Fig 1: Chronic restraint stress (CRS)-induced reduction in the number of adult hippocampal NSCs is prevented by Atg7 deletion. (A) Scheme of mouse breeding and experimental time line for generation of tamoxifen (TAM)-inducible NSC-specific Atg7-NSC cKO mice. (B) Gene dose–dependence of ATG7 immunofluorescence intensities in the DG of the hippocampus. Scale bar: 50 µm. Solid and dotted circles indcate granule neurons and NSCs, respectively. The graph shows quantification of SOX2+ and ATG7+ cells (n = 6). (c) Timeline of the experiment. (D) Representative images of BrdU and SOX2 staining in the subgranular zone (SGZ) of the hippocampus. Scale bar: 20 µm. The graph on the right shows quantification of BrdU+ and SOX2+ cells (n = 6–8 per group). (E) Immunofluorescence of SOX2, NES and VIM in the SGZ of the DG of the hippocampus. Scale bar: 50 µm. Arrows indicate NSCs positive for each marker. ***P < 0.001 for the total SOX2+ cells. ###P < 0.001 for BrdU+ SOX2+ cells. n.s., not significant.
Fig 2: CORT-induced reduction of NSC number and hippocampal dysfuction is prevented in Atg7-NSC cKO mice. (A) Timeline of the CORT injection experiment. (B) Quantification of BrdU+ and SOX2+ cells (n = 4 or 5 per group). **P < 0.01 for the total SOX2+ cells ##P < 0.01 for BrdU+ SOX2+ cells. (C) Nes promoter-driven lentiviral expression of mRFP-EGFP-MAPLC3B in SOX2+ cells in vivo. Scale bar: 20 µm. The graph on the right shows quantification of autophagosomes and autolysosomes (n = 7 or 8 cells per group). (D) Timeline for the measurement of the number of BrdU+ RBFOX3+ double–positive NSCs. (E) Quantification of BrdU+ RBFOX3+ double-positive cells (n = 4 or 5 per group). (f) Elevated plus maze test (n = 4 per group). (g) Y-maze test (n = 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: CRS induces autophagy in adult hippocampal NSCs. (A) Work flow of correlative light electron microscopy (CLEM) imaging. (B) CLEM imaging of SOX2-positive cells at day-4 of CRS (n = 13 cells per group). Representative images of autophagosomes (white arrows) in CRS group are shown at higher magnification. The number of autophagosomes was counted per 5 µm × 5 µm without counting the autophagosomes of whole cells using serial sections. Scale bars: 2 µm. (C) Nes promoter-driven lentiviral expression of mRFP-EGFP-MAP1LC3B in vivo. Virus was injected 4 days prior to CRS and analyzed at day 2 of CRS. Scale bar: 20 µm. The graph on the right shows quantification of autophagosomes and autolysosomes (n = 7 or 8 cells per group). (D and E) Cleaved CASP3 (c.CASP3) staining (D) and TUNEL assay (E) in the DG at day 7 of CRS. STS was injected 12 h before analysis. Arrows indicate c.CASP3-positive cells. Scale bar: 100 µm. ***P < 0.001. n.s., not significant.
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