IL-6 and Cognitive Changes Figure 5. IL-6-induction of Nox2 and subsequent decrease of parvalbumin and GAD67 expression in PV-positive interneurons is NFkB-dependent. Nuclear extracts of brain samples obtained from wild-type or IL-6-deficient animals were analyzed for content of the NFkB p65 subunit by colorimetric quantification of p65 using a commercially available kit (TransAM). A high concentration of p65 was found in nuclear extracts obtained from old wild type mice (a), and this was greatly diminished in nuclear extracts obtained from similar-age IL-6 deficient mice. The specificity of the binding to wild type oligonucleotide was confirmed by competition with either sense (oligo) or mutated oligonucleotides (mutant oligo). ANOVA analysis yielded a significant effect of genotype (F(1,18) = 9.414, P,0.05). (b) Application of IL-6 to neurons in culture induced Nox2 expression (b, top panel) and DHE oxidation (b, bottom panel and c) that was blocked by the NFkB inhibitor SN50. Dissociated primary neuronal cultures were treated with IL-6 (10 ng/ml) for 24 h in the absence or presence of SN50 (5 mM), an inhibitor of NFkB nuclear translocation. Dihydroethidium (DHE, 1 mg/ml) was applied for the last hour as described[18]. After fixation, cells were immunostained for Nox2. * = significant with respect to control. # = significant with respect to IL-6, F(2,346) = 12.826, P,0.05). (d. e) SN50 also prevented the decrease in expression of parvalbumin and GAD67 induced by IL-6 exposure in PV-interneurons (*F(2,409) = 234.7, P,0.05; #F(2,409) = 192.8, P,0.05). doi:10.1371/journal.pone.0005518.g005 system we previously described as responding to ketamine with IL- 6-dependent induction and activation of Nox2[15]. Exposure of these cultures to IL-6 in the absence and presence of SN50 (5 mM), a cell permeable peptide which blocks NFkB nuclear transloca- tion[37], demonstrated that inhibiting NFkB-mediated transcrip- tional events completely prevented IL-6-mediated induction of Nox2 expression and superoxide production (Fig. 5b, c) and prevented the loss of GABAergic phenotype of PV-interneurons (Fig. 5d, e), as demonstrated by loss of PV and GAD67 immunoreactivity. The specificity of SN50 has previously been confirmed in these same cultures by EMSA using an inactive peptide, SN50M.[37] These results suggest that Nox2 activation/ induction is dependent on NFkB-mediated transcription in neurons. IL-6 deficiency, or reduction of neuronal superoxide production, ameliorates impaired spatial learning in normal aging mice The PV-interneuronal GABAergic system is responsible for the control of cortical output and the generation of gamma oscillations, involved in temporal-encoding and storage/recall of information required for the correct functioning of working memory processes[38]. To analyze the possible correlation between the number of PV-interneurons in the old brain and cognitive ability, we subjected old animals to a series of simple memory tasks and compared their performance to that of similar age animals deficient in IL-6 production. Short term spatial memory recognition was tested by analyzing habituation to the spatial distribution of objects in an open field (novel object exploration task, Fig. 6a). Three objects were used for this task: a monkey, a zebra and a horse, for which the mice showed equal preference. There were significant overall genotypic differences in contacting the objects (monkey: F(1,23) = 8.6, P,0.01; zebra: F(1,23) = 5.9, P,0.05; and horse: F(1,23) = 8.7, P,0.01) with IL-6- deficient animals making more overall contacts than wild-type mice. In all cases there were significant effects of trial, with decreases in contacts over trials. In addition, there were significant genotype6trial interactions in exploration of the monkey (F(2,46) = 15.1, P,0.0001), horse (F(2,46) = 5.8, P,0.01), and moderately for the zebra (F(2,48) = 3.1, P = 0.05). IL-6 deficient mice showed a greater degree of habituation to object exploration PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5518