remained fully edited after KA injection in both the brain areas analyzed, indicating that modifications in calcium permeability of AMPA receptors were not involved in KA action. In the hippocampus, we observed an increase in GluR3 and GluR4 R/G level after KA injection in both WT and COX-22/2 mice. Our data suggest that increased editing at the AMPA R/G site, by altering resensitization kinetics, could enhance the receptor response to glutamate, resulting in a synapse operating at an increased gain. The potentially higher frequency of post-synaptic receptor activation due to fast resensitization kinetics, might in this case lead to an increased Ca2+ concentration in the post-synaptic neuron. On the other hand, in the cortex, we found a decrease in the GluR3 R/G site editing, flip form, in vehicle-injected COX-22/2 compared with WT mice. Although no changes in editing levels were observed after KA injection, the observed decrease in GluR3 R/G site editing might cause a selective reduction of conductance for GluR3-containing receptors after KA. Regarding the editing levels of KA receptors, KA injection selectively affected only the GluR6 I/V site in the cortex, indicating that KA slightly decreased cation permeability of the GluR6-containing receptor channels. The observed increase in I/ V GluR6 levels might indicate an excitotoxin-induced decrease in the activity of voltage-gated glutamatergic channels of the KA subtype. It is possible that desensitization at KA-preferring receptors plays an important role in neuroprotection. 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 exposure, suggesting that the effects observed were not due to life-time compensatory changes that may exist in knockout mice. No alteration in the editing reaction was observed after celecoxib treatment, indicating that editing levels are extremely conserved and editing modifications may occur only after a longer treatment or in a situation of life-time inhibition of COX-2 activity as observed in COX-22/2 mice. Furthermore, we observed that COX-2 gene deletion enhanced the neuroinflammatory response to KA. KA increased mRNA expression of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6, iNOS, a marker of oxidative stress, GFAP, a marker for astrocytes, and CD11b, a marker for microglia. These data are consistent with previous reports from our and other groups showing that COX-2 gene deletion or inhibition increases the neuroinflammatory response to endotoxins [42]. iNOS may also contribute to microglia-mediated KA induced neurotoxicity by increasing the production of extracellular reactive oxygen and nitrogen species, which, in turn, stimulate microglial release of pro-inflammatory mediators that, like radical oxygen species, are toxic to neurons. References 1. Minghetti L (2007) Role of COX-2 in inflammatory and degenerative brain diseases. Subcell Biochem 42: 127–141. 2. Kaufmann WE, Worley PF, Pegg J, Bremer M, Isakson P (1996) COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex. Proc Natl Acad Sci U S A 93: 2317–2321. 3. Katori M, Majima M (2000) Cyclooxygenase-2: its rich diversity of roles and possible application of its selective inhibitors. Inflamm Res 49: 367–392. 4. Kaufmann WE, Andreasson KI, Isakson PC, Worley PF (1997) Cyclooxygenases and the central nervous system. Prostaglandins 54: 601–624. 5. Breder CD, Dewitt D, Kraig RP (1995) Characterization of inducible cyclooxygenase in rat brain. J Comp Neurol 355: 296–315. 6. Toscano CD, Ueda Y, Tomita YA, Vicini S, Bosetti F (2008) Altered GABAergic neurotransmission is associated with increased kainate-induced seizure in prostaglandin-endoperoxide synthase-2 deficient mice. Brain Res Bull 75: 598–609. Glutamate Receptors Editing MAP2 gene expression was increased in the hippocampus of COX-22/2 mice after KA. MAP2 exhibits microtubule-stabilizing activity and regulates the microtubule networks in dendrites, resulting in dendrite elongation [65]. Thus, as reported in surviving neurons of medial extended amygdala after status epilepticus [66], KA might induce a process of sprouting and reactive synaptogenesis. In conclusion, we have previously shown that Cox-22/2 mice are more susceptible to KA-induced seizures and neuronal damage [6]. Although some of the changes observed in this model may be the consequence of adaptive modifications due to deletion of the COX-2 gene, chronic administration of the Cox-2 selective inhibitor celecoxib recapitulated these findings [6]. While we cannot directly link changes in glutamate receptor editing to susceptibility to KA, we speculate that altered mRNA editing and expression of glutamate receptors in COX-22/2 mice could cause an imbalance in the interplay between AMPA and NMDA receptors and alterations in glutamatergic neurotransmission. These alterations might contribute, at least in part, to the increased susceptibility of COX-22/2 mice to KA-induced excitotoxicity [6]. Overall, our findings suggest a role of COX-2 in modulating the expression of glutamate receptors and, in turn, regulating glutamatergic neurotransmission. Future studies are warranted to elucidate the molecular mechanisms that underline the interplay between COX-2 gene and the glutamatergic system. Supporting Information Table S1 KA-induced editing of AMPA/KA glutamate receptor subunits in hippocampus and cortex of COX- 2+/+ and COX2/2 mice. Data are Means 6 SEM expressed as %editing level compared to vehicle-injected COX-2+/+ mice. Statistical analysis was performed with Student’s test. (PPTX) Table S2 KA-induced editing of AMPA/KA glutamate receptor subunits in hippocampus and cortex of wild type mice after pretreatment with celecoxib. Data are Means 6 SEM expressed as % editing level compared to vehicle- injected wild type mice. Statistical analysis was performed with Student’s test. (PPTX) Author Contributions Conceived and designed the experiments: LC FB SB. Performed the experiments: LC AB SP CDT CM. Analyzed the data: LC AB. Wrote the paper: LC FB AB SB. 7. Bleakman D, Lodge D (1998) Neuropharmacology of AMPA and kainate receptors. Neuropharmacology 37: 1187–1204. 8. Borbely S, Dobo E, Czege D, Molnar E, Bakos M, et al. (2009) Modification of ionotropic glutamate receptor-mediated processes in the rat hippocampus following repeated, brief seizures. Neuroscience 159: 358–368. 9. Fujikawa DG (2005) Prolonged seizures and cellular injury: understanding the connection. Epilepsy Behav 7 Suppl 3: S3–11. 10. Hou ST, MacManus JP (2002) Molecular mechanisms of cerebral ischemia- induced neuronal death. Int Rev Cytol 221: 93–148. 11. Molinuevo JL, Llado A, Rami L (2005) Memantine: targeting glutamate excitotoxicity in Alzheimer’s disease and other dementias. Am J Alzheimers Dis Other Demen 20: 77–85. 12. Rapoport SI, Bosetti F (2002) Do lithium and anticonvulsants target the brain arachidonic acid cascade in bipolar disorder? Arch Gen Psychiatry 59: 592– 596. PLoS ONE | www.plosone.org 18 May 2011 | Volume 6 | Issue 5 | e19398