Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in finely controlling output functions is currently challenging, primarily due to a lack of techniques to visualize spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed R-GenGAR-DA. More specifically, a circular permutated red fluorescent protein (cpmApple) was inserted into the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated reasonable dynamic range (ΔF/F0 = −50%) and DA affinity (EC50 = 0.7 μM) as well as the highest selectivity for DA over NE (143-fold) amongst available DA biosensors. Due to its high selectivity, R-GenGAR-DA1.2 allowed dual-color fluorescence live imaging for monitoring DA and NE, combined with the existing green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. By enabling precise measurement of DA, as well as simultaneous visualization of DA and NE, the red-DA biosensor R-GenGAR-DA1.2 is promising in advancing our understanding of the interplay between DA and NE in organizing key brain functions.

中文翻译：

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遗传编码的红色荧光多巴胺生物传感器可对多巴胺和去甲肾上腺素进行双重成像

多巴胺（DA）和去甲肾上腺素（NE）是关键的神经调节剂，通常协调作用，调节广泛的脑功能。尽管它们具有生理重要性，但在精细控制输出功能时弄清DA和NE之间的关系目前仍具有挑战性，这主要是由于缺乏以足够高的选择性可视化时空动态的技术。尽管已经开发了遗传编码的荧光生物传感器来检测DA，但它们的选择性差，无法将DA与NE区分开。在这里，我们报道称为R-GenGAR-DA的DA的红色荧光遗传编码GPCR（G蛋白偶联受体）激活报告基因的发展。进一步来说，将圆形排列的红色荧光蛋白（cpmApple）插入人DA受体D1（DRD1）的第三个细胞内环，然后筛选DRD1和cpmApple之间的接头内的突变体。我们开发了两个变体：DA刺激后变亮的R-GenGAR-DA1.1和变暗的R-GenGAR-DA1.2。在可用的DA生物传感器中，R-GenGAR-DA1.2表现出合理的动态范围（ΔF/ F0 = -50％）和DA亲和力（EC50 = 0.7μM），并且相对于NE而言，DA相对于NE的选择性最高（143倍）。由于其高选择性，R-GenGAR-DA1.2允许使用双色荧光实时成像来监测DA和NE，并与现有的绿色NE生物传感器GRABNE1m结合使用，该传感器对DA的NE选择性高（> 350倍）原代培养的HeLa细胞和海马神经元中的SPAR