Fig. 5. The effect of action potential frequency and stimulus duration on autonomous activation of CaMKII. CaMKII autonomy ratio is the ratio of Ca2+-independent activity/total activity. Autonomous activity of CaMKII was measured byin vitro phosphorylation assay in homogenates of neurons electrically stimulated for 5, 15, 45, and 600 sec at 0.1, 1, 3, and 10 Hz. A, Stimulation at 1, 3, and 10 Hz for 15 sec induced statistically significant Ca2+-independent activation of CaMKII compared with unstimulated controls (p < 0.01; t test;n = 24 dishes), but the differences among responses to 1, 3, and 10 Hz were not statistically significant at 15 and 45 sec. The results shown (mean ± SEM) are from five independent experiments (ANOVA; p = 0.02; n= 150 dishes). Inactivation of the enzyme was promoted by long-duration high-frequency stimulation. The duration of stimulation required to reach maximal levels of CaMKII autonomy varied inversely with the frequency of stimulation. Stimulation at very low frequency (0.1 Hz) failed to increase CaMKII autonomous activity significantly.B, Maximal autonomous activation of CaMKII correlated with the number of action potentials delivered at these different frequencies. Within the range of 1–10 Hz, near-maximal autonomous activity was reached after ∼45 action potentials, regardless of stimulus frequency. Statistical analysis by ANOVA (p < 0.0001; n = 178), followed by Fisher's LSD multiple comparison procedure (Table 1) indicates significant increases in CaMKII autonomous activation after 45 action potentials are delivered at all frequencies tested within the range of 0.5–10 Hz, despite large differences in the stimulus duration required to reach 45 action potentials at these different frequencies (i.e., 4.5–90 sec).