Results of the present study demonstrate that Dnmt3a expression is subject to dynamic regulation in NAc by two types of chronic emotional stimuli. Chronic social defeat stress induces a persistent upregulation of Dnmt3a in this brain region. In contrast, cocaine biphasically regulates Dnmt3a expression on a short timescale, whereas with longer-term withdrawal persisting upregulation of Dnmt3a is observed as well. Functional experiments establish that the cocaine-induced downregulation of Dnmt3a enhances cocaine reward, whereas upregulation of Dnmt3a exerts the opposite effect. Likewise, functional experiments in the chronic defeat stress and forced swim paradigms suggest that prolonged upregulation of Dnmt3a in NAc drives depressive-like behavior. Taken together, these data suggest that an appropriate balance of DNA methylation in NAc crucially gates behavioral responses to emotional stimuli—a hypermethylated state dampens responses to rewarding stimuli and heightens responses to aversive stimuli, conversely, a hypomethylated state heightens responses to rewarding stimuli and dampens responses to aversive stimuli.
Our analysis of dendritic spine density on NAc neurons demonstrates that DNA methylation is an essential mediator of cocaine-induced spinogenesis: Dnmt3a overexpression in NAc mimics the cocaine-induced increase in spines, while RG108 infusion into this region blocks cocaine’s action. A key question is how such a role for Dnmt3a in NAc spine regulation relates to the highly dynamic regulation seen for Dnmt3a expression. Since Dnmt3a overexpression alone is sufficient to regulate spine density, and because DNA demethylation is an enzymatically unfavorable chemical reaction and debate still exists regarding the enzymatic basis of active DNA demethylation, we speculate that the transient reduction in Dnmt3a expression, such as what we observe 24 hr after chronic cocaine, is likely not of a sufficient timescale to downregulate spines29
. Rather, we speculate that the transient increase in Dnmt3a expression seen 4 hr after each cocaine exposure leads to the progressive accumulation of DNA methylation and is thereby responsible for the overall induction of NAc dendritic spines. The fact that a highly persistent increase in Dnmt3a expression is seen at 4 weeks of withdrawal, a time point when NAc spine density is robustly induced as revealed by multiple laboratories and methods16–18
, is consistent with our hypothesis. The mechanisms responsible for the complex time course of Dnmt3a regulation in NAc–with early induction, quickly followed by suppression, and then followed by a slowly developing but very sustained induction–is unknown and requires future exploration (see below). Likewise, the persistent induction of Dnmt3a in NAc after chronic social defeat stress raises the novel possibility that NAc spine density may be regulated under these conditions as well, something in fact observed in preliminary investigations30
Our findings that Dnmt3a induction increases NAc spine density, while it attenuates cocaine reward, highlights an important question in the addiction field: What is the behavioral relevance of cocaine’s induction of dendritic spines on NAc neurons? Several conflicting reports exist on this subject22
. Robinson and colleagues positively correlated spine induction with increased locomotor sensitization31
. Three studies that directly manipulated genes (ΔFosB, NFκB, or G9a) in NAc, known to regulate spines, found that manipulations that block spine induction also block cocaine’s behavioral responses5, 23
. However, two other studies yielded conflicting results: blocking CDK5 or activating MEF2 blocks cocaine-induced spinogenesis but enhances cocaine reward17, 32
. The pattern seen for Dnmt3a matches these latter findings. The basis for these paradoxical results is unknown. One possibility is that regulation of different types of spines might exert very different functional effects on NAc neurons and consequently on behavior. A recent study reported highly complex regulation of various spine types over a course of chronic cocaine exposure and withdrawal33
, and we show a selective effect of DNA methylation on the regulation of thin spins. The observations that Dnmt3a induces thin spines, but blunts cocaine’s behavioral effects, raise the possibility that the induction of thin spines actually represents a homeostatic adaptation that serves to oppose the behavioral effects of cocaine. Clearly, this question requires much further investigation.
The electrophysiological function of cocaine-induced, DNA methylation-dependant thin spines remains speculative at this point, however, interesting electrophysiological correlates have been reported under similar cocaine injection regimens. In association with the increase in thin spines, chronic cocaine causes: 1) a reduction in firing rate in the NAc shell34
, 2) synaptic depression (decreased AMPA/NMDA ratio) during early (24 hrs) withdrawal35
, and 3) synaptic potentiation (increased AMPA/NMDA ratio) during late (10–14 days) withdrawal35
. First, some evidence exists for spines driving neuronal firing rate, as it was found recently that the spine density reductions that are associated with dopamine depletion induce a homeostatic response of increased firing of medium spiny neurons36
. Second, synaptic depression, indicated by decreased AMPA/NMDA ratio, may represent an increased pool of AMPA receptor-lacking synapses, which are also known to be regulated by cocaine37
. Since thin spines are thought to represent highly plastic, newly formed spines which lack AMPA receptors, we speculate that cocaine-induced DNA methylation generates such less responsive thin spines38
. Furthermore, It is also possible that, as these spines mature, they may incorporate AMPA receptor and thereby contribute to the increased AMPA/NMDA ratio as well as behavioral sensitization found during later withdrawal. Finally, the influence of DNA methylation on synaptic plasticity is heavily supported in the hippocampal literature. In cultured hippocampal neurons, consistent with our discovery that RG108 reduces cocaine-induced spine density of NAc neurons, Dnmt inhibition causes a marked reduction in functional synapses in an activity-dependant manner as measured by a reduction in mEPSC frequency14
. Moreover, both Dnmt inhibition and conditional knock out of Dnmt1 and Dnmt3a block long-term potentiation, a process thought to involve the formation and/or consolidation of dendritic spines11, 12
. These studies point to the importance of understanding the electrophysiological changes brought about by DNA methylation in the NAc. Such studies will provide key insight into whether cocaine-induced, DNA methylation-dependent changes in thin spines are a cause or consequence of changes in firing rate and/or synaptic depression.
As noted earlier, the mechanisms responsible for cocaine’s short-term biphasic regulation of Dnmt3a mRNA expression are unknown. In hippocampus, Dnmt3a was also found to be rapidly upregulated by fear conditioning, but the expression pattern following this upregulation over longer time points has not been assessed. Since Dnmt inhibition in hippocampus blocks memory formation, it was presumed in this study that Dnmt3a’s transient increase initiates downstream methylation of target genes that ultimately influence fear memory12
. This is analogous to our proposal that the transient increases in Dnmt3a that occur with each cocaine injection cause more lasting regulation of cellular and behavioral plasticity. In our study, the fact that me3K4H3 binding to the Dnmt3a promoter is commensurately associated with Dnmt3a mRNA regulation at both the 4 and 24 hr time points suggests that a histone 3, Lys4 methyltransferase, such as KMT2A (also known as MLL1), may regulate Dnmt3a’s expression. Additionally, me3K4H3 is well known to inversely correlate with DNA methylation at particular gene promoters, raising the intriguing possibility that Dnmt3a may feed back and regulate its own mRNA expression39
. On a general level, one hypothesis that may explain Dnmt3a’s complex regulation could be a transcriptional response to the rapid fluctuations in levels of dopamine or BDNF that are seen with cocaine exposures40
. This is an intriguing possibility since: 1) BDNF protein, like Dnmt3a mRNA, also accumulates with prolonged cocaine withdrawal41
, and 2) chronic social defeat stress induces lasting enhancement of BDNF signaling in NAc26
and, as we show here, also causes lasting increases in Dnmt3a expression in this brain region.
Finally, these data, in conjunction with studies of histone acetylation and methylation1, 5–7, 25
, support an emerging model that epigenetic alterations in NAc have profound effects on the regulation of emotional behavior in models of both drug addiction and depression. The regulation is complex, with different epigenetic mechanisms apparently exerting distinct effects on behavioral endpoints. A promising line of future research would be to assess the behavioral function of additional enzymes that mediate still other forms of epigenetic modifications. In parallel, the gene targets (and their degree of overlap) for these various enzymes remain largely unknown. The elaboration of these targets should help us identify the many ways in which epigenetic mechanisms, including Dnmt3a-mediated DNA methylation, regulate NAc neuronal function to mediate the complex behavioral phenotypes of addiction and depression. It is also important to explore in future studies how Dnmt3a regulation and other epigenetic modifications in NAc contribute to the complex interactions known to exist between cocaine and stress vulnerabilities.
In this study, we found that both chronic cocaine and stress differentially regulate the de-novo DNA methyltransferase, Dnmt3a, in NAc to control dendritic spine plasticity and behavioral responses to these stimuli. We find that Dnmt3a activity in NAc in vivo is necessary for cocaine-induced increases in dendritic spine density selectively for the thin type of spines. Our findings also suggest that the rewarding responses to cocaine negatively correlate with thin spines and also raise the possibility that depressive responses to chronic stress may positively correlate with increased spine density. Taken together, these observations implicate a new epigenetic modification–DNA methylation–in the molecular mechanisms controlling cocaine- and stress-induced structural and behavioral plasticity and could ultimately lead to the development of improved treatments for drug addiction and depression.