A Distinct Layer 1 Astrocyte Program Shapes Perisynaptic Structure and Calcium Signaling in Mouse Motor Cortex

Summary Although layer-specific molecular and morphological diversity among cortical astrocytes is increasingly well established, how these distinct states are linked to specialized calcium signaling and maintained in the adult cortex remains unclear. Here, combining super-resolution structural imaging, two-photon calcium imaging, and analysis of public single-cell and spatial transcriptomic resources, we identify Layer 1 (L1) astrocytes in mouse primary motor cortex as a distinct superficial astrocyte program. These cells occupy compact territories yet contain dense synapse-associated loop-like structures and display frequent, fast, broadly spreading calcium events that engage a large fraction of the territory. Transcriptomic analyses identify Id1 and Id3 as enriched components of this superficial program. CRISPR–Cas9 deletion of Id1 and Id3 in adult astrocytes selectively disrupts L1 and superficial Layer 2/3 (L2/3) astrocytes, expanding territory size, reducing fine-process complexity, and suppressing calcium activity. Thus, adult layer-specific transcriptional programs maintain specialized astrocyte structure and signaling. Graphical Abstract Highlights Layer 1 astrocytes in mouse motor cortex have compact, loop-rich nanoarchitecture Layer 1 astrocytes show fast, frequent, widespread calcium events Single-cell data define a superficial astrocyte state enriched for Id1 and Id3 Adult Id1/Id3 loss selectively disrupts Layer 1 astrocyte structure and calcium In Brief Bhattacharjee et al. , identify a specialized Layer 1 astrocyte state in mouse motor cortex, defined by compact, loop-rich nanoarchitecture and unusually high calcium signaling. Id1 / Id3 deletion selectively disrupts this superficial program, linking layer-specific astrocyte form and function to adult transcriptional control.