Quantification of editing levels The editing level quantification for AMPA GluR2, GluR3 and GluR4 and KA GluR5 and GluR6 transcripts was done by RT- PCR and sequence analysis [33]. Briefly, following amplification of the region containing the editing site, a pool of GluR cDNA was obtained in which both the edited and unedited mRNA forms were co-expressed. The PCR products were sequenced and the edited nucleotide appeared as overlapping A/G peaks: A from unedited transcripts and G from the edited ones [33]. The percentage of edited mRNA molecules in a pool of specific GluR mRNAs can be determined by calculating the peak area of the edited nucleotide (G) versus the sum of G and A peak areas using the software DS gene that permit to analyze the area of the peak. We previously determined that the editing level can be reliably calculated as a function of the ratio between the G peak area and A plus G peaks areas [33]. The nucleotide areas were quantified by the Discovery Studio Gene 1.5 program (Accelrys Inc., San Diego, CA, USA) [43]. The mean values and standard errors from each group of animals were used for statistical analysis. Statistical analysis Editing and expression data were obtained by amplifying the mRNA from each animal. The mean values and standard errors obtained from each group were reported. Editing data were analyzed with Student’s t-test. For RT- PCR results, a two-way ANOVA was performed on the log-transformed DD Ct. Bonferroni’s post-hoc test was used for further comparisons. p values,0.05 were considered statistically significant. Data ob- tained in celecoxib-treated animals were analyzed using a one-way ANOVA, followed by a Bonferroni’s post-hoc test. Results mRNA expression of AMPA and KA glutamate receptor subunits is decreased in COX-22/2 mice after KA injection Using qRT-PCR, we examined the relative changes in the mRNA expression pattern of the AMPA (GluR1–4) and KA (GluR5–7, KA1 and KA2) glutamate receptors subunits in the hippocampus and cortex of vehicle-injected COX-22/2 mice (n = 9) compared with vehicle-injected WT mice (n = 9). Further- more, we analyzed the expression levels of the receptor subunits of KA-injected WT (n = 9) and COX-22/2 mice (n = 9) compared with their respective controls. Also we analyzed the mRNA expression of KA-injected COX-22/2 mice compared with KA- injected WT mice. In the hippocampus (Fig. 1A–I) of vehicle-injected COX-22/2 mice the mRNA expression of GluR2, GluR3 and GluR6 was significantly increased (GluR2: 1.2260.06, p,0.001; GluR3: 1.2660.04, p,0.001; GluR6: 1.3960.05, p,0.001), as compared with vehicle-injected WT mice (Fig. 1B, C; Fig. 1F). These data indicate that COX-2 deletion alters the transcription rate of glutamate receptor genes. KA injection affected the expression pattern of several AMPA and KA receptor subunits both in WT and COX-22/2 mice. Specifically, the mRNA expression of all GluR subunits (except GluR6) from KA-injected WT mice showed a decrease of about 20–50% when compared with vehicle-injected WT mice (Mean 6 SEM; GluR1: 0.7560.04, p,0.001; GluR2: 0.7660.04, p,0.001; GluR3: 0.7960.04, p,0.001; GluR4: 0.7860.05, p,0.01; GluR5: 0.8060.02, p,0.001; GluR7: 0.6860.04, p,0.001; KA1: 0.5060.02, p,0.001; KA2: 0.7360.03, p,0.001) (Fig. 1A–I). Glutamate Receptors Editing Furthermore, KA-injected COX-22/2 mice showed a signifi- cant decrease in the mRNA expression of all AMPA and KA subunits (except GluR7 and KA1) (GluR1: 0.6260.04, p,0.001: GluR2: 0.5660.02, p,0.001; GluR3: 0.5760.02, p,0.001; GluR4: 0.6660.03, p,0.01; GluR5: 0.6760.07, p,0.05; GluR6: 0.7560.02, p,0.001; KA2: 0.860.06, p,0.01), as compared with vehicle-injected COX-22/2 mice (Fig. 1A–I). Small but significant changes of about 20–30% were observed for the GluR1, GluR5 and KA2 subunits (GluR1: 0.8260.06, p,0.01: GluR5: 0.7260.05, p,0.001; KA2: 0.8060.04, p,0.05) between KA- injected COX-22/2 mice and KA-injected WT mice (Fig. 1A, 1E–1I). In the cortex, similarly to the changes observed in the hippocampus, we found an increase in the expression of GluR2, GluR3, GluR6 and GluR7 (GluR2: 1.4260.09, p,0.001; GluR3: 1.2860.02, p,0.001; GluR6: 1.4460.10, p,0.001; GluR7: 1.4060.06, p,0.001) in vehicle-injected COX-22/2 mice com- pared with WT mice (Fig. 2B–C, 2F–G). Furthermore, the mRNA expression of GluR3 and KA1 subunits was decreased in KA-injected WT mice compared to vehicle-injected WT mice (GluR3: 0.7860.05, p,0.001; KA1: 0.7260.03, p,0.01) (Fig. 2C, 2I). Moreover, a down-regulation in the mRNA expression of AMPA GluR2–3 and KA GluR6–7 was observed in KA-injected COX-22/2 mice compared with vehicle- injected COX-22/2 mice (GluR2: 0.7860.02, p,0.001; GluR3: 0.8960.04, p,0.05; GluR6: 0.7460.02, p,0.05; GluR7: 0.8760.02, p,0.05) (Fig. 2B–C, 2F, 2H). mRNA expression of NMDA glutamate receptor subunits is decreased in COX-22/2 mice after KA injection Next, we examined the relative changes in the mRNA expression pattern of the NMDA (NR1, NR2a–d and NR3a–b) glutamate receptors subunits in the hippocampus and cortex of vehicle-injected COX-22/2 mice (n = 9) compared with vehicle- injected WT mice (n = 9). Then, we analyzed the expression levels of the same receptor subunits of KA-injected WT (n = 9) and COX- 22/2 mice (n = 9) compared with their respectively controls. Also we analyzed the mRNA expression of KA-injected COX-22/2 mice compared with KA-injected WT mice. In the hippocampus of vehicle-injected COX-22/2 mice the mRNA expression of all NMDA receptor subunits, except for NR2b, was significantly decreased as compared with vehicle- injected WT mice (NR1: 0.6260.02, p,0.001; NR2a: 0.4860.01, p,0.001; NR2c: 0.5760.02, p,0.001; NR2d: 0.6060.02, p,0.001; NR3a: 0.6760.01, p,0.001; NR3b: 0.4660.12, p,0.05) (Fig. 3A–G). Furthermore, the mRNA expression of all NMDA subunits (except NR2b) from KA-injected WT mice were decreased when compared with vehicle-injected WT mice (NR1: 0.5960.04, p,0.001; NR2a: 0.5560.05, p,0.001; NR2c: 0.5560.04, p,0.001; NR2d: 0.5660.04, p,0.001; NR3a: 0.6860.03, p,0.001; NR3b: 0.5060.09, p,0.05) (Fig. 3A–G). Also, KA-injected COX-22/2 mice showed a significant decrease in the mRNA expression of all NR subunits, except NR1, NR2a and NR3a, as compared with vehicle-injected COX-22/2 mice (NR2b: 0.7160.02, p,0.001: NR2c: 0.4860.1, p,0.001; NR2d: 0.5960.07, p,0.01; NR3a: 0.6160.05, p,0.001) (Fig. 3A–G). A decrease of about 30–50% was observed in the expression of all NR subunits, except NR1 and NR3b, between KA-injected COX- 22/2 and KA-injected WT mice (NR2a: 0.6360.03, p,0.01; NR2b: 0.7060.02, p,0.001; NR2c: 0.4960.14, p,0.001; NR2d: 0.6060.07, p,0.01; NR3a: 0.6060.05, p,0.001) (Fig. 3A–G). In the cortex, the expression of NR1, NR2a, NR2c and NR2d subunits was decreased when we compared vehicle-injected COX-22/2 (NR1: 0.7260.06, p,0.001; NR2a: 0.6760.02, PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19398