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/F0 = − 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.
Highlights
The catecholaminergic neuromodulators dopamine (DA) and norepinephrine (NE) have very high structural similarity, differing only by a single hydroxy group.Dopaminergic projections mainly originate from the ventral tegmental area and the substantia nigra pars compacta [1], whilst noradrenergic projections mainly originate from the locus coeruleus (LC) [2, 3]
Gln 224 of DA1.2 which results in the 66-fold selectivity (Additional file 1: Fig. S7), is located just before cpmApple (Fig. 2a) in a region of the receptor known to move during activation [46]
Distinct active states are characteristic for G protein-coupled receptor (GPCR) signaling through multiple effectors [47], which applies to DA receptor D1 (DRD1) [48, 49]
Summary
Dopaminergic projections mainly originate from the ventral tegmental area and the substantia nigra pars compacta [1], whilst noradrenergic projections mainly originate from the locus coeruleus (LC) [2, 3]. DA is involved in reward [7,8,9], motivation [10], novelty response [11], and motor control [12, 13]. The involvement of DA and NE overlap in many brain functions [14, 15], such as learning and memory [11, 16], arousal [17, 18], and stress response [6, 19]. The prefrontal cortex receives both dopaminergic and noradrenergic projections, and these
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