Figure 1
PAC activation elevates intracellular cAMP levels.
(a) Experimental paradigms for cAMP quantification via immunocytochemistry. (b) Representative images of cAMP fluorescence intensity in cultured granule cells immunostained for PAC (red) and cAMP (green). (c) Representative image of granule cells immunostained for the microtubule associated protein tau (red), cAMP (green) and PAC (blue) in 30 min light group. The images of a PAC negative cell (PAC (−)) are shown on the left, while the images of PAC positive cell (PAC (+)) are shown on the right. Scale bars = 10 μm. (d) Quantification of somatic cAMP fluorescence intensity in cultured granule cells. The cAMP fluorescence intensity was normalized to that of PAC (−) granule cells in each group. cAMP levels returned to basal levels 30 min after the light was turned off (30 min light +30 min dark). **p < 0.01 between indicated groups; Tukey’s test after one-way ANOVA, n = 27–68 cells. (e) Quantification of cAMP fluorescence intensity in the axonal and dendritic neurites of cultured granule cells 30 min after the light stimulation. The cAMP fluorescence intensity was normalized to that of PAC (−) granule cells in each group. **p < 0.01 vs. PAC (−) cells; Tukey’s test after one-way ANOVA , n = 11–19 cells. (f) Intracellular cAMP levels were analyzed using an ELISA of PAC-transfected HEK cell lysates at each time point after blue-light stimulation. **p < 0.01 vs. 0 min and ##p < 0.01 vs. 30 min; Tukey’s test after a one-way ANOVA, n = 4–8 wells.
