Visualization of disassembly of F-actin by cannabinoid agonists in in dendritic spines on mature cortical neurons, leading to reduction in the density of mature spines.

(A) Representative real time images of FRAP experiments. Localized photobleaching over single, individual postsynaptic spines (red circles), led to loss of LifeAct-mCherry labeling of F-actin and progressive recovery of fluorescence with actin polymerization, which was inhibited by ACEA as compared to vehicle control. (B & C) Summary of FRAP values as a function of time expressed as one phase exponential function curves fitted to the respective data sets from neurons treated with ACEA or vehicle either from wild-type cultured cortical neurons (B) or with siRNA-mediated WAVE1 knockdown (C) (13–15 dendritic spines/group from 4 independent culture experiments). (D) Immunoblot example of WAVE1 down-regulation 3 d following siRNA delivery in mature cultured cortical neurons and the corresponding quantification (seven independent culture experiments). (E) Quantitative summary of the magnitude of F-actin recovery in the dendritic spine after photobleaching in neurons from the diverse treatment groups (five independent culture experiments). (F) Representative confocal images of mature cortical neurons transduced with rAAV-CamK-II-GFP virions and treated with ACEA (100 nM) or vehicle for 24 h. GFP-labelled dendritic spines were morphologically classified (lower panels) and quantified. Yellow bars represent 50 μm and white bars represent 5 μm. (G) Quantitative summary of average density of dendritic spines of varying morphology in cortical neurons treated with ACEA (100 nM) or vehicle (n = 20–21 dendrites analyzed from three cultures/group). All graphs represent mean values ± SEM *p < 0.05 as compared to control group and †p < 0.05 as compared to WT neurons, two-way ANOVA (E) or one-way ANOVA (D & G) followed by posthoc Tukey’s test. N.s. stands for “not significant.”