and PFC-dependent cognitive processes [38]. Although PV-neuron loss was observed in all regions of hippocampus, as well as prefrontal cortex, the most pronounced decrease was in CA2-3. Both IL-6-/- mice and C3-treated mice showed preservation of PV-interneurons in CA3, and C3 rescued interneurons in CA1, as well. The CA3a,b region of hippocampus has been shown to be critical for detection of novelty[52], spatial learning[53], and for encoding of new spatial information into short-term memory[52,54–56]. Importantly, impaired spatial learning and memory is consistently reported across studies in older adults, and this is found even in the absence of a dementing illness[53,55]. While CA1 is generally believed to be necessary for encoding of temporal but not spatial information, it is believed to be involved retrieval of spatial information previously encoded by CA3[56,57]. Thus, the behavioral correlates of deficits in CA3 neural circuitry would be decreased novelty recognition and impaired spatial learning[58], and for CA1, decreased spatial working memory[56,57]; and both areas are believed necessary for effective performance on the probe component of the Morris water maze. To determine whether rescue of PV-interneurons was associated with improved cognitive performance, we carried out behavioral testing, and found that aged IL-6-/- mice, which had significant preservation of PV-neurons in CA3, had significantly greater recognition of three novel objects, and then showed greater habituation to compared to wild-type controls. Habituation to a novel environment tests simple learning[52,58] without the need for aversive stimuli, and has been shown to be impaired in animals with lesions to CA3. In addition, while aged wild-type mice showed impaired abilities in finding the escape chamber in the Barnes maze, IL-6-/- mice progressively found the escape chamber more quickly across trials. Thus, IL-6 deficiency appears to decrease learning deficits attributable to CA3 in aged mice. We further evaluated spatial learning and memory in aged mice treated long-term with a SOD mimetic. Mice treated chronically with C3 had significant rescue of PV-interneurons in both CA1 and CA3. Consistent with the importance of these regions to spatial memory, treated mice had significantly better performance on the probe component of the Morris water maze. Of note, a virtual Morris water maze for humans has been developed[59,60] and has revealed that older humans show a nearly identical decline in performance on the probe trial component[59,60]. Taken together, our data suggest that loss of PV-interneurons in the hippocampus could underlie age- related cognitive deficits in older humans. Furthermore, the data suggests that decreasing IL-6 induction of Nox2, or eliminating downstream superoxide production directly can preserve cognitive performance. Increased circulating IL-6 is a highly prevalent finding in older adults, with estimates based on the clinical literature that more than 80% of individuals over the age of 65 will have circulating levels of IL-6 greater than 5 pg/ml. This indicates that a sizeable and growing number of older individuals might be at risk for IL-6- mediated cognitive effects. In addition, our results showing that sustained elevation of peripheral IL-6 results in loss of PV- interneurons with aging provide several clinically-testable hypoth- eses. The PV-interneurons targeted by IL-6 represent the same interneuron population that is selectively vulnerable to injury from commonly-used general anesthetics, and this vulnerability is observed even at low doses of these agents. Thus, the use of general anesthetics in patients with chronic elevation of circulating IL-6 might be predicted to increase the risk of adverse cognitive outcomes after anesthesia. Because treatments which lower plasma IL-6 levels and/or block IL-6 signaling are currently in clinical use for other indications, these agents could be used to test idea that lowering IL-6 may preserve in the integrity of the PV-inhibitory system and prevent associated cognitive deficits. Clinical testing of IL-6 and Cognitive Changes the mechanisms presented here could further contribute to our broader understanding of the role of inflammatory signaling in both physiology and pathology the brain. Methods Materials Antibodies used were as follows: anti-parvalbumin rabbit polyclonal was from Swant (Bellizona, Switzerland), and anti- GAD67 monoclonal from Chemicon (Millipore). Polyclonal anti- Nox2 was from Santa Cruz Biotechnology Inc. (Santa Cruz, California), and monoclonal anti Nox2 was from BD Transduc- tion, or a generous gift of Mark Quinn (Montana State University). Unless otherwise stated, all reagents were from Sigma and tissue culture media from Invitrogen. The spin traps 5-(diisopropox- yphosphoryl)-5-methyl-1-pyrroline-N-oxide (DIPPMPO) and 5- (diethylphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) were from Alexis Biochemicals (San Diego, CA). The NFkB inhibitor, SN50, was from Calbiochem (San Diego, CA). Maintenance of mice, and administration of dihydroe- thidium (DHE), apocynin, IL-6, and the SOD mimetic, C3. Male C57BL/6, gp91phox(-/-) and IL-6(-/-) mice were obtained from Jackson Labsat 8 weeks of age and housed in our facility until 12– 15 weeks (young), or 22–26 months of age (old), when they were used for experiments. DHE was injected as described[27]. Briefly, two serial i.p. injections of freshly prepared dihydroethidium (27 mg/kg) are given at 30 minute intervals. Eighteen hours later, mice are anesthetized with inhaled halothane, and perfused intracardially with cold saline followed by 4% paraformaldehyde in PBS for immunohisto-chemistry experiments, or their brains dissected and forebrains processed for synaptosomal preparations (as described below). Apocynin (5 mg/kg/day) was administered in the drinking water for 7 days. IL-6 injections were 5 mg/kg i.p. on 2 consecutive days as described[15], except for in vivo imaging, in which an additional 5 mg/kg injection was given between the first and last injection. C3 was given in the drinking water at 1 mg/kg/day starting at 12 months of age as described[27] for cell counts, or for 21 days for DHE studies. All animal studies were approved by the Animal Care Program at the University of California, San Diego, and are in accordance the PHS Guide for the Care and Use of Laboratory Animals, USDA Regulations, and the AVMA Panel on Euthanasia. Synaptosomal preparations. Synaptosomes were prepared from adult mouse forebrain as described[18]. Cortical neuronal cultures were prepared form cortices of Swiss Webster E15 mouse embryos and grown on poly-lysine coated coverslips with paraffin ‘‘feet’’ for 21 days as previously described[18]. RT-PCR. RNA was extracted from mouse forebrain using Trizol. After reverse transcription of mRNA (1 mg), the cDNA produced was analyzed for IL-6, IL-1b, and TNFa expression using actin as an internal control. PCR was carried out by CFAR Genomics Core at UCSD. PCR conditions were the following 45 seconds at 95uC, 15 seconds at 45uC, and 1 minute at 72uC for 35 cycles. IL-6 primers: forward: 59-atggatgctaccaaactggat-39 and reverse: 59-tgaaggactctggctttgtct-39. IL-1b primers: 59-caaccaacaa- gtgatattctccatg-39 and reverse: 59-gatccacactctccagctgca-39. TNFa primers: 59-catcttctcaaaattcgagtgacaa-39 and reverse: 59-tgggagta- gacaaggtacaaccc-39. GAPDH primers: forward: 59-gaacatcatcc- ctgcctctactgg-39 and reverse: 59-tccaccaccctgttgctgta-39. Superoxide detection in synaptosomes by electron paramagnetic resonance (EPR) spectroscopy Detection of NADPH-dependent superoxide production in synaptosomal preparations was performed using spin-trapping EPR PLoS ONE | www.plosone.org 10 May 2009 | Volume 4 | Issue 5 | e5518