基因编码乙酰胆碱荧光探针的开发及其应用
作者:Miao Jing, Peng Zhang, Guangfu Wang, Jiesi Feng, Lukas Mesik, Jianzhi Zeng, Huoqing Jiang, Shaohua Wang, Jess C Looby, Nick A Guagliardo, Linda W Langma, Ju Lu, Yi Zuo, David A Talmage, Lorna W Role, Paula Q Barrett, Li I Zhang, Minmin Luo , Yan Song, J Ju
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2018-07-20 17:54:25
一、研究背景
乙酰胆碱作为重要的神经递质,其在中枢神经系统中的功能现如今仍然存在较大争议。进一步了解乙酰胆碱的功能离不开对其在大脑中动态变化的灵敏实时检测,然而现如今的检测方法缺乏足够特异性或灵敏度,无法实现在活体动物中对复杂多变的乙酰胆碱信号进行实时追踪。为了解决该问题,本文通过将循环重排的荧光蛋白与乙酰胆碱受体进行融合,成功将乙酰胆碱受体结合乙酰胆碱后的构象改变转变为荧光信号的变化,从而可以通过成像手段实时检测乙酰胆碱的释放。
二、研究亮点与特色
(1)第一次开发出基因编码的乙酰胆碱荧光探针,可实现在不同动物模型中内源乙酰胆碱释放的灵敏检测。
(2)通过对内源乙酰胆碱释放的分析了解其释放如何受到神经元活性调节,以及其在释放后的空间作用范围。
三、预期社会与经济效益
在基础研究中,乙酰胆碱探针可以实现对于内源乙酰胆碱释放及其调控的精确指示,这有利于了解其在大脑中的分布以及生理学功能。同时,结合在疾病模型中对乙酰胆碱进行检测,可以了解乙酰胆碱与特定疾病之间的关系,为解析疾病的发病机理和可能的药物开发提供依据。
四、下一步工作
未来工作我们将进一步优化乙酰胆碱探针的信噪比,同时将其在多种体系中进行应用,为了解大脑中乙酰胆碱的功能提供重要的信息。
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A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies
作者:Miao Jing, Peng Zhang, Guangfu Wang, Jiesi Feng, Lukas Mesik, Jianzhi Zeng, Huoqing Jiang, Shaohua Wang, Jess C Looby, Nick A Guagliardo, Linda W Langma, Ju Lu, Yi Zuo, David A Talmage, Lorna W Role, Paula Q Barrett, Li I Zhang, Minmin Luo , Yan Song, J Ju
●
2018-07-20 17:54:25
导语
The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.
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