**11. Transgenerational epigenetic inheritance of addiction-like phenotypes**

Perhaps the most intriguing discovery in epigenetics is that epigenetic marks acquired due to experience can be passed along to future generations. Unfortunately, this may include epigenetic changes that make one vulnerable to addiction. The phrase "it runs in the family" is often spoken in social circles regarding the seeming ability of addiction to be inherited. While much is known about inheritance based on classical Mendelian genetic inheritance, much less is known about transgenerational epigenetic inheritance.

Several criteria must be met for transgenerational epigenetic inheritance. First, in order to pass down epigenetic changes across generations, the changes need to be present in the germ cells (i.e., sperm or egg). In other words, the epigenetic changes must occur in future generations independent of behavioral and social transfer, relying only on the molecular transmission of epigenetic information [71]. Second, the behavioral phenotypes need to persist across several generations, depending on the sex and pregnancy status of the parent exposed to the initial environmental trigger. In males and nonpregnant females, an environmental trigger that affects the parent generation (i.e., F0) and their germ cells, will directly impact the next (i.e., F1) generation. This is referred to as multi- or inter-generational inheritance [72]. However, if the behavioral phenotype persists into the third generation (i.e., F2), which had no direct exposure to the trigger, it can be regarded as transgenerational inheritance. With pregnant females, not only is the parent and embryo directly affected, but also the germ cells of the embryo that will develop into the F2 generation. Therefore, the F3 generation must exhibit the phenotype to be considered transgenerational. Third, epigenetic modifications present in the parents need to persist into future generations (see **Figure 5**). Interestingly, most epigenetic marks (particularly DNA methylation) are erased immediately in the embryo following fertilization [73]. Very few exceptions are currently known, but some include imprinted genes (i.e., methylation-induced silencing of genes in one parent's allele and not in the others), certain histone and protamine (i.e., histone-like proteins found in sperm) modifications, and reserve pools of coding and noncoding RNA [72]. Although narrowing the field of investigation, the complex pattern of changes required for transgenerational epigenetic inheritance still remains poorly understood [72, 74].

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

**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*

time point during development.

expression only in *TH*+ cells.

34 Recent Advances in Drug Addiction Research and Clinical Applications

**Figure 5.** Epigenetic inheritance. For pregnant F0 females (top left panel), drug exposure directly affects (red-outlined symbols) both the F0 female and the fetus, including the brain and germ cells of the upcoming F1 generation. Howev‐ er, the F2 generation also receives direct effects of drug exposure from the F0 generation via the germ cells of the F1 generation. For males (top right panel), drug exposure directly affects both the F0 generation and the germ cells that lead to the F1 generation. Therefore, the F3 and F2 generation of the pregnant female and male, respectively, can re‐ ceive transgenerational epigenetic inheritance from the F0 generation without having been in direct contact with drugs of abuse (orange-outlined symbols).

#### **11.1. Drug abuse-related traits passed across generations**

The idea that addiction-like phenotypes can be passed down across generations based on experiences of the parents is compelling in terms of uncovering potential biomarkers that could be used to predict one's risk of developing drug addiction. Only a few studies have investi‐ gated this possibility in the context of drug abuse models. Vassoler and colleagues [75] found that male adult rats with a history of cocaine self-administration passed an addiction-*resist‐ ant* phenotype onto male, but not female, offspring. One potential mediator of these effects is mPFC BDNF expression, where both the male F0 generation's sperm and the mPFC of the male, but not female, offspring exhibited increased H3 acetylation at the BDNF promoter, as well as increased BDNF expression in the mPFC of the male offspring. Consistent with this idea, mPFC BDNF is associated with resilience to drug effects [47, 49, 50]. These data are particularly compelling given that it was the father who received the initial trigger (i.e., drug exposure), thereby avoiding potential confounds of maternal care, social/behavioral transfer, and *in utero* environment changes that may occur following drug exposure in the females. Nonetheless, one cannot rule out potential stress effects during copulation. Gapp et al. [76] avoided this potential confound by isolating the sperm of the affected F0 males and artificially inseminating the F0 females. They found that sperm noncoding RNAs from fathers subjected to early life stress sufficiently passed on molecular and behavioral phenotypes to the next two generations. Interestingly, early life stress is also a strong predictor for developing drug addiction [1]. Other studies have shown that repeated morphine administration in F0 female adolescents produces male offspring that are more sensitive to the analgesic and tolerance effects of morphine [77]. Furthermore, ethanol exposure to F0 males decreases ethanol intake and increases sensitivity to the inhibitory effect of ethanol on anxiety-like behavior in F1 offspring [78]. Reduced methylation at the *Bdnf* promoter was also observed in this study in both the F0 male sperm and in the F1 males' VTA. The VTA sends dopaminergic projections to the NAc and BDNF in the VTA has a facilitating effect on drug-abuse-related behavior [79]. These few examples provide some evidence of multi-generational inheritance of drug abuserelated traits and associated changes to the epigenome. However, further research is required to examine if these traits persist into additional generations and whether blocking or reversing the epigenetic changes in the germline will prevent transmission of these traits. The latter effect would have very exciting implications for approaches to prevent the development of addiction in future generations.
