The peak EPSC amplitude did not correlate

with the rise or decay kinetics (Figure 1D), arguing that the EPSC kinetics did not result from dendritic filtering or inadequate voltage clamp. Nevertheless, we tested whether the EPSC amplitude influenced its kinetics by including a subsaturating concentration of the high-affinity AMPAR antagonist, NBQX (600–800 nM) or recording at Trametinib clinical trial depolarized membrane potentials. Neither of these manipulations affected the results and the data were pooled for analysis (Figure 1; n = 14 and 16, respectively). Additionally, we monitored the series resistance (Rs) during each experiment and rejected experiments when the Rs was unstable. On average, Rs during EPSC2Hz was 102 ± 5.7% of the value during EPSC0.05Hz stimulation (average 2.7 ±

1.4 MΩ; range 1.5–4.9 MΩ; n = 30; p > 0.05). We also considered whether dendritic filtering contributed to the EPSC kinetic slowing. However, there was no correlation between the EPSC rise and decay times as expected if these parameters were significantly filtered by the dendritic cable (data DAPT chemical structure not shown; p = 0.98; Hestrin et al., 1990). Together this suggests that changes in voltage control or in dendritic filtering do not contribute to the kinetic slowing that occurs with increased stimulation frequency. Slowing of the EPSC time course occurred across a range of physiologically relevant frequencies. Although increasing the stimulus frequency to 0.2 Hz had no effect on the EPSC kinetics, further increases to 1, 2, and 4 Hz caused significant prolongation of both the rise and decay times (Figure S1A, available

online; n = 4–9; p < 0.05). Increasing the stimulation frequency to 10 Hz did not further slow the EPSC (data not shown), suggesting that at higher frequencies vesicular depletion may outweigh desynchronization. We also TCL tested whether EPSC slowing requires prolonged repetitive stimulation. The rise time of the third EPSC during a brief 15 Hz train was slower than that of the first EPSC (Figure S1B; 0.62 ± 0.1 ms and 0.41 ± 0.05 ms, respectively; n = 11; p < 0.001) and similar kinetic differences were found when CFs were stimulated with two pulses at 20 Hz (n = 15, not shown). Thus, activity-dependent slowing of EPSCs occurs across physiologically relevant stimulation paradigms. We posit that increasing CF stimulation frequency not only leads to the depletion of the available vesicles but also desynchronizes the timing of vesicle fusion, leading to EPSCs that are smaller in amplitude with slower kinetics. Whereas activity-dependent vesicle depletion reduces the EPSC amplitude, release desynchrony shapes the EPSC time course. Action potentials trigger well-timed phasic vesicle fusion (synchronous release) that is sometimes followed by a smaller elevation in quantal release called delayed release or asynchronous release, which lasts for tens of milliseconds (Barrett and Stevens, 1972, Goda and Stevens, 1994, Isaacson and Walmsley, 1995 and Atluri and Regehr, 1998).