**10. Genetic tools for uncovering epigenetic roles in drug-abuse-related behavior**

While pharmacological approaches have translational value for development of therapeutic agents, efficacy may be compromised by the widespread drug distribution if the effects of an epigenetic manipulation vary depending on the brain region of interest. Also, pharmacological manipulations used to date have widespread effects on the genome, whereas sharpening the mechanism/location targeted may improve desired outcomes. Recent preclinical research has shed light on this area with technologies that selectively manipulate genes in specific brain pathways and cell types.

#### **10.1. Viral vectors**

systemic and intracranial administration of these compounds, where the former has a more

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

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‐

human translational value, while the latter allows for greater brain region specificity.

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

30 Recent Advances in Drug Addiction Research and Clinical Applications

on other drug classes.

drawal.

**9.2. Histone deacetylase inhibitors**

One approach to manipulating a certain gene within a particular brain region is the use of viral vectors. Viral vectors are constructed to be nonreplicative so that they do not produce more viral particles after infecting the cell. They enter the cell through endocytosis and insert a gene of interest (i.e., transgene) into the genome of specific neurons (**Figure 3**). There are many different modes of transfection that vary in length from days to months. In order to achieve high levels of expression in a particular cell type, within the viral vector the transgene is typically downstream from a promoter sequence that is specific to that cell. Thus, upon viral transfection, the cells own transcriptional machinery will recognize and bind to the promoter that will then activate transcription of the transgene. The direction of regulation (i.e., increase vs. decrease expression) is determined by the sequence of the transgene. For instance, an increase in gene expression is obtained by inserting the sequence of the transgene into the viral vector with a strong upstream promoter. In order to decrease gene expression, a couple of methods may be used. One involves transfecting a short-hairpin RNA (shRNA) that is processed into a mature short-interfering RNA (siRNA). siRNAs are similar to miRNAs, except that they are perfectly complementary to the target mRNA and will therefore selectively downregulate only one target gene, in contrast to the multiple targets of most miRNAs. This is referred to as a 'knockdown,' rather than a 'knockout,' as it is preventing translation of the gene rather than completing deleting it from the genome. In order to accomplish a 'knockout' using viral vectors, transgenes that express a new gene editing approach, called the CRISPR-Cas9 system, can be used. The latter uses a guide RNA that is complementary to specific sequences in the DNA (e.g., gene of interest) that directs enzymes to that site and excises the sequence from the DNA, therefore deleting it.

**Figure 3.** Viral-mediated gene transfer. Viral particles are infused into a region of interest and infect local cells through receptor-mediated endocytosis. Once viral particles are released from the vesicle inside the infected cell, viral RNA is reverse-transcribed into DNA (via reverse transcriptase; dark blue) and transported into the nucleus, where it becomes integrated into the genome (via integrase; yellow). By using a strong promoter (orange line) upstream of the transgene, the cell's transcriptional machinery produces an abundance of viral transgene expression in the cell. TF = transcription factor.

Research using viral vectors has furthered our understanding of the impact of epigenetic manipulations on drug-abuse-related behavior. As previously described, DNA methylation is thought to inhibit cocaine abuse-related behaviors in animal models [16, 18, 52]. To test whether *Dnmt3a* expression in the NAc specifically mediates these effects, LaPlant et al. [18] infused viral vectors into this region that either increased or decreased *Dnmt3a* levels. Increas‐ ing NAc *Dnmt3a* expression countered cocaine CPP in mice, while decreasing expression increased this behavior [18]. Interestingly, this same manipulation also increases depressivelike behavior following repeated social stress in mice [18], suggesting the blunted rewarding effects of cocaine may be due to increases in anhedonia. This illustrates the importance of testing the role of epigenetic modulators in both drug abuse and mood disorder models.

Another exciting use of viral vectors is to express synthetically engineered transcription factors that bind to specific sequences in the DNA and regulate histone modifications at one specific gene loci. Heller and colleagues [69] recently used this approach and found that histone acetylation or methylation near the *fosB* gene locus increases or decreases cocaine reward CPP, respectively. Again, this same manipulation produces either anti- or pro-depressive behaviors, respectively, following repeated social stress [69], further demonstrating the complex role of these molecules in both reward and emotional regulation processes. Bidirectional manipula‐ tion of cocaine self-administration in rats has also been demonstrated for miR-212 levels in the dorsal striatum where viral-mediated increases prevent escalation of cocaine self-administra‐ tion, whereas knockdown increases cocaine self-administration [42]. In some cases, decreasing miRNA levels may be needed to attenuate addiction-related behavior. For instance, viralmediated increases in miR-206 expression in the prefrontal cortex create an alcohol-dependent phenotype in rats [47], and, therefore, it is possible that decreasing miR-206 levels in the PFC may be protective against alcoholism. These examples suggest that the development of new therapeutics that target epigenetic mechanisms have potential for treating addiction. Current‐ ly, there are no pharmacological agents for manipulating miRNAs, although development is in the initial stages for their delivery in drug compounds [70]. A future challenge for this avenue of research will be to develop methods of site-selective drug delivery.

#### **10.2. Cre-Lox recombination**

**Figure 3.** Viral-mediated gene transfer. Viral particles are infused into a region of interest and infect local cells through receptor-mediated endocytosis. Once viral particles are released from the vesicle inside the infected cell, viral RNA is reverse-transcribed into DNA (via reverse transcriptase; dark blue) and transported into the nucleus, where it becomes integrated into the genome (via integrase; yellow). By using a strong promoter (orange line) upstream of the transgene, the cell's transcriptional machinery produces an abundance of viral transgene expression in the cell. TF = transcription

32 Recent Advances in Drug Addiction Research and Clinical Applications

Research using viral vectors has furthered our understanding of the impact of epigenetic manipulations on drug-abuse-related behavior. As previously described, DNA methylation is thought to inhibit cocaine abuse-related behaviors in animal models [16, 18, 52]. To test whether *Dnmt3a* expression in the NAc specifically mediates these effects, LaPlant et al. [18] infused viral vectors into this region that either increased or decreased *Dnmt3a* levels. Increas‐ ing NAc *Dnmt3a* expression countered cocaine CPP in mice, while decreasing expression increased this behavior [18]. Interestingly, this same manipulation also increases depressive-

factor.

Another approach to manipulating gene expression is the use of Cre-Lox recombination (**Figure 4**). Cre recombinase is an enzyme that identifies sequences in the DNA called LoxP sites. When Cre recognizes these sites, it catalyzes a reaction that can either excise or invert the DNA sequence contained between the two sites, depending on which direction the LoxP sites are oriented. If the two LoxP sites are in the same direction, Cre will excise the DNA, effectively deleting a gene that is between those two sites. If the LoxP sites are in the opposite direction, Cre will then invert the two LoxP sites along with inverting the flanked DNA sequence. This latter effect allows for gene activation, where a previous nonfunctional inverted gene sequence becomes functional after Cre-Lox mediated-inversion.

Cre-lox recombination is carried out in rodents that are bred to have LoxP sites at specific locations in the DNA that flank a gene of interest (e.g., *Bdnffl/fl*). A viral vector expressing Cre recombinase can then be infused into a specific brain region and Cre-expressing infected cells will recognize the LoxP sites and either excise or invert the flanked gene. This will result in region- and temporal-specific manipulation of gene expression. Another approach with even greater precision involves breeding mice to express Cre in only certain cell types. This is accomplished by breeding rodents that express Cre downstream from a promoter that is specific for only one type or subtype of cells. For instance, Cre can be expressed specifically in catecholamine neurons when used downstream of a promoter for tyrosine hydroxylase (TH). TH is an enzyme involved in synthesizing catecholamines (e.g., dopamine). Only cells with

**Figure 4.** Cre-lox recombination. The top panel illustrates how Cre-lox recombination can result in gene excision, where Cre recombinase (red) recognizes two loxP sites in the same orientation and joins the two ends of DNA, then cleaves one end to join the other, resulting in excision of the flanked gene between the two loxP sites. The middle panel illustrates how Cre recognizes two loxP sites in opposite orientations and inverts the intervening DNA sequence (e.g., Gene X). Gene inversion can be used to turn on a gene that is initially inverted and inactive. The bottom panel depicts how crossbreeding two transgenic mice that express Cre recombinase only in tyrosine hydroxylase-expressing cells (*TH*-Cre) with mice that ubiquitously express Bdnf with flanked loxP sites (*Bdnffl/fl*) results in mice with deficient *Bdnf* expression only in *TH*+ cells.

TH will have the transcriptional machinery to recognize the TH promoter and express Cre. Next, there are two methods for manipulating gene expression in a cell-type-specific manner. The first is to crossbreed two transgenic mice: the one that expresses Cre only in certain cell types (e.g., TH+ neurons) and the other that ubiquitously expresses a LoxP-flanked *Bdnf* gene (i.e., *Bdnffl/fl*). The offspring will no longer express *Bdnf* in TH-expressing cells. A limitation of this technique is that Cre recombination occurs at conception and the transgene is either expressed or deleted permanently. Therefore, changes may occur during development to compensate for the gene modification, making it difficult to know whether subsequent functional differences are due to the gene modification or the compensatory changes that ensued thereafter. Another way to overcome this limitation is to inject a viral vector into a brain region that contains the gene of interest in a plasmid with the gene flanked by inverted LoxP sites. While the virus will infect all the cells in that region, only the cells that are expressing Cre recombinase (e.g., TH+) will recognize the LoxP sites. In this case, Cre recombination will only occur in specific cell types in a particular brain region and, importantly, during a specific time point during development.

Research employing the Cre-Lox recombination approach has shown that the effect of epigenetic mechanisms can be cell-type specific. For instance, the histone methytransferase *G9a* has differential roles in cocaine-related behaviors depending on whether it is expressed in striatal neurons that contain dopamine D1 (D1R) versus D2 (D2R) receptors. *G9a* is down‐ regulated by cocaine in both D1R and D2R-containing neurons; however, Cre-mediated downregulation of *G9a* selectively in D1R-neurons is associated with decreasing cocaine CPP and locomotor behavior in mice, while the opposite effects occur with selective downregulation in D2R-neurons [36]. These effects were observed using both Cre-Lox recombinase procedures described above, providing strong evidence for the cell-type-specific role of *G9a* in cocaine abuse-related behavior.
