(WITHDRAWN) Dissecting Single Acetylcholine Exocytosis Transients and Quantifying Quantal Exocytosis Release of Acetylcholine

Acetylcholine, a critical neurotransmitter involved in many brain functions, lacks methodology to directly and timely monitor quantal exocytosis release of acetylcholine at the presynapse. In this work, we introduce an ultrafast enzyme-based amperometric biosensor for detection of acetylcholine. The sensor is fabricated based on a previous invention in our lab to significantly improve temporal resolution of biosensors. Here carbon fiber microelectrodes are modified using noble metal particles and the enzymes acetylcholine esterase and choline oxidase are co-immobilized and creating an ultrathin layer at the sensor surface. The enzymatic sequential catalytic reaction of acetylcholine producing hydrogen peroxide serve as the electroactive detection for acetylcholine. By placing the biosensor in close contact to neurites of differentiated human cholinergic cells, sub-millisecond recording of exocytotic fusion pore regulation of acetylcholine was recorded. The resulting data revealed a diversity of exocytosis current spike kinetics and dynamics, indicating complex mechanisms for the transient exocytosis fusion regulating acetylcholine release. Categorizing the spikes in terms of lifetime and shape showed a significant difference in relative quantal release. For absolute quantification, electroanalysis of synthetic vesicles pre-loaded with different concentrations of acetylcholine served to create a calibration curve and was used to determine the absolute number of acetylcholine molecules involved in quantal release. Treating the cells with drugs affecting the neurotransmitter loading mechanisms showed that both activity and the quantal release was significantly affected in a predictable manner. This study presents a significant advancement in acetylcholine detection and paves the way for further advancements in research regarding fusion pore regulation of acetylcholine and mechanistic studies of exocytosis involving acetylcholine at single cell level.