Fig 2: IRR-mediated neurogenesis ablation after extinction promoted spontaneous fear recovery. (A) Top, Rats were trained to associate a context with foot-shocks (day 1; conditioning). One day later, rats were trained to associate the same context with the absence of foot-shock (day 2; extinction). The next day, rats were placed in the same context without foot-shocks (day 3; test 1). Then, rats received three IRR or non-IRR sessions during a 35-day period. Finally, rats were placed again in the same context alone to test for fear recovery (day 38; test 2). Bottom, IRR after extinction training promoted spontaneous fear recovery, as indicated by high freezing and low bar presses per minute (inset), when comparing IRR with non-IRR (IRR: n = 9; non-IRR: n = 12). Insets: bar-pressing behavior (press/min) across the different phases of the behavioral protocol. Data are shown for first block (early) and last block (late) of conditioning and extinction (5-minute blocks), and as a single block in test sessions (10-minute block). (B) Rats subjected to IRR showed less number of immature cells (DCX+) per area (mm2) in the dentate gyrus of the hippocampus than non-IRR. (C) Representative immunohistochemically stained immature cells (DCX+; black) in the dentate gyrus of the hippocampus after IRR or non-IRR. Low magnification scale bar = 200 µm and high magnification scale bar = 50 µm. (D) Negative correlation between DCX+/mm2 and percent freezing response in Test 2 (R = -0.58; p < 0.05). Positive correlation between number of DCX+/mm2 and presses per min in Test 2 (R = 0.47; p < 0.05). **p < 0.01, ***p < 0.001. Error bars indicate SEM. IRR: irradiation; Cond: conditioning; Ext: extinction; DCX+: doublecortin positive cells.

Fig 3: 7-day HFD affects cellular complexity of DCX immunolabeled cells in dorsal hippocampus. Schematic experimental design (A). Animal body weight at the beginning of the experiment (1st day of diet, 5 weeks of age) and after 7 days of relative diet (B). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 vs. 1st day corresponding group value. Two-way ANOVA with Tukey’s multiple comparisons test. Representative DCX+ cell 3D morphological reconstruction in the dorsal hippocampus of animals fed with a LFD (Blue) or HFD (Red) for 1 week (C). Sholl intersections profiles of DCX immunolabeled cells of the dentate gyrus of the dorsal hippocampus of animals fed for 7 days with HFD (Red) or LFD (Blue) starting from the fifth week of age (n = 4 animals per group) (D). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 vs. LFD, nested ANOVA on linear mixed-effect model, with animal as a random effect. Summary of morphometric parameters illustrating the total number of bifurcations (E), the total number of branches (F), the total number of terminal tips (G) and the path distance (H) of DCX immunolabeled cells of the dentate gyrus of the dorsal hippocampus of animals fed for 7 days with HFD (Red) or LFD (Blue) starting from the fifth week of age (n = 4 animals per group). Data are represented as Box and Wiskers graph (E–G) or bar graph (mean ± SEM) (H). *p < 0.05, **p < 0.01 vs. LFD, ANOVA on linear mixed-effect model, with animal as a random effect. ImageJ FIJI software (version 1.52) (https://imagej.nih.gov/ij/) was used to produce cell 3D morphological reconstruction. GraphPad Prism 8 (https://www.graphpad.com/) and Power Point were used to generate the figure.

Fig 4: No effect of 7-day HFD on cellular complexity of DCX+ cells in the ventral hippocampus. Representative DCX+ cell 3D morphological reconstruction in the ventral hippocampus of animals fed with a LFD (Blue) or HFD (Red) for 1 week (A). Sholl intersections profiles of DCX immunolabeled cells of the dentate gyrus of the ventral hippocampus of animals fed for 7 days with HFD (Red) or LFD (Blue) starting from the fifth week of age (n = 4 animals per group) (B). Data are presented as mean ± SEM. Summary of morphometric parameters illustrating the total number of bifurcations (C), the total number of branches (D), the total number of terminal tips (E) and the maximum reached extension from soma (F) of DCX immunolabeled cells of the dentate gyrus of the ventral hippocampus of animals fed for 7 days with HFD (Red) or LFD (Blue) starting from the fifth week of age (n = 4 animals per group). Data are represented as Box and Wiskers graph (C–E) or bar graph (mean ± SEM) (F). ImageJ FIJI software (version 1.52) (https://imagej.nih.gov/ij/) was used to produce cell 3D morphological reconstruction. GraphPad Prism 8 (https://www.graphpad.com/) and Power Point were used to generate the figure.

Fig 5: Distribution of nestin (A), Olig2 (B), and DCX (C) in relation to SSEA-4/YKL-40 shown in merged DAPI stained images. The inner subventricular zone (ISVZ) (A1) and ventricular zone (VZ) (A2) of occipital cortex from a 21st wpc human fetus (CRL: 200 mm), double-immunolabeled with antibodies against nestin (red) and SSEA-4 (green), and nuclei labeled with DAPI (blue). The outer VZ which showed no immunoreactivity was left out. Note the abundant SSEA-4 immunopositive cells in ISVZ (arrows) and the very few nestin-positive cells (arrow-heads). In VZ adjacent to the lateral ventricle the majority of the cells are nestin-positive whereas no SSEA-4 positive cells are present (A1). The few yellow strokes in (A1) probably represent closely apposed fibers belonging to SSEA-4 and nestin positive cells in the merged image. In (B) many nuclei of cells in the inner VZ of parietal cortex from a 15th wpc human fetus (CRL: 110 mm), are positive for Olig2 (arrows) whereas very few cell bodies are immunoreactive for YKL-40 (arrowhead) in the outer VZ. The merged images show no overlap. In (C) the ISVZ and outer VZ in the medial wall of the temporal cortex from the same fetus are double immunolabeled with antibodies against DCX (red) and SSEA-4 (green). Note the abundant SSEA-4 immunopositive cells in ISVZ (arrows) and the DCX labeled fibers in both VZ and ISVZ (arrowheads). The merged images show no overlap. Abbreviations: DCX: doublecortin; ISVZ: inner subventricular zone; SVZ: subventricular zone; VZ: ventricular zone. A–C, same magnification. Scale bar: 50 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]