一般演題（ポスター）

一般演題（ポスター） Synapse I

1P-15 Dual imaging of SVs and DCVs exocytosis Nakajima Yui¹，Shinoda Yo¹，Furuichi Teiichi^1,2 ¹Dept. of Appl. Biol. Sci., Fac. of Sci. and Technol., Tokyo Univ. of Sci.，²RIKEN Brain Science Institute Exocytosis of synaptic vesicles（SVs）mediates the release neurotransmitters into synaptic cleft through the sequential process of vesicle docking, priming and fusion, when the action potential arrives at the presynaptic terminals. In neuronal cells, there is another secretory vesicle, called dense-core vesicles（DCVs）that play a major role in the release of neuropeptides and peptide hormones. Very little, however, is known about the regulatory process of DCV exocytosis. To clarify the distinction between SV and DCV exocytosis regarding the underlying mechanisms including subcellular release patterns and stimulus-dependent release kinetics, simultaneous cell imaging of both exocytosis events must be informative. We constructed a pH-sensitive red fluorescent protein mOrange2 fused with the DCV luminal protein chromogranin A（ChgA）,“ChgA-mOrange2”as a fluorescent probe for DCV exocytosis. We also used a pH-sensitive green fluorescent protein pHluorin（pH）fused with the SV membrane protein synaptophysin（SYP）,“SYP-pH”as a fluorescent probe for SV exocytosis. Two probe constructs were co-transfected into rat primary cultured cortical neurons and were subjected to live-cell time-lapse imaging. At present, we successfully detected exocytosis events indicated by ChgA-mOrange2 and SYP-pH, most of which likely showed different subcellular sites and different mode. We will further repeat dual imaging and will present the data on their comparative exocytosis mechanisms.		1P-16 Neuropsin dependent synaptic tagging in vivo Ishikawa Yasuyuki Dept.of Systems Life Engineering, Maebashi Institute of Technology Synaptic plasticity is widely accepted to provide a cellular basis for learning and memory. Synaptic associativity could be involved in activity-dependent synaptic plasticity, because it distinguishes between local mechanisms of synaptic tags and cell-wide mechanisms that are responsible for the synthesis of plasticity-related proteins. An attractive hypothesis for synapse specificity of long-term memory（LTM）is synaptic tagging：synaptic activity generates a tag, which captures the plasticity-related proteins derived outside of synapses. Previously we have been reported that neuropsin, a plasticity-related extracellular protease, was involved in synaptic tag setting. In the present study, we tested the hypothesis that neuropsin was engaged in behavioral tag setting for LTM in vivo. Behaviorally, weak training, which induces short-term memory（STM）but not LTM, can be consolidated into LTM by exposing animals to novel but not familiar environment 1 h before training. We found that neuropsin deficient mouse impaired such transformation short-term into long-term memory. These results suggest neuropsin as a tag setting in vivo.

1P-17 The role of metabotropic glutamate receptor on structural plasticity of dendritic spines in cultured hippocampal neurons Kakegawa Ryoma，Kamasaki Shingo，Todoriki Takashi，Kojima Nobuhiko Graduate school of Life Sciences, Toyo University The morphology of dendritic spines is closely related to higher brain functions. Various external signals including glutamate regulate the spine morphology. The group I metabotropic glutamate receptor（mGluR）is one of regulators of spine morphology, but details of the underlying mechanism still remains unclear. We have demonstrated that drebrin a major F-actin-binding protein in dendritic spines is important for spine morphogenesis and plasticity. Drebrin in its sequence has two binding motifs for Homer that is scaffolding protein of mGluR. We then propose that group I mGluR activity regulates spine morphology through drebrin-Homer interaction in dendritic spines. To elucidate this working hypothesis, using cultured hippocampal neurons, we examine the relationship between mGluR5 activity and localization of mGluR5, drebrin and Homer1 in dendritic spines. Mouse hippocampal neurons were cultured with Banker method. At 21 days in vitro, neurons were incubated with 1mM CHPG, a selective mGluR5 agonist for 15min, then processed for immunocytochemistry for mGluR5, drebrin and Homer. Neurons were also stained with fluorescence-conjugated phalloidin for F-actin staining. Quantitative analyses of confocal microscopic images revealed that the CHPG treatment significantly increased the number of spine in which drebrin and Homer were co-localized. The same treatment affected spine morphology. These findings suggest that mGluR5 activity regulates spine morphology through drebrin-Homer interaction. mGluR has recently been shown to participate in several neuropsychiatric diseases. The elucidation of functional roles of mGluR in spine morphogenesis helps us to gain better understanding of the mechanism of higher brain functions.		1P-18 Deletion of drebrin A impairs hippocampal synaptic plasticity and hippocampus-dependent fear learning in adulthood Yasuda Hiroki¹，Kojima Nobuhiko^2,3，Hanamura Kenji³，Shirao Tomoaki^1,3 ¹ERSC, Med., Gunma Univ.，²Toyo Univ., Life Sci.，³Neuropharm., Med., Gunma Univ. Structural plasticity of dendritic spines, that underlies higher brain function including learning and memory, is dynamically regulated by actin cytoskeleton and its associated proteins. Drebrin A（DA）is an actin binding protein preferentially expressed in the brain and localized highly in dendritic spines of mature neurons. The isoform conversion from drebrin E（DE）to DA and its accumulation into dendritic spines occur during synaptic maturation. We have demonstrated that DA has a pivotal role in spine morphogenesis and plasticity. However, it is not determined which process is required, the accumulation of drebrin（either DE or DA）within spines or the isoform conversion of drebrin. To answer this question we further analyzed mutant mice（named DAKO mice）in which the isoform conversion from DE to DA was disrupted by a deletion of the DA-specific exon. In adult DAKO mouse brain DE continued to be expressed instead of DA. Electrophysiological study using hippocampal slices revealed that LTP induced by high frequency stimulation of CA1 synapses was impaired in older（than 30 week old）DAKO mice, but not in younger（than 8-9 week old）ones. In contrast, LTD was abnormally induced by low frequency stimulation of CA1 synapses in older（than 30 week old）DAKO mice, but not in younger（than 8-9 week old）ones. Unlike the LTD in juvenile mice, this form of LTD does not depend on the NMDA receptor activity, rather depends on metabotropic glutamate receptor 5（mGluR5）activity, suggesting that mGluR5-signaling is altered in old DAKO mice. In parallel with the electrophysiological phenotype these mice exhibit impaired hippocampus-dependent fear memory in an age-dependent manner. The impairment was evident in mice older than 30 week old, but not in mice younger than 10 week old. Thus, our data indicate that the isoform conversion of drebrin is critical and DA is indispensable for normal synaptic plasticity and hippocampus-dependent types of fear memory.

1P-19 EphB Extracellular Phosphorylation Controls Pathological Pain and Synaptic Function of NMDA Receptors Hanamura Kenji^1,2，Sheffler-Collins Sean^2,5，Xia Nan²，Halley Washburn²，Tilu Dipti³，Hassler Shayne⁶，Spellman Daniel⁴，Zhang Guoan⁴，Neubert Thomas⁴，Price Theodore⁶，Dalva Matthew² ¹Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine，²Department of Neuroscience and Farber Institute for Neurosciences, Thomas Jefferson University，³Departments of Pharmacology, The University of Arizona College of Medicine，⁴Department of Pharmacology and Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine，⁵Neuroscience Graduate Group, University of Pennsylvania School of Medicine，⁶School of Behavioral and Brain Sciences, University of Texas at Dallas N-methyl-d-aspartate receptors（NMDARs）are localized to synapses to drive adaptive and maladaptive changes in response to sensory experience. Synaptic organizing proteins control recruitment and retention of NMDARs by inducible intracellular and extracellular interactions. However the mechanisms enabling extracellular interactions are largely unknown. Here we show that synaptic accumulation of GluN2B-containing NMDARs and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a tyrosine in the fibronectin type III（FN3）domain of EphB2. Ligand-dependent extracellular tyrosine phosphorylation drives the EphB-NMDAR interaction and surface retention of EphB2 and NMDARs. In contrast, in the absence of NMDAR, phosphorylation of this tyrosine residue regulates endocytosis and degradation of EphB2. Viral transduction of EphB2 and drug administration demonstrate that extracellular phosphorylation mediates EphB and injury-induced pathological pain behavior. FN3 domains of other synaptic proteins contain a homologous residue suggesting that extracellular phosphorylation is novel candidate mechanism. Together these data identify tyrosine residue in FN3 domain of EphB and extracellular phosphorylation as possible therapeutic targets.		1P-20 NMDA receptors are involved in X-irradiation-induced decrease in drebrin clusters within dendritic spines of cultured hippocampal neurons MIAO SHUCHUAN，Puspitasari Anggraeini，Hanamura Kenji，Koganezawa Noriko，Roppongi Reiko，Shirao Tomoaki Department of Neurobiology and Behavior Therapeutic X-irradiation of the brain possibly causes cognitive impairment, which is associated with synaptic dysfunction. We have reported that cranial 10 Gy X-irradiation has acute effects on fear memory in adult mice. This impairment of the memory was associated with the decrease in immunostaining intensity of an actin-binding protein, drebrin in molecular layer of dentate gyrus in vivo. Drebrin is usually concentrated in dendritic spines, postsynaptic sites of excitatory glutamatergic synapses and correlates well with the severity of cognitive impairment. We have shown that glutamate-induced decrease in drebrin clusters within dendritic spines of cultured neurons is mediated by NMDA receptor activity. However the mechanism regulating this X-irradiation-induced decrease in drebrin immunostaining intensity is unknown. In order to examine whether NMDA receptors is involved in X-irradiation-induced decrease in drebrin in postsynaptic sites, we used primary hippocampal neuronal culture and analyzed the acute effect of X-irradiation on drebrin accumulation within dendritic spines in vitro. The neurons were treated with 50 μ M Amino-5-phosphonovaleric acid（APV；an NMDA receptor antagonist）1 hour before 10 Gy of X-irradiation at 21 days in vitro. The neurons were fixed 8 hours after X-irradiation. Immunocytochemical analysis showed that drebrin cluster density along dendrites significantly decreased 8 hours after X-irradiation. This decrease was blocked by pretreatment with APV. In addition, we also analyzed the cluster density of GluN1 subunit of NMDA receptors. The GluN1 cluster density was significantly increased 8 hours after X-irradiation. Our results suggest that X-irradiation induces decrease in drebrin clusters within dendritic spines by inducing accumulation and activation of NMDA receptors. Antagonists of NMDA receptor may provide a new avenue toward therapeutic tools to mitigate X-irradiation-induced synaptic dysfunction.

1P-21 Effect of histone deacetylase inhibitor on synaptic dysfunction elicited by X-irradiation Hiruma Takashi，Koganezawa Noriko，Puspitasari Anggraeini，Shirao Tomoaki Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine Treatments for cancers have been developing surprisingly due to progresses of radiotherapy techniques. It has been known, however, that cranial irradiation causes cognitive deficits, although the underlie mechanism of such cognitive deficits is still remain unknown. We have been studying the effect of X-irradiation on neurons in vitro, and have reported that the density of dendritic spines was decreased or the changes in spine morphology by X-irradiation. Furthermore, using drebrin, an actin binding protein, as a marker for synaptic function, we found the number of drebrin clusters decreased transiently by X-irradiation. We have also shown that amyloid beta oligomers-induced change of drebrin accumulation is mediated by histone deacetylase (HDAC) (Ishizuka et al., 2014). And some of HDAC inhibitors are known to protect normal cells from radiation-induced damage. In this study we examined if the accumulation change of drebrin by X-irradiation is also mediated by HDAC, and tested the possibility of HDAC inhibitors usage as therapeutic tools to weaken irradiation effects on synaptic function. We used primary hippocampal cultured neurons and suberoylanilide hydroxamic acid (SAHA) as a HDAC inhibitor. Drebrin was used as a marker of synaptic function and a post synaptic marker and Synapsin I was used as a pre synaptic marker and these proteins were analyzed immunocytochemically. The cultured neurons were pretreated with SAHA 1 hour before irradiation and were fixed at 2, 8, and 24 hours after the irradiation. We evaluated the effect of SAHA by counting drebrin and Synapsin I clusters. Although the data was not significantly different due to the small sample number, it suggested that low dose of SAHA blocks the X-irradiation induced transient decrease of drebrin accumulation.		1P-22 The effect of carbon ion irradiation on cell motility in human glioblastoma cell lines Matsumoto Tomoki，Yoshida Yukari，Yakoh Tomoko，Takahashi Akihisa，Nakano Takashi Gunma University Heavy Ion Medical Center This study aimed to investigate the effect of carbon ion（C-ion）irradiation on cell motility in glioblastoma cells. Cell motility was assessed by a wound-healing assay, and the cell survival was evaluated by clonogenic assay.

1P-23 The molecular mechanisms of cell motility by X-ray irradiation in human glioblastoma cell lines Sejimo Yukihiko，Yoshida Yukari，Matsumoto Tomoki，Yakoh Tomoko，Takahashi Akihisa，Nakano Takashi Gunma University Heavy Ion Medical Center This study aimed to investigate the molecular mechanisms of cell motility by X-ray irradiation（X-irradiation）in glioblastoma cells. Human glioblastoma cell lines was used U251 and T98G. Cell motility was assessed by a wound-healing assay, and the cell survival was evaluated by clonogenic assay.