0 to 3.5 Hz (Figure 6A, black). The extent of enhancement ranged from 1.7-fold to 6.7-fold (Figure 6B, black, the ratio of mEPSC frequency 2–6 s EPZ-6438 following tetanic stimulation to basal frequency). In wild-type animals the time course of mEPSC frequency enhancement decayed with a time constant of ∼12 s (Figure 6C). Tetanic stimulation also increased the frequency of spontaneous events in PKCα−/−, PKCβ−/−, and PKCα−/−β−/− mice, as shown in representative experiments in which enhancement was 4.2-fold (increased

from 0.67 to 2.8 Hz), 3.7-fold (0.74 to 2.77 Hz), and 3.9-fold (0.80 to 3.1 Hz), respectively (Figure 6A). There was no significant difference in selleck compound the enhancement of mEPSC frequency among wild-type (3.5 ± 0.3,

n = 15), PKCα−/− (3.5 ± 0.5, n = 10), PKCβ−/− (4.4 ± 0.5, n = 16) and PKCα−/−β−/− groups (4.3 ± 0.6, n = 13) (p = 0.43) (Figures 6B and 6C). These results suggest that at the calyx of Held synapse, PTP and the enhancement of spontaneous release arise from different mechanisms. Calcium-dependent PKCs are crucial to PTP, but they do not mediate tetanus-evoked increases in mEPSC frequency. We tested whether PKCα and PKCβ mediate the increase in mEPSC amplitude that follows tetanic stimulation (He et al., 2009). In wild-type mice, tetanic stimulation altered the distribution of mEPSC sizes, and after tetanic stimulation the fraction of small mEPSCs was reduced and the fraction of large mEPSCs increased, as shown in a representative experiment (Figure 7A). In slices from PKC knockout animals, tetanic stimulation also increased mEPSC amplitude and produced similar effects on the mEPSC distributions, as illustrated in representative experiments from slices from PKCα−/− (Figure 7B), PKCβ−/− (Figure 7C), and PKCα−/−β−/−

(Figure 7D) mice. As shown in the cumulative histograms (Figure 7E), tetanic stimulation significantly increased the mEPSC amplitude in slices from wild-type, PKCα−/−, PKCβ−/−, and PKCα−/− β−/− mice compared to their respective baseline (p < 0.05 for all paired comparisons). On average, enhancement was somewhat smaller in PKCβ−/− (10.1% ± 2.8%), and PKCα−/−β−/− (10.9% ± 4.7%) compared to wild-type (13.1% ± 3.5%) and PKCα–/– (18.7% ± 2.5%), Terminal deoxynucleotidyl transferase but these differences were not statistically significant (p = 0.34). The time courses of the enhancement of mEPSC amplitude in the different genotypes (Figure 7F) can be approximated by single exponential decays with timeconstants of 47 ± 9 s, 39 ± 4 s, 67 ± 17 s, and 35 ± 8 s for wild-type, PKCα−/−, PKCβ−/−, and PKCα−/−β−/− groups, respectively. Phorbol esters activate PKC by binding to the diacylglycerol (DAG) binding site (Newton, 2001), leading to large synaptic enhancement that mimics and occludes PTP (Korogod et al., 2007 and Malenka et al., 1986).