Synaptic Microtubule-Associated Protein EB3 and SRC Phosphorylation Mediate Structural and Behavioral Adaptations During Withdrawal From Cocaine Self-Administration.

Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.

BAZ1B in Nucleus Accumbens Regulates Reward-Related Behaviors in Response to Distinct Emotional Stimuli.

ATP-dependent chromatin remodeling proteins are being implicated increasingly in the regulation of complex behaviors, including models of several psychiatric disorders. Here, we demonstrate that Baz1b, an accessory subunit of the ISWI family of chromatin remodeling complexes, is upregulated in the nucleus accumbens (NAc), a key brain reward region, in both chronic cocaine-treated mice and mice that are resilient to chronic social defeat stress. In contrast, no regulation is seen in mice that are susceptible to this chronic stress. Viral-mediated overexpression of Baz1b, along with its associated subunit Smarca5, in mouse NAc is sufficient to potentiate both rewarding responses to cocaine, including cocaine self-administration, and resilience to chronic social defeat stress. However, despite these similar, proreward behavioral effects, genome-wide mapping of BAZ1B in NAc revealed mostly distinct subsets of genes regulated by these chromatin remodeling proteins after chronic exposure to either cocaine or social stress. Together, these findings suggest important roles for BAZ1B and its associated chromatin remodeling complexes in NAc in the regulation of reward behaviors to distinct emotional stimuli and highlight the stimulus-specific nature of the actions of these regulatory proteins. SIGNIFICANCE STATEMENT: We show that BAZ1B, a component of chromatin remodeling complexes, in the nucleus accumbens regulates reward-related behaviors in response to chronic exposure to both rewarding and aversive stimuli by regulating largely distinct subsets of genes.

Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action.

Sirtuins (SIRTs), class III histone deacetylases, are well characterized for their control of cellular physiology in peripheral tissues, but their influence in brain under normal and pathological conditions remains poorly understood. Here, we establish an essential role for SIRT1 and SIRT2 in regulating behavioral responses to cocaine and morphine through actions in the nucleus accumbens (NAc), a key brain reward region. We show that chronic cocaine administration increases SIRT1 and SIRT2 expression in the mouse NAc, while chronic morphine administration induces SIRT1 expression alone, with no regulation of all other sirtuin family members observed. Drug induction of SIRT1 and SIRT2 is mediated in part at the transcriptional level via the drug-induced transcription factor ΔFosB and is associated with robust histone modifications at the Sirt1 and Sirt2 genes. Viral-mediated overexpression of SIRT1 or SIRT2 in the NAc enhances the rewarding effects of both cocaine and morphine. In contrast, the local knockdown of SIRT1 from the NAc of floxed Sirt1 mice decreases drug reward. Such behavioral effects of SIRT1 occur in concert with its regulation of numerous synaptic proteins in NAc as well as with SIRT1-mediated induction of dendritic spines on NAc medium spiny neurons. These studies establish sirtuins as key mediators of the molecular and cellular plasticity induced by drugs of abuse in NAc, and of the associated behavioral adaptations, and point toward novel signaling pathways involved in drug action.

ΔFosB induction in striatal medium spiny neuron subtypes in response to chronic pharmacological, emotional, and optogenetic stimuli.

The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.

Cocaine Self-administration Alters Transcriptome-wide Responses in the Brain's Reward Circuitry.

BACKGROUND: Global changes in gene expression underlying circuit and behavioral dysregulation associated with cocaine addiction remain incompletely understood. Here, we show how a history of cocaine self-administration (SA) reprograms transcriptome-wide responses throughout the brain's reward circuitry at baseline and in response to context and/or cocaine re-exposure after prolonged withdrawal (WD). METHODS: We assigned male mice to one of six groups: saline/cocaine SA + 24-hour WD or saline/cocaine SA + 30-day WD + an acute saline/cocaine challenge within the previous drug-paired context. RNA sequencing was conducted on six interconnected brain reward regions. Using pattern analysis of gene expression and factor analysis of behavior, we identified genes that are strongly associated with addiction-related behaviors and uniquely altered by a history of cocaine SA. We then identified potential upstream regulators of these genes. RESULTS: We focused on three patterns of gene expression that reflect responses to 1) acute cocaine, 2) context re-exposure, and 3) drug + context re-exposure. These patterns revealed region-specific regulation of gene expression. Further analysis revealed that each of these gene expression patterns correlated with an addiction index-a composite score of several addiction-like behaviors during cocaine SA-in a region-specific manner. Cyclic adenosine monophosphate response element binding protein and nuclear receptor families were identified as key upstream regulators of genes associated with such behaviors. CONCLUSIONS: This comprehensive picture of transcriptome-wide regulation in the brain's reward circuitry by cocaine SA and prolonged WD provides new insight into the molecular basis of cocaine addiction, which will guide future studies of the key molecular pathways involved.

Regulation of BAZ1A and nucleosome positioning in the nucleus accumbens in response to cocaine.

Chromatin regulation, in particular ATP-dependent chromatin remodelers, have previously been shown to be important in the regulation of reward-related behaviors in animal models of mental illnesses. Here we demonstrate that BAZ1A, an accessory subunit of the ISWI family of chromatin remodeling complexes, is downregulated in the nucleus accumbens (NAc) of mice exposed repeatedly to cocaine and of cocaine-addicted humans. Viral-mediated overexpression of BAZ1A in mouse NAc reduces cocaine reward as assessed by conditioned place preference (CPP), but increases cocaine-induced locomotor activation. Furthermore, we investigate nucleosome repositioning genome-wide by conducting chromatin immunoprecipitation (ChIP)-sequencing for total H3 in NAc of control mice and after repeated cocaine administration, and find extensive nucleosome occupancy and shift changes across the genome in response to cocaine exposure. These findings implicate BAZ1A in molecular and behavioral plasticity to cocaine and offer new insight into the pathophysiology of cocaine addiction.

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior.

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

Chronic escalating cocaine exposure, abstinence/withdrawal, and chronic re-exposure: effects on striatal dopamine and opioid systems in C57BL/6J mice.

Cocaine addiction is a chronic relapsing disease with periods of chronic escalating self-exposure, separated by periods of abstinence/withdrawal of varying duration. Few studies compare such cycles in preclinical models. This study models an "addiction-like cycle" in mice to determine neurochemical/molecular alterations that underlie the chronic, relapsing nature of this disease. Groups of male C57BL/6J mice received acute cocaine exposure (14-day saline/14-day withdrawal/13-day saline + 1-day cocaine), chronic cocaine exposure (14 day cocaine) or chronic re-exposure (14-day cocaine/14-day withdrawal/14-day cocaine). Escalating-dose binge cocaine (15-30 mg/kg/injection × 3/day, i.p. at hourly intervals) or saline (14-day saline) was administered, modeling initial exposure. In "re-exposure" groups, after a 14-day injection-free period (modeling abstinence/withdrawal), mice that had received cocaine were re-injected with 14-day escalating-dose binge cocaine, whereas controls received saline. Microdialysis was conducted on the 14th day of exposure or re-exposure to determine striatal dopamine content. Messenger RNA levels of preprodynorphin (Pdyn), dopamine D1 (Drd1) and D2 (Drd2) in the caudate putamen were determined by real-time PCR. Basal striatal dopamine levels were lower in mice after 14-day escalating exposure or re-exposure than in those in the acute cocaine group and controls. Pdyn mRNA levels were higher in the cocaine groups than in controls. Long-term adaptation was observed across the stages of this addiction-like cycle, in that the effects of cocaine on dopamine levels were increased after re-exposure compared to exposure. Changes in striatal dopaminergic responses across chronic escalating cocaine exposure and re-exposure are a central feature of the neurobiology of relapsing addictive states.