Temporal cAMP dynamics regulates axonal morphogenesis.
(a) Experimental paradigms. In some cases, blue light was applied with a protocol of 1 min × 30 times with 4-min intervals. For reagents, either the PKA antagonizing peptide PKI (20 μM) or the Epac blocker ESI-09 (100 μM) was bath-applied 15 min before light exposure for 1 hr. (b) Representative images of cultured granule cells immunostained for tau (green) and PAC (blue). Actin was labeled with rhodamine phalloidin (red). Scale bar = 50 μm. (c) Bar graphs indicating the number of branches. **p < 0.01 vs. PAC w/o light, ##p < 0.01 vs. PAC PKI w/o light and $$p < 0.01 vs. PAC ESI w/o light; Steel-Dwass test after Kruskal-Wallis test, n = 20–30 cells for each group. (d) Bar graphs indicating the length of the primary axon. **p < 0.01 vs. PAC w/o light, ##p < 0.01 vs. PAC PKI w/o light and $$p < 0.01 vs. PAC ESI w/o light; Tukey’s test after a one-way ANOVA, n = 20–30 cells for each group. (e) Intracellular cAMP levels were analyzed with an ELISA of PAC-transfected HEK cell lysates. **p < 0.01 vs. control; Tukey’s test after a one-way ANOVA, n = 4–8 wells. (f) Cumulative probability of the length of each axonal branch. *p < 0.05 and **p < 0.01; Kolmogorov–Smirnov test, n = 150–500 branches from 20–30 cells for each group.