Figure 2.

Effect of selective and prolonged activation of NMDAR on cellular morphology and neuronal death. A, Primary cultured cortical neurons at DIV 12 were subjected to synaptic (50 μm Bic + 2.5 mm 4-AP) or extrasynaptic (50 μm Bic + 2.5 mm 4-AP + 10 μm MK801 followed by 30 μm NMDA application) NMDAR activation for 12 and 24 h. The level of LDH released in the neuronal cell culture medium was determined by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. Results are expressed as mean ± SD from five independent experiments. Histograms represent neuronal cell death normalized to maximum neuronal death (0.1% Triton X-100 for 10 min). Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test. n = 5; **p < 0.01 vs corresponding control; ##p < 0.01 vs extrasynaptic NMDAR stimulation. B, Morphological analysis of eGFP-transfected neuronal cultures. eGFP-labeled cortical neurons (DIV 12; transfection efficiency of 3 to 6%) were treated according to the synaptic protocol or the extrasynaptic protocol described in A for 12 or 24 h. Images represent the projection of z-stack images acquired by confocal microscopy at the indicated times and are representative of the cultures. Scale bar, 20 μm. Arrows are pointing to typical dendritic varicosities. C, Quantification of living cells, dead cells, and varicosity-containing cells in cultures exposed or not to synaptic or extrasynaptic treatment during the indicated times (12 or 24 h). Quantification of the three cell populations was achieved on four independent cultures by counting eGFP-transfected neurons on the overall coverslip under confocal microscope. Histograms represent means ± SD, and statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (n = 4; *p < 0.05). D, Quantification of living cells, necrotic cells, and apoptotic cells in cultures exposed or not to synaptic or extrasynaptic protocol during the indicated times (12 or 24 h). Quantification of the three cell populations was achieved on six independent cultures using a double-stain apoptosis detection kit containing the blue-fluorescent Hoechst 33342 dye and the red-fluorescent PI dye, which is permeant only to dead cells. Counting of living, apoptotic, and necrotic cell populations was performed under a fluorescence microscope. Histograms represent means ± SD and statistical analysis was performed by ANOVA followed by Bonferroni–Dunn's test (n = 6; *p < 0.05). E, The photo is an illustration of apoptotic and living cells stained with Hoechst 33342. Arrows are pointing to typical apoptotic cells exhibiting apoptotic bodies. Scale bar, 20 μm.