Skip to main content
. 2024 Jan 10;44(2):e1393222023. doi: 10.1523/JNEUROSCI.1393-22.2023

Figure 4.

Figure 4.

CaMKIIα knockdown blocks activity-dependent new spine stabilization. A, Images of red fluorescence showing mature spines on dendrites from hippocampal CA1 neurons in slice culture (DIVs 7–9) transfected with dsRed-Express and CaMKIIα shRNA alone (top row) or co-expressed with shRNA-resistant CaMKIIα* variants (middle and bottom rows), showing mature dendritic spines stimulated with HFU at 0 min. B, shRNA-mediated knockdown of CaMKIIα (filled blue; n = 8 spines/8 cells) impaired HFU-induced long-term growth of mature spines that was restored with co-expression of shRNA-resistant GFP-CaMKIIα* (black; n = 14 spines/14 cells) but not with co-expression of monomeric mGFP-CaMKIIα* (gray; n = 10 spines/10 cells). C, Images of red fluorescence showing dendrites on CA1 neurons (DIVs 7–9) expressing a DsRed-Express cell fill and either CaMKIIα shRNA (top row) or CaMKIIα shRNA + shRNA-resistant GFP-CaMKIIα* variants (bottom two rows). HFU-induced new spine (filled arrowheads) stabilization failed following knockdown of CaMKIIα (open arrowhead at 70 min), which was rescued with expression of shRNA-resistant GFP-CaMKIIα*, but not with expression of monomeric shRNA-resistant mGFP-CaMKIIα*. D, Knockdown of CaMKIIα disrupted the stabilization of new spines (filled circles) as compared with unstimulated new spines (open circles) at times beyond 30–40 min (blue; stimulated, 20 spines/20 cells; unstimulated, 103 spines/20 cells). Rescuing with full-length shRNA-resistant CaMKIIα* restored activity-dependent new spine stabilization (black; stim, 18 spines/18 cells; unstim, 98 spines/18 cells), but not shRNA-resistant monomeric mCaMKIIα* (gray; stim, 10 spines/10 cells; unstim, 47 spines/10 cells). E, Activity-dependent new spine stabilization at 70 min (filled bars) was not different from that of unstimulated new spines (open bars) following knockdown of CaMKIIα (black), which was restored when CaMKIIα is rescued with shRNA-resistant CaMKIIα* (blue), but not with shRNA-resistant monomeric mCaMKIIα*. Two-way ANOVA with Bonferroni’s multiple-comparisons test in B, log-rank task in D, and Barnard's exact test in E. *p < 0.05, **p < 0.01, ***p < 0.001.