SUMMARY Retrograde signaling systems are fundamental modes of communication that synapses utilize to dynamically and adaptively modulate activity. However, the inductive mechanisms that gate retrograde communication in the postsynaptic compartment remain enigmatic. We have investigated retrograde signaling at the Drosophila neuromuscular junction, where three seemingly disparate perturbations to the postsynaptic cell trigger a similar enhancement in presynaptic neurotransmitter release. We show that the same presynaptic genetic machinery and enhancements in active zone structure are utilized by each inductive pathway. However, all three induction mechanisms differ in temporal, translational, and CamKII activity requirements to initiate retrograde signaling in the postsynaptic cell. Intriguingly, pharmacological blockade of postsynaptic glutamate receptors, and not calcium influx through these receptors, is necessary and sufficient to induce rapid retrograde homeostatic signaling through CamKII. Thus, three distinct induction mechanisms converge on the same retrograde signaling system to drive the homeostatic strengthening of presynaptic neurotransmitter release.

Summary We have interrogated the synaptic dialog that enables the bi-directional, homeostatic control of pre-synaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). We find that homeo-static depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. We propose that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment.

Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic homeostatic plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, we have developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. We find that PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments.

The endoplasmic reticulum (ER) is an extensive organelle in neurons with important roles at synapses including the regulation of cytosolic Ca, neurotransmission, lipid metabolism, and membrane trafficking. Despite intriguing evidence for these crucial functions, how the presynaptic ER influences synaptic physiology remains enigmatic. To gain insight into this question, we have generated and characterized mutations in the single () ortholog in Esyts are evolutionarily conserved ER proteins with Ca-sensing domains that have recently been shown to orchestrate membrane tethering and lipid exchange between the ER and plasma membrane. We first demonstrate that Esyt localizes to presynaptic ER structures at the neuromuscular junction. Next, we show that synaptic growth, structure, and homeostatic plasticity are surprisingly unperturbed at synapses lacking expression. However, neurotransmission is reduced in mutants, consistent with a presynaptic role in promoting neurotransmitter release. Finally, neuronal overexpression of enhances synaptic growth and the sustainment of the vesicle pool during intense activity, suggesting that increased Esyt levels may modulate the membrane trafficking and/or resting Ca pathways that control synapse extension. Thus, we identify as a presynaptic ER protein that can promote neurotransmission and synaptic growth, revealing the first neuronal functions of this conserved gene family.

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in monoubiquitination. Finally, we show that Peflin, a Ca 2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca 2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.