Fig. 4: 5-HT4R activation boosts the RhoA activity and accumulation of F-actin within individual spines.
a Representative western blot (left) for visualization of active RhoA (RhoA-GTP) (upper), total RhoA (middle), and loading control GAPDH (bottom) in hippocampal neurons (DIV12) treated with BIMU8 or vehicle (veh) for 10 min. Active RhoA was precipitated by pull-down. (Right) Relative RhoA activity calculated as a ratio of RhoA-GTP to total RhoA normalized to GAPDH expression (N = 4 cultures for each group). *p < 0.05 (Mann–Whitney U test). b, c Representative time-lapse confocal images of defined spines (left) in the cerulean-expressing hippocampal neurons co-transfected with FRET-based biosensor RaichuRhoA (b) and LifeAct-mRuby (c). Images were acquired every 2.5 min. After 7.5 min imaging under control conditions (−7.5 min to 0 min), either vehicle or BIMU8 was added to the bath solution and cells were imaged for the further 10 min. Scale bar, 1 µm. Fluorescence intensity for ratiometric changes in the YPet/mTurquoise ratio, reflecting the RhoA activation (b) and LifeAct-mRuby, indicating the F-actin accumulation in the same spines (c), is shown. (Right) Quantification of the YPet/mTurquoise fluorescence intensity ratio (b) and the mRuby fluorescence intensity (c) in control (n = 13 cells) and BIMU8 responding spines (n = 18 cells). *p < 0.05, **p < 0.01 (Mann–Whitney U test). See also Supplementary Fig. 5. d Spine contours for visualizing morphological changes of dendritic spine in control and BIMU8-treated neurons before (−7.5 and 0 min) and after treatment (10 min). e, f Post-hoc immunostaining of hippocampal neurons (the same spines shown as in (b, c) with anti-PSD-95 antibody (e) and quantification of relative PSD-95 staining in spines after stimulation with vehicle or BIMU8 (f). **p < 0.01 (two-tailed unpaired t-test). See also Supplementary Fig. 5. Data are presented as mean ± SEM. In (b, c), data are presented as relative changes in the YPet/mTurquoise ratio (b) and relative changes in F-actin (c).
