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. We found a corresponding variance in the GABA_A receptor reversal potential (E_GABA) for synapses originating from individual interneurons onto a single pyramidal cell. These findings suggest a similar heterogeneity in the cytoplasmic intracellular concentration of chloride ([Cl^-]_i) in individual dendrites. We determined [Cl^-]_i in the murine hippocampus and cerebral cortex of both sexes by (1) two-photon imaging of the Cl^--sensitive, ratiometric fluorescent protein SuperClomeleon; (2) Fluorescence Lifetime IMaging (FLIM) of the Cl^--sensitive fluorophore MEQ (6-methoxy-N-ethylquinolinium); and (3) electrophysiological measurements of E_GABA by pressure application of GABA and RuBi-GABA uncaging. Fluorometric and electrophysiological estimates of local [Cl^-]_i were highly correlated. [Cl^-]_i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters, but were progressively modified after inhibiting the polymerization of the anionic biopolymer actin. These methods collectively demonstrated stable [Cl^-]_i microdomains in individual neurons in vitro and in vivo and the role of immobile anions in its stability. Our results highlight the existence of functionally significant neuronal Cl^- microdomains that modify the impact of GABAergic inputs.SIGNIFICANCE STATEMENT Microdomains of varying chloride concentrations in the neuronal cytoplasm are a predictable consequence of the inhomogeneous distribution of anionic polymers such as actin, tubulin, and nucleic acids. Here, we demonstrate the existence and stability of these microdomains, as well as the consequence for GABAergic synaptic signaling: each interneuron produces a postsynaptic GABA_A response with a unique reversal potential. In individual hippocampal pyramidal cells, the range of GABA_A reversal potentials evoked by stimulating different interneurons was >20 mV. Some interneurons generated postsynaptic responses in pyramidal cells that reversed at potentials beyond what would be considered purely inhibitory. Cytoplasmic chloride microdomains enable each pyramidal cell to maintain a compendium of unique postsynaptic responses to the activity of individual interneurons.