Histone modifications around individual BDNF gene promoters in prefrontal cortex are associated with extinction of conditioned fear

Abstract

Extinction of conditioned fear is an important model both of inhibitory learning and of behavior therapy for human anxiety disorders. Like other forms of learning, extinction learning is long-lasting and depends on regulated gene expression. Epigenetic mechanisms make an important contribution to persistent changes in gene expression; therefore, in these studies, we have investigated whether epigenetic regulation of gene expression contributes to fear extinction. Since brain-derived neurotrophic factor (BDNF) is crucial for synaptic plasticity and for the maintenance of long-term memory, we examined histone modifications around two BDNF gene promoters after extinction of cued fear, as potential targets of learning-induced epigenetic regulation of gene expression. Valproic acid (VPA), used for some time as an anticonvulsant and a mood stabilizer, modulates the expression of BDNF, and is a histone deacetylase (HDAC) inhibitor. Here, we report that extinction of conditioned fear is accompanied by a significant increase in histone H4 acetylation around the BDNF P4 gene promoter and increases in BDNF exon I and IV mRNA expression in prefrontal cortex, that VPA enhances long-term memory for extinction because of its HDAC inhibitor effects, and that VPA potentiates the effect of weak extinction training on histone H4 acetylation around both the BDNF P1 and P4 gene promoters and on BDNF exon IV mRNA expression. These results suggest a relationship between histone H4 modification, epigenetic regulation of BDNF gene expression, and long-term memory for extinction of conditioned fear. In addition, they suggest that HDAC inhibitors may become a useful pharmacological adjunct to psychotherapy for human anxiety disorders.

Figure 1.

Learning-induced histone modifications ±SEM around BDNF gene promoters (A–C) P1 and (D–F) P4. (A) At the BDNF P1, histone H3 acetylation was increased in fear-conditioned animals compared to both other groups (**P < 0.01), and H3 acetylation was decreased in extinguished animals relative to naive controls (*P < 0.05). (B) There were no differences in histone H4 acetylation. (C) Representative gel for the BDNF P1 PCR product. (D) At BDNF P4, histone H3 acetylation was increased in the fear-conditioned without extinction mice compared to naive controls (*P < 0.05). (E) Histone H4 acetylation was significantly increased in extinguished animals compared to both other groups (*P < 0.05), which did not differ from each other. (F) Representative gel for the BDNF P4 PCR product. (G,H) There were no significant learning-induced histone modifications around the promoter for β-tubulin. (I) Representative gel for the β-tubulin PCR product. (n = 4 samples pooled from two animals each.) (N) Naive; (FC-No EXT) fear conditioned without extinction; (EXT) extinction.

Figure 2.

Effect of learning on (A,B) BDNF exon I and (C,D) IV mRNA expression in the prefrontal cortex. (A) Representative quantitative RT-PCR plot for the effect of strong (57 CSs) extinction training on BDNF exon I mRNA expression in the prefrontal cortex. (B) Strong extinction training yielded a significant increase in BDNF exon I mRNA expression. (C) Representative quantitative RT-PCR plot for the effect of strong (57 CSs) extinction training on BDNF exon IV mRNA expression in the prefrontal cortex. (D) Strong extinction training yielded a significant increase in BDNF exon IV mRNA expression. (**P < 0.01, n = 4 samples pooled from two animals each.) (N) Naive; (FC-No EXT) fear conditioned without extinction; (EXT) partial extinction.

Figure 3.

Mean ± SEM percent freezing 7 d after histone deacetylase (HDAC) inhibitor injection before partial extinction training. None of the drugs had any significant effect on pre-CS freezing compared to mice treated with vehicle and partially extinguished. (A) At a dose of 100 mg/kg, valproic acid potentiates long-term memory for the extinction of conditioned fear. (B) Valpromide has no effect on extinction of conditioned fear. (C) At a dose of 1000 mg/kg, sodium butyrate potentiates long-term memory for the extinction of conditioned fear. (*P < 0.05 compared to mice extinguished with vehicle; n = 8–16 mice/group.) (FC-No EXT) Fear conditioned without extinction.

Figure 4.

Chemical structures of compounds used to facilitate extinction.

Figure 5.

Effect of histone deacetylase inhibition by valproic acid (VPA) on learning-induced histone modifications (±SEM) around the P1 promoter of the BDNF gene and on BDNF exon I mRNA expression in the prefrontal cortex. VPA (100 mg/kg) combined with partial extinction training (20 CS presentations) (A) had no significant effect on H3 acetylation around the P1 promoter, (B) but increased histone H4 acetylation relative to all other treatment groups (*P < 0.05, n = 4 samples pooled from two animals each). (Inset) Raw data. (C) Representative quantitative RT-PCR plot for the effect of VPA and partial extinction training on BDNF exon I mRNA expression in the prefrontal cortex. (D) Extinction training yielded a significant increase in BDNF exon I mRNA expression. (*P < 0.05, n = 6 samples pooled from two animals each.) (FC-No EXT) Fear conditioned without extinction; (EXT) partial extinction.

Figure 6.

Effect of histone deacetylase inhibition by valproic acid (VPA) on learning-induced histone modifications (±SEM) around the P4 promoter of the BDNF gene and on BDNF exon IV mRNA expression in the prefrontal cortex. VPA (100 mg/kg) combined with partial extinction training (20 CS presentations) (A) had no effect on H3 acetylation around the P4 promoter of the BDNF gene, (B) but increased histone H4 acetylation relative to all other treatment groups (**P < 0.01, n = 4 samples pooled from two animals each). (Inset) Raw data. (C) Representative quantitative RT-PCR plot for the effect of VPA and partial extinction training on BDNF exon IV mRNA expression in the prefrontal cortex. (D) VPA (100 mg/kg) yielded no significant (ns) increase in exon IV mRNA expression in retention controls, but a significant increase with extinction. (**P < 0.01, n = 6 samples pooled from two animals each.) (FC-No EXT) Fear conditioned without extinction; (EXT) partial extinction.