Abstracts

Rationale: In the central nervous system, transmembrane chloride (Cl-) gradients play a crucial role in regulating inhibitory function generated by Cl--permeable GABAA receptors. A recent study by Glykys et al. (1) suggested that impermeant anionic species in both cytosol and extracellular space may be a primary influence regulating Cl- ion distribution and GABAA receptor mediated synaptic transmission, with transmembrane Cl- transporters serving only a very minor role under normal conditions. In this study, we used two-photon excitation fluorescence lifetime imaging (FLIM) of the halide ion sensing dye, N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide (MQAE), to quantitatively assay the intracellular distribution of Cl- and other anionic species, and determine the relative contributions of impermeant anions and chloride transporters to transmembrane Cl- gradients.Methods: Mouse organotypic hippocampal slice cultures were prepared from P3-8 mice, and maintained for 3 weeks. Cultures were stained with 1 mM MQAE solution for 15 minutes. We used a two-photon microscopy system with a femtosecond laser (SpectraPhysics MaiTai DeepSee) tuned at 780 nm. The emission between 440 nm and 500 nm was collected with a GaAsP PMT. The PMT signal was processed with a Becker-Hickl time-correlated photon counting system for FLIM imaging. Becker-Hickl SPC Image software was used to analyze FLIM data.Results: MQAE fluorescence lifetimes in hippocampal dentate granule cells were measured under control conditions, and in the presence of Cl- transporter inhibitors during GABA bath application. Blocking the potassium-chloride co-transporter, KCC2, with furosemide or bumetanide application induced shorter fluorescence lifetimes due to higher [Cl-]i. In addition to Cl-, the fluorescence lifetime of MQAE was also sensitive to the presence of other anions, with significant effects of HEPES > gluconate > phosphate > bicarbonate, and no effect of small anions such as NO3-. MQAE fluorescence lifetime was estimated to 38 nanoseconds (ns) in pure water, while lifetimes were reduced to 28 ns in the presence of 40 mM phosphate and 17 ns in the presence of 80 mM gluconate ions. MQAE lifetimes with bicarbonate concentration varied from 10 mM to 80 mM were unchanged (24 ns). MQAE lifetimes in dentate granule cell cytosol with minimal chloride ion concentration induced by membrane Cl- permeabilization with tributyltin chloride and nigericin was 6 ns.Conclusions: MQAE lifetime in cytosol was A) significantly altered by furosemide and bumetanide application, and B) was significantly shorter than expected from solution calibrations, suggesting that MQAE lifetime is influenced both by Cl- ion transporters and also by other intracellular impermeant anionic species. (1) Glykys J., Dzhala V., Egawa K., Balena T., Saponjian Y., Kuchibhotla K. V., Bacskai B. J., KahleK. T., Zeuthen T., Staley K.J., Science 343:670-675, 2014. This work is supported by NIH R01NS082046-01.