Astrocytes connect specific brain regions through plastic networks | Nature

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Subjects

- Astrocyte

- Cellular neuroscience

- Light-sheet microscopy

- Molecular neuroscience

- Super-resolution microscopy

Abstract

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 plasticity3,4,5, 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.

Main

Astrocyte intercellular communication is critical to proper CNS function. This communication occurs through gap junctions—membrane channels that connect the cytoplasm of neighbouring cells, enabling them to redistribute resources and share biochemical signals. Studies using mice lacking astrocyte gap junctions have shown that these gap junctions are necessary for memory formation1,2, synaptic plasticity3,4,5, coordination of neuronal signalling6, and closing the visual and motor critical periods7,8. In disease, networks of gap junction-linked astrocytes redistribute metabolic resources across the CNS to protect degenerating neurons11,12. Despite these insights, our understanding of the spatial architecture and functional topology of astrocyte networks remains limited. Existing methods, such as dye diffusion in acute slices or reporter activation in injury models, are inherently constrained to local environments and often disrupt native connectivity. As a result, it remains unclear whether astrocytes form a continuous brain-wide syncytium or operate through discrete, region-specific subnetworks. It is also unclear whether their anatomical connectivity aligns with neuronal networks or establishes an independent framework for long-range, non-neuronal signalling.

Tracing astrocyte gap junction networks

To address this knowledge gap, we developed a vector-based approach to express a fusion protein comprising connexin 43 (Cx43, encoded by Gja1), the main gap junction protein used by astrocytes, and TurboID (TID), a rapid and promiscuous biotinylating enzyme13,14,15, under the shortened Gfap promoter16,17,18 (AAV5-GfaABC1D-Cx43:TID:HA; Fig. 1a). When this fusion protein incorporates into a connexon as any one of its six constituent connexins (Fig. 1b), molecules that flux through the infected astrocyte’s gap junctions are rapidly tagged with small, inert biotin (Fig. 1c). This enables us to detect the infected astrocyte population (through the haemagglutinin (HA) tag on the fusion protein), the in-network astrocytes (by staining biotinylated moieties with streptavidin) and cells that are not in-network with the infected population (gaps in astrocyte tiling; Fig. 1d). As the mouse CNS has minimal-to-no native biotin13,14,15, streptavidin staining provides little background. Moreover, each biotin can bind to only a single streptavidin, eliminating variability introduced through antibody multiplexing and making images more quantitative.

Fig. 1: Visualizing astrocyte gap junctional communication using the astrocyte network tracer.The alternative text for this image may have been generated using AI.Full size image

a, Diagram of the astrocyte network tracer construct. b, Cx43 connexons contain six connexins, only one of which needs to be the fusion protein for TID to reside on the gap junction. c, Infected astrocytes biotinylate molecules that flux through Cx43 gap junctions into adjacent, uninfected cells. d, This volume-fills in-network ast