Failure analysis

revealed a significant enhancement in release probability in larvae overexpressing TOR in muscles (Figure 6C), indicating that a presynaptic mechanism underlies the increase in QC. In addition, we found that the increase in QC due to TOR overexpression was critically dependent on Brp. We have previously demonstrated that removal of one copy of the brp gene does not affect baseline electrophysiological properties ( Tsurudome BIBW2992 price et al., 2010), but it does suppresses the ability of GluRIIA mutants to undergo retrograde compensation ( Figure S5I). Similarly, heterozygosity for brp profoundly inhibited the ability of postsynaptic TOR to induce an increase in QC, providing further evidence that the enhancement in QC in response to postsynaptic TOR overexpression relies on a presynaptic mechanism. These results together provide strong evidence that increased TOR activity can induce a retrograde increase in neurotransmitter release. Consistent with our model, the increase in QC due to TOR overexpression was also critically dependent on eIF4E and S6K, as heterozygosity for eIF4E or S6k greatly reduced the ability of TOR to induce a retrograde increase in QC ( Figures 6A and 6B). These results indicate that TOR’s influence on translation initiation most likely underlies its role as a retrograde regulator

NLG919 nmr of synaptic homeostasis at the NMJ. The strong sensitivity of the TOR-induced retrograde increase in QC to S6K prompted us to test whether muscle overexpression of a constitutively active S6K (S6KSTDE) transgene could mimic the action of TOR (Barcelo and Stewart, 2002). As our results would predict, overexpression of S6KSTDE in muscles led to a similar increase in average EJC amplitude and QC without affecting quantal size, consistent with the idea that TOR exerts its action largely through S6K to induce a retrograde increase in QC (Figure 6D). We also verified that postsynaptic overexpression of S6KSTDE did not lead to

any structural changes (Figures 6F–6O) or changes in mEJC distribution (Figure S5H) and that it indeed enhanced the probability of release presynaptically using failure analysis (Figure 6E). Finally, as our model would predict, we found that heterozygosity found for eIF4E completely suppressed the QC increase due to overexpression of S6KSTDE, suggesting that the aspect of S6K’s function that contributes to the induction of retrograde signaling is critically dependent on the availability/function of the cap-binding protein complex ( Figure 6D). Although our genetic interaction experiments provide strong support that TOR’s action depends on protein translation, we wished to test this issue directly by asking whether the TOR induced increase in QC was sensitive to pharmacological interference with translation. For this we fed control larvae and larvae overexpressing TOR postsynaptically either regular food or food containing 500 mg/ml cycloheximide, a potent inhibitor of translation.