Research Article

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

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Science  29 Jun 2018:
Vol. 360, Issue 6396, eaat4422
DOI: 10.1126/science.aat4422
  • High-resolution dopamine imaging in vivo.

    dLight1 permits robust detection of physiologically and behaviorally relevant dopamine (DA) transients with high sensitivity and spatiotemporal resolution, including dynamic learning-induced dopamine changes in the nucleus accumbens (bottom) and task-specific dopamine transients in the cortex (top).

  • Fig. 1 Development and characterization of dLight1.

    (A) Simulated structure of dLight1 consisting of DRD1 and cpGFP module. (B) Sequence alignment of transmembrane (TM) domain 5 and 6 in β2AR, DRD1, and DRD4. Library design is shown. Amino acid abbreviations: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. (C) Screening result of 585 linker variants. Red and blue vertical bars indicate fluorescence changes (ΔF/F) in response to 10 μM DA; significance values of ΔF/F are shown by colored bars and scale (n = 3 trials, two-tailed t test). (D) Expression of dLight variants in HEK cells. Fluorescence intensity and signal-to-noise ratio of apo and sat state are shown. Scale bars, 10 μm. (E) In situ titration of DA on HEK cells. Data were fitted with the Hill equation (n = 5). (F) Pharmacological specificity of dLight1.1. DRD1 full agonist (dihydrexidine, 295 ± 8%, n = 5); DRD1 partial agonists (SKF-81297, 230 ± 7.7%, n = 5; A77636, 153 ± 7.8%, n = 7; apomorphine, 22 ± 0.8%, n = 6); DRD1 antagonists (SCH-23390, –0.04 ± 0.01%, n = 7; SKF-83566, 0.04 ± 0.03%, n = 7); DRD2 antagonists (sulpiride, 213 ± 5.1%, n = 5; haloperidol, 219 ± 11%, n = 6). Data are means ± SEM. ****P < 0.0001 [one-way analysis of variance (ANOVA), Dunnett posttest]; n.s., not significant.

  • Fig. 2 Imaging electrically evoked and pharmacologically modulated dopamine release in acute dorsal striatum slices.

    (A) Schematics of experimental setup. (B) Single-trial fluorescence response (average in black) in response to a single stimulus (0.5 ms). Images were acquired at 15 Hz using two-photon light at 920 nm. Averaged ΔF/F = 182 ± 21% across seven trials, mean ± SEM. (C) Representative hotspot (ΔF/F) for line scan. Scale bar, 20 μm. (D) Individual fluorescence traces during line scan (500 Hz) in response to a single stimulus (average in black across 13 trials). Inset shows zoomed-in view of the fluorescence plateau. (E) Fluorescence responses to low- and high-frequency stimuli (left, 0.2 Hz; right, 1 Hz). (F) Quantification of data in (E) [relative (fold) change in ΔF/F = 0.506 ± 0.061 at 1 Hz across five trials]. (G) Single-trial fluorescence response in the presence of cocaine (10 μM) triggered by a single stimulus, overlaid with trace without cocaine. (H) Quantification of fold change in peak fluorescence amplitude (1.056 ± 0.095, n = 7, P = 0.056) and duration (3.15 ± 0.213, n = 4). (I) Estimation of released DA concentration (single-trial trace shown). (J) Quantification of fold change in peak fluorescence in the presence of 1 μM quinpirole (0.437 ± 0.052, n = 5), 400 nM sulpiride and 1 μM quinpirole (quin+sulp, 0.926 ± 0.070, n = 5), 1 μM U69,593 (0.838 ± 0.042, n = 4), and 1 μM naloxone (1.022 ± 0.053, n = 4), all bath-applied. (K) Single-trial fluorescence response to either a single pulse (black) or a train of five pulses at 40 Hz (red) in the absence (left) and presence (right) of the nicotinic acetylcholine receptor blocker hexamethonium (200 μM). (L) Quantification of fold change in peak fluorescence response in (K) (Hex/Control: 0.561 ± 0.038, n = 10; control 5stim/1stim: 1.13 ± 0.069, P = 0.06, n = 7; Hex 5stim/1stim: 1.76 ± 0.16, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (paired t test).

  • Fig. 3 Deep brain imaging of DA release triggered by optogenetic stimulation and combined with calcium imaging in freely behaving mice.

    (A) Schematics showing fiber photometry recording of dLight1.1 or control sensor in NAc while stimulating VTA DA neurons by optogenetics. (B) Expression of dLight1.1 in NAc around fiber tip location and ChrimsonR-expressing axons from midbrain. (C) ChrimsonR-expressing TH+DA neurons in VTA. (D) Averaged fluorescence increase in response to optogenetic stimuli (n = 5 mice). (E) Quantification of peak fluorescence at 20 Hz. (F) Fluorescence fold changes relative to 5 Hz. (G and H) Optogenetically induced fluorescence increase of dLight1.1 after systemic administration of saline, D1 antagonist (SCH-23390, 0.25 mg/kg), and DA reuptake inhibitor (GBR-12909, 10 mg/kg) (n = 5 mice). (I) Schematics showing fiber photometry recording of dLight1.1 in NAc and optogenetic stimulation of VTA GABA neurons that inhibits VTA DA neurons. (J and K) Averaged fluorescence decrease in response to optogenetic stimulation at 40 Hz (n = 4 mice) and quantification of mean fluorescence. (L) Dual-color fiber photometry recording of DA release with dLight1.1 and local neuronal activity with jRGECO1a. (M and N) Increase of dLight1.1 (green) and jRGECO1a (magenta) fluorescence during 5% sucrose consumption with lick rate (black, n = 5 mice) and quantification of mean fluorescence. (O and P) Fluorescence decrease in dLight1.1 (green) and increase in jRGECO1a (magenta) during unpredictable footshock delivery (0.6 mA for 1 s, n = 5 mice) and quantification of mean fluorescence. Data shown are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (paired or unpaired t tests for two-group comparisons; one-way ANOVA by post hoc Tukey test for multiple-group comparisons).

  • Fig. 4 Dynamic changes of NAc DA signaling during appetitive Pavlovian conditioning and reward prediction error.

    (A) Pavlovian conditioning procedures involved learning to associate neutral cues (CS; house light and 5-kHz tone) with a sucrose reward (US; 50 μl of 5% sucrose) and subsequent extinction. (B) Change of CS-evoked licks across cue-reward learning (left) and extinction (right). (C and D) dLight1.1 dynamics in response to CS and US in first and last sessions of cue-reward learning, shown in single (gray) and averaged (blue) trials (n = 20 trials) from a single animal (C) or averaged across all trials and animals (n = 5 mice) (D). Lick rate is shown in black. (E) Same as (D) for cue-reward extinction (n = 5 mice). In (D) and (E), dotted lines indicate CS onset, US onset, and CS offset, respectively. (F to H) Evolution of CS-evoked (F) and US-evoked [(G), left] average fluorescence and US-triggered licks [(G), right] across learning and extinction sessions. (H) Quantification of peak fluorescence across learning and extinction. (I) Reward prediction error procedure. (J) Fluorescence response during expected (red) versus unexpected (black) reward consumption (n = 4 mice). (K) Peak fluorescence evoked by expected (red) and unexpected (black) reward consumption. (L) Fluorescence response during expected (red) versus unexpected (brown) reward omission (n = 4 mice). Second and third dotted lines indicate US onset and CS offset, respectively. (M) Mean fluorescence during baseline and after unexpected reward omission. Data are means ± SEM. **P < 0.01 (Pearson correlation coefficient and paired t test).

  • Fig. 5 Spatially resolved imaging of cortical dopamine release during a visuomotor association task.

    (A) Schematics of experimental setup. (B) A trial was initiated when mice were required to stand still for 10 s after a visual cue (blue square). If mice started to run during the stimulus phase (“hit trials”), a water reward was given. In 20% of randomly selected hit trials, the reward was withheld. If no run was triggered by stimulus presentation, the trials were counted as “miss trials.” Erroneous or spontaneous runs during the standstill phase ended the trial (no “Go” cue or reward). (C) Top: Dorsal view of mouse cortex with the chronic cranial window (circle) and imaging location indicated (square). Bottom: Heat map of dLight1.2 expression pattern in layer 2/3 of M1 cortex. The image is overlaid with computationally defined regions of interest (ROIs, ~17 μm × 17 μm). Colored ROIs indicate the type of fluorescence responses observed during the task. (D) Population data (N = 4 mice, n = 19 recording sessions) showing average task-related dLight1.2 transients (bottom) and mouse running velocity (top) aligned to trial/standstill cue onset (0 s). The solid vertical line indicates “Go” cue onset. The dashed line marks the end of the reward expectation phase during unrewarded hit and miss trials. The period during which running velocity–dependent reward consumption occurred is indicated by the horizontal line. Left: ROIs showing significantly increased responses during reward expectation/locomotion. Right: ROIs showing significant fluorescence increases to reward (dark green) but not unexpected reward omission (light green). Shaded areas of ΔF/F traces indicate SD. (E) Population data realigned to running onset (vertical black line). ROIs with “Go” cue responses [(D), left] can be subdivided into ROIs responsive to locomotion in all trials (left) and responsive to reward expectation only (center), with no fluorescence increases during spontaneous runs (pink). P < 0.05 (Wilcoxon test, Bonferroni-corrected for multiple comparisons).

Supplementary Materials

  • Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

    Tommaso Patriarchi, Jounhong Ryan Cho, Katharina Merten, Mark W. Howe, Aaron Marley, Wei-Hong Xiong, Robert W. Folk, Gerard Joey Broussard, Ruqiang Liang, Min Jee Jang, Haining Zhong, Daniel Dombeck, Mark von Zastrow, Axel Nimmerjahn, Viviana Gradinaru, John T. Williams, Lin Tian

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S16
    • Captions for Data S1 to S3
    • References
    Data S1
    Sequences of the constructs used in this study.
    Data S2
    Data info for Figure 1-2.
    Data S3
    Data info for Figure 5.

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