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. 2023 Aug 17;12:e83975. doi: 10.7554/eLife.83975

Figure 2. Optogenetic activation of hindbrain reticulospinal V2a neurons and cardiomyocytes by channelrhodopsins.

(A) Schematic of experimental devices. A larva was embedded in agarose. The hindbrain region or the heart were irradiated with light. Tail (caudal fin) movements and heartbeats were monitored by a high-speed camera with infrared light. (B) Schematic diagram of optogenetic tools used in this study. GC, guanylyl cyclase; AC, adenylyl cyclase; BLUF, sensors of blue-light using FAD. (C) Expression of GtCCR4-3.0-EYFP, GtCCR4-MT, KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, CoChR-tdTomato, and ChrimsonR-tdTomato in the zebrafish hindbrain reticulospinal V2a neurons. 3 dpf (day post fertilization) Tg(vsx2:GAL4FF);Tg(UAS-hsp70l:GtCCR4-3.0-EYFP, GtCCR4-MT-P2A-TagCFP, KnChR-3.0-EYFP, or CrChR2[T159C]-mCherry, myl7:mCherry) larvae were fixed and stained with anti-GFP (EYFP, green), anti-Myc tag (green) or anti-DsRed (RFP, magenta) antibodies. For CoChR and ChrimsonR, fluorescent images of the hindbrain of Tg(vsx2:GAL4FF);Tg(UAS:CoChR-tdTomato, or UAS-hsp70l:ChrimsonR-tdTomato, myl7:mCherry) larvae are shown. Inset: higher magnification images for the boxed areas showing double-labeled neurons. (D) Tail movements of 3-dpf Tg larvae expressing GtCCR4-3.0-EYFP, GtCCR4-MT, KnChR-3.0-EYFP, and CrChR2[T159C]-mCherry, CoChR-tdTomato, and ChrimsonR-tdTomato in the reticulospinal V2a neurons after stimulation of the hindbrain area with LED (0.4 mW/mm2) light with a wavelength of 520 nm (GtCCR4-3.0-EYFP, GtCCR4-MT, ChrimsonR-tdTomato) and 470 nm (KnChR-3.0-EYFP, CrChR2[T159C]-mCherry, CoChR-tdTomato) for 100 ms. Light stimulations started at time 0 s. Typical examples are shown. Scale bars = 150 μm in (C), 5 μm in the insets of (C).

Figure 2—source data 1. Data for Figure 2D, tail movements of Tg larvae expressing ChRs.

Figure 2.

Figure 2—video 1. Tail movements in a larva expressing CrChR2[T159C]-mCherry in reticulospinal V2a neurons.
Download video file (180.1KB, mp4)
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.
Figure 2—video 2. Tail movements in a larva expressing GtCCR4-3.0-EYFP in reticulospinal V2a neurons.
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The hindbrain in a 3-dpf Tg(vsx2:GAL4FF); Tg(UAS-hsp70l:GtCCR4-3.0-EYFP, myl7:mCherry) larva was stimulated with 520 nm light for 100 ms. The timing of light stimulation is indicated by a green circle.
Figure 2—video 3. Tail movements in a larva expressing GtCCR4-MT-P2A-TagCFP in reticulospinal V2a neurons.
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The hindbrain in a 3-dpf Tg(vsx2:GAL4FF);Tg(UAS-hsp70l:GtCCR4-MT-P2A-TagCFP, myl7:mCherry) larva was stimulated with 520 nm light for 100 ms. The timing of light stimulation is indicated by a green circle.
Figure 2—video 4. Tail movements in a larva expressing KnChR-3.0-EYFP in reticulospinal V2a neurons.
Download video file (137.9KB, mp4)
The hindbrain in a 3-dpf Tg(vsx2:GAL4FF);Tg(UAS-hsp70l:KnChR-3.0-EYFP, myl7:mCherry) larva was stimulated with 470 nm light for 100 ms. The timing of light stimulation is indicated by a blue circle.
Figure 2—video 5. Tail movements in a larva expressing CoChR-tdTomato in reticulospinal V2a neurons.
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The hindbrain in a 3-dpf Tg(vsx2:GAL4FF);Tg(UAS:CoChR-tdTomato), larva was stimulated with 470 nm light for 100 ms. The timing of light stimulation is indicated by a blue circle.
Figure 2—video 6. Tail movements in a larva expressing ChrimsonR-tdTomato in reticulospinal V2a neurons.
Download video file (146.1KB, mp4)
The hindbrain in a 3-dpf Tg(vsx2:GAL4FF);Tg(UAS-hsp70l:ChrimsonR-tdTomato, myl7:mCherry) larva was stimulated with 520 nm light for 100 ms. The timing of light stimulation is indicated by a green circle.