Summary 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 NE2m and GRAB NE2h ) with a superior response, high sensitivity and selectivity to NE both in vitro and in 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 NE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo , providing new insights into the physiological and pathophysiological roles of NE.

Monitoring norepinephrine releasein vivousing next-generation GRABNEsensors