iClima: a ratiometric genetically encoded sensor for optical imaging of fast GABAergic chloride currents in vitro and in vivo

Fast GABAergic inhibition in the brain is mediated by chloride currents whose driving force depends on intracellular Cl ⁻ concentration ([Cl ⁻ ] _i ). Resolving the spatiotemporal dynamics of [Cl ⁻ ] _i in neurons requires a sensor combining high Cl ⁻ affinity, pH robustness, and photostability - properties no existing tool achieves simultaneously. Here we introduce iClima (improved Chloride Imaging), a ratiometric genetically encoded sensor with high Cl ⁻ affinity (K _d =3.5 mM at pH 7.2), largely pH-insensitive under physiological conditions, and markedly improved photostability compared to existing sensors. These properties enable dynamic Cl ^- imaging without simultaneous pH correction. We demonstrate that iClima resolves GABA _A -driven Cl ⁻ transients in cultured neurons, detects discrete Cl ⁻ hotspots along dendrites, and captures sensory-evoked Cl ⁻ transients in vivo . Finally, exploiting the spectral compatibility of iClima with GCaMP6f, we simultaneously measure inhibitory Cl ⁻ and excitatory Ca ²⁺ in the same neurons in vivo , revealing that somatic inhibitory drive is orientation-invariant while excitatory output is sharply tuned.