The green fluorescent NE (green-NE) biosensor, GRAB NE1m, has a high selectivity for NE (> 350-fold selectivity for NE over DA). Binding of DA or NE to the sensor induces a conformational change, which couples with a change in the fluorescence of circular-permutated fluorescent protein, such as green fluorescent protein (GFP) for green fluorescence. Recently developed genetically encoded fluorescent biosensors are able to detect extracellular DA or NE with high spatial and temporal resolution, and sensitivity in freely moving animals using in vivo imaging. A method combining sensitivity, specificity, and spatiotemporal resolution is required to satisfactorily answer research questions regarding the timing of release, spatial diffusions, concentrations, and DA/NE ratios. In contrast, fast-scan cyclic voltammetry and a synthetic catecholamine nanosensor both have higher sensitivity and temporal resolution, but cannot distinguish between DA and NE. For example, microdialysis with high-performance liquid chromatography has high sensitivity and selectivity to detect either DA or NE but suffers from poor spatial and temporal resolution. ![]() However, due to technical limitations, little is actually known about these properties with high spatial and temporal resolution within the same preparation. Interactions between DA and NE theoretically depend on the timing of release, spatial diffusions, concentrations, and DA/NE ratios. Furthermore, dysfunction of dopaminergic or noradrenergic systems are thought to be associated with psychiatric disorders and neurodegenerative diseases, such as attention-deficit/hyperactivity disorder (ADHD), schizophrenia, and Parkinson’s disease. ![]() In particular, the prefrontal cortex receives both dopaminergic and noradrenergic projections, and these systems are involved in attention and working memory. In addition, the involvement of DA and NE overlap in many brain functions, such as learning and memory, arousal, and stress response. DA is involved in reward, motivation, novelty response, and motor control. It was discovered recently that noradrenergic LC axons co-released DA along with NE. Dopaminergic projections mainly originate from the ventral tegmental area and the substantia nigra pars compacta, whilst noradrenergic projections mainly originate from the locus coeruleus (LC). The catecholaminergic neuromodulators dopamine (DA) and norepinephrine (NE) have very high structural similarity, differing only by a single hydroxy group. 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. 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 GRAB NE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range ( ΔF/F 0 = − 43%), DA affinity (EC 50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. ![]() We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. 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. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. ![]() Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. 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. Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert.
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