Figure 4.

Evidence for the existence of chloride microdomains from in vitro sCLM imaging. A, sCLM was calibrated in organotypic slice cultures at different concentrations of Cl^– and in the presence of 50 µm of nigericin and 100 µm of tributyltin. With single-cell resolution, we calculated R_max, R_min, and K_d as described in Materials and Methods. Only neurons that responded to different Cl^– concentrations in extracellular media ([Cl^–]_o) were selected for final calculations. The range of values for R_max measured by two-photon microscopy in different calibration experiments was between 1.240 and 1.777. R_min ranged between 0.289 and 0.466. K_d was between 22.31 and 22.99 mm. For DualView imaging, R_max and R_min were 1.640 and 0.984 respectively. B, MEQ was calibrated using Stern–Volmer equation. MEQ was dissolved in the same solution as the internal pipette solution (K-MeSO₄-based) with different concentrations of Cl^–. C, D, Two-photon imaging of an organotypic slice culture showing a hippocampal interneuron (C) and the dendritic variations in [Cl^–]_i (D). E, A CA1 pyramidal cell from a hippocampal organotypic slice-expressing sCLM. F, Top, The rotated neuron in E with a red arrow. Bottom, The magnified pseudocolored [Cl^–]_i maps of different segments of the dendrite. G, Histogram of all pixels derived from the dendrite of the neuron depicted in F. H, CLM-expressing neurons were imaged for 30 min (180 frames at 6 frames/min). The deviation from a white noise power spectrum is plotted against the number of time frames for the mean power spectra of both the neuronal YFP signal and closed-shutter noise. The resulting normalized curves converge toward significantly different values and demonstrate a decreased time variance of the spatial distribution of the YFP signal versus normalized closed-shutter noise.