Acute manganese chloride treatments Acute (30 min) manganese chloride (MnCl2) treatments were performed on 5,000 synchronized L1 per sample, and live worms were scored 24 h later, as previously described [66]. Scores were normalized to percent control (0 mM MnCl2 exposure). Acute dopamine treatments Dopamine (Sigma Chemical Co., St Louis, MO) solutions were prepared in M9 buffer. Acute (30 min) treatments on young L1 worms were first tested from 1 mM to 50 mM DA, guiding us in choosing 10 mM as the working sub-lethal dose. Synchronized L1 were acutely pre-treated with 10 mM DA for 10 min, washed 5 times in 85 mM NaCl solution and subjected to MnCl2 acute treatments. Control worms were pre-treated with M9. Dopamine content measurement Synchronized L1 worms were collected, washed three times in 85 mM NaCl and distributed in tetraplicates of 200,000 L1. Worms were pelleted and the supernatant was removed. The tubes were then immediately frozen in liquid nitrogen and stored at 280uC. For each tube, the worm pellet was re-suspended in lysis buffer containing EDTA to scavenge free metal ions and was sonicated to disrupt cell membranes. Fifty mL of the lysate was used to perform a BCA assay to measure protein levels. Isoproterenol was added as an internal standard to the remaining 250 mL of the lysate, which was applied to the aluminum membrane to bind DA. Collected DA samples were then processed for High Performance Liquid Chromatography (HPLC). To correct for inter-sample variations in the extraction efficiency, the ratio of DA to the internal standard, isoproternol, was estimated, and the total DA content was calculated relative either to protein levels or to the number of worms. Isoprostane content measurement Worms were grown at high densities on 8P-plates. 20,000 synchronized L1 per sample were washed off the plates in 85 mM NaCl, collected in 10 mL 85 mM NaCl and acutely treated (30 min under gentle agitation) with MnCl2. Worms were pelleted and washed three times in 85 mM NaCl and then re-suspended in 85 mM NaCl, 0.5% Triton X-100, 5 mM Tris Buffer pH 6.8, 0.56 protease inhibitor cocktail (Sigma P8340) with zirconia beads, up to 1 mL. Samples were then processed with a Mini beadbeater-16 (Biospec Products, OK, USA) for 7 cycles of 20 s and kept 1 min in ice-cold water after each cycle. 20 mL of supernatant per sample were kept for measurement of protein levels by the Bradford method. 850 mL were added to 10 mL Folch solution and gently shaken every 5 min for 30 min. 2 mL of 0.9% NaCl per tube were added. Tubes were centrifuged at 3,000 rpm for 10 min at 4uC, the aqueous layer was discarded, and the organic phase was dried under nitrogen flow at 37uC. The detailed procedures for the purification and derivatization steps were previously described [174]. ROS measurement Synchronized L1 were acutely treated with MnCl2 as described earlier and washed 4 additional times in M9 buffer. 2979 dichlorodihydrofluorescein diacetate (H2DCF-DA) was added at 1 mM for one hour in the dark. Worms were then washed 4 times in M9 buffer. Worms were frozen and thawed twice and homogenized by sonication and then centrifuged. The superna- tants were transferred to a 96-well plate and their fluorescence levels (excitation: 485 nm; emission: 535 nm) were detected using a FLEXstation III (Molecular Devices, Sunnyvale, California) pre- heated at 37uC. The fluorescence from each well was measured BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity every 20 min for up to 2 h. Here, we report values obtained at 1 h. Fluorescence measurements were normalized to time zero values, and rates of increase in fluorescence (reflecting ROS levels) were expressed as percent control. Measurements were repeated 3 times, each condition was performed in triplicate, and the experiment was repeated in three independent worm preparations for each tested strain. Lifespan experiments Synchronized L1 worms were acutely exposed to MnCl2 concentrations as described earlier. Live and healthy-looking worms (60–66 per condition) were collected on the same day at the late L4 stage and transferred every five days to new OP50-seeded NGM plates. Survival was assessed every two to three days until all worms had died. All tested C. elegans strains were assessed in parallel, and each experiment was performed three times, yielding qualitatively identical results. Plotted curves represent averages of those triplicate independent experiments. Epifluorescence, DIC, and confocal microscopy For each slide, at least 30 worms were mounted on 4% agarose pads in M9 and anaesthetized with 0.2% tricaine/0.02% tetramisole in M9. Fluorescence observations and scoring of neuronal defects were performed with an epifluorescence micro- scope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan) equipped with a Lambda LS Xenon lamp (Sutter Instrument Company) and Nikon Plan Fluor 206 dry and Nikon Plan Apo 606 1.3 oil objectives. Confocal images acquired for illustration or GFP intensity measurement purposes were captured through Plan- Neofluar 406, Plan-Apochromat 636, or Plan-Neofluar 1006 oil objectives with a 1.3, 1.4 and 1.3 apertures, respectively, on a LSM510 confocal microscope (Carl Zeiss MicroImaging, Inc.) scanning every 200 nm for XZ sections. Images were processed with the Zeiss LSM Image Browser 4.0.0.157 software and edited using Photoshop 7.0 (Adobe). Microscopes were housed in air- conditioned rooms (20–22uC). Amphid and phasmid neuron staining was performed following MnCl2 acute treatment by soaking the worms for 2 h in a 10 mg/mL DiI solution prepared with M9, washing off the dye for 1 h in M9 and then recovering the worms on OP50 coated NGM plates. SKN-1::GFP fluorescence measurements SKN-1::GFP transgenic worms were acutely treated as previously described, transferred to OP50-1 seeded NGM plates and imaged 1 h post-treatment. Fluorescence measurements of the SKN-1::GFP signal were performed on complete confocal Z-stack maximal projections of L1 C. elegans ASI neurons. Treated and untreated animals were mounted on the same slide and imaged with the same magnification, gain, offset, pinhole and laser power settings. GFP integral intensity and signal density of the maximal projection of the ASI nuclei were measured with the freeware ImageJ (developed by Wayne Rasband, NIMH, Maryland, USA). Statistics Dose-response lethality curves, longevity curves and histograms for DA, isoprostane or ROS content measurements were generated with GraphPad Prism (GraphPad Software Inc.). We used a sigmoidal dose-response model with a top constraint at 100% to draw the curves and determine the LD50 or the average lifespan values reported in the graphs. Statistical analysis of significance was carried out by one-way ANOVA for the dose- response curves, longevity curves and dopamine measurements; two-way ANOVA was used to measure isoprostane and ROS PLoS Genetics | www.plosgenetics.org 14 August 2010 | Volume 6 | Issue 8 | e1001084