Synapses are the fundamental functional units of the nervous system, but the molecular and cellular interactions that regulate their establishment are largely unknown. Studies using purified retinal ganglion cell neurons (RGCs) showed that astrocytes secrete signals such as thrombospondins that strongly induce excitatory synapse formation. In our preliminary studies, we identified another astrocyte-secreted synaptogenic protein, hevin. Addition of hevin to purified RGC cultures robustly stimulates excitatory synaptogenesis. Moreover, Hevin-null mice have significantly less excitatory synapses that present striking structural defects suggesting that hevin is required for the formation and morphological maturation of synapses in vivo. Astrocytes also express a close homolog of hevin called SPARC. Intriguingly, SPARC is not synaptogenic but specifically inhibits hevin-induced synaptogenesis. These data show for the first time that astrocytes regulate synaptic connectivity not only by stimulating, but also by inhibiting synaptogenesis. Our results signify the exciting possibility that astrocytes, through the regulation of relative levels of hevin and SPARC, can actively control the development and function of synaptic networks in the developing and adult brain. How hevin induces synapse formation and the nature of SPARC's antagonistic function are unknown. Therefore, our objective here is to unravel a novel molecular mechanism of regulation of synaptic development and maintenance by astrocytes through hevin/SPARC signaling. In this application, we will first test the hypothesis that hevin and SPARC regulate synaptic morphology (Aim 1) and formation of dendritic spine synapses in vivo (Aim 2). Second, we will determine the contribution of hevin/SPARC signaling to synaptic function in vivo (Aim 2). Third, we will test the hypothesis that hevin mediates synaptogenesis through interactions with the trans-synaptic adhesion molecules neurexins and neuroligins, whereas SPARC antagonizes hevin by competing for hevin-binding to neuroligins (Aim 3). These studies are important since they will provide new insights into the control of formation, maintenance and function of synapses by astrocytes. These new insights will have a significant positive impact by advancing our molecular and cellular understanding of astrocyte-neuron interactions that orchestrate central nervous system development and function. A deeper mechanistic understanding of synapse formation and how astrocytes participate in this process will lead to the development of innovative approaches to prevent or cure neurological disorders such as autism, depression and addiction. PUBLIC HEALTH RELEVANCE: The proposed study aims to advance our understanding of astrocyte-neuron interactions that orchestrate central nervous system development and function. Understanding how synaptogenesis is regulated is crucial for understanding how our brains are sculpted during development, and how we learn and remember as adults. In addition, knowledge on how synaptogenesis can go awry is required for development of new prevention and treatment strategies against disorders that stem from dysregulated synapse formation such as Alzheimer's disease, epilepsy, autism and drug addiction.