Journal Article

A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro

Nakamoto, Chihiro, Gloriam, David E., Fukata, Yuko, Fukata, Masaki, Takeuchi, Tomonori, Harpsøe, Kasper, Tomizawa, Yoko, Goto, Yuhei, Aoki, Kazuhiro

2021

Overview

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 (EC 50  = 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 GRAB NE1m , 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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Animals / Binding sites / Biomedical and Life Sciences / Biomedicine / Biosensing Techniques / Biosensors / Catecholamines / Cell culture / Detectors / Dopamine / Dopamine - metabolism / Dopamine D1 receptors / Dopamine receptors / Fluorescence / Fluorescence probe / G protein-coupled receptors / GPCR / HeLa Cells / Hippocampal neuron / Hippocampus / Humans / Membrane proteins / Methodology / Mutation / Neuroimaging / Neurology / Neuromodulation / Neurons - metabolism / Neurosciences / Neurotransmitters / Norepinephrine / Norepinephrine - metabolism / Norepinephrine - pharmacology / Phenols / Physiological aspects / Plasmids / Proteins / Psychopharmacology / Red fluorescent protein