Apoptosis was analyzed by TUNEL assay using Click-iT® Plus TUNEL Assay (Life Technologies, Inc., Carlsbad, CA, USA) according to manufacturer’s instruction. Briefly, at the end of the indicated treatments, primary rat cerebral cortical neurons grown on coverslips were incubated with TdT reaction mixture for 2 h at 37°C, followed by 30-min incubation with the Alexa Fluor® 594 dye. Then, the cells were counterstained with DAPI (Sigma-Aldrich) for 20 min and observed under a fluorescence microscope (Leica, Germany). The TUNEL-positive nuclei of six non-overlapping fields per coverslip were counted by a researcher blinded to treatment, and these counts were converted to percentages by comparing the TUNEL-positive counts to the total number of cell nuclei as determined by DAPI counterstaining, that is TUNEL-positive ratio = (number of red nuclei/number of blue nuclei) × 100%.