Genetically-encoded ACh sensors

项目摘要 乙酰胆碱（Acetylcholine，ACh）介导中枢和外周神经系统中的细胞间通讯 系统，以及非神经系统。脑内神经元和非神经元细胞释放的乙酰胆碱 这些系统调节复杂的大脑功能，如注意力、感知、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，包括成瘾、阿尔茨海默病、癫痫、精神分裂症、帕金森病 疾病和抑郁症，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， 在大多数组织和器官中胆碱能传递的调节和确切的功能作用 对乙酰胆碱的认识仍然很少，这主要是由于现有监测乙酰胆碱的工具有限。我们 最近启动的基于基因编码的G蛋白偶联受体激活的开发 通过将环状排列的绿色荧光蛋白（cpGFP）与 毒蕈碱受体我们正在通过大规模定点突变来改进传感器， 筛选我们的初步数据表明，GACh传感器将具有特异性，信噪比 比率、动力学和光稳定性适合于内源性ACh信号的实时成像。 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：目的1是优化和表征GACh传感器。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， 产生更多性能更好的GACh传感器（目标1a）。此外，我们将描述 培养细胞和神经元中GACh传感器的性质（目的1b）。我们希望这些实验 优化GACh传感器的特异性、信噪比、动力学和光稳定性。目标二是 来验证和利用GACh传感器。在我们的初步研究中，我们发现GACh传感器 选择性检测外源性ACh和内源性ACh释放。我们将核实 GACh传感器可以很容易地用于检测两种小鼠不同脑区的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， 并解决了一些关于中枢胆碱能传递的基本问题（目标2b）。我们 我希望这些实验能够证明GACh传感器在监测 内源性乙酰胆碱信号的动力学和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.