دورية أكاديمية
A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro
| العنوان: | A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro |
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| المؤلفون: | Chihiro Nakamoto, Yuhei Goto, Yoko Tomizawa, Yuko Fukata, Masaki Fukata, Kasper Harpsøe, David E. Gloriam, Kazuhiro Aoki, Tomonori Takeuchi |
| المصدر: | Molecular Brain, Vol 14, Iss 1, Pp 1-14 (2021) |
| بيانات النشر: | BMC, 2021. |
| سنة النشر: | 2021 |
| المجموعة: | LCC:Neurology. Diseases of the nervous system |
| مصطلحات موضوعية: | GPCR, Dopamine, Norepinephrine, Fluorescence probe, Hippocampal neuron, Neurology. Diseases of the nervous system, RC346-429 |
| الوصف: | Abstract 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 the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of 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 replaced by 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 a reasonable dynamic range (ΔF/F 0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1756-6606 71434445 |
| Relation: | https://doaj.org/toc/1756-6606 |
| DOI: | 10.1186/s13041-021-00882-8 |
| URL الوصول: | https://doaj.org/article/f81a0da714344452943514f5d04d1252 |
| رقم الأكسشن: | edsdoj.f81a0da714344452943514f5d04d1252 |
| قاعدة البيانات: | Directory of Open Access Journals |
Find this article in full text from Springer Verlag. 
Full Text Finder | تدمد: | 17566606 71434445 |
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| DOI: | 10.1186/s13041-021-00882-8 |