Delayed gramicidin delivery through an intra-pipette capillary facilitates perforated patch recordings

The gramicidin-perforated patch-clamp technique is indispensable for recording neuronal activities without changing the intracellular Cl⁻ concentration. Conventionally, gramicidin contained in the pipette fluid is delivered to the cell membrane by passive diffusion. Gramicidin deposited on the pipette orifice sometimes hampers giga-seal formation, and perforation progresses only slowly. These problems may be circumvented by delivering a high concentration of gramicidin from an intra-pipette capillary after a giga-seal is formed. We herein describe the detailed protocol of this improved method. This protocol would greatly facilitate the investigation of Cl⁻ gradient-dependent neuronal activities.