Article Figures & Data
Figures
- Figure 1.
IHH chamber. The intermittent hypobaric hypoxia chamber is primarily composed of a chamber body made of organic glass (130 × 80 × 80 cm), a vacuum compression pump system, and a computer control system, which can simulate a gradual descent from atmospheric pressure to the target partial pressure of oxygen. ① Computer monitor, ② Air-intake duct, ③ Vacuum-pumping pipeline, ④ Barometer, ⑤ CO2 monitor, ⑥ O2 monitor.
- Figure 2.
IHH ameliorated autistic-like phenotypes in Shank3B−/− mice. A, Experimental protocol. WB, western bolt; P42, postnatal day 42. B, Grooming time in each group following treatment with or without IHH (n = 16–24 mice per group). C–F, Representative heat maps and statistics for the three-chamber sociability test. IHH treatment improved the social preference (C, D) and social novelty abilities (E, F) in Shank3B−/− mice (n = 16–24 mice per group). G, Representative bands of western blot. H, Protein levels of GluR1, GluR2, NR2A, and NR2B in the striatum of Shank3B−/− and control mice following IHH (n = 6–8 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 3.
IHH improved compulsive behaviors in Fmr1−/y mice. A, Experimental protocol. WB, western bolt; P42, postnatal day 42. B, Statistical data in the marble burying test (n = 24–28 mice per group). C, Correct rate in the Y-maze test (n = 12–16 mice per group). D, E, Western blot analysis for GluR1, GluR2, NR2A, and NR2B expression in the medial prefrontal cortex in Fmr1−/y mice and control littermates (n = 3–6 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 4.
IHH enhanced HIF1α signaling in the DRN of C57BL/6J mice. A, Representative images of c-Fos-positive cells in the medial prefrontal cortex (mPFC) and the dorsal raphe nucleus (DRN). Cg1, cingulate cortex, area 1; PrL, prelimbic cortex; IL, infralimbic cortex. B, Relative number of c-Fos-positive cells in different brain regions (n = 3–5 mice per group). NAc, nucleus accumbens; BLA, basolateral amygdala; CeA, central nucleus of the amygdala; DG, dentate gyrus; VTA, ventral tegmental area. C, Relative levels of Hif1a in different brain regions (n = 6 mice per group). Stri, striatum; Amy, Amygdala. D, E, Relative fluorescence intensity of HIF1α in the mPFC and DRN of mice treated with or without IHH (n = 3 mice per group). F–I, Representative western blot bands and statistics of HIF1α protein levels in the DRN (cytoplasm and nucleus parts) of C57BL/6J mice treated with or without IHH (n = 5–8 mice per group). Scale bar = 50 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 5.
IHH activated serotonergic neurons in the DRN. A, Representative confocal images of cells co-stained with anti-c-Fos and anti-Tph2 antibodies. B, The number of c-Fos-positive cells and c-Fos/Tph2 double positive cells in the DRN of adult C57BL/6J mice treated with or without IHH (n = 3 mice per group). C, Schematic diagram and statistics for serotonergic neuron action potentials in the DRN of ePet1tdTomato mice following IHH treatment, compared to that of controls (n = 29–31 cells from 3 mice per group). D, Schematic of the viral injection for expressing rAAV2/9-EF1α-DIO-GCaMP6f in DRNePet1 neurons and optical fiber Ca2+ recordings during IHH training. E, Representative images of GCaMP6f expression in Tph2-positive neurons in the DRN. F, Calcium transients observed in DRNePet1 neurons. Ca2+ imaging was performed during the first 5 min of each stage following IHH training (n = 2 mice). Scale bar = 50 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 6.
DRN infusion of cobalt chloride alleviated autistic-like behaviors in Shank3B−/− mice. A, B, Relative HIF1α protein levels in the dorsal raphe nucleus (DRN) of C57BL/6J mice 24 h after treatment with CoCl2 (200 µM) or artificial cerebrospinal fluid (ACSF) (n = 6 mice per group). C, D, Representative images and statistics for anti-NeuN antibody staining in the DRN following DRN infusion of CoCl2 or ACSF (n = 4–5 mice per group). E, Schematic diagram of cannula insertion into the DRN of Shank3B−/− mice and behavioral tests. CoCl2 (200 µM) was injected into the DRN 24 h before each behavioral test. F, Grooming time (n = 18–23 mice per group). G–J, The three-chamber sociality test (n = 16–20 mice per group). Scale bar = 50 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 7.
Validating the efficacy of Egln1-shRNA in the DRN of ePet1-Cre mice. A, Schematic of viral injection. WB, western blot. B, Representative images of EGFP expression in the dorsal raphe nucleus (DRN) of ePet1-Cre mice 28 d after the viral injection. C, D, Western blots analysis following DRN infusion of Egln1-shRNA or SC (n = 6 mice per group). Scale bar = 50 µm. Data are presented as mean ± SEM. **p < 0.01.
- Figure 8.
Knockdown of Egln1 in the DRNePet1 neurons rescued autistic-like phenotypes in Shank3B−/− mice. A, Schematic of virus injection and behavioral tests. B, Grooming time (n = 8–10 mice per group). C–F, The three-chamber sociality test (n = 8–10 mice per group). G, H, Western blotting for GluR1, GluR2, NR2A, and NR2B expression levels in the striatum of Shank3B−/− mice (n = 6 mice per group). Scale bar = 50 µm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 9.
Downregulation of PHD2 activated the DRNePet1 neurons of Shank3B−/− mice and wild-type littermates. A, B, Prolyl hydroxylase domain-containing protein 2 (PHD2) knockdown increases the spontaneous action potential firing frequency of DRePet1 neurons (n = 16–25 cells from 3 mice per group). C–G, The effect of PHD2 knockdown on the firing rates, amplitudes, spike half-widths, and spike thresholds of depolarizing current-induced action potentials in DRNePet1 neurons (n = 14–24 cells from 3 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 10.
Validating the efficacy of Hif1a-shRNA in the DRN of C57BL/6J mice. A, Schematic of virus injection. B, Representative confocal images of EGFP expression 28 d after dorsal raphe nucleus (DRN) infusion of both rAAV-CMV-Cre and Hif1a-shRNA. C, D, Western blot assay showing that DRN infusion of both rAAV-CMV-Cre and Hif1a-shRNA significantly decreased the protein level of HIF1α in the DRN compared to that of mice injected with both rAAV-CMV-Cre and SC into the DRN (n = 8 mice per group). Scale bar = 50 µm. Data are presented as mean ± SEM. **p < 0.01.
- Figure 11.
Knockdown of Hif1α in DRNePet1 neurons induced compulsive behaviors. A, B, Schematic diagram of virus injection and behavioral tests. C, Grooming test (n = 21–22 mice per group). D, Marble burying test (n = 21–22 mice per group). E–H, Three-chamber sociality test. (n = 24–27 mice per group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 12.
HIF1α knockdown in DRNePet1 neurons reduced their spontaneous activity. A, B, HIF1α knockdown decreased the spontaneous firing frequency of DRNePet1 neurons (n = 28–31 cells from 3 mice per group). C–E, HIF1α knockdown had little effect on the firing rates, amplitudes, spike half-widths, and spike thresholds of depolarizing current-induced action potentials in DRNePet1 neurons (n = 23–25 cells from 3 mice per group). Data are presented as mean ± SEM. ***p < 0.001.
