Next-generation multicolor indicators for in vivo imaging of norepinephrine

Abstract Norepinephrine (NE) is a critical neuromodulator that dynamically shapes brain states and behavior. Genetically encoded fluorescent NE indicators have enabled in vivo visualization of NE release, yet their limited sensitivity and spectral flexibility constrain widespread use in complex experimental paradigms. Here we developed next-generation green and red fluorescent NE indicators, named nLightG2 and nLightR2, respectively. We systematically compared them to other state-of-the-art indicators and found that both indicators improve the detection of endogenous NE release across preparations. Using dual-color photometry, both nLightG2 and nLightR2 could reliably track physiologically relevant NE release along with neuronal activity in different brain areas. Using two-photon imaging, nLightR2 permitted simultaneous dual-color imaging of NE release and astrocytic activity in the hippocampus, while nLightG2 enabled the detection of spatiotemporally discrete NE release events in the visual cortex of awake mice. This improved toolkit will prove useful for dissecting the spatiotemporal complexity of norepinephrine signaling in the brain.