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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 post-OGD VRAC induced from rat hippocampal pyramidal neurons.A. Shows a neuronal recording during a 40 min of OGD perfusion. The OGD first induced a progressive activation of membrane conductance accompanied by a downward shift in the holding currents, and was then followed by an anoxic depolarization (AD) at 25 min after OGD onset. A 40 min OGD treatment typically resulted in an irreversible change in neuronal electrophysiology. For the recordings shown in B-1, C-1 and D-1, the OGD exposure was shortened to 25 min, where the OGD-induced neuronal electrophysiological changes were readily reversible at ∼6 min after withdrawal of OGD (reperfusion). In the reperfusion stage, the voltage step protocol was delivered at the time points of “a” and “b” to obtain the I-V curves (B2, C-2 and D-2, the voltage step induced current traces are not shown). The presence of a strong outwardly rectifying chloride conductance and a progressive increase of conductance in the reperfusion stage were disclosed by the I-V curves shown B-2, where the I-V curves obtained from 6 min and 20 min post-OGD can be compared. Both of the I-V curves showed a strong outward rectification and reversed at −40 mV. During the time period from 6 min to 20 min, the amplitude of the outward currents at +100 mV increased by 32% (1542±116 pA at “a” vs. 2041±65 at “b”). *: indicates a statistical significance of difference at p<0.05. In the recording of C-1, an inhibitor cocktail for Cl- cotransporter and GABAA, i.e., 200 µM furosemide+400 µM DNDS+20 µM bicuculline, was applied to the reperfusion solution that did not prevent the outgrowing of outward chloride currents (C-2, 1460±126 pA at “a” vs. 1857±207 at “b”, p>0.05). The recording in D-1 showed that 100 µM NPPB not only prevented the outward anion conductance from further growing, but actually inhibited the outward currents to below the control level measured at 6 min in the reperfusion stage (1347±156 pA at “a” vs. 1013±89 at “b”).
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pone-0016803-g003: The post-OGD VRAC induced from rat hippocampal pyramidal neurons.A. Shows a neuronal recording during a 40 min of OGD perfusion. The OGD first induced a progressive activation of membrane conductance accompanied by a downward shift in the holding currents, and was then followed by an anoxic depolarization (AD) at 25 min after OGD onset. A 40 min OGD treatment typically resulted in an irreversible change in neuronal electrophysiology. For the recordings shown in B-1, C-1 and D-1, the OGD exposure was shortened to 25 min, where the OGD-induced neuronal electrophysiological changes were readily reversible at ∼6 min after withdrawal of OGD (reperfusion). In the reperfusion stage, the voltage step protocol was delivered at the time points of “a” and “b” to obtain the I-V curves (B2, C-2 and D-2, the voltage step induced current traces are not shown). The presence of a strong outwardly rectifying chloride conductance and a progressive increase of conductance in the reperfusion stage were disclosed by the I-V curves shown B-2, where the I-V curves obtained from 6 min and 20 min post-OGD can be compared. Both of the I-V curves showed a strong outward rectification and reversed at −40 mV. During the time period from 6 min to 20 min, the amplitude of the outward currents at +100 mV increased by 32% (1542±116 pA at “a” vs. 2041±65 at “b”). *: indicates a statistical significance of difference at p<0.05. In the recording of C-1, an inhibitor cocktail for Cl- cotransporter and GABAA, i.e., 200 µM furosemide+400 µM DNDS+20 µM bicuculline, was applied to the reperfusion solution that did not prevent the outgrowing of outward chloride currents (C-2, 1460±126 pA at “a” vs. 1857±207 at “b”, p>0.05). The recording in D-1 showed that 100 µM NPPB not only prevented the outward anion conductance from further growing, but actually inhibited the outward currents to below the control level measured at 6 min in the reperfusion stage (1347±156 pA at “a” vs. 1013±89 at “b”).

Mentions: We next used oxygen-and-glucose deprived (OGD) to simulate ischemic conditions and determine whether VRAC could be activated as a consequence of OGD treatment and the mechanism accounting for it. After perfusion of slices with OGD for 40 min, the neuronal recording first showed a progressive inward shift in the holding currents and increase in membrane conductance (Fig. 3A, n = 3). At 24.7±0.3 min after OGD onset (n = 11), a sudden downward shift in the holding currents and opening of a large membrane conductance was followed, which is a OGD induced neuronal electrophysiological change, termed anoxic depolarization (AD) and typically occurred ∼8 min after treatment of slices with the standard OGD solution [17], [18], [31]. Requiring a much longer OGD stimulation for AD induction should be largely due to the low Na+ and zero Ca2+ ions used in our bath solution, which diminished the influx of Na+ and Ca2+ through their respective voltage-gated channels and ionotropic glutamate receptors [17], [18], [32]. In view of a large downward shift in holding potential, corresponding to a positive shift of membrane potential (Fig. 3A), OGD induced hemichannels opening could also contribute to the AD [33]. Nevertheless, because 40 min OGD treatment always resulted in an irreversible damage to the recordings, we shortened the OGD to 25 min, where the AD could be readily induced but was still reversible (Fig 3B-1). Under this condition, the OGD-induced neuronal electrophysiological changes recovered to the control level within 5–6 min after OGD withdrawal (Fig. 3B-1). Thereafter, an outwardly rectifying current component progressively appeared during the following 20 min post-OGD reperfusion stage (Fig. 3B-1, b). Such a time-dependent increase of post-OGD outward conductance is more clearly shown in the I-V curves in Fig. 3B-2. Specifically, the amplitude of the outward currents was increased by 26% at the end of 20 min compared to the initial 6 min in the post-OGD stage (Fig. 3B1-2, 1542±116 pA at 6 min “a”, 1910±140 pA at 20 min “b”, +100 mV, n = 5). The currents were mostly carried by chloride anions but not hemichannels, because the whole-cell currents were reversed precisely at the EA of −40 mV, and activation of latter should shift reversal potential to around zero potential [33].


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 post-OGD VRAC induced from rat hippocampal pyramidal neurons.A. Shows a neuronal recording during a 40 min of OGD perfusion. The OGD first induced a progressive activation of membrane conductance accompanied by a downward shift in the holding currents, and was then followed by an anoxic depolarization (AD) at 25 min after OGD onset. A 40 min OGD treatment typically resulted in an irreversible change in neuronal electrophysiology. For the recordings shown in B-1, C-1 and D-1, the OGD exposure was shortened to 25 min, where the OGD-induced neuronal electrophysiological changes were readily reversible at ∼6 min after withdrawal of OGD (reperfusion). In the reperfusion stage, the voltage step protocol was delivered at the time points of “a” and “b” to obtain the I-V curves (B2, C-2 and D-2, the voltage step induced current traces are not shown). The presence of a strong outwardly rectifying chloride conductance and a progressive increase of conductance in the reperfusion stage were disclosed by the I-V curves shown B-2, where the I-V curves obtained from 6 min and 20 min post-OGD can be compared. Both of the I-V curves showed a strong outward rectification and reversed at −40 mV. During the time period from 6 min to 20 min, the amplitude of the outward currents at +100 mV increased by 32% (1542±116 pA at “a” vs. 2041±65 at “b”). *: indicates a statistical significance of difference at p<0.05. In the recording of C-1, an inhibitor cocktail for Cl- cotransporter and GABAA, i.e., 200 µM furosemide+400 µM DNDS+20 µM bicuculline, was applied to the reperfusion solution that did not prevent the outgrowing of outward chloride currents (C-2, 1460±126 pA at “a” vs. 1857±207 at “b”, p>0.05). The recording in D-1 showed that 100 µM NPPB not only prevented the outward anion conductance from further growing, but actually inhibited the outward currents to below the control level measured at 6 min in the reperfusion stage (1347±156 pA at “a” vs. 1013±89 at “b”).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037944&req=5

pone-0016803-g003: The post-OGD VRAC induced from rat hippocampal pyramidal neurons.A. Shows a neuronal recording during a 40 min of OGD perfusion. The OGD first induced a progressive activation of membrane conductance accompanied by a downward shift in the holding currents, and was then followed by an anoxic depolarization (AD) at 25 min after OGD onset. A 40 min OGD treatment typically resulted in an irreversible change in neuronal electrophysiology. For the recordings shown in B-1, C-1 and D-1, the OGD exposure was shortened to 25 min, where the OGD-induced neuronal electrophysiological changes were readily reversible at ∼6 min after withdrawal of OGD (reperfusion). In the reperfusion stage, the voltage step protocol was delivered at the time points of “a” and “b” to obtain the I-V curves (B2, C-2 and D-2, the voltage step induced current traces are not shown). The presence of a strong outwardly rectifying chloride conductance and a progressive increase of conductance in the reperfusion stage were disclosed by the I-V curves shown B-2, where the I-V curves obtained from 6 min and 20 min post-OGD can be compared. Both of the I-V curves showed a strong outward rectification and reversed at −40 mV. During the time period from 6 min to 20 min, the amplitude of the outward currents at +100 mV increased by 32% (1542±116 pA at “a” vs. 2041±65 at “b”). *: indicates a statistical significance of difference at p<0.05. In the recording of C-1, an inhibitor cocktail for Cl- cotransporter and GABAA, i.e., 200 µM furosemide+400 µM DNDS+20 µM bicuculline, was applied to the reperfusion solution that did not prevent the outgrowing of outward chloride currents (C-2, 1460±126 pA at “a” vs. 1857±207 at “b”, p>0.05). The recording in D-1 showed that 100 µM NPPB not only prevented the outward anion conductance from further growing, but actually inhibited the outward currents to below the control level measured at 6 min in the reperfusion stage (1347±156 pA at “a” vs. 1013±89 at “b”).
Mentions: We next used oxygen-and-glucose deprived (OGD) to simulate ischemic conditions and determine whether VRAC could be activated as a consequence of OGD treatment and the mechanism accounting for it. After perfusion of slices with OGD for 40 min, the neuronal recording first showed a progressive inward shift in the holding currents and increase in membrane conductance (Fig. 3A, n = 3). At 24.7±0.3 min after OGD onset (n = 11), a sudden downward shift in the holding currents and opening of a large membrane conductance was followed, which is a OGD induced neuronal electrophysiological change, termed anoxic depolarization (AD) and typically occurred ∼8 min after treatment of slices with the standard OGD solution [17], [18], [31]. Requiring a much longer OGD stimulation for AD induction should be largely due to the low Na+ and zero Ca2+ ions used in our bath solution, which diminished the influx of Na+ and Ca2+ through their respective voltage-gated channels and ionotropic glutamate receptors [17], [18], [32]. In view of a large downward shift in holding potential, corresponding to a positive shift of membrane potential (Fig. 3A), OGD induced hemichannels opening could also contribute to the AD [33]. Nevertheless, because 40 min OGD treatment always resulted in an irreversible damage to the recordings, we shortened the OGD to 25 min, where the AD could be readily induced but was still reversible (Fig 3B-1). Under this condition, the OGD-induced neuronal electrophysiological changes recovered to the control level within 5–6 min after OGD withdrawal (Fig. 3B-1). Thereafter, an outwardly rectifying current component progressively appeared during the following 20 min post-OGD reperfusion stage (Fig. 3B-1, b). Such a time-dependent increase of post-OGD outward conductance is more clearly shown in the I-V curves in Fig. 3B-2. Specifically, the amplitude of the outward currents was increased by 26% at the end of 20 min compared to the initial 6 min in the post-OGD stage (Fig. 3B1-2, 1542±116 pA at 6 min “a”, 1910±140 pA at 20 min “b”, +100 mV, n = 5). The currents were mostly carried by chloride anions but not hemichannels, because the whole-cell currents were reversed precisely at the EA of −40 mV, and activation of latter should shift reversal potential to around zero potential [33].

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