Cocaine-induced chromatin remodeling increases brain-derived neurotrophic factor transcription in the rat medial prefrontal cortex, which alters the reinforcing efficacy of cocaine

Abstract

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

Figure 1.

BDNF protein levels are increased in the mPFC after cocaine self-administration. A, A diagram depicting a coronal section of the rat brain at the level of the mPFC (3.20 mm anterior to bregma) (Paxinos and Watson, 1997). The mPFC dissection (shown in black) included the anterior cingulate, prelimbic, and infralimbic cortices. B, Western blot analysis revealed an increase in BDNF protein levels in mPFC. Densitometry results demonstrate a significant increase in BDNF levels in mPFC (saline, n = 4; cocaine, n = 4). C, Similarly, an ELISA demonstrated a significant increase in BDNF levels in the mPFC (saline, n = 4; cocaine, n = 4). IDV, Integrated density value. *p < 0.05, significant differences between saline and cocaine treatments (unpaired t tests,).

Figure 2.

shRNA-induced downregulation of BDNF expression in the mPFC enhances the reinforcing efficacy of cocaine, as assessed with a PR schedule. A, The mean ± SEM BDNF staining intensity per cell (in arbitrary units) in control and AAV10–BDNF shRNA-treated rats. A total of 380 cells were sampled in the control sections and 231 cells from AAV10–BDNF shRNA-treated mPFC. Results indicate that BDNF shRNA produced a 58% reduction in BDNF staining intensity per cell, which was a statistically significant decrease (unpaired t test, p < 0.05) (scrambled, n = 3; shRNA, n = 3). B, Photomicrograph from the mPFC of a control subject. C, Lower magnification of EGFP labeling in the mPFC of an AAV10–BDNF shRNA-treated rat. Scale bar, 100 μm. D–F depict a subset of cells from B. Shown are BDNF labeling (D), EGFP expression (E), as well as superimposed BDNF and EGFP (F). Scale bars, 50 μm. The black arrows point to cells that express EGFP and have low BDNF labeling. In contrast, the white arrows point to cells that do not express EGFP and have higher BDNF expression. G, The mean ± SEM breakpoint, as defined by the cumulative responses and injections per session under the PR schedule for control and BDNF siRNA treatments. *p < 0.05, significant difference between these groups (unpaired t test). H, Time course of cocaine self-administration from representative control and BDNF shRNA-treated subjects. There were seven subjects per treatment in the behavioral experiment.

Figure 3.

Cocaine self-administration selectively increases BDNF exon IV mRNA in the mPFC. BDNF exon I, II, IV, and VI transcripts were assessed in the mPFC of cocaine self-administration rats and yoked saline controls. There was no BDNF exon II expression in the mPFC. *p < 0.05, significant increase in BDNF exon IV-containing transcript 7 d after cocaine self-administration relative to the saline control group (saline, n = 4; cocaine, n = 8) (Tukey's HSD test).

Figure 4.

Increased association of acetylated histone H3 with BDNF exon IV in mPFC after cocaine self-administration. A, Western blots of global AcH3 or H3 levels in cocaine self-administration and yoked saline control subjects. B, Analysis of densitometry values of AcH3 blots demonstrates that there is no significant change in global AcH3 levels (saline, n = 3; cocaine, n = 3). IDV, Integrated density value. C, In contrast, there was a significant increase in AcH3 association with BDNF exon IV promoter in the mPFC of cocaine relative to saline subjects as measured by ChIP (saline, n = 5; cocaine, n = 4) (Tukey's HSD test, *p < 0.05).

Figure 5.

Cocaine self-administration results in increased association of pCREB with BDNF exon IV in the mPFC. A, Western blots showing total pCREB and GAPDH levels in the mPFC. B, Analysis of the Western blot quantification showed no significant change in pCREB levels in mPFC in cocaine self-administration relative to saline controls (saline, n = 3; cocaine, n = 3). IDV, Integrated density value. C, However, ChIP analyses showed that there was a significant increase in the association of pCREB with BDNF promoter IV in the mPFC in the cocaine relative to the saline group (saline, n = 9; cocaine, n = 5) (Tukey's HSD test, *p < 0.05).

Figure 6.

Decreased association of MeCP2 with BDNF exon IV in the mPFC after cocaine self-administration. A, Western blots showing total MeCP2 and GAPDH levels in the mPFC of control and cocaine self-administration rats. B, Analysis of the Western blot densitometry revealed no significant difference between groups for MeCP2 levels in mPFC (saline, n = 3; cocaine, n = 3). IDV, Integrated density value. C, In contrast, there was a significant decrease in the association of MeCP2 with BDNF promoter IV in the mPFC of cocaine versus saline-treated subjects (saline, n = 5; cocaine, n = 5) (Tukey's HSD test, *p < 0.05).