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

Most genetically encoded sensors for mesoscopic cortical imaging target excitatory signals, leaving inhibitory dynamics less understood. Capturing large-scale extracellular GABA activity in vivo is essential for understanding how inhibition shapes cortical processing across brain states. To validate the genetically encoded sensor iGABASnFR2 for mesoscale in vivo imaging of extracellular GABA dynamics and to assess how inhibitory activity reorganizes with sensory input, behavioral state, and pharmacological manipulation. We used wide-field imaging in head-fixed mice expressing iGABASnFR2 via retro-orbital AAV injection. Imaging was performed under anesthesia, during quiet wakefulness, natural sleep (NREM, REM), and after administration of the GABA reuptake inhibitor Tiagabine. We analyzed sensory-evoked and spontaneous GABA signals using seed-pixel correlation and spectral analyses. iGABASnFR2 revealed delayed, modality-specific inhibitory responses to sensory stimulation that were stronger and faster in the contralateral cortex. These responses were conserved between anesthesia and quiet wakefulness. During spontaneous activity, GABAergic networks showed state-dependent reorganization: widespread bilateral synchrony was observed in NREM and wakefulness, while REM sleep showed reduced GABA levels and decoupling. Tiagabine elevated baseline GABA, prolonged sensory responses, and enhanced long-range inhibitory connectivity. iGABASnFR2 enables reliable, real-time imaging of extracellular GABA across large cortical areas. These findings demonstrate its utility for tracking state-dependent inhibitory motifs and highlight the dynamic architecture of cortical inhibition in health and disease.