The first identified neurotransmitter, Acetylcholine (ACh), regulates a diverse array of physiological processes throughout the body, from development, sensation, motor control to higher brain cognitive functions. Despite its importance, further understanding of cholinergic transmission, especially in the complex central nervous system, is greatly hindered by the limitations of available techniques in monitoring ACh dynamics. These techniques, including microdialysis based biochemical detection, electrochemical methods and reporter-gene based assays like TANGO, are either poor in molecular specificity or lack of enough spatial and temporal specificity for in vivo studies. In this work, Dr. Yulong Li’s group developed a family of G-protein-coupled receptor activation-based ACh (GACh) sensors by tapping into the naturally-evoked ACh-sensing GPCR as the scaffold. Through iterative screening and optimization, the novel GACh sensors show excellent sensitivity, specificity, signal-to-noise ratio, and sub-second kinetics, which enabled the robust detection of endogenous ACh release in multiple preparations including acute tissues slices in vitro, the olfactory system of flies and the visual system of mice in vivo. The detection and analysis of endogenous ACh release also revealed firing pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Overall, the novel genetically-encoded GACh sensors provide a user-friendly, broadly applicable tool for precisely and cell-specifically monitoring cholinergic transmission underlying diverse biological processes.


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