Article Figures & Data
Figures
- Figure 1.
Two-photon imaging of [Cl−]i in brain slices. A, Example image of CFP (480 nm) and YFP (530 nm) fluorescence in an organotypic hippocampal culture from a SClm mouse. Scale bar: 20 μm. B, Illustration of Fluorescence Resonance Energy Transfer (FRET) from CFP donor to YFP acceptor of the SClm sensor. FRET values (YFP/CFP fluorescence ratio) decrease with higher chloride concentrations. C, Two-photon imaging of chloride-dependent changes in the FRET ratio (530/480-nm emission) in a WT organotypic hippocampal culture with AAV SClm expression. [Cl−]i was clamped to the indicated external chloride concentration via ionophore treatment. Individual cells are color-coded to their FRET ratios. Scale bar: 20 μm. D, Calibration curve constructed from ionophore experiments (black/gray symbols) and perforated patch (red symbols) data. Data are presented as mean ± SEM. These data were fit by Equation 2, yielding the following fit parameters: Kd = 24.6 mM, Rmax = 1.51, Rmin = 0.12 (red curve; for details, see Materials and Methods). This calibration curve was used to convert FRET ratios into estimated chloride levels in the rest of this study. We also show the individual data points representing individual ionophore experiments (average over 12 cells per experiment). At 5 and 10 mM extracellular chloride FRET values were highly variable (open symbols). As described in Materials and Methods, we excluded these data points from our analysis and resorted to perforated patch recordings (red symbols) for this chloride range. The gray dotted line shows the alternative calibration curve when all ionophore data are included (fit parameters Kd = 8.4; Rmax = 1.35; Rmin = 0.32; without perforated patch data). Electrophysiology data from 11 cells (DIV1–3), 9 cells (DIV8–10), and 13 cells (DIV20–22); SClm data from 11 slices.
- Figure 2.
Monitoring acute changes in [Cl−]i with SClm. A, Two-photon image of CA1 pyramidal neurons in the hippocampus of an acute slice from a SClm mouse. A patch pipette (in white) is attached to cell #1 for a whole-cell recording. Two control cells are indicated with #2 and #3. Scale bar: 20 μm. B, Time course of CFP (upper row) and YFP (lower row) fluorescence right before and during the first minutes after break-in. After break-in (0’; gray) cell #1 rapidly fills with the high chloride internal solution (70 mM KCl) resulting in a decrease in YFP fluorescence. Scale bar: 20 μm. C, Average FRET ratio over time for cells that were infused with 70 mM KCl (n = 2), and neighboring control cells (n = 4). Error bars represents SEM. D, Acute wash-in with muscimol in cultured slices with viral SClm expression caused a decrease in FRET ratio in CA1 pyramidal neurons within 10 min. Scale bar: 20 μm. E, Average FRET ratios over time during wash-in of muscimol (gray area) and control. Data from 84 cells, 7 slices, 4 mice in both groups.
- Figure 3.
Developmental decrease in [Cl−]i continues in organotypic cultures. A, Example of an organotypic hippocampal culture from a SClm mouse at DIV8. Scale bar: 500 μm. In the zoom an example of the FRET ratios determined in the CA1 area. Scale bar: 20 μm. B, Average FRET ratios of CA1 pyramidal cells at DIV2–3 (n = 10 slices of 6 mice), DIV8–10 (n = 14 slices of 8 mice), and DIV21–22 (n = 14 slices of 8 mice). There was a significant increase in FRET ratio over time (p = 0.033 DIV2–3 vs DIV8–10; p = 0.008 DIV2–3 vs DIV21–22; p > 0.99 DIV8–10 vs DIV21–22; KW test). C, Average estimated [Cl−]i as calculated from the FRET ratios in B. There was a significant decrease in [Cl−]i over time (p = 0.006 DIV2–3 vs DIV8–10; p =0.003 DIV2–3 vs DIV21–22; p = 0.93 DIV8–10 vs DIV21–22; one-way ANOVA). D, Cumulative distribution of FRET ratios for individual cells at the three time points. For each slice, 15 cells were randomly selected, making the total of number of plotted cells per condition at least 150 (p < 0.001 DIV2–3 vs DIV8–10 and vs DIV21–22; p = 0.053 DIV8–10 vs DIV21–22; KS test). E, Individual FRET ratios plotted against soma depth in slices at DIV2–3 (n = 186 neurons). Line represents linear regression fit (r = 0.004; p = 0.41). F, Same as E at DIV8–10 (n = 299 neurons). Line represents linear regression fit (r = 0.002; p = 0.48). G, Same as E at DIV21–22 (n = 314 neurons). Line represents linear regression fit (r = 0.001; p = 0.50).
- Figure 4.
Higher [Cl−]i in L2/3 cells in mPFC slices from mice that experienced early life stress. A, Two-photon image of layer 2/3 neurons of the medial PFC of an acute slice of a P9 SClm mouse. Shown is the CFP and YFP fluorescence, and the corresponding FRET ratios. Individual cells are color-coded to their FRET ratios. Scale bar: 20 μm. B, Average FRET ratios from control mice and from mice after ELS. Data from seven mice in both groups (p = 0.55, t test). The average FRET ratios were significantly lower in the ELS condition when analyzed per slice (p < 0.01, t test) or per cell (p < 0.0001, MW test; data not shown). C, Average estimated [Cl−]i as calculated from the FRET ratios in B (p = 0.07, MW test). D, Cumulative distribution of individual FRET ratios in slices from control and ELS mice. For each mouse 50 cells were randomly selected, making a total of 350 plotted cells per condition (p < 0.001; KS test). E, Individual FRET ratios plotted against soma depth in slices from control mice (n = 443 neurons). Line represents linear regression fit (r = 0.035; p < 0.0001). F, Same as E for slices from mice after ELS (n = 470 neurons). Line represents linear regression fit (r = 0.058; p < 0.0001).
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