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Kunihiko Obata - One of the best experts on this subject based on the ideXlab platform.
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respiratory activity in brainstem of fetal mice lacking Glutamate Decarboxylase 65 67 and vesicular gaba transporter
Neuroscience, 2007Co-Authors: Morimitsu Fujii, Yuchio Yanagawa, Akiko Arata, N Kanbarakume, Kenji Saito, Kunihiko ObataAbstract:Abstract The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid Decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking Glutamate Decarboxylase 67. Eur J Neurosci 21:173–178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem–spinal cord blocks prepared from GAD65−/−:67−/− and vesicular GABA transporter (VGAT) −/− mice on embryonic day 14 (E14)–E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65−/−:67−/− preparations but not in VGAT−/− preparations. C4 discharges were observed in the knockout mice on E14–E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a “blind” patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65−/−:67−/− mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT−/− mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.
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Respiratory activity in brainstem of fetal mice lacking Glutamate Decarboxylase 65/67 and vesicular GABA transporter
Neuroscience, 2007Co-Authors: Morimitsu Fujii, Yuchio Yanagawa, Akiko Arata, Kenji Saito, N. Kanbara-kume, Kunihiko ObataAbstract:Abstract The respiratory neural network in the mammalian medulla oblongata shows rhythmic activity before birth. GABA and glycine are considered to be involved in control of respiratory rhythm. Recently we have demonstrated respiratory failure in glutamic acid Decarboxylase (GAD) 67-deficient mice [Tsunekawa N, Arata A, Obata K (2005) Development of spontaneous mouth/tongue movement and related neural activity, and their repression in mouse fetus lacking Glutamate Decarboxylase 67. Eur J Neurosci 21:173–178]. To further evaluate the involvement of GABA and glycine in fetal respiratory function, we studied neural activities in brainstem–spinal cord blocks prepared from GAD65−/−:67−/− and vesicular GABA transporter (VGAT) −/− mice on embryonic day 14 (E14)–E15 and E18. In these knockout mice, the synthesis of GABA and the vesicular release of GABA and glycine are completely absent, respectively. Spontaneous respiratory discharges were observed in the ventral roots at the cervical cord (C) 4 level from wild-type mice but not from the knockout mice on E18. Administration of substance P induced C4 discharges in GAD65−/−:67−/− preparations but not in VGAT−/− preparations. C4 discharges were observed in the knockout mice on E14–E15, although the frequency was lower than that in the wild-type. Neuronal activities in the respiratory network of the E18 brainstem were recorded using a “blind” patch-clamp technique. Expiratory and inspiratory neurons with their characteristic firing patterns were observed in the wild-type fetuses. Strychnine reversed inspiratory-phase hyperpolarization to large depolarization in expiratory neurons. On the other hand, neurons in the same area of the knockout mice fired spontaneously without any rhythm. Substance P induced hyperpolarizing potentials in medullary neurons of GAD65−/−:67−/− mice. Further administration of strychnine induced large depolarizing potentials. Rhythmic activities were not observed in VGAT−/− mice even in the presence of substance P and strychnine. These results indicate that the lack of GABA and glycine impairs the function of the respiratory network in mouse fetuses and the impairment progresses with fetal age.
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altered conditioned fear behavior in Glutamate Decarboxylase 65 null mutant mice
Genes Brain and Behavior, 2003Co-Authors: Oliver Stork, H Yamanaka, Simone Stork, Nobuko Kume, Kunihiko ObataAbstract:We investigated the involvement of the 65 kDa isoform of glutamic acid Decarboxylase (GAD65) and GAD65-mediated γ-aminobutyric acid (GABA) synthesis in the formation and expression of Pavlovian fear memory. To this end, behavioral, endocrine and autonomic parameters were examined during conditioned fear retrieval of mice with targeted ablation of the GAD65 gene (GAD65–/– mice). These mutant mice were found to display specific fear behavior (freezing, escape), as well as autonomic (increased defecation) and endocrine activation (increased plasma corticosterone) during fear memory retrieval. However, freezing was reduced and flight and escape behavior were increased in GAD65–/– mice compared to their wild type and heterozygous littermates, while corticosterone levels and defecation rates did not differ between genotypes. Active defensive behavior of GAD65–/– mice was observed during both auditory cued and contextual retrieval of fear memory, as well as immediately after conditioning. These data indicate a selectively altered behavioral fear response in GAD65–/– mice, most likely due to deficits in threat estimation or the elicitation of appropriate conditioned fear behavior, and suggest that GAD65 is a genetic determinant of conditioned fear behavior. GAD65–/– mice provide a valuable tool to further dissect the GABAergic mechanisms involved in fear and anxiety and to model GABA-related neurological and psychiatric disorders.
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Structure of the mouse Glutamate Decarboxylase 65 gene and its promoter: preferential expression of its promoter in the GABAergic neurons of transgenic mice.
Journal of neurochemistry, 2002Co-Authors: Kimitoshi Makinae, Kunihiko Obata, Takashi Kobayashi, Takayasu Kobayashi, Hideichi Shinkawa, Hiroyuki Sakagami, Hisatake Kondo, Fumi Tashiro, Jun-ichi Miyazaki, Shinri TamuraAbstract:GABA is synthesized by Glutamate Decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.
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Synaptic localization of the 67,000 mol. wt isoform of Glutamate Decarboxylase and transmitter function of GABA in the mouse cerebellum lacking the 65,000 mol. wt isoform
Neuroscience, 1999Co-Authors: Kunihiko Obata, Takaichi Fukuda, Shiro Konishi, H. Mitoma, Toshio KosakaAbstract:Subcellular localization of the 67,000 mol. wt isoform of Glutamate Decarboxylase and neurotransmitter function of GABA were investigated in the cerebellum of the mice lacking the 65,000 mol. wt isoform of Glutamate Decarboxylase. The GABA content decreased by 25% in the cerebellum. Putative GABA-releasing terminals from basket/stellate and Golgi cells were immunostained with Glutamate Decarboxylase-67 antibody. Basket cell-derived inhibitory postsynaptic currents in Purkinje cells and the high potassium-induced release of GABA were not significantly affected. Although previous investigations have suggested that Glutamate Decarboxylase-65 is mainly involved in transmitter synthesis and that Glutamate Decarboxylase-67 is transported to the nerve terminals only after association with Glutamate Decarboxylase-65, the present results indicate that Glutamate Decarboxylase-67 is independently concentrated in the nerve terminals and provides GABA for synaptic transmission in the absence of Glutamate Decarboxylase-65.
Howard Prentice - One of the best experts on this subject based on the ideXlab platform.
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Activation of Brain L-Glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)
Molecular Neurobiology, 2017Co-Authors: Chi-chi Chou, Jigar Pravinchandra Modi, Chen-yu Wang, Pei-chien Hsu, Yi-hsuan Lee, Kai-fa Huang, Andrew H. J. Wang, Changlong Nan, Xupei Huang, Howard PrenticeAbstract:Protein phosphorylation plays an important role in regulating soluble L-glutamic acid Decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.
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Activation of Brain L-Glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)
Molecular neurobiology, 2016Co-Authors: Chi-chi Chou, Chen-yu Wang, Pei-chien Hsu, Yi-hsuan Lee, Kai-fa Huang, Andrew H. J. Wang, Changlong Nan, Xupei Huang, Jigar Modi, Howard PrenticeAbstract:Protein phosphorylation plays an important role in regulating soluble L-glutamic acid Decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform e were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C e (PKCe) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCe, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.
J R Stellar - One of the best experts on this subject based on the ideXlab platform.
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An examination of Glutamate Decarboxylase(65) immunoreactive puncta with respect to rat ventral pallidum neurons after repeated cocaine administration.
Neuroscience letters, 2000Co-Authors: K R De Leon, M S Todtenkopf, J R StellarAbstract:The ventral pallidum is known to have topographically organized reciprocal gamma-aminobutyric acid-ergic projections with the nucleus accumbens, and changes in these connections may play a role in mediating the behavioral sensitizing effect of repeated exposure to cocaine. The present study investigated Glutamate Decarboxylase-65 (GAD(65)) immunoreactivity in the rat ventral pallidum after repeated cocaine administration. Male Sprague-Dawley rats were administered bi-daily injections of 15 mg/kg cocaine or saline vehicle for 5 consecutive days. After 2 or 14 days of withdrawal, ventral pallidal sections were immunocytochemically processed for GAD(65) immunoreactive puncta and counts were made. In both groups, there were no statistically significant differences in the number or density of GAD(65) puncta in medial or lateral portions either in contact with neuronal cell bodies or in the neuropil after 2 or 14 days of withdrawal. The results suggest that there is no alteration in the number of GABAergic boutons expressing GAD(65) immunoreactivity in the ventral pallidum after repeated exposure to cocaine.
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An examination of Glutamate Decarboxylase65 immunoreactive puncta with respect to rat ventral pallidum neurons after repeated cocaine administration
Neuroscience Letters, 2000Co-Authors: K R De Leon, M S Todtenkopf, J R StellarAbstract:Abstract The ventral pallidum is known to have topographically organized reciprocal γ -aminobutyric acid-ergic projections with the nucleus accumbens, and changes in these connections may play a role in mediating the behavioral sensitizing effect of repeated exposure to cocaine. The present study investigated Glutamate Decarboxylase-65 (GAD 65 ) immunoreactivity in the rat ventral pallidum after repeated cocaine administration. Male Sprague–Dawley rats were administered bi-daily injections of 15 mg/kg cocaine or saline vehicle for 5 consecutive days. After 2 or 14 days of withdrawal, ventral pallidal sections were immunocytochemically processed for GAD 65 immunoreactive puncta and counts were made. In both groups, there were no statistically significant differences in the number or density of GAD 65 puncta in medial or lateral portions either in contact with neuronal cell bodies or in the neuropil after 2 or 14 days of withdrawal. The results suggest that there is no alteration in the number of GABAergic boutons expressing GAD 65 immunoreactivity in the ventral pallidum after repeated exposure to cocaine.
Chi-chi Chou - One of the best experts on this subject based on the ideXlab platform.
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Activation of Brain L-Glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)
Molecular Neurobiology, 2017Co-Authors: Chi-chi Chou, Jigar Pravinchandra Modi, Chen-yu Wang, Pei-chien Hsu, Yi-hsuan Lee, Kai-fa Huang, Andrew H. J. Wang, Changlong Nan, Xupei Huang, Howard PrenticeAbstract:Protein phosphorylation plays an important role in regulating soluble L-glutamic acid Decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform ε were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C ε (PKCε) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCε, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.
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Activation of Brain L-Glutamate Decarboxylase 65 Isoform (GAD65) by Phosphorylation at Threonine 95 (T95)
Molecular neurobiology, 2016Co-Authors: Chi-chi Chou, Chen-yu Wang, Pei-chien Hsu, Yi-hsuan Lee, Kai-fa Huang, Andrew H. J. Wang, Changlong Nan, Xupei Huang, Jigar Modi, Howard PrenticeAbstract:Protein phosphorylation plays an important role in regulating soluble L-glutamic acid Decarboxylase (GAD) and membrane-associated GAD activity. Previously, we reported the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 (hGAD65) and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrated that protein kinase A (PKA) and protein kinase C isoform e were the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. In the current study, using MALDI-TOF, a total of four potential phosphorylation sites were identified in GAD65, two of which (threonine-95 (T-95) and Ser-417) were not reported previously. We have identified one specific phosphorylation site, (T95), in hGAD65 that can be phosphorylated by kinase C e (PKCe) using MALDITOF. When T95 is mutated to alanine, hGAD65 could no longer be phosphorylated by PKCe, and the effect of PKC-mediated activation on hGAD65 is abolished. However, when T95 is mutated to glutamic acid, which mimics the phosphorylation status of hGAD65, the activity was greatly increased. An increase of GAD65 activity by 55 % compared to the wild type hGAD65 was observed indicating that mutation of T95 to glutamic acid mimics the effect of phosphorylation. A model depicting the role of phosphorylation of GAD65 in regulation of GABA neurotransmission is presented.
K R De Leon - One of the best experts on this subject based on the ideXlab platform.
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An examination of Glutamate Decarboxylase(65) immunoreactive puncta with respect to rat ventral pallidum neurons after repeated cocaine administration.
Neuroscience letters, 2000Co-Authors: K R De Leon, M S Todtenkopf, J R StellarAbstract:The ventral pallidum is known to have topographically organized reciprocal gamma-aminobutyric acid-ergic projections with the nucleus accumbens, and changes in these connections may play a role in mediating the behavioral sensitizing effect of repeated exposure to cocaine. The present study investigated Glutamate Decarboxylase-65 (GAD(65)) immunoreactivity in the rat ventral pallidum after repeated cocaine administration. Male Sprague-Dawley rats were administered bi-daily injections of 15 mg/kg cocaine or saline vehicle for 5 consecutive days. After 2 or 14 days of withdrawal, ventral pallidal sections were immunocytochemically processed for GAD(65) immunoreactive puncta and counts were made. In both groups, there were no statistically significant differences in the number or density of GAD(65) puncta in medial or lateral portions either in contact with neuronal cell bodies or in the neuropil after 2 or 14 days of withdrawal. The results suggest that there is no alteration in the number of GABAergic boutons expressing GAD(65) immunoreactivity in the ventral pallidum after repeated exposure to cocaine.
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An examination of Glutamate Decarboxylase65 immunoreactive puncta with respect to rat ventral pallidum neurons after repeated cocaine administration
Neuroscience Letters, 2000Co-Authors: K R De Leon, M S Todtenkopf, J R StellarAbstract:Abstract The ventral pallidum is known to have topographically organized reciprocal γ -aminobutyric acid-ergic projections with the nucleus accumbens, and changes in these connections may play a role in mediating the behavioral sensitizing effect of repeated exposure to cocaine. The present study investigated Glutamate Decarboxylase-65 (GAD 65 ) immunoreactivity in the rat ventral pallidum after repeated cocaine administration. Male Sprague–Dawley rats were administered bi-daily injections of 15 mg/kg cocaine or saline vehicle for 5 consecutive days. After 2 or 14 days of withdrawal, ventral pallidal sections were immunocytochemically processed for GAD 65 immunoreactive puncta and counts were made. In both groups, there were no statistically significant differences in the number or density of GAD 65 puncta in medial or lateral portions either in contact with neuronal cell bodies or in the neuropil after 2 or 14 days of withdrawal. The results suggest that there is no alteration in the number of GABAergic boutons expressing GAD 65 immunoreactivity in the ventral pallidum after repeated exposure to cocaine.