Mechanisms of drug addiction

Drug addiction is a debilitating psychiatric disorder that is characterized by compulsive drug seeking and taking despite severe adverse consequences [1-3]. Once an individual becomes addicted to a drug of abuse, there are few effective clinical options, and most addicts relapse within a short period of time. Thus, addiction research focuses on two major outstanding questions. First, what are the neural mechanisms underlying the transition from recreational drug use to a chronically addicted state? Second, what are the mechanisms responsible for the persistence of addictive behaviors even after prolonged drug abstinence? A better understanding of these mechanisms might provide clues into how we can block or even reverse the addicted state and thereby reduce the rate of relapse.

Drug-induced changes in gene expression in key brain reward regions, such as the nucleus accumbens (NAc), prefrontal cortex (PFC) and ventral tegmental area (VTA), represent one mechanism thought to contribute to both of these key questions [1-3]. For example, the transcription factor ΔFosB is induced several fold in the NAc by chronic drug exposure and has been implicated in the transition to an addicted state [4-6]. Altered expression of specific genes, such as activator of G-protein signaling 3 (AGS3) [7] and brain-derived neurotrophic factor (BDNF) [8], has been reported weeks after the last drug experience, and manipulation of these genes in rodents regulates drug relapse behavior [7,9,10]. Genome-wide mRNA analyses have identified many more potential gene targets for drugs of abuse in distinct brain reward regions, and these targets might also contribute to long-lasting behavioral effects [11-16]. Therefore, it has become of great interest to identify the underlying mechanisms by which chronic drug exposure promotes stable changes in gene expression and, ultimately, behavior. Recent evidence has suggested that epigenetic mechanisms – key cellular processes that integrate diverse environmental stimuli to exert potent and often long-lasting changes in gene expression through the regulation of chromatin structure – contribute to these drug-induced transcriptional and behavioral changes [17-19]. This review discusses current progress toward understanding how epigenetic mechanisms are regulated by drugs of abuse in brain reward regions and how such mechanisms might contribute to drug-related behaviors. Ultimately, better understanding of these mechanisms might reveal novel drug targets for the development of improved pharmaceutical interventions.

Epigenetic mechanisms

The word ‘epigenetic’ historically refers to a heritable phenotype not coded by DNA itself but by a cellular process ‘above the genome’. Cellular differentiation is a classic example where epigenetic phenomena have a critical role [20,21]. Because all cells in an organism contain the same genetic information, the ability to form clonal populations of distinct cell types with unique functions (e.g. neurons versus hepatocytes) is achieved by transmitting the correct transcriptional programs from parent to daughter cell. This epigenetic process is in large part coordinated through control of chromatin structure. Increasing evidence indicates that changes in chromatin structure not only mediate these heritable epigenetic phenomena [22] but also that the same types of changes in chromatin occur in mature, post-mitotic neurons [23,24].

Chromatin is made up of DNA and the histone proteins around which the DNA is wrapped. Histones are assembled into an octamer made up of two copies of H2A, H2B, H3 and H4 [25]. These histone proteins, together with DNA, undergo a complex supercoiling process, which results in a highly compact structure in which meters of extended DNA is condensed and organized. This highly condensed structure means that control over gene expression occurs partly by gating access of transcriptional activators to DNA [26,27]. The structure of chromatin, and hence access to the DNA sequence wrapped around it, is highly regulated by post-translational modifications of histones and the DNA itself [28]. Such modifications include acetylation, phosphorylation and methylation of histones, methylation of DNA, and copious others, with each modification either positively or negatively regulating the transcriptional activity of the underlying gene (Table 1). Ultimately, dozens of potential modifications that occur at many distinct histone residues are thought to summate to determine the final transcriptional output of a given gene [29].

Table 1

Histone modifications and their function^a

Drug-induced changes in chromatin structure

Histone acetylation

Acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and DNA, which is thought to relax chromatin structure and make DNA more accessible to transcriptional regulators [28]. Histone acetylation is best characterized on histones H3 and H4: it can occur on lysines 9, 14, 18 and 23 on the N-terminal tail of H3 and at lysines 5, 8, 12 and 16 on the tail of H4. Genome-wide studies have shown that hyperacetylation in promoter regions is strongly associated with gene activation, whereas hypoacetylation is correlated with reduced gene expression [30,31]. This association also exists in the brain in vivo in response to drugs of abuse. Acute exposure to cocaine, for example, which is known to rapidly induce the immediate early genes c-fos and fosb in the NAc, increases histone H4 acetylation on their proximal gene promoters (Figure 1) [17]. Time-course analysis revealed that this modification occurs within 30 min and disappears by 3 h, consistent with the induction kinetics of these immediate early genes. At least for fosb, this increase in histone acetylation is dependent on the HAT cAMP response element-binding protein (CREB)-binding protein (CBP) [18]. Interestingly, despite several control gene promoters where acute cocaine does not affect histone acetylation (β-tubulin, tyrosine hydroxylase, histone H4), acute cocaine does increase global levels of histone H4 acetylation and histone H3 phospho-acetylation (see next section), but not H3 acetylation alone, within 30 min [17,32]. Thus, global changes in histone modifications, which have been observed not only in response to drugs of abuse [17], but also in learning models and in response to environmental enrichment [33,34], might be accounted for by acetylation, and strong induction, of a specific subset of genes.

Figure 1

Regulation of chromatin remodeling by drugs of abuse. Cocaine and amphetamine increase levels of cAMP in the nucleus accumbens (NAc) and activate protein kinase A (PKA). PKA then phosphorylates cAMP-response-element-binding protein (CREB), which allows ...

Repeated cocaine exposure, either forced (investigator) administration or self-administration, is known to induce a distinct set of genes in the NAc (e.g. cdk5 and bdnf), some of which remain elevated for days to weeks [7,8,12,15,35]. Consistent with such stable changes in gene expression, increased histone H3 acetylation was observed on the gene promoters of both cdk5 and bdnf for 1 to 7 days after the final dose of cocaine [17]. Stable changes in histone acetylation and gene expression have been observed for nearly two weeks after withdrawal from cocaine self-administration in the PFC as well. For example, npy (neuropeptide Y) expression was found to be upregulated and its gene promoter hyperacetylated while egr-1 (early growth response 1) was found to be downregulated and hypoacetylated after cocaine withdrawal [36].

Histone methylation

Histone methylation is particularly complex and can exist in mono-, di- (me2) or tri-methylated (me3) states, enabling each state to recruit unique coregulators and exert distinct effects on transcriptional activity [28]. Histone methylation is also unique because each lysine residue has distinct, and often opposite, effects on transcription. For example, H3K4 is highly associated with gene activation, whereas H3K9 and H3K27 are usually associated with repression [27]. However, even this is an oversimplification, as H3K9 is often found downstream of the gene promoter in coding regions along with H3K36 and might be involved in transcriptional elongation [28,39]. Such complexity is reflected in the PFC of adolescent rats, where cocaine was found to induce significant reductions in global levels of both the activating H3K4me3 mark as well as the repressive H3K27me3 mark [40]. One likely explanation is that these global decreases in histone methylation are occurring on distinct gene promoters. Indeed, using the gene-specific technique, chromatin immunoprecipitation (Figure 2a), chronic cocaine exposure increases H3K9me2 on genes where histone acetylation, an activating mark, is not induced. Many of these hypermethylated genes are also downregulated by cocaine (see next section). There is also early evidence that specific histone methyltransferases and demethylases are themselves regulated by cocaine in the NAc [41].

Figure 2

Studying chromatin regulation in brain using chromatin immunoprecipitation. (a) Schematic of the chromatin immunoprecipitation (ChIP) protocol. ChIP is a technique used to quantify how much of a specific DNA sequence is occupied by a given histone modification ...

Although enzymes exist to demethylate histones (Box 1), in a mouse model of depression (social defeat stress), increased H3K27me2 on the bdnf promoter was observed in the hippocampus for at least a month after the final stress [42]. This repressive mark correlated with a significant downregulation of BDNF expression. If the mice were treated with chronic antidepressants after the stress, however, increases in the activating mark, H3K4me2, occurred on the bdnf promoter and BDNF expression returned to normal levels, despite persistent increases in H3K27me2. Although the study of histone methylation in animal models of addiction is in its early phases, these studies together have shown that histone methylation is dynamically regulated by drugs of abuse and other emotional stimuli and has the potential to stably alter gene expression in vivo. It will be important to identify how such long-lasting changes in histone methylation are maintained and whether these mechanisms mediate persistent behavioral deficits.

Role of epigenetic mechanisms in drug-related behaviors

The identification of cocaine-induced alterations in histone acetylation, phosphorylation and methylation in the NAc and other brain areas suggests that such modifications might be involved in regulating behavioral responses to drugs of abuse. Indeed, the first evidence for this came from studies that demonstrated that the pharmacological and genetic manipulation of certain HDACs in the NAc alters levels of histone acetylation in vivo and profoundly affects behavioral sensitivity to cocaine [17]. In the conditioned place preference test, in which an animal learns to associate the rewarding effects of cocaine with a specific environment, either systemic administration of sodium butyrate or trichostatin A, both non-specific HDAC inhibitors, significantly potentiates the rewarding effects of cocaine [17]. Delivery of the more-specific HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) directly into the NAc is sufficient to increase cocaine reward [19]. Similar potentiating effects of HDAC inhibition on drug-related behavior were observed with amphetamine and D1 agonists [47,48]. Consistent with the hypothesis that increased histone acetylation potentiates behavioral sensitivity to cocaine, mice that are deficient in CBP, a HAT, exhibit reduced histone acetylation on the fosb promoter, as well as reduced sensitivity to cocaine [18]. Similarly, reducing histone acetylation in the NAc by virally overexpressing certain HDACs (HDAC4 or HDAC5, but not HDAC9) in the NAc significantly decreases cocaine place conditioning and, at least for HDAC5, this effect requires the C-terminal catalytic deacetylase domain [19]. However, class II HDACs such as HDAC5 associate with class I HDACs (e.g. HDAC3) at this same C-terminal domain [49], so the relative contribution of this interaction, versus any catalytic activity of HDAC5, to its full effects on cocaine reward remains unclear.

The observation that chronic cocaine treatment regulates HDAC5 in the NAc raises the exciting possibility that this class II HDAC is involved in the behavioral transitions that occur between acute and chronic cocaine exposure (e.g. between drug experimentation and compulsive drug use). Specifically, chronic, but not acute, cocaine administration induces HDAC5 phosphorylation and nuclear export in the NAc, actions that block the enzyme's effects on histones. Nuclear export of HDAC5 results in histone hyperacetylation and increased mRNA expression of specific HDAC5 target genes, which would then contribute to sensitized behavioral responses to the drug [19]. An example of such a target gene is the NK1 (neurokinin 1 or substance P) receptor. Consistent with this model, naïve HDAC5-knockout mice display normal rewarding responses to initial cocaine exposures, but become hypersensitive if they are previously exposed to a chronic course of cocaine [19]. Importantly, HDAC5-knockout mice also hypersensitize to other chronic, but not acute, stimuli, including chronic social defeat stress, chronic cardiac stress and chronic neuropathic pain [19,50,51]. These findings, taken together with HDAC inhibitor studies in learning and memory and depression models, suggest that histone acetylation controls the saliency of a wide variety of environmental stimuli [34,42,52]. Whether it is cocaine, stress, pain or memory, pharmacological and genetic manipulations that result in elevated histone acetylation appear to potentiate the respective behavioral responses.

Histone H3 phosphorylation and phospho-acetylation also appear to play key roles in drug-regulated behaviors. As discussed earlier, cocaine rapidly increases global levels of histone H3 phosphorylation and phospho-acetylation in the striatum with similar kinetics to c-fos mRNA induction [17,32]. Moreover, c-fos seems to be highly linked to these modifications because c-fos induction by acute cocaine is potentiated by HDAC inhibitors and entirely blocked by loss of the histone H3 kinase MSK1 [17,32]. MSK1 is a downstream member of the MAP kinase cascade and, as mentioned earlier, is necessary for cocaine-induced H3 phosphorylation. Loss of MSK1 also reduces locomotor responses to cocaine [32], which is consistent with a mechanism involving dysregulation of c-fos [53].

Histone methylation is also regulated by cocaine; however, the behavioral significance of this modification is still under investigation. New inhibitors of histone methyltransferases, in addition to viral-mediated gene transfer, are allowing this question to be directly addressed. For example, preliminary findings suggest that inhibition of a particular H3K9 histone methyltransferase, the expression of which is regulated in the NAc by chronic cocaine, potentiates behavioral responses to the drug [41]. Because inhibition of H3K9 methylation would be expected to enhance gene activity, these results are consistent with studies of histone acetylation [17,19] and indicate that manipulations that increase gene transcription generally promote behavioral sensitivity to drugs of abuse.

The strong behavioral consequences of pharmacological or genetic manipulation of epigenetic mechanisms in the brain in vivo suggests that chromatin-modifying enzymes, such as histone deacetylases or methyltransferases, might be useful targets for the development of new psychiatric treatments. Histone deacetylase inhibitors, for example, have antidepressant-like effects in animal models [42,52]. Consistent with this idea is the recent finding that tranylcypromine, a monoamine oxidase inhibitor (MAOI) used to treat depression, is a much stronger inhibitor of the histone demethylase KDM1 (lysine demethylase 1, formerly LSD1) than it is of either

Interplay between transcription factors and epigenetic mechanisms

In order for environmental stimuli to regulate chromatin structure on the correct set of genes, mechanisms exist to guide the proper chromatin-remodeling enzymes and transcriptional regulators to the right gene locus. Transcription factors serve as a key mechanism by which distinct gene programs are controlled because they bind to highly specific DNA regulatory sequences. These regulatory sequences – termed response elements – serve as an address, so the cell can rapidly initiate specific gene programs that are influenced by a given set of transcription factors. Whereas some chromatin-remodeling enzymes can bind directly to a chromatin mark through specialized protein domains [28], many others get targeted to chromatin by interacting with specific transcription factors. The transcription factor CREB, which plays an essential role in behavioral responses to cocaine [55], was one of the first transcription factors known to direct a chromatin-modifying enzyme to gene promoters. When CREB is phosphorylated, it interacts with CBP, a HAT that helps facilitate target gene activation by acetylating neighboring histones [56].

ΔFosB, mentioned earlier, is another key transcription factor involved in behavioral responses to cocaine. ΔFosB is a stable, truncated splice product of the fosB gene that binds to AP-1 (activator protein 1) sites in the promoter regions of responsive genes [5,6]. Interestingly, ΔFosB activates certain genes (e.g. cdk5) [12,17,35] and represses others (e.g. c-fos under certain conditions) [12,57]. Recent studies have found that at the cdk5 gene, which is upregulated after chronic cocaine [35], ΔFosB binds to the promoter and recruits transcriptional activators, such as the SWI/SNF (switch/sucrose nonfermentable)-remodeling protein BRG1 (BRM/SWI2-related gene 1) (Figure 3a) [17].

Figure 3

Future studies

Drug-induced alterations in chromatin structure have now been implicated in both the pathogenesis and maintenance of the addicted state. An important area for future research is to translate these findings from simple behavioral models, such as conditioned place preference and locomotor responses, to self-administration and relapse paradigms, which better model the human syndrome. Moreover, cocaine and other related stimulant addiction makes up only a small part of the substance abuse problem, making it equally important to focus on other drugs of abuse, such as opiates, nicotine, ethanol, cannabinoids and inhalants (see Box 2). Indeed, progress is being made, as several recent findings have begun to explore other drugs of abuse. In the amygdala, for example, regulation of histone acetylation was observed in ethanol-treated rats, where acute ethanol exposure reduces HDAC activity and increases global levels of histone H3 and H4 acetylation, and withdrawal from chronic ethanol has the opposite effect (increased HDAC activity and reduced histone acetylation) (Figure 1) [59]. Changes in acetylation have also been implicated in the molecular and behavioral responses to an inhalant in Drosophila [60].