Fig. 4

Increased neuronal activity causes pan-nuclear neuronal γH2AX labeling in vivo and in vitro. a, b Mice were analyzed 1 h after they received an intraperitoneal injection of kainate (KA, 20 or 30 mg/kg) or vehicle (saline) at 4–6 months of age. a Representative confocal microscopic images of dentate gyrus sections co-labeled for γH2AX (red) and NeuN (grey). Insets show higher magnification views of the areas outlined by yellow squares. Note the pan-nuclear pattern of the γH2AX labeling. Scale bars: 200 μm; 10 μm (insets). b Quantitation of γH2AX immunofluorescence intensity in the dentate gyrus. Mean levels in saline-treated mice were arbitrarily defined as 1.0. n = 9 mice per group (pooled from 3 independent experiments). ***p < 0.001 by Mann-Whitney test. c, d Primary hippocampal neuronal cultures from mice were pretreated for 1 h with vehicle or tetrodotoxin (TTX, 1 μM) on DIV 14. Vehicle or bicuculline (Bic, 10 μM) was then added to the medium and cultures were incubated for 1 h. c Representative widefield images of neuronal cultures co-labeled for γH2AX (red) and NeuN (grey). Scale bar: 10 μm. d Quantitation of neuronal γH2AX immunofluorescence intensity. For each culture, the mean levels in different wells of saline-treated cultures were arbitrarily defined as 1.0. n = 4 independent cultures from different mice per condition. *p < 0.05 vs. mean of 1.0 (control) by one sample t-test. Bars represent means ± SEM