Our data suggest that the association between prenatal cannabis exposure and addiction vulnerability can be explained, at least in part, by cannabis-induced alterations in the epigenetic regulation of the DRD2 gene in the NAc. Mimicking the human mesolimbic DRD2 expression impairments in a rat prenatal THC exposure model also enabled us to follow the long-term trajectory of disrupted Drd2 regulation, which persisted into adulthood and was associated with increased levels of 2meH3K9. Moreover, prenatal THC-exposed adults showed enhanced opiate reward conditioned place sensitivity that superseded their baseline pre-conditioning aversion that is consistent with increased heroin self-seeking behavior previously documented in animals with similar prenatal treatment (5
The finding that prenatal cannabis exposure disrupts DRD2 gene expression in the human fetal brain is interesting given that D2
R dysregulation has been implicated in addiction risk. Imaging studies of adult subjects with a history of drug abuse have reported reduced levels of D2
Rs in the striatum (12
), and animal models documented that reduction of NAc Drd2 gene enhances drug intake (31
). Human genetic studies have also linked variants of the D2 receptor gene to addiction phenotypes related to alcohol, opiates and psychostimulant abuse (32
). While we do not exclude a genetic contribution to the impairment of the DRD2 mRNA expression observed in the cannabis-exposed fetuses, the complementary animal model supports our contention that adult NAc D2
R regulation is impacted by prenatal THC exposure.
Studies of the human brain are invariably complex especially given the inability to regulate confounds including multi-drug use and it is expected that alcohol and cigarette could affect DRD2. Examining other striatal genes in addition to DRD2, however, can help to provide important information regarding the specificity of cannabis-related alterations. Similar to DRD2, PENK mRNA is also expressed on striatopallidal neurons in both the ventral and dorsal striatum (11
), but striatal PENK expression was strongly influenced by alcohol exposure. The impact of maternal alcohol use on striatal PENK mRNA levels could have masked cannabis-induced effects of PENK mRNA expression considering that reduced NAc PENK mRNA was detected in neonate rats with prenatal THC exposure (5
). However, as compared to cannabis, alcohol was associated with a widespread disturbance of most markers studied particularly in the dorsal striatum. The specificity of the molecular observations to cannabis was also supported by the observation that although cigarette smoking is common in cannabis users, only the PDYN mRNA was directly related to the maternal report of cigarette use, in contrast to the DRD2 expression which was significantly correlated to maternal cannabis intake. Thus, it is possible to dissociate specific drug-related effects in the human fetal brain, especially when substantiated by the use of complementary animal models.
Mimicking cannabis-induced human fetal mesolimbic DRD2 gene expression disturbances in the prenatal THC rat model also provided novel information regarding epigenetic disturbances that could maintain Drd2 gene expression impairments into adulthood. Identifying specific epigenetic mechanisms that may play a role in maintaining cannabis-induced alterations in Drd2 gene expression is important since most studies showing long-term epigenetic disturbances in the offspring due to environmental influences has been related to maternal diet-induced changes (34
) and no data currently exist on such protracted effects of early developmental drug exposure. The majority of epigenetic studies in the drug abuse literature have focused on cocaine-induced changes in chromatin during adolescence or adulthood, rather than during the prenatal period (26
). A particularly interesting observation of our study is the THC-induced increase in 2meH3K9 in a discrete region upstream of the Drd2 TSS. In contrast to 3meH3K4, the 2meH3K9 mark remained unchanged at the Drd1 gene, indicating a developmental regulatory mechanism that might act on specific sequences located between −1.8 and 3 kb upstream of the Drd2 TSS. Developmental regulation of 2meH3K9 has been shown to be important for the appropriate tissue-specific expression of a variety of gene loci not only at promoters but also in broader regulatory regions including enhancers (36
The site-specific increase in 2meH3K9 was accompanied by decreased 3meH3K4 across the analyzed Drd2 genomic fragment. Reduced 3meH3K4 was also detected at the Drd1 gene without significant change in 2meH3K9, suggesting that long-term disruption of 3meH3K4 due to prenatal THC exposure may not on its own be sufficient for altering transcriptional activity. However, the direction of change of both 2meH3K9 (increase) and 3meH3K4 (decrease) at the Drd2 gene is consistent with the observed reduction in Drd2 mRNA expression in adulthood. Antagonistic roles for histone H3 lysine 9 and 4 methylation have been widely documented in gene regulation during brain development (25
). For example, it has been shown using in vitro
biochemical and in vivo
approaches that H3K9 methylation levels can only be increased in the absence of the 3meH3K4 mark due to the specificity of enzymes that modulate H3K9 methylation, the binding of which is blocked by high level 3meH3K4 (38
). Such a mechanism would explain the reduced 3meH3K4 levels induced by THC and the recruitment of the enzymes that modulate 2meH3K9 at the upstream sequence element of the Drd2 gene. As the RNA polymerase II transcription machinery is known to physically and functionally interact with chromatin regions containing 3meH3K4 (39
), the THC-induced reduction in 3meH3K4 could explain decreased Pol II association with the gene and downregulation of Drd2 mRNA production. Identification of the chromatin modifying enzymes that mediate the long-term effect of prenatal THC exposure will be an important subject of future studies.
In conclusion, these data emphasize the sensitive nature of the prenatal period, during which cannabis exposure can set into motion epigenetic alterations that contribute to long-term disturbances of the D2R in adulthood, thereby laying a foundation for increased vulnerability to addiction and potentially other psychiatric disorders.