In wild-type third-instar larvae, less than 10% of all synaptic boutons are smaller than 2 μm2, while the majority of boutons are larger than this ( Figure S1D).

In vglutMN mutants, we observed a dramatic increase in the proportion of boutons smaller than 2 μm2 (small boutons), while the number of synaptic boutons larger than this (typical boutons) in addition to the total number of boutons was reduced compared to controls ( Figure 1P; Table S1). To quantify this shift of bouton sizes, we calculated the ratio of small to typical boutons (bouton size index) and found a 366% (p < 0.001) increase in vglutMN mutants compared to controls GSK126 nmr ( Figure 1Q). These phenotypes were observed with multiple vglut RNAi lines or hypomorphic vglut mutants and could be rescued by transgenic Vglut ( Figures S1E–S1H). Similar to typical

synaptic boutons, Selleckchem Y27632 the small boutons in vglutMN mutants had correctly localized markers for active zones, periactive zones, synaptic vesicles, postsynaptic membranes, and postsynaptic receptor fields ( Figure S2A). Therefore, vglut mutants have synapses with reduced terminal area concomitant with a disproportionally large amount of small synaptic boutons. This result established that even though synaptic transmission is not required for initial embryonic synapse assembly in Drosophila ( Daniels et al., 2006), it is necessary for the subsequent phase of synaptic terminal growth. In vglutMN mutants, both evoked and miniature forms of neurotransmission are inhibited. Because the majority of NT at Drosophila NMJ terminals is via evoked release ( Kurdyak et al., 1994), we next used genetically

encoded peptide toxins to specifically block this form of NT and dissect its contribution to synapse development. Transgenic tetanus toxin light chain (UAS-TeTxLC) cleaves the vSNARE n-Synaptobrevin, which is essential for evoked, but not miniature, synaptic Calpain vesicle release ( Sweeney et al., 1995). Expression of TeTxLC in a subset of MNs eliminated the ability of these NMJ terminals to produce evoked release when the axon was stimulated ( Figures 1C, 1F, and S2B). In contrast, miniature NT was unaffected ( Figures 1C, 1G, S2C, and S2D). As a second independent method of inhibiting evoked release, we generated a transgenic membrane-tethered version of Plectreurys toxin II (UAS-PLTXII), which blocks the Drosophila synaptic N-type voltage-gated calcium channel Cacophony that is essential for evoked release ( Wu et al., 2008). Similar to TeTxLC, expression of PLTXII in MNs dramatically reduced evoked release but did not significantly alter miniature NT ( Figures 1D, 1F, 1G, and S2B–S2D). We assessed the effects of expression of both of these toxins on synaptic terminal development ( Figures 1M and 1N). We found no change of synaptic terminal area, the number of synaptic boutons, or the bouton size index at these terminals compared to controls ( Figures 1O–1Q).