Figure 5.

Using the vesicular ATPase inhibitor bafilomycin to block refilling of vesicles with glutamate caused different rates of decline in EPSCs and mEPSCs. (A) A representative EPSC in control conditions evoked in an HC by a 200-ms depolarizing step from −70 to −10 mV applied to a simultaneously voltage-clamped rod. The depolarizing step evoked an initial fast EPSC due to release from ribbons followed by a second slower peak due to non-ribbon release (Chen et al., 2014). (B) An EPSC evoked in the same rod/HC pair 15 min after application of bafilomycin (7 μM) showed a decrease in amplitude of both EPSC peaks. (C) EPSC amplitude and mEPSC frequency and amplitude were normalized to the average of four responses obtained prior to bafilomycin application at 4 minutes (dashed vertical lines). The 1^st and 2^nd EPSC peaks both declined in control conditions due to rundown. The declines in amplitude of the 1^st (C) and 2^nd (D) EPSC peaks were accelerated slightly but not significantly by bafilomycin (comparing the change in normalized EPSC amplitude of the first 3 vs. last 3 responses from rod/HC pairs in control N=9 vs. bafilomycin N=8: peak 1, P = 0.65; peak 2, P=0.44, unpaired t-tests). In contrast to changes in EPSC amplitude, HC mEPSC frequency (E) and amplitude (F) both declined significantly during application of bafilomycin (average of the first 3 vs. last 3 records in control vs. bafilomycin, frequency: P < 0.04; amplitude: P < 0.04; N = 7 HCs in bafilomycin, N = 8 HCs in control, unpaired t-tests). HC mEPSCs were collected in 10 s trials in which the rod was voltage clamped at −70 mV. EPSC and mEPSC trials alternated every 30 s.