Bf150 86 7

Mice (P40-50) were perfused transcardially with ice-cold ACSF (pH = 7.4) containing (in mM): 127 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 25 glucose, bubbled continuously with carbogen (95% O2 and 5% CO2). Brains were rapidly removed and coronal slices (275 μm) were cut on a VT1000S vibratome (Leica) in oxygenated ice-cold choline-based external solution (pH = 7.8) containing (in mM): 110 choline chloride, 25 NaHCO3, 1.25 NaHPO4, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate. Slices were recovered in ACSF at 34°C for 15 min and then kept at RT before recording.
Recordings were made with a MultiClamp 700B amplifier (Molecular Devices) at RT using 3-5 MOhm glass patch electrodes (Sutter, BF150-86-7.5). Data were acquired using ScanImage software, written and maintained by Dr. Bernardo Sabatini (https://github.com/bernardosabatini/ SabalabAcq). Traces were analyzed in Igor Pro (Wavemetrics). Recordings with a series resistance > 25 MOhms or holding current above −200 pA were rejected.

2-Arachidonylglycerol (10 µM in 0.01% DMSO, Tocris Bioscience, UK) was puff applied for 1s (14–35 Pa) by a homemade time-controlled pressure device. Puff pipettes were made from borosilicate capillaries (tip diameter ranging from 1.5–2 μm; BF150-86-7.5, Sutter Instrument, USA). The following compounds were applied to the bath: 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281; 1µM for in vitro experiments and 0.5mg/kg for in vivo experiments; Sigma–Aldrich, USA), CdCl₂, (100µM, Sigma–Aldrich, St. Louis, MO, USA), Tetrodotoxin (1 µM; Alomone Labs, Israel), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 5 μM in 0.1% DMSO; Tocris Bioscience, UK), 6-N,N-Diethyl-D-β,γ-dibromomethylene ATP trisodium salt (ARL 67156 trisodium salt, 50 μM; Tocris Bioscience, UK).

Whole-cell currents were obtained using a Multiclamp 700B amplifier (Axon instruments, Foster City, CA). Patch pipettes were made from thin-walled borosilicate glass (outer diameter 1.5 mm, internal diameter 0.86 mm; Sutter instrument BF150–86-7.5) and pulled (P-97 puller Sutter Instruments, Novato CA) to resistances of 6–8 MΩ. For Figure 2, the internal solution consisted of (in mM): K-gluconate 130, HEPES 10, BAPTA 10, KCl 10, MgCl₂ 0.9, Mg₂ATP 4, Na₂GTP 0.3, phosphocreatine 20, added 100 μM Alexa-555, with osmolarity 291–295 mOsm and pH adjusted to 7.2 with KOH. For Figure 4, the external solution consisted of (in mM): CsCl 122, MgCl₂ 2, CaCl₂ 10, glucose 5, HEPES 10, with osmolarity 315±5 mOsm and 7.4 pH, adjusted with CsOH. The internal solution consisted of (in mM): Cs-gluconate 100, CsCl 26, MgCl₂ 2, EGTA 1, HEPES 10, with osmolarity 300±5 mOsm and 7.2 pH, adjusted with CsOH. Astrocyte whole-cell currents were evoked from a holding potential of 0 mV and stimulated using a voltage ramp protocol from −100 mV to +100mV for 500 ms after a potential step to −100 mV (500 ms) (Butenko et al. 2012 (link)). Current signals were filtered at 1 kHz low pass filter and digitized with a Digidata 1320 board (Axon instrument). pClamp 10.2 (Axon instrument) was used for data acquisition and storage.

Whole-cell patch clamp recordings were performed with infrared differential interference contrast visualization at room temperature (21–25 °C). The fluorescently labeled neurons were visualized and identified with a microscope equipped with GCaMP6 or red fluorescent protein (mKate2) filter (BX-51WI, Olympus). Recording pipettes (BF150–86-7.5, Sutter Instruments) were pulled in a horizontal pipette puller (P-97, Sutter Instruments) with a tip resistance of 3–5 MΩ. Patch pipettes were filled with a solution containing (in mM): 128 potassium gluconate, 10 Hepes, 10 phosphocreatine sodium salt, 1.1 EGTA, 5 ATP magnesium salt, and 0.4 GTP sodium salt. pH was adjusted to 7.3 with KOH, and osmolarity was adjusted to 300–305 mOsm with sucrose. An axon 700B amplifier (Molecular Devices) was used to record membrane potentials. Signals were low-pass filtered at 5 kHz and sampled at 20 kHz with a Digidata 1550 and Clampex 10.6 (Molecular Devices), and data were stored on a computer for subsequent off-line analysis.