Optogenetic determination of dynamic and cell-type-specific chloride equilibrium potentials

Optogenetics has revolutionized neurobiological research by providing tools for modulating neuronal activity. As these tools utilise light-activated ion fluxes, they afford new opportunities to examine the nature of transmembrane ion gradients. Traditional investigation into the equilibrium potential for chloride (E Cl ) has been limited to studying endogenous chloride-permeable receptors. Here we demonstrate the utility of using a light-activated chloride channel, stGtACR2, to probe somatic E Cl in rodent. This agonist-independent optogenetic strategy is validated in vitro and in vivo , captures differences in E Cl dynamics following manipulations of endogenous chloride fluxes, and reveals distinct resting E Cl across genetically-defined neuronal subpopulations. Using this approach to challenge chloride homeostasis, we uncover cell-specific E Cl dynamics that are supported by the differential expression of endogenous handling mechanisms. Our findings establish an optical method for investigating transmembrane chloride gradients and thereby expand the repertoire of optogenetics.