Fig. 3. Early-life stress in D2+/− mice induced altered expression of BDNF-TrkB signaling in the dorsal striatum. (A) Experimental design for treatment with early-life stress, weaning, and time points for sacrifice. A diagram showing the regions (circles) in the dorsal striatum used for tissue prep (right panel). (B, C) Real-time PCR data showing transcript levels of TrkB, tBdnf, Bdnf1, and Bdnf4 (B), and Mecp2, Hdac1, Hdac2, Hdac3, Hdac4, and Hdac5 (C) in the dorsal striatum of WT and D2 heterozygous KO pups raised normally or after exposure to early-life stress. Tissue samples were prepared on postnatal day 14. n=4~6 animals, 4~6 PCR repeats (TrkB: ELS effect, F(1, 12)=18.12, p=0.0011; genotype effect, F(1, 12)=0.1136, p=0.7419; ELS x genotype interaction, F(1, 12)=9.483, p=0.0095; tBdnf: ELS effect, F(1, 12)=4.962, p=0.0458; genotype effect, F(1, 12)=0.3740, p=0.5523; ELS x genotype interaction, F(1, 12)=0.2762, p=0.6088; Hdac1: ELS effect, F(1, 20)=0.5866, p=0.4527; genotype effect, F(1, 20)=23.25, p=0.0001; ELS x genotype effect; F(1, 20)=6.090, p=0.0227; Hdac3: ELS effect, F(1, 20)=0.0.8137, p=0.3778; genotype effect, F(1, 20)=11.24, p=0.0032; ELS x genotype effect, F(1, 20)=0.1040, p=0.7504). (D, E) Real-time PCR data showing transcript levels of TrkB, tBdnf, Bdnf4 (D), Mecp2, Hdac1, Hdac2, Hdac3, Hdac4, and Hdac5 (E) in the dorsal striatum of WT and D2 heterozygous KO mice raised normally or after exposure to ELS, and sacrificed on postnatal day 70. n=4–6 animals, 3~6 PCR repeats (TrkB: ELS effect, F(1, 14) =276.5, p<0.0001; genotype effect, F(1, 14)=53.77, p<00001; ELS x genotype interaction, F(1, 14)=179.5, p<0.0001; tBdnf: ELS effect, F(1, 12)=2.902, p=0.1142; genotype effect, F(1, 12)=10.83, p=0.0064; ELS x genotype interaction, F(1, 12)=4.888, p=0.0472; Bdn4f: ELS effect, F(1, 8)=2.734, p=0.1369; genotype effect, F(1, 8) =26.33, p=0.0009; ELS x genotype interaction, F(1, 8)=14.65, p=0.0050; Mecp2: ELS effect, F(1, 15)=17.80, p=0.0007; genotype effect, F(1, 15)=7.572, p=0.0148; ELS x genotype effect, F(1, 15)=9.961, p=0.0065; Hdac2; ELS effect, F(1, 16)=20.81, p=0.0003; genotype effect, F(1, 16)=6.247, p=0.0237; ELS x genotype effect, F(1, 16)=12.25, p=0.0030; Hdac3: ELS effect, F(1, 12)=7.365, p=0.0188; genotype effect, F(1, 12)=10.02, p=0.0081; ELS x genotype effect, F(1, 12)=0.08215, p=0.7739; Hdac5: ELS effect, F(1, 12)=0.06437, p=0.8040; genotype effect, F(1, 12)=1.741, p=0.2117; ELS x genotype effect, F(1, 12)=0.7854, p=0.3929). (F~I) Western blots showing TrkB, BDNF, MeCP2 (F), p-AKT, AKT, p-ERK1/2, ERK1/2, p-CREB, CREB and β-Actin (H) levels in the dorsal striatum of WT mice and D2 heterozygous KO mice raised normally or or after exposure to early-life stress, and sacrificed on postnatal day 70. Quantification of Western blots (G, I). n=6~8 animals, 3~5 repeats (TrkB: ELS effect, F(1, 8)=0.6597, p=0.4402; genotype effect, F(1, 8)=20.88, p=0.0018; genotype x ELS interaction, F(1, 8)=7.630, p=0.0246; BDNF: ELS effect, F(1, 8)= 3.428, p=0.1012; genotype effect, F(1, 8)=1.998, p=0.1953; genotype x ELS interaction, F(1, 8)=13.14, p=0.0067; MeCP2: ELS effect, F (1, 16)=5.085, p=0.0385; genotype effect, F(1, 16)=18.71, p=0.0005; genotype x ELS interaction, F(1, 16)=22.01, p=0.0002). Data are presented as mean±SEM. * and ** denote differences between the indicated groups at p<0.05 and p<0.01, respectively (Two-way ANOVA and Holm-Sidak post hoc test).
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