properties are largely determined by subunit composition of the tetrameric receptor, assembled from GluR1–4 AMPA, GluR5–6 KA1 and KA2, NR1 with NR2a–d or NR3a–b, receptor subunits. Molecular diversity of AMPA/KA and NMDA receptors under physiological or pathological conditions is generated by differential spatial-temporal patterns of GluR expression, by alternative RNA splicing and editing and by targeting and trafficking of receptor subunits at dendritic spines [22]. In particular, RNA editing is an important post-transcriptional event in gene modification that alters one or more translation codons, thereby giving rise to functionally distinct proteins from a single gene [23]. The editing positions have been named on the basis of amino acid substitution, such as the Q/R site in AMPA GluR2 and KA GluR5 and GluR6 [23], the R/G site in GluR2, GluR3 and GluR4 [24] and the I/V and Y/C site in GluR6 [25]. These amino acid changes lead to modification of the channel properties: Q/R site profoundly influences the ionic properties of AMPA and KA receptor [26,27] and it may determine the maturation and cellular trafficking of GluR2 [28,29], whereas the R/G site influences the kinetic aspect of channel gating [24,30]. The GluR6 I/V and Y/C editing sites may be involved, together with the Q/R site, in a finer regulation of ion permeability [25]. Unlike GluR2 Q/R site, which is always fully edited, the editing levels of the other sites are developmentally [31] and regionally regulated [32,33]. It is well known that excess release of excitatory neurotransmit- ters, such as glutamate and KA, is an important underlying cause of neuronal damage in cerebral ischemia, epilepsy, Parkinson’s disease, and Alzheimer’s disease [34,35,36]. This type of excita- tion-induced neuronal damage is frequently accompanied by excess calcium influx and followed by generation of reactive oxygen and nitrogen species, which cause damage to intracellular membranes and trigger apoptotic pathways leading to delayed cell death [21]. The aim of this study was to investigate mRNA editing regulation of AMPA/KA receptor subunits and mRNA expression of all ionotropic glutamate receptor subunits (AMPA/KA and NMDA) in the cortex and hippocampus of COX-22/2 and wild type (WT, COX-2+/+) mice after KA exposure, and to determine whether altered editing or expression of these receptors underlies the increased susceptibility of COX-22/2 mice to KA-induced seizure intensity and neuronal damage. Furthermore, we showed that pretreatment with celecoxib for 2 weeks recapitulated the effects on gene expression of some AMPA, KA and most of the NMDA receptor subunits observed in COX-22/2 mice, after KA treatment. We also investigated the neuroinflammatory response of COX-22/2 and WT mice to systemic KA injection by measuring the expression of inflammatory markers in the brain. We demonstrate that COX-22/2 mice show an increase in the gene expression of microglia (CD11b) and astrocyte (GFAP) markers, pro-inflammatory cytokines (TNF-a, IL-1b and IL-6), and inducible nitric oxide syntase (iNOS). Materials and Methods Ethics Statement All animal experiments were performed under an animal protocol (NICHD #08-026) approved by the National Institutes of Health (NIH), National Institute of Child Health and Disease (NICHD) Animal Care and Use Committee, in accordance with the NIH guidelines on the care and use of laboratory animals. Animal housing Six week-old male COX-22/2 and WT mice on a C57Bl/6- 129/Ola background were received from a private NIEHS colony Glutamate Receptors Editing maintained by Taconic Farms (Germantown, NY) separately from their commercially available colony [37,38]. All mice used in this study were progeny derived from heterozygous by heterozygous matings and therefore all contained the same strain and genetic background. Mice were maintained on a 12 h light/dark cycle with free access to food and water intake. For celecoxib pretreatment, WT mice were given free access for two weeks to a diet containing 6000 ppm celecoxib. Celecoxib (CelebrexTM) capsules (400 mg; Pfizer Inc., New York, NY) were obtained from the NIH Division of Veterinary Medicine and were incorporated into feed by Research Diets, Inc. (New Brunswick, NJ) [6]. Kainate injection KA injection was performed as previously described [6]. Briefly, 12–14-week-old male mice were injected intraperitoneally (i.p.) with 10 mg/kg KA (Biomol International, Plymouth Meeting, PA; 2 mg/ml in 0.9% saline) or vehicle (0.9% saline). This dose of KA caused seizures but did not result in fatalities in WT mice [6]. Mice were euthanized after 24 h after KA injection and brains used for molecular analysis were rapidly dissected, frozen in 2-methylbu- tane at 250uC, and stored at 280uC until use. Gene expression Fresh frozen mouse hippocampus and cerebral cortex were processed for RNA extraction using the Qiagen RNeasy Lipid Tissue Mini kit (Qiagen, Valencia, CA), as directed by the manufacturer. RNA quantification and quality control were done using both spectrophotometric analysis and the AGILENT Bioanalyzer 2100 lab-on-a-chip technologies. Retro-Transcription (RT) was done using the Moloney murine leukemia virus-reverse transcriptase (MMLV-RT) (Invitrogen). 2.5 mg of total RNA were mixed with 2.2 ml of 0.2 ng/ml random hexamer (Invitrogen), 10 ml of 56 buffer (Invitrogen), 10 ml of 2 mM dNTPs, 1 ml of 1 mM DTT (Invitrogen), 0.4 ml of 33 U/ml RNasin (Promega), 2 ml MMLV-RT (200 U/ml), in a final volume of 50 ml. The reaction mix was incubated at 37uC for 2 h and then the enzyme heat inactivated at 95u for 10 min. To perform the PCR reactions different amount of the RT product were mixed with 2.5 ml 106 buffer (Polymed), 0.7 ml of 1.5 mM MgCl2, 2.5 ml of 2 mM dNTP, 0.7 ml of each forward and reverse primer, 1.25 U of Taq polymerase in a final volume of 25 ml [39]. Quantitative real-time polymerase chain reaction (RT-PCR) was performed on selected genes AMPA GluR1–4 (Applied Biosystems TaqMan Gene Expression Assay id probes: GluR1 Mm00433- 753_m1; GluR2 Mm00442822_m1; GluR3 Mm00497506_m1; GluR4 Mm00444754_m1); KA GluR5–7, KA1 and KA2 (GluR5 Mm00446882_m1; GluR6 Mm00599860_m1; GluR7 Mm01179- 716_m1; KA1 Mm00615472_m1; KA2 Mm00433774_m1); NMDA NR1, NR2a–d and NR3a–b (NR1 Mm00433800_m1; NR2a Mm00433802_m1; NR2b Mm00433820_m1; NR2c Mm0- 0439180_m1; NR2d Mm00433822_m1; NR3a Mm01341723_m1; NR3b Mm00504568_m1); cytokine (TNFa: Mm00443258_m1; IL-1b: Mm00434228_m1; IL-6: Mm01210733_m1); glial fibrillary acid protein (GFAP: Mm01253033_m1); microglial marker (CD11b: Mm00434455_m1); transcription factor (NF-kB: Mm- 00501346_m1); microtubule associate protein 2 (Mtap2: Mm00- 485230_m1); Nitric oxide species (iNOS: Mm00440485_m1) and phosphoglicerate kinase 1 (pgk1: Mm01225301_m1) as reference genes. PCR reactions were performed using the Applied Biosystems 7500 system. Data were analyzed using the comparative threshold cycle (DD Ct) method [40]. Results were normalized with Pgk1 as the endogenous control, and expressed as fold difference from the vehicle-injected WT mice, as previously reported [41,42]. PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19398