Recording of GABA-Gated Currents.

Electrophysiological recordings in dissociated cells were performed in the whole-cell patch-clamp configuration (Reddy and Jian, 2010; Wu et al., 2013) with an inverted microscope with phase contrast and differential interference contrast optics (model IX71; Olympus, Tokyo, Japan). Physiologic bath solution for dissociated neurons contained 140 mM NaCl, 3 mM KCl, 10 mM HEPES, 2 mM MgCl₂, 2 mM CaCl₂, and 16 mM glucose (pH adjusted to 7.4 with NaOH; osmolarity, 315–325 mOsm/kg). Recording pipette tip resistances were 3–5 MΩ for single-cell recording and were filled with a cesium pipette solution containing 124 mM CsCl, 20 mM tetraethylammonium, 2 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, 0.1 mM GTP, 4 mM ATP (pH adjusted to 7.2 with CsOH; osmolarity, 295–305 mOsm/kg). Currents were recorded using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The membrane capacitance, series resistance, and input resistance of the recordings were monitored by applying a 5-mV (100-millisecond) depolarizing voltage step from a holding potential of −70 mV for dissociated cells. Signals were low-pass filtered at 2 kHz and digitized at 10 kHz with a Digidata 1440A system (Molecular Devices, Sunnyvale, CA). The current values were normalized to cell capacitance (an index of cell size) and expressed as current density (pA/pF). For whole-cell current from isolated single cells, fractional potentiation produced by allosteric modulator was calculated as I_A/I_GABA, where I_GABA was the response of peak amplitude at the application of GABA (3 μM) and I_A is the response of peak amplitude at the coapplication of GABA and the allosteric drug (0.01–1 μM). For fast application of test drugs, the perfusion pipette was positioned < 200 µm away from the cell in the chamber. GABA and allopregnanolone (AP) were applied using a multichannel perfusion system (Automate Scientific, Berkeley, CA). A 2-minute wash with bath solution was instituted after each drug trial to prevent receptor desensitization.