p,0.001; NR2c: 0.7360.02, p,0.001; NR2d: 0.7960.03, p,0.05) with vehicle-injected WT mice (Fig. 4A–G). An increase of about 40% was observed in the expression of the NR2b subunit in vehicle-injected COX-22/2 compared with vehicle-injected WT mice (NR2b: 1.4260.09, p,0.001). Also, the expression of NR1, NR2a, NR2c and NR2d subunits was decreased when we compared KA-injected WT with vehicle-injected WT mice (NR1: 0.7360.04, p,0.01; NR2a: 0.7160.02, p,0.001; NR2c: 0.8060.06, p,0.01) (Fig. 4A–G). A down-regulation in the expression of several NR subunits (NR2b: 0.7460.05, p,0.05; NR2c: 0.7060.05, p,0.01; NR2d: 0.7360.04, p,0.05; NR3b: 0. 5060.10, p,0.05) was observed in KA-injected COX-22/2 mice compared with vehicle-injected COX-22/2 mice (Fig. 4A–G). A significant decrease of about 30– 50% was observed in the expression of NR2a, NR2c and NR2d subunits between KA-injected COX-22/2 mice and KA-injected WT mice (NR2a: 0.6360.03, p,0.01; NR2c: 0.7060.02, p,0.001; NR2d: 0.4960.14, p,0.001) (Fig. 4A–G). Pretreatment with celecoxib altered mRNA expression of AMPA/KA and NMDA glutamate receptor subunits in WT mice after KA injection We examined the relative changes in the mRNA expression of the AMPA (GluR1–4), KA (GluR5–7, KA1 and KA2) and NMDA (NR1, NR2a–d, NR3a–b) glutamate receptor subunits in the hippocampus and cortex of mice treated for two weeks with celecoxib, a COX-2 selective inhibitor, prior to KA injection, in vehicle-injected and KA-injected WT mice (n = 9). In the hippocampus, no changes were observed when we compared vehicle-injected and celecoxib treated mice (data not shown). KA injection affected the expression of all AMPAR, KAR (except GluR6) and NMDAR subunits in both celecoxib untreated controls (GluR1: 0.7860.02, p,0.001; GluR2: 0.7660.03, p,0.001; GluR3: 0.7960.04, p,0.01; GluR4: 0.7860.05, p,0.001; GluR5: 0.8060.02, p,0.001; GluR7: 0.6860.04, p,0.05; KA1: 0.5060.02, p,0.001; KA2: 0.7360.03, p,0.001; NR1: 0.5960.05, p,0.001; NR2a: 0.5560.02, p,0.001; NR2c: 0.5560.05, p,0.001; NR2d: 0.5660.04, p,0.001; NR3a: 0.6860.03, p,0.001; NR3b: 0.5060.09, p,0.001) and cele- coxib-treated WT mice (GluR1: 0.3860.02, p,0.001; GluR2: 0.3260.01, p,0.001; GluR3: 0.2760.01, p,0.001; GluR4: 0.5760.02, p,0.001; GluR5: 0.6060.02, p,0.001; GluR7: 0.5060.02, p,0.001; KA1: 0.5060.01, p,0.001; KA2: 0.3560.01, p,0.001; NR1: 0.3760.01, p,0.001; NR2a: 0.2460.003, p,0.001; NR2b: 0.2760.01, p,0.001; NR2c: 0.3860.02, p,0.001; NR2d: 0.3960.02, p,0.001; NR3a: 0.1860.01, p,0.001; NR3b: 0.2060.03, p,0.001) when com- pared with vehicle-injected WT mice (Fig. 5A–I and Fig. 6A–G). Celecoxib pretreatment significantly decreased the mRNA expression of most iGluRs in KA-injected mice (GluR1: 0.5060.02, p,0.001; GluR2: 0.4160.01, p,0.001; GluR3: 0.3460.01, p,0.001; GluR4: 0.7360.02, p,0.01; GluR5: 0.7660.02, p,0.001; GluR6: 0.8860.02, p,0.01; KA2: 0.4860.01, p,0.001; NR1: 0.6260.02, p,0.05; NR2a: 0.4360.01, p,0.001; NR2b: 0.3060.01, p,0.001; NR3a: 0.2760.02, p,0.001; NR3b: 0.4160.05, p,0.01) (Fig. 5A–I and Fig. 6A–G). In the cortex, no changes were observed when we compared vehicle-injected and celecoxib treated mice (data not shown). KA injection affected the expression of several AMPAR, KAR and NMDAR subunits in both celecoxib-untreated (GluR3: 0.7860.05, p,0.001; KA1: 0.7060.03, p,0.001; KA2: 0.8060.03, p,0.001; NR1: 0.7060.03, p,0.001; NR2a: 0.7460.01, p,0.001; NR2b: 0.8460.04, p,0.05; NR2c: Glutamate Receptors Editing 0.7560.06, p,0.01; NR2d: 0.8360.05, p,0.05) and celecoxib- pretreated mice (GluR4: 0.6160.02, p,0.001; GluR5: 0.8660.03, p,0.05; GluR7: 0.7260.06, p,0.05; KA1: 0.6660.01, p,0.001; KA2: 0.6560.03, p,0.001; NR1: 0.4560.03, p,0.001; NR2a: 0.4760.06, p,0.001; NR2b: 0.6160.03, p,0.001; NR2c: 0.2960.01, p,0.001; NR2d: 0.3760.01, p,0.001; NR3b: 0.4960.12, p,0.05) when com- pared with vehicle-injected WT mice (Fig. 7A–I and Fig. 8A–G). Although the effect was less robust than the one observed in the hippocampus, after KA injection celecoxib-pretreated mice showed reduced cortical mRNA expression of several iGluRs compared with celecoxib-untreated mice (GluR4: 0.6760.03, p,0.001; GluR7: 0.6960.06, p,0.05; KA2: 0.8160.03, p,0.01; NR1: 0.6460.04, p,0.001; NR2a: 0.6360.08, p,0.01; NR2b: 0.7360.04, p,0.01; NR2c: 0.3860.001, p,0.001; NR2d: 0.4260.02, p,0.001). Celecoxib pretreatement increased the expression of GluR3 (1.260.03, p,0.05) in response to KA injection (Fig. 7A–I and Fig. 8A–G). Pattern of in vivo mRNA editing levels of GluRs after KA treatment Since the function of AMPA glutamatergic receptors is modulated by the mRNA editing of the different subunits, we analyzed editing levels of GluR2 Q/R and R/G sites, GluR3 and GluR4 R/G sites, GluR5 Q/R site and GluR6 I/V, Y/C and Q/ R sites in the hippocampus and cortex. The AMPA R/G sites were analyzed in combination with the AMPA splicing variants called flip and flop [24]. In all mice tested the editing level of GluR2 Q/R site was virtually 100%, with no variations due to either KA injection or genotype (data not shown). In hippocampus and cortex, no changes were detected in the R/G site, flip- and flop-isoforms, of GluR2 in either KA- injected WT mice or in vehicle-injected COX-22/2 mice when compared with vehicle-injected WT mice (Table S1, Supplementary data). In the hippocampus, the GluR3 R/G site for the flip isoform showed a small but significant increase after KA injection in WT mice (8.4%, p,0.001) and vehicle-injected COX-22/2 mice (8.4%, p,0.001) when compared to vehicle-injected WT mice (Fig. 9A). In the cortex, we found a decrease (216.4%, p,0.001) in the GluR3 R/G site for the flip variant in vehicle-injected COX-22/2 mice when compared with vehicle-injected WT mice (Fig. 10A). Similarly, in the hippocampus, GluR4 R/G site, flip variant, showed an increase after KA injection in both WT (8%, p,0.05) and COX-22/2 mice (8%, p,0.05) when compared with vehicle- injected WT mice (Fig. 9B). No changes were observed in the editing levels of GluR3 (Fig. 9A and Fig. 10A) and GluR4 R/G site (Fig. 9B and Table S1), flop-isoform, after KA injection or between genotypes in the hippocampus or cortex. No changes by genotype or KA-treatment were found in the editing level of kainate GluR5 Q/R site in the hippocampus or in the cortex (Table S1). GluR6 Y/C and Q/R sites were not altered by KA treatment either in the hippocampus (Table S1) or cerebral cortex (Fig. 10B). However, there was a small but statistically significant increase in the I/V site in the cortex of KA-injected WT mice (p,0.001) and vehicle-injected COX-22/2 mice (p,0.001) when compared with vehicle-injected WT mice (Fig. 10B). Furthermore, we analyzed editing levels of the different subunits in mice pretreated for two weeks with celecoxib. No changes due to KA-treatment or celecoxib were detected either in the hippocampus or cortex (Table S2, Supplementary data). PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19398