In this study, we find that repeated cocaine exposure regulates MEF2 transcription factors to control aspects of long-lasting synaptic and behavioral plasticity. Our findings suggest that chronic cocaine exposure reduces MEF2-dependent transcription to promote increased MSN dendritic spine density in the NAc. Surprisingly, this MEF2-controlled increase in dendritic spine density is associated with reduced behavioral sensitivity to cocaine, suggesting that the strong correlation between increases in NAc spine density and sensitized behavioral responses to cocaine may be functionally uncoupled processes. Our findings suggest that repeated cocaine exposure suppresses MEF2 activity in part by a cAMP/RCS-dependent reduction in calcineurin activity, which regulates MEF2 phosphorylation at the inhibitory Cdk5 sites, P-Ser408/444 (). This inhibition of MEF2 through increased P-Ser408/444 levels is also likely influenced by the previously reported up-regulation of Cdk5 levels and activity in the striatum after chronic cocaine exposure (Bibb et al., 2001
). Finally, by combining in vivo
MEF2 ChIP on genome-wide promoter arrays and gene expression microarrays, we identified a number of putative MEF2-target genes that likely contribute to aspects of cocaine-induced dendritic spine and behavioral plasticity in the NAc.
Model for how cocaine regulates MEF2 in the NAc to alter dendritic spine density and behavioral responses.
Cocaine administration increases the inhibitory Ser408/444 phosphorylation of MEF2A/2D in striatum with a complex time course. A single dose of cocaine is sufficient to induce MEF2 phosphorylation, but is only detected at 24 hours after the first injection. Repeated, daily cocaine injections appear to maintain the levels of MEF2 P-Ser408/444, as it is detected at 4 hours and 24 hours after the last cocaine injection (7-day injection regimen). However, in the absence of reinforcing cocaine injections, we observe that P-S408/444 levels return to baseline by 48 hours after the last injection in the 7-day injection regimen. This indicates that MEF2 inhibitory phosphorylation does not persist in the striatum for as long as the cocaine-induced spine changes that MEF2 activity controls. Moreover, it also suggests that suppression of MEF2 activity by cocaine likely regulates the initiation and maintenance of spine density changes observed during and shortly after chronic cocaine exposure, but may not play a significant role in the maintenance of those spine changes during extended withdrawal periods. In the future, it will be important to explore the regulation and potential role of MEF2 in the maintenance phase of spine plasticity during withdrawal.
Several recent reports have demonstrated an important role for Cdk5 activity in regulating chronic cocaine-induced spine and behavioral plasticity (Benavides et al., 2007
; Bibb et al., 2001
; Norrholm et al., 2003
; Taylor et al., 2007
). We show here that MEF2 potently regulates NAc spine plasticity and, as a key target of Cdk5, may mediate some of its downstream effects on synapse and network plasticity. There are a number of interesting parallels between our MEF2 findings and reports in the literature regarding the function of Cdk5 activity in cocaine-induced spine plasticity and behavioral responses (Norrholm et al., 2003
; Taylor et al., 2007
). Specifically, chronic infusion of roscovitine into the NAc region, which we find increases MEF2 activity in striatal neurons (Supplemental Fig. S3B
), blocks the chronic cocaine-induced increase in NAc MSN dendritic spine density (Norrholm et al., 2003
). Similarly, we find that increasing MEF2 activity in the NAc (via MEF2-VP16 expression) also blocks the cocaine-induced increase in dendritic spine density (), suggesting that downstream inhibition of MEF2 by Cdk5 is a key component of roscovitine’s effect on spine plasticity in the NAc. Another interesting parallel between the Cdk5 and MEF2 studies are the recent findings that daily roscovitine injections into the NAc (Taylor et al., 2007
) or conditional Cdk5 gene deletion in the NAc (Benavides et al., 2007
) enhanced locomotor sensitization to repeated cocaine treatments. Consistent with these observations, we find that enhanced MEF2 activity in the NAc (via MEF2-VP16 expression) increased the locomotor responses to repeated cocaine injections (). Together these studies provide evidence that experimental manipulations that block cocaine-induced increases in NAc dendritic spine density enhance sensitized behavioral responses to cocaine, suggesting that spine density increases are not required for locomotor sensitization and might instead be functioning to antagonize the process of sensitization. It is perhaps not surprising that spine increases are not required for behavioral sensitization since chronic morphine exposure, which elicits long-lasting locomotor sensitization, actually decreases NAc dendritic spine density (Robinson and Kolb, 1999b
). In our study, we cannot directly determine whether lower spine density actually causes increased cocaine sensitivity, per se
, because the proximal manipulation in our study is MEF2 – not the spines themselves. It is possible that MEF2 could function through independent mechanisms to control cocaine behavior and dendritic spine density. Nevertheless, one can conclude from this and previous reports that cocaine-induced increases in NAc dendritic spine density does not appear to be required for behavioral sensitization to cocaine. This functional relationship between cocaine-induced dendritic spines and behavioral responses suggests that the additional NAc dendritic spines may contribute to homeostatic adaptations that counteract the neural plasticity processes that cause sensitized behavioral responses to cocaine, or that MEF2-regulated spine plasticity might represent an independent process that does not significantly impact sensitized behavioral responses to cocaine.
How does regulation of MEF2-dependent transcription control NAc synapse density? In cultured hippocampal neurons, MEF2-dependent transcription is induced by glutamatergic synaptic activity to promote elimination of existing excitatory synapses (Flavell et al., 2006
). As such, it is possible that suppression of MEF2 activity by cocaine in the NAc increases dendritic spine density by reducing the elimination rate of existing synapses rather than increasing the formation rate of new synapses. NAc neurons may therefore rely upon MEF2-dependent transcription to control homeostatic synaptic plasticity after repeated cocaine exposure (Turrigiano, 2007
). Indeed, intrinsic neuronal excitability is reduced in the NAc of chronic cocaine-treated animals (Hu et al., 2004
), and glutamatergic inputs to the NAc may be decreased after extended drug taking as a result of a decreased mPFC function (hypofrontality), which has been observed in both animal models of addiction and in human brain imaging studies of drug addicts (Jentsch and Taylor, 1999
; Volkow et al., 2003
). Therefore, it is interesting to speculate that increased NAc dendritic spine density may function homeostatically to compensate for reduced NAc excitability, and ultimately limit an animal’s sensitivity to the cocaine.
Cocaine regulation of MEF2-dependent transcription ultimately mediates structural and behavioral changes in the NAc through the altered expression of downstream target genes. We utilized genome-wide ChIP-chip technology to identify nearly 900 gene promoters in cocaine-treated NAc tissue where MEF2A binding is enriched. Many of the target genes we identified cluster to cellular functions that regulate structural plasticity, such as F-actin remodeling. The MEF2 target genes that encode the proteins, N-WASP, WAVE3, and profilin 1, are all known to potently regulate cytoskeletal remodeling as well as dendritic spine density (Irie and Yamaguchi, 2002
; Pilpel and Segal, 2005
; Wegner et al., 2008
). Importantly, dysregulation of actin polymerization with latrunculin A or a LIM-kinase peptide antagonist in the NAc promoted cocaine reinstatement behaviors in self-administering rats (Toda et al., 2006
). Therefore, MEF2 gene targets that regulate actin remodeling may play important roles in sensitized cocaine responses, perhaps independent of the spine changes observed after chronic cocaine exposure.
Another functional group of MEF2-bound genes in the NAc clustered to the PI3-kinase/Akt signaling pathway (Supplemental Fig. S9A
). Interestingly, a chemical inhibitor of PI3-kinase delivered ICV (intracerebroventricular) significantly blocked the expression of cocaine locomotor sensitization (Izzo et al., 2002
), suggesting that MEF2-dependent regulation of this signaling pathway may play an important role in locomotor sensitization. Since cocaine suppresses MEF2 activity in the NAc, it was notable that a catalytic subunit of PI3-kinase was one of 82 MEF2 target genes downregulated by chronic cocaine exposure (). Pik3cg
expression is also potently regulated by MEF2 activity in culture, suggesting that the cocaine-induced suppression of MEF2 activity directly contributes to the downregulation of this gene in the NAc. Consistent with a reduction in Pik3cg
mRNA in the NAc after chronic cocaine exposure, we also observed a significant reduction in the phosphorylation state of Akt (Ser473), a key substrate of PI3-kinase activity, in the NAc after chronic cocaine. Moreover, RNAi-based reduction of MEF2 levels in the NAc, which would be expected to reduce Pik3cg
expression and PI3-kinase activity, reduces behavioral sensitivity to cocaine much like the infusion of a PI3-kinase inhibitor.
In this study, we found that cocaine administration regulates MEF2-dependent gene transcription in the NAc to control dendritic spine plasticity and behavioral responses to cocaine. We show here that chronic cocaine reduces MEF2 activity through a novel signaling mechanism involving Cdk5, calcineurin and RCS. We find that reducing MEF2 activity in NAc in vivo is required for cocaine-induced increases in dendritic spine density. Our findings also suggest that behavioral sensitization to cocaine is functionally uncoupled from these cocaine-induced increases in dendritic spine density. Taken together, these observations implicate a new transcription factor in the molecular mechanisms controlling cocaine-induced structural and behavioral plasticity and could ultimately lead to the development of improved treatments for drug addiction.