In the present study, we examined this hypothesis in NADPH oxidase mutant mice (gp91phox-/Y) and wild type littermates by exposing them to IH, and assessed changes in hippocampus- dependent learning and memory tasks. In addition, we also tested other behavioral paradigms for anxiety and depression, since such problems are frequently encountered in patients with sleep apnea [36–41]. Materials and Methods Animals Male hemizygous gp91phox-/Y (B6.129S-Cybbtm1Din/J) mice (20– 22 grams) and C57BL/6J mice (20–22 grams) were purchased from Jackson Laboratories (Bar Harbor, Maine), housed in a 12 hr light/dark cycle (lights on from 7:00 am to 7:00 pm) at a constant temperature (2661uC). Mice were housed in groups of four in standard clear polycarbonate cages, and were allowed access to food and water ad libitum. All behavioral experiments were performed during the light period (between 9:00 am and 12:30 pm). Mice were randomly assigned to either IH or room air (RA) exposures. The experimental protocols were approved by the Institutional Animal Use and Care Committee and are in close agreement with the National Institutes of Health Guide in the Care and Use of. All efforts were made to minimize animal suffering and to reduce the number of animals used. Figure 1 summarizes the timeline of the treatments and the behavioral experiments. After each behavioral paradigm the mice were immediately returned back to their respective exposures. Intermittent Hypoxia Exposures Animals were maintained in 4 identical commercially-designed chambers (300x200x200; Oxycycler model A44XO, BioSpherix, Redfield, NY) operated under a 12 hour light-dark cycle (7:00 am–7:00 pm) for 14 days prior to behavioral testing. Oxygen concentration was continuously measured by an O2 analyzer, and was changed by a computerized system controlling gas outlets, as previously described [6,42,43], such as to generate oxyhemoglobin nadir values in the 65–72% range every 180 seconds. Ambient temperature was kept at 22–24uC. Behavioral Testing The Morris water maze was used to assess spatial reference learning and memory, as well as working memory. The maze protocol is similar to that described by Morris [44] with modifications for mice. The maze consisted of a white circular pool, 1.4 m in diameter and 0.6 m in height, filled to a level of 35 cm with water maintained at a temperature of 21uC (Morris 1984). Pool water was made opaque by addition of 150 ml of non- toxic white tempera paint. A Plexiglas escape platform (10 cm in diameter) was positioned 1 cm below the water surface and placed at various locations throughout the pool. Extramaze cues surrounding the maze were located at fixed locations, and visible to the mice while in the maze. Maze performance was recorded by a video camera suspended above the maze and interfaced with a video tracking system (HVS Imaging, Hampton, UK). NADPH Oxidase and Intermittent Hypoxia Briefly, a standard place-training reference memory task was conducted on mice in the water maze following exposure to 14 days of IH or RA. One day prior to place learning, mice were habituated to the water maze during a free swim. Place learning was then assessed over six consecutive days using a spaced training regimen that has been demonstrated to produce optimal learning in mice [45]. Each training session consisted of three trials separated by a 10 minute inter-trial interval (ITI). On a given daily session, each mouse was placed into the pool from 1 of 4 quasirandom start points (N, S, E or W) and allowed a maximum of 90 seconds to escape to the platform where the mice were allowed to stay for 15 sec. Mice that failed to escape were led to the platform. The position of the platform remained constant during the trials. 24 h following the final training session, the platform was removed for a probe trial to obtain measures of spatial bias. To assess the performance in the water maze, mean escape latencies and swim distance were analyzed. Reference memory. Retention tests were carried out 14 days after acquisition of the task. In the retention test, performance in a single session (two trials) was assessed, and the mean average performance of the two trials was calculated. Elevated plus maze (EPM). The elevated plus maze (EPM) was used to assess anxiety. The basic measure is the animal preference for dark, enclosed places over bright, exposed places [46]. A 60 w light was placed above the apparatus and the test was video taped by an overhead camera. Mice were placed in the center of the maze facing a closed arm, and allowed to explore for 10 min in isolation. Each mouse received one videotaped trial. Mice prefer to enter into closed arms compared to open arms. Time spent in the dark area is viewed as avoidance or anxiety-like behavior. The following parameters were scored: (a) Percent time spent in open and closed arms; (b) number of entries to closed arms; (c) Time spent in the center. An arm entry was defined as the entry of all four feet into either one of the closed arm. Of note, the maze was cleaned with 30% ethanol between trials to remove any odor cues. Forced swimming test (FST). Briefly, mice were indivi- dually forced to swim in an open cylindrical container (diameter 14 cm, height 20 cm), with a depth of 15 cm of water at 2561 uC. The immobility time, defined as the absence of escape-oriented behaviors, was scored during 6 min, as previously described [47,48,49]. Each mouse was judged to be immobile when it ceased struggling, and remained floating motionless in the water, making only those movements necessary to keep its head above water. The average percentage immobility was calculated by a blinded experimenter. NADPH Oxidase Expression. qRT-PCR analysis of p47phox was performed using ABI PRISM 7500 System (Applied Biosystems, Foster City, CA). RNA from frontal cortex in IH and RA exposed mice was prepared with TRIZOL. cDNA synthesis was performed using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Ribosomal 18S rRNA was used as a reference gene to normalize the expression ratios for the gene of interest. Primer sequences were 59-CAGCCA- GCACTATGTGTACA-39 and 59-GAACTCGTAGATCTCG- GTGAA-39 for p47phox (91 bp). One microgram of total RNA Figure 1. Schematic diagram on the sequence of behavioral experiments and exposures to either IH or RA in both wild type and NADPH oxidase knock-out mice. doi:10.1371/journal.pone.0019847.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19847