Characterization of iGABASnFR2 for in vivo mesoscale imaging of intracortical GABA dynamics.

SIGNIFICANCE: Although genetically encoded sensors have advanced the study of cortical excitation, tools for large-scale imaging of inhibition remain limited. Visualizing extracellular gamma-aminobutyric acid (GABA) dynamics in vivo is essential for understanding how inhibitory networks shape brain activity across sensory, behavioral, and pharmacological states. AIM: Our aims are to validate and apply the genetically encoded sensor iGABASnFR2 for wide-field imaging of extracellular GABA and to characterize how cortical inhibition reorganizes across brain states, sensory modalities, and after GABA transporter blockade. APPROACH: We performed mesoscale imaging in head-fixed C57BL/6 mice systemically expressing iGABASnFR2. Recordings were conducted under isoflurane anesthesia, during quiet wakefulness, natural sleep [non-rapid eye movement (NREM) and rapid eye movement], and after administration of the GAT-1 inhibitor tiagabine. We analyzed both sensory-evoked and spontaneous GABA signals using time-series, spectral, and seed-pixel correlation analyses. RESULTS: iGABASnFR2 demonstrated strong and modality-specific GABAergic responses to sensory stimulation, with faster and stronger activation in the contralateral cortex. Although the general spatial patterns of sensory-evoked GABA responses were consistent across anesthesia and quiet wakefulness, the amplitude, timing, and spread of these responses were significantly greater during wakefulness. During spontaneous activity, cortical GABA levels and connectivity modulated by brain state: GABA amplitude and interhemispheric synchrony, were highest during quiet wakefulness but reduced during NREM sleep. Tiagabine elevated baseline GABA levels, abolished stimulus-evoked responses, and enhanced local and long-range inhibitory synchrony. CONCLUSIONS: iGABASnFR2 enables reliable, high-resolution imaging of cortical GABA dynamics in vivo. These results demonstrate that inhibitory signaling is dynamically structured across brain states and can be pharmacologically modulated. This tool offers opportunities to explore the role of inhibition in health and disease at the mesoscale level.