KCC2 drives chloride microdomain formation in dendritic blebbing

Intracellular chloride ion concentration ([Cl^-]_i) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl^-]_i dynamics during N-methyl-d-aspartate (NMDA) excitotoxicity to determine how Cl^- changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl^- dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl^-]_i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl^-]_i subdomains at dendritic blebs in a process that requires Ca²⁺ influx and the subsequent opening of small-conductance Ca²⁺-activated K⁺ (SK) channels. We propose sustained and localized K⁺ efflux increased extracellular K⁺ concentrations ([K⁺]_o) sufficiently at discrete regions to reverse K⁺-Cl^- cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl^-]_i microdomains in blebbing with relevance for multiple neurological disorders.