突起伸展、回路形成 Axonal/Dendritic Growth and Circuit Formation
P1-2-44 自発性脱分極波の阻害によるシナプス回路網形成への影響 Effects of in ovo blockade of the spontaneous depolarization wave on functional synaptogenesis in the developing CNS ○佐藤容子1, 田代結花1, 佐藤勝重2 ○Yoko Momose-Sato1, Yuka Tashiro1, Katsushige Sato2 関東学院大学 人間環境学部 健康栄養学科1, 駒沢女子大学 人間健康学部 健康栄養学科2 Department of Health and Nutrition, College of Human Environmental Studies, Kanto-Gakuin University, Yokohama, Japan1, Department of Health and Nutrition Sciences, Faculty of Human Health, Komazawa Women's University, Tokyo , Japan2 We demonstrated previously that spontaneous activity in the embryonic brain exhibits a large-scale wave of neural depolarization, which propagates over a wide region of the CNS. This wave, referred to as the depolarization wave, is expressed during a specific period of embryogenesis, at which synaptic contacts are established and become functional between sensory nerves and postsynaptic neurons in the CNS. Such a developmental profile has led us to a hypothesis that the depolarization wave might serve as a regulator of synaptogenesis, which is known to be activity-dependent. In the present study, we tested this hypothesis by blocking the depolarization wave in ovo and examining its effects on functional synaptogenesis in the brainstem sensory nucleus, the nucleus of the tractus solitarius (NTS). Although chronic inhibition of the depolarization wave caused a retardation of the embryonic growth, the developmental time course, spatial distribution, and physiological characteristics of postsynaptic responses in the NTS were not significantly affected. The results seem not in favor of the idea that the depolarization wave plays an indispensable role in the formation of functional synaptic networks along the cranial sensory pathway.	P1-2-45 RhoファミリーG蛋白質TC10は細胞膜近傍でのGTP加水分解により小胞の細胞膜融合と突起伸展を促進する TC10 promotes exocytic vesicle fusion and neurite outgrowth by releasing exocyst component Exo70 by its GTP hydrolysis ○鯉沼真吾1,2, 藤田明音1, 安田さや香1, 永井寛之1, 上口裕之3, 和田直之2, 中村岳史1 ○Shingo Koinuma1,2, Akane Fujita1, Sayaka Yasuda1, Hiroyuki Nagai1, Hiroyuki Kamiguchi3, Naoyuki Wada2, Takeshi Nakamura1 東京理科大・生命研1, 東京理科大理工応用生物2, 理研BSI3 RIBS, Tokyo Univ of Sci, Chiba, Japan1, Faculty of Sci and Tech, Tokyo Univ of Sci, Chiba, Japan2, RIKEN BSI, Saitama, Japan3 Neurite outgrowth is essential for both the initial wiring of neuronal networks during development and regeneration. We have previously shown that Rac1 is locally activated at neurite tips in a variety of neuritogenic processes although the mechanisms of Rac1 activation vary widely. It is well known that the promoting effect of Rac1 on neuritogenesis is mainly through cytoskeletal reorganization. However, neurite outgrowth also requires a local increase in the surface area, which relies on anterograde transport of membrane vesicles. Recently, TC10, which is a Rho family GTPase and located to vesicles and the plasma membrane, has been shown to be required for axonal growth. We are pursuing the possibility that the Rac1-TC10 pathway contributes to neurite outgrowth through the membrane addition to neurite tips. In EGF-treated HeLa cells, we previously found that TC10 activity near the plasma membrane dropped immediately before the fusion through the Rac1-mediated pathway. In combination with other functional assays, we have proposed that GTP hydrolysis by TC10 triggers vesicle fusion. By using TC10 FRET sensors, here we have shown that TC10 activity decreased on the plasma membrane in parallel with the extension of cell periphery in a range of neuronal cells including hippocampal neurons. In PC12 cells, NGF or dbcAMP treatment rapidly decreased the level of GTP-TC10. This down-regulation was mediated by Rac1 and radical oxygen species generation. We found that TC10 activity at the vesicles was markedly higher than that at the plasma membrane in NGF-treated PC12 cells. We note that TC10 frequently resided on the vesicles containing Rab11, which is a key regulator of recycling endosomal pathways and has been shown to play a role in neurite outgrowth as well as TC10. We think that the role of GTP hydrolysis by TC10 in membrane fusion might be applicable to neurite outgrowth.
P1-2-46 Moved to Oral Session O2-7-5-4	P1-2-47 プロスタグランジンEP4受容体を介した視床下部神経細胞の突起伸長促進 Facilitation of neurite elongation mediated by prostaglandin EP4 receptor in mouse hypothalamic cell line ○土屋裕義1, 杉本幸彦2, 藤原葉子1, 藤村昭夫1, 輿水崇鏡1 ○Hiroyoshi Tsuchiya1, Yukihiko Sugimoto2, Yoko Fujiwara1, Akio Fujimura1, Taka-aki Koshimizu1 自治医大・医・分子薬理1, 熊大・院薬・生化2 Dept of Mol Pharmacol, Jichi Med Univ, Tochigi1, Dept of Pharm Biochem, Kumamoto Univ Grad Sch of Pharm Sci, Kumamoto2 Prostaglandin E2 (PGE2) has been shown to participate in various physiological and pathological processes in the brain. Recently, the roles of PGE2 in hypothalamus are attracting attention, for example, in fever, arousing effect, stress response and male sexual behavior. PGE2 actions are mediated by specific EP receptors on the plasma membrane. EP receptors are subdivided into four subtypes, EP1, EP2, EP3 and EP4, on the basis of their responses to various agonists and antagonists. Although much knowledge has been accumulated about the critical roles of PGE2 in the central nervous system, little is known about PGE2 actions on the neuronal morphogenesis. In recent study, we investigated the effect of PGE2 on the neurite growth in mouse hypothalamic cell line N37, which expresses EP1 and EP4 receptors, but not EP2 or EP3 receptor. After the differentiation by serum starvation, PGE2 or an EP4 specific agonist ONO-AE1-329 dramatically accelerated the neurite growth in N37. Since EP4 receptor couples to Gs protein, we next analyzed the role of cAMP on the neurite outgrowth in N37 cell. Consequently, the PGE2 elevated intracellular cAMP level and a selective activator of PKA, 6-Bnz-cAMP-AM significantly facilitated neurite elongation. These results indicate that PGE2-induced neurite growth is mediated by EP4 receptor, followed by the activation of cAMP-PKA pathways. Furthermore, studies with specific inhibitors of protein kinase revealed that neurite extension caused by PGE2 is mediated by the activation of CaMKII, PKC, and/or ERK.Present results suggest that PGE2 is required for accelerating neurite projection during hypothalamic development and this PGE2-EP4 siganling in N37 cells provide a new model to investigate the mechanism of the neuronal maturation.
P1-2-48 マーモセット内側前頭前皮質の樹状突起およびスパインの生後発達 Postnatal development of dendritic structures in the medial prefrontal cortex of the marmoset ○佐々木哲也1, 青井宏諭2, 小賀智文2, 藤田一郎2, 一戸紀孝1 ○Tetsuya Sasaki1, Hirosato Aoi2, Tomofumi Oga2, Ichiro Fujita2, Noritaka Ichinohe1 国立精神・神経セ神経研・微細構造1, 大阪大院・生命機能2 Dept of Ultrastructural Study, Nat Inst of Neurosci, NCNP, Tokyo, Japan1, Grad Sch Frontier Biosci, Osaka Univ, Osaka, Japan2 Abnormalities of the processes of spinogenesis and pruning are implicated in several psychiatric disorders. We have been investigating the normal processes of spine formation/pruning in the cerebral cortex using the common marmoset as a primate model. Our previous study showed similarities and differences of developmental profiles of basal dendrites and spines of layer-III pyramidal cells between the primary visual cortex (V1), an inferior temporal area (TE), and a ventrolateral prefrontal cortex (area 12) (Oga et al., 2013). Here we extended our analyses to the medial prefrontal cortex (mPFC), which is suggested to be involved in some developmental disorders. Five marmosets ranging from 3-day old to 2.5-year old were used. Layer-III pyramidal cells in areas 8B/9, 14r, 24 (granular, dysgranular, agranular mPFC, respectively) were intracellularly injected with Lucifer Yellow, and reacted for Diaminobenzidine. Basal dendrites of more than 170 cells were reconstructed, and their morphological features were analyzed. The three areas were generally similar in their developmental time course of dendrites and spines formation. Dendritic length, the number of branching points, and the estimated total spine number reached a peak value at 2-3 months of age, and decreased thereafter toward adults. Neurons in mPFC had more spines than those in areas 12, TE and V1 at any developmental stage studied. The magnitude of the decrease in the number of spines from the peak period to adults differed among the areas (area 8B/9: 47%, area 14r: 29%, area 24: 30%, cf. area 12: 41%). The results suggest that the limbic cortical areas (areas 14r and 24) maintain more spines than the neocortical areas (areas 8b/9 and 12). These features may be related to the vulnerability of this region to developmental disorders. Search for the molecular mechanisms that underlie the developmental changes will provide further clues for an understanding of pathogenesis of developmental disorders.	P1-2-49 ショウジョウバエ感覚ニューロンにおける軸索刈り込みメカニズムの探索 Molecular and cellular dissection of axonal pruning in Drosophila sensory neurons ○森川麗1, 榎本和生1 ○Rei Morikawa1, Kazuo Emoto1 大阪バイオサイエンス研・神経細胞生物学1 Dept Cell Biol, Osaka Biosci Inst1 Establishment of mature neural networks requires remodeling of initially formed circuits through elimination of inappropriate connections, which is in part achieved by axonal pruning. Although considerable progress has been made in understanding the processes that initially form axonal connections, much less is known about molecular and cellular mechanisms by which unnecessary axons are selectively eliminated. One of the obstacles is the limited number of simple and genetically tractable model systems to study axonal pruning. We have been working on neuronal remodeling mechanisms of Drosophila class IV dendrite arborization (C4da) sensory neurons. It has been elucidated that dendrites of C4da neurons are eliminated during the pupal periods in part through caspase-dependent degeneration. Here we report that C4da neurons also provide a good model to study mechanisms of axonal pruning. During the embryonic periods, C4da neurons elaborate synaptic terminal branches that project contralaterally in the ventral nerve cord, a counterpart of the mammalian spinal cord. We found that these axonal branches persist throughout the larval periods, but are pruned within 28 hours after pupariation. We also found that overexpression of caspase inhibitor proteins, which impair the C4da dendrite pruning, did not affect the axonal pruning. In addition, live imaging revealed that the C4da axonal branches appeared to be eliminated through retraction, rather than degeneration. Thus these observations suggest that C4da neurons likely remodel not only the receptive fields but also the axonal connectivity during metamorphosis, and that these pruning events are mediated by distinct mechanisms. To elucidate molecular mechanisms of the C4da axonal pruning, we have begun an RNAi-based genetic screen. We have identified several genes required for the remodeling processes, and will discuss how these genes regulate axonal pruning.
P1-2-50 発生期の第一次視覚野の同側眼優位カラムに特異的に発現している因子の同定およびその因子の片眼遮断に左右されない発現パターン Identification of a molecule specific for ipsilateral ocular dominance (OD) columns in the developing visual cortex and its unaltered expression after monocular deprivation ○冨田江一1, , 由利和也3 ○Koichi Tomita1, Max Sperling2, Kazunari Yuri3, Tobias Bonhoeffer2, Mark Huebener2 生理研/行動・代謝分子解析センター1, マックス・プランク神経生物学研究所2, 高知大・医・解剖3 Cent Genet Anal Behav, NIPS, Okazaki1, MPI of Neurobiology, Muenchen2, Dept Neurobiol & Anat, Kochi Med Sch, Kochi Univ, Kochi3 Ocular dominance (OD) columns, alternating regions of left- and right eye input in the visual cortex of higher mammals, have long been thought to develop from an initially intermingled state by an activity dependent process. While indirect evidence points to potential alternative mechanisms based on molecular cues, direct proof for a molecular difference between left- and right eye columns is missing. Here we show that a molecular chaperone is expressed in a clustered fashion in developing cat visual cortex and these clusters are in precise register with OD columns of the ipsilateral eye as early as postnatal day 16, when OD columns have just appeared. A coincident expression of the molecular chaperone with ipsilateral OD columns is not altered by eye-specific changes in neuronal activity. Importantly, a periodic, clustered expression of the molecular chaperone is already present weeks before OD columns have started to form. Taken together, these results suggest that molecular differences between future left and right eye OD columns, which emerge independently from neuronal activity, may contribute to the segregated termination of eye specific afferents in the developing visual cortex.	P1-2-51 てんかん脳における細胞内cAMPレベルの上昇と軸索分枝形成の関連 cAMP signaling mediates activity-induced robust axonal branching in the epileptic brain ○柴田和輝1, 中原聡一郎1, 田中謙二2, 鍋倉淳一3, 伊関峰生4, 渡辺正勝5, 松木則夫1, 小山隆太1 ○Kazuki Shibata1, Soichiro Nakahara1, Kenji Tanaka2, Junichi Nabekura3, Mineo Iseki4, Masakatsu Watanabe5, Norio Matsuki1, Ryuta Koyama1 東大院・薬・薬品作用学1, 慶應大・医・精神神経科学2, 生理学研究所 生体恒常機能発達機構研究部門3, 東邦大・薬・薬品物理分析学4, 光産業創成大学院大学・光バイオ分野5 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo1, Department of Neuropsychiatry, School of Medicine, Keio University2, Division of Homeostatic Development, National Institute for Physiological Sciences3, Faculty of Pharmaceutical Sciences, Toho University4, The Graduate School for the Creation of New Photonics Industries5 In the epileptic brain, it has been suggested that aberrantly induced axonal collaterals establish abnormal excitatory circuits. Therefore, the inhibition of aberrant axonal branching can be a potential therapeutic strategy against the development of epilepsy. Here we examined the involvement of cAMP signaling in the hyperexcitability-induced sprouting of hippocampal mossy fibers. In a pentylenetetrazol (PTZ)-induced rat model of neonatal seizures, we found that increased cAMP levels in the dentate gyrus precede the mossy fiber sprouting. To examine whether increased levels of intracellular cAMP mediate the mossy fiber sprouting, we utilized hippocampal slice cultures in which the morphology of granule cells was visualized by membrane-targeted GFP. In the hyperexcitable slices cultured chronically with the GABAA receptor antagonist picrotoxin, activity-induced mossy fiber sprouting was blocked by the cAMP antagonist Rp-cAMPs, while the cAMP agonist Sp-cAMPs alone induced the excessive branching of mossy fibers in control cultures. To further confirm the role of intracellular cAMP in the sprouting, we transfected the granule cells with photoactivated adenylyl cyclase (PAC) which enables temporal manipulation of the intracellular cAMP levels. PAC is originally isolated from Euglena gracilis and produces cAMP in response to blue light, and the blue light exposure (470nm) to slice cultures induced aberrant mossy fiber branching. We further suggested that the elevated cAMP-induced sprouting is mediated by exchange protein directly activated by cAMP (EPAC) but not by PKA. Our study suggests that PAC is a useful tool to manipulate cAMP levels in neurons, and that the elevation of intracellular cAMP underlies the aberrant network formation in the epileptic brain.
P1-2-52 BDNFにより誘導されるアクチン結合性SRFコアクチベーターMKL1のリン酸化 : ラット大脳皮質ニューロンの樹状突起形態における549番目セリン残基の影響 BDNF-induced phosphorylation of SRF co-activator MKL1 at serine 549 is involved in the regulation of dendritic morphology in rat cortical neurons ○石橋悠太1, 辻井惇也1, 石川充1, 久保友喜美1, 福地守1, 津田正明1, 田渕明子1 ○Yuta Ishibashi1, Jyunya Tsujii1, Mitsuru Ishikawa1, Yukimi Kubo1, Mamoru Fukuchi1, Masaaki Tsuda1, Akiko Tabuchi1 富山大院　医薬 分子神経生物1 Dept.Biol.Chem., Grad.Sch.of Med.&Pharm. Sci., Univ.Toyama, Toyama, Japan1 Morphological alteration and gene expression in neurons play critical roles in the formation of neuronal circuitry and acquirement of higher brain functions, including learning, memory, cognition, and emotion. Accumulating evidence suggests that cytoskeletal alterations propagate signals into the nucleus where gene expression is regulated and gene expression contributes to provide neurons with proper functional molecules in a spatiotemporal manner. Megakaryoblastic leukemia(MKL) family members, which have actin-binding domains and the transcriptional activation domain, have been suggested to be the bridge molecules for linking regulation of cell cytoskeleton to gene expression. It has been suggested that MKL translocates into the nucleus followed by activating of Rho signaling pathway, and activates SRF-mediated and cytoskeleton-associated gene expression. Our previous studies have shown that MKL1 and MKL2 are highly expressed in the brain and play an important role in activating SRF-mediated gene expression and regulating dendritic morphology in rat cortical neurons. We have found that MKL1 is strongly phosphorylated by BDNF-stimulation and this process is mediated by ERK/MAP kinase pathway. We have speculated that this phosphorylation is involved in regulation of MKL1 functions. In this study, we have shown that the 549th serine of MKL1 was phosphorylated by BDNF-stimulation. Our preliminary study suggested that MKL1 mutant with the substituted alanine at 549th serine residue has the differential effect on dendritic morphology, compared with the wild type. Ongoing studies are aimed at investigating which signaling pathways mediate this phosphorylation and whether this phosphorylation affects the subcellular localization of MKL1 and SRF-mediated gene expression.	P1-2-53 体性感覚野バレル皮質形成の分子メカニズム Molecular mechanisms of the somatosensory barrel cortex development ○下郡智美1, 弓削主哉1, 菊池里美1, 吉田彩1, 禹真美1, ビッキーチュング1, 松居亜寿香1, 益子宏美1, 小川正晴1 ○Tomomi Shimogori1, Kazuya Yuge1, Satomi Kikuchi1, Aya Yoshida1, Mami U1, Chung Vicky1, Asuka Matsui1, Hiromi Mashiko1, Masaharu Ogawa1 理化学研究所　脳センター1 RIKEN BSI, Saitama, Japan1 Correct wiring of neuronal network during the development of the brain is critical matter for its correct function. We therefore looked for developing rodent thalamus-cortex connectivity to reveal its molecular mechanism for correct neuronal circuit formation. We first investigated how the neuronal input from the thalamus changes the cortical area pattern using somatosensory barrel cortex in mouse. We revealed that correct thalamic axon arbor formation and subsequent neuronal input is important for correct barrel formation. To further elucidate molecular mechanism of thalamocortical axon dependent barrel formation, we performed microarray analysis in brain, which does not form the barrel structure. We first transected one side of infraorbital nerves (ION) under the whisker pad at birth and confirmed the barrel structure is lost at postnatal day 6. Next we performed microarray analysis on the barrel less hemisphere and isolated candidate genes. We tested all candidate gene expression by in situ hybridization, and further tested their expression in ION transected barrel cortex. As a consequence, we found 16 molecules that recapitulated microarray result (9, down regulation, 7, up regulation). To further confirm these molecules control barrel development, we employed in utero electroporation to over express or suppress functional protein and found some of the candidate molecules control thalamocrotical patterning, cell aggregation and even control cell identities of barrel septa and hollow. Our results provide molecular framework of the activity dependent barrel cortex development.
P1-2-54 新しい低密度培養法を用いた小脳顆粒細胞におけるGSK3betaの機能解析 Functional analysis of GSK3beta in cerebellar granule neurons by using new method of low-density culture ○久保田健太1, 瀬野岳史1, 小西慶幸1,2 ○Kenta Kubota1, Takeshi Seno1, Yoshiyuki Konishi1,2 福井大院・工・知能システム1, 福井大学生命科学複合研究教育センター2 Department of Human and AI Systems, Univ of Fukui, Fukui1, Research and Education Program for Life Science, Univ of Fukui, Fukui2 [Background] In recent years, the basic molecular systems involved in the polarity formation have becoming clear by studies using the hippocampal pyramidal neurons. However, variety types of neuron are present in brain, and the mechanisms by which these variety neuronal morphologies are established remained to be clarified. Considering that low-density culture method of hippocampal neurons that enable to observe the morphology of individual neurons has largely contributed to the progress this field of study, establishing low-density culture method in other types of neurons would be valuable.[Purpose] In this study, we aimed to establish a new method to maintain the primary culture of cerebellar granule neurons (CGNs) at low-density. In addition, by using this culture system, we analyzed the function of glycogen synthase kinase-3beta (GSK-3beta) in early neuronal morphologies of CGNs. [Result] CGNs require the trophic support for neighboring cells, thus it has been difficult to culture at low-density. We resolved the big issue by co-culturing two different density of CGNs culture so that low-density neuronal culture faces to the high density culture. Immunostaining analysis indicated that the CGN formed a correct polarity in our culture system. When CGNs were treated with lithium chloride (LiCl) or 6-bromoindirubin-3'-oxime (BIO), inhibitor for GSK3beta, the length of axons were decreased. However number of axon was not be altered significantly. Thus, our observations suggest that GSK3beta do not play major role in polarity formation of CGNs.	P1-2-55 コンドロイチン硫酸による軸索伸長と誘導の制御機構 Regulatory mechanism of axon growth and guidance by chondroitin sulfate ○中村真男1, 田村純一2, 北川裕之3, 戸島拓郎1,4, 上口裕之1 ○Masao Nakamura1, Jun-ichi Tamura2, Hiroshi Kitagawa3, Takuro Tojima1,4, Hiroyuki Kamiguchi1 理化学研究所　脳科学総合研究センター　神経成長機構研究チーム1, 鳥取大学　地域学部　地域環境科学科2, 神戸薬科大学　薬学部　薬学科・衛生薬学科3, 科学技術振興機構さきがけ4 Laboratory for Neuronal Growth Mechanisms, RIKEN Brain Science Institute, Saitama, Japan1, Department of Regional Environment, Tottori University, Tottori, Japan2, Department of Biochemistry, Kobe Pharmaceutical University, Kobe, Japan3, PRESTO, Japan Science and Technology Agency, Saitama, Japan4 Chondroitin sulfate (CS) proteoglycans are a family of molecules consisting of a core protein and one or more highly-sulfated CS chains. It is well known that CS polysaccharides are axon growth-inhibitory molecules in the injured central nervous system. Recent studies have identified receptor-protein tyrosine phosphatase σ (RPTPσ) and leukocyte common antigen-related (LAR) as CS receptors. However, intracellular signaling pathways that link these CS receptors with axonal growth cone behaviors remain largely unknown. CS is divided into several subtypes (CS-A, C, D, and E) based on its sulfation pattern. In the present study, we examined the effects of these CS subtypes on growth and guidance of embryonic chicken dorsal root ganglion (DRG) neuronal axons cultured on a laminin substrate. All CS subtypes presented as a substrate inhibited axon growth. Similarly, bath application of CS-D or CS-E solution caused growth cone collapse. Furthermore, application of CS-E gradients through a glass pipette caused growth cone turning away from the CS-E source. This repulsion was abolished by treatment with BAPTA-AM, a cytosolic Ca2+ chelator. Using synthetic CS oligosaccharides, we also identified octasaccharide is the minimum chain length of CS-E for DRG axon growth inhibition. These results indicate that CS chains function as a repulsive factor for axon growth and guidance via intracellular Ca2+ signaling.
P1-2-56 NMDA受容体GluN2Bサブユニットの発現量減少が発達期の皮質脊髄路可塑性のcritical period終了を決定する Closure of the critical period in developmental corticospinal plasticity is determined by the decline of synaptic expression of GluN2B subunit-containing NMDA receptors: biochemical and live imaging study ○磯尾紀子1, 大野孝恵1, 磯脇睦美1, 亀田浩司1, 村部直之1, 三品昌美2, 桜井正樹1 ○Noriko Isoo1, Takae Ohno1, Mutsumi Isowaki1, Hiroshi Kameda1, Naoyuki Murabe1, Masayoshi Mishina2, Masaki Sakurai1 帝京大学　医学部　生理学講座1, 立命館大学2 Dept. of Physiol., Teikyo Univ. Sch. of Med., Tokyo, Japan1, Ritsumeikan Univ.2 In nervous system, once a short time window of developmental plasticity so called "critical period(CP)" is closed, it is no longer re-opened. It is suggested that NMDA receptor(NMDAR) subunit composition is shifted from GluN2B to 2A during development, raising possibility that this shift is involved in the closure of the CP. We previously reconstructed the corticospinal(CS) projection with functional synapses in vitro using the slice co-cultures of the mouse cerebral cortex and spinal cord, where CS axons innervated the entire spinal cord at an early developmental stage and then regressed mostly from the ventral side in an NMDAR-dependent manner. There is CP(6-11 DIV) for this activity-dependent synapse elimination with axonal regression. In this study, we studied through live imaging the developmental dynamics of EYFP tagged channelrhodopsin-labeled CS axons. When the synapse elimination was blocked by APV application during the CP, and thereafter APV was removed, the CS axons on the ventral side were no longer eliminated in wild type mice(WT), while those were eliminated in the GluN2A knockout mice(2AKO) spinal cords. At the end of the CP, the synaptic GluN2B expression levels(synaptic GluN2B) in 2AKO were higher than those in WT spinal cords. However, even after the CP closed, upregulation of the synaptic GluN2B by an mGluR5 antagonist, MTEP induced the synapse elimination in WT. Furthermore, inhibition of the synaptic GluN2B internalization by a casein kinase 2 inhibitor, TBB also resulted in the synapse elimination. Finally, we investigated whether the CP was elongated by suppression of inhibitory-input. Application of a glycine receptor antagonist, strychnine before the end of the CP, also induced the synapse elimination after the CP. These results suggest that the closure of the CP in this type of developmental plasticity is determined by the decline of the synaptic GluN2B.	P1-2-57 プロテオミクス解析によるCaMKKリン酸化基質の同定 Proteomic screening for calcium/calmodulin-dependent protein kinases substrates ○中牟田信一1, 松島彩乃1西岡朋生1, 貝淵弘三1,2 ○Shinichi Nakamuta1, Ayano Matsushima1, Sharmin Aktar1, Tomoki Nishioka1, Kozo Kaibuchi1,2 名古屋大学大学院 医学系研究科 神経情報薬理学1 Department of Cell Pharmacology, Nagoya University, Nagoya, Japan1, JST, CREST, Tokyo2 Polarity is an essential feature of many cell types, including neurons that have structurally and functionally distinct compartments, axon and dendrites. Cytosolic calcium (Ca2+) signals play important roles in the regulation of neuronal morphologies. We have recently reported pivotal roles of inositol 1,4,5-trisphospoate (IP3)-Ca2+-Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) signaling in axon specification (Nakamuta et al., Science Signaling, 2011). However, the downstream of CaMKK signaling remains elusive. We here comprehensively screened CaMKs substrates by treating cortical neurons with the CaMKs activators and inhibitors. The identities of the protein substrates and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the CaMKs inhibitors were regarded as substrates candidates. We identified more than 70 proteins as candidates including regulators of microtubule dynamics. We will show the possible roles of these candidates in axon specification.
P1-2-58 生体内で神経細胞の極性化を制御する微小環境 Microenvironment for Neuronal Polarization in vivo ○難波隆志1, 木部祐士1, 船橋靖広1, 中牟田信一1, 武内恒成2, 貝淵弘三1 ○Takashi Namba1, Yuji Kibe1, Yasuhiro Funahashi1, Shinichi Nakamuta1, Kosei Takeuchi2, Kozo Kaibuchi1 名古屋大学大学院医学系研究科神経情報薬理学講座1, 新潟大学院医歯分子細胞医学分子細胞機能2 Dept. Cell Pharmacol., Nagoya Univ. Grad. Sch. of Med., Nagoya, Japan1, Dept. Med., Niigata Univ., Niigata, Japan2 Neurons are highly polarized cells that have two structurally and functionally distinct compartments, axons and dendrites. In the developing central nervous system, neuron development occurs in a heterogeneous environment composed of extracellular matrices, radial glial cells and neurons themselves. Although many intrinsic factors concerning axon formation have been identified, the microenvironmental cues involved in axon formation remain largely unknown. We show here that the immunoglobulin super family cell adhesion molecules (IgCAMs), previously implicated in the axon fasciculation and guidance, is also required for neuronal polarization in the mammalian cerebral cortex. Knock down of the IgCAM expression by shRNA significantly impairs axon formation. In addition, we found that unpolarized multipolar cells closely contacted with axons of early-born neurons during axon formation. These results suggest that IgCAM-mediated cell adhesion between unpolarized multipolar cells and axons of early-born neurons regulates neuronal polarization in the embryonic cerebral cortex.	P1-2-59 蛍光タンパク発現モデルマウスを用いた小脳抑制性介在ニューロンルガロ細胞の形態学 Morphological analysis of cerebellar interneuron Lugaro cells using with GFP-expressing transgenic mice line ○宮崎太輔1, 田中謙二2, 幸田和久3, 柚崎通介3, 渡辺雅彦1 ○Taisuke Miyazaki1, F Kenji Tanaka2, Kazuhisa Kohda3, Michisuke Yuzaki3, Masahiko Watanabe1 北海道大学大学院　医学研究科　解剖発生学分野1, 慶應大学医学部　精神神経科学2, 慶應大学医学部　神経生理3 Dept Anatomy, Grad Sch Med Hokkaido Univ, Sapporo1, Dept Neuropsychiatry Sch Med Keio Univ, Tokyo2, Dept Physiology Sch Med Keio Univ, Tokyo3 Cerebellar interneuron Lugaro cells (LCs) have fusiform cell bodies and extend horizontal bipolar dendrites beneath the Purkinje cell (PC) layer. Although basic morphological properties of LCs are known from the Golgi impregnation method, detailed neuronal characteristics have been unclear due to the lack of useful neurochemical markers. In the present study, we produced a transgenic mouse line, in which green fluorescent protein (GFP) was specifically expressed in LCs by KENGE (knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction) -tet system. In fluorescent in situ hybridization, most LCs expressed both GAD67 and GlyT2 mRNAs as previously reported, however, a subset of LCs expressed only GAD67 mRNA. In multiple immunofluorescence and double immunoelectron microscopy using with GFP immunolabeling, neuronal tracing, LCs received inhibitory inputs from PC axon collaterals and excitatory inputs from climbing fibers (CFs), mossy fibers and ascending axons of cerebellar granule cells. Furthermore, we demonstrated that LCs formed dendritic meshwork beneath PC layer using cerebellar sections cut parallel with the layer. Interestingly, the extent of dendritic meshwork of given LCs was confined to a certain cerebellar microzone as visualized by aldolaseC immunolabeling, never exceeding the border of microzones. We also investigated projection pattern of LC axons. LCs projected ascending and transverse axons to the molecular layer, where they formed symmetrical synapses with somatodendritic domain of inhibitory interneurons. Especially, ascending axons intensively innervated the soma of basket cells (BCs). Considering that BCs send inhibitory afferents to PCs and PCs in turn do to LCs in the parasagittal plane, these three types of neurons may constitute microzonal inhibitory circuits. Together, these results suggest that LCs receive various cerebellar afferents and construct microzonal local circuitry to regulate the activity of PCs.
P1-2-60 成長円錐旋回運動におけるホスホリパーゼDとホスファチジン酸の機能解析 Functional analysis of phospholipase D and phosphatidic acid in growth cone turning ○肥田友伸1, 富山達也1, 上口祐之1 ○Tomonobu Hida1, Tatsuya Tomiyama1, Hiroyuki Kamiguchi1 理化学研究所　脳総合研究センター　神経成長機構研究チーム1 RIKEN BSI, Saitama, Japan1 Phospholipase D (PLD) is a lipase which catalyzes the hydrolyzesis of phosphatidylcholine to form phosphatidic acid (PA). PA has emerged as a lipid second messenger to regulate cell migration, cell proliferation, and cell differentiation. However, whether PLD-PA signal is involved in growth cone turning has never investigated. In the present study, we examined the role for PLD and PA in growth cone turning using embryonic chick dosal root ganglion neurons. Pharmacological inhibition of PLD significantly inhibited NGF and MAG-induced growth cone attraction but not Sema3A and MAG-induced growth cone repulsion. We next examined whether NGF gradient induced asymmetrical production of PA in growth cones. By using TIRF imaging of PA-binding domain of human Dock2 (PABD) tagged with AcGFP and mRFP, we revealed that NGF gradient induced asymmetrical translocation of PABD to the growth cone membrane. Furthermore, pharmacological inhibition of PLD inhibited the translocation of PABD. We next examined whether asymmetrical production of PA caused growth cone attraction. We revealed that exogenous PA gradient induced growth cone attraction and asymmetrical translocation of PABD to growth cone membrane. We next investigated how PLD and PA regulate growth cone attraction. We have previously reported that Ca2+ signals with CICR trigger growth cone attraction via asymmetric centrifugal vesicle transport and subsequent exocytosis. We revealed that pharmacological inhibition of PLD suppressed NGF-induced exocytosis in growth cones and exogenous PA gradient induced exocytosis in growth cone. Our finding suggests that NGF induces growth cone attraction through asymmetrical production of PA on one side of growth cones membrane in PLD-dependent manner and subsequent exocytosis.	P1-2-61 人工甘味料サッカリンは神経突起伸長を促進する Saccharin promotes neurite extension through enhancing microtubule formation ○室井喜景1, 石井利明1 ○Yoshikage Muroi1, Toshiaki Ishii1 帯広畜産大学1 Dept Basic Vet Med, Univ of Obihiro, Obihiro1 Saccharin is an artificial calorie-free sweetener. Here we report that saccharin promotes neurite extension in the neuronal cell lines. We previously reported that mouse neuroblastoma N1E-115 cells formed neuronal processes under amino acid-free condition. To test whether glucose deprivation also induces neurite outgrowth, we observed the morphology of the cells cultured in glucose-free DMEM. Deprivation of glucose increased the number of the cells bearing neuronal processes in N1E-115 cells. The effect was inhibited by addition of glucose at the concentration of glucose contained in DMEM. The other monosaccharides or sweeteners tested, except saccharin, had no effect on the length of neuronal processes. Saccharin increased the number of the cells bearing longer neuronal processes in a dose-dependent manner. Saccharin also showed the similar effects on PC12 cells treated with nerve growth factor. These results suggest that saccharin may enhance neurite extension regardless of the cells. To determine how saccharin enhances neurite extension, we evaluated the effects of saccharin on microtubule formation after treatment with nocodazole, which destroy microtubule assembly. In both cells, saccharin increased the volume of microtubules formed 30 min after washing out nocodazole. These results suggest that saccharin promotes microtubule formation. We concluded that saccharin potentiates tubulin polymerization to enhance neurite extension.
P1-2-62 Olig2欠損マウスの前脳でみられる神経回路形成の異常 Defects of neural circuit formation in the Olig2 knockout mouse forebrain ○小野勝彦1, 野村真1, 後藤仁志1, 竹林浩秀2, 池中一裕3 ○Katsuhiko Ono1, Tadashi Nomura1, Hitoshi Gotoh1, Hirohide Takebayashi2, Kazuhiro Ikenaka3 京都府立医科大学大学院　医学研究科　神経発生生物学1, 新潟大学大学院　医歯学総合研究科　　神経生物学・解剖学分野2, 自然科学研究機構　生理学研究所　分子神経生理学部門3 Deptf Biol, Kyoto Pref Univ of Med, Kyoto, Japan1, Divi of Neurobiol Anat, Grad Sch Med Dent Sci, Niigata Univ, Niigata, Japan2, Div of Neurobiol Bioinfo, Natl Instit Physiol Sci, Okazaki, Japan3 Olig2 is a transcription factor essential for the generation of oligodendrocyte precursor cells and motor neuron. Olig2 is expressed in the restricted region of the forebrain. However, forebrain-specific function of Olig2 has not been elucidated yet. Especially, it remains elusive whether Olig2 is involved in neural circuit formation in the forebrain. In the present study, we examined developing forebrain of the Olig2-KO mouse. Axon architecture revealed by neurofilament immunohistochemistry is disorganized in the E13.5 thalamus; axons in the wild type dorsal thalamus are extended in parallel to each other along dorsoventral axis, whereas those in the Olig2-KO have winding courses. The axonal disorganization becomes more prominent in the late fetal stages such as E17.5 and E18.5; abnormal thick fasciculi are formed and oriented randomly in the Olig2-KO thalamus. These aberrantly oriented axons are immunoreactive for netrin-G1, and thus they are thalamocortical axons. We next examined topographic reciprocal connections between the cortex and thalamus with lipophilic fluorescent dye. Crystals of DiI or DiA are injected into the frontal, middle and occipital cortices of E17.5 or E18.5 fetal brain. In the wild type, axons labeled from each cortical subdivision extended in segregated courses within the internal capsule, and are arranged in parallel within the thalamus. However, those in the Olig2-KO mouse show overlapping courses. In addition, axons from different cortical subdivisions crossed each other in the thalamus. These observations strongly suggest that Olig2 regulates neural circuit formation between the thalamus and cortex.	P1-2-63 In vivo遺伝子導入法による単一神経細胞からつながる回路の可視化 Visualization of multisynaptic neural pathway connecting from an electroporated single neuron ○飯島友也1, 杉順子1, 杉山清佳1 ○Tomoya Iijima1, Junko Sugi1, Sayaka Sugiyama1 新潟大学 医歯学総合研究科 神経発達研究室1 Lab of Neuronal Development, Graduate School of Medical and Dental Sciences, Niigata University1 Neural circuits are refined by sensory experience in early postnatal life. An excitatory-inhibitory balance modulates a unique time window or 'critical period' for plasticity. A previous study identified the importance of Otx2 homeoprotein for activation of visual cortical plasticity, implicating in particular maturation of Parvalbumin (PV)-cell circuits. Interestingly, Otx2 protein synthesized at the retina and lateral geniculate nucleus (LGN) could transport into the primary visual cortex (V1) along the visual pathway. The experience-dependent transfer of Otx2 prompted us to analyze how this homeoprotein propagates through neuronal circuits. We first attempted to establish the method for in vivo single cell electroporation under a standard stereoscopic microscope. For visualization of synaptic coupling neurons, genes of GFP and wheat germ agglutinin (WGA)-lectin, which encodes a well-known anterograde transsynaptic tracer, were transferred into a single cell in LGN or V1 at 4-week-old mice. Immunostaining revealed that transfected cells contained both exogenous GFP and WGA-lectin. Transsynaptic transports of WGA-lectin were enough to visualize neuronal networks from an originally-electoroporated neuron in visual cortical areas. After electroporation into a layerII/III pyramidal cell, the projection pattern and neuronal circuit were distinct from each neuron, indicating that those pyramidal cells were rather heterogeneous to contribute various cortical processing. Thus, our tracing system may have the advantage of being able to unveil transsynaptic transport of homeoprotein by comparing with WGA-lectin behavior.
P1-2-64 視床軸索分岐におけるNetrin-4-Unc5Bシグナルの役割 Role of Netrin-4-Unc5B Signaling in Thalamocortical Axon Branching ○佐々木健介1, 早野泰史1, 山本亘彦1 ○Kensuke Sasaki1, Yasufumi Hayano1, Nobuhiko Yamamoto1 大阪大院・生命・細胞分子神経生物学1 Grad Sch Frontier Biosci, Osaka Univ, Japan1 During development, thalamocortical (TC) axons form elaborate branches in a lamina-specific and activity-dependent manner. We previously reported that Netrin-4 acts as a branch-promoting factor for TC axons. In the present study, we explored the downstream mechanism of Netrin-4 signaling in axon branching. To investigate Netrin-4 receptor, in situ hybridization for the candidate molecules was performed in 1- to 2-week-old rats, when TC axon branching develops. The results showed that unc5b, dcc and neogenin were strongly expressed in sensory thalamic nuclei, such as the ventrobasal complex of the thalamus and the dorsal part of the lateral geniculate nucleus. To further identify Netrin-4 receptor, binding of Netrin-4-His on HEK293T cells which express the candidate receptors was examined by immunostaining with anti-His-tag antibody. Netrin-4 clearly bound to Unc5B-expressing cells but not Dcc- or Neogenin-expressing cells. We further investigated the function of Unc5B for TC axon branching, using organotypic cocultures of the thalamus and cortex. When thalamic cells were transfected with an unc5b expression plasmid, TC axons formed highly elaborate branches. Conversely, knockdown of Unc5B using an shRNA expression plasmid attenuated TC axon branching. Furthermore, this effect was rescued by co-transfection with shRNA-resistant unc5b. These results suggest that Netrin-4 synthesized by cortical cells promotes axon branching via binding to Unc5B which is expressed in developing sensory TC axons.

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