Neuronal axons have traditionally been considered to be the primary mediators of functional connectivity among brain regions. However, the role of astrocyte-mediated communication has been largely underappreciated. Astrocytes communicate with one another through gap junctions, but the extent and specificity of this communication remain poorly understood. Astrocyte gap junctions are necessary for memory formation1,2, synaptic plasticity3–5, coordination of neuronal signalling6, and closing the visual and motor critical periods7,8. These findings indicate that this form of communication is essential for proper central nervous system development and function. Despite the importance of astrocyte gap junctional networks, studying them has been challenging. Current methods such as slice electrophysiology disrupt network connectivity and introduce artefacts due to tissue damage. Here, we developed a vector-based approach that labels molecules as they are fluxed by astrocyte gap junctions in awake, behaving animals to overcome these limitations. We then used whole-brain tissue clearing9,10 to image these intact, three-dimensional astrocyte networks. We show that multiple astrocyte networks traverse the mouse brain. These networks selectively connect specific regions, rather than diffusing indiscriminately, and vary in size and organization. We observe local networks that are confined to single brain regions and long-range networks that robustly interconnect multiple regions across hemispheres, often exhibiting patterns distinct from known neuronal networks. We also demonstrate that astrocyte networks undergo structural reorganization in the adult brain after sensory deprivation. These findings reveal a mode of communication between distant brain regions that is mediated by plastic networks of gap junction-coupled astrocytes.
