Effects of fractalkine on morphine and/or Tat-induced alterations in microglial motility (A-D) and nitrosative stress (E-F). Morphine and Tat together (Morph+Tat) increased microglial movement in time-lapse images (representative cells circled in red) (A,B), while concurrent fractalkine exposure negated the effects of Morph+Tat (representative cells encircled in red) (B,C). When the vectorial trajectories of individual microglial movement are plotted each hour for 6 h, (i) the sporadic movements of individual cells and (ii) the ability of fractalkine to attenuate the overall motility are appreciated (B). Measurements confirmed that combined Morph+Tat increased microglia motility compared to vehicle, morphine, or Tat exposure alone (*P < 0.05). Addition of exogenous fractalkine (1 μg/ml) completely reversed the effect of Morph+Tat (D) (#P < 0.05 vs. Morph+Tat alone), returning motility to baseline levels. The average movement of ~100 microglia in each treatment group was determined by measuring the distance from nuclear position at 0 h vs. 12 h per treatment in each experiment; values are the mean movement (μm/h) ± SEM from n = 4 experiments. Representative microglia that were tracked are encircled in red; the occasional macrophage/microglial interlopers that appeared and/or disappeared within the observation field and could not be tracked are circled in yellow; scale bar = 20 μm.