PROJECT SUMMARY Astrocytes influence neuronal circuit assembly and function and have been shown to respond, modulate, and drive disease pathogenesis. Despite this, relatively little is known about astrocyte diversity across brain regions, development, and species. However, recent single-cell RNA sequencing studies demonstrate that astrocytes have significant transcriptomic heterogeneity across the brain. Detailed molecular and cellular characterization of these subpopulations is needed to determine whether molecularly-defined astrocyte subpopulations serve distinct functions in both health and disease. Thus, the goal of this project is to use cutting-edge techniques to characterize the transcriptomic, structural, and functional diversity of astrocyte subpopulations across developmental stages and disease-relevant brain regions in the mouse and marmoset brain. By conducting a detailed analysis of over 70,000 astrocyte nuclei across eight brain regions in the young adult marmoset, I have discovered striking regional heterogeneity among astrocytes, particularly between cortical and subcortical regions. In this proposal, I aim to make three significant advances: a) map the evolution of astrocyte regional heterogeneity across development, b) compare this heterogeneity between rodent and non-human primate species, and c) characterize the functional identities of transcriptomically distinct astrocyte subtypes. To address the first two of these, I will conduct single-nucleus RNA sequencing of four brain regions in the embryo, neonate, juvenile, and adolescent marmoset and mouse. To address the third, I will use expansion microscopy to assess whether molecularly-defined astrocyte subtypes have distinct morphologies, ensheathment of synapses, and/or configuration of ion channels at the nanoscale. Additionally, I will image astrocyte calcium dynamics, considered key components of signaling processes used by astrocytes to regulate neuronal networks, in acute brain slices from both mouse and marmoset to characterize the functional diversity of transcriptomically-defined astrocyte subtypes. The proposed study will mark the first cross-species, cross-development, cross-region molecular profile of brain cells using consistent experimental and computational methodology, and will create new knowledge about the development of astrocyte heterogeneity across brain regions in rodents and non-human primates. Importantly, this detailed molecular and cellular characterization of astrocyte subpopulations will facilitate their precise manipulation for basic and translational research on neurodevelopmental and psychiatric disorders. I will carry out this project in the labs of Dr. Guoping Feng and Dr. Ed Boyden in the Brain and Cognitive Sciences Department at the Massachusetts Institute of Technology. The Feng and Boyden labs contain all required equipment for the proposed project. All necessary training regarding required laboratory and computational techniques will be pr...