(A) One-cell example as seen in GLT1-SEP + DIC channel (left), with a selected area (dotted rectangle) illustrating a circular, 2.06 µm wide FRAP spot (dotted circle, right). (B) Diagram, the paired-sample FRAP protocol, in which two trials are carried out in succession, to account for any non-specific, time-dependent drift in FRAP kinetics. Plots, one-cell example of the paired-sample FRAP test, with the first and second trial data are shown in blue and green, respectively; arrow, bleaching pulse (λx2p = 690 nm, 10–15 mW under the objective, duration 46 ms); fluorescence ROI, photobleaching spot as in (A). (C) Diagram illustrating the paired-sample FRAP protocol, which includes both control and glutamate application cycles; FRAP kinetics under glutamate application could be corrected for non-specific drift by using the control cycle data. (D) Left, average time course of the GLT1-SEP FRAP (dots and shade: mean ± 95% confidence interval, here and thereafter) in baseline conditions (black) and upon glutamate application (250 ms puff 200 ms before the photobleaching pulse lured); asterisk, p < 0.05 (n = 69 FRAP spots in N = 13 cells). Right, FRAP time course (mean values) fitted with the Soumpasis FRAP equation for (see main text) for control and glutamate tests. Best-fit GLT1-SEP diffusion coefficient D is shown for control (Cntrl) and glutamate puff (Glu) trials, as indicated. (E) Average FRAP time course in control and glutamate-puff tests carried out with the C-terminus deleted mutant CLT1ΔC-SEP, as indicated (n = 30 FRAP spots in N = 7 cells); other notation as in (D). (F) Average FRAP time course in control and glutamate-puff tests in the presence of AMPA and metabotropic glutamate receptor (mGluR) blockers (n = 34 FRAP spots in N = 7 cells): MPEP (1 mM), LY341495 (30 nM), YM298198 (0.3 µM); NBQX (10 µM) was added to suppress network hyper-excitability under LY341495; other notation as in (D). (G) Average FRAP time course in control conditions and after the ATP pressure puff (100 µM, 250 ms duration 200 ms before bleaching start, no glutamate), as indicated (n = 14 FRAP spots in N = 4 cells); other notation as in (D). (H) Control test: Average FRAP time course in control conditions and under surface cross-linkage by anti-GFP antibody, as indicated (n = 13 FRAP spots in N = 2 cells); other notation as in (D).