Inhibiting vMCs does not induce fear generalization.

(A) Virus injection: The vDG of Calb2-Cre mice were injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry virus. (B) Experimental design: After virus expression, mice were subjected to CFC training under WS in context A. After 3 days, mice were intraperitoneally injected with saline (Saline group) or CNO (CNO group); 40 min later, mice were placed in context B for the fear generalization test, and 1.5 h after testing, mice were perfused for immunostaining. (C) Representative images of mCherry⁺ expression (red) and cFOS⁺ immunostaining (green) in the dDG (top) and vDG (bottom). Left: Saline group. Right: CNO group. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) The Saline and CNO groups showed a similar freezing level in context B during the fear generalization test. (E) The percentage of cFOS⁺ cells. The dDG granular cell layer of the CNO group displayed a significantly lower percentage of activated dDG cells (cFOS⁺) than that of the saline group. (F) Experimental design. Left: The vDG of Calb2-Cre mice were injected with AAV-hSyn-DIO-mCherry virus. Right: After virus expression, mice were subjected to CFC training under WS in context A. After 3 days, mice were intraperitoneally injected with saline (Saline group) or CNO (CNO group), and 40 min later, mice were placed in context B for the fear generalization test, and 1.5 h after testing, mice were perfused for immunostaining. (G) Representative images of mCherry⁺ expression (red) and cFOS⁺ immunostaining (green) in the dDG (top) and vDG (bottom). Left: Saline group. Right: CNO group. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (H) The Saline and CNO groups showed a similar freezing level in context B during the fear generalization test. (I) The percentage of cFOS⁺ cells. The dDG granular cell layer of the CNO group showed a similar percentage of activated dDG cells (cFOS⁺) with that of the saline group. Statistical comparisons were performed using unpaired Student’s t test. Data were presented as mean ± SEM. The underlying data and statistical information in S6 Fig can be found in S1 Data. The mice depicted were created with BioRender.com.

(TIF)