Figure 7. EAATs regulate exocytosis from RBC terminals. A, Schematic of whole-mount retinal preparation. FM1-43-labeled RBC terminals were observed at the level of the red plane with confocal microscopy. B, FM1-43-labeled terminals (left) and the RBC marker αPKC (middle) were colocalized (right). C, In the presence of AP4, no FM1-43 unloading occurred. FM1-43 was unloaded by a CPPG (200 μm) evoked depolarization. In the presence of an EAAT agonist, d-aspartate (1 mm), FM1-43 unloading was reduced. By contrast, in the presence of an EAAT antagonist TBOA (100 μm), FM1-43 unloading was increased. D, Bright light also unloaded FM1-43 from dark-adapted retinas. After FM1-43 loading into RBC terminals, bright-light flashes were applied in the presence or the absence of d-AP4, and then an image of RBC terminals was captured. FM1-43 unloading increased as a function of bright-light flash number (no flashes, 1, n = 56; 2 flashes, 0.87 ± 0.08, n = 12, p > 0.2; 6 flashes, 0.70 ± 0.02, n = 99, *p < 0.001; 10 flashes, 0.63 ± 0.02, n = 48, *p < 0.001). In the presence of AP4, the same flashes did not unload FM1-43 (p > 0.4 vs no flashes; n = 32–57). E, FM1-43 unloading increased with CPPG concentration (in micromolar concentration) (Spearman correlation, 1.0; p < 0.01). Fraction FM1-43 intensities were compared at 4 min, 20 s (AP4 and 0 CPPG, 0.94 ± 0.01, n = 30; 50 CPPG, 0.91 ± 0.02, n = 36; 200 CPPG, 0.54 ± 0.01, n = 94; 600 CPPG, 0.10 ± 0.02, n = 38; 2000 CPPG, 0 ± 0.0, n = 31). CPPG perfusion started at time 0, but because of dead volume, solution exchange occurred in ∼1 min. FM intensity was normalized to maximum terminal brightness level at either time 0 or 20 s for E and F. F, Summary graph shows the effects of an EAAT agonist, d-aspartate, and an EAAT antagonist, TBOA, on the time course of FM1-43 unloading, elicited by CPPG (200 μm). The fraction FM1-43 intensities were compared at 6 min. d-Aspartate reduced CPPG-evoked, FM1-43 unloading almost threefold (fraction control, 0.27 ± 0.03, n = 94, was reduced to 0.80 ± 0.07 by d-aspartate, n = 47; p < 0.001). TBOA increased CPPG-evoked, FM1-43 unloading ∼13-fold (fraction control, 0.27 ± 0.03, n = 94, was increased to 0.02 ± 0.0 by TBOA, n = 103; p < 0.001). For FM1-43 unloading experiments, n = number of terminals from three to five preparations. Scale bar, 5 μm. Error bars indicate SEM.