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Fig. 4
Targeted downregulation of miR-137 in Hcrt neurons consolidates wakefulness. (A) Schematic representation of local injection of AAVs in the lateral hypothalamus of Hcrt:IRES-Cre driver mice. (B) Expression levels of Hcrt in murine brain after rAAV-anti-miR-137, rAAV-anti-miR-665, and rAAV-anti-miR-128 overexpression, specifically in the Hcrt-producing neurons. rAAV-anti-miR-128 was used as a control, as it does not target Hcrt. Mice were euthanized at 5 wk postinjection. Differences between the anti-miR and control groups were analyzed using a one-way ANOVA with Dunnett’s post hoc test. *P = 0.039. (n: control = 5; anti-miR-137 = 5; anti-miR-665 = 6 animals). (C and D) Quantification of vigilance states per 4-h intervals over 24 h. Local expression of anti-miR-137 in Hcrt neurons increases wakefulness (C) and accordingly decreases NREM sleep (D), compared with control mice during the first 4 h of the dark period (ZT12-16). There is a significant increase of 24.1 ± 9.9% in the duration of wakefulness and a reduction of 56.4 ± 22.3% in that of NREM sleep, in anti-miR-137-injected animals during ZT12-16 (anti-miR-137 vs. control: *P < 0.05 for wakefulness and NREM; anti-miR-665 vs. control: P > 0.05 for wakefulness and NREM; one-way ANOVA with Dunnett’s post hoc test. n: control = 7; anti-miR-137 = 5; anti-miR-665 = 6 animals). (E) Distribution of wakefulness bout durations shows significantly longer bouts in anti-miR-137–injected animals compared with controls during ZT12-16 (anti-miR-137 vs. control: **P < 0.01 for 4- to 8-s bouts of wakefulness; *P < 0.05 for >512-s bouts; two-way ANOVA with Dunnett’s post hoc test. n: control = 7; anti-miR-137 = 5; anti-miR-665 = 6 animals). (F) Dynamics of NREM delta (0.75 to 4 Hz) power over 24 h indicates a tendency that anti-miR-137–injected animals have higher EEG delta power (22.8 ± 8.5) in the first percentile of NREM sleep during the dark period compared with controls (anti-miR-137 vs. control: P = 0.063; anti-miR-665 vs. control: P > 0.05; one-way ANOVA with Dunnett’s post hoc test. n: control = 7; anti-miR-137 = 5; anti-miR-665 = 6 animals).