We next asked if increased miniature events are necessary for cpx mutant terminal overgrowth. We reduced miniature NT by expressing Sotrastaurin either
a dominant-negative glutamate receptor subunit (UAS-dGluRDN) ( Schmid et al., 2006) in postsynaptic muscles or an RNAi against vglut (RNAi-Vglut) in the presynaptic MNs of cpx mutants ( Figures 4F and 4G) and controls ( Figures S5D and S5E). Both manipulations did not significantly alter evoked NT in cpx mutants but did decrease miniature NT ( Figures 4F–4I). In both conditions, we found that the aberrant synaptic terminal area and bouton size indexes of cpx mutants were suppressed ( Figures 4J, 4K, 4Q, and 4R). Thus, the inhibition of miniature NT suppressed the terminal overgrowth of cpx mutants while the depletion
of evoked NT did not. Therefore, increased miniature neurotransmission, as found in complexin mutants, is sufficient to promote synaptic terminal growth. cpx mutants had opposing check details synaptic morphological changes to vglutMN and iGluRMUT mutants. This was most apparent in the bidirectional effect upon bouton size, with vglutMN and iGluRMUT mutants having an increase in the proportion of small boutons and cpx mutants oppositely having a decreased fraction of these boutons. During terminal development, new synaptic boutons are added and then expand and may also be eliminated ( Koch et al., 2008 and Zito et al., 1999). A defect in one or more of these steps could potentially result in the changes to bouton sizes we observed
when miniature NT was altered. We sought therefore to visualize the development of individual synaptic boutons by time-lapse live imaging through the transparent cuticle of intact larvae. To do this, we utilized the LexA binary system to express a membrane-localized GFP in the presynaptic terminals of both control and Non-specific serine/threonine protein kinase miniature NT mutants. Beginning 24 hr after hatching, we anesthetized animals every 24 hr for 4 days during larval development and imaged their synaptic terminals, returning them to food media between imaging periods. Using this technique, we found that new synaptic boutons formed continuously throughout the imaging period in control and NT mutant backgrounds at the same rate (Figures S6A and S6B). In control animals, 94.4% (34/36) of newly formed small boutons (<2 μm2) then became progressively larger over time to become typical-sized boutons (>2 μm2) during the imaging period (Figures 5A and 5G). This expansion in size was not perturbed by inhibiting evoked NT using TeTxLC (Figures 5B and 5G). However, in iGluRMUT mutants, where miniature NT was reduced, we found that the enlargement of small boutons was severely retarded compared to iGluRWT animals ( Figures 5C, 5D, and 5H) and only 19.6% (10/51) of small boutons ever expanded to become typical-sized boutons.