The effect of the genetic deletion of P2rx7 on ATP-evoked tritiated (A, C) and endogenous (B) glutamate efflux (D) mRNA expression and (E–I) immunofluorescence staining of the NR2B subunit of the NMDA receptors in acute hippocampal slices.

<p>A/10 mM ATP was used to induce [³H]Glu release from hippocampal slices of P2rx7+/+ and P2rx7−/− mice. After a 60-min preperfusion, the basal extracellular [³H]Glu efflux was lower in P2rx7−/− mice. 6-min perfusion of ATP (10 mM) resulted in a transient increase in the efflux of [³H]Glu in P2rx7+/+ mice, which peaked at 6 min after ATP administration and gradually decreased to baseline levels after 12 min. The ATP-evoked [³H]Glu efflux is substantially decreased in the hippocampus of P2rx7−/− mice, and the residual efflux is abolished by the selective P2X1 receptor antagonist NF449 (100 nM), which was applied at 15 min before ATP perfusion. [³H]Glu release is expressed as a percentage of the amount of radioactivity in the tissue at the sample collection time (fractional release). For the evaluation of the basal tritium outflow, the tritium content of the first four consecutive 3-min samples were taken into account. The curves represent the mean ± SEM of 8–12 identical experiments. B/HPLC analysis. Samples indicated in (A) as S1 and S2 were analyzed. ATP perfusion (10 mM) significantly increases the endogenous Glu level in the effluent. Results are expressed as pmol/3 min. N = 8, ***P<0.001. C/Concentration-response relationship of ATP-evoked [³H]Glu efflux in P2rx7+/+ and P2rx7−/− mice. Experiments were performed according to the protocol shown in (A), using different concentrations of ATP, as indicated in the abscissa. The net ATP-induced release was calculated and expressed as the fractional release (%). The curves represent the mean ± S.E.M. of 4–12 identical experiments. D/Changes in the mRNA expression levels of the NMDA-NR2B receptor in hippocampus obtained from P2rx7+/+ and P2rx7−/− mice. Immediately after the 60-min incubation, the brain slices were removed and total RNA was extracted from the hippocampus and reverse transcribed to cDNA. Quantitative SYBR Green real-time PCR was performed using specific primers, as described in Methods, and cDNA as a template. The experiments were repeated two times with similar results. The expression level of the NR2B receptor was normalized to that of the distinct housekeeping gene, 18S rRNA. The data are displayed as the mean ± SEM. Asterisk indicates significant difference from the P2rx7+/+ mice (*P<0.05, Student’s t-test). E-I/Immunofluorescence staining for NR2B on hippocampal sections of P2rx7+/+ and P2rx7−/− mice. Besides the typical dotted staining most likely presenting NR2B - immunolabelled terminals (arrows, F, H) some staining is observable around cell bodies especially in the pyramidal cell layer (three arrowheads G). The whole staining is more intense in the hippocampal section of P2rx7−/− mouse (G, I). The most intense staining is observed in the CA3 regions (CA3 on E, G and F, H), while stratum oriens (or) shows the least immunoreactivity either in P2rx7+/+ and P2rx7−/− sample. Contrary to P2rx7+/+ staining (E), intense immunofluorescence illustrates hilus region on P2rx7−/− (G) sample. Images acquired at higher magnification (F,H) also show blood vessels (arrowheads), stained at the same level in both sections (background staining). bars: 50 µm in F, H, 500 µm in E, G. I. Immunofluorescence staining intensity for NR2B in P2rx7+/+ and P2rx7−/− mice. Average intensity was quantified with NIH ImageJ program (U.S. National Institutes of Health, Bethesda, MD) and is expressed in arbitrary units. Asterisk indicates significant difference from the wild type mice (*P<0.05, Student’s t-test, n = 3).