Organization of Astrocytic GLT‐1 at Cortical Inhibitory Synapses

Glutamate spillover from excitatory synapses modulates neighboring inhibitory synapses, yet the ultrastructural organization of the major glutamate transporter GLT‐1 at these sites remains poorly defined. Using quantitative pre‐embedding electron microscopy in rat and human cortex, we found that GLT‐1‐positive astrocytic leaflets (ALs) were frequently juxtaposed to morphologically identified symmetric synapses, with similar prevalence across axo‐somatic, proximal axo‐dendritic, and distal axo‐dendritic subtypes. Because inhibitory synapses are embedded in a dense excitatory neuropil, we applied distance‐based phenotyping relative to the nearest asymmetric synapse to define symmetric‐associated GLT‐1+ ALs. Within this population, distal axo‐dendritic symmetric synapses showed shorter AL‐to‐synaptic‐edge distances and were embedded in a tighter local excitatory microenvironment. Post‐embedding immunogold further showed that GLT‐1 was enriched at the plasma membranes of ALs and localized extrasynaptically relative to symmetric synapses. Consistently, symmetric‐associated membrane GLT‐1 and closely spaced GLT‐1/α2 couples (with an interdistance ≤ 50 nm) were preferentially localized within 1000 nm of distal symmetric synapses compared to proximal. Similar organizational features of membrane GLT‐1/α2 couples were observed in human cortex. These findings identify a subtype‐dependent extrasynaptic astrocytic GLT‐1 organization at cortical inhibitory synapses and provide a morphological framework for glutamate‐dependent modulation of inhibitory signaling.