Cortical DNA methylation maintains remote memory

Abstract

A behavioral memory's lifetime represents multiple molecular lifetimes, suggesting the necessity for a self-perpetuating signal. One candidate is DNA methylation, a transcriptional repression mechanism that maintains cellular memory throughout development. We found that persistent, gene-specific cortical hypermethylation was induced in rats by a single, hippocampus-dependent associative learning experience and pharmacologic inhibition of methylation 1 month after learning disrupted remote memory. We propose that the adult brain utilizes DNA methylation to preserve long-lasting memories.

Figure 1. Learning induces persistent DNA methylation of calcineurin in the prefrontal cortex

a, CaN CpG island analyzed and primer location. CaN is hypermethylated in CS animals at 1d and 7d (no retrieval test; 1h: F_{(3, 18)} = 0.27, 1d: F_{(3, 19)} = 5.73, 7d: F_{(3, 25)} = 33.52). * post hoc P ≤ 0.05. CaN’s hypermethylation is significantly greater at 1 and 7d (F_{(2, 20)} = 13.96, # P ≤ 0.01). See Supplementary Table 1 for N’s. b, Amplicon analyzed by BSP (transcription start site – TSS) at 7d. Charts depict number of alleles with 0, 1, 2 or 3 methylated CpGs in the amplicon. c, Single allele cytosine methylation for the 59 CpGs located in the amplicon at 7d (N = 4 per group). Error bars indicate s.e.m.

Figure 2. Cortical DNA methylation persists for at least 30 days

a, 30d after training, CaN is still hypermethylated (F_{(3, 19)} = 4.77).* P ≤ 0.05. b, CaN transcript is decreased in CS at 30d (t₅ = −8.36, * P ≤ 0.001; F_{(2, 15)} = 12.72). c, 2h after retrieval test, CaN protein levels are decreased in CS as well (F_{(3, 30)} = 24.31, * P ≤ 0.001; # P ≤ 0.05). See Supplementary Table 2 for N’s.

Figure 3. Cortical DNA methylation is required for remote memory

a, Intra-ACC infusions of 5-aza (N’s = 14, 13; F_{(1, 26)} = 11.48), zeb (N’s = 7, 7; F_{(1, 13)} = 21.07) or RG108 (N’s = 10, 10; F_{(1, 19)} = 5.17) 30d after training disrupted remote fear memory. * P ≤ 0.05. b, DNMTi interfered with CS-induced CaN methylation (N’s = 6, 6; F_(1,11) = 8.96) and normalized CaN transcript levels (t₈ = 8.34, * P ≤ 0.001). c, Intra-ACC infusions of DNMTi 1d after training had no effect on fear memory (N’s = 7, 8; F_(1,14) = 0.81, P > 0.05). Same animals also expressed normal fear memory 30 days later (F_(1,12) = 0.26, P > 0.05). Experiment was repeated in absence of a test at 2d to confirm lack of damage to ACC. Again, infusions had no effect on fear memory (N’s = 7, 8; F_(1,14) = 0.81, P > 0.05). Error bars indicate s.e.m.