14 mV for CSP-α KO) ( Figure S1A available online) and MEPP frequency was slightly, but not significantly, increased (0.58 ± 0.09 Hz in WT versus 0.83 ± 0.2 Hz in CSP-α KO NMJs) ( Figure S1B). Next, we monitored end-plate potentials (EPP) evoked by nerve stimulation ( Figure 1A) and found a significant decrease in quantal content (QC) (36.4 ± 2.3 for WT and 23 ± 2.1 for KO synapses, p < 0.001 Student's t test) ( Figure 1B), similar to the values in the CSP-α KO mice that do not express spH (40.7 ± 2.7 for WT and 28.6 ± 3.2 for CSP-α KO) ( Figure S1C). Upon stimulation with long depolarizing trains
(100 shocks, at 10, 30, and 100 Hz) ( Figure 1 C), mutant and control terminals Quisinostat concentration displayed initial similar levels of facilitation ( Figure S1D) followed by synaptic depression, that was generally stronger in the mutants ( Figure 1C) (10Hz: 55.0 ± 2.6% WT and 47.0 ±
3.8 CSP-α KO, p = 0.09; 30 Hz: 56.9 ± 2.4% WT and 44.1 ± 3.8% CSP-α KO, p = 0.006; 100 Hz: 48.7 ± 2.3% WT and 38.8 ± 4.6% CSP-α KO, p = 0.045). Interestingly, during the train, recordings from CSP-α KO revealed high fluctuations in EPP amplitude ( Figure 1D), presenting a higher coefficient of variation (CV) for higher stimulation frequencies ( Figure 1E). In contrast, at control recordings the CV was rather constant at different stimulation frequencies. Such a phenotype could be reflecting changes in the probability of release (p) or in the number of release sites (n). To clarify that issue, we investigated quantal properties of neurotransmitter release using binomial buy INCB018424 analysis ( Boyd and Martin, 1956 and Searl and Silinsky, 2003). Amplitude distributions of EPPs (sets of 100 EPPs recorded at every terminal at 0.2 Hz stimulation frequency) ( Figure 1F), were fitted to a binomial distribution to estimate the probability of release (p) Urease and the number of synaptic release sites (n) ( Figure 1G). The probability
of release was similar in WT and CSP-α KO junctions (0.38 ± 0.01 WT versus 0.39 ± 0.02 CSP-α KO). In contrast, the mutant junctions presented a significant reduction in the number of release sites (n) ( Figure 1H) (103.1 ± 12 versus 59.8 ± 6.9 for WT and CSP-α KO, p = 0.006, Student’s t test). If parameter n is regarded as the number of vesicles that are docked and primed, then p refers to the probability that a vesicle is released from the pool ( Miyamoto, 1975 and Schneggenburger et al., 1999). Therefore, the reduced n value at the mutant synapses could be interpreted as a reduction in the number of those vesicles that constitute the readily releasable pool (RRP) due to a defect in priming. Because SNAP-25 is very unstable at central synapses in CSP-α KO mice ( Chandra et al., 2005 and Sharma et al., 2011b), we wondered if the same molecular phenomenon at motor nerve terminals could explain the priming deficit.