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) [58], 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 [87] 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 [82]. 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.