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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Biol Psychiatry. 2019 Jan 14;85(11):904–914. doi: 10.1016/j.biopsych.2018.12.021

Figure 4. Kdm6b siRNA infusion into the CeA provokes anxiety-like behavior and decreases expression of Arc eRNA and mRNA.

Figure 4.

A) Schematic representation of Kdm6b siRNA infusion directly into the central nucleus of amygdala (CeA) of adolescent intermittent saline (AIS)-exposed control adult rats.

B) Elevated plus maze (EPM) exploration test for anxiety-like behaviors in adult rats infused with Kdm6b siRNA, control siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=6 rats in each group). ***p<0.001 by one-way ANOVA [percentage of open arm entries (F2,15=21.8, p<0.001) and percent of time spent in the open arm (F2,15=26.3, p<0.001)] followed by post-hoc Tukey test.

C) Light/dark box (LDB) exploration test for anxiety-like behaviors in adult control rats infused with Kdm6b siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=8 rats in each group). ***p<0.001 by Student’s t-test [time spent in the dark compartment (t (14) =−7.25, p<0.001), time spent in the light compartment (t (14) =7.25, p<0.001)].

D) RNA analysis of Arc enhancer RNA [eRNA (+) and (−) strand] and Arc mRNA transcripts in the amygdala of AIS adult rats infused with Kdm6b siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=7–8 rats in each group). *p<0.05, **p<0.01 by Student’s t-test [eRNA plus (t (14) =2.82, p<0.05) and minus strand (t (14) =2.69, p<0.05) transcripts of Arc eRNA; Arc mRNA (t (12) =3.25, p<0.01)].

E) Chromatin immunoprecipitation (ChIP) analysis of H3K27me3, KDM6B, CREB binding protein (CBP) and acetylated H3K27 (H3K27ac) at the synaptic activity response element (SARE) site of the Arc promoter in the amygdala of AIS adult rats infused with Kdm6b siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=7–8 rats in each group). *p<0.05, **p<0.01, ***p<0.001 by Student’s t-test [H3K27me3 occupancy (t (13) =−3.08, p<0.01), KDM6B (t (13) =3.54, p<0.01), CBP (t (14) =3.77, p<0.01), and H3K27ac (t (14) =2.59, p<0.05) occupancy].

F) ChIP analysis of H3K27me3, KDM6B, CBP, and H3K27ac at the Arc promoter (−441 bp) site in the amygdala of adult AIS rats infused with Kdm6b siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=7–8). **p<0.01, ***p<0.001 by Student’s t-test [H3K27me3 occupancy (t (13) =−3.26, p<0.01), KDM6B (t (13) =4.21, p<0.01), CBP (t (14) =4.56, p<0.001), and H3K27ac (t (14) =5.03, p<0.001) occupancy].

G) ChIP analysis for the E subunit of negative elongation factor (NELF-E) at the Arc promoter (−441 bp) in the amygdala of rats infused with Kdm6b siRNA or vehicle (iFect solution) in the CeA. Data represent mean ± SEM (n=7–8 rats in each group). *p<0.05 by Student’s t-test [t (13) =−2.39, p<0.05].