Efficacy of Synaptic Inhibition Depends on Multiple, Dynamically Interacting Mechanisms Implicated in Chloride Homeostasis
Figure 2
Measurements of [Cl−]i in neurons with MQAE using Fluorescence Lifetime Imaging Microscopy (FLIM).
A. Two-photon excitation fluorescence of MQAE-loaded hippocampal neurons (26 DIV). The mean intensity of MQAE fluorescence within the cell bodies 1 & 2 was significantly different (left), which could be interpreted as indicating different levels of [Cl−]i or different dye uptake and accumulation between the two cells. The lifetime maps of the same cells are shown in the micrograph on the right. Note how, in contrast to intensities, the fluorescence lifetime of both cells were not significantly different indicating that there were no difference in [Cl−]i between the two cells. Values are mean ± S.D. of all pixels in each cell body. B. Measurements of MQAE lifetime at different [Cl−]i inside the cell body after membrane permeabilization and equilibration with [Cl−]o at 8, 15 or 20 mM (N = 73 cells/12 coverslips). According to the Stern-Volmer equation: τ0/τ = 1 + Ksv[Cl−]. The measured Ksv from these data was 32 M−1, consistent with previous reports [86]. C. Effect of increasing concentration of furosemide (to block KCC2) on [Cl−]i in cultured neurons exposed to 100 µM muscimol (to evoke a constant Cl− load by opening GABAAR; N = 75 cells/10 coverslips). D. The selective KCC2 antagonist VU 0240551 caused a dose-dependent significant increase in [Cl−]i (p<0.05), but bumetamide had no significant (n.s.) effect alone or after blocking KCC2 with VU 0240551, indicating lack of significant NKCC1 transport in these cells (N indicated in each bar = cells/coverslips; ***, p < 0.001).