Enhanced Monitoring of Nanosecond Electric Pulse-Evoked Membrane Conductance Changes in Whole-Cell Patch Clamp Experiments.

Abstract

Patch clamp electrophysiology serves as a powerful method for studying changes in plasma membrane ion conductance induced by externally applied high-intensity nanosecond electric pulses (NEPs). This paper describes an enhanced monitoring technique that minimizes the length of time between pulse exposure and data recording in a patch-clamped excitable cell. Whole-cell membrane currents were continuously recorded up to 11ms before and resumed 8ms after delivery of a 5-ns, 6MV/m pulse by a pair of tungsten rod electrodes to a patched adrenal chromaffin cell maintained at a holding potential of -70mV. This timing was achieved by two sets of relay switches. One set was used to disconnect the patch pipette electrode from the pre-amplifier and connect it to a battery to maintain membrane potential at -70mV, and also to disconnect the reference electrode from the amplifier. The other set was used to disconnect the electrodes from the pulse generator until the time of NEP/sham exposure. The sequence and timing of both sets of relays were computer-controlled. Using this procedure, we observed that a 5-ns pulse induced an instantaneous inward current that decayed exponentially over the course of several minutes, that a second pulse induced a similar response, and that the current was carried, at least in part, by Na+. This approach for characterizing ion conductance changes in an excitable cell in response to NEPs will yield information essential for assessing the potential use of NEP stimulation for therapeutic applications.