Here we explore the relationship between presynaptic homeostatic plasticity and proteasome function at the Drosophila neuromuscular junction. First, we demonstrate that the induction of homeostatic plasticity is blocked after presynaptic proteasome perturbation. Proteasome inhibition potentiates release under baseline conditions but not during homeostatic plasticity, suggesting that proteasomal degradation and homeostatic plasticity modulate a common pool of vesicles. The vesicles that are regulated by proteasome function and recruited during homeostatic plasticity are highly EGTA sensitive, implying looser Ca2+ influx-release coupling. Similar to homeostatic plasticity, proteasome perturbation enhances presynaptic Ca2+ influx, readily-releasable vesicle pool size, and does not potentiate release after loss of specific homeostatic plasticity genes, including the schizophrenia-susceptibility gene dysbindin. Finally, we provide genetic evidence that Dysbindin levels regulate the access to EGTA-sensitive vesicles. Together, our data suggest that presynaptic protein degradation opposes the release of low-release probability vesicles that are potentiated during homeostatic plasticity and whose access is controlled by dysbindin.

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Both intracellular pH (pHi) and synaptic cleft pH change during neuronal activity yet little is known about how these pH shifts might affect synaptic transmission by influencing vesicle fusion. To address this we imaged pH- and Ca2+-sensitive fluorescent indicators (HPTS, Oregon green) in boutons at neuromuscular junctions. Electrical stimulation of motor nerves evoked presynaptic Ca2+i rises and pHi falls (∼0.1 pH units) followed by recovery of both Ca2+i and pHi. The plasma-membrane calcium ATPase (PMCA) inhibitor, 5(6)-carboxyeosin diacetate, slowed both the calcium recovery and the acidification. To investigate a possible calcium-independent role for the pHi shifts in modulating vesicle fusion we recorded post-synaptic miniature end-plate potential (mEPP) and current (mEPC) frequency in Ca2+-free solution. Acidification by propionate superfusion, NH4+ withdrawal, or the inhibition of acid extrusion on the Na+/H+ exchanger (NHE) induced a rise in miniature frequency. Furthermore, the inhibition of acid extrusion enhanced the rise induced by propionate addition and NH4+ removal. In the presence of NH4+, 10 out of 23 cells showed, after a delay, one or more rises in miniature frequency. These findings suggest that Ca2+-dependent pHi shifts, caused by the PMCA and regulated by NHE, may stimulate vesicle release. Furthermore, in the presence of membrane permeant buffers, exocytosed acid or its equivalents may enhance release through positive feedback. This hitherto neglected pH signalling, and the potential feedback role of vesicular acid, could explain some important neuronal excitability changes associated with altered pH and its buffering. Synapse 67:729–740, 2013.