Effect of carbonic anhydrase inhibitor on H+- and Cl--transients and epileptiform activity in brain slices of ClopHensor-expressing transgenic mice.

Alterations in H⁺ and Cl- concentrations are implicated in various neurological diseases, including epilepsy. Carbonic anhydrases (CAs) are crucial regulators of pH homeostasis, and certain CA inhibitors possess anticonvulsant properties. In this study, we investigated the effect of the CA inhibitor ethoxzolamide (EZA) on Cl- and H⁺ dynamics in hippocampal slices under control conditions and in an in vitro epilepsy model. We used transgenic mice expressing ClopHensor, a neuron-targeted sensor for simultaneous monitoring of Cl- and H⁺. Local field potential recordings and fluorescence imaging were performed in the CA1 area. Schaffer collateral stimulation evoked synaptic changes in H⁺ and Cl- concentrations, and epileptiform activity was induced by perfusing slices with 100 µM 4-aminopyridine (4-AP). EZA was applied under both control conditions and during the development of ictal-like events. In controls, EZA induced significant neuronal acidification without altering basal intracellular Cl- levels. Synaptically evoked H⁺ and Cl- transients were reduced by EZA in a concentration-dependent manner. In the 4-AP model, ictal-like discharges triggered robust transient increases in both ions, with mean changes of approximately 0.3 pH units and 40 mM for H⁺ and Cl-, respectively. EZA reversibly suppressed this ictal activity and prevented the associated transients, underscoring the CO2/HCO3- system's role in sustaining epileptiform synchronization. These findings not only confirm previous studies linking GABAergic Cl- influx to seizure initiation and propagation but also identify intracellular pH as a potential feedback modulator and reinforce the therapeutic potential of carbonic anhydrase inhibitors for the treatment of epilepsy.