PROJECT SUMMARY Neuronal synaptic connections are essential for executing the distinct tasks that are required for brain homeostasis and function. Disturbances to synaptic biology during development or in mature neuronal networks are a key hallmark of most neurodevelopmental disorders and neurodegenerative diseases, respectively. Thus, a better understanding of the mechanisms that govern the establishment and maintenance of synapses could provide new insights into the origins of these disorders and potential therapeutic opportunities. Extra-neuronal influences, whether via direct cellular contact or released proteins, have been demonstrated to be critical in governing synaptic physiology. Outside of the broad metabolic responsibilities that astrocytes fulfill in the brain during normal physiology, it is becoming increasingly clear that astrocytes are instrumental in modulating neuronal synaptic connections. Specifically, astrocytic interactions with neuronal synapses are increasingly implicated in 1) the establishment of proper synaptic connections and 2) dysfunctions that negatively impact synaptic biology. Our group was the first to document the involvement of alternative Wnt ligand, Norrin, in synaptic properties—a protein long associated with the X-linked genetic disorder Norrie Disease (ND). Namely, Norrin enhanced neuronal firing frequency and network connectivity in in vitro cultures and caused synaptic abnormalities in vivo in adult Norrin-null mice. However, the mechanistic basis of these findings remains unknown, as well as whether Norrin is important in synaptogenesis. Thus, the overall goal of this proposal is to define the role that Norrin plays in synaptic biology, including the establishment of these connections during development and maintenance in adulthood. Additionally, this study will be the first to use a humanized mouse model of ND to provide characterization of the inherent and synaptic properties of ND cortical neurons. These objectives will be pursued with murine models, including Norrin-null mice and human Norrin gene point mutation (V45E) mice. First, I will determine the physiological mechanism by which Norrin mediates enhanced neuronal firing frequency and connectivity using whole-cell patch clamp electrophysiology in in vitro and ex vivo preparations. Additionally, I will use molecular biology and imaging techniques to evaluate Norrin’s effect on synaptic neurotransmitter receptor expression. Secondly, I will investigate the involvement of Norrin in synaptogenesis by utilizing iDISCO, single-cell RNAseq, and RNAscope in the postnatal mouse brain. Using the V45E mice I will characterize the electrophysiological properties of cortical neurons in the mature ND brain. Lastly, utilizing high-content in vitro and in vivo imaging, real-time synaptic dynamics will be evaluated in conditions of functional and dysfunctional Norrin across the synaptogenesis period. This proposal will provide novel insights into Norrin-mediated ...