firmed that VRACs are permeable to taurine, at least under conditions when its molecule is negatively charged [21,22,28]. Nevertheless, there is continuous debate as to whether taurine shares the same permeability pathway with Cl2 and other anionic amino acids [29–31]. In particular, in several cell types and in 3 brain slices, swelling-activated [ H]taurine efflux shows different kinetics and pharmacological properties, when compared to Cl2 (125I2) or D-[3H]aspartate release [32–37]. Although VRAC and VRAC-mediated amino acid fluxes have been extensively studied in cultured cells, there is limited information regarding their properties in intact brain tissue. Several studies, which used perfusion of hypoosmotic medium via microdialysis probes to induce cell swelling in the brain, found increased extracellular levels of taurine and several amino acids, which are known to permeate through VRAC [38–41]. Likewise, Phillis and co-workers found hypoosmotic medium-stimulated release of taurine and the VRAC- permeable amino acids using a cortical cup perfusion technique, and such a release was strongly inhibited by the putative VRAC-blockers DNDS, NPPB, niflumic acid and tamoxifen [42]. In the present work, we used a microdialysis approach in anesthetized animals to compare properties of swelling-activated fluxes of the excitatory amino acids glutamate and aspartate to those of taurine in the rat cortex in vivo. Materials and Methods Animal surgery and microdialysis procedures All animal procedures performed in this work were approved by the institution’s animal care and use committee and adhered to the NIH guidelines for care and use of laboratory animals. Male Sprague-Dawley rats (Taconic Farms), weighing between 325 and 425 g, were allowed free access to food and water. Rats were given atropine sulfate (0.5 mg/kg, i.m.) to reduce respiratory tract fluid secretion, and anesthetized with isoflurane prior to intubation. Intubated rats were mechanically ventilated with a gas mixture of 2.25% isoflurane in 30% O2/balance N2. A saline drip (0.9% NaCl) was administered intraperitoneally throughout the exper- imental procedure to prevent dehydration. Body temperature was monitored throughout the experiment with a rectal probe and was maintained between 36uC and 36.5uC with a heating pad. Animals were placed in a stereotaxic frame and microdialysis probes (2 mm tip, 20 kD cutoff, CMA Microdialysis, North Chelmsford, MA, U.S.A.) were slowly lowered through burr holes into the frontoparietal cortex (from bregma, 1 mm anterior; 64 mm lateral; 2.6 mm down from the dura). Artificial cerebral spinal fluid (aCSF; in mM: 120 NaCl, 2.7 KCl, 1 MgSO4, 1.2 CaCl2, 25 NaHCO3, 0.05 ascorbic acid; pH = 7.3) was perfused at 2 ml/min through the microdialysis probes. After two hours of probe stabilization at least two 20 minute perfusate samples were collected by a CMA-170 refrigerated fraction collector (CMA Microdialysis) to determine baseline amino acid levels before the application of drug or hypoosmotic medium. Hypoosmotic aCSF (in mM: 25 NaCl, 2.7 KCl, 1 MgSO4, 1.2 CaCl2, 25 NaHCO3, 0.05 ascorbic acid; pH = 7.3) was perfused at 2 ml/min for one hour and perfusate samples were collected every 5 minutes. Each rat was implanted with two microdialysis probes placed bilaterally in the cortex, with one probe serving as a control (hypoosmotic solution only) and the probe on the other side (chosen at random) serving as the experimental condition (hypoosmotic solution plus drug). All drugs were delivered through the microdialysis probes. Amino acid analysis in microdialysate samples Dialysate concentrations of the amino acids were determined by reverse-phase high performance liquid chromatography (HPLC) Cortical Amino Acid Release using a Hewlett-Packard Series 1100 HPLC system. Pre-column derivatization of the amino acids was done with o-phthaldialde- hyde/2-mercaptoethanol. The derivatives were separated using a C18 Varian column (4.66100 mm, 3 mm particle diameter). The fluorescence signal was detected by a Hewlett-Packard 1046A programmable fluorescence detector. Amino acid standards were used to calculate the concentrations of the amino acids in the perfusate. Preparation of primary astrocyte cultures Confluent primary astrocyte cultures were prepared from the cerebral cortex of newborn Sprague-Dawley rats as described elsewhere [43], with minor modifications summarized below. Newborn Sprague-Dawley rats were euthanized by rapid decapitation, the cerebral cortices were separated from the meninges and basal ganglia, and tissue was dissociated using the neutral protease Dispase II (Roche Applied Science, Indianapolis, IN, U.S.A.). Dissociated cells were seeded on poly-D-lysine coated 18618 mm glass coverslips (Caroline Biological Supply Co, Burlington, NC, U.S.A.) for efflux experiments, or 12-well tissue culture plates for uptake experiments. Cell cultures were grown for 3–4 weeks in Minimal Essential Medium (MEM) supplemented with 10% heat inactivated horse serum (HIHS), 50 U/ml penicillin and 50 mg/ml streptomycin at 37uC in a humidified atmosphere of 5% CO2/95% air. Culture medium was replaced twice a week. After two weeks of cultivation, penicillin and streptomycin were removed from the culture medium. Immuno- cytochemistry showed $95% of the cells stained positively for the astrocytic marker glial fibrillary acid protein. Preparation of rat cortical synaptosomes Rat cortical synaptosomes were isolated from the cortical tissue of male Sprague-Dawley rats (Taconic Farms) weighing between 180 and 230 g according to [44] with modifications described elsewhere [45]. Final synaptosomal pellets were resuspended in HEPES-buffered medium containing (in mM): 135 NaCl, 3.8 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, 10 HEPES; pH = 7.4. Synaptosomes were incubated for 30–40 min at 37uC in order to allow them to restore transmembrane ion gradients before further use in amino acid release experiments. [3H]Taurine and D-[3H]aspartate efflux assays [3H]Taurine or D-[3H]aspartate efflux measurements were performed in astrocyte cultures as follows. Astrocytes grown on glass coverslips were loaded overnight with either [3H]taurine (4 mCi/ml) or D-[3H]aspartate (4 mCi/ml) in 2.5 ml of MEM containing 10% HIHS in a CO2 incubator set for 5% CO2/95% air at 37uC. Before the start of the efflux measurements, the cells were washed free of extracellular isotope and residual serum- containing medium in HEPES-buffered solution. The basal HEPES-buffered medium contained (in mM): 135 NaCl, 3.8 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, 10 HEPES; pH = 7.4. The coverslips were inserted into a Lucite perfusion chamber which had a depression precisely cut in the bottom to accommodate the coverslip and a Teflon screw top leaving a space above the cells of around 100–150 mm in height. The cells were superfused at a flow rate of 1.2 ml/min in an incubator set at 37uC with isoosmotic or hypoosmotic HEPES- buffered media. To prepare hypoosmotic medium, the concen- tration of NaCl was reduced to 85 mM. The osmolarities of all buffers were checked using a freezing point osmometer (mOsmette, Precision Systems, Natick, MA, U.S.A.) and were 287–290 and 197–200 mOsm for isoosmotic and hypoosmotic media, respec- tively. Superfusate fractions were collected at one minute intervals. PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3543