Figure 5. Prelimbic cortex (PL) engram reactivation correlates with freezing when claustrum (CLA) or insular cortex (INS) inputs are inhibited.

(A) Experimental design. 3 weeks before behavior started, Fos::tTA mice were injected with AAVretro-Cre in PL and AAV-DIO-hM4Di-mCherry in the desired input region, as well as AAV-TRE-GFP in the PL, so that GFP was only expressed in cFos⁺ cells in the absence of doxycycline (Dox). (B) GFP expression at the PL injection site (scale 400 µm). (C) Magnified view in the PL (scale 20 µm) of reactivated engram cells, indicated by white arrows. (D) Representative image of the CLA input region. (E) Experimental timeline (top) and freezing percentage (bottom) during recent memory recall when CLA → PL projections were inhibited during encoding (Cohen’s d=–0.87). (F) Reactivation of PL engram cells (%GFP⁺cFos⁺/GFP⁺) at recent recall for CLA → PL inhibition, correlated with freezing percentage at recent recall for clozapine-N-oxide (CNO) (orange) and vehicle (gray) groups. (G) Representative image of the INS input region. (H) Experimental timeline (top) and freezing percentage (bottom) during recent memory recall when INS → PL projections were inhibited during recent recall (Cohen’s d=–1.33). (I) Reactivation of PL engram cells (%GFP⁺cFos⁺/GFP⁺) at recent recall for INS → PL inhibition, correlated with freezing percentage at recent recall for CNO (orange) and vehicle (gray) groups. (E, H) Stars represent p-values of two-tailed unpaired t-tests between CNO and vehicle groups (*: p≤0.05). (F, I) Correlations assessed with linear regressions, R² and p-values are reported on the graphs. n=11–12 (CLA) or 5–9 (INS) per group.

Figure 5—figure supplement 1. Complementary quantifications for the engram reactivation analysis in the prelimbic cortex (PL).

(A–D) For claustrum (CLA) → PL inhibition during encoding, percentage of (A) GFP⁺ cells, (B) cFos⁺ cells, (C) double positive GFP⁺cFos⁺ cells normalized to chance and (D) reactivation expressed as %GFP⁺cFos⁺/GFP⁺. (E–H) For insular cortex (INS) → PL inhibition during recent recall, percentage of (E) GFP⁺ cells, (F) cFos⁺ cells, (G) double positive GFP⁺/cFos⁺ cells normalized to chance and (H) reactivation.

Figure 5—figure supplement 2. Claustrum (CLA) → prelimbic cortex (PL) inhibition during encoding does not affect remote recall and remote engram reactivation in PL.

(A) Representative image of the CLA input region. (B) Timeline and freezing percentage of CLA → PL inhibition during encoding and tested at remote recall. (C) Reactivation of PL engram cells (%GFP⁺cFos⁺/GFP⁺) at remote recall, correlated with freezing percentage for clozapine-N-oxide (CNO) (orange) and vehicle (gray) groups. Percentage of (D) GFP⁺, (E) cFos⁺, (F) double positives GFP⁺cFos⁺ normalized to chance and (G) reactivation as %GFP⁺cFos⁺/GFP⁺. Correlation assessed with linear regression, R² and p-value are reported on the graphs. n=5–6.

Figure 5—figure supplement 3. Clozapine-N-oxide (CNO) administration per se does not alter memory recall or engram reactivation.

(A) Experimental design. Fos::tTA mice were injected with AAVretro-Cre in PL and AAV-DIO-mCherry in the claustrum (CLA), as well as AAV-TRE-GFP in prelimbic cortex (PL). (B) Experimental timeline and freezing percentage at recent memory recall following CNO or vehicle administration at encoding. (C) Reactivation of PL engram cells (%GFP⁺cFos⁺/GFP⁺) at recent recall showed no correlation with freezing for the CNO (orange) and vehicle (gray) groups. n=7 animals per group.