Dopaminylation of histone H3 in ventral tegmental area regulates cocaine seeking

Abstract

Vulnerability to relapse during periods of attempted abstinence from cocaine use is hypothesized to result from the rewiring of brain reward circuitries, particularly ventral tegmental area (VTA) dopamine neurons. How cocaine exposures act on midbrain dopamine neurons to precipitate addiction-relevant changes in gene expression is unclear. We found that histone H3 glutamine 5 dopaminylation (H3Q5dop) plays a critical role in cocaine-induced transcriptional plasticity in the midbrain. Rats undergoing withdrawal from cocaine showed an accumulation of H3Q5dop in the VTA. By reducing H3Q5dop in the VTA during withdrawal, we reversed cocaine-mediated gene expression changes, attenuated dopamine release in the nucleus accumbens, and reduced cocaine-seeking behavior. These findings establish a neurotransmission-independent role for nuclear dopamine in relapse-related transcriptional plasticity in the VTA.

Fig. 1.. Histone H3 dopaminylation in the VTA is dysregulated by cocaine.

(A) H3 dopaminylation in human postmortem VTAs from cocaine-dependent subjects versus controls. No changes were observed in H3K4me3Q5dop, H3K4me3, H3, or Tgm2 expression (fig. S5A). A.U., arbitrary units. (B) Experimental timeline of cocaine self-administration (SA) followed by tissue collection time points during withdrawal (WD). FR1–5, fixed-ratio 1 to 5. (C) Number of infusions earned in daily 6-hour test sessions in rats self-administering cocaine or saline. (D) Analysis of H3 dopaminylation in the VTAs (0 versus 1 versus 30 days of WD) from rats with extended access to cocaine versus saline (see fig. S5B for full scatter plots). No changes were observed in H3K4me3Q5dop, H3K4me3, H3, or Tgm2 expression (fig. S5, C to E). Data presented as averages ± SEM. See supplementary materials for full figure legends with statistical comparisons.

Fig. 2.. H3Q5dop in the VTA contributes to cocaine-mediated gene expression.

(A) VTA transduced with a lentivirus expressing H3.3Q5A-HA-EF1-RFP (red fluorescent protein) overlayed with a nuclear co-stain [4′,6-diamidino-2-phenylindole (DAPI)]. (B) H3 dopaminylation in VTAs infected with lentiviral vectors. (C) Experimental timeline of self-administration RNA-seq experiment after viral transduction. (D) RRHO map key describing the extent and directionality of overlap between differential gene expression. (E) RRHO comparing differential expression between cocaine-regulated genes. Each pixel represents the overlap between differential transcriptomes, with the significance of overlap of a hypergeometric test color-coded. Coc, cocaine; Sal, saline. (F and G) Overlap of differentially expressed (DEx) PCGs in VTA tissues comparing cocaine versus saline (empty) (F) or cocaine versus saline (H3.3 WT) (G) to H3.3Q5A versus empty or H3.3 WT (cocaine) animals, respectively. (H and I) Heat maps of overlapping genes obtained from RNA-seq data comparing cocaine versus saline (empty) and H3.3Q5A versus empty (cocaine) animals (H) or cocaine versus saline (H3.3 WT) and H3.3Q5A versus H3.3 WT (cocaine) animals (I) using normalized RNA expression values. (J) KEGG 2019 pathway enrichment analysis for the 211 overlapping PCGs displaying reversals in cocaine-mediated gene expression from (G) and (I). LTP, long-term potentiation. Data presented as averages ± SEM.

Fig. 3.. Attenuating H3Q5dop expression in the VTA after extended access to cocaine reduces dopamine release in the ventral striatum.

(A) Frequency of sAPs in VTA dopaminergic neurons from cocaine-naïve rats infected with empty vector, H3.3 WT, or H3.3Q5A. Representative sAP traces are provided. (B) Experimental timeline of cocaine SA FSCV experiments after viral transduction and 1 hour of cocaine seeking. (C to E) Current versus time traces (C) and color plots (D) demonstrating reduced dopamine release (evoked) in the NAc of H3.3Q5A versus empty and H3.3 WT (VTA) expressing animals. Quantified as input-output curves (E) (current versus stimulation intensity). (F) Baseline dopamine (DA) release into the NAc was similarly reduced by H3.3Q5A. Data presented as averages ± SEM.

Fig. 4.. Reducing H3Q5dop in the VTA attenuates cocaine seeking.

(A) Experimental timeline of cocaine SA drug-seeking experiments after viral transduction. (B and C) After 10 days of extended access to cocaine, rats were infected intra-VTA with one of the three viral vectors (on day 11) (B), followed by 30 days of WD and 1 hour of cocaine seeking (C). Data presented as averages ± SEM.