Abstract
Genetically encoded indicators engineered from G-protein-coupled receptors are important tools that enable high-resolution in vivo neuromodulator imaging. Here, we introduce a family of sensitive multicolor norepinephrine (NE) indicators, which includes nLightG (green) and nLightR (red). These tools report endogenous NE release in vitro, ex vivo and in vivo with improved sensitivity, ligand selectivity and kinetics, as well as a distinct pharmacological profile compared with previous state-of-the-art GRABNE indicators. Using in vivo multisite fiber photometry recordings of nLightG, we could simultaneously monitor optogenetically evoked NE release in the mouse locus coeruleus and hippocampus. Two-photon imaging of nLightG revealed locomotion and reward-related NE transients in the dorsal CA1 area of the hippocampus. Thus, the sensitive NE indicators introduced here represent an important addition to the current repertoire of indicators and provide the means for a thorough investigation of the NE system.
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Data availability
DNA and protein sequences for indicators developed in this study were deposited on NCBI (accession numbers ON737776–ON737782) or are available in Supplementary Data 1 of Supplementary Information. DNA plasmids used for viral production have been deposited both with the UZH Viral Vector Facility (https://vvf.ethz.ch/) and on AddGene. Viral vectors can be obtained either from the Patriarchi laboratory, the UZH-VVF or AddGene. Source data are provided with this paper. Due to its large size raw data can be made available only upon reasonable request.
Code availability
Custom MATLAB code is available on https://github.com/patriarchilab/nLightG under a GNU v.3 license.
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Acknowledgements
We thank J.-C. Paterna and the Viral Vector Facility of the Neuroscience Center Zürich (ZNZ) for their help with virus production, S. Schillemeit for expert technical support with histology and K. Assomou and M. Stoeber (University of Geneva) for cloning and providing the LgBit-mini-G-protein fusions and for assistance with NanoBiT complementation assay. We thank C. Lovato for help with perfusion pipette production and S. Schweer for cell culture assistance (both Ruhr University Bochum). The plasmids encoding Beta2AR-SmBit and LgBit-β-arrestin-2, as well as the Alexa-647 labeled M1 anti-FLAG antibody were a kind gift from M. Stoeber (University of Geneva). The rAAV2/9 carrying DNA encoding ChrimsonR-mRuby2 in a double-floxed inverted reading frame under control of the EF1α-promoter (Addgene viral prep 124603-AAV9) was kindly gifted by C. Harvey (Harvard University). The plasmid coding for DRD1-GFP was a kind gift from D. Stamou (University of Copenhagen). The results are part of a project that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement: 891959) (T.P.). We also acknowledge funding from H2020-ICT (grant agreement: 101016787) (T.F and T.P.), the University of Zürich and the Swiss National Science Foundation (grant agreement: 310030_196455) (T.P.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, grant agreement: 178316478-B8) (J.S.W) and the National Institutes of Health (NIH) BRAIN Initiative (grant agreement: U19 NS107464) (T.F.).
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Contributions
T.P. led the study. T.P., V.R. and C.K. conceived the sensor engineering strategy. Z.K., V.R. and C.K. performed molecular cloning, in vitro indicator screening and characterization in HEK293T cells and neurons, and analyzed data under the supervision of T.P. J.D. measured two-photon brightness under the supervision of L.R., B.W. and T.P. T.Z., A.R., L.M.W. and L.S. performed and T.Z. and A.R. analyzed patch-clamp fluorometry experiments. M.A.B. prepared cortical neuronal cultures under the supervision of D.B. Z.K., M.W. and M.H. performed and analyzed imaging experiments in acute brain slices under the supervision of T.P. Z.K., M.W. and M.H. performed and analyzed in vivo tail-lift experiments under the supervision of T.P. S.N.D. and M.W. performed and analyzed optogenetic experiments in vHPC under the supervision of J.B. and T.P. A.D. performed and analyzed optogenetic and photometry experiments in LC and dHPC mice under the supervision of J.S.W. S.C. performed and analyzed in vivo two-photon imaging experiments under the supervision of T.F. T.P., Z.K., V.R., C.K. and X.Z. contributed to writing with input from all authors.
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T.P. is a coinventor on a patent application related to the technology described in this article. All other authors have nothing to disclose.
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Extended data
Extended Data Fig. 1 Development of nLightG and nLightR.
a, Representative images of indicators expressed in HEK293T cells in the absence or presence of NE (10 μM) and heatmaps of fluorescence responses. Scale bar, 15 μm. b, Quantification of basal brightness and dynamic range. n = 21 cells. c, d, Normalized fluorescence intensity dose-response curves of nLightG and nLightR for NE (c) and DA (d) in HEK293T cells. Four-parameter dose-response curve fits determined the EC50 values. n = 3 wells per concentration for each ligand. e, Sequence of RdLight1, dLight1.3b and sperm whale Alpha-1 AR with reference BW numbering. f, g, Dynamic range of red (f) or green (g) indicator variants generated by grafting at different BW registries. The response was measured in HEK293T cells upon addition of NE (10 μM). n = 21 cells. P values were as follows: 1.585×10−8 (f); 5.66–6.30, 4.149×10−17; 5.65–6.31, 9.093×10−7; 5.64–6.32, 2.199×10−8; 5.62–6.34, 4.341×10−14; 5.61–6.35, 3.283×10−14 (g). h, Dynamic range of the red indicator variants with or without ICL2 grafting measured in HEK293T cells. n = 21 cells. P = 2.625×10−5. i, Left, time trace of the relative fluorescent response of nLightR (with or without ICL2 graft). Saturating concentrations of DA (200 μM) and NE (10 μM) were added consecutively. n = 21 cells for each trace. Right, ratio of average fluorescent responses to DA versus NE from traces shown on left. P = 2.292×10−4. j, Same as in (h) but for nLightG and using the ICL2 of dLight1.3b for grafting. P = 0.566. k, Same as in (i) but for nLightG. P = 2.103×10−3. All data are shown as mean ± SEM. All experiments were repeated at least three times with similar results. Mean values were compared using a two-tailed Students t-test with Welch’s correction.
Extended Data Fig. 2 Additional in vitro characterization of nLightG and nLightR.
a, Fluorescence response of GRABNE1m in HEK293T, and quantification of mean responses to ligands. NE (10 μM), trazodone (Trz, 100 nM) and (Yoh (100 nM) were added consecutively. Error bars represent mean ± SEM. n = 11 cells from 3 independent experiments. n.s., P = 0.9902; **** P = 1.374×10−8. b, Left, representative images of cellular brightness for nLightG and GRABNE1m. Scale bars, 20 μm. Right, violin plot comparison of basal brightness between nLightG and GRABNE1m. Medians are thin dotted lines. n = 46, 37 cells for nLightG and GRABNE1m, respectively, from 3 independent experiments. n.s., not significant. P = 0.4839. a-b, Two-tailed Students t-test with Welch’s correction. c, One-photon fluorescence excitation and emission spectra of nLightG in the presence (Sat) or absence (Apo) of NE (100 μM). n = 4 wells. d, same as in (c) for nLightR. e, Two-photon brightness of nLightG in HEK cells. Spectra are normalized to the Apo form at 950 nm. Ratio between Sat and Apo is shown as black dotted line. n = 3 dishes. f, Left, normalized fluorescence dose-response curves of NE in nLightG- and nLightR-expressing neurons. Datapoints were fitted with four-parameter dose-response curves to determine EC50 values. n = 11 cells for nLightG and n = 3 cells for nLightR from three independent experiments. Right, maximal ΔF/F0 response of nLightG- and nLightR-expressing neurons to NE or DA. Both ligands were separately applied at 300 μM concentration on the cells. n = 11, 24 cells for nLightG with NE and DA, respectively, and n = 5 cells for nLightR with NE or DA. Two-tailed Students t-test with Welch’s correction. P values: 3.421×10−9, nLightG; 2.240×10−6, nLightR. mean ± SEM. g, Fluorescence response of nLightG to non-ligand neurotransmitters (10 μM). Welch ANOVA with Dunett’s multiple comparison test. Data are shown as mean ± SEM. n (cells) as follows: 12 for HBSS; 23 for NE; 24 for Epi; 23 for DA; 23 for 5-HT; 26 for Ach; 23 for Ado; 21 for Hist; 22 for GABA; 21 for Glu. P values: NE, P < 10−14; Epi, P < 10−14; DA, P = 0.931×10−6; Ser, P = 0.066; Ach, P = 0.999; Ado, P = 0.705; His, P = 0.999; GABA, P = 0.999; Glu, P = 0.999. h, same as in (g) for nLightR. P values: NE, P < 10−14; Epi, P < 10−14; DA, P < 10−14; Ser, P = 0.887; Ach, P = 0.998; Ado, P = 0.999; His, P = 0.999; GABA, P = 0.999; Glu, P = 0.998. All experiments were repeated at least three times with similar results.
Extended Data Fig. 3 Signaling characterization of nLightG and nLightR.
a, Intracellular calcium signaling for nLightG and swAlpha-1 AR. Calcium activity was measured at baseline conditions, during NE (10 μM) and ionomycin (10 μM). Signals were normalized to ionomycin response. n = 19 and 22 cells for swAlpha-1 AR-jRGECO1a and nLightG-jRGECO1a. b, Statistical analysis of a. Each data point is the mean jRGECO1a response for one cell. Violin plot represents the kernel density estimate of the probability density function for each sample. Two-tailed Students t-test with Welch’s correction. P = 2.887×10−7. c, Same as in a for nLightR. n = 21 and 23 cells for swAlpha-1 AR-GCaMP6s and nLightR-GCaMP6s, respectively. d, Same as in b for c. P = 3.393×10−21. e, Luminescence signal ratios upon ligand stimulation (NE, 10 μM). The ratios were normalized to the baseline luminescence ratio before the addition of NE. Each trace is the average of three independent experiments. f, Same as in e but for nLightR. g, Statistical analysis of data shown in e and f. Two-tailed Students t-test with Welch’s correction. P values were as follows: mini-Gq, swAlpha-1 AR, 7.75×10−3; nLightG, 0.590; nLightR, 0.589; mini-Gs, swAlpha-1 AR, 3.88×10−3; nLightG, 0.281; nLightR, 0.410; mini-Gi, swAlpha-1 AR, 0.126; nLightG, 0.792; nLightR, 0.147; β-arrestin-2, swAlpha-1 AR, 8.97×10−2; nLightG, 0.134; nLightR, 0.377. All data are shown as mean ± SEM. All experiments were repeated three times with similar results.
Extended Data Fig. 4 Pharmacological characterization of nLightG and GRABNE1m in anesthetized mice.
a, Schematic representation of viral injections for photometry recordings of nLightG or GRABNE1m in vHPC during optogenetic stimulation of LC in anesthetized mice. b, Experimental protocol for optogenetic stimulation combined with drug injection during isoflurane anaesthesia. c, Left, average traces of normalized signal changes (ΔF/F0 %) of GRABNE1m photometry recordings in response to three LC optical stimulation protocols (5 Hz) pre- and post- yohimbine injection. Signals were normalized to the average peak value pre-yohimbine. The period of optogenetic stimulation is represented with an orange shade. Right, statistical comparison of peak normalized ΔF/F0 % responses to 5 Hz LC between pre- and post- yohimbine injection. P = 0.0014, n = 7 mice, two-sided one-sample t-test. d, Same as in c for nLightG. n.s., non-significant (P = 0.31), n = 9 mice, two-sided one-sample t-test. All data are shown as mean ± S.E.M.
Extended Data Fig. 5 Processing of fiber photometry data presented in Fig. 4.
a, Processing of an example photometry trace in response to optogenetic stimulation (pink) of the same site (LC). nLightG was excited at wavelengths of 470/10 (‘ligand-dependent’) and 405/10 nm (‘control’), in a temporally interleaved manner. 10 minutes-long, 30 seconds-long, and 5 seconds-long recordings are shown from left to right, dashed boxes indicate magnified regions. Firstly, a 1st-4th degree polynomial fit was applied (black line). Subsequently, signals were divided by this fit, to correct for bleaching and normalize them (center top). Next, signals were smoothed with a moving-average filter of a 100 ms (center bottom). Finally, ΔF/F0 was calculated as the difference between the resulting signals excited at 470/10 and 405/10 nm (bottom). Original signals contained substantial artefacts likely to originate from locomotion and hemodynamics (as seen by the oscillations of 10–13 Hz likely to originate from the animal’s heartbeat37 in the bleaching-corrected unsmoothed trace). These artefacts are substantially reduced by subtracting the 405/10 nm-excited signal from the 470/10 nm-excited signal. b, Heatmap of 20 individual trials of optogenetic stimulation, corresponding to the processing steps shown in a. c, Mean ± standard deviation of the trials shown in b. d, Plot correlating z-scored data across all animals (n = 6) excited at 405/10 nm vs 470/10 nm (Pearson’s correlation coefficient r = 0.61). e/f, Plot correlating z-scored data across all animals (n = 6) excited at 470/10 nm e and 405/10 nm f against the calculated ΔF/F0 (e, Pearson’s correlation coefficient r = 0.61; f, Pearson’s correlation coefficient r = −0.19). g, Individual Pearson’s correlation coefficients from d,e,f (n = 6 animals). Data are shown as mean ± standard deviation.
Extended Data Fig. 6 Running-induced nLightG signals in the mouse hippocampus.
a, Event-triggered averages for individual animals (n = 4) showing the changes in nLightG signal amplitude over the whole field-of-view when the mouse started running for the five consecutive recording days. Signals were recorded using two-photon microscopy. The colors indicate the different animals (subject 1–4). b, Event-triggered averages of running speed of individual animals in the virtual corridor when the mouse started running. Color code as in a. c, Same as in b for lick rate. d, Structural similarity in the time interval displayed in a–c. e, nLightG signal amplitude as a function of running speed for individual animals. f, nLightG signal amplitude as a function of lick rate for individual animals. In a to f, data is presented as (mean ± 95% confidence interval).
Extended Data Fig. 7 nLightG signals associated with reward position in the mouse hippocampus.
a, Event-triggered averages for individual animals (n = 4) showing the changes in nLightG signal amplitude over the whole field-of-view when the mouse crossed the reward position for the five consecutive days of recording. Signals were recorded using two-photon microscopy. The colors indicate the different animals (subject 1–4). b, Event-triggered averages of running speed of individual animals in the virtual corridor when the mouse crossed the reward position. Color code as in a. c, Same as in b for lick rate. d, Structural similarity in the time interval displayed in a–c. e, nLightG signal amplitude as a function of running speed for individual animals. f, nLightG signal amplitude as a function of lick rate for individual animals. In a to f, data is presented as (mean ± 95% confidence interval).
Extended Data Fig. 8 ROI selection for analysis of in vivo two-photon data.
a-b, Representative field-of-view showing hippocampal CA1 neurons expressing nLightG in vivo (same field-of-view as in Fig. 4j). The red solid line in (a) indicates the zoomed in area shown in (b). White boxes indicate regions-of-interest (ROIs) identified within the field-of-view and centered on putative cells using the machine learning algorithm CITE-On30. Scale bar, 50 µm.
Extended Data Fig. 9 Further comparison between our and published indicators.
a, Basal brightness of LightG and LightR indicator constructs plotted against their fluorescence response ΔF/F0 in HEK293T cells. Basal brightness values reflect the average brightness of indicator-expressing HEK293T cells in the ligand-free state. Grafts containing only the ICL3 module of LightG or LightR are represented as triangles. Grafts containing ICL2 and ICL3 modules of LightG or LightR are represented as circles. Previously published indicators are represented as rectangles. b, Absolute changes in fluorescence (ΔF) of LightG (green) and LightR (red) grafts measured in HEK293T cells for a set of ten GPCRs. n = 21 cells from three independent experiments. Data were obtained from the same imaging experiments shown in Fig. 5. c, Heatmap of ΔF for a subset of indicators (those with ΔF > 0.3). Scale bars, 20 µm. All experiments were repeated at least three times with similar results.
Extended Data Fig. 10 Ligand EC50 measurements for a subset of indicators.
Normalized fluorescence dose-response curves of a, AchLightR (AchLightR-DG, hmM3R double graft); b, AchLightG (AchLightG-SG, hmM3R single graft, light green; AchLightG-DG, double graft, dark green); c, HisLightG (hmH4R, double-graft); and d, AdoLightG (hmA2AR, double-graft). All sensors were expressed in HEK293T cells and titrated with their endogenous agonists. Datapoints were fitted with four-parameter dose-response curves to determine the EC50 values. n = 3 wells per concentration for each ligand. All data are shown as mean ± SEM and all experiments were repeated three times with similar results.
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Data 1.
Supplementary Video 1
Measuring the kinetics of nLightG using patch-clamp fluorometry. Fluorescence changes in an outside-out membrane patch from nLightG-expressing HEK293T cells (center). Six consecutive NE applications (5 μM, 1 s) with ultrafast (<0.5 ms) switching of the perfusion pipette (left) are shown. Data were recorded at a frame rate of 100 Hz. The video plays at 0.5× real time.
Supplementary Data 1
List of oligonucleotides used in this study.
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Kagiampaki, Z., Rohner, V., Kiss, C. et al. Sensitive multicolor indicators for monitoring norepinephrine in vivo. Nat Methods 20, 1426–1436 (2023). https://doi.org/10.1038/s41592-023-01959-z
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DOI: https://doi.org/10.1038/s41592-023-01959-z
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