"While emerging body of evidence implicates microglia in synaptic pruning and indicates the role of complement system in mediating phagocytosis of synaptic elements by surrounding microglia, no neuronal signals that label synapses according to their activity and maturity have been identified yet. Here we propose to develop an organotypic hippocampal slice culture system to investigate synaptic pruning during development, and to use it to identify candidate molecular tags that mediate discrimination between weak and strong synapses during circuit maturation. We will test two candidate molecules: ""don’t-eat-me"" signal sialic acid and ""eat-me"" signal phosphatidylserine. Using time-lapse spinning disc microscopy of live organotypic slices, pharmacological manipulations and optogenetics we will observe local neural activity-dependent dynamics of desialyation and phosphatidylserine exposure on synapses. Organotypic hippocampal cultures prepared from genetically modified animals lacking sialidase or phospholipid scramblase as well as their chimeras with wild-type animals will help to validate the role of candidate phagocytic signals in synaptic pruning and to determine whether these signaling molecules are cell-autonomous or disturb the system as a whole. Since aberrant or impaired synaptic pruning is hypothesized to be involved in the pathology of various neurodevelopmental disorders, such as autism and Rett Syndrome, as well a mental illness, such as schizophrenia, uncovering the mechanisms that mediate circuit formation and refinement are crucial for understanding these disorders and to develop new approaches to their treatment. The research proposed here will provide invaluable insight into the cellular and molecular mechanisms by which microglia refine circuits and contribute to synaptic plasticity, help us to better understand how the immune system and brain interact, and how circuit wiring can become pathologically altered."