IL-6 and Cognitive Changes Figure 3. Increased synaptosomal Nox-2 activation and superoxide production in old wild-type mice is absent in old IL-6-/- mice; determination by electrochemical oxygen consumption analysis and EPR spin-trapping spectroscopy. (a) NADPH-induced oxygen consumption is significantly decreased by the Nox inhibitor apocynin (300 mM, black bars). Oxygen consumption by synaptosomal Nox from the cortex of young (3–4 M, n = 9) and old (23–24 M, n = 9) animals at 37uC was induced by the addition of 5 mM NADPH to samples containing 2–5 mg synaptosomal protein. The observed Nox specific activity was significantly higher in IL-6 injected young animals (white bars, p = 0.04, n = 5) and in old brains (light gray bar, p = 0.01, n = 9) and was significantly lower in IL-6-/- old mice (dark gray bar, p = 0.04, n = 4). IL-6 treatment: Two IL-6 i.p. injections (5 mg/kg), separated by 12 hrs were administered to young and old animals before the groups were sacrificed 12 hrs later for synaptosomal isolation. (b) Representative EPR spectra recorded 3 minutes after mixing 5 mM NADPH with 0.2–0.5 mg synaptosomal protein isolated from brain of old mice at 37uC with 70 mM DEPMPO in the absence or in the presence of 200 mM AEBSF or 300 mM apocynin. In each incubation, 10 mM DETC was included to inhibit the SOD enzyme, resulting in enhanced and quantifiable EPR signals. Using computer simulations we concluded that the observed signals are attributable to a mixture of adducts of DEPMPO with hydroxyl radical and lipid derived carbon centered radical and both radicals are derived from superoxide since the signals were completely abolished in the presence of SOD; not shown. (c) Quantification of the observed EPR signals accumulated over the first 3 minutes from NADPH addition, n = 4–9 per group. Superoxide yield in synaptosomes isolated from different groups follows essentially the same activity pattern seen by oxygraphy and shown in (a). The asterisks (*) designate statistical significance by ANOVA followed by Tukey test with respect to young control while (#) indicate statistical significance with respect to old control. EPR settings are described fully in methods. Data are mean 6 SEM. (d) Nox-generated superoxide burst in synaptosomes isolated from IL-6-/- KO mice is significantly lower than that in their old control brains. doi:10.1371/journal.pone.0005518.g003 a hippocampal form of memory, shows significant impairment in both healthy humans and rodents as they age[33], and has been correlated with disruption of glutamatergic transmission in frontal cortex and hippocampus in mice and non-human primates [34,35]. We have recently reported that blockade of NMDA receptors by the drug-of-abuse, and dissociative anesthetic ketamine leads to increased Nox2 activity in brain, and is associated with damage to parvalbumin-positive (PV) GABAergic inhibitory interneurons[18]. The robust induction of Nox2 expression and activity we observed led us to ask whether PV inhibitory interneurons might also be selectively lost during aging, as well. We found a substantial and highly significant loss of PV- interneurons in aged hippocampus (Fig. 4), with the CA2-3 region being the most affected (20% decrease for CA1, main effect of age: F(1,17) = 34.4, P,0.05; 45% decrease for CA2-3: main effect of age: F(1,17) = 378.7, P,0.05; 33%, 17% decrease for DG, main effect of age: F(1,17) = 20.1, P,0.05) (Fig. 5b), as well as a 17% decrease in prelimbic cortex (Fig. S3, P = 0.007 by t-test). To determine whether old mice lacking IL-6, which had lower Nox2 expression and superoxide production in brain would have preservation of PV-interneurons during aging, we carried out analysis in young and old wild-type and IL-6-/- mice. IL-6 deficiency attenuated the age-related loss of PV-interneurons (Fig. 4c); two-way ANOVA analysis of the genotype6age interaction showed a statistically significant effect for area CA3 (F(1,23) = 23.9, P,0.001) and a strong trend for area CA1 (F(1,23) = 3.1, P = 0.09). Rescue of cells in the dentate gyrus was difficult to determine because the small number of PV-interneu- rons in this region. To assess whether a sustained reduction in brain superoxide levels would also preserve PV-interneurons, we analyzed brain slices from mice treated chronically with an SOD mimetic, and found that C3 treatment also prevented the age- related loss of PV-interneurons, as well (Fig. 4b; main effect of treatment for CA1: F(2,22) = 4.9, P = 0.035; main effect of PLoS ONE | www.plosone.org 5 May 2009 | Volume 4 | Issue 5 | e5518