Cortical Amino Acid Release Figure 4. Effect of the anion channel blocker DNDS on hypoosmotic medium-induced amino acid release in the rat cortex. (a–c) Microdialysis probes were perfused on opposite sides of the cortex with hypoosmotic medium (HYPO, 265% osmolarity) in the presence or absence of 10 mM DNDS, given one hour prior to and during one-hour hypoosmotic medium perfusion. The data represent average dialysate levels of glutamate (a), aspartate (b) and taurine (c) +/2SEM from 5 rats. * p,0.05, HYPO vs. HYPO+DNDS (glutamate); ** p,0.01, HYPO vs. HYPO+DNDS (aspartate); *** p,0.001 HYPO vs. HYPO+DNDS (taurine). Significance was determined by repeated measures ANOVA. doi:10.1371/journal.pone.0003543.g004 which are dependent on normal Na+ and K+ gradients [54]. Therefore to rule out the involvement of glutamate transport reversal in mediating hypoosmotic-stimulated excitatory amino acid release we used the broad-spectrum non-transportable glutamate transporter blocker DL-TBOA that blocks all neuronal and glial transporters [55,54]. 500 mM DL-TBOA given 20 min- utes prior to and during hypoosmotic medium perfusion, significantly increased rather than decreased excitatory amino acid release and did not affect taurine levels (Fig. 7a, b), suggesting that normal transport operation is maintained under hypoosmotic conditions and transport reversal is not responsible for the hypoosmotic medium-stimulated excitatory amino acid release. As expected, the in vivo extracellular levels of taurine were not affected by DL-TBOA (data not shown). Although DL-TBOA has been well characterized as an inhibitor of normal glutamate transporter function [55], we wanted to verify DL-TBOA’s effectiveness in blocking glutamate transporters working in the reverse mode. Cultured astrocytes, preloaded with D-[3H]aspartate, were treated for 40 minutes with 1.0 mM ouabain to increase [Na+]i, prior to and during 20 minutes perfusion with an isoosmotic 100 mM [K+]e medium. These treatment conditions have previously been shown to induce the reversal of glutamate transporters in cultured astrocytes [56]. In vitro, 300 mM DL-TBOA given 10 minutes prior to and during 10 mM [K+] perfusion completely blocked glutamate transport reversal-induced D-[3H]aspartate release, verifying that DL- TBOA is an effective inhibitor of glutamate transport reversal (Fig. 7c). Effects of H2O2 on hypoosmotic-stimulated release of excitatory amino acid and taurine in vivo and in vitro We further investigated if reactive oxygen species modulate swelling-sensitive excitatory amino acid release in the brain, as seen in cultured astrocytes (Haskew-Layton et al., 2005). One mM H2O2, administered 20 minutes prior to and during hypoosmotic medium perfusion, did not affect basal levels of glutamate or aspartate but significantly enhanced the swelling-evoked release of both excitatory amino acids (Fig. 8a,b). In contrast, H2O2 did not alter dialysate levels of taurine under hypoosmotic conditions (Fig. 8c), suggesting that excitatory amino acids and taurine release are differentially regulated. To verify that H2O2 does not upregulate glutamate and aspartate release via a VRAC- independent mechanism, we tested the effects of 1 mM H2O2 on amino acid levels in the absence of hypoosmotic medium in a separate set of experiments. As seen in Fig. 8a, b, when superfused under isoosmotic conditions, 1 mM H2O2 did not produce a substantial increase in excitatory amino acid levels but did cause a small gradual upward shift in the baseline. Since H2O2 has been reported to alter glutamate uptake [57], we tested the effects of 1–1,000 mM H2O2 on the excitatory amino acid uptake in cultured astrocytes. H2O2 did not alter the excitatory amino acid uptake in vitro up to the highest concentration tested (data not shown). These data, in conjunction with the lack of a H2O2 effect on the basal levels of excitatory amino acid in vivo, suggest that the effects of H2O2 on hypoosmotic glutamate and aspartate levels are unlikely due to uptake inhibition. In order to model the effects of H2O2 on taurine release in astroglial and neuronal cells, we tested in vitro the effect of 300 mM H2O2 on swelling-activated amino acid release in cultured rat astrocytes and cortical synaptosomes. Astrocytes were simulta- neously preloaded with D-[3H]aspartate and [14C]taurine to reveal any potential differences between release properties of the excitatory amino acids and taurine in the same cells. As in our previous study (Haskew-Layton et al., 2005), H2O2 strongly potentiated the swelling-induced release of D-[3H]aspartate (data not shown), as well as the release of taurine (Fig. 9a) by 2-3-fold. In striking contrast, the same concentration of H2O2 was completely 3 ineffective in potentiating [ H]taurine release in synaptosomes, whether H2O2 added 10 minutes before and during application of hypoosmotic medium (Fig. 9b) or acutely (data not shown). These results suggest that the H2O2-insensitive, hypoosmotic-medium stimulated taurine release observed in vivo may originate from a neuronal compartment. Discussion In the present in vivo study we employed a microdialysis approach to compare properties of the swelling-activated release of major organic osmolytes, the excitatory amino acids glutamate and aspartate and the sulfonic acid taurine, in the rat cortex. Swelling-activated amino acid release may mimic pathological processes that occur in ischemia and several other neuropathol- ogies, where extensive astroglial cell swelling has been detected [7,9]. To model the effects of cell swelling without the contribution PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3543