Integration of single-cell electropermeabilization together with electrochemical measurement of quantal exocytosis on microchips

Abstract

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from quantal exocytosis of oxidizable transmitter from individual vesicles. Using electroporation we have developed an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration can reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization have similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Robust quantal release is elicited upon electroporation in 0 Ca2+/5 mM EGTA in the bath solution. Electropermeabilization-induced transmitter release requires Cl- in the bath solution--bracketed experiments demonstrate a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl- ] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane impermeant substances into a cell.