Figure 2

ADF/cofilin inactivation via Rac1-LIM kinase signalling mediates the increase in stable actin at spines of the barrel cortex in socially isolated rats. Representative western blots of the barrel cortex synaptoneurosomes from non-isolated or isolated rats (a,e, and f), and isolated rats with or without RU486 treatment (b). (a) Phosphorylation of ADF/cofilin (Ser3) was increased in the barrel cortex of isolated rats (n = 34) compared to non-isolated rats (n = 34). (b) The increased phosphorylation of ADF/cofilin in isolated rats was blocked by the administration of RU486 (n = 14) during isolation but not by treatment with vehicle (n = 13). (c) FRAP analysis showing the effect of ADF/cofilin phospho-mutant (S3A) expression on actin dynamics in the spines of the barrel cortex of isolated rats. Representative images and graphs depicting the recovery of the GFP-actin signal before and after photobleaching at spines expressing GFP-actin with RFP-S3A or RFP alone (control) in isolated rats are shown. Representative images shown in the figures were Gaussian- and unsharp mask-filtered. Arrowheads indicate dendritic spines. Scale bar represents 1 µm. (d) Recovery time constant and quantification of the unrecoverable fraction of spines expressing the ADF/cofilin phospho-mutant in isolated rats. The increased unrecoverable actin fraction in isolated rats was prevented in the spines expressing RFP-S3A (n = 27) but not in control spines expressing RFP (n = 21). (e) Phosphorylation of LIM kinase (LIMK) was increased in the barrel cortex of isolated rats (n = 5) compared with the non-isolated controls (n = 7). (f) The increased phosphorylation of LIMK in isolated rats was blocked by the administration of RU486 (n = 7) during isolation but not by treatment with vehicle (n = 8). (g) Rac1 activity (GTP-Rac1) was increased in the barrel cortex of isolated rats (n = 9) compared with the non-isolated controls (n = 9). (h) The increased GTP-Rac1 in isolated rats was blocked by the administration of RU486 (n = 8) during isolation but not by treatment with vehicle (n = 5). The images shown were the cropped blots for the corresponding proteins. The original uncropped images were shown in Supplementary Figure 1. *P < 0.05 (Unpaired Student’s t-test). Error bars represent SEM.