Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories

Abstract

Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata.

Figure 1. Remote Fear Memories Are Resistant to Attenuation despite Using Reconsolidation-Updating Mechanisms

(A) Schematic of the experimental paradigm. For details, see text. (B) Schematic of the massed extinction paradigm. (C) Using the massed extinction paradigm for contextual fear memories, recent memories show no signs of spontaneous recovery (n = 10; repeated-measurements ANOVA; p ≤ 0.0001). (D) Using the same massed extinction paradigm, remote memories spontaneously recover (n = 9; repeated-measurements ANOVA; p ≤ 0.0001). (E) Using the same paradigm but without memory recall, remote memories spontaneously recover (n = 10; repeated-measurements ANOVA; p ≤ 0.0001). (F) Schematic of the spaced extinction paradigm. (G) Using the spaced extinction paradigm for contextual fear memories, recent memories show significant attenuation of fear and no signs of spontaneous recovery (n = 12; repeated-measurements ANOVA; p = 0.0038). (H) Using the same spaced extinction paradigm, remote memories show no signs of attenuation (n = 16). (I) Using the spaced extinction paradigm without memory recall, remote memories show no signs of attenuation (n = 12). Error bars indicate ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 for Tukey's posthoc analysis. See also Figure S1 for results of remote cued fear memories.

Figure 2. The Recall of Remote Memories Is Not Salient Enough to Induce Neuroplasticity-Permitting Histone Acetylation Changes

(A) Representative images of immunohistochemical labelings of acetylated H3K9/14 (“AcH3”) in hippocampal area CA1 1 hr after recent and remote memory recall as compared to behaviorally naive animals. Scale bar, 100 μm. (B) Quantification thereof (n = 3–4 animals each). (C) Quantitative PCR results of the abundance of acetylated H3K9/14 in the promoter region of cFos at the same time points (n = 8 animals each). (D) Quantitative RT-PCR results of the expression of cFos in the hippocampus at the same time points (n = 4–5 animals each). (E) Quantification of the number of cFos-positive cells in the hippocampus at the same time points (n = 3 animals each). (F) Western blot analysis of S-nitrosylation of HDAC2 using the biotin-switch assay and streptavidin precipitation 1 hr after contextual fear conditioning and at different intervals after recent memory recall. (G) Representative pictures of western blot analysis of S-nitrosylation of HDAC2 1 hr after recent and remote memory recall. (H) Quantification thereof (n = 3–4 animals each). (I) Quantitative PCR results of HDAC2 binding to the promoter region of cFos at the same time points (n = 8 animals each). Error bars indicate ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Student's t test. See also Figure S2.

Figure 3. Nitrosylation of HDAC2 Is Critical for Memory Updating during Reconsolidation

(A) Schematic of the experimental paradigm. 1 hr before recent memory recall, L-NAME or its VEH was administered i.p. (B) Using the massed extinction paradigm for contextual fear memories, L-NAME-treated animals show significant spontaneous recovery, whereas VEH-treated ones do not (n = 8 for each treatment; repeated-measurements ANOVA; p ≤ 0.0001 for VEH, p = 0.0034 for L-NAME). (C) Representative pictures of western blot analysis of S-nitrosylation of HDAC2 1 hr after or without recall of recent memories in L-NAME and VEH-treated animals. (D) Schematic of the experimental paradigm. For details, see text. (E) Representative pictures of western blot analysis of S-nitrosylation of HDAC2 1 hr after remote memory recall in molsidomine- or VEH-treated HDAC2^WT or HDAC2^C262/274A-injected animals. (F) Using the massed extinction paradigm for contextual fear memories, HDAC2^WT-treated animals show no signs of spontaneous recovery, whereas HDAC2^C262/274A-injected animals do (n = 7–10 for both groups; repeated-measurements ANOVA; p ≤ 0.0001 for HDAC2^WT, p = 0.01 for HDAC2^C262/274A). Error bars indicate ± SEM. *p ≤ 0.05, **p≤0.01, ***p≤0.001 by Tukey's posthoc. See also Figure S3.

Figure 4. Remote Fear Memories Become Amenable to Attenuation by CI-994

(A) Chemical structure of CI-994, a benzamide-based HDAC inhibitor. (B) Concentration-time curve of CI-994 in C57BL/6 mouse brain after a 30/10/1mg/kg i.p. dose. (C) Pharmacokinetic parameters of CI-994 in C57BL/6 mouse brain (see Extended Experimental Procedures for additional details). (D) Schematic of the experimental paradigm. 1 hr post remote memory recall, either CI-994 or its VEH was administered i.p., and 1 hr later, the different extinction procedures were applied. (E) Using the massed extinction paradigm to the context, VEH-treated animals show significant spontaneous recovery of fear (n = 11; repeated-measurements ANOVA; p = 0.0001). (F) Using the same paradigm, HDACi-treated animals show no spontaneous recovery (n = 16; repeated-measurements ANOVA; p ≤ 0.0053). (G) Using the same paradigm but without memory recall, HDACi-treated animals show significant spontaneous recovery (n = 17; repeated-measurements ANOVA; p ≤ 0.0001). (H) Using the spaced extinction paradigm to the context, VEH-treated animals do not show any attenuation in their fear response (n = 15). (I) Using the same paradigm, HDACi-treated animals show significant reduction in their freezing response immediately and 1 day after extinction and show no signs of spontaneous recovery (n = 16; repeated-measurements ANOVA, p ≤ 0.0001). (J) Using the same paradigm but without memory recall, HDACi-treated animals do not show any attenuation in their fear response (n = 12). Error bars indicate ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Tukey's posthoc. See also Figure S4.

Figure 5. HDACis Prime a Transcriptional Program for Increased Neuroplasticity

(A) Representative images of immunohistochemical labelings of acetylated H3K9/14 (“AcH3”) in hippocampal area CA1 1 hr after recall and 1 hr after completion of extinction in VEH- and HDACi-treated animals. Scale bar, 100 μm. (B) Quantification thereof (n = 3–4 animals each). (C) HDAC2 activity assay showing that CI-994 effectively inhibited hippocampal HDAC2 activity (n = 4 animals each). (D) Heatmap depicting 475 differentially expressed genes (DEGs) determined by RNA-sequencing in the hippocampus between VEH-treated and HDACi-treated animals after remote memory attenuation. Each line represents a DEG, each row the gene expression per animal. Blue and red indicate low and high levels of expression, respectively. (E) Histogram showing the biological processes to which the DEGs belong (orange, number of DEGs per biological process; green, significance of enrichment). (F) Quantitative RT-PCR confirmation of the expression of several neuroplasticity-related genes detected under (D) (n = 5–9 animals each). (H) Quantitative PCR results showing the abundance of AcH3K9/14 at the promoter region of the genes under (F) (n = 5–11 animals each). (I) Quantitative PCR results showing the binding of HDAC2 to the promoter region of the genes under (F) (n = 5–6 animals each). Error bars are ± SEM. *p ≤ 0.05, **p ≤ 0.01 by Student's t test. See also Figure S5 and Table S1.

Figure 6. HDACis Increase Neuroplasticity after Remote Memory Attenuation

(A) Representative scans of [³H]-2DG uptake in coronal brain sections depicting higher metabolic activity in the hippocampus (outlined by white dotted lines) for CI-994-treated animals. (B) Quantification thereof (n = 10 animals each). (C) Field excitatory postsynaptic potential (fEPSP) slopes in hippocampal area CA1 of VEH- and CI-994-treated animals (n = 6 slices from four mice each); sample traces below the point chart represent fEPSPs at 1 min before (gray) and 1 hr after (colored) theta-burst stimulation (TBS). (D) Representative images of MAP2-labeled hippocampal sections. Scale bar, 200 mm. (E) Quantification thereof (n = 3 animals each). (F) Representative images of Golgi-stained hippocampal pyramidal neurons used for Sholl analysis. Scale bar, 100 μm. (G) Quantification of the number of dendritic branches per given distance from the soma (n = 8–12 neurons of 3 animals each). (H) Representative images of Golgi-stained hippocampal pyramidal neurons. Scale bar, 10 μm. (I) Quantification of (H) in terms of number of spines (n = 3 animals each). (J) Quantification of (H) in terms of number of mature versus immature spines (n = 3 animals each). (K) Representative images of hippocampal brain section (stratum radiatum) used for transmission electron microscopy. Arrows point to synapses. Scale bar, 1 μm. (L) Quantification thereof (n = 3–4 animals each). Error bars indicate ± SEM. #p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01 by Student's t test. See also Figure S6.

Figure 7. Working Model

HDACis epigenetically prime the expression of neuroplasticity-related genes (e.g., cFos) to overcome the absence of hippocampal neuroplasticity upon remote memory recall and thereby the resilience of remote fear memories to successful extinction (for details, see text).