FIGURE 4.

Synaptic activity-induced protection against FoxO3a-GFP nuclear translocation after glutamate treatment requires nuclear calcium-CaMKIV signaling. A and B, shown is a quantitative analysis of the percentage of hippocampal neurons with nuclear localized FoxO3a-GFP in unstimulated hippocampal neurons (Control) and hippocampal neurons stimulated for 1 h with 10 μm glutamate in the absence or presence of actinomycin D (ActD; 10 μg/ml). Before application of glutamate, hippocampal neurons were treated for the indicated times with 50 μm bicuculline to induce AP bursting or received no pretreatment. Numbers of cells analyzed: 28930 in A, n = 5; 21598 in B, n = 3. NS, not significant. C, shown is an immunocytochemical analysis of expression of CaMKIV(K75E), CaMBP4, and red fluorescence protein (RFP) in hippocampal neurons transfected with the appropriate expression vectors. Hoechst staining was used to identify nuclei. Representative confocal microscopy images are shown. CaMBP4 localizes to the cell nucleus, whereas CaMKIV(K75E) is expressed in the cytosol as has been described (61). The Scale bar is 10 μm. D, shown is a quantitative analysis of the percentage of hippocampal neurons with nuclear localized FoxO3a-GFP in hippocampal neurons transfected with an expression vector for FoxO3a-GFP or in hippocampal neurons co-transfected an expression vector for FoxO3a-GFP and expression vectors for the indicated proteins. Hippocampal neurons were stimulated for 1 h with 10 μm glutamate or were left unstimulated. Before application of glutamate, hippocampal neurons were treated for 16 h with 50 μm bicuculline to induce AP bursting or received no pretreatment. Numbers of cells analyzed: 7887, no co-transfection; 7245, red fluorescence protein; 7261, CaMKIV(K75E); 5272, CaMBP4. Statistically significant differences (ANOVA followed by Tukey's post hoc test) are indicated with asterisks; ***, p < 0.001. NS, not significant. Bars represent the means ± S.E. (n = 4).