Relation between Release of Catecholamines and FFN511 Studied with Electrochemical Detector Arrays

Abstract

Exocytosis is an essential vesicle fusion process that mediates transmitter release. For high resolution spatiotemporal analysis of single vesicle fusion events, a combinatorial approach of amperometry and fluorescence imaging was employed. Amperometric spikes from physically stimulated bovine chromaffin cells loaded with the Fluorescent False Neurotransmitter FFN511were detected by microfabricated four-electrode electrochemical detector (ECD) arrays. To determine the location of a fusion event leading to amperometric spikes with different quantal size in the different ECD array electrodes, random walk simulations were performed using the actual ECD array geometry. The location of a measured release event was fitted to match the simulated data. To account for drift of the ECD array, the pixel shift of each frame was fitted relative to the image that was used to generate the simulation map. The transparent glass surface in the center of the ECDs allowed the concurrent fluorescence imaging of localized loss of FFN511from secretory vesicles in TIRF microscopy mode. The frequency of amperometric spikes was much higher than that of FFN511 release events. Only the amperometric spikes that were correlated in time and location with a loss of fluorescence event were considered for analysis. The intensity time course of a 3x3 pixel area (480 x 480 nm2) around the fusion site of the fluorescence movie was determined and different fusion events were aligned at the onset of their respective amperometric spikes. Time super-resolution analysis using the recently developed Event Correlated Microscopy (ECOM) method revealed that FFN511 release followed the amperometric spike onset with a mean delay of ∼115 ms, presumably reflecting slow diffusion of FFN while in amperometric detection catecholamine molecules are rapidly consumed during the oxidation process. Supported by NIH grant R01GM121787.