OGD induced VRAC activation requires Na- +loading through glutamate receptors.

<p><b>A</b>. In the presence of 40 µM AP-5 and 30 µM NBQX in the OGD solution to inhibit ionotropic glutamate receptors, the 25 min OGD induced neuronal electrophysiological changes seen in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016803#pone-0016803-g003" target="_blank">Fig. 3A</a> were largely attenuated. Also, the activation of VRAC in the reperfusion stage was significantly inhibited. <b>B</b>. Shows the differences in the outward current amplitudes between 6 min and 20 min post-OGD under the following conditions: 1) control; 2) in the presence of DNDS+ bicuculline +furosemide in the OGD and 3) in the presence of AP-5+NBQX in the OGD solution. * Indicates that the difference between the control and AP-5+NBQX groups was statistically significant at <i>p</i><0.05.</p

The effects of NPPB and DCPIB on OGD induced pyramidal neuron death.

<p>Besides the control group (in aCSF, <b>A</b>), 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>B</b>. OGD); 2) bath solution+100 µM NPPB (<b>C.</b> NPPB); and 3) bath solution+10 µM DCPIB (<b>D.</b> 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 <i>vs</i>. 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 <i>p</i>≤0.01t. <b>†</b>. The difference between the NPPB and DCPIB groups is statistically significant at <i>p</i>≤0.05.</p

Expression of VRAC currents in CA1 pyramidal neurons in hippocampal slices.

<p><b>A</b>, Shows a hypoosmotic medium -activated -chloride conductance (HAC) from a pyramidal neuron. After initial recording in the isoosmotic medium (iso, dashed line) as control, the perfusion was switched to the hypoosmotic medium (hypo, −50 mOsm) for 60 min. The neuronal Na⁺, Ca²⁺ and K⁺ channel conductances were pharmacologically inhibited (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016803#s2" target="_blank">Methods</a>). The cell was held at −40 mV in the resting condition, and a pair of alternate voltage pulses at ±40 mV was delivered to the cell every 15 second. Each test pulse in the pair was 1 second long and was separated from each other by 300 ms at −40 mV resting voltage (see the shadowed inset in <b>A</b> for protocol). Because each series of paired alternate pulses was delivered every 15 s, the time scale bar shown under <b>A</b> includes all the unrecorded time periods, or the duration of alternate pulses induced currents are not proportional to the applied time scale. A progressive increase of chloride conductance was recorded over a 60 min of hypo exposure. <b>B</b>. A whole-cell chloride conductance recording with 30 min of hypo exposure. The HAC slowly inactivated after switching the perfusion to the iso. In the same recording, a voltage step protocol was delivered to the cell at the times indicated as “<b>a</b>”, “<b>b</b>” and “<b>c</b>” that represent the chloride currents at control, HAC and recovery, respectively. The I-V curves in <b>C</b> were at times of “a” and “b” and constructed by plotting the steady-state currents against the applied voltages, ranging from −100 mV to +100 mV in a 20 mV increments (see shadowed inset in <b>B</b>). In all the I-V curves in <b>C</b>, the chloride conductance was outwardly rectifying and reversed at around −40 mV.</p

The post-OGD VRAC induced from rat hippocampal pyramidal neurons.

<p><b>A</b>. 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>B</b>-1, <b>C</b>-1 and <b>D-</b>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 “<b>a</b>” and “<b>b</b>” to obtain the I-V curves (<b>B</b>2, <b>C</b>-2 and <b>D</b>-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>B</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 “<b>a</b>” <i>vs</i>. 2041±65 at “<b>b</b>”). *: indicates a statistical significance of difference at <i>p<</i>0.05. In the recording of <b>C</b>-1, an inhibitor cocktail for Cl^- cotransporter and GABA_A, 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 (<b>C</b>-2, 1460±126 pA at “<b>a</b>” <i>vs</i>. 1857±207 at “<b>b</b>”, <i>p</i>>0.05). The recording in <b>D</b>-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 “<b>a</b>” <i>vs</i>. 1013±89 at “<b>b</b>”).</p

Short-term OGD did not change the levels of caspase-9 and -3.

<p><b>A</b>. Western blot analysis was used to detect caspase-9, caspase-3 and their precursors from lysates of slices pretreated with different conditions as indicated. 100 µg whole cell proteins in each lane were used in western analysis using anti- caspase-9, caspase-3 and GAPDH. <b>B-D</b>. Bar graphs show the expression levels of procaspase-9, procaspase-3 and caspase-3 that are normalized to GAPDH (n = 3). The difference was not statistically significant among different treatment groups in each category shown in <b>B</b>–<b>D</b>.</p

The pharmacology of post-OGD chloride conductance in neurons.

<p>The values in y-axis are the differences of the outward current amplitudes between 6 min and 20 min, <i>I</i>_{20 min} –<i>I</i>_{6 min}, in the post-OGD stage. The outward currents were taken from the voltage step at +100 mV. The post-OGD outward chloride currents were not sensitive to the inhibitor cocktail containing DNDS, bicuculline and furosemide and DCPIB, but sensitive to 100 µM NPPB and 300 µM IAA-94. The latter two inhibitors attenuated the outward currents to the level below the current amplitudes measured at 6 min (445±75 pA in control, −334±72 pA in NPPB and −226±106 in IAA-94). ** Indicates a statistical significance of difference between the control and a tested inhibitor at <i>p</i><0.01.</p