Optogenetic determination of dynamic and cell-type-specific inhibitory reversal potentials.

The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh ) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAA R). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAA R receptor mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo, and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically-defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials, and thereby expand the repertoire of optogenetics. Significance statement The strength of synaptic inhibition in the brain is determined, in part, by the reversal potential of the ionic currents that flow through inhibitory ligand-gated ion channels (EInh ). Estimates of EInh have traditionally used agonists to activate receptors on the cell surface, but this has limitations. Our study presents an optogenetic strategy for performing agonist-independent measurements of EInh in the brain. We demonstrate the effectiveness of the approach in vitro , in vivo, and across different neuronal subtypes. Its excellent temporal control allows for measurements of EInh dynamics, which reveal differences between genetically-defined neuronal subpopulations. This expands the application of optogenetics and affords new opportunities to study synaptic inhibition.