Fig. 7: Astrocyte specific deletion of glucocorticoid receptors (GR) in the LH corrects cellular and behavioural impairments induced by ELS. | Nature Communications

Fig. 7: Astrocyte specific deletion of glucocorticoid receptors (GR) in the LH corrects cellular and behavioural impairments induced by ELS.

From: A divergent astrocytic response to stress alters activity patterns via distinct mechanisms in male and female mice

Fig. 7: Astrocyte specific deletion of glucocorticoid receptors (GR) in the LH corrects cellular and behavioural impairments induced by ELS.The alternative text for this image may have been generated using AI.

A Experimental timeline for combined ELS and genetic deletion of astrocyte GRs in the LH. Separate cohorts of mice subjected to behavioural protocols or IHC. B Viral construct used to achieve astrocyte specific deletion of GR receptors in Nr3c1fl/fl mouse line, scale bar = 25 µm. C–E Validation of AAV2/5-GfaABC1D-Cre-eGFP expression in LH astrocytes. C quantification of orexin neurons (Mann–Whitney test: Naïve = 16 cells ±1.77, (N = 4), Cre-eGFP injected = 19 cells ±1.66 (N = 5), p = 0.381), (D) Quantification of S100β astrocytes (Mann–Whitney test Naïve = 17 cells ±1.20 (N = 6), Cre-eGFP injected = 18.40 cells ±0.759 (N = 5), p = 0.495), (E) Cellular identity of Cre-eGFP and expression specificity of 94.5% in S100β + astrocytes (N = 7). F Representative LH astrocytes expressing control virus (AAV2/5-GfaABC1D-eGFP) or AAV2/5-GfaABC1D-Cre-eGFP in 3D view and 2D slice view of branching structures (branch points & branching processes), 3D volume scalebars = 5 µm, 2D slice scale bar = 2 µm. G Volume of -eGFP control or cre- expressing LH astrocytes from ELS male mice (unpaired t test: eGFP = 4803 μm3 ±342 (N = 3, n = 19 cells), Cre = 4365 μm3, ±529 (N = 3, n = 19 cells), p = 0.491). H Total branch points/volume (unpaired t test: eGFP = 18.18 ±2.08 (N = 3, n = 19 cells), Cre = 31.91 ±4.30 (N = 3, n = 19 cells), p = 0.007). I Normalised Cx43 fluorescence intensity (a.u) in LH of -eGFP control or cre- astrocytes (unpaired t test: eGFP = 71.37Arb.U. ±8.56 (N = 3, n = 14 cells), Cre = 102.6Arb.U. ±11.1 (N = 3, n = 15 cells), p = 0.036), scale bars = 25 µm. J Volume of eGFP control or cre- expressing LH astrocytes from ELS female mice (Mann-–Whitney test: eGFP = 4622 μm3, ±518 (N = 3, n = 21 cells), Cre = 3545 μm3, ±484 (N = 3, n = 16 cells), p = 0.130). K Total branch points/volume (unpaired t test: eGFP = 15.15 ±0.994 (N = 3, n = 21 cells), Cre = 31.06 ±5.27 (N = 3, n = 16 cells), p = 0.0018). L Normalised Cx43 fluorescence intensity (a.u) in LH of -eGFP control or cre-astrocytes (unpaired t test: eGFP = 91.32Arb.U. ±7.46 (N = 3, n = 15 cells), Cre = 158.5Arb.U. ±13.1 (N = 3, n = 15 cells), p < 0.001), scale bars = 25 µm. M Experimental timeline for combined expression of AAV2/5-GfaABC1D-Cre-eGFP and AAV2/8-miniHCRT-tdTomato in Nr3c1-ELS mice, scale bars = 25 µm. N Representative firing rates of orexin neurons in Naive eGFP (N = 3, n = 7), ELS eGFP (N = 3, n = 14) and ELS Cre (N = 3, n = 14) injected male mice. O Restoration of RMP (one-way ANOVA: F = 10.32, p < 0.001) and (P) firing rates (one-way ANOVA: F = 5.78, p = 0.0072) in Naive eGFP (N = 3, n = 7), ELS eGFP (N = 3, n = 14) and ELS Cre (N = 3, n = 17) injected male mice. Q Representative firing rates of orexin neurons in Naïve eGFP (N = 3, n = 12) ELS eGFP (N = 3, n = 11) and ELS Cre (N = 3, n = 18) injected female mice (one-way ANOVA: F = 7.90, p = 0.0014). R Restoration of RMP (one-way ANOVA: F = 7.90, p = 0.0014) and (S) firing rate (one-way ANOVA: F = 8.32, p = 0.001) in Naïve eGFP (N = 3, n = 11), ELS eGFP (N = 3, n = 12), and ELS Cre (N = 4, =16) injected female mice. T, V Running wheel behaviour of eGFP and Cre injected male mice: (T) Distance ran per day, (U) Total distance (one-way ANOVA: F = 1.96, p = 0.173), and (V)Llight cycle activity (one-way ANOVA: F = 3.23, p = 0.001). W–Y Running wheel behaviour of eGFP and Cre injected female mice: (W) Distance ran per day, (X) Total distance (one-way ANOVA: F = 6.02, p = 0.011), and (Y) Light cycle activity (one-way ANOVA: F = 1.24, p = 0.315) N = mice, n = cells. Bar charts = mean ± S.E.M. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

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