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. 2020 Apr 30;9:e54441. doi: 10.7554/eLife.54441

Figure 4. Physiological experiments: iGluSnFR expression slows uptake by cortical astrocytes.

Figure 4.

(A) Synaptic transporter currents (STCs) recorded in cortical astrocytes expressing either iGluSnFR (blue) or tdTomato (red). Traces indicate average responses (mean ± SEM; iGluSnFR: n = 21 cells; tdTomato: n = 15 cells), normalized in amplitude. (B) Responses in A, plotted on a semi-log scale. (C) Rising phases (± SEM) of the responses in A, plotted on an expanded time scale. (D) Summary data showing that STC rise and decay were slower in iGluSnFR+ astrocytes. * indicates p<0.05. (E) Astrocyte Vm (mean ± SEM), as a function of injected current, shows that iGluSnFR+ astrocytes rested at slightly more hyperpolarized potentials compared to tdTomato+ astrocytes. Input resistances (indicated by the slope of the relation) were not different in the two groups, although intercell variability diminished the statistical power of this comparison (power = 0.8 would require n = 250). (F) STCs and iGluSnFR signals measured in the same experiments (mean ± SEM, n = 10 cells). (G) Expressing iGluSnFR in both neurons and glia (‘double’) slowed STCs to a greater extent than when iGluSnFR was expressed in astrocytes only (‘single’). Asterisks indicate p=0.007 (Wilcoxon Rank test between single (n = 15) and double (n = 13)). Decay time constants normalized to average STC decay in control. (H) As in G, but showing the decays of iGluSnFR signals. Asterisks indicate p=0.0003 (Wilcoxon Rank test between single (n = 10) and double (n = 5)). (I) Simulated STC waveforms corresponding to average responses in iGluSnFR+ (blue) and tdTomato+ (red) astrocytes from panel A. (J) Waveforms used to derive STCs in I. In each case a clearance time course (red or blue) was convolved with a filter waveform (gray). This simple example demonstrates how even subtle differences in STC time course can reflect substantial differences in glutamate clearance time course (Diamond, 2005).

Figure 4—source data 1. STC and iGluSnFR imaging experiments.
Measured STC rise and decay values from tdTomato+ and iGluSnFR+ astrocytes (Figure 4D). Measured steady-state membrane potentials recorded under current clamp from tdTomato+ and iGluSnFR+ astrocytes (Figure 4E). STC decay data from astrocytes in singly and doubly infected slices, normalized to the average control STC decay in each case (Figure 4G). iGluSnFR signal decay data from astrocytes in singly and doubly infected slices, normalized to the average control STC decay in each case (Figure 4H).