Simultaneous detection of vesicular content and exocytotic release with two electrodes in and at a single cell

Abstract

We developed a technique employing two electrodes to simultaneously and dynamically monitor vesicular neurotransmitter storage and vesicular transmitter release in and at the same cell. To do this, two electrochemical techniques, single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC), were applied using two nanotip electrodes. With one electrode being placed on top of a cell measuring exocytotic release and the other electrode being inserted into the cytoplasm measuring vesicular transmitter storage, upon chemical stimulation, exocytosis is triggered and the amount of release and storage can be quantified simultaneously and compared. By using this technique, we made direct comparison between exocytotic release and vesicular storage, and investigated the dynamic changes of vesicular transmitter content before, during, and after chemical stimulation of PC12 cells, a neuroendocrine cell line. While confirming that exocytosis is partial, we suggest that chemical stimulation either induces a replenishment of the releasable pool with a subpool of vesicles having higher amount of transmitter storage, or triggers the vesicles within the same subpool to load more transiently at approximately 10–20 s. Thus, a time scale for vesicle reloading is determined. The effect of L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor to dopamine, on the dynamic alteration of vesicular storage upon chemical stimulation for exocytosis was also studied. We found that L-DOPA incubation reduces the observed changes of vesicular storage in regular PC12 cells, which might be due to an increased capacity of vesicular transmitter loading caused by L-DOPA. Our data provide another mechanism for plasticity after stimulation via quantitative and dynamic changes in the exocytotic machinery.