The above experiments were conducted using a high dose of d-fenfluramine (50 mg/kg) that is comparable with our previously published results (60). Mice tolerated this dose of d-fenfluramine well with 100% survival rate. To examine if the response of FoxOs to d-fenfluramine is dose-dependent, mice were also treated with different doses of d-fenfluramine (0, 1, 5, 10, 30 mg/kg) for 2 hr (Figure 1e). Except FoxO1 in the cerebral cortex, which was more sensitive to a lower dose of d-fenfluramine (1 mg/kg), a dose range of 10-30 mg/kg was sufficient to induce a 50% increase in FoxO1 phosphorylation in the hippocampus and FoxO3a phosphorylation in both cerebral cortex and hippocampus. To assure maximal effect in all experiments, we then used 50 mg/kg d-fenfluramine for all experiments and data analysis in this study.
