High sensitivity imaging of intracellular Cl ^- reveals the spatiotemporal evolution of GABAergic chloride fluxes in vitro and in vivo

Fast inhibition in the brain is primarily mediated by chloride currents through the GABA _A and glycine receptors. The driving force of these currents depends on the intracellular concentration of chloride ([Cl ^- ] _i ) that is an important determinant of inhibitory drive and neuronal processing. In physiological conditions, baseline [Cl ^- ] _i is modulated on a rapid timescale by Cl ^- fluxes through inhibitory synapses. The spatiotemporal dynamics of the [Cl ^- ] _i landscape provides important information on the nature, localisation and kinetics of inhibitory inputs. The study of these processes with sufficient spatial and temporal resolution in large neuronal populations requires in vivo imaging, but no existing tool has the required sensitivity for Cl ^- and photostability. Here we introduce iClima (improved Chloride Imaging), a novel ratiometric genetically encoded sensor with high Cl⁻ affinity (K _d =3.5 mM at pH 7.2) and high resistance to photobleaching. Critically, iClima has a pKa of 7.84 that renders it largely pH-insensitive under physiological conditions. We demonstrate that iClima can detect Cl ^- transients in response to GABA _A activation in vitro , and to sensory stimulation in pyramidal neurons in the mouse visual cortex, in vivo . Finally, by exploiting the spectral properties of iClima and of the Ca ²⁺ sensor GCaMP6f, we demonstrate measurements in the same cell of both Ca ²⁺ and Cl ^- transients. Together, these results establish iClima as a versatile platform for dissecting inhibitory circuit dynamics, from the subcellular scale of synaptic Cl⁻ microdomains to the network scale of seizure-related Cl⁻ dysregulation.