Figure 6.
Treatment of neurons with C3 transferase, but not Y27632, blocks cypin-promoted increases in dendrite number without affecting cypin protein expression. Hippocampal neurons were transfected with constructs encoding GFP or cypin–GFP at DIV 10, treated with the appropriate drug at DIV 11 for 24 h, and processed for immunostaining at DIV 12. A, GFP images of representative neurons. Scale bar, 10 μm. B, Average number of primary and secondary dendrites in transfected neurons. n values are as follows: in GFP group: none, n = 35; C3, n = 22; Y27632, n = 20; both, n = 18; and in cypin group: none, n = 24; C3, n = 15; Y27632, n = 15; both, n = 13. #p < 0.05, ##p < 0.01, ###p < 0.001 by ANOVA followed by Bonferroni's multiple comparisons test. **p < 0.01 by ANOVA followed by Dunnett's multiple comparisons test compared with GFP. C, Left, Sholl analysis of hippocampal neurons. Overexpression of cypin significantly increases number of intersections in the range 20–55 μm compared with expression of GFP (red circles; p < 0.01 at 30–45 μm, p < 0.05 at other points). Treatment with C3 transferase does not change dendritic arbor complexity, even combined with expression of cypin (blue x). Middle, Neurons overexpressing cypin and treated with Y27632 (blue x) show a significant increase in number of intersections at 5–75 μm from the soma (except at 10 μm; p < 0.05 at 15, 25–35, 60, and 75 μm, p < 0.01 at other points). Neurons treated with Y27632 (green triangles) alone show increased number of intersections at 35–75 μm from the soma (p < 0.05 at 35 and 75 μm, p < 0.01 at other points). p values were determined by Kruskal–Wallis test followed by Dunn's multiple comparisons test compared with GFP. Right, Neurons overexpressing cypin (blue x) or GFP (green triangles) and treated with both inhibitors show no significant change in number of intersections from that of control (GFP). D, Hippocampal neurons were treated with C3 transferase or Y27632 at indicated concentrations at DIV 11 for 24 h. Representative Western blot of three individual experiments is shown. E, Quantitative analysis shows that treatments do not change is cypin protein expression (p > 0.05 by ANOVA). Error bars indicate SEM.