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complexin recruits calcium sensor synaptotagmin 1 to the SNARE-driven fusion machinery for synaptic vesicle exocytosis
Abstracts However, the temporal and spatial distribution patterns of extrasynaptic glutamate have remained elusive. Here we directly imaged and quantified the extrasynaptic glutamate dynamics using a fluorescent indicator of glutamate (EOS, glutamate (E) optical sensor). We found that physiologically relevant patterns of synaptic inputs induce the micromolar level of extrasynaptic glutamate pooling in acute brain slices. This demonstrates that physiological synaptic activities generate significant extrasynaptic glutamate dynamics that are sufficient for activating extrasynaptic glutamate receptors. doi:10.1016/j.neures.2009.09.713
P2-b13 Input-specific remodeling of postsynaptic density (PSD) proteins in Purkinje cell spines Toshimitsu Fuse, Haruhiko Bito Department Neurochem, University Tokyo Grad. Sch. Med., Tokyo, Japan Mature Purkinje cells receive segregated excitatory inputs from one single climbing fiber (CF) and numerous parallel fibers (PFs). Although the composition of synaptic proteins differs depending on the incoming fibers, the molecular mechanisms underlying such input-specific specification remain poorly understood. We found that Shank1A/Synamon is abundant at the PSD of PF synapses but is hardly detected at CF synapses in the mature cerebellum. To identify a potentially causal link between CF activity and reduced Shank1A, we applied stimuli that mimic CF activity, either by opening P/Q-type Ca2+ channels (55 mM KCl) or AMPA receptors (0.5 M AMPA), and measured synaptic Shank1A, in mature cultured Purkinje neurons that lack CF-like inputs. Indeed, Shank1A clusters decreased robustly, and this was abolished in the presence of MG132, suggesting that Shank1A is actively degraded by the ubiquitin proteasome system. Interestingly, treatment with either -CTX MVIIC or -agaIVA suppressed this degradation, consistent with the role of sustained Ca2+ -influx via P/Q-type Ca2+ channels in limiting Shank1A content. doi:10.1016/j.neures.2009.09.714
P2-b14 Dynamics of synaptic vesicle exocytosis at the level of single synapses revealed by optical glutamate imaging Hirokazu Sakamoto, Shigeyuki Namiki, Sho Iinuma, Kenzo Hirose Department Neurobiol, University of Tokyo, Tokyo, Japan Exocytosis of synaptic vesicles normally occurs at presynaptic release site. However, the number of release sites (Nsite ) and the number of releasable vesicles (Nves ) at a central synapse, which typically contains only one active zone, have not been revealed. In this study, we applied optical glutamate imaging to directly measure glutamate released from single hippocampal synapses. Fluctuation analysis based on a binomial model and deconvolution analysis enables us to estimate Nsite and Nves , respectively. Interestingly, although Nsite and Nves strongly correlated, Nves is usually larger than Nsite . This result indicates that limited vesicles in releasable pool are released by a single presynaptic action potential. Furthermore, the release rate per vesicle was fast at synapses which had relatively large Nsite . Therefore, Nsite is a key determinant of dynamics of synaptic vesicle exocytosis, and contributes to short term synaptic plasticity at the level of single synapses. doi:10.1016/j.neures.2009.09.715
P2-b15 The role of the N-terminal domain of glutamate receptor ␦2 in synapse formation Takeshi Uemura, Masayoshi Mishina Department of Mol. Neurobiol. & Pharmacol., University of Tokyo, Tokyo, Japan Glutamate receptor (GluR) ␦2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in long-term depression (LTD) induction at parallel fiber (PF)–PC synapses, motor learning, and the matching and connection of PF–PC synapses in developing and adult cerebella. However, it remains unsolved how GluR␦2 regulates PF–PC synapse formation. Here we show that 293T cells expressed GluR␦2 recruit presynaptic proteins by co-cultured cerebellar neurons. In addition, FM1-43 uptake was observed on the surface of these cells, suggesting that GluR␦2 induces functional presynaptic differentiation. In contrast, the chimera receptor in which the extracellular N-terminal domain (NTD) of GluR␦2 is replaced with the NTD of GluR␣1 could not recruit presynaptic proteins. Furthermore, beads bearing the NTD of GluR␦2 recruit presynaptic proteins. These results suggest that GluR␦2 triggers presynaptic differentiation by direct interaction with presynaptic components through the NTD. doi:10.1016/j.neures.2009.09.716
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P2-b16 Identification of interneuron subtypes driving glutamateindependent “proto-afterdischarge” in rat hippocampal slices Yoko Tsukamoto 1 , Yoshikazu Isomura 1,2 , Michiko Imanishi 1 , Taihei Ninomiya 1 , Yuchio Yanagawa 3 , Tomoki Fukai 2 , Masahiko Takada 1 1 3
Tokyo Met Inst Neurosci,Tokyo, Japan; Gunma Univ, Gunma, Japan
2
RIKEN BSI, Saitama, Japan;
Strong electrical stimulation induces low-frequency synchronous oscillations (afterdischarge) in rat hippocampal CA1 slices. Recently, we found that short and local network oscillations (“proto-afterdischarge”) persisted in the presence of glutamate receptor antagonists. The glutamate-independent proto-afterdischarge was abolished by a GABAA receptor antagonist or a gap-junction inhibitor, indicating that an interneuron network may play a crucial role in the generation of proto-afterdischarge. Here, we identified interneuron subtypes driving the protoafterdischarge by whole-cell recordings from GABAergic interneurons visualized in VGAT-Venus rats. Although most of interneurons became silent under the glutamateblockade condition, fast-spiking (FS) cells in str. pyramidale (e.g., basket cells) showed proto-afterdischarge activity, suggesting that FS cells may contribute to rhythm generation of hippocampal afterdischarge. doi:10.1016/j.neures.2009.09.717
P2-b17 Synaphin/complexin recruits calcium sensor synaptotagmin 1 to the SNARE-driven fusion machinery for synaptic vesicle exocytosis Hiroshi Tokumaru 1 , Chigusa Shimizu-Okabe 1 , Takumi Shinohara 1 , Teruo Abe 2 1 2
Facul. Pharmaceutical Sci. Tokushima Bunri Uviversity, Kagawa, Japan; Department Celluler Neurobiol. Brain Res. Insti., Niigata, Japan
Binding of the cytosolic protein synaphin (also called complexin) to the SNARE complex is critical for synaptic vesicle exocytosis. We have previously shown that synaphin (Syp) directly interacts with native synaptotagmin 1 (Syt1), a major Ca sensor for fast neurotransmitter release (Brain Cell Biol. 36, 173-189, 2008). We further examined the interaction using recombinant Syt1 expressed in COS cells. Like native Syt1, recombinant Syt1 bound to the C-terminal region (91–124) of Syp, and the binding was greatly reduced by replacing the seven consecutive glutamic acids in the region by alanines (E108-114A). Syt1 binding to GST-Syp increased dramatically upon addition of SNAREs. Together with our previous results, these findings suggest that Syp recruits Syt1 to the SNARE-driven fusion machinery and synergistically functions with Syt1 in mediating fast synaptic vesicle exocytosis. doi:10.1016/j.neures.2009.09.718
P2-b18 Parvalbumin/GABA cells in the monkey cerebral cortex: gap junctions, chandelier- and basket-terminals Akiko Yamashita 1 , Takao Oishi 2 , Narumi Katsuyama 1 , Masato Taira 1 , Motoharu Hayashi 2 1
Nihon University School of Medicine, Japan; Kyoto University, Japan
2
Primate Research Institute,
In mammalian cerebral cortex, chandelier and basket cells are parvalbumin (PV)/GABA cells. PV immunohistochemistry showed that the numbers of terminals of chandelier and basket cells were higher in monkeys and humans than in rats and cats. PV cells in rats and cats are connected electrically each other by gap junctions (Fukuda et al., 2006). Double-labeling immunohistochemistry for PV and connexin 36 (Cx36), a marker protein of gap junctions, detected small number of Cx36 puncta on the dendrites of PV cells in the monkey cerebral cortex. This indicates that cortical PV cells in monkeys connected electrically by gap junctions, but the density of gap junctions was low. The properties of PV/GABA cells in monkey cerebral cortex may be different from those in rat and cat cortices doi:10.1016/j.neures.2009.09.719
P2-b19 Caffeine-induced synaptic enhancement under blockade of adenosine A1 receptors at the hippocampal mossy fiber synapse Ikuma Sato, Haruyuki Kamiya Department of Neurobiology, Hokkaido University School of Medicine, Sapporo, Japan Previous study demonstrated that application of caffeine, a drug which induces Ca2+ release from ryanodine-sensitive stores, robustly enhances EPSPs at the hippocampal mossy fiber synapse. This suggests that ryanodine receptors play a significant role in regulating presynaptic Ca2+ dynamics at this synapse. However, since caffeine
P2-b13 Input-specific remodeling of postsynaptic density (PSD) proteins in Purkinje cell spines Toshimitsu Fuse, Haruhiko Bito Department Neurochem, University Tokyo Grad. Sch. Med., Tokyo, Japan Mature Purkinje cells receive segregated excitatory inputs from one single climbing fiber (CF) and numerous parallel fibers (PFs). Although the composition of synaptic proteins differs depending on the incoming fibers, the molecular mechanisms underlying such input-specific specification remain poorly understood. We found that Shank1A/Synamon is abundant at the PSD of PF synapses but is hardly detected at CF synapses in the mature cerebellum. To identify a potentially causal link between CF activity and reduced Shank1A, we applied stimuli that mimic CF activity, either by opening P/Q-type Ca2+ channels (55 mM KCl) or AMPA receptors (0.5 M AMPA), and measured synaptic Shank1A, in mature cultured Purkinje neurons that lack CF-like inputs. Indeed, Shank1A clusters decreased robustly, and this was abolished in the presence of MG132, suggesting that Shank1A is actively degraded by the ubiquitin proteasome system. Interestingly, treatment with either -CTX MVIIC or -agaIVA suppressed this degradation, consistent with the role of sustained Ca2+ -influx via P/Q-type Ca2+ channels in limiting Shank1A content. doi:10.1016/j.neures.2009.09.714
P2-b14 Dynamics of synaptic vesicle exocytosis at the level of single synapses revealed by optical glutamate imaging Hirokazu Sakamoto, Shigeyuki Namiki, Sho Iinuma, Kenzo Hirose Department Neurobiol, University of Tokyo, Tokyo, Japan Exocytosis of synaptic vesicles normally occurs at presynaptic release site. However, the number of release sites (Nsite ) and the number of releasable vesicles (Nves ) at a central synapse, which typically contains only one active zone, have not been revealed. In this study, we applied optical glutamate imaging to directly measure glutamate released from single hippocampal synapses. Fluctuation analysis based on a binomial model and deconvolution analysis enables us to estimate Nsite and Nves , respectively. Interestingly, although Nsite and Nves strongly correlated, Nves is usually larger than Nsite . This result indicates that limited vesicles in releasable pool are released by a single presynaptic action potential. Furthermore, the release rate per vesicle was fast at synapses which had relatively large Nsite . Therefore, Nsite is a key determinant of dynamics of synaptic vesicle exocytosis, and contributes to short term synaptic plasticity at the level of single synapses. doi:10.1016/j.neures.2009.09.715
P2-b15 The role of the N-terminal domain of glutamate receptor ␦2 in synapse formation Takeshi Uemura, Masayoshi Mishina Department of Mol. Neurobiol. & Pharmacol., University of Tokyo, Tokyo, Japan Glutamate receptor (GluR) ␦2 selectively expressed in cerebellar Purkinje cells (PCs) plays key roles in long-term depression (LTD) induction at parallel fiber (PF)–PC synapses, motor learning, and the matching and connection of PF–PC synapses in developing and adult cerebella. However, it remains unsolved how GluR␦2 regulates PF–PC synapse formation. Here we show that 293T cells expressed GluR␦2 recruit presynaptic proteins by co-cultured cerebellar neurons. In addition, FM1-43 uptake was observed on the surface of these cells, suggesting that GluR␦2 induces functional presynaptic differentiation. In contrast, the chimera receptor in which the extracellular N-terminal domain (NTD) of GluR␦2 is replaced with the NTD of GluR␣1 could not recruit presynaptic proteins. Furthermore, beads bearing the NTD of GluR␦2 recruit presynaptic proteins. These results suggest that GluR␦2 triggers presynaptic differentiation by direct interaction with presynaptic components through the NTD. doi:10.1016/j.neures.2009.09.716
S143
P2-b16 Identification of interneuron subtypes driving glutamateindependent “proto-afterdischarge” in rat hippocampal slices Yoko Tsukamoto 1 , Yoshikazu Isomura 1,2 , Michiko Imanishi 1 , Taihei Ninomiya 1 , Yuchio Yanagawa 3 , Tomoki Fukai 2 , Masahiko Takada 1 1 3
Tokyo Met Inst Neurosci,Tokyo, Japan; Gunma Univ, Gunma, Japan
2
RIKEN BSI, Saitama, Japan;
Strong electrical stimulation induces low-frequency synchronous oscillations (afterdischarge) in rat hippocampal CA1 slices. Recently, we found that short and local network oscillations (“proto-afterdischarge”) persisted in the presence of glutamate receptor antagonists. The glutamate-independent proto-afterdischarge was abolished by a GABAA receptor antagonist or a gap-junction inhibitor, indicating that an interneuron network may play a crucial role in the generation of proto-afterdischarge. Here, we identified interneuron subtypes driving the protoafterdischarge by whole-cell recordings from GABAergic interneurons visualized in VGAT-Venus rats. Although most of interneurons became silent under the glutamateblockade condition, fast-spiking (FS) cells in str. pyramidale (e.g., basket cells) showed proto-afterdischarge activity, suggesting that FS cells may contribute to rhythm generation of hippocampal afterdischarge. doi:10.1016/j.neures.2009.09.717
P2-b17 Synaphin/complexin recruits calcium sensor synaptotagmin 1 to the SNARE-driven fusion machinery for synaptic vesicle exocytosis Hiroshi Tokumaru 1 , Chigusa Shimizu-Okabe 1 , Takumi Shinohara 1 , Teruo Abe 2 1 2
Facul. Pharmaceutical Sci. Tokushima Bunri Uviversity, Kagawa, Japan; Department Celluler Neurobiol. Brain Res. Insti., Niigata, Japan
Binding of the cytosolic protein synaphin (also called complexin) to the SNARE complex is critical for synaptic vesicle exocytosis. We have previously shown that synaphin (Syp) directly interacts with native synaptotagmin 1 (Syt1), a major Ca sensor for fast neurotransmitter release (Brain Cell Biol. 36, 173-189, 2008). We further examined the interaction using recombinant Syt1 expressed in COS cells. Like native Syt1, recombinant Syt1 bound to the C-terminal region (91–124) of Syp, and the binding was greatly reduced by replacing the seven consecutive glutamic acids in the region by alanines (E108-114A). Syt1 binding to GST-Syp increased dramatically upon addition of SNAREs. Together with our previous results, these findings suggest that Syp recruits Syt1 to the SNARE-driven fusion machinery and synergistically functions with Syt1 in mediating fast synaptic vesicle exocytosis. doi:10.1016/j.neures.2009.09.718
P2-b18 Parvalbumin/GABA cells in the monkey cerebral cortex: gap junctions, chandelier- and basket-terminals Akiko Yamashita 1 , Takao Oishi 2 , Narumi Katsuyama 1 , Masato Taira 1 , Motoharu Hayashi 2 1
Nihon University School of Medicine, Japan; Kyoto University, Japan
2
Primate Research Institute,
In mammalian cerebral cortex, chandelier and basket cells are parvalbumin (PV)/GABA cells. PV immunohistochemistry showed that the numbers of terminals of chandelier and basket cells were higher in monkeys and humans than in rats and cats. PV cells in rats and cats are connected electrically each other by gap junctions (Fukuda et al., 2006). Double-labeling immunohistochemistry for PV and connexin 36 (Cx36), a marker protein of gap junctions, detected small number of Cx36 puncta on the dendrites of PV cells in the monkey cerebral cortex. This indicates that cortical PV cells in monkeys connected electrically by gap junctions, but the density of gap junctions was low. The properties of PV/GABA cells in monkey cerebral cortex may be different from those in rat and cat cortices doi:10.1016/j.neures.2009.09.719
P2-b19 Caffeine-induced synaptic enhancement under blockade of adenosine A1 receptors at the hippocampal mossy fiber synapse Ikuma Sato, Haruyuki Kamiya Department of Neurobiology, Hokkaido University School of Medicine, Sapporo, Japan Previous study demonstrated that application of caffeine, a drug which induces Ca2+ release from ryanodine-sensitive stores, robustly enhances EPSPs at the hippocampal mossy fiber synapse. This suggests that ryanodine receptors play a significant role in regulating presynaptic Ca2+ dynamics at this synapse. However, since caffeine