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An immunocytochemical study for the local GABAergic system in rat trigeminal ganglion
Abstracts / Neuroscience Research 71S (2011) e108–e415
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in vitro and in vivo. Cultured astrocytes were activated by polyI:C treatment in vitro, leading to an increase in mRNA levels of IFITM3 as well as inflammatory cytokines. When primary cultured neurons were treated with the conditioned-medium of polyI:C-treated astrocytes (polyI:C-ACM), neurite outgrowth and spine formation were diminished. The neurodevelopmental abnormalities of cultured neurons induced by polyI:C-ACM were alleviated when astrocytes were prepared from ifitm3−/− mice. Furthermore, aberrant MAP2 expression in the frontal cortex in vivo as well as memory impairment was evident in polyI:C-treated wild-type mice, but such neurodevelopmental and behavioral abnormalities were not induced in polyI:C-treated ifitm3−/− mice. These results suggest a role for IFITM3 in neurodevelopmental disorders associated with perinatal immune activation. Research fund: Grant-in-Aid for JSPS Fellows.
metric analysis of microglia labeled by intracellular injection in fixed slices. Using hierarchical clustering analysis, three groups were disclosed. The first type was mainly composed of microglia from sham control mice, and the cells showed a characteristic ramified morphology of resting microglia. The second type was composed of microglia from D3 and D7 mice, and the cells showed bushy or hypertrophic appearance. The third type was mainly composed of microglia from D14 mice. Interestingly, the shape of microglia on D14 was intermediate between hypertrophic and ramified. Taken together, our findings indicate that microglia in the axotomized hypoglossal nucleus might be proliferative on D3, fully activated on D7, and rather deactivated on D14.
doi:10.1016/j.neures.2011.07.960
P3-c09 An immunocytochemical study for the local GABAergic system in rat trigeminal ganglion
P3-c07 Distinct roles of HPC-1/STX1A and STX1B in neuronal survival Takefumi Kofuji 1 , Tomonori Fujiwara 2 , Masumi Sanada 2 , Tatsuya Mishima 2 , Kimio Akagawa 2
1 Radioisotope Laboratory, Kyorin University School of Medicine, Tokyo, Japan 2 Deparment of Cell Physiology, Kyorin University School of Medicine, Tokyo, Japan
The membrane protein syntaxin1 is believed to play an important role in the exocytosis of synaptic vesicles. In neurons, two types of syntaxin1 isoforms, HPC-1/syntaxin1A (STX1A) and syntaxin1B (STX1B), both of which are transcribed from distinct genes and thought to have similar functions as neuronal t-SNARE, are predominantly expressed. Previously, we generated the gene knockout mice for STX1A or STX1B. Interestingly, STX1A null mutant mice normally developed (Fujiwara et al., J. Neurosci., 2007), but STX1B null mutant mice were died within 2 weeks after birth, suggesting that STX1A and STX1B might have distinct roles in neuronal function. In this study, we examined the dissociated culture neurons from STX1A- and STX1B-null mutant mice. On poly-l-lysine-coated coverglass, dissociated STX1A-deficient neurons normally survived compared with WT neurons, but STX1B-deficient neurons died suddenly after 7–8 days in vitro (DIV). However, the number of synaptopysin puncta and that of main branching were not different between survived STX1A- and STX1B-deficient neurons. Interestingly, on WT glial feeder layers, STX1B-deficient neurons normally survived. However, glial feeder layers from STX1B null mutant mice have no survival effect unlike glial feeder layers from STX1A null mutant mice. Furthermore, conditioned medium from WT and STX1A-deficient glial feeder layers had survival effect for STX1B-deficient neurons, while conditioned medium from STX1Bdeficient glial feeder layers did not support the survival. These data suggest that STX1A and STX1B might have distinct roles in neuronal survival which might be supported by glia function lacking in STX1B null mutant mice. Implication of these different roles in STX1A and STX1B will be discussed. doi:10.1016/j.neures.2011.07.961
P3-c08 Time course alterations in immunophenotypic and morphological features of microglia following hypoglossal axotomy in mice Jun Yamada 1 , Shozo Jinno 2 1 Department of Anatomy and Neurobiology, Graduate School of Medical Sciences, Kyushu University 2 Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University
Microglia, the resident immune cells of the brain, become activated in response to various noxious stimuli. The peripheral nerve transection paradigm allows systematic and detailed study of microglial reactions and neuronal cell death. Here, we quantitatively examined the time course alterations in immunophenotypic and morphological features of microglia following hypoglossal axotomy in mice. First, we analyzed the antigen expression profiles of microglia in the hypoglossal nucleus with normal nerve (sham control) and nucleus with transected nerve from the same animal 3 days (D3), 7 days (D7) and D14 after axotomy. In the sham control mice, virtually no microglia expressed a dividing cell marker, proliferating cell nuclear antigen (PCNA). The expression ratios of PCNA in microglia significantly increased on D3 (90%), while the ratios showed an immediate decline (less than 10%) on D7, D14 and D28. Voltage-dependent potassium (Kv) channels 1.3 and 1.5 were rarely observed in microglia of sham control and D3 mice, but about 80% of microglia expressed Kv1.3 and Kv1.5 on D7, and almost all microglia expressed Kv1.3 and Kv1.5 on D14. The expression ratios of Kv1.3 and Kv1.5 were declined on D28 (40%). Then, we carried out morpho-
doi:10.1016/j.neures.2011.07.962
Hana Hayasaki 1 , Toshiki Morishima 2 , Yoshiro Sohma 3 , Sachiko Yoshida 4 , Atsuo Fukuda 2 , Yoshinori Otsuki 1 1
Department of Anatomy, Osaka Medical College, Osaka, Japan 2 Dept. Physiol., Hamamatsu Univ. Sch. Med 3 Dept. Pharmacol., Keio Univ. Sch. Med 4 Dept. Material Sci., Toyohashi Univ. Tech GABAergic system has been intensively studied in the central nervous system, however not in the trigeminal ganglion (TG) in which synapse does not exist. We have previously identified GABA in both neural cell bodies and satellite cells (SC) and an expression of functional GABA receptors in neural cell bodies in rat TG (1). Based on a dynamic observation for GABA released to extracellular space from dispersed TG cells as well as sliced TG tissues using a novel GABA imaging system (2), we proposed a GABA handling system between neuronal cell bodies and SCs which might play an important physiological role in TG (3). In this study, to confirm our hypothesis further, we investigated intracellular GABA and related molecules especially in the dispersed cell culture of TG using immunocytochemistry. In the dispersed TG cell cultures, neurons and SCs were identified by NeuN and GFAP staining, respectively. The dispersed neurons, but not SC, were immunopositive for a glutamic acid decarboxylase (GAD). After 12 h incubation in a GABA-free medium, SCs showed a lower GABA signal intensity than neurons, whereas they showed a similar signal intensity after 12 h incubation in a medium containing 100 M GABA. We also found neurons, not SCs, were immunopositive for GABA transporter 3 (GAT3). These findings are consistent with our hypothesis that neuronal cell bodies synthesize and spontaneously release GABA via GAT and satellite cells uptake and release GABA via DNDS-sensitive pathway (3). Research fund: KAKENHI (21591990).
References (1) Hayasaki, et al., 2006. Eur. J. Neurosci. 23, 745–757. (2) Morishima, et al., 2010. Neurosci. Res. 67, 347–353. (3) Hayasaki, et al., 2010. NEURO2010 , pp. P2–b24.
doi:10.1016/j.neures.2011.07.963
P3-c10 Involvement of TRPM2 channel in mechanisms underlying microglial activation Hisashi Shirakawa , Ayaka Kusano, Takahito Miyake, Masakazu Konno, Takayuki Nakagawa, Shuji Kaneko Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto, Japan Microglia, the resident immunocompetent cells in the central nervous system (CNS), play an important role in maintaining brain homeostasis. However, under pathological conditions, microglia become activated, indicated by an altered morphology and release of various inflammatory mediators including nitric oxide (NO) and cytokines/chemokines, involved in CNS disease pathogenesis. Although these functions of activated microglia are conjunction with Ca2+ signaling, there is not enough explanation about Ca2+ mobilization in microglia. Transient receptor potential melastatin 2 (TRPM2), a Ca2+ -permeable nonselective cation channel, is highly expressed in brain and immune cells including microglia. We previously found that TRPM2 deficiency impaired mechanical allodynia in sciatic nerve injuryinduced neuropathic pain model and NO production in lipopolysaccharide (LPS)/interferon gamma (IFN␥)-activated microglia. Here, we focused on the mechanisms underlying LPS/IFN␥-induced microglial activation. Ca2+ imaging experiments revealed that LPS/IFN␥ stimulation gradually increased intracellular Ca2+ concentrations in wild-type (WT) mice-derived microglia whereas no elevation was observed in TRPM2-knockout (KO) mice-derived
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in vitro and in vivo. Cultured astrocytes were activated by polyI:C treatment in vitro, leading to an increase in mRNA levels of IFITM3 as well as inflammatory cytokines. When primary cultured neurons were treated with the conditioned-medium of polyI:C-treated astrocytes (polyI:C-ACM), neurite outgrowth and spine formation were diminished. The neurodevelopmental abnormalities of cultured neurons induced by polyI:C-ACM were alleviated when astrocytes were prepared from ifitm3−/− mice. Furthermore, aberrant MAP2 expression in the frontal cortex in vivo as well as memory impairment was evident in polyI:C-treated wild-type mice, but such neurodevelopmental and behavioral abnormalities were not induced in polyI:C-treated ifitm3−/− mice. These results suggest a role for IFITM3 in neurodevelopmental disorders associated with perinatal immune activation. Research fund: Grant-in-Aid for JSPS Fellows.
metric analysis of microglia labeled by intracellular injection in fixed slices. Using hierarchical clustering analysis, three groups were disclosed. The first type was mainly composed of microglia from sham control mice, and the cells showed a characteristic ramified morphology of resting microglia. The second type was composed of microglia from D3 and D7 mice, and the cells showed bushy or hypertrophic appearance. The third type was mainly composed of microglia from D14 mice. Interestingly, the shape of microglia on D14 was intermediate between hypertrophic and ramified. Taken together, our findings indicate that microglia in the axotomized hypoglossal nucleus might be proliferative on D3, fully activated on D7, and rather deactivated on D14.
doi:10.1016/j.neures.2011.07.960
P3-c09 An immunocytochemical study for the local GABAergic system in rat trigeminal ganglion
P3-c07 Distinct roles of HPC-1/STX1A and STX1B in neuronal survival Takefumi Kofuji 1 , Tomonori Fujiwara 2 , Masumi Sanada 2 , Tatsuya Mishima 2 , Kimio Akagawa 2
1 Radioisotope Laboratory, Kyorin University School of Medicine, Tokyo, Japan 2 Deparment of Cell Physiology, Kyorin University School of Medicine, Tokyo, Japan
The membrane protein syntaxin1 is believed to play an important role in the exocytosis of synaptic vesicles. In neurons, two types of syntaxin1 isoforms, HPC-1/syntaxin1A (STX1A) and syntaxin1B (STX1B), both of which are transcribed from distinct genes and thought to have similar functions as neuronal t-SNARE, are predominantly expressed. Previously, we generated the gene knockout mice for STX1A or STX1B. Interestingly, STX1A null mutant mice normally developed (Fujiwara et al., J. Neurosci., 2007), but STX1B null mutant mice were died within 2 weeks after birth, suggesting that STX1A and STX1B might have distinct roles in neuronal function. In this study, we examined the dissociated culture neurons from STX1A- and STX1B-null mutant mice. On poly-l-lysine-coated coverglass, dissociated STX1A-deficient neurons normally survived compared with WT neurons, but STX1B-deficient neurons died suddenly after 7–8 days in vitro (DIV). However, the number of synaptopysin puncta and that of main branching were not different between survived STX1A- and STX1B-deficient neurons. Interestingly, on WT glial feeder layers, STX1B-deficient neurons normally survived. However, glial feeder layers from STX1B null mutant mice have no survival effect unlike glial feeder layers from STX1A null mutant mice. Furthermore, conditioned medium from WT and STX1A-deficient glial feeder layers had survival effect for STX1B-deficient neurons, while conditioned medium from STX1Bdeficient glial feeder layers did not support the survival. These data suggest that STX1A and STX1B might have distinct roles in neuronal survival which might be supported by glia function lacking in STX1B null mutant mice. Implication of these different roles in STX1A and STX1B will be discussed. doi:10.1016/j.neures.2011.07.961
P3-c08 Time course alterations in immunophenotypic and morphological features of microglia following hypoglossal axotomy in mice Jun Yamada 1 , Shozo Jinno 2 1 Department of Anatomy and Neurobiology, Graduate School of Medical Sciences, Kyushu University 2 Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University
Microglia, the resident immune cells of the brain, become activated in response to various noxious stimuli. The peripheral nerve transection paradigm allows systematic and detailed study of microglial reactions and neuronal cell death. Here, we quantitatively examined the time course alterations in immunophenotypic and morphological features of microglia following hypoglossal axotomy in mice. First, we analyzed the antigen expression profiles of microglia in the hypoglossal nucleus with normal nerve (sham control) and nucleus with transected nerve from the same animal 3 days (D3), 7 days (D7) and D14 after axotomy. In the sham control mice, virtually no microglia expressed a dividing cell marker, proliferating cell nuclear antigen (PCNA). The expression ratios of PCNA in microglia significantly increased on D3 (90%), while the ratios showed an immediate decline (less than 10%) on D7, D14 and D28. Voltage-dependent potassium (Kv) channels 1.3 and 1.5 were rarely observed in microglia of sham control and D3 mice, but about 80% of microglia expressed Kv1.3 and Kv1.5 on D7, and almost all microglia expressed Kv1.3 and Kv1.5 on D14. The expression ratios of Kv1.3 and Kv1.5 were declined on D28 (40%). Then, we carried out morpho-
doi:10.1016/j.neures.2011.07.962
Hana Hayasaki 1 , Toshiki Morishima 2 , Yoshiro Sohma 3 , Sachiko Yoshida 4 , Atsuo Fukuda 2 , Yoshinori Otsuki 1 1
Department of Anatomy, Osaka Medical College, Osaka, Japan 2 Dept. Physiol., Hamamatsu Univ. Sch. Med 3 Dept. Pharmacol., Keio Univ. Sch. Med 4 Dept. Material Sci., Toyohashi Univ. Tech GABAergic system has been intensively studied in the central nervous system, however not in the trigeminal ganglion (TG) in which synapse does not exist. We have previously identified GABA in both neural cell bodies and satellite cells (SC) and an expression of functional GABA receptors in neural cell bodies in rat TG (1). Based on a dynamic observation for GABA released to extracellular space from dispersed TG cells as well as sliced TG tissues using a novel GABA imaging system (2), we proposed a GABA handling system between neuronal cell bodies and SCs which might play an important physiological role in TG (3). In this study, to confirm our hypothesis further, we investigated intracellular GABA and related molecules especially in the dispersed cell culture of TG using immunocytochemistry. In the dispersed TG cell cultures, neurons and SCs were identified by NeuN and GFAP staining, respectively. The dispersed neurons, but not SC, were immunopositive for a glutamic acid decarboxylase (GAD). After 12 h incubation in a GABA-free medium, SCs showed a lower GABA signal intensity than neurons, whereas they showed a similar signal intensity after 12 h incubation in a medium containing 100 M GABA. We also found neurons, not SCs, were immunopositive for GABA transporter 3 (GAT3). These findings are consistent with our hypothesis that neuronal cell bodies synthesize and spontaneously release GABA via GAT and satellite cells uptake and release GABA via DNDS-sensitive pathway (3). Research fund: KAKENHI (21591990).
References (1) Hayasaki, et al., 2006. Eur. J. Neurosci. 23, 745–757. (2) Morishima, et al., 2010. Neurosci. Res. 67, 347–353. (3) Hayasaki, et al., 2010. NEURO2010 , pp. P2–b24.
doi:10.1016/j.neures.2011.07.963
P3-c10 Involvement of TRPM2 channel in mechanisms underlying microglial activation Hisashi Shirakawa , Ayaka Kusano, Takahito Miyake, Masakazu Konno, Takayuki Nakagawa, Shuji Kaneko Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto, Japan Microglia, the resident immunocompetent cells in the central nervous system (CNS), play an important role in maintaining brain homeostasis. However, under pathological conditions, microglia become activated, indicated by an altered morphology and release of various inflammatory mediators including nitric oxide (NO) and cytokines/chemokines, involved in CNS disease pathogenesis. Although these functions of activated microglia are conjunction with Ca2+ signaling, there is not enough explanation about Ca2+ mobilization in microglia. Transient receptor potential melastatin 2 (TRPM2), a Ca2+ -permeable nonselective cation channel, is highly expressed in brain and immune cells including microglia. We previously found that TRPM2 deficiency impaired mechanical allodynia in sciatic nerve injuryinduced neuropathic pain model and NO production in lipopolysaccharide (LPS)/interferon gamma (IFN␥)-activated microglia. Here, we focused on the mechanisms underlying LPS/IFN␥-induced microglial activation. Ca2+ imaging experiments revealed that LPS/IFN␥ stimulation gradually increased intracellular Ca2+ concentrations in wild-type (WT) mice-derived microglia whereas no elevation was observed in TRPM2-knockout (KO) mice-derived