Rsc 200

To preliminary test functional expression of glutamate and GABA receptors in neuronal cultures, we first used the whole cell patch-clamp recordings using EPC10 amplifier and PatchMaster software (HEKA electronics, Germany). Experiments were performed at RT (21-23oC). Cells were held in voltage-clamp mode at −70 mV continuously perfused with the extracellular basic solution (BS) contained (in mM): 152 NaCl, 2.5 KCl, 2 CaCl₂, 10 HEPES, 10 d-Glucose pH 7.4 adjusted with NaOH. Intracellular solution contained: 130 CsCl, 5 MgCl₂, 10 HEPES, 5 EGTA, 0.5 CaCl₂, 2 Mg-ATP, 0.5 Na-GTP, 5 KCl with pH 7.2 adjusted by CsOH. Patch pipets had a resistance 4–5 MOhm. The agonists GABA (100 μM) or glutamate (100 μM together with 10 μM Glycine) were applied for 2 s via the fast local perfusion system (RSC-200, BioLogic, France).

Patch clamp recording of HEK293 cells expressing pdP2X7 constructs was performed as previously described (Karasawa and Kawate, 2016 (link)). Briefly, HEK293 cells were transfected with 1 μg of the pdP2X7 constructs using FuGENE6 (Promega, Madison, WI). Cells were used 18–32 hr after transfection for measuring the pdP2X7 receptor activities using the whole cell patch clamp configuration. Membrane voltage was clamped at −60 mV with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA), currents were filtered at 2 kHz (eight-pole Bessel; model 900BT; Devices, Ottawa, IL), and the recording data were sampled at 10 kHz using a Digidata 1440A and pCLAMP 10.5 software (Molecular Devices, Sunnyvale, CA). The extracellular solution contained 147 mM NaCl, 10 mM HEPES, 13 mM Glucose, 2 mM KCl, 0.1 mM CaCl₂, (pH 7.3). The pipette solution contained 147 mM NaCl, 10 mM HEPES, 10 mM EGTA, which was adjusted to pH7.0 using NaOH. Extracellular solutions were rapidly exchanged to the solutions containing 1 mM ATP using a computer-controlled perfusion system (RSC-200; Bio-Logic, France).

ATP-induced whole-cell currents were recorded at -60 mV using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). The recordings were captured and stored using the Digidata 1322A and pCLAMP9 software. The cell culture was perfused with an extracellular solution that contained the following: 142 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM D-Glucose, adjusted to pH 7.3 with 1 M NaOH. The patch electrodes were filled with a solution containing the following: 154 mM CsCl, 11 mM EGTA, and 10 mM HEPES, adjusted to pH 7.2 with 1.6 M CsOH. The control and ATP-containing solutions were applied via a perfusion system (RSC-200, BIOLOGIC, Claix, France).

The intracellular pipette solution was composed of (in mM): CsCl 150, MgCl₂ 1, HEPES 10, BAPTA 10, CsOH to pH 7.3. The culture dish was washed and filled with the control extracellular solution (in mM): NaCl 165, MgCl₂ 1, CaCl₂ 1, MES 10, HEPES 6, NaOH to pH 9.5, then HCl to pH 7.5. Lower pH values were reached by adding to this solution HCl 2 M in water (and more pH 7.5 solution to return pH up if necessary). Various solutions were applied by gravity, near the cell recorded, using either a multiway perfusion system converging to a single tip (50–100 μL/min, exchange time # 1 s) for ancient experiments (Figures 1b, 2, 3, and most of Figures 4 and 5), or a computer‐driven moving‐head multichannel system (RSC‐200; BioLogic, France), exchange time <30 ms in our conditions (Figures 1a, 7, 8 and part of Figures 4 and 5). A 3 M KCl, 5 g/L agar bridge was used to connect the reference electrode to the dish solution. Transmembrane voltage was clamped most of the time to a constant value of −20 mV (occasionally −30 or −40 mV) [except during various non‐detailed controls]. Electric current flowing through the membrane from extracellular to intracellular face (inward current) is counted negative, and represented downwards in the figures.