These results are consistent with miniature neurotransmission also being necessary for synaptic ultrastructural maturation in addition to morphological expansion. We next sought to establish the nature of the developmental signal induced by miniature neurotransmission. Synaptic structure can be influenced by factors that act locally at the individual terminal level (e.g., synaptic adhesion factors; Davis

and Goodman, 1998) or throughout the whole neuron (e.g., transcriptional regulation; McCabe et al., 2003). Action potentials and evoked NT affect the entire synaptic terminal. In contrast, we surmised that the effects of miniature NT might be spatially see more restricted to individual active zones and therefore could act locally to regulate bouton maturation. To test this hypothesis, we examined the synaptic terminals generated by the type Ib motor neuron RP3 (Hoang and Chiba, 2001). The single axon of this neuron bifurcates to produce synaptic terminals concurrently on two postsynaptic targets: muscle 6 and muscle 7 (Figure 7A). The ratio of synaptic boutons produced at each muscle is stereotyped (Davis and Goodman, 1998),

and these muscles are not electrically coupled with each other (Ueda and Kidokoro, 1996), facilitating independent manipulation of NT. We used Gal4 drivers expressed either in muscle 6 alone (but not muscle 7) (Figures 7A and S7A) or in both muscles to dissect if miniature NT signaling acts locally at terminals or throughout the neuron. Similar to other synapses, reduction of miniature Thiazovivin manufacturer TCL NT by iGluRMUT reduced typical bouton numbers ( Figures 7B, 7C, and 7F) and increased the fraction of small boutons ( Figure 7G) at both of the RP3 MN terminals on muscles 6 and 7 compared to controls, though the area of these terminals could not be accurately measured due their complex spatial arrangement.

When we overexpressed a wild-type iGluR transgene (UAS- dGluRWT) in both postsynaptic muscles of iGluRMUT mutants, we restored normal miniature NT at both terminals ( Figure S7B). This also fully rescued bouton numbers and the bouton size index at both terminals ( Figures 7D, 7F, and 7G). We next expressed UAS-dGluRWT only in muscle 6 of iGluRMUT mutants. This increased miniature NT at muscle 6 terminals without altering NT at muscle 7 ( Figure S7B). When we examined the morphology of both terminals, we found that bouton numbers and bouton size were restored at terminals at muscle 6 ( Figures 7E–7G). In contrast, however, the terminals at muscle 7 were not rescued ( Figures 7E–7G). Because both terminals are produced by a single neuron, this result suggested that the effect of reduced miniature NT on synaptic bouton maturation is localized to individual terminals. In a complementary experiment, we examined the suppression of cpx mutants using a similar strategy.