(A) Experimental design, injection of AAVretro-GFP in the PL for input tracing, and quantification of activation by cFos immunostaining 3 weeks later at either contextual fear conditioning (CFC) encoding (blue), recent (green), or remote (red) recall. Brains were collected 90 min after the behavior session. (B) Representative image of AAVretro-GFP injection site in the PL region of the medial prefrontal cortex (mPFC). Scale: 500 µm. (C–Z) For each region: Representative image of PL input tracing, magnified view of GFP and cFos at encoding, recent, and remote time points (all scales: 20 µm); quantifications of cFos in PL projections and total cFos in the input region, expressed as fold change to the no shock control group. Note that cFos in PL projections values were first normalized by chance level for each animal (see Figure 3—figure supplement 1). (C–F) EC (C, scale 500 µm), (E) cFos in EC → PL (one-way ANOVA, F(2,25)=8.153, p=0.0019) and (F) total cFos. (G–J) RSPag (G, scale 400 µm), (I) cFos in RSPag → PL (one-way ANOVA, F(2,35)=3.275, p=0.0497) and (J) total cFos (one-way ANOVA, F(2,35)=3.275, p=0.0497). (K–N) INS (K, scale 500 µm), (M) cFos in INS → PL (one-way ANOVA, F(2,27)=5.405 p=0.0106) and (N) total cFos (one-way ANOVA, F(2,35)=4.583, p=0.0171). (O–R) vCA1 (O, scale 400 µm), (Q) total cFos and (R) cFos in vCA1 → PL. (S–V) basolateral amygdala (BLA) (S, scale 500 µm), (U) cFos in BLA → PL (one-way ANOVA, F(2,28)=4.922, p=0.0147) and (V) total cFos. (W–Z) claustrum (CLA) (W, scale 500 µm), (Y) cFos in CLA → PL (one-way ANOVA, F(2,34)=4.502, p=0.0184), and (Z) total cFos (one-way ANOVA, F(2,35)=3.833, p=0.0313). Stars represent p-values of Tukey’s multiple comparisons tests (*: 0.01 < p < 0.05; **: 0.001 < p < 0.01), hashtag signs represent p-values of two-tailed one-sample t-tests comparing the difference to 1, which represents levels of the no shock controls (#: p≤0.05; ##: 0.001 < p ≤ 0.01; ###: p≤0.001). n=9–13 animals per group.