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.