Fig. 1: Extracellular ATP and P2Y1Rs are required for glutamate-induced neuronal death.

a Rat hippocampal neurons were exposed to glutamate (100 µM) for 30 min, which induced a significant increase in cell death (PI—propidium iodide incorporation) 24 h later. The mean ± SEM percentage of dead cells was quantified from 8 to 12 cultures analyzing 300 cells per culture. Scale bar, 200 µm. ***p < 0.001 one-way ANOVA with Sidak’s test for time-matched comparisons. b Glutamate-induced neuronal death at 30 and 100 µM. *p < 0.05 and **p < 0.01 one-way ANOVA with Dunnet’s test vs. untreated. c Glutamate exposure induced a sustained increase of the extracellular levels of ATP. Data are expressed as the fold change in the levels of ATP in the culture media (mean ± SEM) vs. untreated; *p < 0.05 and **p < 0.01, one-sample t test (hypothetical value of 1). d Glutamate-induced neuronal death was not observed either in the presence of apyrase (5 U/mL) or PPADS (10 µM; P2R antagonist); e it persisted in the presence of Brilliant Blue G (BBG; 100 nM; P2X7R antagonist) or TNP-ATP (10 μM; P2X1-3-containing receptors antagonist); f was prevented by Reactive Blue 2 (RB2; 10 μM; P2YR-preferring antagonist), by MRS2179 (10 µM; P2Y1R antagonist); and g by MRS2500 (0.1 and 1 µM; P2Y1R antagonist). h In the presence of apyrase, ADPβS (5 µM; agonist of P2Y1,12,13Rs) restored glutamate-induced neuronal death, an effect prevented by MRS2179. Data are mean ± SEM percentage of dead cells quantified from 3 to 8 cultures analyzing 300 cells per culture. *p < 0.05 and ***p < 0.001 one-way ANOVA with Sidak’s test for glutamate vs. untreated