PROJECT SUMMARY Acetylcholine (ACh) mediates cell-to-cell communication in the central and peripheral nervous systems, as well as non-neuronal systems. ACh released by neuronal and non-neuronal cells in these systems regulates complex brain functions, such as attention, perception, associative learning, and sleep/awake states, and various biological processes in other tissues and organs, including the heart, liver and pancreas. Dysregulation of cholinergic transmission is linked to a number of neurological diseases, including addiction, Alzheimer’s disease, epilepsy, schizophrenia, Parkinson’s disease and depression, as well as many other health problems, including cardiovascular diseases, obesity, diabetes, immune deficiency and cancer. Despite the significance of ACh in physiological and pathological conditions, the precise regulations and exact functional roles of cholinergic transmission in the majority of tissues and organs remain poorly understood, due primarily to the limitations of available tools for monitoring ACh. We recently initiated development of genetically-encoded G-protein-coupled receptor activation-based sensors for ACh (GACh) by coupling a circular permutated green fluorescent protein (cpGFP) with a muscarinic receptor. We are improving the sensors with large-scale site-directed mutagenesis and screening. Our preliminary data suggest that GACh sensors will have specificity, signal-to-noise ratio, kinetics and photostability suitable for real-time imaging of endogenous ACh signals. Here, I propose to complete the development and validation of GACh sensors following two specific aims: Aim 1 is to optimize and characterize GACh sensors. In our pilot work, we constructed a family of GACh sensors. We plan to use large-scale site-directed mutagenesis and screening to generate more GACh sensors with better performance (Aim 1a). Moreover, we will characterize the properties of GACh sensors in cultured cells and neurons (Aim 1b). We expect these experiments to optimize the specificity, signal-to-noise ratio, kinetics and photostability of GACh sensors. Aim 2 is to validate and utilize GACh sensors. In our preliminary study, we found that GACh sensors selectively detect exogenously applied ACh and endogenously released ACh. We will verify whether GACh sensors can be easily employed to detect ACh signals in various brain regions of both mice and rats (Aim 2a). Finally, we plan to explore the applications of GACh sensors in vitro and in vivo, and address a few fundamental questions about central cholinergic transmission (Aim 2b). We expect these experiments to testify the general applicability of GACh sensors in monitoring the dynamics of endogenous ACh signals and reveal some key features of cholinergic transmission.