Unique actions of GABA arising from cytoplasmic chloride microdomains

Developmental, cellular, and subcellular variations in the direction of neuronal Cl − currents elicited by GABA A receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (E GABA ) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl − ([Cl − i ]). We determined [Cl − ] i by: 1) two-photon imaging of the Cl − sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl − sensitive fluorophore MEQ; and 3) electrophysiological measurements of E GABA . These methods collectively demonstrated stable [Cl − ] i microdomains in individual neurons in vivo . Fluorometric and electrophysiological estimates of local [Cl − ] i were highly correlated. [Cl − ] i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl − microdomains that modify the impact of GABAergic inputs.