Whole Cell Patch Clamp Recordings

Micropipettes of 5 to 7 MΩ were pulled from borosilicate tubing (WPI B150F-4) with a commercial puller (PC-10 Narishige; London, UK) and filled with an internal solution (pH: 7.2–7.3; osmolality: 290–300 mOsm) containing (in mM) 112.5 K-gluconate, 1 EGTA, 10 Na⁺-HEPES, 5 MgATP, 1 GTP, 0.1 leupeptin, 10 phosphocreatine, 4 NaCl, 17.5 KCl, 0.5 CaCl₂, and 1 MgCl₂ (Sigma-Aldrich).

All recordings were performed using a commercial patch amplifier (EPC 10–2; HEKA, Lambrecht/Pfalz, Germany). Action potential frequencies and resting membrane potential were measured in current clamp configuration. Membrane resistance was obtained from the value provided by the amplifier software which uses holding current, holding voltage, and access resistance for its calculation. Thereafter, fast and slow delayed rectifier (FDR and SDR, respectively) and I_A potassium currents were measured in voltage clamp configuration. FDR, SDR, and I_A currents were isolated as described previously (Farajnia et al., 2012). Briefly, Na⁺ and Ca²⁺ currents as well as spontaneous GABAergic currents were eliminated by application of tetrodotoxin (0.5 μM; Tocris bioscience), cadmium (25 μM), and bicuculine (20 μM; Sigma-Aldrich), respectively. Afterward, tetraethylammonium-Cl⁻ (TEA) in low and high concentrations (1 and 20 mM; Sigma-Aldrich) was applied to block FDR and SDR currents, respectively. Current traces were evoked by 400 ms progressively depolarizing voltage pulses (−50 to +60 mV, 10 mV increments), following a prepulse at −100 mV for 100 ms. FDR currents were isolated by subtracting the current traces acquired in the presence of TEA (1 mM) from the ones obtained in control solution. In addition, digital subtraction of traces in 20 mM TEA from traces obtained in 1 mM TEA (blocking FDR) resulted in isolation of SDR. To characterize I_A currents, identical voltage protocol (−60 to +60 mV, 150 ms, 10 mV increments) was applied but with two different prepulses to activate (−90 mV, 150 ms) or inactivate (−45 mV, 150 ms) the current. I_A current was isolated by subtracting the traces lacking the I_A current from traces containing the current. Peak current amplitude was determined in steady state at the end of the test pulse for SDR and FDR currents and the maximum value of the trace was used as the peak value for the transient I_A current. The kinetics of the currents was not analyzed.