Functional study of the role of SAPAP3 postsynaptic density protein on dorsolateral striatal cholinergic interneurons

项目摘要/摘要 强迫症(OCD)是一种神经精神疾病，排在前10名和前15名最致残 根据2017年《柳叶刀》杂志的数据，女性和男性的疾病发病率分别为 美国为2.3%。强迫症在人类中的症状特征是强迫性思维模式。 以及强迫性运动行为。突触缺陷与精神和神经发育有关 疾病，包括强迫症，但与疾病相关的突触蛋白对细胞、回路和 人们对行为输出的理解并不完全。SAPAP3是一种突触蛋白，其突变是相关的 在人类身上被诊断为强迫症。小鼠SAPAP3结构性缺失导致强迫性运动梳理 行为，通过长期服用选择性5-羟色胺再摄取抑制剂(SSRI)和 纹状体定位的SAPAP3重新表达。因此，强迫症的SAPAP3缺失模式有症状 以及治疗有效性，并可用于研究细胞和回路水平的纹状体功能障碍 在这种强迫性运动表型的背后。初步证据显示纹状体胆碱能中间神经元 (CHIS)可能是该模型中广泛存在的纹状体失调的一个因素。这些细胞显示增加 在SAPAP3缺失的纹状体中诱发乙酰胆碱(ACh)的释放，这可以调节纹状体回路 通过无数亚型广泛表达的ACh受体。这一提议结合了电生理学， 光遗传学和双光子扫描激光显微镜(2PLSM)来测试最重要的假设，即 CHI的功能失调与纹状体ACh释放和强迫运动行为有关 缺少SAPAP3的型号。 在(目标1)中，将使用体外脑片的膜片钳电生理学来比较固有的CHI功能， 并用脑片免疫组织化学方法探讨突触ACh的释放机制。这将揭开 SAPAP3缺失对CHI释放ACh的内在功能特性的影响(目标2)意志 利用病毒光遗传和ACh传感器结构、电生理学和2PLSM来测试特定输入 突触诱发的纹状体ACh释放的变化。最后，(目标3)将有选择地拯救SAPAP3 在CHI中表达，以测试该模型纹状体ACh释放中断和强迫症样表型是否固有 由SAPAP3驱动-这些细胞中的缺失。这将告知CHI靶向治疗是否足以 修改这种类似强迫症的电路和行为表型。

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.