Effects of ELS on anxiety-like behavior. (A)

Timeline of ELS and behavioral assays. (B) Serum corticosterone levels are increased during adolescence, following ELS. P=0.01, Naïve N=9, ELS N=11 mice. (C) Representative movement tracks of naive (grey) and ELS (green) mice in open field task. (D) No significant difference in centre/periphery ratio in open field following ELS. P=0.39, Naïve N=9, ELS N=14 mice. (E) Total distance travelled during open field test was increased in ELS mice. P=0.04, Naïve N=9, ELS N=14 mice. (F) Representative locomotor tracks in elevated plus maze in naïve and ELS mice, black segment represents closed arms with grey arms representing open arms. (G) ELS mice had a reduced open/closed arm ratio. P=0.02, Naïve N=18, ELS N=11 mice. (H) Total distance travelled during elevated plus maze test was increased by ELS. P=0.02, Naïve N=9, ELS N=11 mice. See Table S1 for detailed statistical summaries.

ELS alters fear memory, synaptic plasticity, and neural excitability. (A)

Timeline of auditory discriminative fear conditioning paradigm. (B) ELS did not affect % Freezing to neutral and aversive auditory cues during learning. Naïve N=20, ELS N=17. (C) % time freezing during presentation of neutral auditory cue (CS-; p=0.0002) and aversive cue (CS+; p=0.99). Naïve N=20, ELS N=17. (D) Discrimination Index was impaired by ELS. P=0.004. Naïve N=20, ELS N=17. (E) Schematic representation of electrode placement in slice electrophysiology experiment (top) with DIC image below. (F) Representative EPSP traces from naïve (top) and ELS (bottom) brain slices during baseline (black trace) and after LTP stimulation (grey trace). Scale bars = 0.5mV and 10ms. (G) LTP was impaired by ELS. P=0.002. Naïve N=15, ELS N=13. (H) Diagram of procedure for c-fos staining experiments. (I) Brain slices containing lateral amygdala (LA) were selected for c-Fos staining. (J) Region of interested extracted from full amygdala image. Scale bars = 10μm. (K) c-Fos positive cell density was increased by ELS. P<0.0001. Naïve n=39 slices N=10 mice, ELS n=37 slices N=10mice. See Table S1 for detailed statistical summaries.

Long-term astrocyte dysfunction after early-life stress. (A)

Representative immunostaining of GFAP, S100b, GR, DAPI, and GR/DAPI merge in Naïve and ELS conditions. Scale bars = 20μm (B) Nuclear/cystolic GR ratio in astrocytes was increased after ELS. P=0.026. Naïve N=8, ELS N=8. (C) Representative slide scan image of immunostaining for astrocyte proteins GFAP and Cx43. (D) Representative immunostaining of GFAP, Cx43, GLT-1, and DAPI in naïve and ELS conditions. Scale bars = 50μm (E) GFAP staining was reduced in ELS mice. p=0.02, Naïve N=11, ELS N=12. (F) Cx43 staining was decreased in ELS mice. p=0.008. Naïve N=11, ELS=14. (G) GLT-1 expression was unchanged by ELS. P=0.8. Naïve N=6, ELS N=16. See Table S1 for detailed statistical summaries.

Astrocyte dysfunction mimics the effects of ELS on behavior, synapses, and neural excitability. (A)

Representative image showing localisation of viral vector in lateral amygdala. (B) Viral constructs used to manipulate astrocyte function. (C) Astrocyte dysfunction did not affect % Freezing to neutral and aversive auditory cues during learning. eGFP N=12, CalEx N=11, dnCx43 N=17 mice. (D) % time freezing during presentation of neutral auditory cue (CS-) was increased with astrocyte dysfunction (eGFP vs CalEx: p<0.0001; eGFP vs dnCx43: p=0.029; CalEx vs dnCx43: p=0.0005) with no impact on % Freezing to aversive cue (CS+). eGFP N=12, CalEx N=11, dnCx43 N=17 mice. (E) Discrimination Indexes were impaired by astrocyte dysfunction. eGFP vs CalEx: p<0.0001; eGFP vs dnCx43: p=0.013; CalEx vs dnCx43: p=0.03. eGFP N=12, CalEx N=11, dnCx43 N=17 mice. (F) Astrocyte dysfunction occluded the induction of LTP. eGFP vs CalEx: p=0.04; eGFP vs dnCx43: p=0.02; CalEx vs dnCx43: p=0.88. eGFP N=5, CalEx N=9, dnCx43 N=16. (G) Representative c-Fos staining in the lateral amygdala in control (eGFP), CalEx, and dnCx43 conditions. Scale bars = 10μm. (H) c-Fos positive cell density was increased with astrocyte dysfunction. eGFP vs CalEx: p<0.0001; eGFP vs dnCx43: p=0.0005; CalEx vs dnCx43: p=0.88. eGFP n=17 slices N=5 mice, CalEx n=17 slices N=5 mice, dnCx43 n=12 slices N=5 mice. See Table S1 for detailed statistical summaries.

(A) Learning phase (conditioning) of auditory discriminative fear conditioning in male and female naïve mice. Female N=7, Male N=8. (B) Memory recall in of CS+ and CS- in male and female naïve mice. Female N=7, Male N=8. (C) Auditory discrimination in male and female naïve mice. Female N=7, Male N=8. (D) Normalised fEPSP slope showing synaptic response before and after theta burst stimulation (grey vertical bar) in naïve and ELS mice. (E) Representative Z-stack projection of c-FOs staining in entire LA and BLA in naive. (F) Z-stack projection of c-FOs staining in entire LA and BLA in ELS. Scale bars = 100μm. See Table S1 for detailed statistical comparisons.

(A) Representative image of fluorescent reporter expression in lateral amygdala following AAV injection. (B) No effect of astrocyte manipulation on open arm/closed arm duration in EPM. eGFP N=7, CalEx N=9, dnCx43 N=9 mice. (C) No effect of astrocyte manipulation on distance travelled in EPM. eGFP N=7, CalEx N=9, dnCx43 N=9 mice. (D) Representative Z-stack projection of c-FOs staining in entire LA and BLA in eGFP injected mouse 90minutes after conditioning. (E) Representative Z-stack projection of c-FOs staining in entire LA and BLA in CalEx injected mouse 90minutes after conditioning. (F) Representative Z-stack projection of c-FOs staining in entire LA and BLA in dnCx43 injected mouse 90minutes after conditioning. Scale bars = 100μm