Abstract Brain wiring makes us who we are, but our understanding of the wiring mechanism is still in its infancy. Synapses, the structural units for transmitting electrochemical signals between neurons, form the basis of brain wiring and the specificity of synaptic connections determines brain function. During development, the nervous system acquires an excess of synapses that undergoes refinement, to optimize the signal-to-noise ratio. Developmental brain wiring is refined in part through synaptic pruning, which eliminates weak synapses allowing for strengthening of those retained. Synapse formation and elimination also persist in the mature nervous system through experience-dependent structural synaptic plasticity, which is the basis of learning. Therefore, synaptic formation and pruning are crucial not only in shaping neural circuits during development but also in regulating synaptic plasticity in response to experience and memory. Defects in synaptic pruning and maintenance have been implicated in neurodevelopmental disorders and neurodegeneration. Although synapses functionally connect neurons, the glial support cells such as microglia and astrocytes carry out the process of synapse pruning. For example, genetic manipulation of microglial complement and fractalkine receptor pathways in mice has conclusively demonstrated their involvement in synapse pruning. TAMs, a family of three related receptors, are important for astrocytic (but apparently not microglial) synaptic pruning. However, the full spectrum of molecular components involved in this process remains to be defined. GPR56 is a member of the adhesion-GPCR family, conserved between rodents and humans. Our unpublished preliminary studies showed (1) deleting microglial Gpr56 from mouse microglia leads to a significantly increased retinal innervation in dorsal lateral geniculate ganglion (dLGN); (2) GPR56 binds both phosphatidylserine (PtdSer) and the Gla domain of GAS6; and (3) deleting Gas6 leads to a significantly reduced synaptic density in dLGN. TAM receptors require adaptor protein for their interaction with PtdSer and GAS6 is one such adaptor. However, triple deletion of TAM receptors leads to an opposite phenotype from Gas6 knockout. Therefore, we argue that GAS6 does not function through TAM receptors in astrocyte- mediated synaptic pruning. Instead, taking together our preliminary data and others' published results, we hypothesize that GPR56 regulates microglia-mediated developmental synapse refinement by binding to PtdSer and that GAS6 modulates the degree of microglial GPR56 mediated synaptic pruning by competing GPR56- PtdSer interaction. Our present proposal is designed to test this hypothesis using genetic models, electrophysiological analysis, as well as biochemical and cellular assays. The success of the proposed research will establish a novel mechanism and signaling pathway in regulating developmental synapse refinement. We intend to lay the foundation to investigate t...