Fig. 4.
Environmental enrichment (EE) reversed the increased 5′-cytosine methylation in the promoter region of Bdnf exon IV induced by neonatal anesthesia. Significantly increased hippocampal DNA methyltransferase 1 (DNMT1) was observed in the rats with neonatal anesthesia, which was significantly decreased by EE (A, n = 6, 6, 7, and 7 rats in each group, Kruskal-Wallis statistic = 13.5, P < 0.0037). Increased methyl-cytosine-phosphate-guanine–binding protein 2 (MeCP2) in the precipitated complex pulled down by polyclonal DNMT1 antibody in the hippocampal lysates was observed in the rats with neonatal anesthesia, which was reduced by EE (B, n = 6, 6, 7, and 7 rats in each group, F3, 22 = 55.3, P < 0.0001). Significantly increased occupancy of DNMT1 in the Bdnf, but not Gapdh promoter region, was observed in the rats with neonatal exposure to anesthesia, which was significantly decreased by EE (C, n = 6 rats in each group, F3, 20 = 8.9, P = 0.0006). Chromatin immunoprecipitation (ChIP) study with polyclonal antibody against 5′-methylcytosine showed a significant increase in cytosine methylation in the Bdnf exon IV, but not Gapdh, promoter region of the modeled rats, which was also reversed by EE (D, n = 6 rats in each group, F3, 20 = 8.2, P = 0.0009). **P < 0.01; ***P < 0.001. Data represent mean ± SD.