Figure 6. Optogenetic activation of hindbrain reticulospinal V2a neurons with BeGC1-EGFP.
(A) Expression of BeGC1-EGFP in the hindbrain reticulospinal V2a neurons. 3-dpf Tg(vsx2:GAL4FF);Tg(UAS:BeGC1-EGFP, myl7:mCherry);Tg(UAS:RFP) larvae were fixed and stained with anti-GFP (EGFP, green) or anti-DsRed (RFP, magenta) antibodies. Inset: higher magnification images for the boxed area showing double-labeled neurons. In the inset, fluorescence signal intensities were modified to compare the subcellular localization of the tools. (B) Tail movements of 3-dpf Tg larvae expressing BeGC1 in the reticulospinal V2a neurons after stimulation with light (0.4 mW/mm2) at 520 nm for 500 ms. The stimulation started at time 0 s. A typical induced tail movement is shown. (C) Light stimulation-dependent locomotion rates of 3-dpf BeGC1-expressing larvae or non-expressing sibling control larvae. Six consecutive stimulation trials were analyzed for eight BeGC1-expressing and eight non-expressing larvae. The average locomotion rates for each larva are shown. Wilcoxon rank-sum test (BeGC1-EGFP vs. control, p=0.00608). (D–F) Latency (D), duration (E), and strength (F) of induced tail movements in the BeGC1-expressing larvae. The time from the start of light stimulation to the first tail movement was defined as latency (s), and the time from the start of the first tail movement to the end of that movement was defined as duration (s). The maximum distance that the caudal fin moved from the midline divided by body length reflected its strength. Scale bars = 150 μm in (A), 10 μm in the insets of (A). **p<0.01. Means and SEMs are indicated.
