For immunostaining, cultured neurons were fixed at indicated times in PBS containing 4% paraformaldehyde/4% sucrose for 20 minutes at 37°C. The neurons were then permeabilized with 0.1% Triton X-100 (Sigma)/PBS for 5 minutes and blocked with 0.5% fish skin gelatin (Sigma)/PBS for 1 hour, both at 27°C. The cells were then stained for somatodendritic markers using polyclonal rabbit anti-microtubule-associated protein 2 antibody (1:150; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, followed by incubation with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1:200; Jackson Immuno Research Labs, Inc., West Grove, PA, USA) at 37°C for 2 hours. The samples were washed with PBS, dewaxed, and mounted on slide glasses. The stained samples were imaged under an epifluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a 20 × objective lens. The images were obtained with Axiocam MRm CCD camera (Zeiss) and AxioVision software (Zeiss). The toxic effects of aspartic acid on the neurons were evaluated by Live/Dead Assay kit (L-3224; Molecular Probe, Eugene, OR, USA) and the percent of damaged neuron number/total neuron numbers was calculated[63 (link)]. Neuronal viability was evaluated and compared with cultured neurons not exposed to aspartic acid.