An imbalance in molecular signaling cascades and transcriptional regulation in nucleus accumbens (NAc) medium spiny neuron (MSN) subtypes, those enriched in dopamine D1 versus D2 receptors, is implicated in the behavioral responses to psychostimulants. To provide further insight into the molecular mechanisms occurring in MSN subtypes by cocaine, we examined the transcription factor early growth response 3 (Egr3). We evaluated Egr3 because it is a target of critical cocaine-mediated signaling pathways and because Egr3-binding sites are found on promoters of key cocaine-associated molecules. We first used a RiboTag approach to obtain ribosome-associated transcriptomes from each MSN subtype and found that repeated cocaine administration induced Egr3 ribosome-associated mRNA in NAc D1-MSNs while reducing Egr3 in D2-MSNs. Using Cre-inducible adeno-associated viruses combined with D1-Cre and D2-Cre mouse lines, we observed that Egr3 overexpression in D1-MSNs enhances rewarding and locomotor responses to cocaine, whereas overexpression in D2-MSNs blunts these behaviors. miRNA knock-down of Egr3 in MSN subtypes produced opposite behavioral responses from those observed with overexpression. Finally, we found that repeated cocaine administration altered Egr3 binding to promoters of genes that are important for cocaine-mediated cellular and behavioral plasticity. Genes with increased Egr3 binding to promoters, Camk2␣, CREB, FosB, Nr4a2, and Sirt1, displayed increased mRNA in D1-MSNs and, in some cases, a reduction in D2-MSNs. Histone and the DNA methylation enzymes G9a and Dnmt3a displayed reduced Egr3 binding to their promoters and reduced mRNA in D1-MSNs. Our study provides novel insight into an opposing role of Egr3 in select NAc MSN subtypes in cocaine action.

Altered brain energy homeostasis is a key adaptation occurring in the cocaine-addicted brain, but the effect of cocaine on the fundamental source of energy, mitochondria, is unknown. We demonstrate an increase of dynamin-related protein-1 (Drp1), the mitochondrial fission mediator, in nucleus accumbens (NAc) after repeated cocaine exposure and in cocaine-dependent individuals. Mdivi-1, a demonstrated fission inhibitor, blunts cocaine seeking and locomotor sensitization, while blocking c-Fos induction and excitatory input onto dopamine receptor-1 (D1) containing NAc medium spiny neurons (MSNs). Drp1 and fission promoting Drp1 are increased in D1-MSNs, consistent with increased smaller mitochondria in D1-MSN dendrites after repeated cocaine. Knockdown of Drp1 in D1-MSNs blocks drug seeking after cocaine self-administration, while enhancing the fission promoting Drp1 enhances seeking after long-term abstinence from cocaine. We demonstrate a role for altered mitochondrial fission in the NAc, during early cocaine abstinence, suggesting potential therapeutic treatment of disrupting mitochondrial fission in cocaine addiction.

Summary Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens, a primary brain reward region, are seen at early vs. late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local-synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce “metaplasticity” in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner.

Cocaine addiction is a life-long relapsing disorder that results from long-term adaptations within the brain. We find that Activin-receptor signaling, including the transcription factor Smad3, is upregulated in the rat nucleus accumbens shell following withdrawal from cocaine. Direct genetic and pharmacological manipulations of this pathway bidirectionally alter cocaine seeking, while governing morphological plasticity in nucleus accumbens neurons. These findings reveal that Activin/Smad3 signaling is induced following withdrawal from cocaine, and such regulation may be a key molecular mechanism underlying behavioral and cellular plasticity in the brain following cocaine self-administration.

There is evidence that visual stimuli used to signal drug delivery in self-administration procedures have primary reinforcing properties, and that drugs of abuse enhance the reinforcing properties of such stimuli. Here, we explored the relationships between locomotor activity, responding for a visual stimulus, and self-administration of methamphetamine (METH). Rats were classified as high or low responders based on activity levels in a novel locomotor chamber and were subsequently tested for responding to produce a visual stimulus followed by self-administration of a low dose of METH (0.025 mg/kg/infusion) paired with the visual stimulus. High responder rats responded more for the visual stimulus than low responder rats indicating that the visual stimulus was reinforcing and that operant responding for a visual stimulus has commonalities with locomotor activity in a novel environment. Similarly, high responder rats responded more for METH paired with a visual stimulus than low responder rats. Because of the reinforcing properties of the visual stimulus, it was not possible to determine if the rats were responding to produce the visual stimulus, METH or the combination. We speculate that responding to produce sensory reinforcers may be a measure of sensation seeking. These results indicate that visual stimuli have unconditioned reinforcing effects which may have a significant role in acquisition of drug self-administration, a role that is not yet well understood.

Exposure to psychostimulants results in structural and synaptic plasticity in striatal medium spiny neurons (MSNs). These cellular adaptations arise from alterations in genes that are highly implicated in the rearrangement of the actin-cytoskeleton, such as T-lymphoma invasion and metastasis 1 (Tiam1). Previous studies have demonstrated a crucial role for dopamine receptor 1 (D1)-containing striatal MSNs in mediating psychostimulant induced plasticity changes. These D1-MSNs in the nucleus accumbens (NAc) positively regulate drug seeking, reward, and locomotor behavioral effects as well as the morphological adaptations of psychostimulant drugs. Here, we demonstrate that rats that actively self-administer cocaine display reduced levels of Tiam1 in the NAc. To further examine the cell type-specific contribution to these changes in Tiam1 we used optogenetics to selectively manipulate NAc D1-MSNs or dopamine receptor 2 (D2) expressing MSNs. We find that repeated channelrhodopsin-2 activation of D1-MSNs but not D2-MSNs caused a down-regulation of Tiam1 levels similar to the effects of cocaine. Further, activation of D2-MSNs, which caused a late blunted cocaine-mediated locomotor behavioral response, did not alter Tiam1 levels. We then examined the contribution of D1-MSNs to the cocaine-mediated decrease of Tiam1. Using the light activated chloride pump, eNpHR3.0 (enhanced Natronomonas pharaonis halorhodopsin 3.0), we selectively inhibited D1-MSNs during cocaine exposure, which resulted in a behavioral blockade of cocaine-induced locomotor sensitization. Moreover, inhibiting these NAc D1-MSNs during cocaine exposure reversed the down-regulation of Tiam1 gene expression and protein levels. These data demonstrate that altering activity in specific neural circuits with optogenetics can impact the underlying molecular substrates of psychostimulant-mediated behavior and function.

Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1 induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1-important for synaptic connections during development-as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine.histone PARylation | drug addiction | medium spiny neurons

Background Drug addiction is defined as a chronic disease characterized by compulsive drug seeking and episodes of relapse despite prolonged periods of drug abstinence. Neurobiological adaptations, including transcriptional and epigenetic alterations in the nucleus accumbens, are thought to contribute to this life-long disease state. We previously demonstrated that transcription factor SMAD3 is increased following seven days of withdrawal from cocaine self-administration. However, it is still unknown which additional factors participate in the process of chromatin remodeling and facilitate the binding of SMAD3 to promoter regions of target genes. Here we examined the possible interaction of BRG1—also known as SMARCA4, an ATPase-containing chromatin remodeler—and SMAD3 in response to cocaine exposure. Methods The expression of BRG1, as well as its binding to SMAD3 and target gene promoter regions, was evaluated in the nucleus accumbens and dorsal striatum of rats using Western blotting, co-immunoprecipitation, and chromatin immunoprecipitation techniques, respectively, following withdrawal from cocaine self-administration. Rats were assessed for cocaine-seeking behaviors following either intra-accumbal injections of the BRG1 inhibitor PFI3 or viral-mediated overexpression of BRG1. Results Following withdrawal from cocaine self-administration, BRG1 expression and complex formation with SMAD3 are increased in the nucleus accumbens, resulting in increased binding of BRG1 to the promoter regions of Ctnnb1, Mef2d, and Dbn1. Intra-accumbal infusion of PFI3 attenuated, while viral overexpression of Brg1 enhanced, cocaine-reinstatement behavior. Conclusion BRG1 is a key mediator of the SMAD3-dependent regulation of cellular and behavioral plasticity mediating cocaine seeking following a period of withdrawal.