Glutamate and neuronal activity regulate GLT‐1 surface diffusion in brain slices. A: Example of astrocyte expressing GFP‐GLT‐1‐V5 and imaged 3–5 days after transfection. Scale bar, 10 μm. B: Zoomed region on an astrocytic process expressing GFP‐GLT‐1‐V5 (B1), QDs (B2), QDs overlaid with GFP‐GLT‐1‐V5 (B3) and representative QD trajectories (B4). Instantaneous diffusion coefficients (C,E,G) and MSDt plot of QD‐tagged GLT‐1 (D,F,H). C,D: In dissociated culture (black, median D = 0.067 μm2/s; n = 783 trajectories) and in slice cultures (red, median = 0.021 μm2/s; n = 325 trajectories), median D is significantly decreased in slice cultures compared with in dissociated cultures (P = 3.5 × 10−80, Mann‐Whitney test). D,E: In soma (black, median D = 0.027 μm2/s; n = 108 trajectories) and in processes (red, median D = 0.021 μm2/s; n = 325 trajectories), median D is significantly decreased in processes compared with in soma (P = 0.005, Mann‐Whitney test). G,H: After 2 min of glutamate 100 μM (red, median D = 0.033 μm2/s; n = 234 trajectories), and after 20 min of 4‐AP 1 mM (blue, median D = 0.032 μm2/s; n = 174 trajectories), median D is significantly increased in both treatment conditions compared with control (P
glu = 1.15 × 10−10 and P
4‐AP = 0.03, Mann‐Whitney test).