シナプス Synapse
P2-1-1 シナプス伝達の忠実性はプレシナプス塩化物イオン濃度によって決まる Fidelity of synaptic transmission depends upon presynaptic chloride concentrations ○大山千尋1, 堀哲也1,2, 江口工学2, 高橋智幸1,2 ○Chihiro Ohyama1, Tetsuya Hori1,2, Kohgaku Eguchi2, Tomoyuki Takahashi1,2 同志社院・脳科学1, 沖縄科学技術大学院大学2 Brain Science, Doshisha Univ Grad Sch, Kyoyo1, Okinawa Institute of Science and Technology Grad Univ, Okinawa2 After releasing neurotransmitter, synaptic vesicles are retrieved by endocytosis, and refilled with neurotransmitter to be reused for the next round of synaptic transmission. At glutamatergic presynaptic terminals, vesicles are refilled with glutamate by vesicular glutamate transporters using electrochemical gradient produced by vacuolar-type H+-ATPase. In isolated vesicles, magnitudes of glutamate uptake biphasically depend on presynaptic chloride concentrations ([Cl-]), with less uptake both at low and high concentrations (Naito & Ueda, 1985). Likewise, at the calyx of Held glutamatergic presynaptic terminals, vesicles are optimally refilled at 30mM [Cl-], but refilling is less efficient at lower or higher [Cl-] concentrations (Hori & Takahashi, 2012). To address whether presynaptic [Cl-] influence fidelity of synaptic transmission, we recorded postsynaptic action potentials in response to presynaptic stimulations at 20-200 Hz in a cell-attached mode at the calyx of Held-MNTB synapse. The synaptic fidelity depended upon both the frequency and length of stimulation. When the presynaptic [Cl-] was reduced or elevated, the synaptic fidelity declined. Even at the lowest or highest [Cl-], membrane capacitance change in response to a depolarizing pulse remained unchanged, indicating that [Cl-] has no effect on exocytosis or endocytosis of synaptic vesicles. We conclude that presynaptic chloride concentration affects the fidelity of synaptic transmission via modulating the rate of glutamate uptake into vesicles at the excitatory synapse.	P2-1-2 SNARE膜融合活性のシナプトタグミンによる抑制からのCa2+依存性解除 Ca2+-dependent activation of SNARE-mediated membrane fusion by removing synaptotagmin ○増本年男1, , 大守伊織1, 道上宏之1西木禎一1, 松井秀樹1 ○Toshio Masumoto1, David Tareste2,3, Iori Omori1, Hiroyuki Michiue1, Thierry Galli2,3, Tei-ichi Nishiki1, Hideki Matsui1 岡山大学大学院 医歯薬学総合研究科 細胞生理学1 Department of Physiology, Okayama University, Okayama, Japan1, INSERM ERL U950, Membrane Traffic in Neuronal and Epithelial Morphogenesis, Paris, France2, Institut Jacques Monod, UMR 7592, CNRS, Université Paris Diderot, Paris, France3 During neurotransmitter release, a Ca2+ sensor, synaptotagmin synchronizes vesicle exocytosis that is mediated by SNARE complexes consisting of t-SNAREs and v-SNARE. However, it is still unknown that how Ca2+-bound synaptotagmin triggers SNARE-mediated membrane fusion. To address this, we analyzed the interaction between synaptotagmin and SNARE complexes and its effect on membrane fusion. All of recombinant proteins were expressed in E. coli and purified by chromatography. t-SNARE heterodimer (syntaxin/SNAP-25) and v-SNARE were separately reconstituted into liposomes and incubated at 4°C overnight to allow the proteins to form a trans conformation but not to fuse membranes. A cytosolic fragment of synaptotagmin were incubated with trans-SNARE liposomes at 4°C and subjected to density gradient centrifugation to recover the fragment bound onto liposomes in the top layer. Quantitative immunoblotting analysis using known amounts of recombinants as a standard revealed that the top layer contained 0.7 mol of synaptotagmin and 1.2 mol of SNAP25 per mol of synataxin. The binding of synaptotagmin to the trans-SNARE complex was inhibited to 50% by 1 mM Ca2+ at 37°C. These results indicate that synaptotagmin binds stoichiometrically to the trans-SNAREs in the absence of Ca2+ and these proteins dissociated from each other by Ca2+. Next, we examined effects of synaptotagmin binding on SNARE-mediated fusion by fluorescence dequenching-based fusion assay using fluorophores NBD and rhodamine. When fluorescently labeled v-SNARE liposomes were incubated with unlabeled t-SNARE liposomes, the NBD fluorescence increased, indicating the fusion between v- and t-SNARE liposomes. Synaptotagmin inhibited SNARE-mediated liposome fusion by 65% in the absence of Ca2+. This inhibition was partially (50%) reversed by 1 mM Ca2+. Thus, we conclude that synaptotagmin initially binds to trans-SNARE complexes and acts as a clamp preventing SNARE-mediated spontaneous fusion until Ca2+ influx.
P2-1-3 シナプス接着分子LRRTM2の細胞内領域による細胞表面および樹状突起への局在制御 The cytoplasmic domain regulates the cell surface trafficking and dendritic targeting of a synaptic cell adhesion molecule, LRRTM2 ○湊原圭一郎1, 藤吉好則2, 土井知子1 ○Keiichiro Minatohara1, Yoshinori Fujiyoshi2, Tomoko Doi1 京都大院・理・生物物理1, 名古屋大・細胞生理学研究センター2 Dept. Biophys., Kyoto Univ., Kyoto, Japan1, Cellular and Structual Physiology Institute, Nagoya Univ., Nagoya, Japan2 Synaptically localized adhesion molecules, such as neurexins and neuroligins, play important roles in the formation, maturation and function of the synaptic connection. Our previous proteomic analyses have found out leucine-rich repeat transmembrane proteins (LRRTMs), highly enriched in the postsynaptic density (PSD) fraction of the rat forebrain. Recent studies reveal that LRRTMs bind to presynaptically localized neurexin-1β and induce synapse formation as neuroligin does. The mechanism involved in the synaptic targeting of LRRTM2, however, remains unclear. To investigate the roles of the LRRTM2 cytoplasmic domain in the synaptic localization, we performed a sequential deletion assay. It turned out that the deletion of 6 residues in the middle of the C-terminus resulted in the increase of LRRTM2 contained in the CHAPS-solubilized post-nuclear supernatant of HEK cells and hippocampal neurons. Biotinylation experiments of intact HEK cells expressing the mutant revealed about 10-fold higher surface level of the mutant than that of wild-type LRRTM2. These data indicated that the deletion of this critical region increased the localization of LRRTM2 in the plasma membrane. In cultured hippocampal neurons, immunostaining of the wild-type myc-LRRTM2 showed the punctate staining colocalized with postsynaptic marker, PSD-95 and the specific localization to the dendrites and soma. However, the deletion of these critical 6 residues caused a diffused staining pattern throughout the cell including the axon. These results suggest that the deleted cytoplasmic region of LRRTM2 is responsible for trafficking to the cell surface and targeting to the dendrite, possibly by common molecular mechanisms.	P2-1-4 シナプス前終末におけるAtg9A陽性膜小胞の準精製と特徴づけ Semi-purification and characterization of Atg9A-positive membranes from presynaptic terminals ○山口隼司1, 七尾友久1, 佐々木光穂1, 小池正人1, 内山安男1 ○Junji Yamaguchi1, Tomohisa Nanao1, Mitsuho Sasaki1, Masato Koike1, Yasuo Uchiyama1 順天堂大院・医・神経生物学・形態学講座1 Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan1 Basic autophagy plays an essential role in the metabolic maintenance of neurons including axons and presynaptic regions. Disruption of autophagy in the axon has been shown to result in axonopathy. However, it is still unknown how autophagosomes are formed in the axon and its terminal because we do not have much information about the precise localization of autophagy machinery proteins. To identify the mechanism of autophagy in the axon and its terminal, we focused on Atg9A which is the only transmembrane protein among Atg proteins. We have previously shown that Atg9A is co-localized with marker proteins for initial segments and axon terminals of Purkinje cells. The present study employed immunoelectron microscopy using ultrathin cryosections and a subcellular fractionation method to identify and characterize Atg9A-positive membranes. Immunoelectron microscopic observations of presynaptic regions in deep cerebellar nuclei obtained from adult mouse brains found that gold particles indicating Atg9A were often localized on membranes of synaptophysin-positive vesicle-like structures, which were relatively small in size and sometimes tubular in profiles, compared to synaptic vesicles. Moreover, subcellular fractionation experiments were performed using normal adult mouse brains to purify the Atg9A-positive vesicles in axon terminals. Western blot analyses showed that protein bands corresponding to Atg9A and LC3-II were enriched in LP2 fractions, which richly contained presynaptic membrane structures such as synaptic vesicles. Indeed, immunoelectron microscopy revealed that abundant small vesicular structures labeled by gold particles for Atg9A were present in the LP2 fractions. These results suggest that use of Atg9A-positive vesicles from the presynaptic region may be a powerful tool to identify autophagy machinery proteins required for autophagosome formation in the axon.
P2-1-5 アファディンの海馬神経細胞におけるシナプス形成への関与 Afadin is involved in synaptogenesis of hippocampal neurons ○丸尾知彦1,3, 豊嶋大作1,3, 萬代研二1,3, 富樫英1,3, 井上貴仁1,3, 山本昆明1,3, 三好淳3,4, スプリヤントイルワン2,3, 森正弘2,3, 高井義美1,3 ○Tomohiko Maruo1,3, Daisaku Toyoshima1,3, Kenji Mandai1,3, Hideru Togashi1,3, Takahito Inoue1,3, Hideaki Yamamoto1,3, Jun Miyoshi3,4, Irwan Supriyanto2,3, Masahiro Mori2,3, Yoshimi Takai1,3 神戸大学大学院医学研究科分子細胞生物学1, 神戸大学大学院医学研究科神経生理学2, JST-CREST3, 大阪府立成人病センター研究所分子生物学部門4 Div Mol Cell Biol Dep of Biochem and Mol Bio, Kobe Univ, Kobe, Japan1, Div Neurophysiol Dep of Physiol Cell Bio, Kobe Univ, Kobe, Japan2, JST-CREST, Kobe, Japan3, Dep Mol Bio, Osaka Med Cent Cancer and Cardiovascul Diseases, Osaka, Japan.4 Mossy fiber-CA3 pyramidal cell synapses in the hippocampus undergo dynamic remodeling, which is implicated in learning and memory. Afadin and its binding cell adhesion molecules (CAMs) nectin-1 and nectin-3 as well as N-cadherin are especially concentrated at these synapses. Although it has been well established that afadin is crucial for the formation of multiple cell-cell junctions between epithelial cells, mechanistic insights into roles of afadin in synaptogenesis have still largely remained elusive. In this study, we investigated the roles of afadin in synaptogenesis in the hippocampus. In the mice of which afadin gene was conditionally ablated before synaptogenesis, the CAMs, nectin-1, nectin-3, N-cadherin, and the synaptic components, such as VGLUT1, bassoon, and PSD-95, were hardly accumulated in the CA3 stratum lucidum. In cultured afadin-deficient hippocampal neurons, the number of mushroom-shaped spines was decreased. The accumulation of CAMs, synaptic molecules, and surface AMPA-type glutamate receptors at afadin-deficient synapses was also impaired. In addition, both pre- and post-synaptic functions were significantly attenuated as estimated by paired patch-clamp recordings and synaptotagmin-antibody uptake assay. These results indicate that afadin is involved in the initiation of synaptogenesis in the hippocampus.	P2-1-6 マウス脊髄におけるGABA排出機構の発達変化 Distinct development of GABA removing system in the ventral and dorsal horns in the mouse spinal cord ○高山千利1, 金正泰1, 小坂祥範1, 清水千草1, 新崎綾1 ○Chitoshi Takayama1, Jeongtae Kim1, Yoshinori Kosaka1, Chigusa Shimizu-Okabe1, Aya Niizaki1 琉球大学　医学研究科　分子解剖学1 Dept Mol Anat, Univ of Ryukyu, Okinawa, Japan1 In the mature central nervous system (CNS), gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter, negatively regulating glutamatergic excitatory transmission. Released GABA is quickly removed from the synaptic cleft by membrane-type GABA transporters. In the present study, to reveal the time-course of the GABA-removing system development, we immunohistochemically examined the localization of GABA transporter-1 (GAT-1) and GAT-3 in the embryonic and postnatal mouse spinal cord. GAT-1 was localized within the GABAergic terminals in the dorsal horn after embryonic day 15 (E15) when GABAergic synapses were formed. In the ventral horn, on the other hand, only few GAT-1-positive dots were detected during development, although numerous GABAergic synapses were transiently formed from E15 to postnatal day 7 (P7). GAT-3 was first localized within processes of the radial glial cells after E12, and became localized within the processes of astrocytes, which sealed the synapses after E17. These results suggested as follows; (1) extracynatpically released GABA was first removed by GAT-3 into the radial glia, (2) at the transient synapse in the ventral horn, GABA was removed by only GAT-3 into astrocytes, (3) GABA was removed by GAT-1 and GAT-3 into presynapses and astrocytes, respectively in the permanent GABAergic synapses.
P2-1-7 神経発達過程におけるNB-2/Contactin-5及びNB-3/Contactin-6とRPTPγの相互作用解析 Interaction of NB-2/Contactin-5 and NB-3/Contactin-6 with RPTPγ during neurodevelopment ○中野悟司1, 渡邉和忠1,2, 霜田靖1 ○Satoshi Nakano1, Kazutada Watanabe1,2, Yasushi Shimoda1 長岡技術科学大学 生物系1, 長岡工業高等専門学校2 Dept Bioeng, Nagaoka Univ Tech. Niigata, Japan1, Nagaoka Natl Coll Tech, Nagaoka, Niigata, Japan2 NB-2/Contactin-5 and NB-3/Contactin-6 are GPI-anchored cell adhesion molecules belonging to the immunoglobulin superfamily. We have shown that NB-2 and NB-3 were localized at the presynaptic terminals and that the number of glutamatergic synapses was reduced in NB-2 KO and NB-3 KO mice. NB-2 and NB-3 might play an important role in synapse formation, though the mechanism remains unknown. It has been recently reported that the receptor protein tyrosine phosphataseγ (RPTPγ) interacts with NB-2 and NB-3 in vitro. Both NB-2 and NB-3 have a neurite outgrowth-promoting activity. By contrast, it was reported that RPTPγ inhibits NGF-stimulated neurite outgrowth in PC12D cells. Then we hypothesize that the interaction of NB-2/NB-3 with RPTPγ might regulate neurite outgrowth. Here we examined co-localization of NB-2/NB-3 and RPTPγ in the mouse brain. First, immunohistochemical staining showed that RPTPγ was localized in the cerebral cortex, hippocampus, thalamus, inferior colliculus and cerebellum, where NB-2 and NB-3 are known to be localized. Next, biochemical analysis revealed that NB-2/NB-3 and RPTPγ were enriched in the pre-synaptic membrane fraction which was isolated from the synaptosomal fraction of developing brain. Furthermore, immunofluorescence of hippocampal and cortical primary neuron showed that RPTPγ was localized at the presynaptic sites. These results suggest that NB-2/NB-3 and RPTPγ might form cis complexes in the developing brain. We are now examing whether NB-2 and NB-3 have any effects on RPTPγ-dependent regulation of neurite outgrowth.	P2-1-8 グルタミン酸イメージング技術を用いた神経伝達物質放出の分子基盤の解析 Analyzing Molecular Basis Underlying Neurotransmitter Release by Glutamate Imaging Technique ○金原直也1, 坂本寛和1, 太向勇1, 並木繁行1, 廣瀬謙造1 ○Naoya Kimpara1, Hirokazu Sakamoto1, Isamu Taikou1, Shigeyuki Namiki1, Kenzo Hirose1 東京大学大学院医学系研究科神経生物学教室1 Dept. Neurobiol., Univ. of Tokyo, Tokyo1 Neurotransmitter release from presynaptic terminals is essential for information processing in brain. Neurotransmitter is released from refined area called active zone which is composed of a large protein complex containing active zone proteins such as Bassoon, Piccolo, Munc13, Rim1, Erc1 and RBPs. Although these molecules are suggested to be important for neurotransmitter release, how these molecules regulate neurotransmitter release is not fully understood due to technical limitation in precise quantification of released neurotransmitter. In this study, we aimed to understand how active zone proteins regulate neurotransmitter release by developing a new technique for evaluating released neurotransmitter. In this technique, we measured glutamate released from single synapses by fluorescence glutamate imaging technique in cultured hippocampal neurons, and then determined so-called presynaptic parameters including the number of readily releasable vesicles (RRP), release probability. To evaluate the role of active zone proteins in glutamate release, we knocked down active zone proteins and evaluated presynaptic parameters. Through our analysis, we obtained the results below; 1) The number of RRP was reduced by the knockdown of Munc13 or Rim1. 2) Release probability was reduced by the knockdown of Erc1. 3) Both the number of RRP and the release probability was reduced by the knockdown of RBPs. 4) Neither the number of RRP nor release probability was changed by the knockdown of Bassoon or Piccolo. From these results, we conclude that each active zone protein has their own spectra in regulating presynaptic parameters.
P2-1-9 光学測定法によるマウス扁桃体外側核の神経応答に対するカンナビノイド類の作用の解析 Effects of cannabinoids on the neuronal responses in the mouse amygdala analyzed by voltage-sensitive dye imaging ○原宏士朗1,2, 藤枝智美1,3, 入江智彦1, 三輪秀樹4,5, 岡淳一郎2, 白尾智明3, 花尻(木倉)瑠理6, 合田幸広6, 栗原正明7, 関野祐子1 ○Kojiro Hara1,2, Tomomi Fujieda1,3, Tomohiko Irie1, Hideki Miwa4,5, Jun-Ichiro Oka2, Tomoaki Shirao3, Ruri Kikura-Hanajiri6, Yukihiro Goda6, Masaaki Kurihara7, Yuko Sekino1 国立医薬品食品衛生研究所・薬理1, 東京理科大・薬・薬理2, 群馬大院・医・神経薬理3, 群馬大院・医・遺伝発達行動4, 独立行政法人科学技術振興機構 CREST5, 国立医薬品食品衛生研究所・生薬6, 国立医薬品食品衛生研究所・有機化学7 Div Pharmacol, NIHS, Tokyo1, Fac Pharm Sci, Tokyo Univ of Sci, Chiba2, Dept Neurobiol and Behav, Gunma Univ, Grad Sch Med, Maebashi3, Dept Genet and Behav Neurosci, Gunma Univ, Grad Sch Med, Maebashi4, JST, CREST, Tokyo5, Div pharmacognosy phytochemistry and narcotics, NIHS, Tokyo6, Div Organic Chemistry, NIHS, Tokyo,7 To understand the effects of cannabinoids on emotional behaviors, it is necessary to elucidate how they modulate neuronal responses in the amygdala. Our previous studies with voltage-sensitive dye imaging have shown that the external capsule stimulation induces wide spread of a long-lasting hyperpolarizing signals (LLH), which is mediated by GABA receptors, in the lateral amygdala (LA) of the mouse amygdala slice preparation. In this study, we first investigated the effects of a cannabinoid receptor type 1 (CB1R) agonist WIN 55,212-2 (WIN) on synaptic transmission in the LA. WIN (1 μM) suppressed the amplitude of optical response corresponding to depolarization signals and the LLH to 85.2 ± 3.6% and 48.9 ± 7.3% respectively. The reduction of LLH was from direct effects of WIN on the inhibitory synaptic transmission in the LA. In addition, WIN prolonged the depolarization duration, indicating that WIN can increase the excitation level of neurons in the LA. Further, we performed the same experiments with other CB1R agonists, JWH-018, Δ9-tetrahydrocannabinol (Δ9-THC; a natural origin cannabinoid), and cannabicyclohexanol (CCH; a Δ9-THC mimetic synthetic compound). JWH-018 showed similar effects as WIN. However, CCH and Δ9-THC showed different effects on synaptic transmission in the LA. These four agonists can be divided into two groups according to their chemical structures. Namely, WIN and JWH-018 are synthetic compounds belong to naphthoylindoles and showed similar effects on the synaptic transmissions in the LA, whereas Δ9-THC and CCH belong to another group, and showed different effects on those in the LA. Therefore, it is suggested that those two groups of the CB1R agonists have different structure-activity relationships in the LA.	P2-1-10 セロトニンによるラット前脳基底核コリン作動性ニューロンへのGABA遊離の抑制 Inhibition of GABA release onto cholinergic neurons in the rat basal forebrain by 5-HT ○西條琢真1,2, 籾山俊彦1 ○Takuma Nishijo1,2, Toshihiko Momiyama1 東京慈恵医大・薬理1, 慶應大院・薬・薬理2 Dept Pharm, Jikei Univ Sch of Med, Tokyo1, Dept Pharm, Keio Univ Faculty of Pharm, Tokyo2 The modulatory roles of 5-HT in GABAergic transmission onto basal forebrain cholinergic neurons were investigated in P12-19 rat brain slices using whole-cell patch-clamp technique. Cholinergic neurons in the basal forebrain were identified with Cy3-192IgG injected into the lateral ventricles 3-7 prior to the experiments. Pharmacologically isolated GABAA receptor-mediated inhibitory postsynaptic currents (IPSC) were evoked by focal electrical stimulation within the basal forebrain. Bath application of 5-HT reduced the amplitude of the evoked GABAergic IPSCs in a concentration-dependent manner. A 5-HT1B receptor agonist, CP93129, also suppressed the IPSCs, whereas a 5-HT1A receptor agonist, 8-OH-DPAT, had no effect. NAS-181, a 5-HT1B receptor antagonist antagonized the 5-HT-induced suppression of IPSCs, whereas NAN-190, a 5-HT1A receptor antagonist did not. 5-HT (1 μM) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution. This effect remained unchanged when extracellular Ca2+ was replaced by Mg2+ (5 mM). In the presence of Ca2+ channel blocker, ω-Aga-TK, ω-CgTX, or SNX-482, 5-HT could still inhibit the IPSCs. These findings suggest that activation of presynaptic 5-HT1B receptor on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in a Ca2+-independent manner.
P2-1-11 グルタミン酸作動性シナプス伝達を制御するアセト酢酸誘導体の構造的特徴 Structural features of acetoacetate derivatives regulating glutamatergic synaptic transmission ○門脇敦志1, 勝孝1, 森山芳則2, 井上剛1 ○Atsushi Kadowaki1, Takashi Katsu1, Yoshinori Moriyama2, Tsuyoshi Inoue1 岡山大院・医歯薬・生体分子解析学1, 岡山大院・医歯薬・生体膜生化学2 Dept of Biophys Chem, Grad sch of Med Dent and Pharm Sci, Okayama Univ1, Dept of Membrane Biochem, Grad sch of Med Dent and Pharm Sci, Okayama Univ2 A high-fat, low-carbohydrate (ketogenic) diet has been used to treat intractable epilepsy. The diet treatment increases a ketone body (acetoacetate) in the brain. Acetoacetate regulates vesicular glutamate transporters, reduces glutamatergic synaptic transmission and suppresses epilepsy. However, the effect of acetoacetate on glutamatergic synapses is relatively weak. In this study, we therefore explored structural features responsible for the reduction of the synaptic transmission. We made patch-clamp recording from pyramidal cells in the hippocampal CA1 region, and stimulated fibers of Schaffer collateral to evoke glutamatergic EPSCs. When the ketone group of acetoacetate (β-ketobutyrate) was moved to alpha position, the inhibitory effect on the EPSC amplitude increased. Changing the α-ketobutyrate to α-ketoacetate retained the inhibitory effect. Furthermore, the addition of a phenyl group to the α-ketoacetate increased the inhibitory effect. When the ketone group of the phenylacetate was replaced with several types of halogen groups, the inhibitory effect increased in the following order: a fluoro group, a chloro group, a bromo group, showing that increasing the size of the halogen groups increases the inhibitory effect. In the same manner, the phenylacetate with an ethyl group inhibited the EPSC amplitude more strongly than that with a methyl group. To address how the phenylacetate derivatives inhibit the synaptic transmission, we then recorded miniature EPSCs. The phenylacetate derivatives inhibited the miniature EPSC amplitude more strongly than acetoacetate, suggesting that the phenylacetate derivatives regulate the synaptic vesicles containing glutamate. These results show that the phenylacetate with a relatively bulky functional group at alpha position is essential for the reduction of glutamatergic synaptic transmission.	P2-1-12 小脳顆粒細胞の軸索から神経活動依存的に分泌されるCbln1の動態解析 Time-lapse fluorescent imaging of cultured cerebellar granule cells reveals activity-dependent secretion of Cbln1 from axons ○井端啓二1, 柚崎通介1 ○Keiji Ibata1, Michisuke Yuzaki1 慶應大学　医学部　生理学1 Dept Physiol, Keio Univ, Tokyo1 Cbln1 is essential for formation of synapses between parallel fibers (axons of granule cells) and Purkinje cells in the cerebellum. Cbln1 is secreted from granule cells and binds to its pre- and postsynaptic receptors, neurexin and the delta2 glutamate receptor, respectively. However, how and where Cbln1 is released from granule cells has remained unclear. Here, we showed that Cbln1 proteins were released from parallel fibers in an activity-dependent manner. We expressed Cbln1 tagged with pH-sensitive GFP (super-ecliptic-pHluorin; SEP) in cultured granule cells. A punctate pattern SEP-Cbln1 fluorescence was observed in transfected granule cells. Using pH-modifying reagents, we confirmed that SEP-Cbln1 was localized on the cell surface as well as in intracellular organelles. These puncta were partially colocalized with VAMP2, a synaptic vesicle and dense-core vesicle marker, confirming synaptic localization of Cbln1. In addition, some Cbln1 puncta were localized at non-synaptic sites in axons. Electrical field stimulations induced rapid increases in fluorescence intensities; indicating that SEP-Cbln1 in the acidic compartments was exposed to the neutral extracellular space. This rapid increase in SEP signals was completely blocked in the media containing tetrodotoxin, It was also significantly inhibited by chelating extracellular calcium by addition of EGTA. These results indicate that Cbln1 was secreted from acidic vesicles in an activity- and calcium-dependent manner. Since cerebellar granule cells fire at high frequencies up to several hundred Hz in vivo, endogenous Cbln1 may be abundantly present on parallel fibers to maintain synaptic connection with Purkinje cells.
P2-1-13 シナプス前終末でのリサイクリングプールサイズの算出 Estimation of the recycling pool size at the calyx of Held presynaptic terminal ○堀哲也1,2, 赤羽正紀1, 高橋智幸1,2 ○Tetsuya Hori1,2, Masaki Akahane1, Tomoyuki Takahashi1,2 同志社大学　脳科学研究科　神経生理学1, 沖縄科学技術大学院大学　細胞分子シナプス機能ユニット2 Dept Neurophysiol, Doshisha Univ Fac Life and Med Sci, Kyoto1, Cellular and Molecular Synaptic Function Unit, Okinawa Inst Sci Tech Grad Univ2 At the presynaptic terminal, after exocytosis of synaptic vesicles, fused vesicle membranes are re-internalized by endocytosis, refilled with neurotransmitter and recycled for reuse. For a sustained trans-synaptic informational flow, it is essential to maintain the recycling pool of vesicles, but the mechanism regulating this pool size is unclear. In hippocampal autaptic culture, Ikeda & Bekkers (2009) estimated the size per synaptic bouton as 100, using the vacuolar ATPase blocker bafilomycin A1 (BafA1). This number is comparable to that per active zone (AZ) estimated using GTPγS at the calyx of Held of rats (Yamashita et al, 2005). Vesicle refilling can be blocked by BafA1 or whole-cell washout of glutamate at the calyx of Held presynaptic terminal (Hori & Takahashi, 2012). We asked whether the recycling pool size is regulated by presynaptic activity. Bath-application of BafA1 or washout of glutamate gradually reduced the amplitude of evoked EPSCs (eEPSCs), and the frequency of miniature EPSCs (mEPSCs) in parallel. The reduction rate of eEPSCs comprised both the time-dependent and use-dependent components, the former likely caused by a passive leakage of glutamate from vesicles, and the latter by recycling of non-refilled vesicles. The recycling pool size, estimated by dividing the time integral of eEPSCs by that of a mEPSC, was 28/AZ at 0.02 Hz and 526/AZ at 20 Hz stimulations. Neither BafA1 nor glutamate washout affected presynaptic membrane capacitance change. We conclude that neuronal activity can regulate the pool size of recycling vesicles.	P2-1-14 シナプス小胞内へのグルタミン酸取込み機構における小胞内バッファリングキャパシティの重要性 Important features of luminal buffering capacity of the synaptic vesicles for understanding the mechanism of vesicular glutamate transport ○江頭良明1, 相川義勝1, 高瀬美樹1, 高森茂雄1 ○Yoshihiro Egashira1, Yoshikatsu Aikawa1, Miki Takase1, Shigeo Takamori1 同志社大院・脳科学・神経膜分子機能1 Lab. of Neural Memb. Biol., Grad. Sch. of Brain Sci., Doshisha Univ., Kyoto1 Glutamate uptake into synaptic vesicles (SVs) depends on the H+ electrochemical gradient (Δψ and ΔpH) generated by the vacuolar-type H+-ATPase, but its precise mechanism remains controversial. It has been suggested that glutamate uptake mainly depends on Δψ since biochemical studies in the purified SVs showed that vesicle acidification is resulted from but does not precede glutamate uptake. Here we tested whether SVs acidification is indeed associated with glutamate loading in the physiological condition by using cultured neurons expressing synaptophysin-pHluorin. We compared vesicle reacidification kinetics and resting vesicular pH between WT- and VGLUT1-KO synapses and found that glutamatergic vesicles can be acidified with similar kinetics both in the presence and absence of glutamate loading, even though an accumulation of glutamate sets the basal vesicular pH slightly lower (˜0.2 pH unit). To reconcile previous biochemical observations and present results, we measured a buffering capacity (BC) of the SVs, which can affect the magnitude of the vesicle acidification monitored by acridine orange that has been used in biochemical studies. We found that BC of VGLUT1-KO SVs was much smaller than those of WT- and heterozygous-SVs, whereas the difference was largely reduced when glutamate was depleted from the SVs. This implies that an increase in luminal BC during glutamate uptake can causes an influx of more H+ by secondarily activating the V-ATPase, thereby facilitating the change in acridine orange signal which might not precisely reflect changes in luminal pH. Moreover, glutamate uptake assay using VGLUT1-liposomes revealed that VGLUT activity mostly depended on buffering condition inside liposomes but not Δψ, supporting the unanticipated notion that ΔpH is the major driving force for glutamate uptake. Taken together, our present study suggests that glutamate is transported into the SVs according to ΔpH that is kept largely constant during glutamate uptake.
P2-1-15 ラット側坐核内抑制性局所神経回路におよぼすコリン作動性修飾 Muscarinic suppression of inhibitory synaptic transmission among medium spiny neurons in rat nucleus accumbens ○山本清文1, 鰕原賀子1,2, 越川憲明1, 小林真之1 ○Kiyofumi Yamamoto1, Katsuko Ebihara1,2, Noriaki Koshikawa1, Masayuki Kobayashi1 日本大学歯学部薬理学教室1, 日本大学歯学部摂食機能療法学教室2 Department of Pharmacology, Nihon University School of Dentistry1, Department of Dysphagia Rehabilitation, Nihon University School of Dentistry2 Medium spiny neurons (MSNs) in the nucleus accumbens (NAc) project to adjacent MSNs, and form local circuits of lateral inhibition in the NAc. Cholinergic interneurons in the NAc play important roles in regulation of neural activities of MSNs (Ebihara et al., 2013). However, it is still unknown how acetylcholine modulates inhibitory synaptic transmission from MSNs to MSNs. To elucidate the mechanisms of cholinergic modulation of MSN to MSN connections, we performed paired whole-cell patch-clamp recordings from MSNs in rat NAc shell of slice preparations, and recorded unitary inhibitory postsynaptic currents (uIPSCs). Bath application of carbachol (10 μM) suppressed uIPSCs in amplitude by ~44%. The paired-pulse ratio and failure rate of uIPSCs were increased from 0.86 to 1.50 and 32.0% to 59.4%, respectively. Therefore, it is likely that acetylcholine reduces release probability of GABA from synaptic terminals of MSNs. The suppressive effects of carbachol on uIPSCs were antagonized by preapplication of atropine, and pilocarpine (1-10 μM) mimicked the carbachol-induced suppression of uIPSC, suggesting that acetylcholine suppresses GABA release from MSNs via muscarinic receptors. The uIPSC suppression by presynaptic muscarinic receptors in MS neurons may potentiate GABAergic outputs from the NAc by suppressing lateral inhibition.	P2-1-16 ラット大脳新皮質におけるnon-NMDA受容体活動に依存する20Hzオシレーション Non-NMDA receptor activity-dependent oscillations at the frequency of 20 Hz in the neocortex of rats ○吉村弘1,2, 須貝外喜夫2, 姚陳娟1, 長谷川敬展1, 赤松徹也1, 加藤伸郎2 ○Hiroshi Yoshimura1,2, Tokio Sugai2, Chenjuan Yao1, Takahiro Hasegawa1, Tetsuya Akamatsu1, Nobuo Kato2 徳島大・院・ヘルスバイオ・口腔分子生理1, 金沢医大・医・生理2 Dept Mol Oral Physiol, Inst Health Biosci, Univ Tokushima Grad Sch, Tokushima, Japan1, Dept Physiol, Kanazawa Med Univ, Ishikawa, Japan2 NMDA receptor and non-NMDA receptor play key roles in synaptic transmission in the brain. Previous studies revealed that synaptic efficiencies of these receptors are enhanced by caffeine that acts as a stimulant of the brain. We have found that neocortical neurons have the ability to generate membrane potential oscillations at the frequency of 8-10 Hz, by using rat brain slices, applying caffeine to extracellular medium. The caffeine-assisted oscillations required NMDA receptor activities. In the present study, we investigated how non-NMDA receptor activities participate in generation of the oscillations. Field potential recordings were made from layer II/III of the visual cortex slices. Low-frequency electrical stimulation under caffeine-application elicited long-lasting postsynaptic response, and oscillations at the frequency of 8-10 Hz were emerged at late phase of the response. The oscillatory phases were NMDA receptor-activity dependent, since blockade of NMDA receptor by either application of D-AP5 or increase in extracellular concentration of magnesium abolished the oscillatory phase. During the process of disappearance of the oscillation by blockade of NMDA receptor, oscillations at the frequency of 20 Hz began to appear at early phase of the response. The 20 Hz oscillations were abolished by application of CNQX, showing that 20 Hz oscillations were dependent on non-NMDA receptor activities. These results suggest that non-NMDA receptor activity-dependent early phase oscillation may contribute early phase depolarization of membrane potential, resulting in reduction of magnesium block and generation of NMDA receptor activity-dependent oscillations.
P2-1-17 ビオチン標識ω-Aga IV A トキシン結合を用いて透過型電子顕微鏡で可視化したCalyx of Held シナプスにおけるP/Q-type Ca2+ チャネル P/Q-type Ca2+ channels visualized with biotinylated ω-Aga IV A toxin in transmission electron microscopy at the calyx of Held synapse ○中西節子1, 高橋智幸1 ○Setsuko Nakanishi1, Tomoyuki Takahashi1 沖縄科学技術大学院大学　細胞分子シナプス機能ユニット1 Cellular & Molecular Synaptic Function Unit, Okinawa Institute of Science and Technology Graduate University1 The calyx of Held is a giant axosomatic synapse in the brainstem, functioning as a fast auditory relay. Transmitter release at this synapse depends entirely upon P/Q-type Ca2+ channels after hearing onset. To elucidate molecular/structural mechanisms underlying presynaptic functions, it is essential to identify the localization site of P/Q-type Ca2+ channels. Recently developed P/Q-type Ca2+ channel antibody has been used for freeze fracture replica EM, but is not usable for thin section transmission EM studies. &omega-agatoxin IVA, is a peptide toxin from funnel-web spider venom, which specifically binds to P/Q type Ca2+ channels' extracellular S3-S4 loop of domain IV of Cav2.1 subunit, thereby blocking P/Q-type Ca2+ channel-dependent functions. We have examined whether &Omega-Agatoxin IV A specifically binds to Cav2.1 in the chemically fixed tissues using biotinylated-&omega-Agatoxin IV A and visualized with nanogold labeled streptavidin in transcardialy fixed brainstem slices. The ultrastructure of &omega-Agatoxin IV A binding sites were compared with immunoelectron microscopy using a specific antibody to the &alpha-1A subunit of voltage-gated calcium channels. &omega-Agatoxin IV A binding sites visualized with nanogold labeled streptavidin localized on the outer presynaptic plasma membrane at the active zone with intervals of 40 - 60 nm. Immunoelectron microscopy gave a similar localization pattern. Thus, this histochemical method of labeling Cav2.1 will provide a new tool for studying the molecular architecture of presynaptic terminals.	P2-1-18 多細胞高速カルシウムイメージング法を用いた線条体 GABA 作動性ニューロンの機能的神経結合の検討 The functional neuronal connections of the striatal GABAergic neurons were revealed by the multicellular fast calcium imaging ○菊地琴美1, 田村篤史2,3, 森一生3, 八尾寛1,2, 柳川右千夫2,4, 小山内実2,3 ○Kotomi Kikuchi1, Atsushi Tamura2,3, Issei Mori3, Hiromu Yawo1,2, Yuchio Yanagawa2,4, Makoto Osanai2,3 東北大院・生命科学・脳機能解析1, 東北大院・医・医用画像工学3, 群馬大院・医・脳神経発達統御4 Tohoku Univ. Grad. Sch. Life Sci. Sendai, Japan1, JST CREST, Tokyo, Japan2, Tohoku Univ. Grad. Sch. Med. Sendai, Japan3, Gunma Univ. Grad. Sch. Med. Maebashi, Japan4 The striatum receives excitatory inputs from the cortex and sends inhibitory outputs to the other nuclei of the basal ganglia. The striatum consists of the GABAergic projection neurons (medium spiny neuron, MSN), GABAergic interneurons and cholinergic interneurons, and more than 95% neurons are GABAergic. However, the functional neuronal connections of the striatal neurons had not been clear. Therefore, we measured the multicellular spontaneous activities of the striatal GABAergic neurons by the multicellular fast calcium imaging. To identify the GABAergic neurons, we used GAD67-GFP knock-in mouse. To clarify the contribution of the cortical inputs to the striatum, we used the two types of the slice preparations, the cortico-striatal slice and the striatal slice. Previously, we reported that the frequency of activities of the GABAergic neurons was about 0.005 Hz under the control condition. However, the recording time was too short to improve reliability of recording data in the previous report (1-2 minutes). Thus, in this study, we extended the recording time by 10 minutes in single recording period. Since the frequencies of activities of the GABAergic neurons were increased in the 10 minutes recording condition in both the cortico-striatal and the striatal slice, some activities were missed due to the short recording time in the previous study. In the condition of perfusing Mg2+ - free, 5 mM K+ saline, the activities of the GABAergic neurons in the striatal slice were increased than those of the cortico-striatal slice. This result suggested that MSN might receive the excitatory input and the inhibitory input, simultaneously. Thus, a same pyramidal neuron or pyramidal neurons which exhibit synchronous activity innervated both MSN and the interneuron which made inhibitory synaptic connection to that MSN.
P2-1-19 生理的な感覚刺激や脳幹光刺激による脊髄GABAニューロン賦活化のin vivoパッチクランプ解析 In vivo patch-clamp analysis of spinal GABAergic excitation by naturalistic sensory and optogenetic brain stem stimulation ○古江秀昌1,3, 神野尚三2, 井本敬二1,3 ○Hidemasa Furue1,3, Shozo Jinno2, Keiji Imoto1,3 生理研・神経シグナル1, 九州大院・医・形態機能形成2, 総研大・生命科学3 Dept Information Physiol, NIPS, Okazaki1, Dept Dev Mol Anat, Grad Sch Med Sci, Kyushu Univ, Fukuoka2, Sch Life Sci, Grad Univ Adv Study, Okazaki3 Previous patch-clamp recordings from adult spinal cord slices showed that plastic changes in the chloride gradient of superficial dorsal horn neurons resulted in a reduction in GABA-mediated inhibition in neuropathic pain models. However, the physiological significance of spinal GABAergic transmission in nociceptive modulation remains to be determined. To address this issue we made in vivo analyses of GABAergic spinal responses elicited by naturalistic sensory stimulation or optogenetic stimulation of the locus coeruleus of the brain stem. In vivo whole-cell patch-clamp recordings were made from superficial dorsal horn neurons. Under voltage clamp conditions, superficial dorsal horn neurons exhibited spontaneous inhibitory postsynaptic currents (IPSCs). Cutaneous innocuous touch stimulation elicited a barrage of IPSCs and inhibited action potentials elicited by noxious stimulation. The receptive field for touch-evoked IPSCs was larger than that for noxious stimulation-evoked excitatory responses. Immunohistochemical analysis together with slice patch experiments revealed that small-sized afferent fibers made a direct synaptic contact with spinal GABAergic neurons. A selective activation of locus coeruleus neurons in the brain stem with optogenetic approaches also activated spinal GABAergic neurons. The descending GABA activation was mediated through α1 receptors. The present results suggest that tactile cutaneous stimulation and pontospinal noradrenergic activation increase inhibitory synaptic responses in the superficial spinal dorsal horn to modulate noxious transmission.	P2-1-20 気流応答性ニューロンにおける樹状突起カルシウム応答による方向選択的入力分布の解析 Input-distribution based on directional selectivity of dendritic Calcium responses to wind stimulus ○三谷瑠里子1, 小川宏人2 ○Ruriko Mitani1, Hiroto Ogawa2 北海道大院・生命・生命システム1, 北海道大院・理・生物科学2 Biosystem Sci, Grad Sch Life Sci, Hokkaido Univ, Hokkaido, Japan1, Dept Bio Sci, Fac Sci, Hokkaido Univ, Hokkaido, Japan2 Spatiotemporal pattern of synaptic inputs on dendrites is crucial for processing and integration of sensory information in a single neuron. Recently, in vivo dendritic Ca2+ imaging in cortical neurons has revealed local synaptic activities evoked by various kinds of sensory stimulus. But, relationship between distribution of synaptic inputs on whole dendritic abor and contribution of individual input-site for response property of neurons is still unclear. To address this question, we used wind-sensitive system of the cricket. The receptor organs for this system are two antenna-like appendages called cerci at the rear of the abdomen, covered with about 750 mechanosensory hairs. Each sensory neuron innervating the hair sensillum displays distinct directional selectivity. Eight pairs of projection neurons identified as giant interneurons (GIs) in the terminal abdominal ganglion directly receive excitatory synaptic inputs from the mechanosensory afferents, and convey directional information to the thoracic and cephalic ganglia. The directional selectivity of GIs is formed by integration of the synaptic inputs from mechanosensory afferents, but the relationships between dendritic distribution of the synaptic inputs and their directional selectivity is unknown. In this research, we examine the input-distribution on dendrites of GIs using calcium-imaging technique. Supra-threshold air-current with 200-ms duration induced large Ca2+ increase all over the dendrite, but the Ca2+ response at the distal branch differed in directional selectivity from the voltage response. Further, subthreshold short puff evoking just EPSPs caused Ca2+ transient at local dendritic branch but not at spike initiation zone, which displayed various directional selectivity. These results suggest that the synaptic inputs coding various stimulus directions are distributed on the GI's dendrite and are integrated to form the directional tuning property specific to the each type of GIs.
P2-1-21 小脳抑制性シナプス前末端カルシウムチャネルサブタイプスイッチ機構 A mechanism underlying developmental switch of calcium channel subtypes triggering neurotransmitter release ○三木崇史1, 平井宏和2, 高橋智幸1,3 ○Takafumi Miki1, Hirokazu Hirai2, Tomoyuki Takahashi1,3 同志社大学・生命医科学・神経生理1, 群馬大学院・医・神経生理2, 沖縄科学技術大学院・細胞分子シナプス機能3 Dept Neurophysiol, Doshisha Univ, Kyoto1, Dept Neurophysiol, Gunma Univ, Gunma2, Cellular and Molecular Synaptic Function, OIST, Okinawa3 Neurotransmitter release is triggered by Ca2+ influx through multiple types of voltage-dependent Ca2+ channels (VDCC) in the presynaptic terminal. At many central synapses, during the early postnatal period, N-type channels together with P/Q-type channels mediate transmitter release, but as animals mature, neurotransmission becomes entirely P/Q-type-dependent. The mechanism underlying this developmental switch is not known. To address this question, we have developed a cerebellar slice culture system, where inhibitory transmission from Purkinje cells to neurons in the deep cerebellar nuclei is mediated by N- and P/Q-types. Blocking neuronal activity by adding tetrodotoxin (TTX) into culture media abolished the N-to-P/Q-type VDCC switch. Likewise, reducing extracellular Ca2+/ Mg2+ concentration ratio from 2.3 to 0.3 blocked the VDCC switch. However, co-addition of BDNF with TTX rescued the switch. Furthermore, the TrkB receptor blocker K252a significantly attenuated the switch. We conclude that activation of TrkB receptor by BDNF released by neuronal activity plays a key role in the developmental switch of VDCC subtypes in the presynaptic terminal.	P2-1-22 5-HT2A受容体の活性化による、NMDA受容体依存的な樹状突起スパインからのドレブリンの消失を誘導する 5-HT2A receptor activity induces the disappearance of drebrin from dendritic spines in NMDA receptor dependent manner ○六本木麗子1, 白尾智明1 ○Reiko Roppongi1, Tomoaki Shirao1 群馬大学大学院　医学系研究科　神経薬理学1 Dept Neurobiol&behav, Gunma Univ Grad Sch of Med, Gunma1 Accumulation of drebrin-F-actin complex at postsynaptic sites plays a pivotal role in synaptic plasticity, such as long term potentiation and homeostatic synaptic accumulation of glutamate receptors. We have previously shown that NMDA receptor activation induces rapid disappearance of drebrin from dendritic spines (Sekino et al., 2006). The 5-HT receptors are known to be involved in several psychiatric disorders, and the modulatory role of 5-HT2A receptor in spine morphogenesis has been recently reported (Jones K.A et al., 2009). In this study, we examined the effect of 5-HT2A/2C receptor agonist (±-2,5-dimethoxy-4-iodoamphetamine hydrochloride: DOI) on the accumulation of drebrin at postsynaptic sites using primary cultured neurons prepared from mouse hippocampi. We found that the drebrin cluster density at dendritic spines was not changes in 10 min after DOI (1 μM) treatment, but significantly declined after 15 min. In contrast, the cluster densities of Synapsin I and PSD-95 were not changed either in 15 min after DOI treatment, or later. Western blotting analysis showed that DOI treatment did not affect drebrin expression level in neurons. We then analyzed whether glutamate receptor is involved in the DOI-induced drebrin disappearance or not, because 5-HT2A receptor stimulation enhanced pre-synaptic glutamate release (Aghajanian, G.K. and Marek, G.J. 1999). Since NMDA receptor agonist APV inhibited the DOI-induced drebrin disappearance, it is suggested that 5-HT2A receptor activation induces the disappearance of drebrin-F-actin complex via NMDA receptor activation.
P2-1-23 活性化依存的なグリシン受容体の動的・機能的変化 Activation-dependent modulation of glycine receptor dynamics and function ○中畑義久1,2, 石橋仁1,2, 鍋倉淳一1,2,3 ○Yoshihisa Nakahata1,2, Hitoshi Ishibashi1,2, Junichi Nabekura1,2,3 生理学研究所・生体恒常機能1, 総研大・生命科学・生理科学2 Div. Homeostatic Development, Natl Inst for Physiol Sci (NIPS), Okazaki, JAPAN1, Dept Physiol Sci, School Life Sci, Graduate Univ for Advanced Studies (SOKENDAI), Hayama, Japan2, CREST, JST3 Glycinergic synapses play an essential role in inhibitory neurotransmission in the spinal cord and the brainstem. For the functional neurotransmission, postsynaptic localization of suitable receptors is required. The clustering of postsynaptic glycine receptors is believed to be established at immature stages, with the localization of receptors at postsynaptic sites decreased in the chronic presence of strychnine, a selective glycine receptor antagonist (Levi et al., 1998; Kirsch and Betz, 1998). However, these studies used immunostaining of fixed cultured cells, and hence were unable to investigate the functional state of receptors localized at postsynaptic sites. In the present study, we focused on the dynamics and functional modulation of glycine receptors in the living cells by using live cell imaging and electrophysiological techniques. We applied fluorescence recovery after photobleaching (FRAP) to superecliptic pHluorin (SEP)-tagged glycine receptors expressed in primary cultured neurons (14-35 div) derived from mice spinal cord. The fluorescence recovery rate was higher with chronic strychnine, indicating that glycine receptor localization is less stable under chronic strychnine treatment. To distinguish any contribution of lateral diffusion, we applied quantum dot imaging. The results showed that the mobility of glycine receptors at synapse was higher with chronic treatment of strychnine. However, it was lower than the control level after removal of the antagonist. We also applied electrophysiological patch-clamp technique to correlate these results with the functional glycinergic transmission in individual neurons. The amplitude of miniature glycinergic currents was gradually increased after washing out of strychnine in chronic treatment group, but not in the control group. Overall, our results suggest that the activation of glycine receptor itself regulates receptor dynamics and functional neurotransmission in individual neurons even at matured synapses.	P2-1-24 脊椎動物特異的シナプス接着分子複合体netrin-G1-NGL-1とnetrin-G2- NGL-2による回路選択的シナプス可塑性制御 Trans-synaptic interactions of netrin-G1-NGL-1 and netrin-G2-NGL-2 differentially control synaptic plasticity in selective circuits ○松川浩1, 重本隆一3, 糸原重美1 ○Hiroshi Matsukawa1, Rafael Lujan2, Qi Zhang1, Ryuichi Shigemoto3, Shigeyoshi Itohara1 理研・BSI・行動遺伝1, 脳形態解析・生理研3 Lab for Behav Gene, BSI, RIKEN, Saitama, Japan1, Dept Ciencias Medicas, Univ Castilla-La Mancha, Albacete, Spain2, Div Cereb Struct, NIPS, Okazaki, Japan3 Netrin-G1 and netrin-G2, vertebrate-specific membrane-anchored proteins of the UNC-6/netrin family, preferentially distribute on axons of distinct circuits in the brain, which determines the selective distribution of their receptors (NGL-1 and NGL-2) within specific sub-dendritic segments of target neurons in a cortical layer-specific manner (Akiyoshi-Nishimura et al., PNAS, 2007). The physiologic roles of these trans-neuronal ligand-receptor interactions, however, are poorly understood. Here, we performed electron microscopic and electrophysiologic analyses of the hippocampus, in which netrin-G1-NGL-1 and netrin-G2-NGL-2 interactions occur in a circuit-selective manner, in the CA1 and dentate gyrus regions. Immuno-electron microscopy (iEM) revealed that netrin-Gs and NGLs were localized on the excitatory presynaptic and postsynaptic membranes of the selective circuits, respectively. Mice with deletions of either netrin-G1 or netrin-G2 showed opposing alterations in activity-dependent long-term and short-term synaptic plasticity; i.e., attenuated synaptic plasticity in netrin-G1 knockout (KO), and augmented synaptic plasticity in netrin-G2 KO. Interestingly, the corresponding cognate receptor NGL KO mice showed similar synaptic alterations. iEM provided further suggestive evidence that loss of NGL-1 or NGL-2 disrupted synaptic localization of their ligands, netrin-G1 and netrin-G2, respectively. These results suggest that trans-synaptic netrin-Gs-NGLs interactions play important roles in modulating synaptic efficacy, and that the subcellular localization of netin-Gs and NGLs at presynaptic and postsynaptic membranes, respectively, are initiated and/or maintained in a molecularly inter-dependent manner.
P2-1-25 Phldb2は樹状突起スパインの成熟および化学的LTD誘導後のシナプスでのAMPA受容体のエンドサイトーシスを制御する Phldb2 regulates the maturation of dendritic spines and AMPA receptor endocytosis under chemical long-term depression ○謝敏かく1,2, 八木秀司1,2, 猪口徳一1,2,3, 岡雄一郎1,2,3, 黒田一樹1,2, 柚崎通介4, 松田信爾4, 白尾智明5, 石川保幸6, 佐藤真1,2,3 ○Min-Jue Xie1,2, Hideshi Yagi1,2, Tokuichi Iguchi1,2,3, Yuichiro Oka1,2,3, Kazuki Kuroda1,2, Michisuke Yuzaki4, Shinji Matsuda4, Tomoaki Shirao5, Yasuyuki Ishikawa6, Makoto Sato1,2,3 福井大　医　組織細胞形態学・神経科学領域1, 福井大　生命科学複合研究教育センター2, 福井大　子どものこころの発達研究センター3, 慶應大　医　神経生理4, 群馬大院　医　神経薬理5, 奈良先端大院　バイオサイエンス　神経機能6 Dept Morphol Physiol, Div Cell Biol Neurosci, Univ of Fukui1, Res Edu Program Life Sci, Univ of Fukui2, Res Center Child Mental Dev, Univ of Fukui3, Dept Neurophysiol Sch Med, Univ of Keio4, Dept Neurobiol Behav, Univ of Gunma5, Div Funct Neurosci, NAIST, Nara6 Synapse function and plasticity depend on the morphology of dendritic spine. Dendritic filopodium is highly dynamic structure, known as a premature form of the dendritic spine. Here, we report that phldb2 (pleckstrin homology-like domain, family B, member 2), one binding partner for a well-known actin-cross-linking protein Filamin A, works as a positive regulator of spine maturation. We generated Phldb2-deficient mice and found that a proportion of immature spines (filopodia and thin spines) increased in the hippocampus in vivo, which is consistent with our previous observations with Phldb2 knocked-down cultured hippocampal neurons. We observed high expression of drebrin A, which is a neuron-specific F-actin binding protein known as a molecule that is involved in spine maturation, as well as PSD-95 (postsynaptic density-95) in the hippocampus of our Phldb2-deficient mice. We then confirmed that Phldb2 was capable of interacting with drebrin A and PSD-95. We also observed that the overexpression of Phldb2 suppressed the elongation of dendritic spines induced by drebrin A overexpression in the cultured hippocampal neurons. Furthermore, the mobility of PSD 95 in the spine decreased in the Phldb2 knockout mice. These results suggest that Phldb2 regulates spine maturation via drebrin A and PSD-95. Next, we asked whether or not Phldb2 is involved in synaptic plasticity. To address this issue, we employed a NMDA-induced chemical LTD system and examined the surface/total AMPA receptor 2 (AMPAR2) ratio decreases or not by the LTD induction because the endocytosis of AMPA receptor plays a critical role for synaptic plasticity. The surface/total AMPAR2 ratio did not decrease much in the cultured hippocampal neurons taken from the Phldb2-knockout mice compared with the control. Therefore, it is probable that Phldb2 plays an important role for the maturation of dendritic spines and for the synaptic plasticity.	P2-1-26 FILIP関連分子によるMyosin-IIbを介した神経細胞のスパイン形成の制御とシナプス可塑性における機能解析 FILIP-related molecule controls spine maturation and synaptic function in the hippocampal neuron via non-muscle myosin IIb ○黒田一樹1,2, 八木秀司1,4, 謝敏かく1,2, 岡雄一郎1,2,3, 猪口徳一1,2,3, 佐藤真1,2,3 ○Kazuki Kuroda1,2, Hideshi Yagi1,4, Min-Jue Xie1,2, Yuichiro Oka1,2,3, Tokuichi Iguchi1,2,3, Makoto Sato1,2,3 福井大学　医学部　医学科　形態機能医科学講座　組織細胞形態学・神経科学領域1, 福井大学　生命科学複合研究教育センター2, 福井大学　子どものこころの発達研究センター3, 兵庫医科大学　医学部　解剖学　神経科学部門4 Division of Cell Biology and Neuroscience, Department of Morphological and P hysiological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan1, Research and Education Program for Life Science, University of Fukui, Fukui, Japan2, Research Center for Child Mental Development, University of Fukui, Fukui, Japan3, Department of Anatomy and Neuroscience, Hyogo College of Medicine, Hyogo, Japan4 Dendritic spines are small actin-rich structures and the primary post-synaptic sites of excitatory neurotransmission in the brain. The actin cytoskeleton is essential for spine maturation as well as for synaptic plasticity and memory formation. Non-muscle myosin IIb plays a major role for in the regulation of actin dynamics in dendritic spines. However, how myosin IIb directly alters cytoskeletal dynamics through ATPase-driven contraction of actin networks and how myosin IIb function is regulated the during dendrite spine maturation are still poorly understood. Recently, we found that one FILIP (Filamin A-Interacting Protein)-related molecule, FRM, was a binding partner of myosin IIb and was expressed in the hippocampal and neocortical neurons. When endogenous FRM was knocked down in cultured hippocampal neurons, it inhibited spine shortening for spine maturation and changed the ratio of NMDA receptor expressions on spines. These data suggest that FRM is a new myosin IIb modulator that controls spine maturation and synaptic function in the hippocampus as well as in the cerebral cortex. We observed that FRM is involved in spine morphogenesis and maturation, and showed differential expression compared with FRM in the brain. Considering the importance of actomyosin dynamics during the spine maturation, we hypothesized that FILIP family is capable of modulating myosin IIb activity and regulates spine morphogenesis and synaptic plasticity.
P2-1-27 海馬CA3錐体細胞樹状突起におけるシナプス活動の時空間構造 Spatiotemporal structure of synaptic inputs onto dendrites of CA3 pyramidal neurons ○小林千晃1, 松木則夫1, 池谷裕二1 ○Chiaki Kobayashi1, Norio Matsuki1, Yuji Ikegaya1 東京大院・薬・薬品作用学1 Lab Chem. Pharmacol, Grad Sch, Univ of Tokyo, Tokyo, Japan1 Although dendrites were believed to collect excitatory synaptic inputs linearly from connected neurons and convey information to soma, recent studies have demonstrated that dendrites integrate excitatory synaptic inputs in a nonlinear manner, indicating that neurons process synaptic inputs depending on where and when their dendritic trees receive synaptic inputs. Therefore, the spatiotemporal patterns of synaptic inputs are important for understanding the mechanism of information processing in neural circuits. It has been technically difficult to record the spatiotemporal structure of synaptic inputs. We developed functional multi-spine calcium imaging and visualized synaptic activity into single neurons with high temporal resolution. We imaged dendritic trees in a confocal plane of approximately 150×150 µm2, which covered up to hundreds of spines. We found that dendrites intermittently received synchronized synaptic inputs in CA3 pyramidal neurons. These synchronizations engaged variable sets of spine activities on the same and different dendritic branches. The synaptic input patterns, even though the number of activated synapses is identical, may cause different levels of somatic depolarization.	P2-1-28 母体肥満が子どものシナプス発達に及ぼす影響の解析 Maternal Obesity Impairs Synaptic Development of Mouse Offspring ○畑中悠佑1, 和田圭司1, 株田智弘1 ○Yusuke Hatanaka1, Keiji Wada1, Tomohiro Kabuta1 （独）精神・神経医療研究センター・神経研究所・疾病四部1 Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry1 There are many environmental factors which influence an individual's health. Because of their inevitability throughout life, we focus on maternal and dietary environment. Maternal obesity induces the enhancement of oxidative stress and low concentration of BDNF in the brain of the offspring, as a result, it impairs hippocampal neurogenesis and spatial learning. Nutritional disturbance during fetal stage of offspring seems to be critical for its development, however, these adverse effects are transient during early life of the offspring. In order to detect other effects of maternal obesity in later life of its offspring, here, we focused on the dendritic spine, which is postsynaptic structure and is known to be susceptible to a lot of changes. To investigate the effects of maternal obesity on the dendritic spine of the offspring, we fed mothers with high-fat diet and its offspring with control diet. Using two-photon laser scanning microscopy, we performed in vivo imaging of dendritic spines in the somatosensory cortex, and demonstrated that spine turnover of offspring from dams consumed high-fat diet was higher than that of offspring from control mothers. Because immature spines have high turnover ratio, maternal high-fat diet consumption during critical periods in the development of the fetus might cause developmental delay of the neuronal circuit. Interestingly, this effect on the dynamics of the dendritic spine did not disappear even in later life of the offspring. In addition, when we fed both mothers and its offspring with high-fat diet, the number and the size of the dendritic spine were dramatically decreased. These pathological phenotypes of the dendritic spine became worse with aging. These results suggest that maternal obesity may impair development of the dendritic spine, and high-fat diet intake after birth cause worse effects.
P2-1-29 海馬歯状回における空間情報処理メカニズム Spatio-temporal information processing in hippocampal dentate granule cells ○早川博章1, 上條中庸1, 佐村俊和2, 相原威1,2 ○Hirofumi Hayakawa1, Tadanobu Kamijo1, Toshikazu Samura2, Takeshi Aihara1,2 玉川大学大学院　脳情報研究科1, 玉川大学　脳科学研究所2 Grad School of Brain Sci., Tamagawa Univ.,Tokyo1, Brain Sci. Ins., Tamagawa Univ., Tokyo2 Granule cells in rat hippocampal dentate gyrus have the input though the lateral perforant path (LPP) with non-spatial information, smell etc., and the input though the medial perforant path (MPP) with spatial information, place. Inputs from layer II in the entorhinal cortex (EC) are projected to the lateral dendrite (LD) and medial dendrite (MD) of dentate granule cells though the LPP and the MPP, respectively. A previous study suggested that these synaptic connections have different properties. The pared-pulse facilitation (PPF) was observed at LPP-LD synapse. On the other hand, the pared-pulse depression (PPD) was observed at MPP-MD synapse. In addition, it has been found that memory is improved by the presence of smell information in human brain. Therefore, there is a possibility that the processing of spatial information is influenced by non-specific information in dentate granule. However, it is not clear. First, to investigate the frequency dependence of the information processing on LD and MD in dentate granule cells, input stimuli to LD and MD were applied at different frequencies. As the result, the LD and MD showed sustained and transient responses depending on the stimulus frequency, respectively. Next, data fitting for our physiological experiment was performed using a dynamical synapse model with three states so that suitable parameters of the model were fixed. In addition, Gaussian random inputs or regular inputs were applied to PD or DD in NEURON simulator with the fixed synapse dynamic synapse model. The simulation results showed that MD synapse had high sensitivity of pattern discrimination to the theta burst input, and LD input had enhancement effect on the pattern discrimination at MD. These results suggest that inputs with non-spatial information to DD might increase the membrane potential so that might enhance a temporal-pattern discrimination of spatial (place) information to MD in hippocampal granule cell.	P2-1-30 ラット後部帯状皮質における機能的回路網解析 Optical analysis of functional connectivity in the rat granular retrosplenial cortex ○仁木島健一1,2,3, 黒谷亨2,3, 岡ノ谷一夫1,2,3 ○Ken'ichi Nixima1,2,3, Tohru Kurotani2,3, Kazuo Okanoya1,2,3 東京大学大学院　総合文化研究科　広域科学専攻1, 日本科学技術振興機構ERATO　岡ノ谷情動情報プロジェクト2, 理化学研究所　脳科学総合研究センター 情動情報連携チーム3 Graduate School of Arts and Sciences, The University of Tokyo, Tokyo1, ERATO Okanoya Emotional Information Project, Japan Science and Technology Agency, Saitama, Japan2, Emotional Information Joint Research Laboratory, Riken Brain Science Institute, Saitama, Japan3 The rodent granular retrosplenial cortex (GRS) has dense connections with the anterior thalamic nuclei (ATN) and the hippocampal formation, playing a crucial role in some learnings or memories. Distinctive neurons in the GRS superficial layers has been reported to elicit a late-spiking (LS) firing property and form prominent dendritic bundles, co-localizing with patches of ATN terminations in layer 1a. However, previous researches mainly addressed to the anatomical aspects and the detailed functional connectivity remains still unclear.In this study, we conducted optical recordings of the neural activities from rat GRS slices, induced by the electrical stimulations (layers 1, 2, 5 of the GRS, and the subiculum), using voltage sensitive dyes (Di-4-ANEPPS). Layer 1a stimulation first evoked signal transmission to layers 2-4 and then to layer 5, and the horizontal signal propagation within layers 2-4 (and 5). In contrast, the subiculum stimulation induced neural responses mainly in the GRS deep layers rather than superficial layers. Simultaneous extracellular recordings of field potentials suggested that the major components of optical signals attributes to the (poly)synaptic responses, and they were extinguished by the perfusion of glutamate antagonists (DNQX, DL-APV). Interestingly, even after purfusing glutamate antagonists, monosynaptic inhibitly responses could be detected mainly within layers 2-4, which were abolished by the application of 20μM Bicuculline.These results suggest that signal inputs from the ATN to GRS layer1a were transmitted to layer 5 neurons, relayed by superficial neurons even including LS neurons. Also, horizontal connections within these layers might produce polysynaptic signals, which enables GRS deep layer neurons to integrate the modified signal inputs from the ATN with the direct signal inputs from the subiculum. Such function of the GRS circuits can be helpful to encode a various time delays for some forms of association learnings.
P2-1-31 ヒストン脱アセチル化酵素阻害剤とallopregnanoloneの樹状突起スパインに対する影響 Effect of histone deacetylase (HDAC) inhibitor and allopregnanolone on dendritic spine ○清水英雄1, 石塚佑太1, 高木瑛子1, 白尾智明1 ○Hideo Shimizu1, Yuta Ishizuka1, Eiko Takagi1, Tomoaki Shirao1 群馬大学大学院医学系研究科神経薬理学1 Dept Neurobiol & Behav, Gunma Univ Grad Sch Med1 It is known that various stresses, such as physical stress and psychological stress, cause neurological disorders including depression. Although the molecule mechanism of pathogenesis of these disorders is not yet clarified, the impairment of synapses is thought to be common pathological change of these disorders. Recent studies reported that administration of suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, to depression model mice modulated susceptibility and adaptation to stress; suggesting HDAC activation is involved in the synapse impairment. Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is a derivative of progesterone that is produced in both the periphery and the central nervous system (CNS) via enzymatic conversions of progesterone. APα has been shown to be stress sensitive in rat models, with CNS levels rising quickly following acute stress. In this study we examined whether HDAC and APα are involved in the impairment of dendritic spine function. We prepared primary hippocampal culture according to Banker's method, and then treated the cultured neurons with SAHA. At 6 hours after the treatment, the cells were fixed and immunostained with the antibodies against synaptic protiens, such as Syanpsin I, a presynaptic marker; drebrin, a postsynaptic actin cytoskeleton marker; and PSD-95, a postsynaptic density marker. Then we measured the cluster density of these proteins along dendrites. The cluster of each marker was defined as a round staining region with a peak fluorescent level that was twofold greater than the averaged fluorescent level of the dendrites. 1 µM SAHA treatment for 24 hours decreased number of drebrin clusters, but not 0.3 µM. On the other hand, 1 µM SAHA treatment for 6 hours had no effect on number of these clusters. We then analyzed the effect of APα on the dendritic spines. APα treatment for 48 hours increased the number of drebrin clusters. This study suggests that allopregnanolone can be one of the biomarker of depression.	P2-1-32 DCLKは樹状突起の成長を促進して興奮性シナプスの成熟を抑制する Doublecortin-like kinase enhances dendritic remodeling and negatively regulates synapse maturation ○柏木有太郎1, 申義庚1, 栗生俊彦2, 岩崎広英1, 田中輝幸3, 古泉博之4岡部繁男1 ○Yutaro Kashiwagi1, Euikyung Shin1, Toshihiko Kuriu2, Hirohide Iwasaki1, Teruyuki Tanaka3, Hiroyuki Koizumi4, Joseph G Gleeson4, Shigeo Okabe1 東京大院・医・神経細胞生物1, 徳島文理大・香川薬・病態生理学2, 東京大院・医・発達医科学3, カリフォルニア大サンディエゴ校・医・神経遺伝4 Dept Cellular Neurobiol, Univ of Tokyo, Tokyo1, Dept Neurophysiol, Sch Pharmaceutical Sci, Tokushima Bunri Univ, Kagawa2, Dept Dev Med Sci, Univ of Tokyo, Tokyo3, Dept Neurogenetics, UCSD, USA4 Doublecortin-like kinases (DCLKs) are chimeric proteins of the N-terminal Doublecortin (DCX)-like domain and the C-terminal Ca/CaM-dependent protein kinases (CaMKs)-like domain. Our previous works revealed that DCLK1 enhances dendritic growth and suppresses postsynaptic functions in cultured hippocampal neuron. These results suggest that coordination of dendritic growth and synaptogenesis by DCLKs may play an important role in tuning the balance between network remodeling and stabilization of connectivity in postnatal brain. To elucidate the roles of DCLKs in vivo, we first studied the effects of DCLK gene elimination by knockout or RNAi-based knockdown. Pyramidal neurons in DCLK1 knockout mice and those expressing DCLK1 shRNA showed reduced dendritic complexity and larger dendritic spines. We next tested whether further deletion of DCLK2 may have an additive effect on both enhancement of dendritic complexity and inhibition of spine growth. When DCLK1 was downregulated by shRNAs, absence of DCLK2 did not induce further reduction of dendritic complexity. However the shRNA effect on spine morphology was further enhanced in the absence of DCLK2. These results indicate that suppression of spine growth is DCLK-dependent and suggest that the dosage of multiple DCLK genes contributes to spine volume regulation.
P2-1-33 ニワトリ胚毛様体神経節杯状シナプス前終末における光遺伝学的操作・計測 Optogenetic probing and manipulation of the calyx-type presynaptic terminal in the embryonic chick ciliary ganglion ○江川遼1, 細島頌子1,2,5, 侯旭濱4, 加藤秀理1,2, 石塚徹1,2, 仲村春和1, 八尾寛1,2,6 ○Ryo Egawa1, Shoko Hososhima1,2,5, Xubin Hou4, Hidetaka Katow1,2, Toru Ishizuka1,2, Harukazu Nakamura1, Hiromu Yawo1,2,6 東北大学大学院 生命科学研究科 脳機能解析分野1, 戦略的創造研究推進事業2, 東北大・国際高等研究教育機構3, 新潟大・医歯学4, 日本学術振興会5, 東北大・脳神経科学コアセンター6 Dept Dev Biol and Neurosci, Tohoku Univ Grad Sch of Life Sci, Sendai1, JST, CREST, Tokyo2, Tohoku Univ IAREO, Sendai3, Dept of Med and Dent Sci, Niigata Univ, Niigata4, JSPS, Tokyo5, Center for Neurosci, Tohoku Univ Grad Sch of Med, Sendai6 The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite of the importance of the recent optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods have been difficult to target specifically the calyx-type presynaptic terminals. Here, we evaluated the method to manipulate the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of Edinger-Westphal nucleus with EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and found dozens of the presynaptic terminals and axons in CG at E8-14 were labeled with EGFP fluorescence. When the Brainbow construct that contains membrane tethered fluorescent proteins was introduced together with Cre expression construct, the color coding of each presynaptic axon facilitated dissociation of inter-tangled projections during developmental re-organization period of synaptic connections. With the simultaneous expression of one of chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system that removes the obstacles hindered the molecule-based study of the calyx-type presynaptic terminal would provide an experimental platform to unveil the molecular mechanisms underlying the morphology, physiology and development of synapses.	P2-1-34 The neurotrophin–3 receptor TrkC mediates structural and functional development of calyceal giant synapses in dissociated culture of auditory brainstem neurons ○Dimitar Dimitrov1, Hiroshi Takagi1, Laurent Guillaud1, Setsuko Nakanishi1, Naoto Saitoh2, Tomoyuki Takahashi1,2 Cellular & Molecular Synaptic Function Unit, OIST Graduate University, Okinawa, Japan1, Laboratory of Molecular Synaptic Function, Department of Neurophysiology, Faculty of Life and Medical Sciences, Graduate School of Brain Sciences, Doshisha University, Kyoto, Japan2 Synaptic function progresses in three steps–initial contact, adhesion, and organization of presynaptic and postsynaptic proteins towards functional maturation. A growing body of evidence in the last decade indicates that each of these steps is mediated by signaling molecules and their receptors in the synaptic cleft. We have recently developed the calyx of Held–like giant synapse in a culture system of neurons dissociated from the MNTB and aVCN regions of the auditory brainstem of mice. Using imaging, electrophysiology, molecular and genetic approaches in combination, we investigated a mechanism specifically involved in giant synapse formation. We have found that neurotrophin–3 (NT3) and its high–affinity receptor TrkC are required for both the structural and functional development of the calyceal synapse in culture. From 3D structures of GFP–labeled presynaptic terminals, growing calyces can be categorized into 4 stages. In cultures, with no added NT3, the number of calyceal synaptic contact (stage 1) markedly declined and most remaining failed to develop beyond stage 2. This effect of NT3 deficiency is enhanced when an NT3 antibody was further added to the culture media to block endogenous NT3. In contrast, TrkC antibody raised against its LRR region in culture media had no effect on structural development, but strongly attenuated synaptic efficacy, with markedly reduced EPSC amplitude. We conclude that TrkC mediates the formation of giant calyceal synapses and plays a critical role in their structural and functional development.
P2-1-35 Withdrawn	P2-1-36 Rho-kinase accelerates synaptic vesicle endocytosis by linking PKG activity to phosphatidylinositol-4,5-bisphophate synthesis ○Zacharie Taoufiq1, Kohgaku Eguchi1, Tomoyuki Takahashi1,2 Okinawa Institute of Science and Technology Graduate University1, Laboratory of Molecular Synaptic Function, Doshisha University Graduate School of Brain Science2 Rho-kinase plays diverse roles in cell motility mainly via phosphorylation of myosin light chain kinase. In developing neuronal system, Rho-kinase is involved in neuronal migration, neurite outgrowth and retraction. Rho-kinase is highly expressed in mature neurons, but its physiological roles are poorly understood. Here we report that Rho-kinase plays an essential role in the signal cascade for the homeostatic control of synaptic vesicle recycling in presynaptic terminals. Vesicles consumed by excessive exocytosis can be compensated by accelerating vesicle endocytosis via a retrograde feedback mechanism, which involves NO released from postsynaptic cells, presynaptic PKG and a lipid, phosphatidylinositol-4,5-bisphophate (PIP2)(Eguchi et al, 2012 Neuron). Direct application of a Rho-kinase inhibitor into the calyx of Held presynaptic terminal in the rat brainstem slice inhibited this feedback mechanism, with its effect mimicking and occluding that of PKG inhibitors, but reversed by a direct loading of PIP2. In contrast, intraterminal application of a Rho activator counteracted the inhibitory effect of the PKG inhibitor on vesicle endocytosis. This Rho activator increased the PIP2 content in brainstem synaptosomes and counteracted the down-regulatory effect of the PKG inhibitor on the PIP2 synthesis. Both the PKG inhibitor and Rho-kinase inhibitor attenuated the PIP2 content in brainstem synaptosomes, with their effects being occluded to each other. We conclude that Rho-kinase plays a pivotal role linking presynaptic PKG activity to PIP2 synthesis for the maintenance of homeostatic balance of vesicle exocytosis and endocytosis in the nerve terminals.

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