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Intracellular taurine inhibits KCC2 transporter activity
Abstracts / Neuroscience Research 68S (2010) e109–e222
report subthreshold responses of EPSP by applying strong or weak inputs simultaneously and separately, under various input timing and at various spatial distances. Spatially, when the spatial distance between paired stimuli was less than 15 m, nonlinear amplification in EPSP summation was observed. Temporally, in the time window (−20 to +20 ms), supralinear amplification was observed. These results are closely related to the spatiotemporal-characteristic for the associative LTP without BPAP. doi:10.1016/j.neures.2010.07.2066
P1-a22 Purinergic autocrine regulation of CA3 pyramidal neurons: ATP release through pannexin-1 channels Masahito Kawamura 1,2 , David N. Ruskin 2 , Susan A. Masino 2 1
Dept Pharmacol, Jikei Univ Sch Med, Tokyo, Japan 2 Neurosci Prgm/Psychol Dept, Trinity College, Hartford, CT, USA
It is well known that activation of purinoceptors modulates neuronal activity in the CNS, and several conditions, including hypoglycemia and ischemia, alter the extracellular concentration of ATP and adenosine. In the hippocampus, ATP release from astrocytes via gap junction hemichannels is thought to be one of the sources of extracellular ATP and adenosine. It has been reported that hippocampal neurons express various gap junction proteins, yet ATP release from hippocampal neurons is not well determined. Using wholecell patch clamp recordings from acute rat hippocampal slices preparations we found that the reduced extracellular glucose caused ATP release from CA3 pyramidal neurons via pannexin-1 channels. Reduced glucose (from 11 mM to 3 mM) caused an adenosine A1 receptor-induced outward current in the CA3 pyramidal neurons of rats. The amplitude of the outward current was dose-dependent based on intracellular ATP concentration. It was significantly smaller when cells were loaded with the same concentration of intracellular adenosine, indicating that ATP is released directly from the recorded pyramidal neurons. Non-selective gap junction blocker carbenoxolone or pannexin-1 mimetic blocking peptide 10 panx significantly inhibited the outward current. Together, these results suggest that ATP is released directly from CA3 pyramidal neurons through pannexin-1 channels upon reducing of extracellular glucose; the subsequent activation of adenosine A1 receptors decreases CA3 excitability in an autocrine manner.
of taurine transporter, shifted EGABA of the cells to the negative direction. Taken together, intracellularly incorporated taurine may be implicated in developmental regulation of KCC2 activity. The detail effect is further studied. doi:10.1016/j.neures.2010.07.2068
P1-a24 Observation of developmental change of glutamate operation to glutamate or GABA release using enzymelinked photoassay device Takahito Jimbu 1 , Genta Yaegashi 1 , Kei Maruya 1 , Shigeki Itoh 1 , Naohiro Hozumi 2 , Sachiko Yoshida 1 1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 Aichi Inst. of Technology, Toyota
Glutamate and ␥-amino butyric acid (GABA) play important roles in cerebellar differentiations and functions. In the mature cortex, they are released from synapses and taken up by transporter molecules. In developing cortex, although neuronal synapses are not enough to regulate the neurotransmitters, immature could release and regulate cortex glutamate and GABA in order to improve them to develop. We have reported glutamate and GABA releases using enzyme-linked photo assay systems. GABA release was observed underneath the Purkinje cell layer and in the external granular layer (EGL), where no GABAergic neurons located. This ambient GABA supposed to be released from glial cells, induced glutamate release in the EGL, while it was suppressed by applied glutamate. In this study, we reported the function of glutamate and its analogues through the development of cerebellar cortex to release of the transmitters. In the first postnatal week, the glutamate transporter antagonist inhibited the decrement of GABA release with glutamate. The suppression of GABA release with glutamate and glutamate release induced by GABA were observed at the same date. In the next week, GABA release was promoted by AMPA, and then GABA-induced glutamate release disappeared. Glutamate release turned to be induced by glutamate or its analogues. Using the enzyme-linked photo-device, we could observed spike and shot glutamate release stimulated by AMPA. We would discuss the temporal and spatial change of transmitter release mechanism during cerebellar development. doi:10.1016/j.neures.2010.07.2069
doi:10.1016/j.neures.2010.07.2067
P1-a23 Intracellular taurine inhibits KCC2 transporter activity Koichi Inoue 1 , Tomonori Furukawa 1 , Tatsuro Kumada 1 , Junko Jamada 2 , Tianying Wang 1 , Atsuo Fukuda 1 1
e113
2
Dept Physiol, Hamamatsu Univ Sch of Med, Hamamatsu Dept Neurophysiol, Hirosaki Univ Sch of Med, Hirosaki
K+ -Cl− cotransporter 2 (KCC2) is a neuron-specific ion transporter which effluxes Cl− out of cells, and relatively low intracellular Cl− concentration ([Cl− ]i ) elicits inhibitory action of neurotransmitters such as GABA and glycine. Lowering [Cl− ]i takes place along with developmental upregulation of KCC2, but some cases are also reported that KCC2 does not function to extrude Cl− in spite of its apparent expression. Taurine is another inhibitory neurotransmitter and abundant in mammalian brains, especially in embryonic stages. Here we examined the effect of taurine on KCC2 activity. Rat embryonic cerebral cortices were dissected and KCC2-expressing plasmids were then transfected before plating for primary culture. Transfected cells were either untreated or treated with taurine for 3 days and then whole-cell patch-clamp recording was performed with low Cl− -pipette solution to estimate reversal potential of GABA (EGABA ). EGABA of cells without taurine was −49.5 ± 0.4 mV, while that of cells with taurine was −45.3 ± 0.3 mV (p < 0.05), suggesting that taurine inhibited KCC2 transporter activity. When staurosporine, a broad spectrum kinase inhibitor, was applied to taurine-treated KCC2-expressing cells, EGABA of the cells shifted to the negative direction, but this shift was neither obtained from taurine-untreated KCC2-expressing cells nor taurine-treated KCC2-unexpressing cells. These results suggest that KCC2 activity was inhibited by taurine in a kinase activity-dependent manner. Unexpectedly, strychinine and bicuculline, blockers of glycine and GABAA receptor, respectively, did not affect EGABA of KCC2-expressing cells when they were added along with taurine. Instead, application of GES, an inhibitor
P1-a25 Endogenous neurosteroids potentiate excitatory synaptic transimission of granule cells in the rat hippocampal dentate gyrus Motoki Tanaka , Masahiro Sokabe Dept Physiol, Nagoya Univ Grad Sch of Med, Nagoya, Japan Many studies have demonstrated that neurosteroids promote learning and memory through modulations of the synaptic transmission in the hippocampus. However, as most of the studies were done with exogenously applied neurosteroids, how much degree endogenous neurosteroids contribute to the phenomena is poorly understood. To investigate the contribution of endogenous neurosteorids to the function of synapses in the central nervous system, we measured electrophysiologically the effect of aminoglutethimide (AG), an inhibitor of steroid biosynthesis, on the neuronal activities in the dentate gyrus of rat hippocampal slices. AG markedly decreased the basal synaptic transmission and the induction of activity dependent long-term potentiation. However, paired-pulse ratio was not affected by AG, indicating that the presynaptic transmitter release keeps its function without endogenous neurosteroids. By contrast, AG significantly reduced NMDA receptor-mediated EPSC, but not GABA receptor-mediated IPSC in granule cells. These results suggest that endogenous neurosteroids operate as potent positive modulators on the excitatory synaptic transmission by potentiating the function of postsynaptic neurons in the rat dentate gyrus under physiological condition. doi:10.1016/j.neures.2010.07.2070
report subthreshold responses of EPSP by applying strong or weak inputs simultaneously and separately, under various input timing and at various spatial distances. Spatially, when the spatial distance between paired stimuli was less than 15 m, nonlinear amplification in EPSP summation was observed. Temporally, in the time window (−20 to +20 ms), supralinear amplification was observed. These results are closely related to the spatiotemporal-characteristic for the associative LTP without BPAP. doi:10.1016/j.neures.2010.07.2066
P1-a22 Purinergic autocrine regulation of CA3 pyramidal neurons: ATP release through pannexin-1 channels Masahito Kawamura 1,2 , David N. Ruskin 2 , Susan A. Masino 2 1
Dept Pharmacol, Jikei Univ Sch Med, Tokyo, Japan 2 Neurosci Prgm/Psychol Dept, Trinity College, Hartford, CT, USA
It is well known that activation of purinoceptors modulates neuronal activity in the CNS, and several conditions, including hypoglycemia and ischemia, alter the extracellular concentration of ATP and adenosine. In the hippocampus, ATP release from astrocytes via gap junction hemichannels is thought to be one of the sources of extracellular ATP and adenosine. It has been reported that hippocampal neurons express various gap junction proteins, yet ATP release from hippocampal neurons is not well determined. Using wholecell patch clamp recordings from acute rat hippocampal slices preparations we found that the reduced extracellular glucose caused ATP release from CA3 pyramidal neurons via pannexin-1 channels. Reduced glucose (from 11 mM to 3 mM) caused an adenosine A1 receptor-induced outward current in the CA3 pyramidal neurons of rats. The amplitude of the outward current was dose-dependent based on intracellular ATP concentration. It was significantly smaller when cells were loaded with the same concentration of intracellular adenosine, indicating that ATP is released directly from the recorded pyramidal neurons. Non-selective gap junction blocker carbenoxolone or pannexin-1 mimetic blocking peptide 10 panx significantly inhibited the outward current. Together, these results suggest that ATP is released directly from CA3 pyramidal neurons through pannexin-1 channels upon reducing of extracellular glucose; the subsequent activation of adenosine A1 receptors decreases CA3 excitability in an autocrine manner.
of taurine transporter, shifted EGABA of the cells to the negative direction. Taken together, intracellularly incorporated taurine may be implicated in developmental regulation of KCC2 activity. The detail effect is further studied. doi:10.1016/j.neures.2010.07.2068
P1-a24 Observation of developmental change of glutamate operation to glutamate or GABA release using enzymelinked photoassay device Takahito Jimbu 1 , Genta Yaegashi 1 , Kei Maruya 1 , Shigeki Itoh 1 , Naohiro Hozumi 2 , Sachiko Yoshida 1 1 Department of Environmental and Life Sciences, Toyohashi University of Technology 2 Aichi Inst. of Technology, Toyota
Glutamate and ␥-amino butyric acid (GABA) play important roles in cerebellar differentiations and functions. In the mature cortex, they are released from synapses and taken up by transporter molecules. In developing cortex, although neuronal synapses are not enough to regulate the neurotransmitters, immature could release and regulate cortex glutamate and GABA in order to improve them to develop. We have reported glutamate and GABA releases using enzyme-linked photo assay systems. GABA release was observed underneath the Purkinje cell layer and in the external granular layer (EGL), where no GABAergic neurons located. This ambient GABA supposed to be released from glial cells, induced glutamate release in the EGL, while it was suppressed by applied glutamate. In this study, we reported the function of glutamate and its analogues through the development of cerebellar cortex to release of the transmitters. In the first postnatal week, the glutamate transporter antagonist inhibited the decrement of GABA release with glutamate. The suppression of GABA release with glutamate and glutamate release induced by GABA were observed at the same date. In the next week, GABA release was promoted by AMPA, and then GABA-induced glutamate release disappeared. Glutamate release turned to be induced by glutamate or its analogues. Using the enzyme-linked photo-device, we could observed spike and shot glutamate release stimulated by AMPA. We would discuss the temporal and spatial change of transmitter release mechanism during cerebellar development. doi:10.1016/j.neures.2010.07.2069
doi:10.1016/j.neures.2010.07.2067
P1-a23 Intracellular taurine inhibits KCC2 transporter activity Koichi Inoue 1 , Tomonori Furukawa 1 , Tatsuro Kumada 1 , Junko Jamada 2 , Tianying Wang 1 , Atsuo Fukuda 1 1
e113
2
Dept Physiol, Hamamatsu Univ Sch of Med, Hamamatsu Dept Neurophysiol, Hirosaki Univ Sch of Med, Hirosaki
K+ -Cl− cotransporter 2 (KCC2) is a neuron-specific ion transporter which effluxes Cl− out of cells, and relatively low intracellular Cl− concentration ([Cl− ]i ) elicits inhibitory action of neurotransmitters such as GABA and glycine. Lowering [Cl− ]i takes place along with developmental upregulation of KCC2, but some cases are also reported that KCC2 does not function to extrude Cl− in spite of its apparent expression. Taurine is another inhibitory neurotransmitter and abundant in mammalian brains, especially in embryonic stages. Here we examined the effect of taurine on KCC2 activity. Rat embryonic cerebral cortices were dissected and KCC2-expressing plasmids were then transfected before plating for primary culture. Transfected cells were either untreated or treated with taurine for 3 days and then whole-cell patch-clamp recording was performed with low Cl− -pipette solution to estimate reversal potential of GABA (EGABA ). EGABA of cells without taurine was −49.5 ± 0.4 mV, while that of cells with taurine was −45.3 ± 0.3 mV (p < 0.05), suggesting that taurine inhibited KCC2 transporter activity. When staurosporine, a broad spectrum kinase inhibitor, was applied to taurine-treated KCC2-expressing cells, EGABA of the cells shifted to the negative direction, but this shift was neither obtained from taurine-untreated KCC2-expressing cells nor taurine-treated KCC2-unexpressing cells. These results suggest that KCC2 activity was inhibited by taurine in a kinase activity-dependent manner. Unexpectedly, strychinine and bicuculline, blockers of glycine and GABAA receptor, respectively, did not affect EGABA of KCC2-expressing cells when they were added along with taurine. Instead, application of GES, an inhibitor
P1-a25 Endogenous neurosteroids potentiate excitatory synaptic transimission of granule cells in the rat hippocampal dentate gyrus Motoki Tanaka , Masahiro Sokabe Dept Physiol, Nagoya Univ Grad Sch of Med, Nagoya, Japan Many studies have demonstrated that neurosteroids promote learning and memory through modulations of the synaptic transmission in the hippocampus. However, as most of the studies were done with exogenously applied neurosteroids, how much degree endogenous neurosteroids contribute to the phenomena is poorly understood. To investigate the contribution of endogenous neurosteorids to the function of synapses in the central nervous system, we measured electrophysiologically the effect of aminoglutethimide (AG), an inhibitor of steroid biosynthesis, on the neuronal activities in the dentate gyrus of rat hippocampal slices. AG markedly decreased the basal synaptic transmission and the induction of activity dependent long-term potentiation. However, paired-pulse ratio was not affected by AG, indicating that the presynaptic transmitter release keeps its function without endogenous neurosteroids. By contrast, AG significantly reduced NMDA receptor-mediated EPSC, but not GABA receptor-mediated IPSC in granule cells. These results suggest that endogenous neurosteroids operate as potent positive modulators on the excitatory synaptic transmission by potentiating the function of postsynaptic neurons in the rat dentate gyrus under physiological condition. doi:10.1016/j.neures.2010.07.2070