Context-dependent localization and expression of glutamate and GABA and their machinery in microglia

Microglia are central regulators of brain homeostasis and synaptic architecture, but whether they, like astrocytes, act as reservoirs and handlers of glutamate and GABA is unknown. Using post-embedding immunogold electron microscopy, we provide the first ultrastructural evidence that individual microglial processes contain GABA and glutamate. At the nanoscale, glutamate-positive microglial processes were preferentially apposed to glutamatergic terminals, whereas GABA-positive processes showed no consistent spatial relationship to GABAergic boutons. At the microscale, spatial transcriptomics revealed that microglial somata expressing GABA-handling genes are enriched in GABAergic neuron-rich regions, while microglia expressing glutamate-handling genes are enriched in glutamatergic neuron-rich regions. Analysis of four single-cell and single-nucleus RNA-sequencing datasets (6,594,428 cells, including 320,683 microglia) demonstrated conserved expression of glutamate and GABA transport and metabolism genes in microglia across species, corroborated at the protein level by proteomics and in situ by confocal microscopy. Functionally, acute neuroinflammation selectively depleted microglial GABA content by 57% while leaving glutamate unchanged, indicating differential vulnerability of these amino acid pools. Together, these multimodal data identify microglia as previously unrecognized reservoirs of the brain's principal inhibitory and excitatory transmitters, with spatially adapted handling machinery, and implicate them as potential contributors to excitation-inhibition homeostasis within the quad-partite synapse.