The number of T bar-tethered synaptic vesicles in elp3 is increased, and we tested whether a larger pool of synaptic vesicles is immediately ready for fusion in the mutants. First,

Lumacaftor order we used fluctuation analysis to estimate the number of release-ready vesicles in controls and elp3 mutants. Given that EJC amplitudes in elp3 mutants and controls saturate at high calcium ( Figure S5A), we performed this analysis in the presence of a rapidly dissociating competitive receptor antagonist γ-D-glutamylglycine (γ-DGG) that has been used at the Drosophila larval NMJ before ( Pawlu et al., 2004). As shown in Figure S5A, EJC amplitudes recorded in 5 mM external calcium AZD2281 concentration are reduced by 38% when incubated in 10 mM γDGG, and also mEJC amplitudes (recorded in 0.5 mM Ca2+) are smaller both in controls (without γDGG 1.08 ± 0.05 nA; with γDGG 0.68 ± 0.05 nA; Figure S5B) as well as in elp3 mutants (without γDGG 1.29 ± 0.07 nA; with γDGG 0.97 ± 0.03 nA; not shown). While in other systems application of γDGG results in a stronger inhibition of the postsynaptic response ( Foster and Regehr, 2004), our data are in line with previous results at the Drosophila NMJ ( Pawlu et al., 2004) and indicate

that γDGG at least in part prevents postsynaptic receptor saturation in high calcium concentrations. Recordings in the presence of the drug will thus allow us to assess neurotransmitter release while partly suppressing glutamate receptor saturation in controls and mutants. We then recorded EJC amplitudes in γDGG and different calcium concentrations and extracted quantal parameters from parabolic fits from EJC variance versus EJC mean amplitude plots (Figures S5C and S5D) (Foster and Regehr, 2004). Our data

indicate a larger release-ready pool in elp3 mutants compared to controls (controls, 512.7 ± 35.1 quanta; elp3, 592.2 ± 41.3 quanta; p < 0.05). Also, we find a similar release probability (Pr) in controls and mutants in low calcium concentrations (Ca2+): (0.3 mM) control 0.08 ± 0.001 and elp3 0.13 ± 0.01; (0.4 mM) control 0.20 ± 0.01 and elp3 0.17 ± 0.01; and (0.6 mM) control 0.27 ± 0.02 and elp3 0.24 ± BCKDHB 0.03. Similarly, in 3 mM calcium our analyses indicate a similar Pr (control, 0.98 ± 0.02; elp3, 0.96 ± 0.02), but under these conditions, postsynaptic receptor desaturation by γDGG may be incomplete ( Figure S5A), confounding our estimations of the release-ready pool and Pr in the mutants. Nonetheless, in high calcium, Pr is invariably high, and differences in Pr, if any, between elp3 mutants and controls remain small. To independently evaluate presynaptic release properties, we also measured transmission during a short train of high-frequency stimulation (500 ms, 100 Hz) in 5 mM external calcium, ensuring a high Pr (Figure 6A).