The specific aim of this project is to identify, via a functional high-throughput screening (HTS) approach, small molecule positive allosteric modulators (PAMs) and/or allosteric agonists of the M1 muscarinic acetylcholine receptor (mAChR) that are cell permeable, possess submicromolar potency, and show greater than 10-fold selectivity over the other mAChRs (M2-M5). The currently identified probe ML169 (CID-44475955) can be used for in vitro molecular pharmacology and electrophysiology experiments to study the receptor trafficking profile, and the role of selective M1 receptor activation by this unique M1 PAM chemotype. Use of this probe alongside the authors' initially identified M1 PAM probe, ML137 (CID-44251556) could improve understanding of the M1 signaling pathway and elucidate the difference, if any, between high and low ACh fold-shift compounds due to their different pharmacological characteristics. This probe possesses high selectivity versus M2-M5, as well as a large panel of GPCRs, ion channels and transporters. While in vivo studies are possible, it has not been investigated for such uses. Probes developed from these efforts will greatly advance the current state of the art by aiding in the understanding of M1's role in cell-based physiology, and may extend the clinical understanding of psychotic and cognitive symptoms associated with neurodegenerative disorders like Alzheimer's Disease and schizophrenia.