At the end of each experiment, the isotope remaining in the cells was extracted with a solution containing 2% sodium dodecyl sulfate (SDS) plus 8 mM EDTA. Four ml Ecoscint scintillation cocktail (National Diagnostics, Atlanta, GA, U.S.A.) was added and each fraction was counted for [3H] in a Tri-Carb 1900TR Liquid Scintillation Analyzer (PerkinElmer, Boston, MA, U.S.A.). Percent fractional isotope release for each time point was calculated by dividing radioactivity released in each 1-min interval by the radioactivity left in the cells (the sum of all the radioactive counts in the remaining fractions up to the beginning of the fraction being measured, plus the radioactivity left in the cell digest). In a few experiments, astrocytes were simultaneously loaded with D-[3H]aspartate (2 mCi/ml) and [14C]taurine (1 mCi/ml) to compare properties of swelling-activated fluxes of excitatory amino acids and taurine in one cell preparation. In these instances [3H] and [14C] radioactivity was determined in the same perfusate samples using a Tri-Carb 1900TR Liquid Scintillation Analyzer and double-label DPM software. To measure taurine release in synaptosomal preparations, synaptosomal suspensions were loaded with [3H]taurine (0.5 mCi/ ml) for 1 hour at 37uC in basal HEPES-buffered solution. The extracellular isotope was washed by adding 9 volumes of ice-cold medium containing (in mM): 243 sucrose, 5 KCl, 1.2 MgSO4, 10 HEPES, 10 glucose; pH = 7.4. Synaptosomes were sedimented (10,000 g, 2 min at 2uC) and resuspended in the same sucrose medium, which prevents spontaneous synaptosome depolarization at low temperatures. Aliquots of [3H]taurine-loaded synaptosomes (,0.2–0.3 mg protein) were injected in glass tubes containing 4.5 mL of HEPES-buffered basal, low [NaCl] hypoosmotic, or low [NaCl] isoosmotic media, as specified in the Results section. After 5-min incubation at 37uC, taurine efflux was terminated by rapid vacuum filtration through GF/C glass microfiber filters (What- man-GE Healthcare, Florham Park, NJ, U.S.A.). Filters were placed in scintillation vials containing a 4 ml Ecoscint scintillation cocktail and counted for radioactivity remaining in the synapto- somes. Relative taurine efflux values (% loaded/5 min) were calculated by comparing the radioactivity in experimental samples to isotope content in samples filtered through GF/C without incubation at 37uC (‘‘0 time’’). [3H]Taurine and D-[3H]aspartate uptake assay Cultured astrocytes for these experiments were grown in 12-well tissue culture plates according to the cell culture method described in the previous section. Serum-containing medium was washed out, and the cells were incubated for 30 minutes at 37uC with basal HEPES-buffered medium containing 0.5 mCi/mL of [ 3 H]taurine or D-[3H]aspartate plus 10 mM of unlabeled taurine or L-glutamate, respectively. Following the 30-min incubation period cells were washed four times with ice-cold physiological phosphate buffered solution. Cells were then lysed with 2% SDS plus 8 mM EDTA. The isotope content in the lysate was used as a measure of taurine ([3H]taurine) or L-glutamate (D-[3H]aspartate) uptake. Four ml Ecoscint scintillation cocktail was added to each lysates and [3H] was counted in a Liquid Scintillation Analyzer. Statistical analysis The statistical significance of the differences in the amino acid release and uptake were determined with ANOVA or repeated measures ANOVA, as specified throughout the text and in figure legends. For the in vivo experiments, planned comparisons were performed with repeated measures ANOVA to determine differ- ences in amino acid release only during hypoosmotic medium Cortical Amino Acid Release exposure. Origin 7.5 (OriginLab, Northampton, MA) and Statistica 6.1 (StatSoft, Tulsa, OH) were used for statistical analysis. Chemicals Cadmium chloride (CdCl2), hydrogen peroxide (H2O2), man- nitol and ouabain were purchased from Sigma (St. Louis, MI, U.S.A). [3H]Taurine or D-[3H]aspartate were from GE Health- care-Amersham (Buckinghamshire, U.K.). DL-Threo-b-benzylox- yaspartic acid (DL-TBOA) was obtained from Tocris (Ellisville, MI, U.S.A.). 4,49-dinitrostilbene-2,29-disulfonic acid, disodium salt (DNDS) and all cell culture reagents were from Invitrogen (Carlsbad, CA, U.S.A.). All other chemicals including amino acid standards for the HPLC experiments were purchased from Sigma or Aldrich (Milwaukee, WI, U.S.A.) and were the highest purity available. Results Differences in kinetics of cortical amino acid and taurine release in response to perfusion of hypoosmotic medium or low NaCl isoosmotic medium In order to examine volume-sensitive amino acid release in vivo, rat cortices were perfused via a microdialysis probe with hypoosmotic medium in which [NaCl] was reduced from 120 to 25 mM NaCl (65% reduction in osmolarity; medium also contained 25 mM NaHCO3 and other salts as specified in the Methods section). We used a larger reduction in medium osmolarity compared to that typically employed in vitro to account for the fact hypoosmotic media perfused via microdialysis probes are gradually diluted with the extracellular fluids upon their diffusion in the brain. Hypoosmotic medium initiated substantial increases in the levels of VRAC-permeable glutamate, aspartate and taurine (Fig. 1a–c). In the same experiments the extracellular levels of the VRAC-impermeable amino acids, asparagine and glutamine, were either downregulated (glutamine) or not altered (asparagine) by the hypoosmotic medium (Fig. 1d–e). Increases in the extracellular levels of glutamate and aspartate had similar kinetics. Dialysate levels of both amino acids peaked at 15 minutes (,6.5- and ,5-fold increases over baseline, for glutamate and aspartate, respectively), then quickly decreased to levels which were only 2-3-fold higher than the basal release, with additional recovery observed after switching to isoosmotic medium (Fig. 1a, b). In contrast, in the same samples, the swelling-activated release of taurine was consistently delayed by 5 minutes versus excitatory amino acids, had a substantially slower inactivation, and never recovered after returning to isoosmotic conditions (Fig. 1c). To determine if the increases in glutamate, aspartate and taurine levels were due to changes in osmolarity or a consequence of reduced [NaCl]e, the same rats were simultaneously perfused, via microdialysis probes placed in the contralateral cortex, with low [NaCl] (25 mM) medium that was made isoosmotic by the addition of 167 mM mannitol. The isoosmotic low [NaCl] medium failed to induce an increase in dialysate concentrations of glutamate and aspartate (Fig 1a, b). In the same samples, however, we found a large increase in the extracellular levels of taurine in response to isoosmotic [NaCl]e reduction, which was not statistically different from the hypoosmotic-stimulated aug- mentation (Fig 1c). In order to understand the nature of the low [NaCl]-induced taurine release, we exposed cultured astrocytes and isolated nerve endings (synaptosomes) prepared from rat cortical tissue to low [NaCl] media made isoosmotic with mannitol. In striking contrast to the in vivo microdialysis data, cultured astrocytes preloaded with 3 [ H]taurine failed to show any increase in taurine release levels PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3543