Abstract
Genetically encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. RdLight1 can be combined with GFP-based sensors with minimal interference and shows high photostability, permitting prolonged continuous imaging. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell-type-specific neuronal activity and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. By enabling multiplexed imaging of dopamine with other circuit components in vivo, RdLight1 opens avenues for understanding many aspects of dopamine biology.
This is a preview of subscription content, access via your institution
Relevant articles
Open Access articles citing this article.
-
Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors
Communications Biology Open Access 10 June 2022
-
Changes in striatal dopamine release, sleep, and behavior during spontaneous Δ-9-tetrahydrocannabinol abstinence in male and female mice
Neuropsychopharmacology Open Access 27 April 2022
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Get just this article for as long as you need it
$39.95
Prices may be subject to local taxes which are calculated during checkout
Data availability
All DNA plasmids and viruses used in this study have been deposited in NCBI (accession numbers MK751449 and MK751450) and ADDGENE, and can be obtained either from the Tian laboratory at UC Davis, Addgene or the Canadian neurophotonics platform viral (https://tools.neurophotonics.ca/) core under a material-transfer agreement. All source data present in this manuscript are available from https://github.com/lintianlab/RdLight1. Source data are provided with this paper.
Code availability
Custom MATLAB code is available via https://github.com/lintianlab/RdLight1.
References
Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, eaat4422 (2018).
Sun, F. et al. A genetically encoded fluorescent sensor enables rapid and specific detection of dopamine in flies, fish, and mice. Cell 174, 481–496.e19 (2018).
de Jong, J. W. et al. A neural circuit mechanism for encoding aversive stimuli in the mesolimbic dopamine system. Neuron 101, 133–151.e7 (2019).
Mohebi, A. et al. Dissociable dopamine dynamics for learning and motivation. Nature 570, 65–70 (2019).
Zhao, Y. et al. An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011).
Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. Elife 5, e12727 (2016).
Kruss, S. et al. High-resolution imaging of cellular dopamine efflux using a fluorescent nanosensor array. Proc. Natl Acad. Sci. USA 114, 1789–1794 (2017).
Beyene, A. G. et al. Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor. Sci. Adv. 5, eaaw3108 (2019).
Wachter, R. M., Elsliger, M. A., Kallio, K., Hanson, G. T. & Remington, S. J. Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6, 1267–1277 (1998).
Orm, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Ade, K., Wan, Y., Chen, M., Gloss, B. & Calakos, N. An improved BAC transgenic fluorescent reporter line for sensitive and specific identification of striatonigral medium spiny neurons. Front. Syst. Neurosci. 5, 32 (2011).
Eichel, K. et al. Catalytic activation of β-arrestin by GPCRs. Nature 557, 381–386 (2018).
Kebabian, J. W. et al. A-77636: a potent and selective dopamine D1 receptor agonist with antiparkinsonian activity in marmosets. Eur. J. Pharmacol. 229, 203–209 (1992).
Oe, Y. et al. Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance. Nat. Commun. 11, 471 (2020).
Marvin, J. S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10, 162–170 (2013).
Villette, V. et al. Ultrafast two-photon imaging of a high-gain voltage indicator in awake behaving mice. Cell 179, 1590–1608.e23 (2019).
Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Witten, I. B. et al. Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron 72, 721–733 (2011).
Hamid, A. A. et al. Mesolimbic dopamine signals the value of work. Nat. Neurosci. 19, 117–126 (2016).
Stujenske, J. M., Spellman, T. & Gordon, J. A. Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Rep. 12, 525–534 (2015).
Broussard, G. J. et al. In vivo measurement of afferent activity with axon-specific calcium imaging. Nat. Neurosci. 21, 1272–1280 (2018).
Pettibone, J. R. et al. Knock-in rat lines with Cre recombinase at the dopamine D1 and adenosine 2a receptor loci. Eneuro https://doi.org/10.1523/ENEURO.0163-19.2019 (2019).
Hnasko, T. S. et al. Vesicular glutamate transport promotes dopamine storage and glutamate corelease in vivo. Neuron 65, 643–656 (2010).
Schultz, W. Dopamine reward prediction-error signalling: a two-component response. Nat. Rev. Neurosci. 17, 183–195 (2016).
Kalivas, P. W. & Duffy, P. Dopamine regulation of extracellular glutamate in the nucleus accumbens. Brain Res. 761, 173–177 (1997).
Nicola, S. M., Taha, S. A., Kim, S. W. & Fields, H. L. Nucleus accumbens dopamine release is necessary and sufficient to promote the behavioral response to reward-predictive cues. Neuroscience 135, 1025–1033 (2005).
Marvin, J. S. et al. Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat. Methods 15, 936–939 (2018).
Kazemipour, A. et al. Kilohertz frame-rate two-photon tomography. Nat. Methods 16, 778–786 (2019).
Liu, R., Li, Z., Marvin, J. S. & Kleinfeld, D. Direct wavefront sensing enables functional imaging of infragranular axons and spines. Nat. Methods 16, 615–618 (2019).
Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).
Gerfen, C. R. & Surmeier, D. J. Modulation of striatal projection systems by dopamine. Ann. Rev. Neurosci. 34, 441–466 (2011).
Planert, H., Berger, T. K. & Silberberg, G. Membrane properties of striatal direct and indirect pathway neurons in mouse and rat slices and their modulation by dopamine. PLoS ONE 8, e57054 (2013).
Ishikawa, A., Ambroggi, F., Nicola, S. M. & Fields, H. L. Dorsomedial prefrontal cortex contribution to behavioral and nucleus accumbens neuronal responses to incentive cues. J. Neurosci. 28, 5088–5098 (2008).
Burgos-Robles, A., Bravo-Rivera, H. & Quirk, G. J. Prelimbic and infralimbic neurons signal distinct aspects of appetitive instrumental behavior. PLoS ONE 8, e57575 (2013).
Ambroggi, F., Ishikawa, A., Fields, H. L. & Nicola, S. M. Basolateral amygdala neurons facilitate reward-seeking behavior by exciting nucleus accumbens neurons. Neuron 59, 648–661 (2008).
Bamford, N. S., Wightman, R. M. & Sulzer, D. Dopamine’s effects on corticostriatal synapses during reward-based behaviors. Neuron 97, 494–510 (2018).
Rice, M. E. & Cragg, S. J. Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res. Rev. 58, 303–313 (2008).
Hnasko, T. S., Hjelmstad, G. O., Fields, H. L. & Edwards, R. H. Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J. Neurosci. 32, 15076–15085 (2012).
McFarland, K. & Kalivas, P. W. The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior. J. Neurosci. 21, 8655–8663 (2001).
Hioki, H. et al. Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Ther. 14, 872–882 (2007).
Feng, J. et al. A genetically encoded fluorescent sensor for rapid and specific in vivo detection of norepinephrine. Neuron 102, 745–761.e8 (2019).
Jing, M. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat. Biotechnol. 36, 726–737 (2018).
Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator. Nat. Methods 16, 171–174 (2019).
Ravotto, L., Duffet, L., Zhou, X., Weber, B. & Patriarchi, T. A bright and colorful future for G-protein coupled receptor sensors. Front. Cell. Neurosci. 14, 67 (2020).
Ke, M.-T., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat. Neurosci. 16, 1154–1161 (2013).
Quan, J. & Tian, J. Circular polymerase extension cloning. Methods Mol. Biol. 1116, 103–117 (2014).
Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).
Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013).
Rasmussen, S. G. F. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).
Chen, Y. et al. Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement. Protein Cell 4, 299–309 (2013).
Bender, B. J. et al. Protocols for molecular modeling with Rosetta3 and RosettaScripts. Biochemistry 55, 4748–4763 (2016).
Kotowski, S. J., Hopf, F. W., Seif, T., Bonci, A. & von Zastrow, M. Endocytosis promotes rapid dopaminergic signaling. Neuron 71, 278–290 (2011).
Patriarchi, T. et al. Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators. Nat. Protoc. 14, 3471–3505 (2019).
Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013).
Ellis, B. et. al. flowCore: basic structures for flow cytometry data. R package version 1.52.1. (2019).
Tewson, P. H., Martinka, S., Shaner, N. C., Hughes, T. E. & Quinn, A. M. New DAG and cAMP sensors optimized for live-cell assays in automated laboratories. J. Biomol. Screen 21, 298–305 (2016).
Acknowledgements
This work was supported by NIH DP2MH107056 (L.T.); BRAIN Initiative awards U01NS090604, U01NS013522 (L.T. and M.V.Z), U01NS103571 (L.T) and U01NS094375 (J.B.); a Rita Allen Young Investigator Award (L.T.) and R01DA045783 (J.D.B.); the Olga Mayenfisch Foundation (T.P.); and the Novartis Foundation for medical-biological research (T.P.). We thank D. Jullie (UCSF) for advice and providing striatal neuronal culture. We thank J. Zhang and C.-H. Chen for advice on FACS experiments.
Author information
Authors and Affiliations
Contributions
T.P., A. Mohebi, J.S., M.V.Z., J.D.B. and L.T. wrote the manuscript. T.P. developed the sensors and performed in vitro sensor characterization, in vitro multiplex imaging experiments and part of the viral injections for two-photon imaging in brain slices. J.S. performed part of the viral injections, tissue histology, electrophysiology and two-photon imaging and bleaching experiments. A. Mohebi performed in vivo photometry and optogenetic experiments. A. Marley and M.V.Z. performed TIRF microscopy, cAMP measurements and β-arrestin recruitment assays. R.L. generated the structural models of the sensors and performed FACS characterization of sensor coexpression. C.D. performed viral injections and characterization of the sensor’s photoactivation properties. K.P. and B.W. participated in experimental planning for in vivo characterization. C.M.D. and G.O.M performed dissociated neuronal culture. All authors analyzed the data.
Corresponding authors
Ethics declarations
Competing interests
L.T. and G.O.M. are co-founders of Seven Biosciences.
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 Outline and summary of the experiments.
Outline and summary.
Extended Data Fig. 2 Development of RdLight1.
a, Fluorescence emission spectra of dLight1.3b variants carrying red-shifting mutations in their GFP component. Fluorescence emission was measured in the 450-650 nm wavelength range both in the absence of DA (dotted lines) and upon addition of 100 μM DA to the medium (continuous lines). n = 4 cells from 3 cultures. b, Representative image showing membrane expression profile of the initial variant of the red dopamine sensor, carrying the original amino acid linkers from JRGECO1a. The experiments were repeated at least twice with similar results. c, linker screening results from a library of 264 variants in which the two aminoacid pairs flanking cpmApple were randomized (as shown in inset). Red and blue vertical bars indicate fluorescence changes (∆F/F) in response to 100 μM DA; significance values of ∆F/F are shown by colored bars and scale. n = 3 trials, two-tailed t test. d, optimization of the RdLight1 fluorescence response was achieved by selectively mutating residue F129 into a subset of amino acids. Fluorescence change (∆F/F) was measured in HEK293 cells in response to bath-applied 100 μM DA. n = 4 cells from 2 cultures. **p<0.01, ****p<0.0001, two-tailed t test. The experiments were repeated at least twice with similar results. Data are shown as mean ± SEM.
Extended Data Fig. 3 Characterization of RdLight1 photoactivation by blue light.
Confocal imaging of RdLight1 in transfected HEK293 cells. a, b, Frame scan imaging performed in the presence of both blue (488 nm) and green (561 nm) light after (a) or before (b) bath-application of DA. Blue light did not significantly increase the fluorescence response of RdLight1 to DA. Average ∆F/F=-0.006±0.003 (baseline), 0.53±0.03(DA), 0.69±0.06(DA+blue light, One-way Anova, ***P=0.0005, n.s P=0.1224, n=5 cells from two cultures (a). Average ∆F/=0.002±0.001 (baseline+blue light), 0.83±0.06(DA+blue light), 0.69±0.08(DA), One-way Anova, ***P=0.0007, n.s P=0.0678, n=5 cells from two cultures, mean±SEM (b). c, Representative line scan imaging at acquisition rate of 1000 Hz. Bursts of 488nm laser (150 ms duration at 1Hz) was applied followed by application of DA. Laser powers used were: 24779.4 W/cm2 for 561nm laser, 16373.7 W/cm2 for 488nm laser. Experiments were performed six times with similar results.
Extended Data Fig. 4 Molecular specificity of RdLight1.
a, b, Titration curves are shown for the response to DA or NE both in HEK293 cells and in cultured neurons. Data points were fit with a one-site specific binding curve to obtain EC50 values. Data are shown as mean ± SEM. n = 7 cells from 2 cultures for both HEK293 cells and neurons. The response of RdLight to NE at 1mM is significantly lower than that to DA (P=4.8x10 -8, paired t-test (two sided). c, The specificity of RdLight1 was tested by performing titrations of a panel of different neurotransmitters on the sensor expressed in HEK293 cells. Quantification of responses to each concentration of ligand are shown as mean ± SEM. n = 12 cells from 2 cultures. Neurotransmitters tested were: norepinephrine (NE), epinephrine (EPI), acetylcholine (ACH), glutamate (GLU), histamine (HIS), serotonin (5HT). The experiments were repeated at least twice with similar results.
Extended Data Fig. 5 Cellular signaling characterization of RdLight1.
a, cAMP response curve to a titration of the D1 agonist SKF81297 in HEK293 cells transfected with RdLight1 compared to human D1-dopamine receptor (DRD1, positive control) or enhanced green fluorescence protein (GFP, negative control). No significant cAMP response was produced by RdLight1 (n=3 independent experiments, **p=0.0033, ***p=0.00053, one-way ANOVA, Dunnett’s post-hoc test). b, cAMP response curves of a cell line endogenously expressing DRD1 (U2OS). Overexpression of RdLight1 did not alter the endogenous cAMP response to SKF81297 (n=3 independent experiments, p=0.944, two-way ANOVA). c, Expression of RdLight1 (red) in cultured primary striatal neurons did not affect cAMP responses to vehicle, SKF81297 or the adenylate cyclase activator Forskolin, compared to mock-transfected conditions (black), as measured by the green genetically encoded cAMP sensor cADDis (n=10 neurons from three cultures, P=0.744 (unpaired t-test, two-tailed). d, TIRF imaging of RdLight1 response (red) and of a static Alexa-647-conjugated anti-Flag antibody label to delineate sensor expression on cell surface (far-red channel). Quantification of RdLight1 response to a DRD1 agonist (SKF81297), which was immediately abolished by applying antagonist (SCH23390) (n=3 independent experiments). e, RdLight1 internalization is significantly reduced compared to wild type DRD1 as assayed via flow cytometry (% internalized receptor: RdLight1, 4.82 ± 6.42 %; DRD1, 31.9 ± 2.74 %, n=4 independent experiments, p=0.0006 (unpaired t-test, two-tailed). f, g, Representative TIRF microscopy images of HEK293 cells transfected with beta-arrestin-2 and DRD1 or RdLight1. DRD1 and RdLight1 were labeled with an Alexa-647 conjugated anti-flag antibody. Conditions before and after addition of DRD1 agonist SKF81297 are shown. Experiments were performed three times with similar results. h, i, Quantification of beta-arrestin-2 recruitment on the cell membrane from f, g. Under these conditions RdLight1 triggers significantly lower beta-arrestin-2 recruitment than DRD1. RdLight1, 6.8 ± 4.2 (n=8 cells); DRD1, 1.3 ± 0.5 (n=7 cells), P=0.0029 (unpaired t-test, two-tailed). All values shown are mean±SD.
Extended Data Fig. 6 Multiplex imaging of drug selectivity at two dopamine receptor subtypes and characterization of RdLight1 and nLight1.3 coexpression.
a, Combined fluorescence emission spectra of DRD1-based (dLight1.5) and DRD1-based (RdLight1) sensors used for multiplex imaging experiments. Fluorescence emission was measured in the 450-700 nm wavelength range both in the absence of DA (dotted lines) and upon addition of 100 μM DA to the medium (continuous lines). n = 4 cells from 3 cultures. b, c, Dose-response curves were obtained by performing multiplex imaging during titrations of the DRD1-selective agonist A77636 or the DRD2-selective agonist Quinpirole on a coculture of two HEK293 cell populations expressing either RdLight1 (red) or dLight1.5 (green). Maximal fluorescence responses at each applied concentration were quantified and plotted. n = 6 cells from 3 cultures. Data are shown as mean ± SEM. d–f, Characterization of RdLight1 and nLight1.3 coexpression. d, Flow cytometry analysis of cells transfected with RdLight1 (n=13928 cells), nLight1.3(n=25798 cells), or both (n=33049 cells) showed that co-transfection significantly increased both basal green and basal red-fluorescence. Violin plots show the distribution of the log value of fluorescence, on top of the boxplot of the same data. P=2.2X10-16 Two-sided Student’s t-test. e, f, In a perfusion experiment, confocal time-lapse imaging revealed that the response of nLight1.3 to NE when expressed alone (n=8) can be slightly decreased when cotransfected with RdLight1 (n=7 cells), which could possibly due to increased basal green fluorescence from RdLight1 co-transfection. A slight increase in the amplitude of response in RdLight1 was noticed when co-transfected (n=7 cells), but not significant. P=0.04 and 0.16, Two-sided Student’s t-test for the value when NE or DA applied. Traces were plotted as mean +/- sem, with center line as mean value and shade the s.e.m.
Extended Data Fig. 7 Photobleaching of RdLight1 under two-photon illumination.
Two-photon bleaching measurements of RdLight were acquired from acute brain slices. Fluorescence of RdLight was excited at 1020 nm with a Ti: sapphire laser (Ultra II, Coherent) that was focused by an Olympus 40×, 0.8NA water immersion objective. Emitted fluorescence photons were separated by a 620/60 nm filter. Scan area was 60 μm×60 μm, and the scan rate was 30 frames/s. Light intensity was kept at 8.7 mW across measurements. Burst electrical stimulation was performed with a metal bipolar electrode that has a tip spacing of 255 μm (PI2ST30.01A5, Microprobes for life science) every 100 s. Amplitude of the pulses was 4 V, width was 0.3 ms and a total number of 40 pulses was applied at a frequency of 80 Hz. As shown in the figure, photobleaching was continuously measured in a 1 h duration. Overall fluorescence in all the 3 tested slices declined about 17.2±3.1%, mean ± SEM. The experiments were repeated in 3 slices from two mice with similar results.
Extended Data Fig. 8 Injection coordinates for in vivo experiments.
a, List of all in vivo experiments in which RdLight1 was expressed in NAc simultaneously with a green indicator. b, Representative stitched image for each experiment collected on an epifluorescence microscope using a 4x objective. All sections (except for the D1-Cre example) are collected in horizontal planes. The D1-Cre example is in the coronal plane. Each section shows the most ventral section with a fiber punch hole. Scale bar: 1 mm. c, Overlaid reconstruction map of the estimate location of recordings for each experiment. Histology results were not available for one subject in the TH-Cre experiment since the subject died prior to the end of the experiment. Brain atlas outlines in this figure were reproduced with permission from (Paxinos and Watson, 2005).
Extended Data Fig. 9 Additional control experiments for in vivo imaging with iGluSnFR.
To further validate the dip observed in the glutamate signal in NAc following the reward click in Fig. 4, we performed two complementary experiments. a, Only iGluSnFR was expressed in NAc and imaged using a 400 μm fiber during the pavlovian approach task. b, c, similar patterns to Fig. 4b,c, demonstrating a dip following the reward click was observed when only iGluSnFR was expressed and monitored. d–f, To control for pH changes or other artifacts, we expressed a variant of iGluSnFR (SF-iGluSnFR.R75A) with a mutation to the binding sites that destroyed glutamate binding, along with RdLight1. While similar to Fig 4b,c RdLight1 signal was preserved, the dip in the glutamate signal disappeared. Experiments for each panel were repeated independently for n=2 rats with similar results.
Extended Data Fig. 10 In vivo signal-to-noise ratio comparison between RdLight1 and dLight1.3b, and effect of dLight1.1 or RdLight1 expression in NAc on motivated approach behavior.
a, Peak response to an unpredicted click signaling immediate reward delivery. Signals were collected using fiber photometry across n=6 rats with dLight1.3b expressed in NAc and n=7 rats with RdLight1 expressed in the same coordinates. RdLight1 signals demonstrated significantly higher signal-to-noise ratio (SNR). A one-sided t-test was used to assess statistical significance (p=8.04x10^-3). Box plot shows the mean and quartiles of the data with whiskers representing the range of observations. b, To investigate the effect of dopamine indicators on behavior, we compared two cohorts of rats expressing either dLight1.1 (n=8) or RdLight1 (n=6) in NAc to a control group (n=6) expressing neither. Similar to the experimental paradigm for the in vivo photometry experiments, rats are placed in an operant chamber with a speaker and a reward delivery port. An auditory click indicating an immediate delivery of a sucrose pellet is played at random intervals. Rats with either of the indicators expressed in NAc show no difference to the control group in their latency to approach and retrieve the reward following the click. A kolmogorov-smirnov test was used to compare the latency distributions.
Supplementary information
Supplementary Information
Supplementary Note.
Supplementary Video 1
Multiplex imaging of drug-target engagement in HEK293 cells. The movie shows live multiplex confocal imaging of two populations of HEK293 cells that were individually transfected for dLight1.5 or RdLight1 and then cocultured. The fluorescence response of the two sensors to different DA-receptor drugs are shown. The following drugs, in chronological order, were sequentially bath-applied to the cells: A77636, quinpirole, SCH-23390 and sulpiride. Scale bar, 10 μm. Images were acquired at 3.13 s per frame. The experiments were repeated three times with similar results
Supplementary Video 2
Multiplex imaging of NE and DA in a neuron–astrocyte coculture. Multiplex confocal imaging of neurons and astrocytes, related to Fig. 1g,h. Cells were grown in a coculture and transduced together with the following AAVs: AAV1.GFAP.nLight1.3 and AAV.hSynapsin1.RdLight1. The cells were imaged 14 d after AAV transduction. During imaging, DA, NE, the DRD1 antagonist SCH-23390 and the B2AR antagonist ICI118551 were sequentially applied to the cell bath while the fluorescence of both sensors was being recorded. The molecular specificity of the two sensors allowed the in vitro optical dissection of DA from NE, while the subtype-specific antagonists selectively reversed the fluorescence responses. Images were acquired at 3.13 s per frame. The experiments were repeated three times with similar results.
Source data
Source Data Fig. 1
Fluorescent fold change calculated from raw data and used to plot graphs displayed in the figure
Source Data Fig. 2
Fluorescent signals of RdLight1, iGluSnFR and GCaMP6 responses to field stimuli
Source Data Fig. 3
Fluorescent signals of RdLight1 and GCaMP in response to light stimuli or behavior
Source Data Fig. 4
Fluorescent signals of RdLight1, iGluSnFR and GCaMP in response to behavior
Source Data Extended Data Fig. 2
Emission scan and sensor screening
Source Data Extended Data Fig. 3
Fluorescence signals of RdLight in response to blue/yellow light illumination
Source Data Extended Data Fig. 4
In situ titration of RdLight1 and specificity
Source Data Extended Data Fig. 5
cAMP production in three types of cells, internalization and β-arrestin recruitment assay
Source Data Extended Data Fig. 6
Emission spectrum, in situ titration of RdLight1 and nLight1.3 to ligands and drugs
Source Data Extended Data Fig. 7
Fluorescence signal of RdLight in response to field stimuli in acute striatal slice
Source Data Extended Data Fig. 9
Fluorescent traces comparing iGluSnFR, iGluSnFR(mut) and RdLight1 responses to unpredicted reward cue
Source Data Extended Data Fig. 10
Fluorescent traces comparing dLight SNR to RdLight in response to unpredicted reward cue
Rights and permissions
About this article
Cite this article
Patriarchi, T., Mohebi, A., Sun, J. et al. An expanded palette of dopamine sensors for multiplex imaging in vivo. Nat Methods 17, 1147–1155 (2020). https://doi.org/10.1038/s41592-020-0936-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-020-0936-3
This article is cited by
-
A genetically encoded sensor measures temporal oxytocin release from different neuronal compartments
Nature Biotechnology (2023)
-
Surface modification of MoS2 nanosheets by single Ni atom for ultrasensitive dopamine detection
Nano Research (2023)
-
A fluorescent sensor for spatiotemporally resolved imaging of endocannabinoid dynamics in vivo
Nature Biotechnology (2022)
-
Pushing the frontiers: tools for monitoring neurotransmitters and neuromodulators
Nature Reviews Neuroscience (2022)
-
A genetically encoded sensor for in vivo imaging of orexin neuropeptides
Nature Methods (2022)
