iGABASnFR Imaging Reveals that GABA Clearance is rapidly overwhelmed in the Cerebral Cortex

Both phasic and tonic GABAergic activity in the cortex, is shaped by the clearance of GABA from the extracellular space by GABA transporters (GATs). However, estimates of the time-course of GAT activity from GAT currents, GABA_A, and GABA_B electrophysiological recordings range from tens of milliseconds to multiple seconds. These estimates may reflect receptor properties rather than extracellular GABA dynamics. Understanding how long GABA is free in the extracellular space is key to understanding how inhibition controls activity, but has been technically challenging. Here, we utilize the fluorescent GABA sensor, iGABASnFr, to enable us to optically assay extracellular GABA dynamics following bursts of synaptic activity. In the mouse cerebral cortex of either sex, we show that GABA rapidly disperses from release sites via diffusion, with very little role for active transport by GATs. Following diffusion from the sites of release, GABA then accumulates in the extracellular space, where GATs require hundreds of milliseconds to remove it. For areas of high density GABA release or during bursts of activity, this clearance is further slowed. Continued GABA release and summation in the extracellular space for bursts of activity contrasts with recorded Inhibitory post-synaptic current (IPSC) signals, which rapidly depress with additional stimuli, suggesting a disconnect between IPSCs and extracellular GABA. Lastly, the resulting extracellular accumulation of GABA can act hetero-synaptically by activating and/or desensitizing GABA_A receptors. Together, this raises new questions about how GABAergic inhibition works to shape network function and suggests a spatiotemporal continuum of extracellular GABA.Significance Statement We use combined iGABASnFr neurotransmitter imaging, electrophysiology, and modeling to show that with trains of synaptic activity, GABA clearance is slowed and GABA rapidly accumulates in the extracellular space. Additionally, we demonstrate that GABA largely diffuses from release sites rather than being captured by GABA transporters and that these transporters have only a small role in shaping extracellular GABA dynamics in the mouse cortex. Lastly, we show that extracellular GABA accumulation can heterosynaptically desensitize GABA_A receptors, modulating local phasic inhibition.