Fig 5. Suppressed vMCs activity parallels fear generalization.

(A) Experimental design. Left: Administration of 4-OHT to Fos-CreER^T2 × Ai9 F1 mice activates permanent expression of a reporter in neurons activated around the time of the injection. To trace dDG afferent projections, the AAV2/retro-hSyn-EGFP virus was injected into the dDG of F1 mice. Right: After virus expression, mice were subjected to CFC training under WS or SS in context A. After 3 days, mice were intraperitoneally injected with 4-OHT and tested in context B. Four days later, mice were perfused. (B) Representative images of tdTomato⁺ (red) cells labeled in context B and dDG afferent projecting neuron expressed EGFP (green). Nuclei were stained with DAPI (blue). The image on the left was from the WS group, and the image on the right was from the SS group. From top to bottom, the brain regions were the entorhinal cortex, MS, MnR, and vDG. Scale bar, 100 μm. (C) Representative image of Calb2⁺ (red) and cFOS⁺ (green) immunostaining in the vDG. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) The proportion of vMCs (Calb2⁺) to all cells (DAPI⁺). A similar percentage of vMCs among all cells in the vDG hilus between the WS and SS groups. (E) The proportion of activated vMCs (Calb2⁺ cFOS⁺) to all cells (DAPI⁺). A lower percentage of activated vMCs among all cells in the hilus of the SS group. (F) The proportion of activated cells (cFOS⁺) to all cells (DAPI⁺). A similar percentage of activated cells among all cells in the vDG hilus between the WS and SS groups. (G) The proportion of activated vMCs (Calb2⁺ cFOS⁺) to all vMCs (Calb2⁺). A lower percentage of activated vMCs among vMCs in the vDG hilus of the SS group. (H) The proportion of activated vMCs (Calb2⁺ cFOS⁺) to all activated cells (cFOS⁺). A lower percentage of activated vMCs among all activated cells in the vDG hilus of the SS group. (I) Pearson correlation test was used to assess the correlation between the percentage of activated vMCs among vMCs (X-axis: Calb2⁺ cFOS⁺ of Calb2⁺ %) and activated dDG cells (Y-axis: cFOS⁺/DAPI %) among DAPI⁺ cells in the dDG. R = Pearson correlation coefficient. (J) The proportion of activated vDG granular cells (cFOS⁺ cells among DAPI⁺ cells in the vDG granular cell layer). A similar percentage of activated vDG granular cells between the WS and SS groups. In Fig 5D–5H and 5J, statistical comparisons were performed using unpaired t tests. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. The underlying data and statistical information in Fig 5 can be found in S1 Data. The mice depicted were created with BioRender.com. CFC, contextual fear conditioning; dDG, dorsal dentate gyrus; MnR, median raphe; MS, medial septum; SS, strong shock; vDG, ventral dentate gyrus; WS, weak shock.