DNA methylation regulates the extinction of fear memory

Figure 1 N-phthalyl-L-tryptophan (RG108) inhibited the extinction of fear memory. A: The DNA methylation level in hippocampus was significantly decreased after 2 hours of RG108 injection (unpaired t-test, T₉ = 4.291, P < 0.01); B: Experimental procedures. Vehicle, n = 10; RG108, n = 12; C: Fear. cond, freezing levels of the last conditioned stimulus and unconditional stimulus pairing; Rec B, averaged freezing levels during four CS during recall. Significantly higher freezing levels in F. cond than those during recall in contexts B in vehicle group but not in RG108 group (two-way RM ANOVA with multiple comparisons, F. cond vs Rec B, vehicle, T₂₀ = 2.564, P < 0.05; RG108, T₂₀ = 0.2606, P = 0.7971) and vehicle group showed significant lower freezing level in Rec B compared with the RG108 group (T₄₀ = 2.683, P < 0.05); D-F: Freezing levels during the entire experimental course. Recall in Context B, significantly higher freezing level in RG108 group (two-way RM ANOVA, F_{(1, 20)} = 9.063, P < 0.01). ^aP < 0.05; ^bP < 0.01; ^cP< 0.001.

Figure 2 DNA methyltransferase-3A and dendritic spine density changed after RG108 injection. A: Experimental procedures; B: Significant decreased in the density of DNA methyltransferase-α (Dnmt3a)-positive cells in the RG108 group, compared to vehicle group (n = 4 mice, at least 3 sections per mouse; unpaired t-test, T₆ = 3.617, P < 0.05); C: Sample images of Dnmt3a staining; D: Sample images of Golgi staining; E: The dendritic spine density was decreased in RG108 group (axons from at least 3 mice per group, unpaired test, T₁₆ = 3.844, P < 0.01). ^aP < 0.05; ^bP < 0.01; ^cP< 0.001.

Figure 3 Calcium responses in dorsal dentate gyrus neurons during condition stimulus presentation. A and B: Experimental procedures, neurons in dorsal dentate gyrus (dDG). Scale bars, 100 μm; C-F: Averaged population Ca²⁺ responses showed gradual reduction during condition stimulus (CS) presentations and quantification of changes in Ca²⁺ responses with area from the 20 s CS presentations; n = 3; E: Two-way RM ANOVA, F_(1,4) = 0.3651, P = 0.5783; F: Two-way RM ANOVA, with multiple comparisons, F_(1,4) = 3.888, P = 0.1199; first 10 s, T₈ = 0.9554, P = 0.3674; post 10 s, T₈ = 2.800, ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 4 L-methionine promote the fear memory extinction. A: The DNA methylation level in hippocampus was significantly decreased after 7 days L-methionine (L-M)/vehicle injection (Unpaired t-test, T₈ = 3.409, P < 0.01); B: Experimental procedures. Vehicle group (7 days vehicle injection), n = 9; L-M group (7 days L-M injection), n= 10; C: F. cond, freezing levels of the last conditioned stimulus and unconditional stimulus (CS-US) pairing; Rec B, averaged freezing levels during four CS during recall. Significantly higher freezing levels in F. cond than those during recall in contexts B (two-way RM ANOVA, F. cond vs Rec B, vehicle, T₁₇ = 4.935, P = 0.0001; L-M, T₁₇ = 7.266, P < 0.0001); D-F: Freezing levels during the entire experimental course; E: Ext, significantly decreased freezing level in extinction procedure in the L-M group [two-way RM ANOVA, F_(1,17) = 5.353, P < 0.05]. No significant difference was found regarding fear conditioning Day 1 (E) and recall in Context B on Day 10 (F); G: Experimental procedures. Vehicle group, n = 12; L-M, n = 12; H: F. cond, freezing levels of the last CS-US pairing; Rec B, averaged freezing levels during four CS during recall. Significantly higher freezing levels in F. cond than those during recall in contexts B (two-way RM ANOVA, F. cond vs Rec B, vehicle, T₂₂ = 7.433, P < 0.0001; L-M, T₂₂ = 8.909, P < 0.0001); I-K: Freezing levels during the entire experimental course; E: Ext, significantly decreased freezing level in the first day of extinction procedure in L-M group [two-way RM ANOVA, F_(1,22) = 8.798, P < 0.01]. No significant difference was found on fear conditioning Day 1 (I) and recall in Context B on Day 10 (K). P < 0.05. ^aP < 0.05; ^bP < 0.01; ^cP< 0.001; ^dP< 0.0001.