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The next-generation norepinephrine (NE) indicators, nLightG2 & nLightR2 !

Release time：2026-03-26 09:23:09

Norepinephrine (NE) is a key neuromodulator in the central nervous system, involved in regulating arousal, memory formation, stress responses, and other brain functions. The precise monitoring of its spatiotemporal release dynamics is crucial for understanding neural circuit mechanisms. Traditional detection methods suffer from insufficient specificity, low spatiotemporal resolution, and limited sensitivity. Existing genetically encoded fluorescence indicators (GEFIs), such as GRABNE and the first-generation nLight, have narrow dynamic ranges and poor performance of red indicators, limiting their use in complex experiments.

On February 26, 2026, the Tommaso Patriarchi team from the Department of Pharmacology and Toxicology at the University of Zurich published an article titled "Next-generation multicolor indicators for in vivo imaging of norepinephrine" in Nature Methods. The article reports the development and application of the next-generation green fluorescence nLightG2 and red fluorescence nLightR2 norepinephrine indicators. Through point mutations and C-terminal truncation optimization of previous indicators, these two indicators exhibit significant improvements in brightness and dynamic range. They also possess excellent NE specificity, reversibility, and sub-second kinetics. In vitro, brain slice, and in vivo experiments have successfully achieved precise monitoring of NE release in behaviors such as sleep, fear learning, and virtual navigation, supporting multi-path multiplexing with two-photon imaging and dual-fiber recording methods.

https://www.nature.com/articles/s41592-026-03006-z

Development of the Next-Generation nLight Indicators

The next-generation nLight indicators (nLightG2 and nLightR2) are based on the previous nLightG/nLightR, introducing six and three point mutations, respectively, from the corresponding modified dopamine indicators, along with a C-terminal truncation of 55 amino acids. Additionally, ligand-insensitive control indicators (nLightG2-ctr/nLightR2-ctr) were constructed. In the characterization of these indicators in vitro (HEK293T cells, primary cortical neurons), the two new indicators demonstrated a significant enhancement in dynamic range (nLightG2 reached 2350% ± 46%, nLightR2 reached 740% ± 7%), with high specificity for NE (NE/DA selectivity of 31-32 times), excellent reversibility, sub-second kinetics, improved baseline brightness and surface expression, and stability within the pH range of 6-8. The apparent molecular brightness was significantly better than GRAB (NE2m), and the dynamic range enhancement primarily resulted from increased brightness in the NE-bound state.

Figure 1 | In vitro characteristics of next-generation nLight indicators

In Vitro Brain Slice Validation of the Indicators

To compare the new indicators with their parent indicators, two-photon microscopy was used to assess their performance in acute brain slices (Figure 2a). First, nLightR2 and nLightR were validated: iontophoretic application of exogenous NE to brain slices showed that 5 ms NE pulses at different currents induced fluorescence increases in both, with nLightR2 exhibiting significantly higher fluorescence peak ∆F/F₀ than nLightR (Figure 2b-d). For detecting endogenous NE release triggered by electrical stimulation, simulating the physiological scenario of in vivo NE release (Figure 2e-g), the fluorescence amplitude detected by nLightR2 was significantly higher than nLightR (Figure 2e), with a larger ∆F/F₀ above baseline and stronger spatial quantification capability (Figures 2f, g).

For comparison of the green indicators nLightG and nLightG2 (Figure 2h): at different currents, nLightG2 showed larger fluorescence response amplitudes to exogenous NE (Figures 2h, i) and better spatial detection (Figures 2j, k). When detecting endogenous NE, nLightG2's fluorescence peak amplitude (Figure 2l) and signal area (Figures 2m, n) were significantly greater than nLightG. Both indicators exhibited similar dynamic characteristics under the same NE pulses (Figures 2o, p). Overall, nLightR2 and nLightG2 provide improved sensitivity in detecting NE release compared to their parent indicators in two-photon imaging.

Figure 2 | In vitro brain slice validation of NE indicators using two-photon microscopy

In Vivo Performance Validation of the Next-Generation nLight Indicators

To verify the enhanced in vivo performance of the indicators, a tail suspension (TL) test was used to compare nLightR2 and nLightR, as this paradigm can rapidly induce a large release of NE. The results showed that nLightR2 outperformed nLightR. Optogenetically triggered NE release was used to stimulate LC-NE neurons in Dbh-cre mice (Figures 3a-c). After LC optogenetic stimulation, fluorescence increased significantly in the nLightR2 group under 555/30 nm excitation, accompanied by pupil dilation, with no signal in the control channel (Figures 3d-e). The response of nLightR2 was significantly higher than nLightR2-ctr, while no significant difference was observed with nLightR. Under 405 nm excitation, no differences were observed between the indicators, and the extent of pupil dilation was consistent across groups, confirming that the LC activation levels were equivalent. This demonstrated that nLightR2 can monitor NE release triggered by optogenetic stimulation in the dHPC, while nLightR cannot.

To evaluate the in vivo performance of nLightG2, NE release in the pBLA during a conditioned fear reflex experiment was recorded using fiber optic methods (Figures 3g-i). In this paradigm, the pairing of CS (tone) and US (footshock) induces fear responses and NE release in the amygdala, and after conditioning, CS alone can trigger fear. After 4-6 weeks of rAAV-mediated indicator expression in the pBLA, fluorescence was recorded during baseline (CS only), associative (CS+US), and re-exposure (CS only) phases (Figure 3i). All three indicators detected NE release, but nLightG2 showed higher signal amplitudes in all phases (Figures 3j-q). It was found that nLightG2 had a stronger peak response to CS than nLightG and GRAB (NE2m) (Figure 3p), confirming its superior sensitivity in detecting NE release during associative learning stages. The peak signal for footshock-induced NE release was significantly greater with nLightG2 than nLightG and GRAB (NE2m), indicating its higher sensitivity in detecting NE release following aversive stimuli (Figure 3q).

Figure 3 | In vivo validation of NE indicators using fiber optic methods in behavioral experiments

In Vivo Multiplexed Detection of NE and Neural Activity

NE is released in both awake and sleep states. To test nLightR2's ability to report NE release dynamics under different sleep-wake states, NREM sleep was focused—known for LC neuron activity peaks about every 50 seconds, related to sleep regulation, with varying amplitudes and durations. To assess whether nLightR2 can report NE release triggered by these activity peaks, Dbh-cre mice were co-transduced with jGCaMP8f and nLightR2 (or the mutant nLightR2-ctr) in LC-NE neurons (Figure 4a), with EEG/EMG electrodes implanted and dual-fiber photometry used to record sleep-wake states (Figure 4b). All three indicators expressed well at the LC site and could be simultaneously detected through fiber optics with wavelength specificity (Figure 4c); nLightR2 fluorescence tracked LC activity peaks (Figure 4d), while the mutant showed no stable signal (Figure 4e). During NREM sleep, the average LC activity peak was calculated, and the corresponding average fluorescence waveform for nLightR2 showed accurate tracking of LC activity (Figures 4f-h), with a significantly higher cross-correlation coefficient with jGCaMP8f (Figure 4i).

To combine nLightG2 with the red calcium indicator PinkyCaMP to monitor activity changes during a conditioned fear reflex (Figures 4j-l): excitatory neurons were transduced with CaMKiia-PinkyCaMP, and all neurons were transduced with nLightG2 (or the mutant), followed by fiber optic detection in aBLA (Figure 4j). Both indicators expressed well (Figure 4k). Calcium peaks induced by CS (PinkyCaMP) and NE release (nLightG2) were higher during re-exposure compared to baseline, and NE levels continued to rise; the mutant showed no such phenomena, indicating that nLightG2 signals reflect true NE release in aBLA without interference from artifacts (Figures 4m-o). Quantification showed that both PinkyCaMP and nLightG2 significantly increased their peak values triggered by CS during each phase, while nLightG2-ctr showed no change (Figure 4p). During the associative phase (with 10 US+CS pairings), US triggered sharp calcium responses with PinkyCaMP and sustained NE release with nLightG2 (>40 seconds); the mutant only showed a slight signal decrease, indicating the presence of a small non-NE-related component (Figures 4q-t). In summary, the next-generation indicators enable multiplexed in vivo detection of NE and neural activity through fiber optic methods.

Figure 4 | nLightG2 and nLightR2 for studying NE release and neuronal activity using in vivo dual-fiber photometry

Multiplexed Two-Photon Imaging of NE and Intracellular Calcium Signals During Spatial Navigation

To reduce interference from cell-autofluorescence in two-photon recording, cell-type specific promoters were used to express nLightR2 in CA1 neurons and GCaMP6f in astrocytes, enabling spatial separation of the two indicators and synchronous recording of NE in CA1 pyramidal cells and calcium signals in astrocytes (Figures 5a-d). Two-photon image segmentation (Figure 5c): NE signals were measured using a cell-sized region of interest (ROI), while GCaMP6f signals were segmented using astrocyte-specific deep learning software. Event-triggered averaging analysis showed that NE signals increased continuously during running, with a brief rise in NE signals after crossing the reward location (Figures 5d-g), consistent with results from mice expressing only nLightR2. GCaMP6f responses in astrocyte ROIs showed behavioral dependence (Figures 5i-l): a sustained positive ΔF/F₀ response during running (Figure 5i), and heterogeneous responses after crossing the reward location (Figure 5k). ROI analysis confirmed that, during running and after crossing the reward location, NE signals in ROI pairs exhibited high Pearson correlation coefficients (Figures 5f, 5h). Astrocytic GCaMP6f signals in ROI pairs also showed behavior-dependent correlation (Figures 5j-l). Exploring the relationship between the two, it was found that after crossing the reward location, astrocytic GCaMP6f signals correlated with adjacent NE signals, but no such correlation was observed during running (Figures 5m, n). The correlation coefficient between NE and astrocytic GCaMP6f signals depended on the animal's behavior, with significantly higher correlations during the reward phase compared to the running phase (Figure 5o). This behavior-dependent correlation was independent of the ROI distance (Figure 5o). In summary, astrocytic GCaMP6f activity during the reward phase showed higher correlation with NE signals than during running, and the amplitude of its response increased with the amplitude of adjacent NE signals.

Figure 5 | Synchronization of local NE release and astrocytic calcium dynamics in awake mice during spatial navigation using two-photon imaging

Using nLightG2 to Image Local Norepinephrine Dynamics in the Cortex of Awake Mice

To monitor the dynamics of NE in the cortex of awake mice and compare nLightG2 with GRAB (NE2m) and nLightG2-ctr, the indicators were expressed in layers 2/3 of the VC. Two-photon imaging was performed on head-fixed mice on a rotating wheel (Figure 6a). In nLightG2 mice, strong and spatially confined instantaneous NE signals were observed, which responded to approaching stimuli and were visible during the inter-period of the gray screen (spontaneous NE release) (Figures 6b, c); no such signal was observed in nLightG2-ctr mice, confirming that it accurately reports local NE release (Figures 6b, c). The signal was a transient ΔF/F₀ increase, localized to specific sub-regions within the imaging field of view (FOV), forming a mosaic distribution of transiently activated microdomains (Figures 6d-g). Fluorescence changes induced by approaching stimuli were detected in all nLightG2 mice. Among 2200 grid ROIs, only nLightG2 showed significant differences in ΔF/F₀ distribution before and after stimulation (Figures 6h, i), with no significant changes observed in nLightG2-ctr and GRAB (NE2m) (Figures 6h, i). Paired comparison confirmed that approaching stimuli induced a significant increase in nLightG2 signal, while changes were minimal for GRAB (NE2m) and nLightG2-ctr (Figures 6h, i). The probability of strong responses to stimuli was significantly higher for nLightG2 than for nLightG2-ctr, with a wider peak z-score distribution and higher signal variability between ROIs, indicating that nLightG2 has superior sensitivity, dynamic range, and ability to capture spatial/discrete signals compared to GRAB (NE2m) and the control (Figures 6h, i).

When comparing signals during rest and forced activity periods (Figures 6j, k), nLightG2 mice exhibited significantly higher NE signals during activity, while GRAB (NE2m) modulation was weak. The signals during the rest-activity phase were significantly correlated for both nLightG2 and GRAB (NE2m), and moderately correlated for nLightG2-ctr, likely due to non-specific signals (Figure 6j). The receiver operating characteristic (ROC) curve analysis showed that nLightG2 better distinguished between rest and activity phases than GRAB (NE2m). Using generalized linear model (GLM) analysis, the contributions of sensory stimuli and behavioral states were separated (Figures 6l, m), quantifying their effects on NE variability. Data analysis showed that NE dynamics reflected real-time mouse behavior and instantaneous sensory events (Figures 6l, m). In summary, nLightG2 significantly outperforms GRAB (NE2m) in in vivo sensitivity and dynamic range and is a superior tool for analyzing endogenous noradrenergic signals in vivo.

Figure 6 | nLightG2 reveals spatially structured neuronal activity dynamics in the VC of mice during sensory stimuli and behavioral state transitions

Conclusion

The next-generation indicators significantly outperform previous versions in terms of dynamic range, sensitivity, and specificity. The red indicator nLightR2 addresses the performance deficiencies of red-spectrum GEFIs. Supporting multiplexing with dual-fiber photometry and two-photon imaging, they enable the simultaneous monitoring of NE release and neuronal/glial cell activity.

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Tel： +8618971215294

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The next-generation norepinephrine (NE) indicators, nLightG2 & nLightR2 !

Development of the Next-Generation nLight Indicators

In Vitro Brain Slice Validation of the Indicators

In Vivo Performance Validation of the Next-Generation nLight Indicators

In Vivo Multiplexed Detection of NE and Neural Activity

Multiplexed Two-Photon Imaging of NE and Intracellular Calcium Signals During Spatial Navigation

Using nLightG2 to Image Local Norepinephrine Dynamics in the Cortex of Awake Mice

Conclusion

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The next-generation norepinephrine (NE) indicators, nLightG2 & nLightR2 !

Development of the Next-Generation nLight Indicators

In Vitro Brain Slice Validation of the Indicators

In Vivo Performance Validation of the Next-Generation nLight Indicators

In Vivo Multiplexed Detection of NE and Neural Activity

Multiplexed Two-Photon Imaging of NE and Intracellular Calcium Signals During Spatial Navigation

Using nLightG2 to Image Local Norepinephrine Dynamics in the Cortex of Awake Mice

Conclusion

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