# Next-generation multicolor indicators for in vivo imaging of norepinephrine

## TL;DR

Scientists developed improved red and green fluorescent indicators to better track norepinephrine release in the brain of awake mice.

## Contribution

Next-generation red and green fluorescent indicators for norepinephrine with enhanced sensitivity and dual-color imaging capabilities.

## Key findings

- nLightG2 and nLightR2 improve detection of endogenous norepinephrine release across preparations.
- Dual-color photometry enables tracking of NE release and neuronal activity in different brain areas.
- Two-photon imaging allows simultaneous imaging of NE release and astrocytic activity in the hippocampus.

## 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.

Improved red and green indicators for norepinephrine and their characterization are reported. These indicators allow detection of norepinephrine release in awake behaving mice in dual-color fiber photometry and two-photon imaging applications.