Thin walled borosilicate glass

Recordings used methods similar to those described [23 (link)]. Experiments were performed 24–72 h post-plating at 22 °C and across multiple days (controlling for cell health and expression efficiency). All reagents were purchased from Sigma, unless otherwise noted. Patch pipettes were fabricated from thin-walled borosilicate glass (World Precision Instruments) using a horizontal puller (Sutter Instruments) to give a resistance of 2–8 MΩ when filled with intracellular solution (120 mM KCl/2 mM MgCl₂/10 mM EGTA/10 mM HEPES, NaOH adjusted to pH 7.2, 315 mOsm). Extracellular solution contained: 161 mM NaCl/3 mM KCl/1 mM MgCl₂/1.5 mM CaCl₂/10 mM HEPES/6 mM d-glucose, NaOH adjusted to pH 7.4 (320–330 mOsm). A rapid solution changer (BioLogic Science Instruments) connected to a infusion pump (KD Scientific) delivered GABA and benzodiazepine solutions.

Currents were recorded using whole-cell patch clamp method. Cells were settled on the bottom of a 0.5 ml perfusion chamber in the bath solution. Patch glass pipettes were pulled using thin-walled borosilicate glass (World Precision Instruments). The pipettes had inner diameters of 1.5 μm and resistances of 2 MΩ when filled with solution. An Axopatch 200B amplifier and pCLAMP10 (Molecular Devices) were used for data acquisition and analysis. Data were sampled at 20 kHz and filtered at 5 kHz. Series resistance (Rs) was compensated by 80%, and leak subtraction was not used. All currents were elicited from a holding potential of –80 mV by depolarizing steps to voltages between –70 and +70 mV in 10 mV increments for 200 ms. A repolarizing step to −30 mV was applied for 250 ms before returning to the holding potential. To construct the voltage–current (I-V) relationships, the maximal currents during depolarizing steps were plotted against depolarizing voltages in each cell and summarized in each of the groups. The bath solution contained 135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). The pipette solution consisted of 135 mM KCl, 5 mM MgATP, 5 mM EGTA, and 10 mM HEPES (pH 7.2 with KOH). Patch clamp experiments were performed at room temperature (22 ± 1 °C).