic expression of the Pcdh isoforms in single neurons.
P3-1-79 プロトカドヘリン多様性の機能解析に資する抑制性ニューロン赤色標識マウス Inhibitory neuron-specific tdTomato mouse for research on protocadherin diversity ○金子涼輔¹, 平野敬三², 岡山厚², 糸賀康人², 柳川右千夫¹, 八木健² ○Ryosuke Kaneko¹, Keizo Hirano², Atsushi Okayama², Yasuto Itoga², Yuchio Yanagawa¹, Takeshi Yagi² 群馬大・医¹, 大阪大・生命機能² Gunma Univ Grad Sch of Med¹, Grad Sch of Front Biosci, Osaka Univ² The brain contains a huge number of neurons that have diverse characteristics participating in discrimination between individual neurons. It has been speculated that clustered protocadherins (Pcdhs), which encode cadherin-related transmembrane proteins as gene clusters in vertebrate genome, could provide these kinds of neuronal diversity. The murine clustered Pcdhs are further classified into three subfamilies: Pcdh-α (14 genes), Pcdh-β (22 genes), and Pcdh-γ (22 genes). Their loss of function in mice revealed that the Pcdh-α and -γ play important roles in neuronal survival, axonal projection, synaptic connectivity, and several brain functions including learning and memory. The Pcdh-α, -β and -γ genes exhibit random and combinatorial expression in each neurons, generating more than million single-neuron diversities (Nat Genet, 2005; J Biol Chem, 2006; Front Mol Neurosci, 2012). However it still remains unclear what role does the single-neuron diversity generated by Pcdhs play in neural circuit formation. In the present study, we are focusing on molecular functions of Pcdhs in local inhibitory circuit of basket cell-to-Purkinje cell in mouse cerebellum as a model system. In situ hybridization analysis revealed that both of basket cells and Purkinje cells show scattered expression patterns of Pcdhs, suggesting that the single-neuron diversity can play some role at both of these neurons. In order to genetically ablate Pcdhs in the inhibitory circuit, several lines of clustered Pcdhs deficient mice have been generated. Finally, in order to examine morphological abnormalities of the inhibitory circuit in the clustered Pcdhs deficient mice, we have developed BAC transgenic mouse, VGAT-stop-tdTomato mouse. This mouse genetically labels individual basket cells with red fluorescence after induction of Cre activity using Gl4-CreER mouse and tamoxifen injection. We are currently using these genetic methods to assess the roles of Pcdhs in mediating neural circuit formation.	P3-1-80 中枢におけるリン酸化stathmin1の機能解析 Phosphorylated stathmin1 regulates neurite elongation ○山田浩平¹, 谷口学³, 松崎伸介^2,3, 高村明考¹, 服部剛志⁴, 遠山正彌^2,5, 片山泰一² ○Kohei Yamada¹, Manabu Taniguchi³, Shinsuke Matsuzaki^2,3, Hironori Takamura¹, Tsuyoshi Hattori⁴, Masaya Tohyama^2,5, Taiichi Katayama² 大阪大院・連合小児・子どものこころの分子統御機構研究センター¹, 大阪大院・連合小児・分子生物遺伝², 大阪大院・医・神経機能³, 大阪大院・医・分子精神⁴, 近畿大・医・分子脳科学⁵ Center for Children's Mental Development, Osaka University, Osaka¹, Dept Mol Brain Sci, Osaka University, Osaka², Dept Anatomy NeuroSci, Osaka University, Osaka³, Dept Mol Mental, Osaka University, Osaka⁴, Dept Mol Brain Sci, Kinki University⁵ Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide involved in a variety of brain function. Mice lacking the Adcyap1 gene encoding PACAP (Adcyap1^–/–) and PACAP specific receptor, PAC1 deficient mice exhibit marked behavioral abnormalities. In order to clarify the molecular mechanism of PACAP dependent psychiatry disorders, we have first attempted to isolate the PACAP deficiency-regulated gene in the dentate gyrus of the mice using differential display method. Expression of stathmin1 is up-regulated in the dentate gyrus both at the levels of mRNA and protein. In addition we have found that increased expression of stathmin1 in the neurons of the SGC and primary cultured hippocampal neurons induces the abnormal arborization of the processes both in vivo and in vitro. In this time, we found that phosphorylated stathmin1 regulates neurite elongation. These results suggest that up-regulation of phosphorylated stathmin1 may create abnormal neuronal circuits that cause abnormal behavior.
P3-1-81 神経細胞での形態および小胞輸送におけるLMTK1活性の役割 The role of LMTK1 activity in morphology and vesicle transport in neurons ○漆原智己¹, 高野哲也¹, 吉岡望², 浅田明子¹, 斎藤太郎¹, 福田光則², 友村美根子³, 久永真市¹ ○Tomoki Urushibara¹, Tetsuya Takano¹, Nozomu Yoshioka², Akiko Asada¹, Taro Saito¹, Mitsunori Fukuda², Mineko Tomomura³, Shin-ichi Hisanaga¹ 首都大学東京都市教養学部生命科学コース神経分子機能研究室¹, 東北大学大学院生命科学研究科生命機能科学専攻², 明海大学歯学部歯学部薬医学研究室³ Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa, Tokyo, Japan¹, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Japan², MPL, Meikai University School of Dentistry, Sakado, Japan³ Neurons are cells that constitute the signaling network in brain of animals. They have two types of long processes, axon and dendrites, different in structure and function, through which neurons communicate each other. The formation of axon and dendrites is a complicated process including cytoskeletal rearrangement and membrane traffic. The molecular mechanism of the neurite outgrowth has been under extensive investigation. While a role of cytoskeletons is well documented, membrane supply to neurites has not been addressed sufficiently. Recently, we reported that LMTK1 controls axonal outgrowth negatively by inhibiting Rab11 activity in a Cdk5-dependent manner (Takano et al., 2012). Rab11 is a small GTPase involved in recycling endosomal trafficking. LMTK1 is a novel Ser/Thr kinase that is highly expressed in mammalion neurons. Down regulation of LMTK1 resulted in enhanced axonal outgrowth and the inhibitory activity of LMTK1 was regulated by phosphorylation with Cdk5-p35, a proline-directed Ser/Thr kinase specifically expressed in neurons. However, we did not find any differences in axon trajectory between WT and LMTK1 KO mouse brains, although cultured neurons prepared from LMTK1 KO brains had longer axon than those from WT mouse brains. Then, we subjected and observed cerebral cortex of mouse brains at postnatal day 7 or three month stained by the Golgi method. The number and total length of dendrites were increased in cortical neurons of LMTK1 KO mice more than those of WT mice. However, the difference was not found in brains of three month-old mouse. The results suggest that LMTK1 regulates dendrite outgrowth during the early stage of dendrite formations. Now, we are investigating the role of the kinase activity in dendrite formations, and would like to show the results at the meeting.	P3-1-82 成長円錐における網羅的なリン酸化タンパク質の探索 Comprehensive searching of phosphorylated protein of the growth cone ○河崎麻実¹, 武内恒成^1,2, 野住素広¹, 五十嵐道弘¹ ○Asami Kawasaki¹, Kosei Takeuchi^1,2, Motohiro Nozumi¹, Michihiro Igarashi¹ 新潟大学大学院　医学系研究科　分子細胞機能¹, 新潟大・超域研究機構² Division of Molecular and Cellular Biology, Graduate School of Medical Sciences, Niigata University, Niigata¹, Trans-Disciplinary Research Program, Niigata University, Niigata² The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous. The signaling mechanisms operating the growth cone behavior are very important to understand the neuronal network formation, however, these are poorly understood so far particularly in the mammalian CNS because of their complicated molecular basis. We reported that 17 species of the functional maker protein through the proteomics of growth cones (PNAS 2009), indicating that the comprehensive proteomic analysis is quite useful techniques for the growth cone research from the molecular aspects. Recently, the technical advances achieve that the in vivo phosphorylation sites in a given system are comprehensively determined using phosphoproteomic analysis. Based upon this powerful technology, to clarify the kinase substrates of the growth cones, we applied this method to the growth cone fraction and determined more than 1,000 phosphorylation sites. Among them, we focused GAP-43, a classical marker of the growth cone, and identified that GAP-43 was highly phosphorylated at Ser96, and at Thr172. By using specific antibodies of phospho-specifc GAP43 at these sites, we also demonstrated that these sites were highly phosphorylated not only in the developing brain but also in the regrowing axons of the spinal cord. In in silico examinations, Ser96 and Thr172 are within sequence contexts suitable to proline-directed kinases such as JNKs which are reported to be critical to axon elongation, and JNKs inhibitor (SP600125) inhibited the phosphorylation of these residues. These results suggest that JNK-dependent phosphorylation of GAP-43 is one of the important signaling involved in axonal generation and regeneration in in vivo. We are now going to investigate a role of JNK phosphorylation of GAP43 at Ser96 and Thr172 in neural circuit formation.
P3-1-83 神経活動依存的に放出されるタウリンは大脳皮質の辺縁帯における興奮性の神経伝達を促進する Activity dependent release of taurine facilitates the excitatory neurotransmission in the marginal zone of developing cerebral cortex ○熊田竜郎¹, 千太哲¹, 中村直人², 古川智範¹, 山田順子³, 秋田天平¹中原大一郎², 福田敦夫¹ ○Tatsuro Kumada¹, Taizhe Qian¹, Masato Nakamura², Tomonori Furukawa¹, Junko Yamada³, Tenpei Akita¹, Welner Kilb⁴, Heiko, J. Luhmann⁴, Daiichiro Nakahara², Atsuo Fukuda¹ 浜松医科大学医学部神経生理学¹, 浜松医科大学医学部心理学², 弘前大学院医学研究科神経生理学³, Instit of Physiol and Pathophysiol, Johannes Gutenberg-University⁴ Dept Neurophysiol, Hamamatsu Univ Sch Med, Hamamatsu¹, Dept Psychol and Behavioral Neurosci, Hamamatsu Univ Sch Med, Hamamatsu, Japan², Dept Neurophysiol, Hirosaki Univ Grad Sch Med, Hirosaki, Aomori, Japan³, Instit of Physiol and Pathophysiol, Johannes Gutenberg-University, Mainz, Germany⁴ In the developing cerebral cortex, the marginal zone (MZ) consisting of early-generated neurons such as Cajal-Retzius (CR) cells plays important roles in cell migration and lamination. There is accumulating evidence of widespread excitatory neurotransmission mediated by GABA in the MZ. CR cells express not only GABA_A receptors but also α2/β subunits of glycine receptors (GlyR) and exhibit GlyR-mediated depolarizations due to high intracellular Cl^- concentration. The physiological roles of the GlyR and its endogenous agonists during neurotransmission in the MZ are, however, remain unknown. To address this, we performed optical imaging using a voltage-sensitive dye on tangential slices of the MZ of postnatal day (P) 0-3 rats. An optical signal due to action potentials was evoked by a single electrical stimulus, and the signal spread out radially over the MZ. The amplitude of the signal was not affected by glutamate receptor blockers, but suppressed by either GABA_A receptor antagonists or GlyR antagonist. Combined application of GABA_A and GlyR blockers nearly abolished the signal. Bumetanide also reduced the signal, suggesting that the Cl^- uptake mediated by NKCC cotransporters promotes the GABA_A and glycine receptor-mediated depolarizing or excitatory actions. An in vitro microdyalysis analysis revealed that GABA and taurine, but not Gly or glutamate, were released in the MZ in response to the electrical stimulus. The release of taurine was abolished by addition of voltage-sensitive Na⁺ channel blocker. Immunohistochemistry and immunoelectron microscopy indicated that taurine was stored both in CR cells and in non-CR cells in the MZ, but not localized in presynaptic structures. The results suggest that activity-dependent release of endogenous taurine facilitates the excitatory neurotransmission through activation of GlyR in the MZ.	P3-1-84 Teneurin-4は神経突起形成とシナプス形成を促進する Teneurin-4 Positively Regulates Neural Cell Process Formation and Synaptogenesis ○鈴木喜晴^1,2, 沼川忠広³, チョウジョシュア², デベガスサナ^2,4, 水庭千尋¹, 関本佳織¹, 安達直樹³, 功刀浩³, 平澤恵理⁴, 山田吉彦², 赤澤智宏¹ ○Nobuharu Suzuki^1,2, Tadahiro Numakawa³, Joshua Chou², Susana de Vega^2,4, Chihiro Mizuniwa¹, Kaori Sekimoto¹, Naoki Adachi³, Hiroshi Kunugi³, Eri Hirasawa⁴, Yoshihiko Yamada², Chihiro Akazawa¹ 東京医科歯科大学大学院保健衛生学研究科分子生命情報解析学¹, 米国国立保健衛生研究所歯学頭蓋研究所², 国立精神・神経医療研究センター神経研究所疾病研究第三部³, 順天堂大学医学部神経学講座／大学院老人性疾患病態・治療研究センター⁴ Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, TMDU, Tokyo¹, NIDCR, NIH, Bethesda, MD, USA², Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, Tokyo³, Research Institute for Diseases of Old Age and Department of Neurology, Faculty of Medicine, Juntendo Univ., Tokyo⁴ Teneurin-4 is a transmembrane protein highly expressed in the central nervous system. However, its function is poorly understood. In this study, we seek to understand the function of teneurin-4 in neuronal cells. During neurite outgrowth assay of the neuroblastoma cell line Neuro-2a, teneurin-4 expression was localized and accumulated at the tip of cellular processes, such as the growth cones. Knockdown of teneurin-4 expression in Neuro-2a cells decreased the length of neurites and the density of filopodia-like protrusions. On the contrary, teneurin-4 overexpression, promoted the filopodia-like cell protrusion formation. In addition, we look at the activation of Cdc42 and Rac1, key molecules for neural cell process formation. Knockdown and overexpression of teneurin-4 reduced and elevated, respectively, the activation of both molecules. Inhibition of N-WASP, an activator of filopodia formation downstream of Cdc42, blocked the formation of the protrusions by teneurin-4-overexpression. These results suggest that teneurin-4 promotes cell process formation through the activation of the Cdc42/N-WASP and Rac1 pathways. Since cell process formation is required for the initial stage of synaptogenesis, we examined teneurin-4 function in synapse formation. We found that the knockdown and overexpression of teneurin-4, reduced and increased, respectively, the expression of both pre- and post-synaptic proteins during the early stage of the primary cortical neuronal culture. Moreover, the teneurin-4 knockdown inhibited the release of glutamate after the stimulus with high potassium solution in the cortical neuron culture. Therefore, teneurin-4 is required for the expression of synaptic proteins and for the glutamate release. Taken all together, our findings show that teneurin-4 positively regulates the cellular process formation and synaptogenesis in neuronal cells.
P3-1-85 DINE欠損運動神経の骨格筋支配の発生学的異常 Aberrant muscle innervation of motoneurons in Damage Induced Neuronal Endopeptidase (DINE) deficient mice ○永田健一^1,2, 桐生寿美子³, 斉藤貴志¹, 木山博資³, 西道隆臣¹ ○Kenichi Nagata^1,2, Sumiko Kiryu-Seo³, Takashi Saito¹, Hiroshi Kiyama³, Takaomi C. Saido¹ 理研BSI・神経蛋白制御¹, 日本学術振興会², 名古屋大院・医・機能組織³ Lab for Proteolytic Neurosci, RIKEN BSI, Saitama, Japan¹, JSPS, Tokyo, Japan², Dept Functional Anat and Neurosci, Nagoya Univ, Grad Sch Med, Nagoya, Japan³ Damage-induced neuronal endopeptidase (DINE) is a membrane bound metalloprotease, which belongs to NEP family. DINE is specifically expressed in neuronal cells from early embryonic period, and remarkably induced in response to various types of neuronal injuries. We previously revealed that DINE-deficient mice died immediately after birth due to respiratory failure, and reported that muscle innervation of their phrenic nerves, which is responsible for respiratory control, was drastically decreased. However, it remains unclear that other motor nerves also have aberrant innervation. In order to examine the abnormalities of motor neuron innervation throughout embryonic body, we generated DINE deficient mice carrying motor neuron specific expression of GFP by crossing with Hlxb9 GFP Tg mice, and compared DINE-deficient and control embryos by using confocal microscopy. At E12.5, the arborization of motor nerve terminal in forelimb from DINE-deficient mice was somewhat decreased, although the brachial plexus was properly formed similar to that of wild type littermate controls. The abnormalities of the motor nerve arborization were much severer at later stage. Number of neuromuscular junctions in forelimb was significantly decreased. Furthermore, similar phenotype was also detected in the muscles of the hindlimb. Taken together, we conclude that DINE is an essential molecule for proper branching and normal guidance of motor axons en route to their muscle targets.	P3-1-86 ニワトリ胚の毛様体神経節で見られる軸索刈り込みにカスパーゼが関与しているという仮説を検証する Involvement caspases in the axon pruning during calyx-type synaptogenesis of chick ciliary ganglion ○加藤秀理¹, 江川遼¹, 細島頌子¹, 石塚徹^1,2, 八尾寛^1,2,3 ○Hidetaka Katow¹, Ryo Egawa¹, Shoko Hososhima¹, Toru Ishizuka^1,2, Hiromu Yawo^1,2,3 東北大学　生命科学研究科　脳機能解析分野¹, 東北大学脳神経科学コアセンター³ Graduate School of Life Sciences, Tohoku University, Sendai¹, CREST, JST², Center of Neuroscience, Tohoku university Graduate school of Medicine³ Axonal branches of neurons were formed in excess during early developmental stages, but decreased in an appropriate number thereafter before birth (axon pruning).Here, we investigated a hypothesis that caspase system is involved in the axon pruning using calyx-type synapses in the chick ciliary ganglion as a model.White Leghorn embryonated chick eggs at the developmental stages E8, E11 and E14 were used. The native expression of caspase-3 and caspase-6 were immunohistochemically investigated. Plasmids containing a construct of each caspases tagged with EGFP or mCherry were made to over-express the labeled caspases and introduced mid brain of E2 embryo using a conventional electroporation method.In the E8-E14 ciliary ganglia, caspase-3-immunoreactivity was found in small cells and membrane-like structures that are reminiscent of calyx-type presynaptic terminal. On the other hand, caspase-6-immunoreactivity was found in axon-like structures and calix-type presynaptic terminals. The labeled caspase-3 was distributed throughout axons, and accumulated in bouton-type presynaptic terminals. The labeled caspase-6 was found in calyx-type presynaptic terminals and dot-like structures.The immunoreactivity and the overexpression study suggested that caspase-3 is distributing in the axons and presynaptic terminals. On the other hand, caspase-6 is suggested to distribute in the calyx-type presynaptic terminals and terminal axons. The caspase system is possibly involved in axon pruning during synaptogenesis in the chick ciliary ganglion.
P3-1-87 アクチンとプロテインホスファターゼ1に結合するタンパク質scapinin/phactr3の脳発達における発現：神経選択的発現と樹状突起形態への役割 Developmental and neuron-enriched expression of scapinin/Phactr3, an actin- and a protein phosphatase 1 (PP1)-binding protein, in the developing rat brain: the role in dendritic morphology ○田渕明子¹, 皆藤真季¹, 袴田知之¹, 石川充¹, 菊池啓悦¹, 宮田智陽¹, 福地守¹, 津田正明¹ ○Akiko Tabuchi¹, Maki Kaito¹, Tomoyuki Hakamata¹, Mitsuru Ishikawa¹, Keietsu Kikuchi¹, Tomoaki Miyata¹, Mamoru Fukuchi¹, Masaaki Tsuda¹ 富山大学大学院　医学薬学研究部（薬学）分子神経生物学研究室¹ Dept. Biol. Chem., Grad. Sch. of Med. & Pham. Sci., Univ. Toyama, Toyama, Japan¹ Actin-based morphological alteration of neurons plays essential roles in the construction of neuronal circuit and the formation of synapses for neuronal plasticity. Scapinin (nuclear scaffold-associated PP1-inhibiting protein)/Phactr3 (phosphatase and actin regulator) has been reported as an actin- and a protein phosphatase 1-binding protein. Several studies have shown that the mRNA expression of scapinin/phactr3 is enriched in the brain. However, little is known about the regulation of its mRNA expression during brain development, the specific roles in dendritic morphology of neurons or the existense of glial cells. Here we have demonstrated that scapinin/phactr3 mRNA is up-regulated in the developing brain and is selectively enriched in neurons, but not in glial cells. Overexpression of scapinin/phactr3 in cortical neurons increases the dendritic number, but conversely, the expression of scapinin/phactr3 small interfering RNA decreases it, suggesting that scapinin/phactr3 promotes dendritic complexity. Ongoing experiments regarding the effect of scapinin/phactr3 on dendritic spine morphology and the distinct function of actin-binding and PP1-binding domains will also be discussed in this study.	P3-1-88 初期神経活動による大脳皮質神経回路形成の制御 Role of early intracortical activity in the maturation of neocortical circuits ○ブルマントーステン¹, 花嶋かりな¹ ○Torsten Bullmann¹, Carina Hanashima¹ RIKEN Center for Developmental Biology¹ Lab for Neocortical Dev, RIKEN CDB, Kobe¹ The mammalian neocortex is comprised of diverse arrays of neurons that are radially organized into six major layers and tangentially grouped into areas. In the mature cortex, each cortical area receives unique input and generates specific output connections that dictate its functional specialization. Cortical excitatory neurons (glutamatergic) and associated inhibitory interneurons (GABAergic) contribute to the creation of sensory representation of the physical world within each functional area. During the prenatal period, the formation of early neuronal networks depends largely on genetic information encoded within the cell by transcription factors. Subsequently, spontaneous and sensory-driven oscillatory activities are important for shaping cortical circuitry and are considered as the major determinant in refining cortical columns. Here, using transgenic mice and in utero electroporation, I investigate the roles of oscillatory activities for the maturation of neocortical circuit. For this purpose, I take advantage of the fact that the time of electroporation determines the layer into which the transgene is introduced, e.g. E10.5 for subplate neurons and E13.5 for layer IV neurons. The transgenes (including dominant negative mutations) allow to manipulate neuron excitability, intrinsic resonance frequency, electrical coupling, synaptic transmission, axonal formation and the shift from excitatory to inhibitory GABAergic transmission. Furthermore, conditional Nkx2.1 knockout mice are utilized to ablate GABAergic interneuron populations. These approaches enable to determine how intrinsic activity as well as early input from the subplate neurons and interneurons influences the maturation of layer IV neurons and their integration into the barrel field of the somatosensory cortex.
P3-1-89 Development of layer 1 neurons in the mouse neocortex ○Jian Ma¹, Xing-Hua Yao¹, Ying-Hui Fu¹, Yong-Chun Yu¹ Institue of Neurobiology, Fudan University, Shanghai, China¹ Layer 1 of the neocortex harbors a unique group of neurons that play crucial roles in synaptic integration and information processing. Although extensive studies have characterized the properties of layer 1 neurons in the mature neocortex, it remains unclear how these neurons progressively acquire their distinct morphological, neurochemical and physiological traits. In this study, we systematically examined the dynamic development of Cajal-Retzius cells and interneurons in layer 1 during the first two postnatal weeks. Cajal-Retzius cells gradually disappeared from the layer 1 and underwent morphological degeneration after birth. The majority of interneurons abruptly expressed the distinct chemical markers around postnatal day 8, and expressed at least one of six chemical markers, including GABA_ARδ, which preferentially labeled neurogliaform cells. Layer 1 interneurons were divided into two subtypes according to firing pattern: late-spiking (LS) and burst-spiking (BS) neurons. LS neurons preferred to transcript GABA_ARδ, whereas BS neurons preferred to transcript vasoactive intestinal peptide (VIP). We found that both LS and BS neurons exhibited a rapid growth in terms of the electrophysiology and morphology properties during the first postnatal week. Our results provide new insights into the molecular, morphological and functional development of the superficial layer of the neocortex.	P3-1-90 IP3 receptor/channel and axonal guidance ○Carmen Chan¹, Hiroki Akiyama¹, Katsuhiko Mikoshiba², Hiroyuki Kamiguchi¹ Lab. for Neuronal Growth Mechanisms, RIKEN Brain Science Institute¹, Lab. for Developmental Neurobiology, RIKEN Brain Science Institute² Naturally occurring diffusible factors, such as trophic factors, netrin, myelin-associated glycoproteins (MAG), etc, act as guidance cues for developing axons. In vivo and in vitro, these factors form gradients (highest concentration at the source) that attract or repel growth cones. The attractive or repulsive turning response often depends on the generation of localized calcium (Ca2+) elevation inside growth cones, either from extracellular sources, or internal storage (mainly the endoplasmic reticulum). One of the two classes of Ca2+ channels that release Ca2+ from internal storage is the inositol 1,4,5-trisphosphate (IP3) receptor.Our lab has previously shown that after nerve growth factor (NGF) bound to and activated surface receptors, the resultant generation of IP3 formed a gradient across the growth cone (high on the side facing NGF, i.e. the 'near side'). It activated IP3 receptors on the endoplasmic reticulum to release Ca2+ into the cytoplasm, in turn creating a gradient of Ca2+. Such asymmetric Ca2+ elevation enabled attractive growth cone turning.In vertebrates, there are three separate genes coding for three IP3 receptor subtypes. My data showed that IP3 receptor type III (IP3RIII) is responsible for the turning response. Currently I am investigating the mechanism of IP3RIII-depending growth cone turning. Preliminary data suggest that, surprisingly, growth cones lacking IP3RIII are still able to generate Ca2+ elevation in response to NGF. However, IP3RIII is necessary for the asymmetry in Ca2+ response: Ca2+ elevation in growth cones from IP3RIII knockout mice spread to the far side, in contrast to wildtype growth cones in which Ca2+ elevation is localized to the near side only.
P3-1-91 Draxin is a bi-functional guidance molecule showing a concentration dependent transition from growth promotion to inhibition ○Md. Asrafuzzaman Riyadh¹, Yohei Shinmyo¹, Mahmud Hossain¹, Giasuddin Ahmed¹, Iftekhar Bin Naser¹, Hideaki Tanaka¹ Department of Developmental Neurobiology, Kumamoto University¹ The adult pattern of neural connectivity is established during development when axonal growth cone, follow stereotypical routes to their synaptic targets. The responsiveness of developing axons to guidance cues is subject to extensive modulation such that growth cones may react to the same guidance cue differently depending on the developmental context in which the cue is encountered. Draxin, a recently identified axon guidance molecule is essential for forebrain commissure formation. Previous reports showed draxin could act as a repulsive guidance molecule. Here we show that draxin can mediate a concentration dependent transition from growth promotion to inhibition.We performed dissociated culture of thalamic neurons isolated from E14.5 mouse embryos in the presence of different concentration of draxin-AP and control-AP. When 10nM draxin-AP was used it promoted robust outgrowth of neurite compared to same amount of control-AP but when higher amount of draxin-AP (50nM) was used, neurite growth inhibition was observed. In order to mimic the in vivo condition thalamic neurons were grown over the carpet layer made of FI-293 or FI-293-mouse draxin cells and a robust neurite outgrowth was observed in the neurons that were grown over FI-293-mouse draxin. We are now trying to determine the receptor/s that is responsible for the attractive and repulsive function of draxin. This concentration dependent switch from positive to negative responses may be applicable to provide a general principle to explain how cell migration or axon guidance can be directed to a specific location within a gradient.

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