An optimized acetylcholine sensor for monitoring in vivo cholinergic activity

The ability to directly measure acetylcholine (ACh) release is an essential step towards understanding its physiological function. Here, we optimized the GRAB(ACh) (GPCR-Activation‒Based-ACh) sensor to achieve substantially improved sensitivity in ACh detection, as well as reduced downstream coupling to intracellular pathways. The improved version of the ACh sensor retains the sub-second response kinetics, physiological-relevant affinity and precise molecular specificity to ACh of its predecessor. Using this sensor, we revealed compartmental ACh signals in the olfactory center of transgenic flies in response to external stimuli including odor and body shock. Using fiber photometry recording and two-photon imaging, our ACh sensor also enabled sensitive detection of single-trial ACh dynamics in multiple brain regions in mice in a variety of behaviors.