基因编码的去甲肾上腺素、肾上腺素荧光探针的开发及其在体的应用

Norepinephrine (NE) and Epinephrine (Epi), important biogenic monoamine neurotransmitters, are involved in many crucial physiological processes in diverse organs. However, the precise function and regulation of adrenergic and noradrenergic transmission in the majority of tissues remains poorly understood due partly to the limitations of the available techniques for monitoring their release. .In this proposal, we aimed to develop a family of G-protein-coupled receptor activation-based Epi/NE sensors (GRAB-NE) with specificity, kinetics, signal-to-noise ratio and photostability suitable for monitoring dynamics of endogenous NE and Epi in vitro and in vivo. First, we would demonstrate the feasibility of generating a genetically-encoded fluorescent indicator for NE by coupling the conformational change of naturally-evolved adrenergic receptors to a generation of fluorescent signals from a circular-permutated EGFP. In addition, based on this prototype NE sensor, we plan to tune its affinity suitable for physiological concentration, further improve its signal-to-noise ratio and insulate its coupling to downstream signals. Moreover, we will apply our sensors to the nervous and cardiac systems to study the dynamics of NE and Epi. Finally, by generating transgenic mice that specifically express NE sensors in defined subsets of neurons, we would aim to understand at the circuit level of NE's dynamics and regulation during animal behaviors, such as attention, stress and escape.

去甲肾上腺素和肾上腺素作为生命所必需的单胺类神经递质，在诸多器官中都参与极为关键的生理过程。由于检测技术的局限性，人们对大部分组织器官中肾上腺素及去甲肾上腺素突触传递的功能和调节还知之甚少。.本项目旨在开发一系列基于G蛋白偶联受体激活的快速、特异、高信噪比的去甲肾上腺素、肾上腺素荧光探针（GRAB-NE）。首先，我们将肾上腺素能受体激活时的构象变化与循环重排的绿色荧光蛋白光学信号偶联，验证构建遗传编码的去甲肾上腺素荧光探针的可行性。此外，我们将改造这种去甲肾上腺素探针的原型，使其具有不同的配体亲和力，高信噪比和较少的下游信号偶联。我们还计划将我们开发的去甲肾上腺素荧光探针应用于神经系统和心脏系统，用于研究去甲肾上腺素和肾上腺素动态变化。通过构建特异性表达去甲肾上腺素探针的转基因小鼠，我们可以进一步了解去甲肾上腺素能神经环路释放的去甲肾上腺素调节动物行为（如注意力，压力和逃避等）的作用机制。

去甲肾上腺素和肾上腺素作为生命所必需的单胺类神经递质，在诸多器官中都参与极为关键的生理过程。由于检测技术的局限性，人们对大部分组织器官中肾上腺素及去甲肾上腺素突触传递的功能和调节还知之甚少。本项目通过开发一系列基于G蛋白偶联受体激活去甲肾上腺素、肾上腺素荧光探针（GRAB-NE），实现快速、特异、高信噪比地离体或在体检测。首先，我们将肾上腺素能受体激活时的构象变化与循环重排的绿色荧光蛋白光学信号偶联，构建遗传编码的去甲肾上腺素荧光探针。此外，我们改造这种去甲肾上腺素探针的原型，使其具有不同的配体亲和力，高信噪比和较少的下游信号偶联。我们还将我们开发的去甲肾上腺素荧光探针应用于神经系统和心脏系统，用于研究去甲肾上腺素和肾上腺素动态变化。通过构建特异性表达去甲肾上腺素探针的转基因动物以及应用于自由移动的活体小鼠，我们进一步了解去甲肾上腺素能神经环路释放的去甲肾上腺素调节动物行为（如注意力，压力和逃避等）的作用机制。

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