regulation through the Jak/STAT (janus kinase/signal transducers and activators of transcription) pathway, and the transcription factor SOCS3 (suppressor of cytokine signaling-3). An alternative signaling pathway involves cleavage of the full-length membrane- bound IL-6R to produce the soluble sIL-6R, which binds to IL-6 and can then activate gp130 signaling directly, even in cells lacking membrane IL-6 receptors(reviewed in [2]). Our understanding of the biology of IL-6 in the nervous system is more limited. Brain expression of IL-6 has been shown to mediate the sickness behavior, a syndrome of neurocognitive changes in response to acute infection which includes lethargy, confusion, and cognitive deficits[12]. Emerging evidence, however, suggests a more complex role for brain IL-6 in CNS function, including modulation of glutamatergic neurotransmission[13,14]. We have recently reported in a mouse model of schizophrenia that administration of the dissociative anesthetic and drug-of- abuse, ketamine, to young mice increased brain expression of IL-6 and NADPH oxidase (Nox2)[15]. Nox2, originally described as the neutrophil respiratory burst oxidase, generates the reactive oxygen species (ROS), superoxide, through one-electron reduction of oxygen by NADPH. Recent studies on genetically modified mice suggest that elevated superoxide in brain may be a critical mediator of age-related cognitive deficits[16]. Overexpression of the cytosolic superoxide dismutase (SOD1), or the extracellular SOD (EC-SOD), but not the mitochondrial MnSOD (SOD2), reverses age-associated impairment of long-term potentiation (LTP), and prevents loss of spatial memory[16]. In addition, aged gp91phox2/2 mice, which are deficient in Nox2, have better cognitive performance on memory tasks than age-matched wild- type controls[17]. It is plausible, therefore, that a cytosolic and/or extracellular source of superoxide production such as Nox2 might be an important mediator of age-related cognitive deficits. Nox2 activation in neurons can result in dysfunction of the fast- spiking, parvalbumin (PV)-expressing subpopulation of inhibitory GABAergic interneurons[18], required for coordinated firing of excitatory neurons in cortex and hippocampus, and known to be important for the encoding and retrieval of hippocampal- dependent memory[19,20], as well as for the generation of spatial and temporal cues. These interneurons are also involved in attentional-shift abilities, a function attributed to the pre-frontal cortex[21]. Age-associated loss of cortical and hippocampal GABAergic function has been reported for rats, primates, humans, and dogs[22–25], and is believed to contribute to aging-related cognitive deficits. However, the processes underlying these age- related changes in GABAergic inhibitory systems are not clear. In the present study, we wished to determine whether IL-6 is a mediator of age-related changes in brain, and if so the underlying mechanisms that are regulated by IL-6. Results Aging increases IL-6 in plasma and brain Multiple clinical studies have found that plasma IL-6 levels are significantly elevated in older humans[3], with nearly undetectable levels in young adults and levels generally .5 pg/ml in older subjects. To determine whether similar changes occurred in old C57BL6 mice, plasma IL-6 concentrations were measured in randomly-selected healthy old (24 mo) and young mice (4 mo) by ELISA. Levels in old mice were nearly 15-fold higher than young (Fig. 1a). To analyze whether increased plasma IL-6 levels would translate to elevation of the interleukin in brain, we measured brain IL-6 mRNA by RT-qPCR (Fig. 1b) and found increased levels in old C57BL6 mice, as previously found for comparably aged Balb/c mice[26]. IL-6 and Cognitive Changes Increased Nox2 protein expression in brain of aged animals is attenuated in IL-6-/- mice We have previously determined that Nox2 is the main source of neuronal superoxide production in a mouse model of schizophre- nia[18]. Moreover, we recently demonstrated that IL-6 can directly induce and activate Nox2 protein expression in cultured neurons[15], but in that study in young mice, acute peripheral injection of IL-6 failed to induce brain Nox2 expression. In the context of chronically elevated peripheral IL-6 in aged animals, on the other hand, we wished to determine whether aging was associated with an increase in Nox2 expression in brain, by analyzing Nox2 protein expression in young (4 month old) and old (24 month old) mouse brain. Confocal images of double- immunostaining for Nox2 and the neuronal marker MAP2 in the CA1 region of the hippocampus demonstrated significantly higher protein expression in old animals (Fig. 1c). There were rare GFAP-positive astrocytes which also expressed Nox2. We also found almost no Nox2-expressing cells that also were positive for CD11b (activated microglia). Thus, more than 95% or all Nox2- expressing cells in aged mice were Map2-postive neurons. The specificity of the antibody was assessed by staining of equivalent brain regions from Nox2-deficient (gp91phox-/-) animals (Fig. 1c bottom panels). To quantify the age-related increase in Nox2 expression, and assess whether IL-6 was involved in this process, we determined levels of Nox2 protein in young and old wild-type (C57BL/6) and IL-6-deficient (IL-6-/-) animals. A substantial increase in Nox2 protein was observed in extracts prepared from old wild-type mice, which was absent in similar age IL-6-deficient animals (Fig. 1d, e). Age-related increased superoxide production in brain in vivo is attenuated by inhibition of NADPH oxidase or by treatment with a superoxide dismutase mimetic, and is emulated by increased levels of plasma IL-6 To confirm that elevation of brain IL-6 and induction of Nox2 described above led to increased Nox2-dependent superoxide production in vivo, we assessed superoxide levels using confocal imaging of dihydroethidium (DHE) oxidation to its fluorescent product (ox-DHE) in fixed brain slices prepared from DHE- injected young mice, and from DHE-injected old mice that had been treated for 7 days prior to DHE with either no drug, or with the NADPH oxidase inhibitor, apocynin, in their drinking water. Fluorescence from oxidized DHE in neurons from cortex and hippocampus was quantified as previously described[18,27]. Increased neuronal superoxide production observed in old animals was significantly attenuated by apocynin treatment (Fig. 2a, b), strongly supporting the role of Nox2 in this increase. In addition, the requirement for IL-6 in this increase was confirmed by showing that old IL-6-/- mice have significantly lower superoxide levels compared to old wild-type mice (Fig. S1). We also determined that systemic administration of the SOD-mimetic, C3, for three weeks was sufficient to reduce ongoing superoxide production in aged wild-type animals (Fig. 2a and b). DHE oxidation was also evaluated in live mice using a GE eXplore Optix whole animal fluorescence scanner, which allows visualiza- tion of DHE oxidation in the intact animal. This technique also demonstrated increased superoxide production in old versus young mice, as well as in mice injected directly with IL-6 (Fig. 2c). We next determined that increasing plasma IL-6 levels by direct peripheral injection of IL-6 enhances Nox2 expression and superoxide production in brain. Repeated intraperitoneal injection of IL-6 (5 mg/kg i.p. every 12 hours times 3) in mice significantly increased expression of Nox2 protein in brain (Fig. 2d, top) and PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5518