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

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

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

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

Dependence of taurine and glutamate uptake on extracellular [Na⁺] in cultured astrocytes.

<p>Taurine and glutamate transport rates were measured in primary astrocyte cultures using [³H]taurine and d-[³H]aspartate. Extracellular concentrations of amino acids were adjusted to 10 µM using unlabeled taurine or l-glutamate. To compare glutamate versus taurine uptake, the values were normalized to uptake levels under basal conditions ([Na⁺]_o = 135 mM). Note that under basal conditions absolute d-[³H]aspartate uptake rate (nmols/mg protein) was ∼5-fold higher compared to taurine. Data are the mean values ±SEM of three experiments from each group.</p

Effect of H₂O₂ on hypoosmotic medium induced amino acid release in the cortex.

<p>(a–c) Two microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 1 mM H₂O₂ given 20 minutes prior to and during one-hour hypoosmotic medium perfusion. The data represent the average dialysate levels ±SEM of glutamate (a), aspartate (b) and taurine (c) from 9 rats. ** p<0.01 HYPO vs. HYPO+H₂O₂. In separate experiments, rats were perfused with 1 mM H₂O₂ alone (N = 5).</p

Effect of hypoosmotic or isoosmotic low [NaCl] medium on amino acid levels measured in the rat cortex <i>in vivo</i>.

<p>(a–c) Microdialysis probes, implanted in the rat frontoparietal cortex, were perfused with hypoosmotic medium (−95 mM NaCl, −65% osmolarity) or isoosmotic low NaCl medium (−95 mM NaCl +167 mM mannitol) for one hour. In these experiments, the rat brain was perfused with both the hypoosmotic and isoosmotic medium on opposite sides of the cortex. The data represent average dialysate levels of glutamate (a), aspartate (b), and taurine (c) ±SEM from 5 rats. ** p<0.01, hypoosmotic vs. isoosmotic low [NaCl], repeated measures ANOVA. (d–e) In several experiments dialysate levels of glutamine (d, N = 5), and asparagine (e, n = 3) were additionally measured on the “hypoosmotic” side of the brain.</p

Effect of H₂O₂ on swelling-activated taurine release from cultured astrocytes and cortical synaptosomes.

<p>(a) The effect of H₂O₂ on swelling-activated [¹⁴C]taurine release from cultured astrocytes. Astrocytes were preloaded overnight with [¹⁴C]taurine and D-[³H]aspartate. 300 µM H₂O₂ was added to the media 10 min before and during exposure to hypoosmotic medium. For clarity, only [¹⁴C]taurine release is shown. The data are the mean values ±SEM of three experiments. **p<0.01 vs. hypotonic control. (b) The effect of H₂O₂ on swelling activated [³H]taurine release from rat cortical synaptosomes. Integral [³H]taurine release was measured for 5 minutes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003543#s2" target="_blank">Material and methods</a>. 300 µM H₂O₂ was present in media 10 min before and during measurements of taurine release. Data are the mean values ±SEM of three independent experiments performed in quadruplicate. ***p<0.001 vs. basal release.</p