Abstracts

Rationale: Differences in cytoplasmic chloride concentrations ([Cl-]i) of neurons lead to oppositely directed chloride flow, GABA signaling, and anticonvulsant effects. Subcellular variations in the direction of Cl- currents elicited by GABAA receptor activation have been reported for over 10 years, but the reasons for this variance remain controversial and poorly understood. We tested whether subcellular variance in cytoplasmic Cl- concentrations underlies these findings. Methods: We determined the local [Cl-]i by 3 different highly sensitive methods: 1. Two-photon imaging of SuperClomeleon, a ratiometric Cl--sensitive fluorophore consisting of two fluorescent proteins CFP and YFP, joined by a short polypeptide linker which allows FRET-based imaging. 2. Fluorescence Lifetime IMaging (FLIM) of a Cl--sensitive dye, MEQ (6-methoxy-N-ethylquinolinium iodide) delivered to the cytoplasm via whole-cell recording pipette. Unlike SuperClomeleon, MEQ is insensitive to pH, while for both fluorophores the Cl--sensitive signal is independent of the concentration of dye. 3. Electrophysiological measurements of the reversal potential of membrane currents elicited by local application of GABA (EGABA) using whole-cell and gramicidin-perforated patch-clamp recordings and calculation of [Cl-]i based on Nernst equation. Results: All three methods show evidence of [Cl-]i microdomains in dendrites of individual neurons. In addition, there are highly significant correlations between [Cl-]i measured by fluorescent imaging and FLIM with [Cl-]i calculated based on EGABA and Nernst equation. Our data demonstrate that [Cl-]i varies in different segments of dendrites, that the borders of these microdomains are highly stable over a course of an hour, and that stability is unaffected by pharmacological inhibition of cation-chloride cotransporters. To determine whether the Cl- microdomains participate in regulation of inhibitory synapses, we recorded single pyramidal cells by whole-cell patch clamping while stimulating different inhibitory interneurons by puffing glutamate to their cell bodies. Our data indicate that there is a wide range in EGABA at different interneuron-pyramidal cell synapses. Conclusions: These findings are novel and point to the presence of functionally significant dendritic Cl- microdomains that regulate the impact of GABAergic inputs. In addition, they provide new insight into neuronal chloride homeostasis and the existence of additional regulatory mechanisms for the stability of cytoplasmic Cl- concentrations. Funding: These studies were supported by NINDS grant R01NS040109.