E Spinal Slice Was Placed In A Recording Chamber Perfused With
E spinal slice was placed in a recording chamber perfused with Krebs solution. Astrocytes in the spinal dorsal horn laminae I and II were first labeled by the astrocyte specific dye, sulforhodomine 101 (100 M) , which was pressure-injected into the spinal slice through a pipette with a picospritzer [29,58,59]. The identified astrocyte was patched using borosilicate glass recording electrodes (resistance, 4? M) filled with (in mM) 145 potassium-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP and 0.1 Na-GTP (pH 7.3, 290 ?300 mOsm) [51,86]. GTCs were recorded at a holdingYan et al. Molecular Pain (2015) 11:Page 14 ofpotential of -80 mV in voltage clamp mode in the presence of blockers of GABAA receptor (10 M bicuculline), glycine receptor (5 M strychnine), AMPA/kainate receptors (10 M DNQX), NMDA receptor (25 M D-AP5), and tetrodotoxin (1 M) at 35 [51,56]. GTCs were evoked by puffing 50 M L-glutamate onto the recorded astrocyte through a glass pipette connected to a Picospritzer controlled by a computer. mEPSCs and GTCs were recorded using Axopatch 700B amplifiers, digitized at 10 kHz, and analyzed off-line. Access resistance within the range of 10?0 M was monitored continuously throughout the experiments. The recording was abandoned when the access resistance changed more than 20 . The frequency and amplitude of mEPSCs from 3 min before, during, and after the perfusion of tested drugs were analyzed and averaged using a peak detection program (MiniAnalysis; Synaptosoft Inc., Decatur, GA). Four sweeps of GTCs were averaged and the mean amplitude and charge transfer of GTCs  were measured. All the drugs were applied through bathperfusion unless otherwise indicated.In vitro measurement of glutamate uptake activitybuffer. The glutamate uptake activity was determined by incubating the synaptosome preparation with a solution containing [3H] L-glutamic acid (0.4 Ci/mmol) at 37 for 5 min. The reaction was terminated by filtering the synaptosomes through a Whatman GF/B filter presoaked with the same buffer solution. The filter was then transferred to a vial containing scintillation cocktail and the radioactivity, which reflects glutamate PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26713783 uptake activities, in the final samples was measured by a liquid scintillation counter (Beckman, LS6500).Western blot PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27220073 experimentsSynaptosome preparations were prepared from the spinal tissue. The glutamate uptake activity in the synaptosome preparation was measured according to previous publications [51,88]. To investigate the effects of paclitaxel on glutamate transporter activities, the L4 5 spinal cord was exposed by laminectomy and the spinal dura was excised in rats anesthetized with urethane (1.3?.5 g/kg, i.p). The heart rate, breathing, and core temperature of the animals were constantly monitored and maintained within normal limits . Paclitaxel was applied onto the L4-L5 spinal segments through a piece of cotton soaked with paclitaxel (concentration: 2 ng/ml) in aCSF at 35 for 30 min. Rats in the control group receiving vehicles in the same fashion. Immediately after the treatment, the dorsal half of the L4-L5 spinal segments was isolated. Synaptosome preparations were prepared immediately after the spinal tissue was isolated according to the protocol published [50,51]. The spinal tissue was homogenized in icecold buffer solution containing: 0.5 mM EDTA, 0.5 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, PluriSIn 1 0.32 M sucrose, 5 g/ml pepstatin, 5 g/ml aprotinin, 20 g/ml trypsin inhibitor, 4.