Poster Sessions

Poster Sessions イオンチャンネル、シナプス

1P-01 Analysis of effects by rTMS on Glutamate, GABA and Glycine transporters on the mouse brain. Tetsurou Ikeda，Nobuyuki Nukina SNP Team. BSI.RIKEN rTMS is a noninvasive technique to induce electric current in the brain and is supposed to be beneficial for the treatment of patients with depression, schizophrenia and neurodegenerative disorders. We reported previously that rTMS modulates monoamine transporter, dopamine receptor 2, circadian rhythm-related genes and HSP70. However, the mechanisms underlying the effects of rTMS are still unclear. We analyzed the changes in mRNA expression in mouse brain that occurred after rTMS with real time PCR. Following 20 days of rTMS, many genes were differentially expressed in the mouse brain. Up-regulation of Glutamate transporters (EAAT4, GLAST and GLT1), GABA transporters (GAT1 and GAT4) and Glycine transporters (GYLT1 and GLYT2) mRNA expression levels were observed after 20 days rTMS. Surprisingly, with 10 days rest after 20 days rTMS, up-regulation of Glutamate transporters (EAAC1, EAAT4, GLAST and GLT1), GABA transporters (GAT1, GAT2, GAT3 and GAT4) and Glycine transporters (GYLT1 and GLYT2) mRNA expression levels were observed. ATF6 and GRP78 (Bip) mRNA expression levels were up-regulated after transient and chronic rTMS. IRE1, Perk and XBP1 mRNA expression levels were down-regulated after transient and chronic rTMS. Interestingly, ATF6, GRP78 (Bip), IRE1, IRE1 α, Perk and XBP1 mRNA expression levels were down-regulated with 10 days rest after 20 days rTMS. In PC12 cells, an up-regulation of GRP78 (Bip) mRNA and subsequent cell-protective effects were observed after acute and chronic rTMS. These results indicated that the modulation of several genes may be involved in the therapeutic mechanisms of acute and chronic rTMS for patients with neuropsychiatric disorders.		1P-02 Gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography Kozo Hamada¹，Hideyuki Miyatake²，Akiko Terauchi¹，Katsuhiko Mikoshiba¹ ¹Lab for Dev Neurobiol, BSI, RIKEN，²Nano Med Eng Lab, RIKEN The inositol 1,4,5-trisphosphate receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The brain dominant type 1 IP3R (IP3R1) is genetically causative for spinocerebellar ataxia and is implicated in the etiology of Alzheimer’s and Huntington’s diseases. The IP3 binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse IP3R1 in the absence and presence of IP3. Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature helical domain interfacing the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the helical domain. These results suggest that the curvature helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel.

1P-03 Effectiveness of amidino-TAPA against morphine-resistant neuropathic pain Hirokazu Mizoguchi，Chizuko Watanabe，Akira Otowa，Shinobu Sakurada Dept Physiol Anat, Fac Pharmaceut Sci, Tohoku Med Pharmaceut Univ Amidino-TAPA is a selective μ-opioid receptor agonist and its spinal antinociception is mediated through the activation of distinct μ-opioid receptors MOR-1K and MOR-1L, amidino-TAPA-sensitive but morphine-insensitive splice variants. Most notable character of amidino-TAPA is its effectiveness against morphine-resistant neuropathic pain. In the present study, the analgesic mechanism of amidino-TAPA against the neuropathic pain was investigated. The neuropathic pain model was developed in mice, according to the method by Seltzer and his colleagues. The analgesic effect was measured using the von Frey filament at 7 days after the nerve ligation. After the nerve ligation, the spinal analgesic effect of morphine was suppressed in ipsilateral paw in compared in contralateral paw. In contrast, amidino-TAPA showed same magnitude of spinal analgesic effect in both paws. In mice the μ-opioid receptor splice variants containing exon-1 of MOR-1 gene had been knocked-down, the spinal analgesic effect of amidino-TAPA was attenuated in both paws with the same magnitude. In contrast, the spinal analgesic effect of amidino-TAPA was more strongly attenuated in ipsilateral paw than in contralateral paw under the condition that MOR-1K or MOR-1L had been knocked-down. After the nerve ligation, mRNA expression of exon-1-containing splice variants, but not MOR-1K or MOR-1L, were reduced in ipsilateral side DRG in compared in contralateral side DRG. In conclusion, the reduction of spinal analgesic effect of morphine after nerve ligation may be caused by the reduction of the morphine-sensitive splice variants in DRG. On the contrary, the spinal analgesic effect of amidino-TAPA is maintained after the nerve ligation, since amidino-TAPA-sensitive splice variants in DRG are maintained.		1P-04 Response of GABAergic system to the axotomy of rat facial nerve Kazuyuki Nakajima¹，Reika Kikuchi¹，Makoto Hamanoue²，Shinichi Kohsaka³ ¹Dept Sci and Eng, Soka Univ，²Dept Phys, Med, Toho Univ，³National Institute of Neurosci The responses of inhibitory neurons/synapses to motoneuron injury in the cranial nervous system remain to be elucidated. In this study, we analyzed GABAA receptor (GABAAR) and GABAergic neurons in transected rat facial nucleus. Immunoblotting revealed that GABAARα1 levels in axotomized facial nucleus decreased significantly 3-14 days post-insult, and low levels remained for 5 weeks. GABAARβ2,3 levels reduced transiently 5-7 days post-insult, but the levels restored at 5 weeks post-insult. Immunohistochemical methods indicated that the GABAARα1-expressing cells were motoneurons. We next examined specific components of GABAergic neurons, including glutamate decarboxylase (GAD) for synthesizing GABA, vesicular GABA transporter (VGAT) for packing GABA into the vesicles, and GABA transporter-1 (GAT-1) for GABA reuptake. Immunoblotting indicated that GAD, VGAT, and GAT-1 levels decreased transiently from 5 to 14 days post-insult, but returned to the control levels at 5 weeks post-insult. Although GABAARα1  levels in transected nucleus did not return to their control levels for 5 weeks post-insult, administration of glial cell line−derived neurotrophic factor at the cut site significantly ameliorated the reductions. Through these findings, we verified that the injured facial motoneurons suppressed the levels of GABAARα1 during 5 weeks post-insult, presumably due to the deprivation of neurotrophic factor. On the other hand, the levels of GAD, VGAT, and GAT-1 in GABAergic interneurons were transiently reduced at 3-14 days post-insult, but those levels recovered at 4-5 weeks post-insult.

1P-05 Seizure phenotype in syntaxin1B gene ablated mice was associated with GABAergic system Takefumi Kofuji^1,2，Tomonori Fujiwara²，Tatsuya Mishima²，Kimio Akagawa² ¹RI lab, Kyorin Univ Sch Med，²Dept Cell Physiol, Kyorin Univ Sch Med Two types of syntaxin1 (STX1) isoforms, HPC-1/syntaxin1A (STX1A) and syntaxin1B (STX1B), are co-expressed in neurons and are thought to have similar functions in exocytosis of synaptic vesicles. We previously reported that STX1A null mutant mice developed normally and showed normal fast synaptic transmission of glutamate or GABA. However, secretion of monoamine, such as serotonin or dopamine, or neuropeptide, such as oxytocin, was reduced in STX1A null mutant mice. On the other hand, fast synaptic transmissions of glutamate and GABA were impaired in STX1B null mutant mice, which was lethal within 2 weeks after birth. Therefore, STX1A and STX1B might have distinct roles in synaptic transmission. We also reported that STX1B null mutant mice exhibited motor function impairment and tended to be seizure phenotype. Recently, mutations in STX1B gene have been shown to cause a broad spectrum of fever-associated epilepsy syndromes. However, little is known how STX1B is related to its seizure phenotype. In this study, in order to examine involvement of STX1B in seizure, we assessed for susceptibility to seizures induced by administration of pentylenetetrazol (PTZ) or kainic acid in STX1B gene ablated mice. We found that STX1B heterozygote mutant mice showed high susceptibility to the above drugs. PTZ-induced seizures were blocked by anti-convulsant drug, such as valproic acid and phenytonin. We also found that GABA uptake through GABA transporter was affected in STX1B null glial cells. STX1 is thought to regulate the activity of GABA transporter and those cellular localization. Thus, we further examined how STX1B affected the GABAegic system causing seizure. Implication of STX1B roles in seizure phenotype will be discussed.		1P-06 Isoform-dependent regulation of drebrin dynamics in dendritic spines Kenji Hanamura，Shirao Tomoaki Dept Neurobiol. Behav. Grad Sch Med, Gunma Univ Dendritic spines have stable filamentous actin (F-actin) and dynamic F-actin. The formation of stable F-actin plays a pivotal role in spine formation. Drebrin binds to and stabilizes F-actin in dendritic spines. Interestingly, the conversion of the drebrin E isoform to drebrin A occurs in parallel with synapse formation, suggesting that this conversion promotes synapse formation via F-actin accumulation. In this study, we measured the dynamics of GFP-tagged drebrin E (GFP-DE) and drebrin A (GFP-DA) in cultured hippocampal neurons by fluorescence recovery after photobleaching analysis. We found that GFP-DA has a larger stable fraction than GFP-DE. The stable drebrin fraction reflects its accumulation in dendritic spines, therefore the isoform conversion may increase the amount of stable F-actin in dendritic spines. The stable fraction was dependent on the drebrin A-specific sequence “Ins2”, located in the middle of the drebrin protein. In addition, F-actin depolymerization with latrunculin A significantly reduced the stable GFP-DA fraction. These findings indicate that preferential binding of drebrin A to F-actin than drebrin E causes higher stable fraction of drebrin A in dendritic spines, although the F-actin-binding ability of purified drebrin E and drebrin A are comparable in vitro. Therefore, we suggest that drebrin isoform conversion from drebrin E to drebrin A in dendritic spines mediates the accumulation of stable F-actin, which is formed by drebrin.

1P-07 Elimination of glutamatergic synapses by microglia in the basal ganglia outputs of Parkinsonism model rats Junya Tanaka，Hitomi Aono，Mohammed E Choudhury，Kazuya Miyanishi，Yuka Kigami Dept Mol Cell Physiol, Grad Sch Med, Ehime Univ Neurological symptoms of Parkinson’s disease (PD) do not become apparent until the most DArgic neurons degenerate. Therefore, it has long been considered some compensatory mechanisms should exist. So far, many compensatory mechanisms have been proposed such as increased DA turnover or increased sensitivity for DA at the receptor level. In this study, we noticed that microglial cells in the substantia nigra pars reticulata (SNr) and the globus pallidus (GP) displayed more activated morphology than those in the SNc in a 6-OHDA-induced rat hemi-PD model. The activated microglia engulfed pre- and post-synaptic elements, including glutamate receptors into their phagosomes. Microglia in the SNr and GP phagocytosed red fluorescent DiI that was injected into the subthalamic nucleus (STN) as an anterograde tracer. Rat primary microglia increased their phagocytic activities in response to glutamate, while expressing mRNA encoding phagocytosis-related factors. Dexamethasone overcame the stimulating effect of glutamate for cultured microglial cells. Subcutaneous administration of dexamethasone to the PD model rats suppressed microglial activation in the SNr, resulting in aggravated motor dysfunctions, while expression of mRNA encoding glutamatergic, but not GABAergic, synaptic elements increased. Expression of glutamate transporters such as GLT-1 or GLAST did not change in the PD model brain. These findings suggest that microglia in the SNr and GP become activated and selectively eliminate glutamatergic synapses from the STN in response to increased glutamatergic activity. Thus, microglia may be involved in a negative feedback loop in the indirect pathway of the basal ganglia to compensate for the loss of dopaminergic neurons in PD brains.		1P-08 Further characterization of autistic-like behaviors in kirrel3-knockout mice Tomoko Hisaoka¹，Tadasuke Komori¹，Hiroaki Gyobu¹，Toshio Kitamura²，Yoshihiro Morikawa¹ ¹Department of Anatomy & Neurobiolgy, Wakayama Medical University，²Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo A synaptic cell adhesion molecule, kirrel3, plays important roles in the regulation of neural circuit formation, including axonal fasciculation and synapse formation. Recent genetic studies have identified mutations of KIRREL3 gene in patients with autism spectrum disorder (ASD). We have reported that adult kirrel3-knockout (kirrel3^-/-) mice show autistic-like behaviors, such as social recognition deficits, communication deficits, and repetitive thigmotaxis. To establish kirrel3^-/- mice as a mouse model of ASD, we further characterized autistic-like behaviors of kirrel3^-/- mice. Because ASD symptoms often exist from early childhood, we conducted the separation-induced USV recordings for wild-type and kirrel3^-/- pups. At postnatal day 7, the number of USVs emitted by kirrel3^-/- pups significantly increased than that of wild-type pups, indicating that kirrel3^-/- mice showed early onset of social communicative abnormalities. In the olfactory habituation/dishabituation test, kirrel3^-/- mice showed poor abilities to discriminate different social odors, which may cause social recognition deficits. Associated symptoms that appear in high percentage of autistic patients include hyperactivity, anxiety, and cognitive disability. In the light-dark transition test and the elevated plus maze test, kirrel3^-/- mice exhibited hyperactivity and normal anxiety-related behaviors. In addition, kirrel3^-/- mice exhibited normal spatial learning and memory in the Morris water maze test and normal short-term fear memory in the passive avoidance test. These findings suggest that kirrel3^-/- mice are a valuable model of ASD with hyperactivity. This work was supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (15K09873).

1P-09 FILIP-related molecule binds to NMDA receptor and controls spine morphology and synaptic function of the hippocampal neuron. Kazuki Kuroda^1,2，Hideshi Yagi⁴，Min-Jue Xie^2,3，Yugo Fukazawa^1,2，Yuichiro Oka^5,6，Tokuichi Iguchi⁶，Makoto Sato^5,6 ¹Division of Brain Structure and Function, Department of Morphological and Physiological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan，²Research and Education Program for Life Science, University of Fukui, Fukui, Japan，³Research Center for Child Mental Development, University of Fukui, Fukui, Japan，⁴Department of Anatomy and Cell Biology, Hyogo College of Medicine, Hyogo, Japan，⁵Department of Child Development, United Graduate School of Child Development, Osaka University, Osaka, Japan，⁶Department of Anatomy and Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan 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-2b plays a major role for regulation of actin dynamics in the dendritic spines. However, how myosin-2b directly alters cytoskeletal dynamics through ATPase-driven contraction of actin networks and how myosin-2b function is regulated during the spine maturation are still poorly understood. We found that one FILIP (Filamin A-Interacting Protein)-related molecule, LUZP1, was a binding partner of myosin-2b and was expressed in the hippocampal and neocortical neurons. We next examined the effects of altered endogenous LUZP1 expression in cultured hippocampal neurons: the knockdown of LUZP1 expression induced the spine length shortening and changed the ratio of cell surface and total expressing NMDA receptor. Furthermore, we found that LUZP1 was interacted with NMDA receptor. Recently, we generated LUZP1 conditional knockout mice in which loxP sites flank exon 4 in the LUZP1 allele for understanding of the roles of LUZP1 in the hippocampus and the cerebral cortex. When LUZP1 conditional knockout mice crossed with Emx1-Cre mice, these LUZP1 mutant mice showed the anxiety-like behavior compared with the control mice. These data suggest that LUZP1 is a novel myosin-2b modulator that controls synaptic function in the hippocampus as well as in the cerebral cortex.		1P-10 Expression of kirrel3 in the mouse dorsal root ganglia Tadasuke Komori¹，Tomoko Hisaoka¹，Hiroaki Gyobu¹，Toshio Kitamura²，Yoshihiro Morikawa¹ ¹Department of Anatomy & Neurobiology, Wakayama Medical University，²Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo A mammalian homolog of the Drosophila gene kirre (kin of irregular chiasm C-roughest), kirrel3, belongs to the immunoglobulin superfamily and is involved in axonal fasciculation and synapse formation. Previously, we have reported that kirrel3 is expressed in the neurons of dorsal root ganglia (DRGs) and may play a role in targeting of proprioceptive neurons on muscle spindles (Komori et al., J Comp Neurol, 2008). To gain further insights into the functional roles of kirrel3 in the DRGs, we generated kirrel3-deficient mice, in which kirrel3 gene was replaced with the lacZ reporter gene. X-gal staining in heterozygous kirrel3-deficient (kirrel3LacZ/+) mice revealed that the expression of kirrel3 was observed in the DRG neurons, but not in satellite cells. In addition, 35.4 ± 1.5% of total DRG neurons was kirrel3-positive and these neurons were widely distributed among all size ranges. Furthermore, kirrel3 was expressed in almost 80% of TrkB-positive mechanoreceptive neurons in the adult DRGs. During development, expression of kirrel3 was first detected at embryonic day (E) 11.5, gradually increased, and reached the maximum levels (approximately 58% of total DRG neurons) between E17.5 and postnatal day (P) 7. More than 80% of TrkB-positive mechanoreceptive neurons contained kirrel3 in the DRGs of mice from E13.5 to P7. Therefore, kirrel3 was expressed in mechanoreceptive neurons as well as in proprioceptive neurons in the DRGs, suggesting the involvement of kirrel3 in axonal targeting, synapse formation, and transmission of sensory information of these neurons.This work was supported by Grant-in-Aid for Scientific Research (B) (No. 22390036) from Japan Society for the Promotion of Science (JSPS).