2.4. Recording of GABA-gated currents

Whole-cell patch-clamp configuration was used for electrophysiological recordings in dissociated cells (Reddy and Jian, 2010). Experiments were performed at room temperature (22–24 °C). The recording chamber was fixed into the stage of an inverted microscope with phase-contrast and differential interference contrast optics (model IX71; Olympus, Tokyo, Japan). The physiological bath solution for the recording of dissociated neurons was composed of (in mM): 140 NaCl, 3 KCl, 10 HEPES, 2 MgCl₂, 2 CaCl₂, and 16 glucose (pH adjusted to 7.4 with NaOH, osmolarity, 315–325 mOsm/kg). Cells were visualized and images were acquired through video camera CCD-100 (Dage-MTI, Michigan City, IN) with FlashBus Spectrim 1.2 software (Pelco, Clovis, CA). Recording pipettes were pulled from capillary glass tubes (King Precision Glass, Claremont, CA) using a P-97 Flaming-Brown horizontal puller (Sutter Instrument Company, Novato, CA). The pipette tip resistances were 4 to 6 MΩ for recording. The recording pipettes were filled with a cesium pipette solution containing (in mM): 124 CsCl, 20 tetraethylammonium, 2 MgCl₂, 10 EGTA, 10 HEPES, 0.1 GTP, 4 ATP (pH adjust 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-ms) 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 Digidata 1440A system. 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, current response was expressed as the percentage of potentiation produced by GABA or GABA+GX. For fast application of test drugs, the perfusion pipette was positioned <200 µm away from the cell in the chamber. GABA, GX, and Zn²⁺ (1–30 μM) were applied using a multi-channel perfusion system (Automate Scientific, Berkeley, CA). A two-minute wash with bath solution was instituted after each drug trial in order to prevent receptor desensitization.