Perforated patch recording

Our protocol was adapted from Heigele et al. (47). Fresh gramicidin (Sigma-Aldrich) stock solution [20 mg/ml in dimethyl sulfoxide (DMSO)] was prepared daily. Stock solution (2.5 μl) was added to 1 ml of warm (35°C) KCl internal (final concentration, 50 μg/ml). The solution was then vortexed for 1 min, sonicated for 15 min, and, lastly, filtered using a 0.2-μm filter. The solution was used within 2 hours. Recording pipettes (4 to 6 megohms) were tip filled with gramicidin-free KCl internal solution and then backfilled with the gramicidin solution. When approaching cell, small positive pressure was applied until the cell was patched. Seal properties were monitored measuring the current responses to square voltage steps (−5 mV, 150 ms; 0.1 Hz). Eighty- to 150-megohm access resistances could be attained within 10 to 15 min. Lucifer yellow (0.1%) was included in the internal solution to visually confirm in real time the integrity of the membrane. If any lucifer yellow staining of the cell body was observed during or after completion of the recordings, then data were discarded. All perforated patch recordings were performed at 27° to 30°C in the presence of 3 mM kynurenic acid. GABAergic synaptic currents were elicited by extracellular electrical stimulation (0.2 to 1.0 mA, 0.2 ms) using a bipolar electrode positioned in layer 1 of the mPFC, 100 to 300 μm apart from the recorded cell. Current/voltage (I/V) curves were obtained measuring the current at different holding voltages (−100 to −20 mV, 20-mV steps). For each voltage, 5 to 10 traces were averaged. If unclamped action potentials were recorded during any sweep, then that sweep was excluded from the analysis. The current peaks were plotted versus the holding potentials, and the I/V curves were obtained by fitting the data points using second-degree polynomial functions (using Clampfit 10, Axon Instruments).