Project Summary/ Abstract Though synapses are formed between neurons, these structures are contacted with, ensheathed, and regulated by astrocytes. Period of neuronal synaptic connectivity and that of astrocyte maturation overlaps in developing brain. Neuronal signals instruct astrocyte differentiation and morphological maturation whereas astrocytes provide metabolic and trophic factors to support neuronal survival and growth. However, molecular mechanisms and signals that regulate neuron-astrocyte interactions and their role in neuronal circuit assembly and functions are largely unknown. We and others have previously shown that astrocytes modulate specific neural circuit formation, function and plasticity by several secreted proteins including hevin (SPARCL1), thrombospondins (TSPs), glypicans and norrin. While hevin is needed for assembly and plasticity of VGlut2+ (vesicular glutamate transporter 2) thalamocortical connections, TSPs facilitate VGlut1+ synapse formation. Intriguingly, expression of these synaptogenic proteins is developmentally regulated and are also altered in brain pathologies. Although a significant amount of research has been done to identify the neuronal receptors and mechanism of synapse formation by astrocyte-secreted synaptogenic factors SPARCL1 and TSPs, we do not know the signals and mechanisms that regulate their expression in astrocytes. We have recently found that neuronal contact stimulates expression of SPARCL1 and TSP4 via Sphingosine- 1-Phosphate (S1P)-S1P Receptor 1 (S1PR1). We also found that S1PR1 is primarily expressed by astrocytes and is localized to the fine astrocytic processes near and around the synapses and drives astrocyte morphological complexity and morphogenesis. Although, S1P-S1PR signaling is a drug target for many neurological disorders, its fundamental role in neuron-glia interactions and neuronal circuit assembly is not known. Our proposed studies will provide novel insight into the neuron-astrocyte bidirectional communication through S1P-S1PR1 axis in establishing synaptic connectivity and functions. Our detailed mechanistic studies will identify new signaling pathway downstream of S1P-S1PR1 axis in regulating calcium dynamics, glutamate sensing and expression of SPARCL1 and TSP4 in astrocytes. These studies will also advance our knowledge of how neurons regulate astrocyte development, morphogenesis and function. Moreover, these studies will decipher the mechanistic link between levels of S1P and the expression of SPARCL1 and TSP4 and clarify on the fundamental role of S1P/S1PR1 axis in the developing and diseased brain. This proposal thus is poised to provide novel mechanisms of targeting S1P/S1PR1 axis in alleviating neuropathologies.