Transient PAC activation induces axonal branching and elongation.
(a) Experimental paradigms for the axonal morphology assay. Rp-cAMPS was bath-applied at 100 μM for 1 hr 15 min before the light exposure. (b) Representative images of cultured granule cells immunostained for mCherry (red), tau (green) and Prox1 (blue). Scale bar = 50 μm. (c) Bar graphs indicating the number of branches. **p < 0.01 vs. PAC w/o light and ##p < 0.01 vs. PAC + 30 min light; Steel-Dwass test after Kruskal-Wallis test, n = 30 cells for each group. (d) Bar graphs indicating the length of the primary axon. **p < 0.01 vs. PAC w/o light and ##p < 0.01 vs. PAC + 30 min light; Tukey’s test after a one-way ANOVA, n = 30 cells for each group. (e) Cumulative probability of the length of each axonal branch. *p < 0.05 and **p < 0.01; Kolmogorov–Smirnov test, n = 200–600 branches from 30 cells for each group. (f) Representative images of a PAC- and tdTomato-expressing granule cell before (left) and 1 h after (right) focal light stimulation. The cyan circle indicates the stimulated region. Magnified images of areas inside the yellow boxes are shown below. The white arrow indicates a newly formed branch. Scale bars = 20 μm. (g) A bar graph showing the probability of new branch formation around the stimulated region is shown. The probability was higher near or inside the stimulation site. **p < 0.01 between indicated groups, Tukey’s test after a one-way ANOVA, n = 55 trials from 10 cells.