When we expressed dominant-negative UAS-dGluRDN in both postsynaptic muscles of these mutants, the excessive miniature NT at both terminals was strongly inhibited ( Figure S7C). In both terminals, the aberrant number and size ratio of synaptic boutons were also suppressed ( Figures 7J, 7L, and 7M). In contrast, when UAS-dGluRDN was expressed only in muscle 6 of cpx mutants, miniature NT, bouton number, and bouton size index were only suppressed at the terminal on this muscle and not at the terminal on

muscle 7 ( selleck kinase inhibitor Figures 7K–7M and S7C). Together, these experiments demonstrated that the effect on synapse maturation of increasing or decreasing miniature neurotransmission is via a mechanism that acts locally at synaptic terminals. To determine the molecular mechanism through which miniature neurotransmission regulates bouton maturation, we next carried out a candidate mutant screen of molecules that were (1) linked to synapse morphological development and (2) likely to have localized activity at terminals. Among these candidates was Trio, a member of the evolutionarily conserved Dbl homology family of GEFs (Miller et al., 2013). trio mutants had previously been reported to have defective synaptic terminal growth ( Ball et al., 2010), and Trio has been linked to the local regulation of the neuronal cytoskeleton

SCH 900776 manufacturer ( Miller et al., 2013). We confirmed that trio mutants had reduced numbers of synaptic boutons ( Ball et al., 2010) ( Figure S8A). We additionally found that trio mutants had reduced terminal area accompanied by large increase in the proportion

of small boutons ( Figures 8A, 8B, 8D, and 8E) very reminiscent of synaptic terminals when miniature NT is reduced ( Figure 8C). All of these trio mutant synaptic phenotypes were fully rescued by presynaptic expression of transgenic Trio (UAS-Trio) ( Figures 8D and 8E). When we examined the ultrastructure of the abundant small boutons in trio mutants, we found rudimentary T-bar structures ( Figure 8G) of reduced size similar to those observed in the small boutons of miniature NT mutants ( Figures 8H, S8C, and S8D). However, when we measured miniature NT in these mutants, we found it was unchanged compared to controls ( Figure S8E), consistent with previous reports ( Ball et al., 2010). This indicated that the synaptic terminal phenotypes Thymidine kinase in trio mutants did not originate from defective NT. However, the similarity of trio mutant synaptic morphology phenotypes to miniature NT mutant phenotypes suggested that Trio could be part of a molecular pathway triggered by miniature events. Pursuing this hypothesis, we next tested the genetic interaction of miniature NT mutants with trio mutants. We first examined if Trio is required for the terminal overgrowth and bouton size alteration of cpx mutants. Double null mutants of cpx and trio had similarly increased miniature NT to cpx mutants alone ( Figure S8F).