Our results indicated Pfizer Licensed Compound Library that while the inhibition of both evoked and miniature neurotransmission in vglutMN mutants perturbed synaptic development, blocking evoked release alone was not detrimental. We therefore hypothesized that miniature NT could be particularly required for synapse development or alternatively that synapse development relied upon the total amount of NT regardless of whether it was derived from evoked or miniature events. To discriminate between these hypotheses, we sought genetic conditions where miniature NT could be preferentially reduced

versus evoked NT. To do this, we took advantage of the phenomena of synaptic homeostasis that occurs at both Drosophila and mammalian synapses ( Davis, 2013 and Turrigiano, 2012). When postsynaptic ionotropic glutamate receptors (iGluRs) are reduced at Drosophila NMJ synapses, presynaptic terminals increase the number of synaptic vesicles released Alectinib concentration (quantal content) per action potential in order to maintain synaptic strength ( Frank et al., 2006 and Petersen et al., 1997). We exploited this process in mutant combinations where iGluR function was severely inhibited

to specifically reduce miniature NT. As a starting point, we employed iGluR mutants (Schmid et al., 2006) where the expression levels of endogenous glutamate receptor subunits were severely depleted (Figure S3A). In order to avoid disrupting the synaptic scaffolding functions of iGluRs, we combined these mutants with genomic promoter-driven 17-DMAG (Alvespimycin) HCl rescuing transgenes. These transgenes produced either a wild-type glutamate receptor subunit (iGluRWT combination) or a subunit where the glutamate binding region was mutated ( Schmid et al., 2006), rendering the receptor nonfunctional (iGluRMUT combination) ( Figure S3A). Synaptic levels of both iGluRWT and iGluRMUT receptor clusters were similar when

measured using an independent obligate iGluR subunit (dGluRIIC) ( Figures S3B–S3D). We then measured NT in these mutants. iGluRWT terminals had similar miniature NT to controls ( Figures 2A, 2B, 2F, S3F, and S3G). In contrast, iGluRMUT terminals had severely reduced miniature NT with a 96% (p < 0.001) reduction of the mEPSP integral ( Figures 2C, 2F, S3F, and S3G) compared to controls. Miniature NT defects in iGluRMUT mutants were fully rescued by postsynaptic expression of a wild-type iGluR subunit (UAS-dGluRWT) ( Figures 2D and 2F). Though both iGluRWT and iGluRMUT had reduced evoked NT compared to background controls, importantly, they had similar evoked NT to each other ( Figures 2A–2C, 2E, and S3E). As predicted, this was due to an increase in quantal content at iGluRMUT terminals compared to iGluRWT terminals ( Figure S3H). To determine if this homeostatic compensation occurred throughout larval synaptic development, we also measured NT of iGluRWT and iGluRMUT first-instar larval terminals.