Gramicidin-perforated patch recordings

Gramicidin-perforated patch recordings allow electrical access to neurons without disturbing the native Cl⁻ concentration (Kyrozis and Reichling, 1995). The intracellular pipette solution contained the following (in mm): 140 KCl, 5 EGTA, 10 HEPES, 2 MgCl₂, and 0.5 CaCl₂, and pH was adjusted with KOH to 7.4, and osmolarity was measured at between 280 and 290 mOsm/L. The use of a high Cl⁻ concentration (145 mm, equal to the extracellular Cl⁻ concentration) allowed us to detect when a perforated patch broke into whole-cell mode, which would result in a Cl⁻ equilibrium potential at ∼0 mV. The tip of the patch pipette was filled with the high Cl⁻ solution and then backfilled by syringe with the same solution containing gramicidin dissolved in DMSO with a final concentration of 5–25 μg/ml. The liquid junction potential was 5 mV, and data were corrected accordingly. The initial series resistance after gigaohm seal formation exceeded 100 MΩ, but could decline down to 20–40 MΩ within 30 min, at which time data acquisition began. Recordings were discarded if the perforated patch ruptured, as indicated by an abrupt drop in series resistance and the measure of IPSC reversal potential (E_rev) at ∼0 mV.