Figure 7.

PSD-95 binding is essential for stabilizing cypin-promoted dendrites. A, Hippocampal neurons were transfected with cDNAs encoding the indicated constructs (N-terminally tagged with GFP) at 10 DIV and the number of primary and secondary dendrites were assessed at 12 (white bars) and 17 DIV (black bars). Overexpression of cypin resulted in increased primary and secondary dendrite number at both 12 and 17 DIV; however, overexpression with cypin-PDZ, a mutant that cannot interact with PSD-95, resulted in a transient increase in dendrite number at 12 but not 17 DIV. n values are as follows: 12 DIV, GFP, 33; cypin, 30; cypin-PDZ, 24; 17 DIV, GFP, 20; cypin, 29; cypin-PDZ, 12. *p < 0.05; ** p < 0.01; ***p < 0.001 by Kruskal–Wallis test followed by Dunn's multiple comparisons test as compared with GFP control. ^XX p < 0.01; ^XXX p < 0.001 versus GFP at 12 DIV. ^O p < 0.05; ^OOO p < 0.001 versus GFP at 17 DIV. B, Statistics collected for the samples of neurons in the different populations (GFP, cypin, cypin-PDZ). Left, 12 DIV; right, 17 DIV. The proportion of primary dendrites that branch is the same for all neurons (top row) for both time points (p = 0.17 and 0.49 respectively). The number of excess secondary branches is higher for neurons that overexpress cypin than GFP and cypin-PDZ. p < 1 × 10⁻¹⁶ for cypin and p = 0.8 for cypin-PDZ versus GFP control at 12 DIV; p = 2.8 × 10⁻⁷ for cypin and p = 0.37 for cypin-PDZ versus GFP control at 17 DIV. C, Hippocampal neuronal cultures were treated with PSD-95 antisense ODN or sense controls until 17 DIV. The increase in the number of secondary dendrites by treatment with PSD-95 antisense ODNs at 12 DIV was lost by 17 DIV. Note that there was a significant increase in the number of primary dendrites when cultures were treated with PSD-95 antisense compared with sense control at 17 DIV. *p < 0.05 by Mann–Whitney test. Error bars indicate SE.