IN VIVO DETECTION OF NOREPINEPHRINE USING NEXT-GENERATION MULTICOLOR FLUORESCENT INDICATORS IN FIBER PHOTOMETRY AND 2-PHOTON MICROSCOPY

Norepinephrine (NE) is a key neuromodulator that regulates brain states and behavior in most animals. Although genetically encoded fluorescent NE indicators have allowed monitoring of NE release in vivo, their relatively low sensitivity and limited spectral options have restricted their application in more complex experimental settings. We thus developed the next generation of green and red fluorescent NE indicators, termed nLightG2 and nLightR2. Through systematic comparisons with existing NE indicators, we showed that both nLightG2/R2 detect more effectively artificial and endogenous NE release across multiple experimental preparations. In dual-color fiber photometry experiments, we successfully multiplexed NE dynamics using nLightG2/nLightR2 alongside neuronal activity. Using nLightR2 and jGCaMP8 in locus coeruleus (LC) during sleep-wake cycles, we observed that nLightR2 followed LC activity on a surge-by-surge basis. Using nLightG2 and PinkyCaMP in BLA, we found that neuronal activity and NE weakly correlate across time but strongly correlate during foot shock presentation. Interestingly, these signals showed different kinetics: foot shock-driven BLA activity returns to baseline after 5s while NE stays elevated for more than 40s. Multiplexing was also possible with two-photon imaging: we were able to simultaneously observe NE release (nLightR2) together with astrocytic activity (GCaMP6f) in the hippocampus (CA1) in awake head-fixed mice navigating a virtual reality. Finally, nLightG2 allowed visualization of spatially and temporally discrete NE release events in the visual cortex of awake mice presented during spontaneous locomotion and upon presentation of looming stimuli. Together, these improved indicators provide a powerful toolkit for probing norepinephrine’s spatiotemporal signaling in the brain.