Prolonged Hyperactivity Elicits Massive and Persistent Chloride Ion Redistribution in Subsets of Cultured Hippocampal...

Chloride ions play a critical role in neuronal inhibition through the activity of chloride-permeable GABA_A receptor channels. Ion transporters, chloride channels, and immobile ion species tightly regulate intracellular chloride concentrations. Several studies related to epilepsy suggest that chloride extrusion function may decrease in an activity-dependent manner. Consequently, it is crucial to investigate whether intense neuronal activity, as observed during status epilepticus, could lead to sustained increases in intracellular chloride levels in neurons, which in turn could contribute to epilepsy-associated hyperexcitability. This study utilized the chloride sensitive indicator (6-Methoxyquinolinio) acetic acid ethyl ester bromide (MQAE) combined with fluorescence lifetime imaging (FLIM) to examine whether application of the convulsant, pilocarpine, a muscarinic acetylcholine receptor agonist, could induce synchronous epileptiform activity and elevate intracellular chloride concentrations in hippocampal slice cultures. Using a Gaussian mixture model, we identified a multimodal distribution of intracellular chloride levels among neurons, with a significant subset of these cells exhibiting massive and prolonged (days) chloride accumulation. The combination of multicellular imaging and statistical analysis served as a powerful tool for studying the emergence of multiple, distinct populations of neurons in pathological conditions, in contrast to homogeneous populations evident under control conditions.