2.6. Electrophysiological Recordings

The presence of GABA_A-receptor-mediated Cl⁻ currents was assessed by using a port-a-patch planar patch-clamp system (Nanion Technologies, Munich, Germany) in the whole-cell, voltage-clamp configuration, at room temperature (22 °C), as described in [45]. Cultured cells (2–3 days after plating) were detached with Detachin and suspended at a cell density of 1–5 × 10⁶ cells/mL in an external recording solution containing (in mM): 145 NaCl, 2.8 KCl, 2 MgCl₂, 10 CaCl₂, 10 HEPES, 10 D-glucose (pH = 7.4). Suspended cells were placed on the NPC© chip surface, and the whole cell configuration was achieved. Internal recording solution, containing (in mM) 10 CsCl, 110 CsF, 10 NaCl, 10 HEPES, 10 EGTA (pH = 7.2, adjusted with CsOH), was deposited in recording chips, having resistances of 3–5 MΩ. To test the ability of GABA_A receptors to conduct inward Cl⁻ currents, 100 µM GABA or 30 µM muscimol was added to the external solution. The bioelectrical response to agonist stimulation was recorded in the voltage-clamp mode at a holding potential of −70 mV, as described in [17], by using an EPC-10 patch-clamp amplifier (HEKA, Munich, Germany). Immediately after the whole-cell configuration was established, the cell capacitance and the series resistances (<10 MΩ) were measured. During the recordings, these two parameters were measured, and if exceeding ≥10% with respect to the initial value, the experiment was discontinued [45]. Liquid junction potential and capacitive currents were cancelled using the automatic compensation of the EPC-10 [46]. Data were filtered at 10 kHz and sampled at 5 kHz.