Figure 3

Two phases of vesicle release from type III boutons overlap with larval ecdysis. We observed fluorescence changes in the type III boutons of CCAP>ANF-EMD larvae. Six stages were chosen that broadly extend over the duration of the 2^nd larval ecdysis: 'DMH', which precedes the onset of ecdysis; 'DVP', which coincides with the initiation of ETH release; 'FE', which signifies the completion of ecdysis; '+2 hours' after FE; '+3 hours' after FE; and 'L3', wandering 3^rd instar larvae. For DVP, we selected animals at the earliest point when the new vertical plates were recognizable. At each stage, the fluorescence intensity of all visible type III boutons from 4 animals was measured and converted to fluorescence intensity averages (see Methods), in arbitary units (top panel). Representative type III boutons from each stage are shown below the corresponding fluorescence intensity measurements with accompanying asterisks representing single-factor ANOVA results for consecutive stages (bottom panels). The results significantly show two waves of ANF-EMD release, before DVP and after FE, as shown by an asterisk. For *, p < 0.005. Error bars indicate +/- SEM. Scale bar = 10 μm.