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A study on the phenotype of syntaxin1B knockout mice
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-c19 A study on the phenotype of syntaxin1B knockout mice
Tomonori Fujiwara 1 , Masumi Sanada 1 , Takefumi Kofuji 2 , Tatsuya Mishima 1 , Kimio Akagawa 1 1
Cell Physiology, Kyorin univercity school of medicine, Tokyo, Japan 2 Radio Isotope Laboratory, Kyorin univercity school of medicine, Tokyo, Japan Syntaxin1 is thought to regulate 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 believed to have similar function 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 (2007 Fujiwara et al J. Neurosci.), but STX1B null mutant mice were dead within 2 weeks after birth, suggesting that STX1A and STX1B might have distinct roles in neuronal function. In this study, we analyzed the phenotypes of STX1B knockout mice in detail. The fast synaptic transmission in cultured hippocampal neurons derived from neonatal STX1B null mutant mice was impaired unlike in the case of STX1A null mutant mice. We have also analyzed STX1B knockout mice phenotype using heterozygote. Interestingly, STX1B heterozygous mutant mice exhibited severe seizure phenotype unlike STX1A heterozygous mutant mice. It was also observed that STX1B heterozygous mutant mice exhibited several abnormal behavioral profiles. Additionally, LTP in hippocampal slices was reduced in STX1B heterozygous mutant mice. Implication of these abnormalities in STX1B knockout mice will be discussed. doi:10.1016/j.neures.2010.07.1572
e355
sequential actions of cyclooxygenases (COX) and PGE synthases, and binds to one of four PGE receptors, EP1, EP2, EP3 and EP4 for its actions. However, where PGE2 is produced and where it acts for functional hyperemia, remain elusive. To address this issue, we first examined the distribution of COX2, a COX isoform critical for functional hyperemia in the brain. COX-2 was expressed in pyramidal neurons in all tested strains (C57BL/6, FVB, DBA/2, BALB/c, 129X1/Sv, C3H/He). Notably, we could not detect COX-2 expression in astrocytes in any of these strains. Neither COX-1 expression was detected in astrocytes, except in FVB mice. These data suggest that neurons, rather than astrocytes, are the primary source of PGE2 at least in physiological condition. We further characterized prostanoid receptors involved in functional hyperemia. Using laser-capture microdissection and RT-PCR, we identified expression of EP1, EP3 and EP4, but not EP2, in the perivascular area of the brain. Given a role of cAMP signaling in smooth muscle relaxation, we focused on EP4, a Gs-coupled receptors. Immunohistochemistry confirmed EP4 expression in smooth muscles of cerebral arteriole as well as in neurons. These signals were abolished in EP4-deficient mice. Using optical imaging, EP4-deficient mice were impaired in functional hyperemia in somatosensory cortex following electrical plantar stimulation. In sum, our data suggest that PGE2 and EP4 mediate signaling from neurons to arterioles in functional hyperemia. doi:10.1016/j.neures.2010.07.1574
P3-c22 Localization of tissue plasminogen activator and plasminogen in the mouse hypothalamus Yuki Taniguchi , Shoko Morita, Naoko Inoue, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology
P3-c20 Identification of Tomoregulin-1 as a novel addicsinassociating factor Taku Arano 1,2 , Mitsushi J. Ikemoto 1,2 1
Biomedical Research Institute, AIST, Ibaraki, Japan 2 Grad. School of Sci. Toho University, Chiba, Japan Addicsin is a negative modulator of Na+-dependent neural glutamate transporter EAAC1. Addicsin is considered to have various physiological functions by forming a multimeric hetero-complex. However, its functions remain largely unknown. To examine its novel physiological function, we tried to identify potential factors associating with mouse addicsin by performing a yeast two-hybrid screen from a prey cDNA library prepared from the 17–day-old mouse embryos using a full-length of addicsin cDNA as bait. From 3.5×106 yeast transformants, we finally isolated 47 positive clones which clearly displayed ␣-galactosidase activity. Of these positive clones, two clones yielded an identical cDNA sequence encoding mouse Tomoregulin1(TR-1), which is known as a type-I transmembrane protein with short and highly conserved cytoplasmic tail. Immunoprecipitation assay using COS7 cells demonstrated that TR-1 bound directly to addicsin and that the Cterminal region located at amino acid 144–188 of addicsin was important role for the formation of addicsin-TR-1 hetero-complex. On the other hand, addicsin interacted with the region except a short cytoplasmic C-terminal region of TR-1. Immunohistochemical assay revealed that both addicsin and TR-1 were expressed in neurons and showed the same expression profile in the mouse matured brain such as hippocampus. These results strongly suggest that addicsin directly associates with TR-1. Previous reports demonstrate that TR-1 has a possibility for exerting a trophic effect on neurons because TR-2, a homologue of TR-1, can promote survival of hippocampal and mesencephalic neurons in primary culture. These data support the idea that formation of addicsin-TR-1 complex may modulate the survival of neurons. The novel function of addicsin will be discussed. doi:10.1016/j.neures.2010.07.1573
P3-c21 Roles of prostaglandin E2-EP4 signaling in functional hyperemia in the brain Tomohiro Aoki 1 , Tomoyuki Furuyashiki 1 , Akira Takatsuki 2 , Akitoshi Seiyama 2 , Shuh Narumiya 1 1 Department of Pharmacology, Kyoto University Graduate School of Medicine, Japan 2 Department of Medical Devices for Diagnoses, Kyoto University Graduate School of Medicine, Kyoto, Japan
In the brain, neuronal activity induces functional hyperemia, a rapid and local increase in blood flow, mainly due to dilation of cerebral arterioles. It has been proposed that synthesis of prostaglandin (PG) E2, a bioactive lipid, by astrocytes is critical in this process. PGE2 is synthesized from arachidonic acid by
Tissue plasminogen activator (tPA) and plasminogen are known to have numerous brain functions such as learning, memory, anxiety behavior, and ischemia-induced neuronal death, little attention has yet been given to their expression in the hypothalamus. In the present study, we investigated the localization of tPA and plasminogen in the adult mouse hypothalamus using light microscopic immunohistochemistry. Western blot analysis revealed that tPA and plasminogen antibodies specfically recongnized tPA and plasminogen, respectively. The immunoreactivity of tPA was observed at OVLT, LSV, MnPO, PVN, SFO, BST, Sch, median eminence, and Arc, and localized at neuornal dendrites and somata. The immunoreactivity of plasminogen was seen at VDB, HDB, OVLT, PVN, SON, PVN, SFO, median eminence, and Arc, and localized mainly at somata. Higher magnification observation showed that tPA and plasminogen immunoreactivities were seen as numerous punctuate structures in somata and dendrites of hypothalamic neurons, suggesting vesicular localization of tPA and plasminogen. The distribution pattern in the hypothalamus was thus different between tPA and plasminogen. The present study demonstrated the localizationof tPA and plasminogen in the mammalian hypothalamus. doi:10.1016/j.neures.2010.07.1575
P3-c23 Vesicular localization of tPA and plasminogen in barin neurons Seiji Miyata , Naoko Inoue, Shoko Morita, Atsushi Hourai Department of Applied Biology, Kyoto Institute of Technology Although the tissue plasminogen activator (tPA) and plasminogen contribute to numerous brain functions such as learning, memory, anxiety behavior, and ischemia-induced neuronal death, little attention has yet been given to the subcellular localization of tPA and plasminogen in the brain. In the present study, we investigated the vesicular localization of tPA and plasminogen in the adult mouse brain using light and electron microscopic immunohistochemistry. In the hippocampus, tPA immunoreactivity was particularly strong at dendrites of the CA3 pyramidal and dentate gyrus neurons. Plasminogen immunoreactivity was strong at somata of parvalbumin (PV)-positive GABAergic interneurons and moderate at somata of the CA1-3 pyramidal and dentate gyrus neurons. In the cerebral cortex, tPA immunoreactivity was strong at somata and moderate at dendrites of the layer II/III and V neurons, but tPA immunoreactivity of PV-positive somata was weaker as compared to that of the neighboring pyramidal neurons. Plasminogen immunoreactivity was evident at somata of the layer II/III and layer V neurons, but plasminogen immunoreactivity of PV-positive somata was stronger as compared to that of the neighboring pyramidal neurons. Higher magnification observation showed that tPA and plasminogen immunoreactivities were seen as numerous punctuate structures in somata and dendrites of the hippocampal and cerebral neurons. An electron microscopic study fur-
P3-c19 A study on the phenotype of syntaxin1B knockout mice
Tomonori Fujiwara 1 , Masumi Sanada 1 , Takefumi Kofuji 2 , Tatsuya Mishima 1 , Kimio Akagawa 1 1
Cell Physiology, Kyorin univercity school of medicine, Tokyo, Japan 2 Radio Isotope Laboratory, Kyorin univercity school of medicine, Tokyo, Japan Syntaxin1 is thought to regulate 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 believed to have similar function 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 (2007 Fujiwara et al J. Neurosci.), but STX1B null mutant mice were dead within 2 weeks after birth, suggesting that STX1A and STX1B might have distinct roles in neuronal function. In this study, we analyzed the phenotypes of STX1B knockout mice in detail. The fast synaptic transmission in cultured hippocampal neurons derived from neonatal STX1B null mutant mice was impaired unlike in the case of STX1A null mutant mice. We have also analyzed STX1B knockout mice phenotype using heterozygote. Interestingly, STX1B heterozygous mutant mice exhibited severe seizure phenotype unlike STX1A heterozygous mutant mice. It was also observed that STX1B heterozygous mutant mice exhibited several abnormal behavioral profiles. Additionally, LTP in hippocampal slices was reduced in STX1B heterozygous mutant mice. Implication of these abnormalities in STX1B knockout mice will be discussed. doi:10.1016/j.neures.2010.07.1572
e355
sequential actions of cyclooxygenases (COX) and PGE synthases, and binds to one of four PGE receptors, EP1, EP2, EP3 and EP4 for its actions. However, where PGE2 is produced and where it acts for functional hyperemia, remain elusive. To address this issue, we first examined the distribution of COX2, a COX isoform critical for functional hyperemia in the brain. COX-2 was expressed in pyramidal neurons in all tested strains (C57BL/6, FVB, DBA/2, BALB/c, 129X1/Sv, C3H/He). Notably, we could not detect COX-2 expression in astrocytes in any of these strains. Neither COX-1 expression was detected in astrocytes, except in FVB mice. These data suggest that neurons, rather than astrocytes, are the primary source of PGE2 at least in physiological condition. We further characterized prostanoid receptors involved in functional hyperemia. Using laser-capture microdissection and RT-PCR, we identified expression of EP1, EP3 and EP4, but not EP2, in the perivascular area of the brain. Given a role of cAMP signaling in smooth muscle relaxation, we focused on EP4, a Gs-coupled receptors. Immunohistochemistry confirmed EP4 expression in smooth muscles of cerebral arteriole as well as in neurons. These signals were abolished in EP4-deficient mice. Using optical imaging, EP4-deficient mice were impaired in functional hyperemia in somatosensory cortex following electrical plantar stimulation. In sum, our data suggest that PGE2 and EP4 mediate signaling from neurons to arterioles in functional hyperemia. doi:10.1016/j.neures.2010.07.1574
P3-c22 Localization of tissue plasminogen activator and plasminogen in the mouse hypothalamus Yuki Taniguchi , Shoko Morita, Naoko Inoue, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology
P3-c20 Identification of Tomoregulin-1 as a novel addicsinassociating factor Taku Arano 1,2 , Mitsushi J. Ikemoto 1,2 1
Biomedical Research Institute, AIST, Ibaraki, Japan 2 Grad. School of Sci. Toho University, Chiba, Japan Addicsin is a negative modulator of Na+-dependent neural glutamate transporter EAAC1. Addicsin is considered to have various physiological functions by forming a multimeric hetero-complex. However, its functions remain largely unknown. To examine its novel physiological function, we tried to identify potential factors associating with mouse addicsin by performing a yeast two-hybrid screen from a prey cDNA library prepared from the 17–day-old mouse embryos using a full-length of addicsin cDNA as bait. From 3.5×106 yeast transformants, we finally isolated 47 positive clones which clearly displayed ␣-galactosidase activity. Of these positive clones, two clones yielded an identical cDNA sequence encoding mouse Tomoregulin1(TR-1), which is known as a type-I transmembrane protein with short and highly conserved cytoplasmic tail. Immunoprecipitation assay using COS7 cells demonstrated that TR-1 bound directly to addicsin and that the Cterminal region located at amino acid 144–188 of addicsin was important role for the formation of addicsin-TR-1 hetero-complex. On the other hand, addicsin interacted with the region except a short cytoplasmic C-terminal region of TR-1. Immunohistochemical assay revealed that both addicsin and TR-1 were expressed in neurons and showed the same expression profile in the mouse matured brain such as hippocampus. These results strongly suggest that addicsin directly associates with TR-1. Previous reports demonstrate that TR-1 has a possibility for exerting a trophic effect on neurons because TR-2, a homologue of TR-1, can promote survival of hippocampal and mesencephalic neurons in primary culture. These data support the idea that formation of addicsin-TR-1 complex may modulate the survival of neurons. The novel function of addicsin will be discussed. doi:10.1016/j.neures.2010.07.1573
P3-c21 Roles of prostaglandin E2-EP4 signaling in functional hyperemia in the brain Tomohiro Aoki 1 , Tomoyuki Furuyashiki 1 , Akira Takatsuki 2 , Akitoshi Seiyama 2 , Shuh Narumiya 1 1 Department of Pharmacology, Kyoto University Graduate School of Medicine, Japan 2 Department of Medical Devices for Diagnoses, Kyoto University Graduate School of Medicine, Kyoto, Japan
In the brain, neuronal activity induces functional hyperemia, a rapid and local increase in blood flow, mainly due to dilation of cerebral arterioles. It has been proposed that synthesis of prostaglandin (PG) E2, a bioactive lipid, by astrocytes is critical in this process. PGE2 is synthesized from arachidonic acid by
Tissue plasminogen activator (tPA) and plasminogen are known to have numerous brain functions such as learning, memory, anxiety behavior, and ischemia-induced neuronal death, little attention has yet been given to their expression in the hypothalamus. In the present study, we investigated the localization of tPA and plasminogen in the adult mouse hypothalamus using light microscopic immunohistochemistry. Western blot analysis revealed that tPA and plasminogen antibodies specfically recongnized tPA and plasminogen, respectively. The immunoreactivity of tPA was observed at OVLT, LSV, MnPO, PVN, SFO, BST, Sch, median eminence, and Arc, and localized at neuornal dendrites and somata. The immunoreactivity of plasminogen was seen at VDB, HDB, OVLT, PVN, SON, PVN, SFO, median eminence, and Arc, and localized mainly at somata. Higher magnification observation showed that tPA and plasminogen immunoreactivities were seen as numerous punctuate structures in somata and dendrites of hypothalamic neurons, suggesting vesicular localization of tPA and plasminogen. The distribution pattern in the hypothalamus was thus different between tPA and plasminogen. The present study demonstrated the localizationof tPA and plasminogen in the mammalian hypothalamus. doi:10.1016/j.neures.2010.07.1575
P3-c23 Vesicular localization of tPA and plasminogen in barin neurons Seiji Miyata , Naoko Inoue, Shoko Morita, Atsushi Hourai Department of Applied Biology, Kyoto Institute of Technology Although the tissue plasminogen activator (tPA) and plasminogen contribute to numerous brain functions such as learning, memory, anxiety behavior, and ischemia-induced neuronal death, little attention has yet been given to the subcellular localization of tPA and plasminogen in the brain. In the present study, we investigated the vesicular localization of tPA and plasminogen in the adult mouse brain using light and electron microscopic immunohistochemistry. In the hippocampus, tPA immunoreactivity was particularly strong at dendrites of the CA3 pyramidal and dentate gyrus neurons. Plasminogen immunoreactivity was strong at somata of parvalbumin (PV)-positive GABAergic interneurons and moderate at somata of the CA1-3 pyramidal and dentate gyrus neurons. In the cerebral cortex, tPA immunoreactivity was strong at somata and moderate at dendrites of the layer II/III and V neurons, but tPA immunoreactivity of PV-positive somata was weaker as compared to that of the neighboring pyramidal neurons. Plasminogen immunoreactivity was evident at somata of the layer II/III and layer V neurons, but plasminogen immunoreactivity of PV-positive somata was stronger as compared to that of the neighboring pyramidal neurons. Higher magnification observation showed that tPA and plasminogen immunoreactivities were seen as numerous punctuate structures in somata and dendrites of the hippocampal and cerebral neurons. An electron microscopic study fur-