Next-generation multicolor indicators for in vivo imaging of norepinephrine

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Norepinephrine (NE) is a crucial neuromodulator influencing brain states and behavior.
• Existing genetically encoded fluorescent NE indicators have limitations in sensitivity and spectral flexibility.
• Advanced tools are needed to study NE signaling in complex experimental settings.

Purpose of the Study:

• To develop and characterize next-generation green and red fluorescent NE indicators, nLightG2 and nLightR2.
• To compare the performance of nLightG2 and nLightR2 against existing state-of-the-art indicators.
• To demonstrate the utility of these new indicators for in vivo NE monitoring.

Main Methods:

• Development of novel green (nLightG2) and red (nLightR2) fluorescent NE indicators.
• Systematic comparison of indicator performance across various preparations.
• Application of dual-color photometry to track NE release and neuronal activity.
• Utilizing two-photon imaging for simultaneous NE and astrocytic activity monitoring, and for detecting discrete NE release events.

Main Results:

• nLightG2 and nLightR2 exhibit improved sensitivity for detecting endogenous NE release compared to existing indicators.
• Both indicators reliably track physiologically relevant NE release alongside neuronal activity using dual-color photometry.
• nLightR2 enables simultaneous dual-color imaging of NE and astrocytic activity in the hippocampus.
• nLightG2 allows detection of spatiotemporally discrete NE release events in the mouse visual cortex.

Conclusions:

• The developed nLightG2 and nLightR2 indicators represent a significant advancement in NE sensing technology.
• These tools enhance the ability to study the spatiotemporal dynamics of NE signaling in the brain.
• The improved toolkit will facilitate deeper understanding of NE's role in various brain functions and behaviors.