Analyzing Fusion Pore Dynamics and Counting the Number of Acetylcholine Molecules Released by Exocytosis

Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, quantitative methods with adequate spatiotemporal resolution for recording a single exocytotic ACh efflux are lacking. In this study, we introduce an ultrafast amperometric ACh biosensor that enables 50 kHz electrochemical recording of spontaneous single exocytosis events at axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric traces revealed seven distinct current spike types, each displaying variations in shape, time scale, and ACh quantities released. This finding suggests that exocytotic release is governed by complex fusion pore dynamics in these cells. The absolute number of ACh molecules released during exocytosis was quantified by calibrating the sensor through the electroanalysis of liposomes preloaded with varying ACh concentrations. Notably, the largest quantal release involving approximately 8000 ACh molecules likely represents full exocytosis, while a smaller release of 5000 ACh molecules may indicate partial exocytosis. Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a larger quantity of ACh released and a higher frequency of exocytosis events. Therefore, this ACh sensor provides a means to monitor minute amounts of ACh and investigate regulatory release mechanisms at the single-cell level, which is vital for understanding healthy brain function and pathologies and optimizing drug treatment for disorders.