Fig 2: Prenatal corticosterone exposure triggers anxiety/depression-like behavior and spatial memory dysfunction by suppressing neurogenesis in mice. (a–f) Four weeks after exposure to maternal vehicle or corticosterone (10 mg/kg) at E14, mice underwent behavior tests, dexamethasone suppression test, and were sacrificed for IHC at P23. a Open field test was performed to assess anxiety-like behavior for 10 min. Relative time at the periphery/center and total distance explored were determined. Mice exposed to prenatal corticosterone were more active in the peripheral regions. n = 6. b Mice were presented to the forced swim test for 6 min. Immobile and mobile activities were determined during the last 4 min. Mice exposed to prenatal corticosterone showed more immobility. n = 5. c The mice were subjected to Y-maze test to evaluate spatial memory function. Mice exposed to prenatal corticosterone showed cognitive deficits. n = 6. d Before the dexamethasone suppression test, the blood was collected. Blood was extracted again 1 h after dexamethasone (50 µl/kg) was injected. Plasma corticosterone was determined by ELISA. Plasma corticosterone release was not well suppressed by dexamethasone in mice exposed to prenatal corticosterone. n = 5. ## indicates p < 0.01 versus plasma corticosterone of dexamethasone-injected control mice. (e, f) Slide samples of both dorsal and ventral hippocampus for IHC were collected and the quantification analysis was performed in SGZ. The white square part of the images was enlarged. e For BrdU labeling, mice were injected intraperitoneally with BrdU (100 mg/kg) 24 h before sacrifice. The hippocampus was immunostained with BrdU (green), c-Fos (red), and DAPI (blue). The ratio of c-Fos- to BrdU-positive cells was decreased by prenatal corticosterone. Scale bars, 100 µm (magnification × 200). n = 5. f The hippocampus was immunostained with NeuN (green), DCX (red), and DAPI (blue). The ratio of DCX- to NeuN-positive cells was increased by prenatal corticosterone. Scale bars, 100 µm (magnification × 200). n = 5. All immunofluorescence images are representative. Two technical replicates from each animal (n = 5) were performed in results of IHC. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided unpaired student’s t test (Fig. 1a–c , e, f). Two-sided two-way ANOVA (Fig. 1d). *,**indicates p < 0.05 and p < 0.01 versus control, respectively

Fig 3: Glucocorticoid suppresses neurogenesis by reducing astrocytic HMW FGF2 and its downstream INFS. a After exposure to prenatal vehicle or corticosterone (10 mg/kg) at E14, the hippocampus from mice at P1 was collected. RNA sequencing was performed using RNA extracted from the hippocampal tissue. The scatter plot represents the log-fold changes between the mice exposed to prenatal vehicle (control) and the mice exposed to prenatal corticosterone. n = 3. (b, c) Five days after astrocytic differentiation, human NSCs were treated with cortisol (1 µM) for 48 h, and then the media were collected. b The growth factor antibody array was performed using the extracted media. n = 5. Red rectangles indicate significantly downregulated levels of growth factors between control and cortisol. The table indicates the location of detected factors in membrane. c Human FGF2 levels were measured using ELISA. FGF2 levels were decreased by cortisol-treated ACM. n = 5. (d–g) 5 days after astrocytic differentiation, human NSCs were treated with cortisol (1 µM) for 48 h and the ACM were collected. d The neurons differentiated from human NSCs (DIV 7) were treated with ACM and FGF2 (2 ng/ml) for 5 days. Then, the cells were immunostained with DCX (green) and NeuN (red). The ratio of DCX-positive cells to NeuN-positive cells was analyzed by flow cytometry. Upregulated ratio of DCX to NeuN-positive cells by cortisol-treated ACM was normalized by FGF2 treatment. n = 5. (e–g) The neurons (DIV 7) were treated with ACM and FGF2 for 24 h as appropriate. (e) Protein levels of FGFR1 in subcellular fraction samples were detected by western blotting. The ß-actin was used as a loading control for cytosol. The lamin A/C and pancadherin were used as a loading control for nucleus and membrane, respectively. Only nuclear FGFR1 levels were downregulated by cortisol-treated ACM, recovered by FGF2. n = 5. f The cells were immunostained with FGF2 (green), FGFR1 (red), and DAPI (blue). Pearson’s correlation coefficient between FGF2 and FGFR1 in nuclear region was quantified. Cortisol-treated ACM reduced correlation coefficient value, recovered by FGF2. Scale bars, 20 µm (magnification × 1000). n = 5. g Subcellular fraction was done and FGFR1 was co-immunoprecipitated with FGF2 in nuclear parts. HMW FGF2 levels in immunoprecipitated samples were quantified, which were reduced by cortisol-treated ACM. n = 5. All immunofluorescence images are representative. n = 5 from independent experiments with two technical replicates each. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided unpaired student’s t test (Fig. 4c, g). Two-sided two-way ANOVA (Fig. 4d, e, f). **indicates p < 0.01 versus control and ##indicates p < 0.01 versus cortisol, respectively

Fig 4: Building an automated analysis tree to quantify AAV transduction. (A) Brain sections were stained with Hoechst and immunostained for NeuN and GFP and images were captured on the Opera Phenix. Whole brain composites were generated by stitching together a panel of images with a 10% overlap. This was performed for 19 optical slices captured throughout the Z-plane at 1.2 µm intervals. (B) Each channel was analysed separately to identify positive signals for cell nuclei (Hoechst, blue), neurons (NeuN, orange) and AAV transduced cells (GFP, green). The Harmony software pseudo-labels positive signals with a coloured border as shown in the lower right-hand side of the thumbnails. (C) Positive signals for NeuN and GFP were further gated based on user input and machine learning to remove false positives. A combination of these gated outputs were used to identify NeuN+, GFP+ and NeuN+/GFP+ cells. (D) Data from each optical slice throughout the Z-stack were combined to locate neurons and transduced cells throughout the entire section. (E–G) The percentage of the Hoechst+ cells found in each section that were NeuN+, GFP+ or both NeuN+/GFP+ for each mouse strain. (H) The automated analysis of the data collected from all brain sections was reanalyzed using less stringent gating criteria for GFP+. Allowing more cells to be registered as GFP+ increased the transduction efficiency in the C57BL/6J mice. However, a small percentage of the Hoechst+ cells in the CBA/Ca sections were also identified as being GFP+, indicating that this increased signal corresponded to background. Each data point is an individual brain section with each of the 3 C57BL/6J and 3 CBA/Ca mice delineated by distinctive symbols: circle, square or triangle. Scale bar = 1 mm for whole brain imaging, 25 µm for thumbnails.

Fig 5: Suppressed INFS by cortisol downregulates neuroligin 1 and subsequent neurogenesis. (a–h) 5 days after astrocytic differentiation, human NSCs were treated with cortisol (1 µM) for 48 h and the ACM were collected. (a, b) The neurons differentiated from human NSCs (DIV 7) were incubated with ACM as appropriate for 24 h a The mRNA expressions were analyzed by real-time PCR. The genes for neuroligin 1/3, neurexin 2a/ß, and neurexin 3ß were downregulated by cortisol-treated ACM. n = 5. b DNA was immunoprecipitated with the IgG, RNA polymerase, and FGFR1 antibody. The samples were amplified with primers of GAPDH, NLGN1, NLGN3, NRXN2, and NRXN3 genes. Relative optical density of gel bands was quantified by using image J. Cortisol-treated ACM reduced the binding of FGFR1 to the NLGN1 promoter. n = 5. c The neurons differentiated from human NSCs (DIV 6) were transfected with pcDNA3.1/c-eGFP or pcDNA3.1/FGFR1-c-eGFP for 24 h prior to ACM treatment for 48 h. Actinomycin D (500 ng/ml) was pretreated for 30 min before ACM treatment as appropriate. Expression levels of neuroligin 1 were detected by western blotting. Overexpression of FGFR1 recovered neuroligin 1 expression, but actinomycin D did not reverse the effect of cortisol-treated ACM on neuroligin 1 expression. $$indicates, p < 0.01 versus cortisol with pcDNA3.1/FGFR1-c-eGFP transfection. n = 5. (d–g) The neurons differentiated from human NSCs (DIV 6) were transfected with pcDNA3.1/c-eGFP or pcDNA3.1/NLGN1-c-eGFP for 24 h prior to ACM treatment. (d–f) The ACM were administered to conditioned neurons for 48 h. d The neurons were immunostained with GluR1/2 (blue) and PSD95 (red). Pearson’s correlation coefficient between GluR1/2 and PSD95 was quantified. Overexpression of NLGN1 recovered GluR1/2 trafficking into the postsynaptic compartment. Scale bars, 20 µm (magnification × 1000). n = 5. e The neurons were stimulated with a high K+ buffer for destaining. Time-lapse imaging was performed over 90 s at 1 s intervals with an Eclipse Ts2™ fluorescence microscopy. Recycled synaptic vesicles were normalized by NLGN1 overexpression. n = 5. f The neurons were immunostained with MAP2 (red) and DAPI (blue). Sholl analysis was performed for analyzing neurite outgrowth. Overexpression of NLGN1 recovered neurite outgrowth, which was suppressed by cortisol-treated ACM. Scale bars, 50 µm (magnification × 400). n = 5. g ACM were administered to the conditioned neurons for 5 days. The neurons were then immunostained with DCX (green) and NeuN (red). The ratio of DCX-positive cells to NeuN-positive cells was analyzed by flow cytometry. Overexpression of NLGN1 recovered the decreased neurogenesis by cortisol-treated ACM. n = 5. h The differentiated neurons (DIV 7 or 21) were treated with ACM as appropriate. Neuroligin 1 expressions were detected by western blotting. Neuroligin 1 levels in differentiating neurons (DIV 7) were reduced by cortisol-treated ACM. n = 5. All immunofluorescence images are representative. n = 5 from independent experiments with two technical replicates each. Quantitative data are presented as a mean ± S.E.M. The representative images were acquired by SRRF imaging system. Two-sided unpaired student’s t test was conducted in (Fig. 6a, b, h). Two-sided two-way ANOVA was conducted in (Fig. 6c, d, g). *, **indicates p < 0.05, p < 0.01 versus control, respectively. #, ##indicates p < 0.05, p < 0.01 versus cortisol, respectively