Figure 1. NMDA receptor overactivation results in a transient depletion of ATP and delayed apoptosis associated with induction of the BH3-only protein Bim. (A) Model of transient NMDA receptor activation. CGNs were treated with 100 µM glutamate/10 µM Gly for 10 min, or mouse neocortical neurons were treated with 100 µM NMDA/10 µM Gly for 5 min. The onset of injury occurs over a time frame of 4–24 h. (B) Time course of delayed excitotoxic apoptosis. CGNs were treated with 100 µM glutamate/10 µM Gly for 10 min and subsequently stained with Hoechst, and condensed pyknotic nuclei were quantified at the indicated time points. (C) Cell shrinkage during excitotoxic apoptosis. Differential interference contrast images of CGNs at the indicated time points after glutamate treatment. (D) Excitotoxic apoptosis is associated with a DCD. Ca2+ levels were monitored with Fluo-4 and time-lapse microscopy after treatment with 100 µM glutamate/10 µM Gly for 10 min. (E) Individual traces of TMRM fluorescence changes in CGNs as monitored using time-lapse microscopy after treatment with 100 µM glutamate/10 µM Gly for 10 min. (F) Cellular ATP levels in CGNs after treatment with 100 µM glutamate/10 µM Gly for 10 min. ATP levels were normalized to sham-treated neurons. (B and F) Data represent means ± SEM from n = 4 cultures. These experiments were repeated twice with similar results. *, P < 0.05 compared with sham-treated controls. (G) Real-time qPCR analysis of expression of bim, bmf, puma, and bid mRNA at 16 h after glutamate treatment relative to β-actin mRNA levels. (H) Real-time qPCR analysis of bim mRNA expression after glutamate treatment at 16 and 24 h after excitation relative to β-actin mRNA levels. (G and H) Expression levels were normalized to sham-treated cells, and data are represented as means ± SEM from n = 3 independent cultures. *, P < 0.05 compared with sham-treated controls. (I) CGNs transfected with control or bim siRNA for 72 h and subsequently treated with 100 µM glutamate/10 µM Gly for 10 min. Condensed pyknotic nuclei were quantified 24 h after excitation. Data represent means ± SEM from n = 4 cultures. *, P < 0.05. This experiment was repeated once with similar results. (right) The expression levels of Bim were assessed by Western blotting 72 h after siRNA transfection. Probing for β-actin served as a loading control. Bar, 10 µm.
Figure 1.

NMDA receptor overactivation results in a transient depletion of ATP and delayed apoptosis associated with induction of the BH3-only protein Bim. (A) Model of transient NMDA receptor activation. CGNs were treated with 100 µM glutamate/10 µM Gly for 10 min, or mouse neocortical neurons were treated with 100 µM NMDA/10 µM Gly for 5 min. The onset of injury occurs over a time frame of 4–24 h. (B) Time course of delayed excitotoxic apoptosis. CGNs were treated with 100 µM glutamate/10 µM Gly for 10 min and subsequently stained with Hoechst, and condensed pyknotic nuclei were quantified at the indicated time points. (C) Cell shrinkage during excitotoxic apoptosis. Differential interference contrast images of CGNs at the indicated time points after glutamate treatment. (D) Excitotoxic apoptosis is associated with a DCD. Ca2+ levels were monitored with Fluo-4 and time-lapse microscopy after treatment with 100 µM glutamate/10 µM Gly for 10 min. (E) Individual traces of TMRM fluorescence changes in CGNs as monitored using time-lapse microscopy after treatment with 100 µM glutamate/10 µM Gly for 10 min. (F) Cellular ATP levels in CGNs after treatment with 100 µM glutamate/10 µM Gly for 10 min. ATP levels were normalized to sham-treated neurons. (B and F) Data represent means ± SEM from n = 4 cultures. These experiments were repeated twice with similar results. *, P < 0.05 compared with sham-treated controls. (G) Real-time qPCR analysis of expression of bim, bmf, puma, and bid mRNA at 16 h after glutamate treatment relative to β-actin mRNA levels. (H) Real-time qPCR analysis of bim mRNA expression after glutamate treatment at 16 and 24 h after excitation relative to β-actin mRNA levels. (G and H) Expression levels were normalized to sham-treated cells, and data are represented as means ± SEM from n = 3 independent cultures. *, P < 0.05 compared with sham-treated controls. (I) CGNs transfected with control or bim siRNA for 72 h and subsequently treated with 100 µM glutamate/10 µM Gly for 10 min. Condensed pyknotic nuclei were quantified 24 h after excitation. Data represent means ± SEM from n = 4 cultures. *, P < 0.05. This experiment was repeated once with similar results. (right) The expression levels of Bim were assessed by Western blotting 72 h after siRNA transfection. Probing for β-actin served as a loading control. Bar, 10 µm.

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