This transformative award application is designed to test an integrative hypothesis about astrocyte function across the brain while extending the current limits of state-of-the-art technologies in cellular imaging and spatial transcriptomics. The team of investigators with cellular, molecular, systems, theoretical/computational neuroscience and biomedical engineering expertise will take a multipronged approach to test the central hypothesis that astrocytes serve the function of integrating neuromodulator signals in circuits across the brain. Astrocytes are by some accounts the most common cell in the brain. They have been historically viewed as support cells for neural circuits, but emerging evidence suggests that they are active participants in circuit functions. This is especially interesting because astrocytes are affected by stress, aging, and many diseases of the central nervous system. Remarkably, astrocytes express receptors for serotonin, norepinephrine, dopamine, and acetylcholine; the four neuromodulator systems that arise from deep structures and innervate most brain circuits. These neuromodulator systems have considerable overlapping circuit functions suggesting that a mechanism for integrating their inputs is important for disambiguating how each neuromodulator exerts its contribution to the circuit functions. This project will test the possibility that astrocytes provide such an integrator function. The investigators will use state of the arts techniques and develop new ones to test how astrocytes are affected by neuromodulators and how these effects impact circuit functions and behavior in two brain systems. In vivo studies will test the hypothesis on a cell population level manipulating each of the neuromodulator circuits while testing astrocyte responses to all four, circuit physiology, and behavior. Ex vivo studies will focus on single cell differences in astrocytes by combining functional studies that parallel in vivo ones. These spatially resolved functional studies will be aligned with spatial single cell transcriptomics providing gene expression data to parallel functional data at scale. Stress and aging are known to impact astrocytes and neuromodulator functions and adversely affect many neuropsychiatric diseases. The team will lastly establish how stress and aging impact the ability of astrocytes to integrate neuromodulator signals using their cutting edge techniques. Completing these ambitious studies will unequivocally test a transformative idea for an organizing principle around astrocyte function throughout the brain and inform how dysfunction of these cells can be contributing to diseases of the central nervous system.