Monitoring norepinephrine releasein vivousing next-generation GRAB_NEsensors

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter, yet researches using prototype NE sensors were limited by their low sensitivities. Here, we developed next-generation versions of GPCR activation-based NE sensors (GRAB_NE2mand GRAB_NE2h) with a superior response, high sensitivity and selectivity to NE bothin vitroandin vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell type‒specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRAB_NEsensors are valuable tools for monitoring the precise spatiotemporal release of NEin vivo, providing new insights into the physiological and pathophysiological roles of NE.