Next-generation GRAB sensors for monitoring dopaminergic activity in vivo

Abstract

Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent G-protein-coupled receptor-activation-based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors. In response to extracellular DA, both the red and green GRABDA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics and nanomolar-to-submicromolar affinity. Moreover, the GRABDA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Coexpressing red GRABDA with either green GRABDA or the calcium indicator GCaMP6s allows tracking of dopaminergic signaling and neuronal activity in distinct circuits in vivo.

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Fig. 1: Development of red fluorescent DA sensors and second-generation green fluorescent DA sensors.
Fig. 2: Characterization of GRABDA sensors in HEK293T cells and cultured rat cortical neurons.
Fig. 3: GRABDA sensors can be used to measure DA release in acute mouse brain slices.
Fig. 4: In vivo two-photon imaging of DA dynamics in Drosophila using GRABDA sensors.
Fig. 5: GRABDA sensors can detect optogenetically induced nigrostriatal DA release in freely moving mice.
Fig. 6: GRABDA sensors can be used to measure dopaminergic activity in the mouse NAc during sexual behavior.

Data availability

Plasmids for expressing the sensors used in this study and the sequences were available from Addgene (https://www.addgene.org/Yulong_Li/, catalog nos. 140553, 140554, 140555, 140556, 140557, 140558). Source data are provided with this paper.

Code availability

The custom MATLAB codes and TDT programs are available from https://github.com/pdollar/toolbox and https://github.com/bd125/GRAB_DA_Fig6_Code.

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Acknowledgements

This work was supported by the Beijing Municipal Science & Technology Commission (grant no. Z181100001318002), the Beijing Brain Initiative of Beijing Municipal Science & Technology Commission (grant no. Z181100001518004), Guangdong Grant ‘Key Technologies for Treatment of Brain Disorders’ (grant no. 2018B030332001), the General Program of the National Natural Science Foundation of China (project nos. 31671118, 31871087 and 31925017) and the NIH BRAIN Initiative (grant no. NS103558); and by grants from the Peking-Tsinghua Center for Life Sciences and the State Key Laboratory of Membrane Biology at Peking University School of Life Sciences to Y.L.; the NIH (grant nos. R01MH101377 and R21HD090563) and an Irma T. Hirschl Career Scientist Award to D.L.; and the Intramural Research Program of the US NIH/NIEHS (grant no. 1ZIAES103310) to G.C. We thank Y. Rao for sharing the two-photon microscope and X. Lei at PKU-CLS for providing support for the Opera Phenix high-content screening system.

Author information

Affiliations

Authors

Contributions

Y.L. supervised the study. F.S. and Y.L. designed the study. F.S., Y. Zhuo, Y. Zhang and C.Q. performed the experiments related to developing, optimizing and characterizing the sensors in cultured HEK293T cells and neurons with help from J.F. and H.D. F.S. and T.Q. performed the surgery and two-photon imaging experiments related to the validation of the sensors in acute brain slices. J. Zeng, X.L., Y.W. and K.T. performed the two-photon imaging experiments in transgenic flies. J. Zhou performed the fiber photometry recordings during optogenetics in freely moving mice under the supervision of G.C. B.D. performed the fiber photometry recordings in the mouse NAc during sexual behavior under the supervision of D.L. All authors contributed to the data interpretation and analysis. F.S. and Y.L. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Dayu Lin or Guohong Cui or Yulong Li.

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Competing interests

F.S. and Y. L. have filed patent applications whose value might be affected by this publication.

Additional information

Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The development of red fluorescent DA sensors and second-generation green fluorescent DA sensors.

a, Schematic illustration showing the design and optimization of the red fluorescent GRABDA sensors. b, The response to 100 μM DA measured for red fluorescent DA sensor variants during steps 1‒3. The variant with the highest fluorescence change (named rDA0.5) was then sequentially mutated as shown to generate rDA1m, rDA1h, and rDA-mut. c, Schematic illustration showing the design and optimization of the green fluorescent GRABDA sensors. d, Normalized ΔF/F0 in response to 100 μM DA measured for green fluorescent DA sensor variants, normalized to the first-generation DA1h sensor. DA2h was then mutated as shown to generate DA2m and DA-mut. The superscripts in the insets of b,d are based on the Ballesteros–Weinstein numbering scheme54, indicating the mutation sites in the D2R. Source data

Extended Data Fig. 2 The sequences of GRABDA sensors and the residues related to affinity-tuning, cpRFP and cpEGFP optimization.

a,b, The sequences of rGRABDA1m (a) and GRABDA2m (b). The residues related to affinity-tuning, cpRFP (a) and cpEGFP (b) optimization are marked.

Extended Data Fig. 3 Characterization of the sensors in HEK293T cells.

a, b, Schematic illustration showing the local perfusion system. Scale bars, 10 μm. c,d, Representative traces showing the response to DA (left) and subsequent addition of Halo (right). The traces were the average of 3 different regions of interest (ROIs) on the scanning line, shaded with ± s.e.m.. Each trace was fitted with a single-exponential function to determine τon (left) and τoff (right). Similar results were observed for 7–10 cells. e,f, Group summary of τon and τoff. n = 10, 7, 9, 8, 10, 8, 10, 8 cells for rDA1m (τon), rDA1m (τoff), rDA1h (τon), rDA1h (τoff), DA2m (τon), DA2m (τoff), DA2h (τon), DA2h (τoff). g–i, Excitation and emission spectra for the indicated sensors in the absence and presence of DA. j, Photostability of rDA1m and rDA1h (in the presence of 100 μM DA) and the indicated fluorescent proteins was measured using 1-photon and 2-photon microscopy. Each photobleaching curve was fitted with a single-exponential function to determine the time constant. 1-photon, n = 12 cells each. 2-photon, n = 10, 10, 9, 10 cells for rDA1m, rDA1h, jRGECO1a, tdTomato. Two-tailed Student’s t-test was performed. 1-photon, P = 0.9755 between rDA1m and rDA1h; P = 2.72 × 10−5 between rDA1m and mCherry; P = 7.10 × 10−9 between rDA1m and mRuby3; P = 7.90 × 10−10 between rDA1m and tdTomato; P = 1.95 × 10−9 between rDA1m and mScarlet; P = 1.28 × 10−5 between rDA1h and mCherry; P = 2.50 × 10−9 between rDA1h and mRuby3; P = 2.66 × 10−10 between rDA1h and tdTomato; P = 6.75 × 10−10 between rDA1h and mScarlet. 2-photon, P = 0.0963 between rDA1m and rDA1h; P = 0.0511 between rDA1m and jRGECO1a; P = 0.0139 between rDA1h and jRGECO1a; P = 2.82 × 10−11 between rDA1m and tdTomato; P = 1.71 × 10−10 between rDA1h and tdTomato; P = 2.96 × 10−6 between jRGECO1a and tdTomato. Data are presented as the mean ± s.e.m. in e,f,j (bar graph). *P  < 0.05; ***P < 0.001. Source data

Extended Data Fig. 4 The response of GRABDA sensors to different compounds.

a, The normalized dose-response curves for DA and NE in sensor-expressing HEK293T cells. n = 3 wells with 200‒800 cells/well. b, The ΔF/F0 in sensor-expressing cells in response to the indicated compounds applied at 1 μM. n = 3 wells for rDA1h in response to NE, 5-HT, Oct, Gly and l-DOPA. n = 4 wells for the others. Each well contains 200–1200 cells. Data are presented as the mean ± s.e.m.. Data replotted from Fig. 2a. Source data

Extended Data Fig. 5 The minimal coupling of GRABDA sensors to downstream Gi pathway and β-arrestin pathway.

a,b, Normalized ΔF/F0 in sensor-expressing cells in response to DA, with or without the pre-bathing of GTPγS. n = 3 wells with 500‒3000 cells/well. c,d, The representative trace of ΔF/F0 (c) and the group summary of normalized ΔF/F0 (d) in rDA1m-expressing neurons during a 2-hour treatment of 100 μM DA. n = 9 neurons. For the group summary, the averaged ΔF/F0 of each neuron during the 2-hour DA treatment is normalized to 1. Two-tailed Student’s t-test was performed. P = 2.10 × 10−21 between baseline and 0 min; P = 2.99 × 10−17 between 120 min and Halo; P = 1.24 × 10−5 between 0 min and 120 min. e,f, Similar to c and d except that rDA1h was expressed in cultured neurons. n = 11 neurons. Two-tailed Student’s t-test was performed. P = 1.87 × 10−6 between baseline and 0 min; P = 3.43 × 10−17 between 120 min and Halo; P = 0.1519 between 0 min and 120 min. g,h, Similar to c and d except that DA2m was expressed in cultured neurons. n = 15 neurons. Two-tailed Student’s t-test was performed. P = 2.48 × 10−39 between baseline and 0 min; P = 7.42 × 10−35 between 120 min and Halo; P = 0.3322 between 0 min and 120 min. i,j, Similar to c and d except that DA2h was expressed in cultured neurons. n = 17 neurons. Two-tailed Student’s t-test was performed. P = 1.14 × 10−52 between baseline and 0 min; P = 9.80 × 10−38 between 120 min and Halo; P = 0.0061 between 0 min and 120 min. k, Top, schematic illustration depicting the in vivo perfusion experiment. Bottom, the fluorescence image of a transgenic fly expressing DA2m in MB KCs. Scale bar, 50 μm. l,m, Representative images (l, top), trace (l, bottom) and group summary (m) of ΔF/F0 in response to the 1-hour perfusion of 1 mM DA followed by 100 μM Halo in a transgenic fly expressing DA2m in MB KCs. n = 3 flies. Scale bar, 25 μm. Two-tailed Student’s t-test was performed. P = 0.0382 between baseline and 10 min; P = 0.0293 between 60 min and Halo; P = 0.5289, 0.5593, 0.9559, 0.8537, 0.6346, 0.6530, 0.2760, 0.1649, 0.1547, 0.1152, 0.1044 between 5 min and 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, respectively. Data are presented as the mean ± s.e.m.. in a,b,d,f,h,j,m. *P  < 0.05; **P < 0.01; ***P < 0.001. Source data

Extended Data Fig. 6 Comparison between dLight and GRABDA.

a, Representative bright-field and fluorescence images acquired before (baseline) and after application of DA in sensor-expressing HEK293T cells. Similar results were observed for more than 20 cells. Scale bar, 50 μm. b, Representative traces of ΔF/F0 in response to 100 μM DA followed by either 10 μM SCH or 10 μM Halo. Similar results were observed for more than 30 cells. c, Normalized dose-response curves. n = 3 wells with 100‒500 cells/well. d–f, Group summary of the peak ΔF/F0 (d), relative brightness (green/red ratio, GR ratio) (e), and signal-to-noise ratio (SNR) (f) in response to 100 μM DA. d, n = 73, 62, 61, 20 cells for dLight1.1, dLight1.2, DA2m, dLight1.3b. e, n = 77, 66, 20, 60 cells for dLight1.1, dLight1.2, dLight1.3b, DA2m. f, n = 74, 63, 61 cells for dLight1.1, dLight1.2, DA2m. Two-tailed Student’s t-test was performed. d, P = 2.10 × 10−48 between dLight1.1 and DA2m; P = 1.31 × 10−12 between dLight1.2 and DA2m; P = 1.22 × 10−10 between dLight1.3 and DA2m. f, P = 4.09 × 10−22 between dLight1.1 and DA2m; P = 1.13 × 10−33 between dLight1.2 and DA2m. g–i, Dose-response curves (g), relative brightness (h), and fold change of SNR (i) for dLight1.3b and DA2m. n = 20 cells each. j-m, Similar to a-f, except that dLight1.1 and DA2m were expressed in cultured neurons. m, left, n = 30, 28 cells for dLight1.1, DA2m. m, right, n = 30 cells each. Scale bar, 50 μm. Two-tailed Student’s t-test was performed. m, left, P = 4.43 × 10−8; right, P = 3.59 × 10−8. n, Schematic illustration depicting the location of the Drosophila olfactory mushroom body (MB). o, Fluorescence images of the MB using 2-photon microscopy at the indicated laser power settings. Enhanced-contrast images at 15% laser power are shown. Fluorescence is shown in grayscale, with saturated pixels shown in red. Similar results were observed for 4–5 flies. Scale bars, 10 μm. p-r, Representative traces (top) and group summary of relative brightness during odorant application (p), body shock (q), and DA perfusion (r). p,r, n = 5 flies each. q, n = 5, 4 flies for DA2m, dLight1.3b. Average traces (bold) overlaid with single-trial traces (light) from one fly are shown for representation in p,q. Data are presented as the mean ± s.e.m. in c,d,e,f,g,h,i,l,m,p,q,r. ***P < 0.001. Source data

Extended Data Fig. 7 Expressing GRABDA2m or GRABrDA1m sensors shows no significant effect on cAMP or calcium signaling respectively in vivo.

a, Schematic illustration depicting the experimental setup. be, Schematic illustrations depicting the experimental strategy (b,d), representative fluorescence images and ΔF/F0 traces (c,e) in flies expressing the cAMP sensor Pink-Flamindo (b,c) or co-expressing Pink-Flamindo and DA2m (d,e) in MB KCs. The ROIs for measuring the γ2-γ3 compartments in the MB are indicated by dashed white lines. Scale bars, 25 µm. f, Group summary of peak ΔF/F0. n = 9, 7 flies for Pink Flamindo alone, Pink Flamindo & DA2m. Two-tailed Student’s t-test was performed. P = 0.7332. gj, Schematic illustrations depicting the experimental strategy (g, i), representative fluorescence images and ΔF/F0 traces (h,j) in flies expressing the calcium sensor GCaMP5 (g,h) or co-expressing GCaMP5 and rDA1m (i,j) in MB KCs. The ROIs for measuring the MB media lobe are indicated by dashed white lines. Similar results were observed for 7 flies. Scale bars, 25 µm. k,l, Group summary of GCaMP5 peak ΔF/F0 and time constants. n = 7 flies each. Two-tailed Student’s t-test was performed. k, P = 0.607. l, P = 0.601, 0.735 for τon, τoff. Average traces (bold) overlaid with single-trial traces (light) from one fly are shown for representation in c, e,h,j. Data are presented as the mean ± s.e.m. in f,k,l. Source data

Extended Data Fig. 8 Optogenetically induced nigrostriatal DA release in freely moving mice is not affected by desipramine or yohimbine.

ac, Average traces of ΔF/F0 in mice expressing rDA1m and EGFP (a), rDA1h and EGFP (b), or DA2h and tdTomato (c) in the dorsal striatum. Where indicated, the experiments were conducted in mice treated with either the norepinephrine transporter blocker desipramine or the α2-adrenergic receptor antagonist yohimbine. d–f, Group summary of ΔF/F0 and τoff for the experiments shown in a-c, respectively. n = 30 trials from 6 hemispheres of 6 mice for rDA1m. n = 15 trials from 3 hemispheres of 3 mice for rDA1h, n = 25 trials from 5 hemispheres of 4 mice for DA2h. Two-tailed Student’s t-test was performed. d, left, P = 0.1614; right, P = 0.9836. e, left, P = 0.9018; right, P = 0.6605. f, left, P = 0.6489; right, P = 0.2322. Average traces shaded with ± s.e.m. are shown in ac. Data are presented as the mean ± s.e.m. in d-f. Source data

Extended Data Fig. 9 Dual-color recording of DA dynamics and striatal neural activity using DA2m and jRGECO1a in freely moving mice.

a, Schematic illustration depicting the experimental strategy. b, Representative traces showing the fluorescence responses of DA2m and jRGECO1a. c, The zoom-in traces from b during a 25 s recording. d, The cross-correlation between the fluorescence responses of DA2m and jRGECO1a during a 2 min recording. n = 8 hemispheres of 5 mice. Average traces shaded with ± s.e.m. are shown. Source data

Extended Data Fig. 10 The DA signal in the mouse NAc during sexual behavior.

a, Schematic illustration depicting the experimental strategy. b, c, Representative traces (b) and group summary (c) of ΔF/F0 measured from left and right hemispheres during the indicated stages of mating. n = 3 mice. F4,16 = 80.92, P < 10−6 for row factor and F1,4 = 0.1224, P = 0.7441 for column factor by two-way ANOVA. Bonferroni’s multiple comparisons test was performed between groups, P > 0.9999, P > 0.9999, P > 0.9999, P > 0.9999, P > 0.9999. d, Representative traces of the concurrent Z-score signals of rDA1m and DA2h during the indicated stages of sexual behavior. Similar results were observed for 3 mice. e, Average post-stimulus histograms showing the Z-score signals of rDA1m and DA2h aligned to the onset of the indicated mating events. n = 3 mice. Average traces shaded with ± s.e.m. are shown. f, Group summary of the Z-scores measured for rDA1m and DA2h during the indicated mating events. n = 3 mice. F4,16 = 13.02, P = 6.6 × 10−5 for row factor and F1,4 = 0.001, P = 0.9797 for column factor by two-way ANOVA. Bonferroni’s multiple comparisons test was performed, P > 0.99, P > 0.99, P > 0.99, P > 0.99, P > 0.99. g,h, The representative fluorescence signal (g) and group analysis (h) in the green channel when the excitation light is delivered at 470 nm alone (g, left), at 590 nm alone (g, center) or at 470 nm and 590 nm simultaneously (g, right). n = 3 mice. F2,4 = 531.6, P = 3.1 × 10−5 by one-way ANOVA. Tukey’s multiple comparisons test was performed between groups, P = 3.1 × 10−5, P = 2.6 × 10−5, P = 0.4904. i,j, Similar to g and h except the fluorescence signal in the red channel is analyzed. n = 3 mice. F2,4 = 414.2, P = 2.3 × 10−5 by one-way ANOVA. Tukey’s multiple comparisons test was performed between groups, P = 4.8 × 10−5, P = 4.6 × 10−5, P = 0.9738. Data are presented as the mean ± s.e.m. in c,f,h,j. ***P < 0.001. Source data

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Comparison of GRABDA sensors in reporting mushroom body DA dynamics in response to multiple stimuli.

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Sun, F., Zhou, J., Dai, B. et al. Next-generation GRAB sensors for monitoring dopaminergic activity in vivo. Nat Methods 17, 1156–1166 (2020). https://doi.org/10.1038/s41592-020-00981-9

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