Photocurrent amplitude at − 60 mV was plotted as a function of light power. 511 nm light (A), 469 nm light (B, C), and 590 nm light (D) were illuminated. Filled circle: peak photocurrent; open circle: steady-state photocurrent (n = 5, 6). Error bars indicate standard errors of the mean (SEMs).

Figure 1—figure supplement 1—source data 1.

Light power dependency of photocurrent amplitude of GtCCR4-3.0 (A), KnChR (B), CrChR2[T159C] (C), CoChR (D), and ChrimsonR (E).

Photocurrent amplitude at − 60 mV was plotted as a function of light power. 511 nm light (A), 469 nm light (B, C), and 590 nm light (D) were illuminated. Filled circle: peak photocurrent; open circle: steady-state photocurrent (n = 5, 6). Error bars indicate standard errors of the mean (SEMs).

Figure 1—figure supplement 1—source data 1.

Tail movements in a larva expressing CrChR2[T159C]-mCherry in reticulospinal V2a neurons.

The hindbrain in a 3-dpf Tg(vsx2:GAL4FF); Tg(UAS-hsp70l:CrChR2[T159C]-mCherry, myl7:mCherry) larva was stimulated with 470 nm light for 100 ms. The timing of light stimulation is indicated by a blue circle.

Latency of locomotion in ChR-expressing and non-expressing larvae.

The hindbrain of 3-dpf larvae expressing GtCCR4-3.0-EYFP, GtCCR4-MT, or KnChR-3.0-EYFP, and non-expressing sibling control larvae was irradiated with light (520 nm for GtCCR4-3.0-EYFP, GtCCR4-MT and 470 nm for KnChR-3.0-EYFP) for 100 ms. Six consecutive stimulation trials were analyzed for eight rhodopsin-expressing and non-expressing control larvae of each line (48 trials for each condition). Latency was measured as the tail movements observed within 8 s after the onset of light stimulation and plotted in a graph. The number of tail movements was 28 and 7 for GtCCR4-3.0-EYFP and the control, 24 and 4 for GtCCR4-MT and the control, and 25 and 4 for KnChR-3.0-EYFP and the control, respectively.

Figure 3—figure supplement 1—source data 1.

The hindbrain of 3-dpf larvae expressing GtCCR4-3.0-EYFP, GtCCR4-MT, or KnChR-3.0-EYFP, and non-expressing sibling control larvae was irradiated with light (520 nm for GtCCR4-3.0-EYFP, GtCCR4-MT and 470 nm for KnChR-3.0-EYFP) for 100 ms. Six consecutive stimulation trials were analyzed for eight rhodopsin-expressing and non-expressing control larvae of each line (48 trials for each condition). Latency was measured as the tail movements observed within 8 s after the onset of light stimulation and plotted in a graph. The number of tail movements was 28 and 7 for GtCCR4-3.0-EYFP and the control, 24 and 4 for GtCCR4-MT and the control, and 25 and 4 for KnChR-3.0-EYFP and the control, respectively.

Figure 3—figure supplement 1—source data 1.

Latency (A) and duration (B) of evoked tail movements. The hindbrain area of 3-dpf Tg larvae expressing GtCCR4-3.0, KnChR-3.0, and CrChR2[T159C] and control larvae (non-expressing sibling larvae) were irradiated by light of different light intensities. Six consecutive stimulation trials were analyzed for eight rhodopsin-expressing and non-expressing (control) larvae of each Tg line. One-way ANOVA with Tukey’s post hoc test (latency GtCCR4-3.0-EYFP 0.4 mW/mm² vs. 0.2 mW/mm², p=0.00571; KnChR-3.0-EYFP 0.4 mW/mm² vs. 0.2 mW/mm², p=0.00553; 0.4 mW/mm² vs. 0.1 mW/mm², p=5.26e-06; 0.2 mW/mm² vs. 0.1 mW/mm², p=0.00469). **p<0.01, ***p<0.001, ns: not significant. Means and SEMs are indicated.

Figure 3—figure supplement 2—source data 1.

(A) Light stimulation-dependent locomotion rates of 3-dpf Tg larvae expressing CoChR-tdTomato and ChrimsonR-tdTomato. The hindbrain area was irradiated with light (0.4 mW/mm²) with a wavelength of 470 nm (CoChR-tdTomato) or 520 nm (ChrimsonR-tdTomato) for 100 ms. Six consecutive stimulation trials were analyzed for eight rhodopsin-expressing and eight non-expressing (control) larvae of each Tg line. The average locomotion rates for each larva are shown. Wilcoxon rank-sum test (CoChR-tdTomato vs. control, p=0.000205; ChrimsonR vs. control, p=0.0509). (B–D) Latency (B), duration (C), and strength (D) of tail movements. Welch’s t-test (strength CoChR-tdTomato vs. ChrimsonR; p=0.00463). (E) Rate of locomotion induced with CoChR-tdTomato by light of various light intensities. **p<0.01, ***p<0.001, ns: not significant. Means and SEMs are indicated.

Figure 3—figure supplement 3—source data 1.

The heart area of Tg(myl7:GAL4FF);Tg(UAS-hsp70l:GtCCR4-3.0-EYFP, myl7:mCherry) was stimulated with 520 nm light for 100 ms. The timing of light stimulation is indicated by a green circle.

Latency to cardiac arrest (A) and time to resumption of heartbeats (B) after light stimulation with ChRs. The heart area of 4-dpf Tg larvae expressing GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, or GtACR1-EYFP was irradiated with light (520 nm for GtCCR4, GtACR1; 470 nm for KnChR, CrChR2) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm². Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. One-way ANOVA with Tukey’s post hoc test (time to resumption of heartbeat GtACR1-EYFP 0.5 mW/mm² vs. 0.05 mW/mm², p=0.0240). *p<0.05, ns: not significant. Means and SEMs are indicated.

Figure 5—figure supplement 1—source data 1.

Cardiac arrest induced with channelrhodopsins (ChRs) by light of various intensities.

Latency to cardiac arrest (A) and time to resumption of heartbeats (B) after light stimulation with ChRs. The heart area of 4-dpf Tg larvae expressing GtCCR4-3.0-EYFP, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, or GtACR1-EYFP was irradiated with light (520 nm for GtCCR4, GtACR1; 470 nm for KnChR, CrChR2) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm². Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. One-way ANOVA with Tukey’s post hoc test (time to resumption of heartbeat GtACR1-EYFP 0.5 mW/mm² vs. 0.05 mW/mm², p=0.0240). *p<0.05, ns: not significant. Means and SEMs are indicated.

Figure 5—figure supplement 1—source data 1.

(A) Cardiac arrest rate. The heart area of 4-dpf Tg larvae expressing CoChR-tdTomato or ChrimsonR-tdTomato was irradiated with light (470 nm for CoChR; 520 nm for ChrimsonR) for 100 ms at a strength of 0.5, 0.2, or 0.05 mW/mm². Six consecutive stimulation trials were analyzed for four rhodopsin-expressing larvae of each Tg line. (B, C) Latency to cardiac arrest (B) and time to resumption of heartbeats (C) after light stimulation with optogenetic tools. One-way ANOVA with Tukey’s post hoc test was used for statistical analysis. ns: not significant. Means and SEMs are indicated.

Figure 5—figure supplement 2—source data 1.

The hindbrain in a 3-dpf Tg(vsx2:GAL4FF);Tg(UAS-hsp70l:BeGC1-EGFP, myl7:mCherry) larva was stimulated with 520 nm light for 500 ms. The timing of light stimulation is indicated by a green circle.

The hindbrain of 3-dpf larvae expressing bPAC or OaPAC, and non-expressing sibling control larvae was irradiated with 470 nm light for 500 ms. Six consecutive stimulation trials were analyzed for eight PAC-expressing and eight non-expressing control larvae of each line (48 trials for each condition). Latency was measured as the tail movements observed within 30 s after the onset of light stimulation and plotted in a graph. The number of the tail movements were 32 and 13 for bPAC and the control, and 26 and 13 for OaPAC and the control.

Figure 7—figure supplement 1—source data 1.

Latency of locomotion in PAC-expressing and non-expressing larvae.

The hindbrain of 3-dpf larvae expressing bPAC or OaPAC, and non-expressing sibling control larvae was irradiated with 470 nm light for 500 ms. Six consecutive stimulation trials were analyzed for eight PAC-expressing and eight non-expressing control larvae of each line (48 trials for each condition). Latency was measured as the tail movements observed within 30 s after the onset of light stimulation and plotted in a graph. The number of the tail movements were 32 and 13 for bPAC and the control, and 26 and 13 for OaPAC and the control.

Figure 7—figure supplement 1—source data 1.

(A) Expression of bPAC-MT or OaPAC-Flag in cardiomyocytes. 4-dpf larvae expressing bPAC-MT or OaPAC-Flag were fixed and stained with anti-Myc or Flag (green), and anti-DsRed (mCherry, magenta) antibodies. (B, C) Heartbeats (HBs) monitored by luminosity (AU), changes (B), and relative HB frequency (C) of bPAC-expressing larvae. The heart area of Tg larvae expressing bPAC was irradiated with light (0.5 mW/mm²) of 470 nm for 5 s at the indicated periods. Similar results were obtained from four Tg larvae. A typical example from one larva is shown in (B), and average HB frequency of the first or second trial showing a typical pattern for the four larvae is shown in (C). The larvae showed induced bradycardia in the third through sixth trials. Scale bar = 100 μm in (A).

Figure 7—figure supplement 2—source data 1.