Cocaine reward is reduced by decreased expression of receptor-type protein tyrosine phosphatase D (PTPRD) and by a novel PTPRD antagonist.

Receptor-type protein tyrosine phosphatase D (PTPRD) is a neuronal cell-adhesion molecule/synaptic specifier that has been implicated in addiction vulnerability and stimulant reward by human genomewide association and mouse cocaine-conditioned place-preference data. However, there have been no reports of effects of reduced expression on cocaine self-administration. There have been no reports of PTPRD targeting by any small molecule. There are no data about behavioral effects of any PTPRD ligand. We now report (i) robust effects of heterozygous PTPRD KO on cocaine self-administration (These data substantially extend prior conditioned place-preference data and add to the rationale for PTPRD as a target for addiction therapeutics.); (ii) identification of 7-butoxy illudalic acid analog (7-BIA) as a small molecule that targets PTPRD and inhibits its phosphatase with some specificity; (iii) lack of toxicity when 7-BIA is administered to mice acutely or with repeated dosing; (iv) reduced cocaine-conditioned place preference when 7-BIA is administered before conditioning sessions; and (v) reductions in well-established cocaine self-administration when 7-BIA is administered before a session (in WT, not PTPRD heterozygous KOs). These results add to support for PTPRD as a target for medications to combat cocaine use disorders. 7-BIA provides a lead compound for addiction therapeutics.

Dnmt3a2 in the Nucleus Accumbens Shell Is Required for Reinstatement of Cocaine Seeking.

Epigenetic mechanisms have gained increasing attention as regulators of synaptic plasticity and responsiveness to drugs of abuse. In particular, it has been shown that the activity of the DNA methyltransferase 3a (Dnmt3a) mediates certain long-lasting effects of cocaine. Here we examined the role of the Dnmt isoforms, Dnmt3a1 and Dnmt3a2, within the nucleus accumbens (NAc) on transcriptional activity of immediate early genes (IEGs) and acute and long-lasting responsiveness to cocaine and cocaine conditioned cues. Using primary striatal cultures, we show that transcription of Dnmt3a2, but not that of Dnmt3a1, is activated by dopamine D1 receptor signaling and that knockdown of Dnmt3a2 using viral vector-mediated expression of Dnmt3a2-specific shRNAs impairs induction of the IEGs, Arc, FosB, and Egr2 Acute cocaine administration increases expression of Dnmt3a2 but not that of Dnmt3a1 in the NAc shell. In contrast, in the NAc core, expression of Dnmt3a1 and Dnmt3a2 was unaffected by cocaine administration. shRNA-mediated knockdown of Dnmt3a2 in vivo impairs the induction of IEGs, including Egr2 and FosB indicating that Dnmt3a2 regulates cocaine-dependent expression of plasticity genes in the rat NAc shell. Cocaine self-administration experiments in rats revealed that Dnmt3a2 regulates drug cue memories that drive reinstatement of cocaine seeking as well as incubation of this phenomenon within the NAc shell. Dnmt3a2 does not influence the primary reinforcing effects of cocaine. Thus, Dnmt3a2 mediates long-lasting cocaine cue memories within the NAc shell. Targeting Dnmt3a2 expression or function may interfere with cocaine craving and relapse.SIGNIFICANCE STATEMENT In humans, drug craving can occur in response to conditioned cues, even after extended periods of abstinence. In rats, cue-induced cocaine seeking has been shown to increase progressively during the first 2 months of abstinence from drug self-administration. This phenomenon, referred to as incubation of cocaine seeking, is consistent with the hypothesis that in humans craving increases over time and remains high following prolonged abstinence. Those long-lasting behavioral changes are likely to be mediated by epigenetic effects and neuroplastic changes within the mesolimbic brain reward system. Here we show that a specific isoform of DNA-methyltransferases in the NAc shell regulates drug cue memories that drive reinstatement of cocaine seeking after both early abstinence and incubation of cocaine craving.

Role of Src Family Kinases in BDNF-Mediated Suppression of Cocaine-Seeking and Prevention of Cocaine-Induced ERK, GluN2A, and GluN2B Dephosphorylation in the Prelimbic Cortex.

Models of relapse have demonstrated that neuroadaptations in reward circuits following cocaine self-administration (SA) underlie reinstatement of drug-seeking. Dysregulation of the pathway from the prelimbic (PrL) cortex to the nucleus accumbens is implicated in reinstatement. A single BDNF infusion into the PrL cortex following a final cocaine SA session results in attenuation of reinstatement of cocaine-seeking. Inhibiting BDNF's receptor, TrkB, ERK/MAP kinase activation, or NMDA receptors blocks this attenuating effect, indicating that the interaction between glutamate-mediated synaptic activity and TrkB signaling is imperative to BDNF's suppressive effect on drug-seeking. Src family kinases (SFKs) are involved in both NMDA-mediated activation of TrkB- and TrkB-mediated tyrosine phosphorylation of NMDA receptors. We hypothesized that infusion of the SFK inhibitor, PP2, into the PrL cortex prior to a BDNF infusion, immediately after the end of the last cocaine SA session, would block BDNF's ability to suppress reinstatement of cocaine-seeking in rats with a cocaine SA history. PP2, but not the negative control, PP3, blocked BDNF's suppressive effect on context-induced relapse after 1 week of abstinence and cue-induced reinstatement after extinction. As previously reported, infusion of BDNF into the PrL cortex blocked cocaine SA-induced dephosphorylation of ERK, GluN2A, and GluN2B-containing receptors. Inhibition of SFKs using PP2 blocked BDNF-mediated phosphorylation of GluN2A, GluN2B, and ERK. These data indicate that SFK activity is necessary for BDNF-mediated suppression of cocaine-seeking and reversal of cocaine-induced dephosphorylation of key phosphoproteins in the prefrontal cortex related to synaptic plasticity.

Inhibition of DNA methyltransferases regulates cocaine self-administration by rats: a genome-wide DNA methylation study.

DNA methylation is a major epigenetic process which regulates the accessibility of genes to the transcriptional machinery. In the present study, we investigated whether modifying the global DNA methylation pattern in the brain would alter cocaine intake by rats, using the cocaine self-administration test. The data indicate that treatment of rats with the DNA methyltransferase inhibitors 5-aza-2'-deoxycytidine (dAZA) and zebularine enhanced the reinforcing properties of cocaine. To obtain some insights about the underlying neurobiological mechanisms, a genome-wide methylation analysis was undertaken in the prefrontal cortex of rats self-administering cocaine and treated with or without dAZA. The study identified nearly 189 000 differentially methylated regions (DMRs), about half of them were located inside gene bodies, while only 9% of DMRs were found in the promoter regions of genes. About 99% of methylation changes occurred outside CpG islands. Gene expression studies confirmed the inverse correlation usually observed between increased methylation and transcriptional activation when methylation occurs in the gene promoter. This inverse correlation was not observed when methylation took place inside gene bodies. Using the literature-based Ingenuity Pathway Analysis, we explored how the differentially methylated genes were related. The analysis showed that increase in cocaine intake by rats in response to DNA methyltransferase inhibitors underlies plasticity mechanisms which mainly concern axonal growth and synaptogenesis as well as spine remodeling. Together with the Akt/PI3K pathway, the Rho-GTPase family was found to be involved in the plasticity underlying the effect of dAZA on the observed behavioral changes.

Pharmacological modulation of protein kinases as a new approach to treat addiction to cocaine and opiates.

Drug addiction shares brain mechanisms and molecular substrates with learning and memory processes, such as the stimulation of glutamate receptors and their downstream signalling pathways. In the present work we provide an up-to-date review of studies that have demonstrated the implication of the main memory-related calcium-dependent protein kinases in opiate and cocaine addiction. The effects of these drugs of abuse in different animal models of drug reward, dependence and addiction are altered by manipulation of the mitogen-activated protein kinase (MAPK) family, particularly extracellular signal regulated kinase (ERK), calcium/calmodulin-dependent kinase II (CaMKII), the protein kinase C (PKC) family (including PKMζ), cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase G (PKG), the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target mammalian target of Rapamycin (mTOR), cyclin-dependent kinase 5 (Cdk5), heat-shock proteins (Hsp) and other enzymes and proteins. Research suggests that drugs of abuse induce dependence and addiction by modifying the signalling pathways that involve these memory-related protein kinases, and supports the idea that drug addiction is an excessive aberrant learning disorder in which the maladaptive memory of drug-associated cues maintains compulsive drug use and contributes to relapse. Moreover, the studies we review offer new pharmacological strategies to treat opiate and cocaine dependence based on the manipulation of these protein kinases. In particular, disruption of reconsolidation of drug-related memories may have a high therapeutic value in the treatment of drug addiction.

Poly(ADP)-Ribose Polymerase-1 Inhibitors as a Potential Treatment for Cocaine Addiction.

As of 2008, according to the National Survey on Drug Use and Health, nearly 1.4 million Americans met the Diagnostic and Statistical Manual of Mental Disorders criteria for dependence or abuse of cocaine (in any form) in the past 12 months. However, there are no treatments for cocaine use disorders approved by the Federal Drug Administration (FDA). Alterations in gene regulation contribute significantly to the changes that occur in the brain, both structurally and functionally, and the resultant addictive phenotype that occurs with chronic exposure to drugs of abuse. The Emerging Targets of Cocaine Use Disorders meeting sought to explore novel targets for the treatment of stimulant use disorder. The evidence for a role of one novel target, Poly(ADP)-ribose polymerase-1 (PARP-1), was presented at the meeting and will be summarized in this review.

Histone Deacetylases as Potential Targets for Cocaine Addiction.

Drug-induced changes in gene expression likely contribute to long-lasting structural and functional alterations in the brain's reward circuitry and the persistence of addiction. Modulation of chromatin structure through covalent histone modifications has emerged as an important regulator of gene transcription in brain and increasing evidence suggests that misregulation of histone acetylation contributes to the establishment and maintenance of aberrant neuronal gene programs and behaviors associated with cocaine or amphetamine exposure. In this review, we summarize evidence supporting a role for histone acetylation in psychostimulant-induced plasticity and discuss findings from preclinical studies investigating histone deacetylase (HDAC) action and the use of small-molecule HDAC inhibitors (HDACis) to correct drug-mediated transcriptional dysregulation.

Synaptic plasticity mediating cocaine relapse requires matrix metalloproteinases.

Relapse to cocaine use necessitates remodeling excitatory synapses in the nucleus accumbens and synaptic reorganization requires matrix metalloproteinase (MMP) degradation of the extracellular matrix proteins. We found enduring increases in MMP-2 activity in rats after withdrawal from self-administered cocaine and transient increases in MMP-9 during cue-induced cocaine relapse. Cue-induced heroin and nicotine relapse increased MMP activity, and increased MMP activity was required for both cocaine relapse and relapse-associated synaptic plasticity.

The effect of active and passive intravenous cocaine administration on the extracellular signal-regulated kinase (ERK) activity in the rat brain.

According to a current hypothesis of learning processes, recent papers pointed out to an important role of the extracellular signal-regulated kinase (ERK), in drug addiction. We employed the Western blotting techniques to examine the ERK activity immediately after cocaine iv self-administration and in different drug-free withdrawal periods in rats. To distinguish motivational vs. pharmacological effects of the psychostimulant intake, a "yoked" procedure was used. Animals were decapitated after 14 daily cocaine self-administration sessions or on the 1st, 3rd or 10th extinction days. At each time point the activity of the ERK was assessed in several brain structures, including the prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens. Passive, repeated iv cocaine administration resulted in a 45% increase in ERK phosphorylation in the hippocampus while cocaine self-administration did not change brain ERK activity. On the 1st day of extinction, the activity of the ERK in the prefrontal cortex was decreased in rats with a history of cocaine chronic intake: by 66% for "active" cocaine group and by 35% for "yoked" cocaine group. On the 3rd day the reduction in the ERK activity (25-34%) was observed in the hippocampus for both cocaine-treated groups, and also in the nucleus accumbens for "yoked" cocaine group (40%). On the 10th day of extinction there was no significant alteration in ERK activity in any group of rats. Our findings suggest that cortical ERK is involved in cocaine seeking behavior in rats. They also indicate the time and regional adaptations in this enzyme activity after cocaine withdrawal.

Long-term reduction of cocaine self-administration in rats treated with adenoviral vector-delivered cocaine hydrolase: evidence for enzymatic activity.

A new pharmacokinetic approach treating cocaine addiction involves rapidly metabolizing cocaine before it reaches brain reward centers using mutated human butyrylcholinesterase (BChE) or cocaine hydrolase (CocH). Recent work has shown that helper-dependent adenoviral (hdAD) vector-mediated plasma CocH reduced the locomotor-activating effects of cocaine and prevented reinstatement of cocaine-seeking behavior up to 6 months in rats. The present study investigated whether hdAD-CocH could decrease ongoing intravenous cocaine (0.4 mg/kg) self-administration. The hdAD-CocH vector was injected into self-administering rats, and after accumulation of plasma CocH, there was a dramatic reduction in cocaine infusions earned under a fixed ratio 1 schedule of reinforcement that lasted for the length of the study (>2 months). Pretreatment with the selective BChE and CocH inhibitor iso-OMPA (1.5 mg/kg) restored cocaine intake; therefore, the decline in self-administration was likely due to rapid CocH-mediated cocaine metabolism. Direct measurements of cocaine levels in plasma and brain samples taken after the conclusion of behavioral studies provided strong support for this conclusion. Further, rats injected with hdAD-CocH did not experience a deficit in operant responding for drug reinforcement and self-administered methamphetamine (0.05 mg/kg) at control levels. Overall, these outcomes suggest that viral gene transfer can yield plasma CocH levels that effectively diminish long-term cocaine intake and may have potential treatment implications for cocaine-dependent individuals seeking to become and remain abstinent.

Experimental treatments for cocaine toxicity: a difficult transition to the bedside.

Cocaine is a commonly abused illicit drug that causes significant morbidity and mortality. Although there is no true antidote to cocaine toxicity, current management strategies address the life-threatening systemic effects, namely hyperthermia, vasospasm, and severe hypertension. Clinicians rely on rapid cooling, benzodiazepines, and α-adrenergic antagonists for management, with years of proven benefit. Experimental agents have been developed to more effectively treat acute toxicity. Pharmacodynamic approaches include antipsychotics that are thought to interfere with cocaine's actions at several neurotransmitter receptors. However, these medications may worsen the consequences of cocaine toxicity as they can interfere with heat dissipation, cause arrhythmias, and lower the seizure threshold. Pharmacokinetic approaches use cocaine-metabolizing enzymes, such as butyrylcholinesterase (BChE), cocaine hydrolase (CocH), and bacterial cocaine esterase (CocE). Experimental models with these therapies improve survival, primarily when administered before cocaine, although newer evidence demonstrates beneficial effects shortly after cocaine toxicity has manifested. CocE, a foreign protein, can induce an immune response with antibody formation. When enzyme administration was combined with vaccination against the cocaine molecule, improvement in cocaine-induced locomotor activity was observed. Finally, lipid emulsion rescue has been described in human case reports as an effective treatment in patients with hemodynamic compromise because of cocaine, which correlates well with its documented benefit in toxicity due to other local anesthetics. A pharmaceutical developed from these concepts will need to be expedient in onset and effective with minimal adverse effects while at the same time being economical.

The selective dopamine ß-hydroxylase inhibitor nepicastat attenuates multiple aspects of cocaine-seeking behavior.

Although norepinephrine (NE) does not typically modulate cocaine self-administration under traditional schedules of reinforcement, it is required for different inducers of the reinstatement of cocaine-seeking behavior via activation of multiple adrenergic receptor subtypes. We predicted that blockade of NE synthesis would attenuate all known modalities of reinstatement and showed previously that the selective dopamine ß-hydroxylase inhibitor, nepicastat, had no effect on either maintenance of operant cocaine self-administration maintained on a fixed-ratio 1 schedule or reinstatement of food seeking but did abolish cocaine-primed reinstatement. In the present series of studies, we first evaluated the dose-dependent effect of nepicastat (5, 50, or 100 mg/kg) on novelty-induced locomotor activity and found that it blunted exploration only at the highest dose. Next, we assessed the ability of nepicastat (50 mg/kg) to reduce breakpoint responding for cocaine on a progressive ratio schedule and reinstatement induced by drug-associated cues and stress. We found that nepicastat significantly lowered the breakpoint for cocaine, but not for regular chow or sucrose, and attenuated cue-, footshock-, and yohimbine-induced reinstatement. Combined, these results indicate that nepicastat can reduce the reinforcing properties of cocaine under a stringent schedule and can attenuate relapse-like behavior produced by cocaine, formerly cocaine-paired cues, and physiological and pharmacological stressors. Thus, nepicastat is one of those rare compounds that can reduce reinforced cocaine seeking as well as all three reinstatement modalities, while sparing exploratory behavior and natural reward seeking, making it a promising pharmacotherapy for cocaine addiction.

Extracellular signal-regulated kinase in the basolateral amygdala, but not the nucleus accumbens core, is critical for context-response-cocaine memory reconsolidation in rats.

The reconsolidation of cocaine memories following retrieval is necessary for the sustained ability of a cocaine-paired environmental context to elicit cocaine seeking. Extracellular signal-regulated kinase (ERK) is an intracellular signaling molecule involved in nucleus accumbens core (NACc)-mediated reconsolidation of Pavlovian cocaine memories. Here, we used a rodent model of drug context-elicited relapse to test the hypothesis that ERK would be similarly required for the reconsolidation of context-response-cocaine memories that underlie drug context-induced reinstatement of instrumental cocaine-seeking behavior, with a focus on the NACc and on the basolateral amygdala (BLA), another important locus for the reconsolidation of cocaine memories. We show that the mitogen-activated protein kinase (MEK)/ERK1/2 inhibitor, U0126 (1.0 µg/0.5 µl/hemisphere), microinfused bilaterally into the BLA--but not the NACc--immediately after brief re-exposure to a previously cocaine-paired context (that is, cocaine-memory reactivation), significantly attenuated subsequent drug context-induced cocaine seeking relative to vehicle (VEH). This effect in the BLA was associated with a transient inhibition of ERK1/2 phosphorylation, and it depended on memory reactivation given that U0126 administered following exposure to a novel context did not alter subsequent cocaine seeking. Furthermore, similar to U0126, baclofen+muscimol-induced (B+M; 106.8/5.7 ng/0.5 µl/hemisphere) neural inactivation of the NACc, following cocaine-memory reactivation, failed to alter subsequent cocaine seeking. These findings demonstrate that ERK activation in the BLA, but not the NACc, is required for the reconsolidation of context-response-cocaine associative memories. Together with prior research, these results suggest that contextual drug-memory reconsolidation in Pavlovian and instrumental settings involves distinct neuroanatomical mechanisms.

Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction.

Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine's pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE's potential. Innovative computational methods based on the protein's structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction.

Accelerating cocaine metabolism as an approach to the treatment of cocaine abuse and toxicity.

One pharmacokinetic approach to the treatment of cocaine abuse and toxicity involves the development of compounds that can be safely administered to humans and that accelerate the metabolism of cocaine to inactive components. Catalytic antibodies have been developed and shown to accelerate cocaine metabolism, but their catalytic efficiency for cocaine is relatively low. Mutations of human butyrylcholinesterase and a bacterial cocaine esterase found in the soil of coca plants have also been developed. These compounds accelerate cocaine metabolism and antagonize the behavioral and toxic effects of cocaine in animal models. Of these two approaches, the human butyrylcholinesterase mutants show the most immediate promise as they would not be expected to evoke an immune response in humans.

Role of dopamine D1 receptors in the activation of nucleus accumbens extracellular signal-regulated kinase (ERK) by cocaine-paired contextual cues.

Exposure to drug-paired cues can trigger addicts to relapse into drug seeking. Although the molecular mechanisms underlying cue-elicited cocaine seeking are incompletely understood, the protein kinase extracellular signal-regulated kinase (ERK) is known to have an important role. Psychostimulants and their associated cues can activate ERK in medium spiny neurons of the nucleus accumbens core (AcbC). These medium spiny neurons can be classified according to their projections (to ventral pallidum and/or substantia nigra) and by their mRNA expression. The present experiments were designed to determine which distinct set of AcbC projection neurons expresses phosphorylated ERK (pERK) in response to cocaine-paired contextual cues. Combined use of the retrograde label Flurogold with immunohistochemical staining of pERK was used to show that the AcbC pERK accompanying preference for cocaine-paired contexts occurs in both the accumbens (Acb)-nigral and Acb-pallidal projections. The gene expression characteristics of the neurons expressing pERK in response to cocaine-paired cues was further investigated using combined in situ hybridization and immunocytochemistry to show that AcbC pERK+ cells correspond to D1, but not preproenkephalin, mRNA+ cells. Furthermore, intra-AcbC infusion of the D1-antagonist SCH23390 attenuated cue-induced AcbC pERK expression. In aggregate, these results indicate that (i) the D1-expressing AcbC neurons evidence long-term plasticity related to drug-cue memories and (ii) local dopamine D1 receptors are necessary for the expression of cocaine-paired cue-induced pERK in these AcbC neurons.

Inhibition of neuronal nitric oxide synthase prevents alterations in medial prefrontal cortex excitability induced by repeated cocaine administration.

RATIONALE: The medial prefrontal cortex (mPFC), a forebrain region that regulates cognitive function and reward-motivated behaviors, has been implicated in the neuropathological mechanisms of drug addiction and withdrawal. In cocaine-abstinent human addicts, neuronal activity of the mPFC is increased in response to cocaine re-exposure or drug-associated cues. Additionally, repeated cocaine exposure alters the membrane properties and ion channel function of mPFC pyramidal neurons in drug-withdrawn rats, leading to an increased firing in response to excitatory stimuli. Nitric oxide (NO), a diffusible neuromodulator of neuronal excitability, may play a role in initiating and maintaining behavioral effects of psychostimulants. However, the role of NO in the mechanisms by which cocaine affects membrane excitability is not well clarified. OBJECTIVES: In this study, we attempted to determine whether inhibition of neuronal nitric oxide synthase (nNOS) altered the changes induced by repeated cocaine exposure and withdrawal. METHODS: Visualized whole-cell current clamp recordings in brain slices containing the mPFC of rats administered (once per day for 5 days) with either vehicle (10% Cremophor EL in saline 0.9%), cocaine (15 mg/kg, i.p.), or cocaine and the nNOS inhibitor 7-NI (50 mg/kg, i.p.) were employed. RESULTS: We found that nNOS inhibition prevented cocaine sensitization and the increased membrane excitability of pyramidal cells, evidenced by an increased number of evoked spikes and reductions in inward rectification observed after short-term withdrawal from cocaine. CONCLUSIONS: These findings suggest that NO plays an important role in chronic cocaine-induced deregulation of the mPFC activity that may contribute to the development of behavioral sensitization and cocaine withdrawal.

Striatal microRNA controls cocaine intake through CREB signalling.

Cocaine addiction is characterized by a gradual loss of control over drug use, but the molecular mechanisms regulating vulnerability to this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with a history of extended access to cocaine. Striatal miR-212 decreases responsiveness to the motivational properties of cocaine by markedly amplifying the stimulatory effects of the drug on cAMP response element binding protein (CREB) signalling. This action occurs through miR-212-enhanced Raf1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (transducer of regulated CREB; also known as CRTC). Our findings indicate that striatal miR-212 signalling has a key role in determining vulnerability to cocaine addiction, reveal new molecular regulators that control the complex actions of cocaine in brain reward circuitries and provide an entirely new direction for the development of anti-addiction therapeutics based on the modulation of noncoding RNAs.