Fig. 4: The contribution of P2Y1Rs to glutamate-induced neuronal death decreases with increasing severity of excitotoxic conditions.

a Representative images of rat hippocampal primary cultures showing immunoreactivity for P2Y1Rs in neurons (MAP2—dendritic marker; SMI31—axonal marker) and astrocytes (GFAP⁺). Scale bars, 20 µm. b Primary hippocampal cultures incubated with AraC (2 µM) at DIV2–5 to inhibit astrocyte proliferation presented a reduced number of GFAP⁺ cells (DIV7), as depicted in the representative images (scale bar, 50 µm) and quantified in the left histogram. In cultures incubated with AraC, the exposure to glutamate (100 µM) for 30 min induced a significant increase in neuronal death (PI incorporation) observed 24 h later, which was attenuated by the presence of MRS2500 (1 µM). Data are mean ± SEM percentage of viable cells vs. respective control, quantified from four different cultures analyzing 300 cells per culture per condition. c The exposure of rat cultured hippocampal neurons to NMDA (100 µM) for 30 min induced a decrease in cell viability 24 h later evaluated both by MTT reduction (upper graph) and PI incorporation (lower graph), which was not observed in the presence of MRS2500 (1 µM). The decrease in cell viability observed 24 h after the exposure to NMDA (100 µM) for 12 h was not modified by MRS2500. Upper graph—data are mean ± SEM of the percentage of MTT reduction vs. respective control, quantified from 5–8 different cultures, analyzing triplicates per condition per culture. Lower graph—data are mean ± SEM of the percentage of dead cells (PI incorporation) vs. respective control quantified from four cultures analyzing 300 cells per condition per culture. *p < 0.05, **p < 0.01, and ***p < 0.001 one-sample t test (hypothetical value of 100); ^##p < 0.01 unpaired t test; ^&p < 0.05, ^&&p < 0.01, and ^&&&p < 0.001 two-way ANOVA with Sidak’s multicomparison test