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Volume regulated anion channel currents of rat hippocampal neurons and their contribution to oxygen-and-glucose deprivation induced neuronal death.

Zhang H, Cao HJ, Kimelberg HK, Zhou M - PLoS ONE (2011)

Bottom Line: The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC.In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker.Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. zhanghq_04@yahoo.com

ABSTRACT
Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM) and NPPB (100 µM), but not to tamoxifen (10 µM). Using oxygen-and-glucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

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The effects of NPPB and DCPIB on OGD induced pyramidal neuron death.Besides the control group (in aCSF, A), the hippocampal slices were randomly divided into three groups after 25 min OGD treatment to receive the following post-OGD treatment: 1) in bath solution (B. OGD); 2) bath solution+100 µM NPPB (C. NPPB); and 3) bath solution+10 µM DCPIB (D. DCPIB). The fluorescence density of the TO-PRO-3-I staining is proportional to the neuronal death. Between the aCSF control and the OGD group, the neuronal death increased by 5.3 fold (3.2±0.6 in aCSF vs. 16.9±1.9 in OGD, n = 13). The neuronal death was reduced to 6.0 ±0.5 (n = 20) by 100 µM NPPB, and to 9.4±1.1 (n = 20) by 10 µM DCPIB. Both NPPB and DCPIB were added in the reperfusion bath solution to inhibit post-OGD VRAC. All the fluorescence intensity values are arbitrary. **. The difference between the OGD and NPPB or DCPIB groups is statistically significant at p≤0.01t. †. The difference between the NPPB and DCPIB groups is statistically significant at p≤0.05.
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pone-0016803-g006: The effects of NPPB and DCPIB on OGD induced pyramidal neuron death.Besides the control group (in aCSF, A), the hippocampal slices were randomly divided into three groups after 25 min OGD treatment to receive the following post-OGD treatment: 1) in bath solution (B. OGD); 2) bath solution+100 µM NPPB (C. NPPB); and 3) bath solution+10 µM DCPIB (D. DCPIB). The fluorescence density of the TO-PRO-3-I staining is proportional to the neuronal death. Between the aCSF control and the OGD group, the neuronal death increased by 5.3 fold (3.2±0.6 in aCSF vs. 16.9±1.9 in OGD, n = 13). The neuronal death was reduced to 6.0 ±0.5 (n = 20) by 100 µM NPPB, and to 9.4±1.1 (n = 20) by 10 µM DCPIB. Both NPPB and DCPIB were added in the reperfusion bath solution to inhibit post-OGD VRAC. All the fluorescence intensity values are arbitrary. **. The difference between the OGD and NPPB or DCPIB groups is statistically significant at p≤0.01t. †. The difference between the NPPB and DCPIB groups is statistically significant at p≤0.05.

Mentions: The impact of OGD-induced VRAC activation on neuronal death was determined by TO-PRO-3-I staining of CA1 pyramidal neurons in the presence and absence of NPPB in the OGD solution as NPPB shown the strongest inhibition on post-OGD VRAC currents. Although DCPIB did not inhibit neuronal VRAC in the present study, a potent inhibition of DCPIB on VRAC currents of cultured astrocytes promoted us to include this inhibitor in this study as a control. In the presence of NPPB or DCPIB, the TO-PRO-3-I fluorescence density was reduced from 16.9±1.9 (n = 13) to 6.0±0.5 (n = 15) and 9.4±1.1 (n = 19), respectively (p<0.01, Fig. 5A–C). Thus the neuroprotection produced by NPPB was more pronounced compared to DCPIB (p<0.05, Fig. 6D). Nevertheless, the fact that DCPIB was also able to produced a sizable neuroprotection implies a different VRAC target of DCPIB action, such as inhibition of astrocyte VRAC [26]. However, the inhibition of neuronal VRAC by NPPB does contribute significantly to the overall neuroprotection under cerebral ischemia conditions.


Volume regulated anion channel currents of rat hippocampal neurons and their contribution to oxygen-and-glucose deprivation induced neuronal death.

Zhang H, Cao HJ, Kimelberg HK, Zhou M - PLoS ONE (2011)

The effects of NPPB and DCPIB on OGD induced pyramidal neuron death.Besides the control group (in aCSF, A), the hippocampal slices were randomly divided into three groups after 25 min OGD treatment to receive the following post-OGD treatment: 1) in bath solution (B. OGD); 2) bath solution+100 µM NPPB (C. NPPB); and 3) bath solution+10 µM DCPIB (D. DCPIB). The fluorescence density of the TO-PRO-3-I staining is proportional to the neuronal death. Between the aCSF control and the OGD group, the neuronal death increased by 5.3 fold (3.2±0.6 in aCSF vs. 16.9±1.9 in OGD, n = 13). The neuronal death was reduced to 6.0 ±0.5 (n = 20) by 100 µM NPPB, and to 9.4±1.1 (n = 20) by 10 µM DCPIB. Both NPPB and DCPIB were added in the reperfusion bath solution to inhibit post-OGD VRAC. All the fluorescence intensity values are arbitrary. **. The difference between the OGD and NPPB or DCPIB groups is statistically significant at p≤0.01t. †. The difference between the NPPB and DCPIB groups is statistically significant at p≤0.05.
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Related In: Results  -  Collection

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pone-0016803-g006: The effects of NPPB and DCPIB on OGD induced pyramidal neuron death.Besides the control group (in aCSF, A), the hippocampal slices were randomly divided into three groups after 25 min OGD treatment to receive the following post-OGD treatment: 1) in bath solution (B. OGD); 2) bath solution+100 µM NPPB (C. NPPB); and 3) bath solution+10 µM DCPIB (D. DCPIB). The fluorescence density of the TO-PRO-3-I staining is proportional to the neuronal death. Between the aCSF control and the OGD group, the neuronal death increased by 5.3 fold (3.2±0.6 in aCSF vs. 16.9±1.9 in OGD, n = 13). The neuronal death was reduced to 6.0 ±0.5 (n = 20) by 100 µM NPPB, and to 9.4±1.1 (n = 20) by 10 µM DCPIB. Both NPPB and DCPIB were added in the reperfusion bath solution to inhibit post-OGD VRAC. All the fluorescence intensity values are arbitrary. **. The difference between the OGD and NPPB or DCPIB groups is statistically significant at p≤0.01t. †. The difference between the NPPB and DCPIB groups is statistically significant at p≤0.05.
Mentions: The impact of OGD-induced VRAC activation on neuronal death was determined by TO-PRO-3-I staining of CA1 pyramidal neurons in the presence and absence of NPPB in the OGD solution as NPPB shown the strongest inhibition on post-OGD VRAC currents. Although DCPIB did not inhibit neuronal VRAC in the present study, a potent inhibition of DCPIB on VRAC currents of cultured astrocytes promoted us to include this inhibitor in this study as a control. In the presence of NPPB or DCPIB, the TO-PRO-3-I fluorescence density was reduced from 16.9±1.9 (n = 13) to 6.0±0.5 (n = 15) and 9.4±1.1 (n = 19), respectively (p<0.01, Fig. 5A–C). Thus the neuroprotection produced by NPPB was more pronounced compared to DCPIB (p<0.05, Fig. 6D). Nevertheless, the fact that DCPIB was also able to produced a sizable neuroprotection implies a different VRAC target of DCPIB action, such as inhibition of astrocyte VRAC [26]. However, the inhibition of neuronal VRAC by NPPB does contribute significantly to the overall neuroprotection under cerebral ischemia conditions.

Bottom Line: The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC.In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker.Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. zhanghq_04@yahoo.com

ABSTRACT
Volume-regulated anion channels (VRAC) are widely expressed chloride channels that are critical for the cell volume regulation. In the mammalian central nervous system, the physiological expression of neuronal VRAC and its role in cerebral ischemia are issues largely unknown. We show that hypoosmotic medium induce an outwardly rectifying chloride conductance in CA1 pyramidal neurons in rat hippocampal slices. The induced chloride conductance was sensitive to some of the VRAC inhibitors, namely, IAA-94 (300 µM) and NPPB (100 µM), but not to tamoxifen (10 µM). Using oxygen-and-glucose deprivation (OGD) to simulate ischemic conditions in slices, VRAC activation appeared after OGD induced anoxic depolarization (AD) that showed a progressive increase in current amplitude over the period of post-OGD reperfusion. The OGD induced VRAC currents were significantly inhibited by inhibitors for glutamate AMPA (30 µM NBQX) and NMDA (40 µM AP-5) receptors in the OGD solution, supporting the view that induction of AD requires an excessive Na(+)-loading via these receptors that in turn to activate neuronal VRAC. In the presence of NPPB and DCPIB in the post-OGD reperfusion solution, the OGD induced CA1 pyramidal neuron death, as measured by TO-PRO-3-I staining, was significantly reduced, although DCPIB did not appear to be an effective neuronal VRAC blocker. Altogether, we show that rat hippocampal pyramidal neurons express functional VRAC, and ischemic conditions can initial neuronal VRAC activation that may contribute to ischemic neuronal damage.

Show MeSH
Related in: MedlinePlus