PROJECT SUMMARY/ABSTRACT Obsessive Compulsive Disorder (OCD) is a neuropsychiatric disease listed in top 10 and top 15 most disabling illnesses in women and men, respectively, according to the Lancet in 2017 and has a predicted prevalence of 2.3% in the United States. OCD is characterized symptomatically in humans by obsessive thought patterns and compulsive motor behaviors. Synaptic deficits have been linked to psychiatric and neurodevelopmental diseases, including OCD, but the influence of disease-linked synaptic proteins on cellular, circuit, and behavioral outputs are incompletely understood. SAPAP3 is a synaptic protein, whose mutation is correlated with OCD diagnosis in humans. Constitutive SAPAP3 deletion in mice produces compulsive motor grooming behaviors, which are rescued by chronic administration of selective serotonin reuptake inhibitors (SSRIs) and striatum-localized SAPAP3 re-expression. The SAPAP3-deletion model of OCD, therefore, has symptomatic as well as treatment validity and can be used to investigate cellular- and circuit-level striatal dysfunctions underlying this compulsive motor phenotype. Preliminary evidence highlights striatal cholinergic interneurons (ChIs) as a likely contributor of widespread striatal dysregulation in this model. These cells display increased evoked release of Acetylcholine (ACh) in the SAPAP3-lacking striatum, which can modulate striatal circuits through myriad subtypes of widely expressed ACh receptors. This proposal combines electrophysiology, optogenetics, and 2-photon scanning laser microscopy (2PLSM) to test the overarching hypothesis, that functional dysregulation of ChIs is tied to maladaptive striatal ACh release and compulsive motor behavior in the SAPAP3-lacking model. In (Aim 1), patch-clamp electrophysiology in ex vivo brain slices will be used to compare intrinsic ChI function, and brain slice immunohistochemistry will be used to probe synaptic ACh release machinery. This will uncover SAPAP3 deletion’s impacts on intrinsic functional properties related to ACh release by ChIs. (Aim 2) will leverage viral optogenetic and ACh sensor constructs, electrophysiology, and 2PLSM to test for input-specific changes to synaptically evoked striatal ACh release. Finally, (Aim 3) will selectively rescue SAPAP3 expression in ChIs to test if this model’s disrupted striatal ACh release and OCD-like phenotype are intrinsically driven by SAPAP3-deletion in these cells. This will inform whether ChI-targeted therapies may be sufficient to modify this OCD-like circuit and behavioral phenotype.