Memory strength gates the involvement of a CREB-dependent cortical fear engram in remote memory

Abstract

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.

Fig. 1

CFC enhanced neuronal activity in the mPFC. a Left: experimental design of groups used to assess Fos expression. Home-cage (HC; n = 4), context exposure only (Ctx; n = 6), contextual fear conditioning (CFC; n = 6). Lightning bold indicates foot-shock (1US). Right: illustration of a coronal brain section indicating the mPFC region (red) where Fos⁺ neurons were analyzed. b Representative examples of Fos⁺ cells (green) in all groups. c Percentage of Fos⁺ cells in each group. One-way ANOVA F_(2,13) = 126.3, p < 0.0001. Post-hoc Bonferroni test: HC vs. Ctx *p < 0.0001, HC vs. CFC *p < 0.0001, Ctx vs. CFC *p = 0.032. Scale bar = 50 µm. Bar graph shows mean + s.e.m. Source data are provided as a Source Data file

Fig. 2

Viral-TRAP enables inducible activity-dependent tagging of mPFC neurons. a Schematic representation of the viral-TRAP method. A mixture of AAV-Fos::CreER^T2 and Cre-dependent AAV (e.g. AAV-hSyn::DIO-hM4Di-mCherry) is bilaterally infused into the mPFC. The Fos promoter is activated by neuronal activity, resulting in CreER^T2 expression. Systemic injection of 4-hydroxytamoxifen (4TM) allows translocation of CreER^T2 into the nucleus enabling irreversible recombination of the Cre-dependent vector and expression of hM4Di-mCherry driven by the human Synapsin (hSyn) promoter. fmi = forceps minor of the corpus callosum. b Experimental design of groups used to validate viral-TRAP. Home-cage (HC) −4TM (n = 4), HC + 4TM (n = 7), Contextual fear conditioning (CFC) + 4TM (n = 6). 4TM was injected systemically 2 h after CFC on day 0 and all groups were killed 4 days later. c Expression of hM4Di-mCherry in mPFC. fmi = forceps minor of the corpus callosum. ML = midline. Left: scale bar = 250 µm; Right: scale bar = 100 µm. d Percentage of hM4Di-mCherry⁺ cells in mPFC. One-way ANOVA: F_(2,14) = 12.3, p = 0.001; post-hoc Bonferroni test: CFC vs. HC −4TM, p = 0.001, CFC vs. HC + 4TM, *p = 0.007. e Patch-clamp recordings of hM4Di⁺ and mCherry⁺ (control) cells before and after CNO application. ACSF = artificial cerebrospinal fluid. f Resting membrane potential changes for mCherry⁺ (n = 5) and hM4Di⁺ (n = 7) neurons. Wilcoxon signed rank test, hM4Di⁺: Z = −2.37, *p = 0.018; mCherry⁺: Z = −0.41 p = 0.69. g Rheobase changes for mCherry⁺ (n = 5) and hM4Di⁺ (n = 7) neurons. Wilcoxon signed rank test, hM4DI⁺: Z = −2.38 *p = 0.018; mCherry⁺: Z = −1.63, p = 0.10. All bar graphs show means + s.e.m. Source data are provided as a Source Data file

Fig. 3

CFC-tagged mPFC neurons are selectively involved in remote memory expression. a Experimental design. mPFC neurons activated during CFC were tagged with hM4Di-mCherry or mCherry. Both groups received CNO before a recent memory test on day 4. b CNO did not affect freezing levels on day 4. Unpaired t-test: t₁₂ = 1.169, p = 0.265, mCherry (n = 7), hM4Di (n = 7). c Experimental design. Groups received CNO before a remote memory test on day 30. d CNO reduced freezing of the hM4Di group compared with mCherry on day 30. Unpaired t-test: t₁₄ = 2.36, *p = 0.033, n = 8 per group. e Percentage of hM4Di⁺ neurons in recent and remote groups. Unpaired t-test: t₁₃ = 1.413, p = 0.18. n.s. = not significant. f Experimental design. mPFC neurons activated by context B were tagged with hM4Di-mCherry or mCherry. Mice received CNO before a remote memory test in the CFC context. g CNO did not affect freezing in the CFC context. Unpaired t-test: t₁₂ = 0.381, p = 0.71, mCherry (n = 7), hM4Di (n = 7). h Percentage of hM4Di-mCherry⁺ neurons tagged by context B exposure. i mPFC neurons activated during 3US CFC were tagged with hM4Di-mCherry or mCherry. j CNO did not affect freezing on day 30. Unpaired t-test: t₁₁ = 0.016, p = 0.988, mCherry (n = 7), hM4Di (n = 6). k Percentage of hM4Di⁺ neurons tagged during 3US CFC. All bar graphs show means+s.e.m. Source data are provided as a Source Data file

Fig. 4

Stimulation and reactivation of CFC-tagged mPFC neurons. a Experimental design. mPFC neurons activated during CFC were tagged with hM3Dq-mCherry. Freezing levels were assessed after vehicle (VEH) and CNO treatment in context B (recent) and C (remote). b CNO-enhanced freezing at recent and remote time-points. Repeated measures ANOVA, treatment: F_(1,7) = 13.1, p = 0.009 (n = 8 mice). c On day 33, mice received VEH or CNO, remained in their home-cage and were perfused 2 h later. d hM3Dq-mCherry and Fos expression in mPFC after VEH or CNO treatment. White outlined arrowheads indicate hM3Dq-mCherry⁺/Fos⁻ cells; white filled arrowheads indicate hM3Dq-mcherry⁺/Fos⁺ cells. e Percentage of hM3Dq-mcherry⁺ cells that expressed Fos after VEH or CNO. Mann–Whitney U = 0, p = 0.017 (n = 4 per treatment). f Experimental design. mPFC neurons were tagged with mCherry after CFC and re-exposed to the conditioning context 4 or 30 days later. g Example of colocalization of mCherry⁺ and Fos⁺ cells in the mPFC. Yellow outlined arrowheads indicate mCherry⁺/Fos⁻ cells; yellow filled arrowheads indicate mCherry⁺/Fos⁺ cells. h Percentage of Fos⁺ cells within the mCherry⁺ and mCherry⁻ populations. Two-way repeated measures ANOVA revealed a significant Time-point x Population interaction: F_(1,8) = 93.601, p < 0.001. Post-hoc Bonferroni test: Remote mCherry⁺ vs. mCherry⁻ *p < 0.0001; Recent mCherry⁺ vs. Remote mCherry⁺ p < 0.0001; Recent mCherry⁻ vs. Remote mCherry⁺ p < 0.0001; n = 5 per group. i mPFC neurons were tagged with mCherry after 3US CFC and re-exposed to the conditioning context 30 days later. j Example of colocalization of mCherry⁺ and Fos⁺ cells in the mPFC. Yellow outlined arrowheads indicate mCherry⁺/Fos⁻ cells; yellow filled arrowheads indicate mCherry⁺/Fos⁺ cells. k Percentage of Fos⁺ cells within the mCherry⁺ and mCherry⁻ populations. Paired t-test: t₆ = 1.186, p = 0.281 (n = 7 mice). All bar graphs show means + s.e.m. Scale bars = 50 µm. Source data are provided as a Source Data file

Fig. 5

Stimulation of retrieval-tagged mPFC neurons. a Experimental design. mPFC neurons were tagged with hM3Dq-mCherry after recent retrieval in the CFC context and freezing was subsequently assessed in context B and C after VEH and CNO treatment. b Repeated measures ANOVA did not reveal differences in freezing levels between VEH and CNO sessions at both time-points (treatment: F_(1,7) = 0.169, p = 0.69; n = 8 mice). c Experimental design. mPFC neurons were tagged with hM3Dq-mCherry after remote retrieval in the CFC context and freezing was subsequently assessed in context C. d CNO induced freezing compared with VEH. Paired t-test, t₆ = 3.56, *p = 0.012 (n = 7 mice). All bar graphs show means + s.e.m. Source data are provided as a Source Data file

Fig. 6

Disruption of CREB function in mPFC engram cells impairs remote fear memory. a Mice received AAV-Fos::CreER^T2 combined with AAV-hSyn::DIO-EGFP-mCREB into the mPFC. b Representative expression of EGFP-mCREB in mPFC in mice that remained in their home-cage (HC −4TM) and mice that underwent CFC without and with 4TM treatment. Mice were killed 4 days later. Scale bar = 100 µm. fmi = forceps minor of the corpus callosum. ML = midline. c Experimental design. mPFC neurons activated during CFC were tagged with EGFP-mCREB and memory was assessed on day 4 and 30 in the conditioning context. d On day 4, freezing did not differ between mCREB and control mice. Unpaired t-test: t₁₃ =  0.159, p = 0.876. mCherry (n = 8), mCREB (n = 7). e Freezing was significantly reduced in the mCREB mice compared with control mice. Unpaired t-test: t₁₃ = 2.669, *p = 0.019. mCherry (n = 7), mCREB (n = 8). All bar graphs show means + s.e.m. Source data are provided as a Source Data file