On-Chip Electroporation Results in Calcium- and Chloride- Stimulated Exocytosis Assayed with Planar Electrochemical Microelectrodes

Abstract

We are developing microfabricated devices consisting of arrays of electrochemical electrodes in order to increase the throughput of single-cell measurements of quantal exocytosis from neuroendocrine cells and to develop technology that allows simultaneous electrochemical detection and fluorescence imaging of single fusion events. One component of this effort is to develop on-chip methods for stimulating exocytosis from select cell population on the chip. We have demonstrated that the same electrochemical microelectrode can be used to electroporate an adjacent cell and then measure quantal exocytosis using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration, can reliably trigger exocytosis in Ca2+-containing bath solutions using gold and diamond-like-carbon electrodes. Electrodes fabricated from Indium-Tin-Oxide were less effective at eliciting exocytosis via electroporation. Higher rates of exocytosis were observed with bath solution containing higher [Ca2+]e, and experiments with semi-transparent electrodes and the Ca2+ indicator fura-4F demonstrate a rise in [Ca2+]i upon electrical stimulation. Little or no exocytosis was observed in Ca2+-free (5 mM EGTA) solutions with glutamatic acid as the major anion. Surprisingly, exocytosis could be elicited upon electroporation in 0 Ca / 5 mM EGTA solutions if the bath contained high Cl- concentrations (140 mM NaCl or KCl). This is surprising since the same solution does not induce exocytosis when loaded into the cell through a patch pipette during whole-cell recording. We conclude that electric fields sensitize granules to undergo exocytosis in response to Cl-. Supported by NIH R01 NS048826.