Figure 1.

KA-induced SE triggers changes in global hippocampal DNA methylation levels and expression of genes encoding for DNMT and TET methylating enzymes. A) Diagram of experimental setup and an illustration of the method for sub-dissection of hippocampal regions using the fissure markings of the hippocampus. Dotted lines indicate cuts made to dissect hippocampal regions: CA1, CA3, and the DG region. B) 5-mC DNA methylation levels remained unchanged in all hippocampal regions assessed during SE but were significantly increased later in areas CA1 and CA3 of the hippocampus from epileptic animals compared to controls. However, in the DG region of the hippocampus, 5-mC DNA methylation levels were significantly decreased following epilepsy onset (CA1 SE, t₍₁₃₎ = 0.68, p = 0.51, n = 7-8; CA1 epileptic, t₍₁₄₎ = 2.34, p = 0.03, n = 8; CA3 SE, t₍₁₃₎ = 1.17, p = 0.26, n = 7-8; CA3 epileptic, t₍₁₆₎ = 2.12, p = 0.05, n = 9; DG SE, t₍₁₃₎ = 0.94, p = 0.36, n = 7-8; DG epileptic, t₍₁₁₎ = 3.65, p = 0.003, n = 6-7). C) 5-hmC DNA methylation levels were not significantly altered in area CA1 of the hippocampus during SE or following the onset of epilepsy. 5-hmC DNA methylation levels were significantly decreased in hippocampal area CA3 during SE and later returned to baseline levels in the hippocampus of epileptic rats compared to controls. 5-hmC DNA methylation levels were unchanged in the DG region during SE but were significantly decreased in the DG region of the epileptic hippocampus relative to controls (CA1 SE, t₍₉₎ = 0.28, p = 0.78, n = 4-7; CA1 epileptic, t₍₁₀₎ = 0.01, p = 0.99, n = 5-7; CA3 SE, t₍₁₁₎ = 3.90, p = 0.003, n = 6-7; CA3 epileptic, t₍₉₎ = 0.93, p = 0.37, n = 5-6; DG SE, t₍₁₂₎ = 0.20, p = 0.84, n = 6-8; DG epileptic, t₍₁₀₎ = 3.15, p = 0.01, n = 5-7). D) DNMT1 mRNA levels were not significantly altered in hippocampal area CA1 during SE or in the epileptic hippocampus compared to controls. DNMT1 mRNA levels were significantly decreased in hippocampal area CA3 during SE, while no changes were observed in area CA3 of the epileptic hippocampus compared to controls. DNMT1 mRNA levels were significantly decreased in the DG region during SE, while no change occurred in the DG region of the epileptic hippocampus relative to controls (CA1 SE, t₍₁₃₎ = 0.92, p = 0.37, n = 7-8; CA1 epileptic, t₍₁₁₎ = 0.10, p = 0.93, n = 6-7; CA3 SE, t₍₁₃₎ = 2.28, p = 0.04, n = 7-8; CA3 epileptic, t₍₁₁₎ = 1.94, p = 0.08, n = 6-7; DG SE, t₍₁₃₎ = 3.14, p = 0.008, n = 7-8; DG epileptic, t₍₁₀₎ = 0.35, p = 0.73, n = 6). E) DNMT3a mRNA levels were significantly decreased in area CA1 at during SE and at the epileptic stage compared to controls. DNMT3a mRNA levels in area CA3 were unchanged during SE and significantly decreased later in the epileptic hippocampus compared to controls. DNMT3a mRNA levels were unchanged in the DG region during SE and in the epileptic hippocampus relative to controls (CA1 SE, t₍₁₃₎ = 2.40, p = 0.03, n = 7-8; CA1 epileptic, t₍₁₅₎ = 3.248, p = 0.005, n = 8-9; CA3 SE, t₍₁₂₎ = 0.40, p = 0.70, n = 6-8; CA3 epileptic, t₍₁₆₎ = 2.56, p = 0.02, n = 9; DG SE, t₍₁₃₎ = 0.60, p = 0.57, n = 7-8; DG epileptic, t₍₁₃₎ = 0.99, p = 0.34, n = 6-9). F) DNMT3b mRNA levels were unchanged in hippocampal area CA1 during SE and in area CA1 of the epileptic hippocampus compared to controls. DNMT3b mRNA levels were unchanged in area CA3 during SE while being significantly increased in area CA3 of the epileptic hippocampus compared to controls. DNMT3b mRNA levels remained unchanged in the DG region during SE and in the DG region of the epileptic hippocampus relative to controls (CA1 SE, t₍₁₃₎ = 0.80, p = 0.44, n = 7-8; CA1 epileptic, t₍₁₅₎ = 0.62, p = 0.54, n = 8-9; CA3 SE, t₍₁₂₎ = 0.37, p = 0.72, n = 6-8; CA3 epileptic, t₍₁₄₎ = 2.33, p = 0.04, n = 8; DG SE, t₍₁₃₎ = 0.69, p = 0.50, n = 7-8; DG epileptic, t₍₁₁₎ = 0.32, p = 0.76, n = 5-8). G) No changes in TET1 mRNA levels were observed in area CA1 during SE or in the epileptic hippocampus compared to controls. TET1 mRNA levels were significantly decreased in area CA3 and the DG region during SE relative to controls. (CA1 SE, t₍₁₃₎ = 0.14, p = 0.90, n = 7-8; CA1 epileptic, t₍₁₀₎ = 0.56, p = 0.59, n = 6; CA3 SE, t₍₁₃₎ = 2.50, p = 0.03, n = 7-8; CA3 epileptic, t₍₁₀₎ = 0.85, p = 0.42, n = 6; DG SE, t₍₁₃₎ = 3.14, p = 0.008, n = 7-8; DG epileptic, t₍₁₀₎ = 0.49, p = 0.63, n = 6). Student unpaired t-test, *p<0.05, **p<0.01. Error bars are SEM.