Inhibition of endocytosis with dynasore caused an immediate decline in fast, ribbon-related components of EPSCs but not slower components. (A) Top traces: EPSCs recorded from an HC voltage clamped at −60 mV evoked by a 200-ms depolarizing step from −70 to −10 mV. Bottom traces: ICa recorded simultaneously in the presynaptic rod. Passive currents were removed by P/8 subtraction of the rod membrane current. The voltage changes applied to the rod are shown at the bottom (Rod Vh). The first pair of traces at the left were obtained in control conditions, and the second pair of traces were obtained 60 s later with no intervening stimulation after starting application of dynasore (80 µM). The third pair of traces was obtained after a 7-min application of dynasore. (B) Maintained application of dynasore (80 µM) caused an initial rapid decline in the fast component of HC EPSCs. The graph plots the amplitude of fast, ribbon-mediated EPSCs as a function of time. Responses were normalized to the mean amplitude measured during the first four control EPSCs in both control (n = 10) and dynasore-treated retinas (n = 14). (C) Slow EPSC components showed a more gradual decline in amplitude. Slow EPSCs in dynasore-treated rod/HC pairs (n = 14) declined more rapidly than control responses (n = 13). These differences attained statistical significance 7–11 min after beginning application of dynasore (*, P = 0.0054, ANOVA; control, n = 14; dynasore, n = 13). (D) Paired comparisons between control responses and test responses obtained within 1–2 min of dynasore application. Test responses obtained after dynasore treatment were normalized to the amplitude of prior control responses in the same cell pair. Experiments in which dynasore was applied for both short and long periods were included in this comparison. Fast EPSCs were significantly inhibited (dynasore/control: 0.82 ± 0.038, n = 22; ***, P = 0.0001, one sample t test), but there was no significant change in slower EPSC components (dynasore/control: 1.03 ± 0.059, n = 23; P = 0.61) or presynaptic ICa (0.995 ± 0.022, n = 20; P = 0.83). (E) Examples of spontaneous mEPSCs recorded from an HC in control conditions and after 9 min of treatment with dynasore. (F and G) Plots of the frequency (F) and amplitude (G) of mEPSCs normalized to the first three control responses (control, n = 10; dynasore, n = 11). The vertical dashed lines show the time that dynasore application was started. Error bars represent ±SEM.
