Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes

Photorelease of caged Ca2+ is a uniquely powerful tool to study the dynamics of Ca2+-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca2+, together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (similar to 20 mu m by 20 mu m). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca2+ cage NP-EGTA and Ca2+ indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca2+, a uniform rise of [Ca2+](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca2+](i) and a '' priming '' effect of sub-stimulatory [Ca2+](i) on the response of cells to subsequent [Ca2+](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca2+ is a suitable stimulation technique for higher-throughput studies of Ca2+-dependent exocytosis on transparent electrochemical microelectrode arrays.