Nuclear factor κB-dependent histone acetylation is specifically involved in persistent forms of memory

Abstract

Memory consolidation requires gene expression regulation by transcription factors, which eventually may induce chromatin modifications as histone acetylation. This mechanism is regulated by histone acetylases and deacetylases. It is not yet clear whether memory consolidation always recruits histone acetylation or it is only engaged in more persistent memories. To address this question, we used different strength of training for novel object recognition task in mice. Only strong training induced a long-lasting memory and an increase in hippocampal histone H3 acetylation. Histone acetylase inhibition in the hippocampus during consolidation impaired memory persistence, whereas histone deacetylase inhibition caused weak memory to persist. Nuclear factor κB (NF-κB) transcription factor inhibition impaired memory persistence and, concomitantly, reduced the general level of H3 acetylation. Accordingly, we found an important increase in H3 acetylation at a specific NF-κB-regulated promoter region of the Camk2d gene, which was reversed by NF-kB inhibition. These results show for the first time that histone acetylation is a specific molecular signature of enduring memories.

Figure 1.

Strong training induces a persistent recognition memory and histone acetylation increment during consolidation. a, Diagram outlining the experimental design. TR3 (n = 10), TR10 (n = 9), and TR15 (n = 9) groups received 3, 10, or 15 min of training, respectively. b, Graph representing the mean ± SEM of DI% for each group. **p < 0.01 in a one-way ANOVA, followed by the Duncan post hoc test. c, Diagram outlining the experimental design. TR3 (n = 11), TR10 (n = 11), and TR15 (n = 9) groups as in a and b, plus a nontrained HAB group (n = 9). Animals were killed 1 h after training. Graph represents the mean ± SEM of acetyl H3 levels in the hippocampus estimated by Western blot normalized to total H3 levels. *p < 0.05, one-way ANOVA, followed by the Duncan post hoc test.

Figure 2.

Administration of a HAT inhibitor in the hippocampus impairs long-lasting recognition memory and the histone acetylation increment during consolidation. a, Diagram outlining the experimental design. Left: Mean ± SEM of DI% for the 24 h test of the TR15+HATi (n = 11) and TR15+Veh (n = 6) groups. Right: Mean ± SEM of DI% for the 7 d test of the TR15+HATi (n = 8) and TR15+Veh groups (n = 9). **p < 0.01, Student's t test. b, Diagram outlining the experimental design. Animals were killed 1 h after training (n = 6 per group). Graph represents the mean ± SEM of acetyl H3 levels in hippocampus estimated by Western blot normalized to total H3 levels. *p < 0.05 Student's t test. c, Example of cannula position (cresyl violet stained).

Figure 3.

Administration of HDAC inhibitors in the hippocampus enhanced memory persistence by the induction of histone acetylation. a–c, Diagrams outlining the experimental design. a, Graph representing the mean ± SEM of DI% for the TR3+NaB (n = 7) and TR3+Veh (n = 8) groups. *p < 0.05, Student's t test. b, Graph representing the mean ± SEM of DI% for the TR3+TSA (n = 11) and TR3+Veh (n = 10) groups. **p < 0.01, Student's t test. c, Graph representing the mean ± SEM of DI% for the TR3+NaB (n = 7), TR3+Veh (n = 7), and TR15+Veh (n = 8) groups. *p < 0.05, one-way ANOVA, followed by a Duncan post hoc test. d, Graph representing the mean ± SEM of acetyl H3 levels in hippocampus estimated by Western blot normalized to total H3 levels. Animals were killed 1 h after training. *p < 0.05, Student's t test. n = 10.

Figure 4.

Hippocampal NF-κB activity is required for the histone acetylation increment during consolidation of object recognition memory. a–c, Diagrams outlining the experimental design. a, Graph representing the mean ± SEM of DI% for the TR15+Dec (n = 11) and TR15+mDec (n = 10) groups. *p < 0.05, Student's t test. b, Graph representing the mean ± SEM of DI% for the TR15+Dec (n = 7) and TR15+mDec (n = 7) groups. *p < 0.05). c, Graph representing the mean ± SEM of acetyl H3 levels in hippocampus estimated by Western blot normalized to total H3 levels. Animals were killed 1 h after training. *p < 0.05, Student's t test.

Figure 5.

Acetylation of a promoter region that included an NF-κB consensus sequence is specifically increased after strong training. a, Diagram outlining the experimental design. Left upper diagram: NF-kB-binding sites identified within 1 kbp promoter upstream sequences of the Zif268 gene (GenBank Gene ID: 13653). Left graph: mean ± SEM of the fold change relative to input fraction of histone H3 acetylation at the Zif268 promoter in the hippocampus 1 h after training obtained from a ChIP assay. Right upper diagram: Bona fide NF-κB-binding site identified within 1 kbp promoter upstream sequences of the Camk2d gene (GenBank Gene ID: 108058). Right graph: Mean ± SEM of the fold change relative to input fraction of histone H3 acetylation at the Camk2d promoter in the hippocampus 1 h after training obtained from a ChIP assay. *p < 0.05, one-way ANOVA, followed by a Duncan post hoc test. For this study, 2 independent experiments were performed, with 4 animals per group. b, Diagram outlining the experimental design. Graph represents the mean ± SEM of the fold change relative to input fraction of p65 levels at the Camk2d promoter in the hippocampus 1 h after training obtained from ChIP assay. *p < 0.05, Student's t test. For this study, 2 independent experiments were performed with 4 animals per group. c, Diagram outlining the experimental design. Graph represents the mean ± SEM of the fold change relative to input fraction of H3 acetyl levels at the Camk2d promoter in the hippocampus 1 h after training and decoy injection obtained from ChIP assay. *p < 0.05, Student's t test. For this study, 2 independent experiments were performed with 4 animals per group.

Figure 6.

Camk2d expression is specifically induced after strong training. Diagram outlines the experimental design. Graphs represent the mean ± SEM of gene expression levels in hippocampus of Camk2d (left) and βactin (right) measured by real-time PCR for each group (HAB, TR10, and TR15). Animals were killed 3 h after training. *p < 0.05, one-way ANOVA, followed by a Duncan post hoc test.