Figure 4. Local optogenetic stimulation conveys guidance instructions to regenerating axons.

(A, B) Regenerating axons prefer to regrow away from the original trajectory, with only a minority of axons finding the correct path. (A) Representative images of axons retracting or bifurcating at 24 hr AI. At 48 hr AI in WT, regenerating axons extend dorsally, ventrally, or both directions. The injury site is marked by the dashed circles and regenerating axons are marked by arrowheads. Axons are outlined with dashed green lines. Scale bar = 20 μm. (B) Light stimulation fails to increase the percentage of axons regrowing towards the right direction. The percentage of axons extending towards the correct trajectory (ventral + bidirectional) were analyzed by Fisher's exact test, p=0.7554, p=0.1729, p=0.6097, p=0.7638. WT (light) N = 26, optoRaf (light) N = 26, optoRaf (dark) N = 25, optoAKT (light) N = 45, optoAKT (dark) N = 28 neurons. (C, D) Restricted local activation of optoRaf or optoAKT significantly increases the relative regeneration ratio. The ratio is defined to weigh the regeneration potential of the ventral branch against the dorsal branch. (C) A single pulse of light stimulation delivered specifically on the ventral axon branch at 24 hr AI (blue flash symbol) is capable of promoting the preferential extension of regenerating axons in optoRaf or optoAKT expressing larvae. The injury sites are demarcated by the dashed red circles and regenerating axons are marked by arrowheads. Axons are outlined with dashed green lines. Blue flash symbols show the restrict light delivery to the ventral branch. (D) Qualification of the relative regeneration ratio of v'ada. WT (dark) N = 32, WT (local 5 min) N = 32, WT (Con. on) N = 33, optoRaf (dark) N = 32, optoRaf (local 5 min) N = 35, optoRaf (Con. on) N = 36, optoAKT (dark) N = 33, optoAKT (local 5 min) N = 34, optoAKT (Con. on) N = 36 neurons. Data are mean ± SEM, analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test, *p<0.05, ***p<0.001.