Figure 5. Forced-swim test indicates gp91phox_/Y mice are not depressed following IH. gp91phox_/Y exposed to IH show less immobility as compared to C57BL6/J mice exposed to IH. *P,0.05. See text for more details. doi:10.1371/journal.pone.0019847.g005 Discussion OSA is a clinical condition that is now recognized as having important neurobehavioral consequences that stem, at least in part, from pathological inflammatory processes that are associated with the disease [51–53]. During sleep, patients with OSA undergo repeated periods of IH, which essentially correspond to recurring episodes of hypoxia and reoxygenation, leading to increased levels of ROS that are considered as contributing to end- organ injury, including the CNS [54–57]. In the present study, we provide conclusive results suggesting that increased activity of NADPH oxidase-mediated oxidative stress pathways mediates, at least in part the effects of IH on cognitive and behavioral functions. Reactive oxygen species (ROS) can be generated from various subcellular compartments, including mitochondria, the cellular membrane, lysosomes, peroxisomes, and the endoplasmic reticulum [24,58–61]. For example, NADPH oxidase [24,62,63], xanthine oxidase [63], phospholipase A2 [64], lipoxygenases and cyclooxygenase [58], and cytochrome P450 [65] have all been identified as sources of ROS in various subcellular compartments under both physiological and pathological conditions. Although NADPH oxidase is primarily expressed in phagocytic cells, increasing evidence suggests that various subunits of NADPH oxidase are also expressed in nonphagocytic cells such as sympathetic ganglion neurons and cortical neurons [66–68], thereby supporting the conceptual framework that neurons in general express NADPH oxidase [69]. In this context, NADPH oxidase has been implicated in conditions that remotely resemble hypoxia-reoxygenation [70–76]. Activation of NADPH oxidase leads to generation of the superoxide ion (O22), a ROS which can be converted to the highly reactive hydroxyl radical and to peroxynitrite, a highly damaging RNS [77,78]. To date, five NADPH oxidase enzyme (NOX) isoforms have been identified (NOX 1–5), and localization studies have shown that of the five NOX enzymes, the NOX2 and NOX4 isoforms are highly localized in the hippocampus CA1 and cerebral cortex [79,80]. Our study provides insight into the functional role of NADPH oxidase activation and its contribution to intermittent hypoxia NADPH Oxidase and Intermittent Hypoxia induced cognitive deficits. Our studies in the Morris Water Maze revealed preservation of spatial learning and memory in gp91phox_/Y mice as compared to the wild type mice after expo- sures to intermittent hypoxia. Furthermore, swim speed was not different between the groups, demonstrating that the differences were not due to differences in locomotor ability or coordination. The Morris Water Maze is a hippocampal-dependent test of learning and memory, and thus the enhanced performance on the Morris Water Maze of the gp91phox_/Y mice after IH, but not in normoxic conditions, is likely reflective of the attenuation of oxidative stress and enhanced neuronal survival in the hippocampus. As would be anticipated from such considerations, we indeed found that IH exposures markedly increased the expression and activity of NADPH oxidase in wild type mice, thereby confirming the assumption that conditions mimicking the oxygenation patterns of sleep apnea induce activation of NADPH oxidase in cortical brain regions. While we can not infer from our current findings on the cellular source of NADPH oxidase contribution to IH-induced cognitive and behavioral deficits, the near complete abrogation of such deficits in the gp91phox_/Y mice clearly and conclusively assigns a critical role for NADPH oxidase in this context. Moreover, NADPH oxidase inhibition is neuroprotective of hippocampus CA1 pyramidal cells 7 days after ischemia [81– 83]. Of note, we have previously shown that the IH-induced cognitive and behavioral deficits are attenuated by reductions in oxidative stress and inflammatory signaling cascades through pharmacological interventions [9,10], as well as through attenu- ation of oxidative stress via targeted genetic manipulations of manganese superoxide dismutase, platelet-activating factor recep- tor, or nitric oxide synthase [16,18,19]. Depressive and anxiety symptoms are frequent in OSA patients [40,84,85]. Moreover, depression may account for the fatigue seen in OSA patients, even after OSA severity has been controlled [38]. The prefrontal cortex is particularly vulnerable to sleep disruption, and hypoxemia further creates an unfavorable cellular environ- ment for the restorative processes to occur [2]. The elevated plus- maze is the most frequently utilized animal model for assessing anxiety-like behaviors [86,87] since it enables researchers to observe the conflict between two innate rodent behaviors, namely the avoidance of open space exposure as countering the tendency to explore novel environments [87]. Our results show that intermittent hypoxia modified anxiety-like behavior in wild type mice. In contrast, gp91phox_/Y exposed to intermittent hypoxia showed preserved performances in this test, suggesting that regions underlying these behavioral patterns are susceptible to IH, most likely via the oxidant stress mediated by activation of NADPH oxidase. Our findings are not surprising considering the previous reports on the palliative effect of apocynin, a putative NADPH oxidase antagonist, on neuronal viability in the context of intermittent hypoxia [88]. Furthermore, Veasey and collaborators further expanded on the critical role played by NADPH oxidase in the injury to locus coeruleus neurons and the excessive sleepiness that developed as a consequence of IH during sleep [89,90]. The cumulative evidence supports the assumption that the structural and neurobehavioral consequences of IH exposures in adult rodents involve a number of interrelated pathways, namely glutamate excitoxicity, oxidative stress, mitochondrial dysfunction, up-regulation of pro-inflammatory mediators, and altered regula- tion of pro- and anti-apoptotic gene cascades [91–93]. However, the explicit mechanistic involvement of NADPH oxidase in the CNS end-organ injury was not thoroughly explored. We propose that activation of NADPH oxidase by IH would be lead to the observed increases in oxidative stress markers. Earlier findings from our laboratory showed that intermittent hypoxia increases PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19847