Genetically-encoded ACh sensors

项目总结 乙酰胆碱(ACh)介导中枢神经和外周神经的细胞间通讯 系统，以及非神经系统。脑内神经细胞和非神经细胞释放ACh 这些系统调节复杂的大脑功能，如注意力、知觉、联想学习、 和睡眠/清醒状态，以及其他组织和器官中的各种生物过程，包括 心脏、肝脏和胰腺。胆碱能传递的失调与许多 神经系统疾病，包括上瘾、阿尔茨海默病、癫痫、精神分裂症、帕金森氏症 疾病和抑郁，以及许多其他健康问题，包括心血管疾病， 肥胖、糖尿病、免疫缺陷和癌症。 尽管ACh在生理和病理条件下具有重要意义，但准确的 胆碱能传递在大多数组织器官中的调节及其确切的功能作用 仍然知之甚少，主要是由于可用于监测ACH的工具的限制。我们 基于基因编码的G蛋白偶联受体激活的最新研究进展 环状置换绿色荧光蛋白(CpGFP)与乙酰胆碱酯酶(GACH)的偶联 毒鼠强受体。我们正在用大规模的定点突变来改进传感器和 放映。我们的初步数据表明，GACH传感器将具有特异性、信噪比 适合内源性ACh信号实时成像的比率、动力学和光稳定性。 在这里，我建议完成以下两个具体的GACH传感器的开发和验证 目的：目标1是对GACH传感器进行优化和表征。在我们的试点工作中，我们构建了一个 GACH传感器系列。我们计划使用大规模的定点突变和筛选来 生成更多性能更好的GACH传感器(目标1a)。此外，我们还将描述 培养细胞和神经元中GACH感受器的特性(目标1b)。我们预计这些实验将 优化GACH传感器的专属性、信噪比、动力学和光稳定性。目标2是 验证和利用GACH传感器。在我们的初步研究中，我们发现GACH传感器 选择性地检测外源ACh和内源性ACh的释放。我们将核实是否 GACH传感器可以很容易地检测到两只小鼠大脑不同区域的ACh信号 和大鼠(目标2a)。最后，我们计划探索GACH传感器在体外和体内的应用， 并解决了关于中枢胆碱能传递的几个基本问题(目标2b)。我们 预计这些实验将证明GACH传感器在监测环境中的普遍适用性 内源性ACh信号的动态变化，揭示胆碱能传递的一些关键特征。

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.