spectroscopy as described[15,18]. In brief, synaptosomes (,0.1– 0.2 mg synaptosomal protein) were mixed with 70 mM DEPMPO (5-(diethylphosphoryl)-5-methyl-1-pyrroline-N-oxide) and appropri- ate combinations of substrates/inhibitors, and loaded into a 50 ml- glass capillary, which is inserted into the EPR cavity of a MiniScope MS200 Benchtop spectrometer, maintained at 37uC. Previous control experiments have confirmed that the EPR signals are substrate specific, acquired only in the presence of synaptosomes, and not due to redox cycling of reagents used[18]. EPR signals accumulated during the first 6 minutes after addition of NADPH were measured. To be able to detect all superoxide generated, the SOD inhibitor diethyldithiocarbamic acid (DETC; 5 mM) was included in the assay mixture. When analyzing the effects of direct addition of IL-6 on superoxide production in synaptosomes, IL-6 (100 ng/ml, final) was added to synaptosomes 5 minutes before Nox oxidase activity was initiated by addition of substrate (NADPH,5 mM final). EPR conditions were: microwave power, 5 mW; modulation amplitude, 2 G; modulation frequency, 100 kHz; sweep width, 150 G centered at 3349.0 G; scan rate, 7.5 G s21, with each spectrum representing the average of 5 scans. Immunocytochemistry. Fixation of neurons in culture was performed as described[61]. For dual-label immunostaining, coverslips were incubated in 2% normal goat serum (NGS) containing the following primary antibodies (Ab): anti-GAD67 monoclonal (1:1000), anti-Nox2 rabbit polyclonal (1:200), anti- parvalbumin rabbit polyclonal (1:4000), and incubated for 2 h at 37uC. Specific binding was detected by incubation for 45 min at room temperature with a 1:1000 dilution of AlexaFluor secondary Abs (568: red; 488: green). Immunohistochemistry. Brains were sliced using a vibratome into 50 mm coronal sections. As previously described[27], 6 sequential slices, encompassing either the prelimbic region (from Bregma 2.0 to 1.3), or dorsal hippocampal formation, were processed by floating-section fluorescence immunohistochemistry to detect Nox2 and MAP2[18]. For PV-interneuron counting, slices were processed as before and incubated with anti-Parvalbumin antibody (1:5000) for 48 h, washed in PBS and developed using a Vectastain ABC kit according to manufacturer’s instructions Confocal microscopy, live animal fluorescence imaging, and image analysis. Mounted slices or coverslips were evaluated for fluorescence under settings for 488 and 568 excitation on a LSM510 Meta multiphoton laser confocal microscope using 106 or 406 water immersion objective. Oxidized DHE fluorescence was analyzed using Ex l 543 nm, Em l.590 nm. Imaging for PV-interneuron counting was performed using a transmitted light. All PV-positive interneurons were counted in the regions described in Figs 5 and suppl. 2. Image analysis of the neuronal population in primary cultures was essentially as described [61]. Briefly, coverslips are scanned to obtain 200–400 neurons (approx. 26–30 images captured per coverslip per condition using a 406 water immersion objective). Each image analyzed consists of a stack of 16 0.2 mm Z-stage images taken from the base of the neurons and across 3.2 mm depth. When analyzing PV-interneurons in particular, the coverslips are scanned to obtain images as before but all PV- interneurons on the coverslip are imaged. Experiments were performed in duplicate coverslips of neurons prepared from the same dissection across at least three different dissections. Confocal microscope settings were maintained constant for each series of experiments so that the resulting images could be analyzed by densitometry and the treatment-dependent changes in fluorescence compared and expressed as % of control (for primary cultures) or saline (for in vivo) conditions. For each experiment, IL-6 and Cognitive Changes both in vitro and in vivo, controls (saline) were processed in parallel such that variability due to antibody batch or laser power could be avoided. Neurons in the images were then analyzed for their somatic median green and red fluorescence intensity per cell using MetaMorph (linear scale of 0–255 pixel intensity units). Live animal imaging of oxidized DHE (ox-DHE) fluorescence in brain was carried out as described[62]. Briefly, anesthesia was induced in mice injected 16 hours previously with DHE using inhaled isofluorane. Mice were then placed in a eXplore OptixTM- MX2 system whole animal scanner (Advanced Research Tech- nologies, Inc., Montreal, Canada), and maintained on isofluorane anesthesia by nose-cone. Fluorescence images were obtained by raster-scanning a region over brain using a step size of 0.5 mm with 1 second integration time. Behavioral testing. The novel object habituation test was adapted from the Novel Object/Location test as described[63]. To habituate each mouse to the testing environment, they were individually placed in an empty testing chamber (a 51 cm651 cm639 cm opaque Plexiglas box) for 5 minutes prior to the beginning of testing. Each mouse was then moved to a holding cage, and three novel objects (plastic toy animals glued to a 464 in Plexiglas base) were placed in three corners of the testing chamber. After a brief interval, the mouse was placed back into the chamber for another 5 minutes; exploration was assessed by analyzing the number of contacts, defined as nose-first approaches to within 2–4 cm of an object, with a given object in a trial, and habituation was assessed by comparing the number of contacts made with each object over the course of two subsequent trials, three in all. Between animals, the testing chamber, as well as the objects, were cleaned with AirX44 disinfectant and odor counteractant (AirX Laboratories; Folcroft, PA), to remove olfactory cues. One week following novel object exploration, the Barnes maze test was performed as described[64]. The Barne’s maze was carried out as previously described[65]. The Morris water maze was carried out as described in detail[27]. Statistical analysis. For analysis of fluorescence, all intensity values were normalized by the mean obtained for the control treatments (medium exchange alone for cultures, saline injection for animals), processed in parallel with the experimental group, and expressed as a percent of this mean. To obtain the mean fluorescence/cell/animal, percent values were averaged across six slices from the same animal, or from the same culture plating in the case of primary cultures on coverslips. The mean fluorescence intensity/cell/animal (or per experiment in the case of primary cultures) was then used to calculate the mean and standard deviation per group. These were then used for statistical analysis using SigmaStat software. Values obtained per experiment were analyzed by one- or two-way ANOVA as indicated in each figure, followed by Tukey’s post-hoc test for multiple comparisons, with significance set at P,0.05. Two-way ANOVA followed by Tukey’s post-hoc analysis was used to determine significance between genotypes across time for the Novel Object task and the Barnes Maze, or between ages and treatment groups for the Morris water maze, and F and P values for these comparisons are included in the appropriate figure legend or results section. Supporting Information Figure S1 Superoxide imaging in IL-6-/- brain. Superoxide levels are substantially lower in the hippocampal CA1 and cortex regions of IL-6-/- mouse. Confocal images were acquired as described for Figure 2a. Found at: doi:10.1371/journal.pone.0005518.s001 (10.58 MB TIF) PLoS ONE | www.plosone.org 11 May 2009 | Volume 4 | Issue 5 | e5518