**9. Pharmacological manipulations of epigenetic mechanisms**

in miRNA expression can therefore lead to widespread changes in gene expression and alteration in several cellular signaling cascades. Other types of noncoding RNAs include: (1) PIWI-interacting RNA (piRNA), which regulates sperm development, (2) small nuclear RNA (snRNA), which regulates mRNA splicing, and (3) long noncoding RNA (lncRNA), which has

**Figure 2.** MicroRNA processing and function. MicroRNAs are transcribed similarly to protein-coding RNAs, except they form a stem-loop structure following transcription (i.e., pri-miRNA). The enzyme, Drosha, trims the ends of the stem (i.e., pre-miRNA) to prepare for exportation from the nucleus via Exportin 5. Once in the cytoplasm, Dicer cleaves the loop of the pre-miRNA that produces a double-stranded RNA. One strand (i.e., mature miRNA) is chosen to be incorporated into the miRNA-induced silencing complex (miRISC). Upon binding to a complementary sequence in the 3′ untranslated region of a target mRNA, either translational repression, deadenylation, or endonucleolytic cleavage

widespread effects on chromatin modification and transcription [41].

may occur. All three mechanisms lead to decreases in protein product.

28 Recent Advances in Drug Addiction Research and Clinical Applications

Pharmacological agents that target specific epigenetic machinery have been used to further understand the role of epigenetic mechanisms in the effects of drugs of abuse and to explore their potential use as treatments for drug addiction. Most preclinical studies have utilized both systemic and intracranial administration of these compounds, where the former has a more human translational value, while the latter allows for greater brain region specificity.

#### **9.1. Methyl supplementation and DNMT inhibitors.**

DNA methylation can be altered pharmacologically by using methionine or DNMT inhibitors. Methionine is an amino acid commonly found in diet, where methionine metabolism yields methyl groups that serve as donors for methylating DNA. DNMT inhibitors exert the opposite effect by preventing DNMT from catalyzing DNA methylation. Daily, systemic administration of methionine has been shown to reduce both the rewarding and motivating effects of cocaine in rodents [18, 52]. In contrast, intracranial administration of a DNMT inhibitor (i.e., RG108) into the NAc increases the rewarding effects of cocaine [18]. However, this same manipulation decreases drug-seeking behavior following a prolonged abstinence period [53]. These findings suggest that the effect of DNA methylation in the NAc may depend on whether or not there has been a period of abstinence following cocaine exposure. Indeed, our lab and others have shown that dynamic changes occur during forced abstinence from cocaine in animal models, and that these changes can result in opposing effects of pharmacological challenge on cocaine abuse-related behavior depending on whether the manipulation occurs during active drug intake versus abstinence [54–56]. It should be noted that work using DNA methyl supple‐ mentation and DNMT inhibitors has primarily been done with cocaine and needs to be tested on other drug classes.

#### **9.2. Histone deacetylase inhibitors**

The removal of an acetyl group from a histone is catalyzed by histone deacetylases (HDACs). This reaction results in condensing the chromatin and repressing transcription. HDAC inhibitors prevent this reaction from occurring, thereby maintaining DNA accessibility. There are five different classes of HDACs (e.g., I, IIa, IIb, III, and IV) and each class contains multiple HDAC enzymes (e.g., HDAC1, HDAC8, SIRT1, etc.). HDAC inhibitors range in their selectivity for specific HDAC classes. Drugs that target both class I and II HDACs (e.g., Tricostatin A, sodium butyrate, and SAHA) have been found to enhance cocaine locomotor sensitization [21, 57, 58], cocaine and opiate CPP [21, 58, 59], and cocaine self-administration [60] when admin‐ istered systemically prior to cocaine exposure. In contrast, administration of HDAC com‐ pounds *following* cocaine exposure attenuates cocaine CPP [61]. Similarly, these compounds appear to produce mixed effects with alcohol, with some reporting increases [62] and others reporting decreases [63, 64] in consumption. While these effects were found during active drug administration, HDAC inhibitors have also been shown to alleviate anxiety symptoms during alcohol withdrawal [30, 65]. Additionally, several studies have found that the effects of the class I/II HDAC inhibitors were specific to drug self-administration, as no effects were found with these drugs on food reinforcement [60, 63, 64]. Collectively, it appears that class I/II HDAC inhibitors can produce both increases and decreases in drug-abuse-related behavior, and that the effects may vary depending on whether testing occurs during drug exposure or with‐ drawal.

More consistent effects have been observed with selective HDAC inhibitors. For instance, the selective class I HDAC inhibitor, MS-275, decreases both alcohol and cocaine abuse-related behavior in rodents [63, 64, 66, 67]. Also, the highly selective HDAC3 inhibitor, RGF-P966, decreases cocaine CPP [68]. These findings suggest that the use of more selective HDAC inhibitors may improve behavioral outcomes.
