Fig. 1

Glutamate-induced neurotoxicity depends on NMDARs. a-f Neurons from wildtype (WT) mice were exposed to different concentrations of glutamate for 15 min at DIV13. Relative levels of survival were quantified by alamarBlue assay 24 h later. Cultures were treated with vehicle or different antagonists starting 1 h before the glutamate exposure: a non-competitive NMDAR antagonist MK801 (20 μM), b competitive NMDAR antagonist APV (50 μM), non-competitive GluN2B-selective NMDAR antagonists c Ro 25–6981 (1 μM) or d ifenprodil (10 μM), e competitive AMPAR antagonist DNQX (20 μM), or f sodium channel blocker tetrodotoxin (TTX, 1 μM). The boxplots represent the distribution of the differences in mean fluorescence between antagonist- vs. vehicle-treated neurons at each dose across independent experiments. The lower and upper ends of the boxes represent the 25th and 75th quartile of each distribution, respectively. The horizontal line in each box represents the median. The ends of the whiskers terminate at the farthest points that are within 1.5 times the inter-quartile range (difference between upper and lower ends of the box). Individual dots shown in some of the panels represent outliers that fell outside the range defined by the whiskers. Consequently, the curve in a indicates that the survival-promoting effects of MK801 became more and more evident as glutamate concentrations increased, whereas the bell-shaped curves in b–d indicate that, at the particular concentrations used, the respective antagonists were protective at moderate, but not higher, concentrations of glutamate. Numbers of independent experiments (n) with cumulative well numbers per condition in parentheses: a 4 (24–32), b 10 (76–80), c 9 (68–72), d 8 (56–64), e 9 (70–72), and f 4 (30–32). When comparing mean differences across all doses within any given panel, a one-sided, one-sample t-test revealed significant differences between experimental and control conditions in (a, P < 0.05), (b, P < 0.0001), (c, P < 0.001), and (d, P < 0.001). g, h Neuronal cultures were treated with glutamate at DIV13 and fixed 24 h later, followed by immunostaining for the neuronal marker NeuN and the astroglial marker glutamine synthetase and nuclear staining with Hoechst33342. g Representative photomicrographs of immunolabeled cultures imaged on an ArrayScan XTI Live High Content Platform. Scale bar: 200 μm. h Number of Hoechst33342-positive cells per well that were immunoreactive for NeuN or glutamine synthetase. n = 4 independent experiments, each of which included 8–16 wells per condition. Data in h are means ± SEM