Abstract The nervous system is remarkably complex and malleable in terms of developmental and learning-related plasticity. Homeostatic signaling systems, operating at the level of individual neurons and neural circuits, act to maintain the function of individual nerve cells and neural circuitry, thereby ensuring robust and stable brain function. Defective homeostatic signaling is directly linked to the cause and progression of neurological diseases including epilepsy, schizophrenia, Autism Spectrum Disorders (ASD), and neurodegeneration. The molecular design and implementation of homeostatic signaling in the nervous system is only just beginning to emerge. We use the Drosophila neuromuscular junction (NMJ) as a model synapse to delineate the molecular mechanisms governing homeostatic control of synaptic transmission. At the Drosophila NMJ (a glutamatergic synapse), inhibition of postsynaptic glutamate receptors leads to a compensatory increase in presynaptic neurotransmitter release to maintain stable synaptic strength. This phenomenon is called Presynaptic Homeostatic Plasticity, and is evolutionarily conserved in organisms ranging from fly, to mouse, and to human. Presynaptic homeostatic plasticity is initiated by a reduction of glutamate receptor function at the postsynaptic side, but is expressed as an enhancement of presynaptic neurotransmitter release. Therefore, retrograde signaling is required to offset the postsynaptic perturbation, and to restore muscle excitation to its initial baseline level. We previously demonstrated that α2δ-3, an auxiliary subunit of presynaptic calcium channels, is required for presynaptic homeostatic plasticity. Loss of α2δ-3 blocks both the rapid induction and sustained expression of homeostatic plasticity, due to a failure to potentiate presynaptic calcium influx. α2δ proteins reside at the extracellular face of presynaptic release sites, an ideal location for mediating rapid, homeostatic signaling. But how the presynaptic α2δ-3 protein functions as part of this retrograde signaling system, to receive and relay information across the synapse, remains to be elucidated. By using the α2δ-3 protein as bait, we have identified putative α2δ-3 binding partners localized in the postsynaptic compartment with mass-spectrometry method. We hypothesize that the biochemical interaction between presynaptic α2δ-3, and its postsynaptic binding partners, are critical for the transsynaptic homeostatic plasticity mechanisms necessary to stabilize synaptic physiology. We propose to first perform formal genetic and biochemical analyses, to study the function of the putative retrograde signaling molecules have been identified. Second, we will perform functional studies to explore the molecular and cellular mechanisms underlying retrograde signaling in presynaptic homeostatic plasticity, through electrophysiological, biochemical, calcium imaging, and super-resolution imaging methods. Together, the results of these studies will advanc...