Our results provide the first evidence for a selective role of the nuclear specific Class I HDAC, HDAC1, in mediating cocaine-induced behavioral plasticity and suggest a novel interplay among activating and repressive histone PTMs in the transcriptional regulation of cocaine-induced molecular adaptations. We show that local knockout of HDAC1, as well as chronic and continuous infusion of MS-275, a pharmacological inhibitor highly selective in vitro for HDAC1, in NAc suppressed cocaine-induced locomotor sensitization. This effect was not seen with local knockout of other Class I isoforms, HDACs 2 and 3. Knockout of these different HDACs resulted in distinct patterns of altered expression of other HDACs, but only knockdown of HDAC1 affected expression of isoforms previously implicated in behavioral responses to cocaine. We further show that both repeated cocaine and chronic HDAC inhibition alone increased global levels of activating histone acetylation in the NAc but that, in combination, these treatments induced repressive histone methylation through H3K9ac binding at the promoters of specific KMTs and the subsequent induction of these enzymes. Analysis of promoter occupancy 4 hours after repeated cocaine revealed the selective association of HDAC1 with the G9a and GLP promoters in NAc, suggesting a mechanism for HDAC1’s unique effects on repressive histone methylation and cocaine action. Further studies are needed to understand the molecular steps by which HDAC1, but not other Class I HDACs, is recruited to these KMT promoters. Finally, we identify GABAA receptor subunits as functional targets for both cocaine-induced decreases in H3K9me2 and cocaine plus MS-275 increases in this repressive methyl mark. Together, our findings suggest a dynamic interplay among different types of histone modifications in the adult brain, identify HDAC1 as a novel target for drug-induced plasticity, and reveal a role for, and mechanism by which, GABAA receptors in NAc are altered to mediate physiological changes important for the behavioral effects of cocaine.
Several studies to date have identified altered patterns of histone acetylation in NAc as important mediators of psychostimulant-induced behavioral and molecular plasticity. HDACs tightly regulate histone acetylation and both non-selective HDAC inhibition and manipulations of specific Class II or III isoforms can alter psychostimulant-induced adaptations1,3–5,10–16
. Class I HDACs differ from other classes of HDACs in that they are nuclear specific, demonstrating very little nuclear-cytoplasmic shuttling, and evidence suggests highly specific biological actions of the different isoforms within this class27,28
. Recent reports have revealed that Class I HDAC isoforms 1–3 regulate memory and mediate responses to antipsychotic drugs29–32
, suggesting a potentially distinct role, as yet unexplored, for such enzymes in drug addiction as well. The selective role for HDAC1 reported here may seem surprising given evidence of regulatory cross talk between HDACs 1-3, such that loss of HDAC1 in embryonic stem cells causes a compensatory upregulation of HDACs 2 and 333
. However, such a compensatory mechanism is thought to be incomplete33
. Moreover, we show a very different pattern of compensation in adult NAc. Local knockdown of HDAC1 led to decreased HDAC2
expression, which is in agreement with previous findings that loss of HDAC1 leads to an overall reduction in total HDAC activity in non-nervous tissues33
. Interestingly, HDAC1 knockdown also caused an upregulation in HDAC5
expression. Previous findings have implicated both SIRT1 and HDAC5 in mediating behavioral responses to cocaine. SIRT1
is upregulated in NAc following repeated exposure to cocaine and SIRT inhibition decreases behavioral responses to the drug5
. Conversely, nuclear levels of HDAC5 are downregulated in NAc by repeated cocaine and loss of HDAC5 results in cocaine hypersensitivity16
. While further work is required to determine the extent to which such compensatory regulation of SIRT1 and HDAC5 might contribute to the behavioral effects of HDAC1 knockdown, our observation of selective HDAC1 binding to H3K9 KMTs suggests an important direct role for HDAC1. As noted, evidence for distinct functions of Class I HDAC isoforms in the adult mouse brain has been reported recently in a study comparing HDAC1 and 2 contributions to cognitive processes. Although HDACs 1 and 2 form functional heterodimers and are often found in the same protein complexes14
, memory formation was impaired with overexpression of HDAC2 but not HDAC1 in the hippocampus29
. Our findings show an opposite dissociation of function and implicate HDAC1 in NAc in cocaine-induced plasticity. These results thereby provide new insight into the selective roles of Class I HDACs in the adult mouse brain.
The effects of pharmacological inhibition of HDACs on psychostimulant-induced plasticity appear to depend on the timecourse of HDAC inhibition. Studies employing co-administration procedures in which inhibitors are given acutely, just prior to psychostimulant administration, report heightened behavioral responses to the drug1,3,4,13,14
. In contrast, experimental paradigms like the one employed here, in which HDAC inhibitors are administered more chronically, for several days prior to psychostimulant exposure, show inhibited expression3
or decreased acquisition of behavioral adaptations to drug5,11,12
. The clustering of seemingly discrepant results based on experimental methodologies is interesting in light of our present findings. Both HDAC inhibitors and psychostimulants increase global levels of histone acetylation in NAc. Thus, when co-administered acutely, these drugs may have synergistic effects, leading to heightened transcriptional activation of psychostimulant-regulated target genes. In contrast, when a psychostimulant is given in the context of prolonged, HDAC inhibitor-induced hyperacetylation, homeostatic processes may direct AcH3 binding to the promoters of genes (e.g., G9a
) responsible for inducing chromatin condensation and gene repression (e.g., via H3K9me2) in order to dampen already heightened transcriptional activation. Our present findings thus demonstrate clear cross talk among histone PTMs and suggest that decreased behavioral sensitivity to psychostimulants following prolonged HDAC inhibition might be mediated through decreased activity of HDAC1 at H3K9 KMT promoters and subsequent increases in H3K9me2 and gene repression. The same complexity has been reported previously with local knockdown of HDAC5 in the NAc16
. While many genes were induced, just as many were repressed, and among the induced genes were transcriptional repressors such as SUV39H1
Previous studies examining chromatin remodeling in NAc underlying drug-induced behavioral plasticity have focused solely on histone PTMs known to occur on active, euchromatic regions of the genome (e.g. H3K14ac, H3K9ac, H3K9me2, H3K4me3). More recent evidence suggests that cocaine exposure additionally causes a de-repression of previously silenced chromatin (heterochromatin) in NAc: repeated cocaine reduced global levels of H3K9me3, a histone PTM that is associated specifically with silenced, non-coding regions of the genome, which mediated a decrease in total heterochromatin levels in NAc23
. Our present findings, that chronic MS-275 plus repeated cocaine increase H3K9me3 and expression of SUV39H1
, the enzyme that catalyzes this modification, suggest that regulation of non-coding regions of the genome may also be important for MS-275’s suppression of cocaine-induced behavioral activation.
Numerous studies to date have implicated glutamatergic plasticity in the brain’s reward circuitry in the development and maintenance of addictive-like behaviors. Studies have shown that altered glutamate receptor subunit levels and altered glutamate transmission in NAc are important molecular and physiological changes underlying the development of cocaine-induced behavioral sensitization34–39
. Further evidence suggests that glutamate receptor subunits are targets for the transcription factors ΔFosB and phospho-CREB, and that their genes display promoter hyperacetylation, providing one mechanism for the sustained changes in glutamatergic transmission in NAc in response to repeated cocaine5,14,39,40
. More recent studies have begun to reveal a role for GABAA
receptor subunits in NAc in mediating cocaine-induced behavioral adaptations. Repeated cocaine increases the frequency of GABAA
receptor-mediated mIPSCs in striatonigral medium spiny neurons, and α2 subunit-containing GABAA
receptors are required for the induction of cocaine behavioral sensitization25,26
. Furthermore, ChIP-chip analyses have identified several GABAA
receptor subunits as direct, physiological targets for increased ΔFosB and phospho-CREB binding following repeated cocaine5
. Here, we provide further evidence for GABAergic plasticity in NAc as a potential mediator of cocaine behavioral plasticity and identify chromatin remodeling as a regulator of cocaine’s effect on GABAA
receptor subunit expression. Although GABAA
receptor subunit expression, as well as inhibitory tone on NAc medium spiny neurons, was increased by either cocaine or MS-275 alone, only the cocaine-induced increase was associated with a behavioral phenotype. The absence of enhanced locomotor activity following MS-275 treatment alone suggests that, although increases in GABAA
subunit expression and functional synaptic changes are necessary for the expression of cocaine-induced locomotor activation, they are not sufficient. Several studies to date have identified putative target genes for cocaine-induced promoter hyperacetylation and transcriptional upregulation1,5,14
. Only recently has cocaine-induced decreases in repressive H3K9me2 promoter binding been linked to the transcriptional upregulation of genes important in mediating behavioral adaptations to cocaine6,41
. Our data provide evidence linking cocaine-induced increases in GABAA
receptor subunit expression to decreased binding of H3K9me2 at these gene promoters. Although combined treatment of MS-275 and repeated cocaine did cause a significant increase in H3K9ac binding at GABAA
subunit gene promoters, we found a much more prominent increase in repressive H3K9me2 enrichment, suggesting that the normalization of GABAA
subunit expression and inhibitory tone on NAc medium spiny neurons under these conditions was mainly driven by a reversal of cocaine-induced transcriptional de-repression.
The finding that MS-275 with repeated cocaine increased both H3ac and H3me2 on Lys9 residues at GABAA
subunit gene promoters deserves further comment. The NAc is composed primarily of two distinct subpopulations of medium spiny projection neurons, those expressing dopamine D1- and those expressing dopamine D2-receptors, and these different neuronal populations exhibit different molecular adaptations in response to repeated cocaine42–44
. In the present study, we did not distinguish between these different neuronal subtypes, thus it is possible that the increases in H3ac and H3me2 at the same Lys residue reported here are occurring in different neuronal populations. Future experiments are required to address this and alternative possibilities.
The interaction between cocaine and MS-275 reported here is noteworthy. Either cocaine or MS-275 treatment alone caused global increases in H3 acetylation and increases in GABAA
subunit gene expression, but when combined, these treatments caused increases in global repressive H3K9me2, most likely driven by a loss of HDAC1 and a subsequent gain in H3ac at H3K9 KMT promoters, that prevented cocaine-induced increases in GABAA
subunit gene expression and inhibitory tone in NAc (Supplementary Fig. 5
). The results highlight a unique mode of biological regulation that provides further insight into mechanisms of chromatin regulation in the adult brain. Results of the present study also specifically inform the mechanisms underlying the prolonged actions of HDAC inhibitors in NAc and broaden current knowledge of molecular and chromatin endpoints for novel addiction treatments.