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Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca 2+ -permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg 2+ block and Ca 2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca 2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain. SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction., (Copyright © 2021 the authors.)

Heightened sensation seeking is associated with an increased risk of substance use disorder in clinical populations. In rats, sensation seeking is often examined by measuring locomotor reactivity to a novel environment. So-called high responders (HR) acquire self-administration of psychostimulants more quickly and consume higher amounts of drug compared to low responder (LR) rats, indicating that the HR trait might confer a stronger addiction propensity. However, studies of addiction-like behaviors in HR vs LR rats have typically utilized self-administration paradigms that do not dissociate individual differences in the hedonic/reinforcing and motivational properties of a drug. Moreover, little attention has been given to whether HR rats are more susceptible to drug-access conditions that promote a state-dependent addiction phenotype. We report that on a behavioral economics task, HR rats have higher preferred brain-cocaine levels compared to LR rats but do not differ with respect to their demand elasticity for cocaine. In contrast, when tested on an intermittent access schedule of cocaine self-administration, which has been shown to promote several addiction-related endophenotypes, HR rats exhibit greater escalation of intake and more drastic reductions in cocaine demand elasticity. Together, these data indicate that the HR trait does not confer higher extant addiction behavior, but rather that this phenotype is associated with a propensity for addiction that remains dormant until it is actuated by intermittent drug intake. These findings reveal a 'trait' (HR) by 'state' (intermittent drug intake) interaction that produces a strong addiction-like phenotype. This article is part of the special issue on 'Vulnerabilities to Substance Abuse'., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cue exposure therapy (CET) reduces craving induced by drug-associated cues in individuals with substance use disorders. A preclinical model of CET, cue extinction, similarly reduces cue-induced cocaine seeking in rodent self-administration models; however, those models may not capture the habitual or compulsive aspects of drug use. Thus, the effectiveness of cue extinction was tested in male and female rats trained to self-administer cocaine using second-order (SO) or fixed-ratio (FR) schedules of reinforcement to facilitate dorsolateral striatum (DLS) dopamine-dependent or -independent response strategies, respectively. Cue extinction significantly reduced drug seeking in FR-trained rats, replicating prior results, but was ineffective in SO-trained rats. SO-trained rats also showed increased indices of glutamate signaling in the DLS relative to FR-trained rats, despite comparable levels of cocaine intake. Furthermore, in SO-trained rats, antagonism of AMPA receptors in the DLS rescued the efficacy of cue extinction to reduce drug seeking. Finally, the effectiveness of cue extinction was also revealed in SO-trained rats when they were taught to perform a new, non-habitual response for cocaine cue presentation. Overall, our findings indicate that habit-like drug seeking leads to plasticity in the DLS and behavior that is resistant to cue extinction, but that the effects of cue extinction are restored when DLS glutamatergic signaling is blocked. These results have implications for the effectiveness of clinical cue exposure therapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Drug addiction is a chronic disease defined by a complex set of characteristics, including loss of control over drug intake and persistent drug craving, which primarily affects a small percentage of people who try drugs. Although many models have been developed to study individual aspects of drug use, there is great translational value in having an animal model that encompasses multiple aspects of the human disease, including the variation in severity observed in humans. Here, we describe an intermittent access model of cocaine self-administration that produces a subset of rats that display many of the core features of addiction, including escalation of drug intake, a binge-like pattern of drug use, robust locomotor sensitization, and high levels of drug-seeking during cue-induced reinstatement. This group is compared with rats that have the same drug history but do not develop this pattern of drug-taking and drug-seeking, as well as rats that undergo a traditional continuous access paradigm. Finally, we observe that high levels of cocaine consumption produce long-term changes in intracellular calcium signaling in the dorsomedial striatum.

Rationale: Virtually all cocaine self-administration studies have used a "unit dose" as a reinforcing stimulus; the subject is a passive recipient of an experimenter-selected dose., Objectives: The present experiments examined the consequence of requiring the subject to actively determine the dose and speed of each injection., Methods: A two-lever procedure was used in which responding on a progressive ratio (PR) schedule provided access to cocaine on a hold down (HD) schedule. With HD, the pump is turned on for the duration that the lever is held down, thus the dose and speed of injection is determined by the behavior of the subject. The procedure allows for the evaluation of both drug taking and drug seeking responses., Results: The results were qualitatively different from PR self-administration studies using unit dose. The self-administered HD dose varied across the session; the self-administered dose was found to inversely correlate with drug levels at the time of access. Importantly, the 2 L-PR-HD procedure identified a subpopulation of subjects that showed extremes in both drug seeking and drug taking. Subjects at the top end of the distribution displayed unprecedented final ratios (> 900) and rapidly self-administered very large doses (> 1.4 mg; ~ 4.2 mg/kg). Manipulation of drug-taking variables (HD access duration and concentration of drug in the pump) showed that the immediacy of a cocaine bolus, not the duration of access, is the major determinant of drug seeking., Conclusions: Incorporating a consummatory response into a PR procedure provides a unique perspective on the interactions of drug-seeking and drug-taking.

3,4-Methylenedioxypyrovalerone (MDPV) is a new psychoactive substance (NPS) considered to be a cocaine-like psychostimulant. The substitution of an established illicit drug as cocaine with an NPS is a pattern of use reported among drug users. The aim of this study was to investigate the relationship between cocaine and MDPV in the reinstatement of the conditioned place preference (CPP) paradigm, in order to establish whether there is cross-reinstatement between the two psychostimulants. Four experimental groups of male OF1 mice were subjected to the CPP paradigm: MDPV-MDPV, Cocaine-Cocaine, Cocaine-MDPV, and MDPV-Cocaine. The first drug refers to the substance with which the animals were conditioned (cocaine 10 mg/kg or MDPV 2 mg/kg) and the s to the substance with which preference was reinstated. In parallel, G9a, ΔFosB, CB1 receptor, CDK5, Arc and c-Fos were determined in ventral striatum. MDPV induced CPP at doses from 1 to 4 mg/kg. Although 2 mg/kg MDPV induced a stronger psychostimulant effect than 10 mg/kg cocaine, both doses seemed to be equivalent in their rewarding properties. However, memories associated with MDPV required more time to be extinguished. MDPV and cocaine restore drug-seeking behavior with respect to each other, although relapse into drug-taking is always more pronounced with the conditioning drug. The fact that MDPV-treated mice show increased ΔFosB protein levels correlates with its longer extinction time and points to the activation of neuroplasticity mechanisms that persist for at least 12 days. Moreover, in these animals, a priming-dose of cocaine can trigger significant neuroplasticity, implying a high vulnerability to cocaine abuse., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier Inc.)

The greatest difficulty in treating cocaine addiction is the enormous rates of relapse, which occur despite immense negative consequences. Relapse risks are even greater in addicts with comorbid depression, perhaps because they use drugs to alleviate depressive symptoms. Only a few preclinical studies have examined this comorbidity, mostly exploring depressive-like effects following drug exposure. We examined rats from two different depression-like models: (a) chronic-mild-stress (CMS), which respond to antidepressant medications and (b) depressed-rat-line (DRL), a genetic model of selective breeding, which is less responsive to antidepressant medications. We tested addictive behaviors in a cocaine self-administration procedure, including the "conflict model," where drug-seeking and relapse encounter adverse consequences: an electrified grid in front of the drug-delivering lever. Following behavioral testing, we explored a potential association between behavioral outcomes and protein expression of brain-derived neurotrophic factor (BDNF). We found that DRL rats self-administer more cocaine compared with both CMS and controls, while CMS and control groups did not differ significantly. Notably, DRL but not CMS rats, displayed higher rates of relapse than controls, and expressed higher levels of BDNF in the prelimbic cortex (PLC). Potential translation of these results suggest that medication-resistant depressed patients tend to consume more drugs and are more susceptible to relapse. The increase in PLC BDNF levels is consistent with previous rat models of depression, and concomitantly, with its suggested role in promoting cocaine-seeking., (© 2019 Society for the Study of Addiction.)

We recently introduced an animal model to study incubation of drug craving after prolonged voluntary abstinence, mimicking the human condition of relapse after successful contingency management treatment. Here we studied the role of the nucleus accumbens (NAc) in this model. We trained rats to self-administer a palatable solution (sucrose 1% + maltodextrin 1%, 6 h/day, 6 days) and methamphetamine (6 h/day, 12 days). We then evaluated relapse to methamphetamine seeking after 1 and 15 days of voluntary abstinence, achieved via a discrete choice procedure between the palatable solution and methamphetamine (14 days). We used RNAscope in-situ hybridization to quantify the colabeling of the neuronal activity marker Fos, and dopamine Drd1- and Drd2-expressing medium spiny neurons (MSNs) in NAc core and shell during the incubation tests. Next, we determined the effect of pharmacological inactivation of NAc core and shell by either GABA A and GABA B agonists (muscimol + baclofen, 50 + 50 ng/side), Drd1-Drd2 antagonist (flupenthixol, 10 µg/side), or the selective Drd1 or Drd2 antagonists (SCH39166, 1.0 µg/side or raclopride, 1.0 µg/side) during the relapse tests. Incubated methamphetamine seeking after voluntary abstinence was associated with a selective increase of Fos expression in the NAc core, but not shell, and Fos was colabeled with both Drd1- and Drd2-MSNs. NAc core, but not shell, injections of muscimol + baclofen, flupenthixol, SCH39166, and raclopride reduced methamphetamine seeking after 15 days of abstinence. Together, our results suggest that dopamine transmission through Drd1 and Drd2 in NAc core is critical to the incubation of methamphetamine craving after voluntary abstinence.

Rationale: Cocaine use disorder (CUD) remains difficult to treat with no FDA-approved medications to reduce relapse. Antagonism of metabotropic glutamate receptor 5 (mGlu5) has been demonstrated to decrease cocaine-seeking but may also further compromise cognitive function in long-term cocaine users., Objectives: Here we assessed the effect of repeated administration of negative or positive allosteric modulators (NAM or PAM) of mGlu5 on both cognitive performance and (context+cue)-primed cocaine-seeking after prolonged abstinence (≥ 45 days)., Methods: Adult male Sprague-Dawley rats underwent 6 days of short-access (1 h/day) and 12 days of long-access (6 h/day) cocaine self-administration. Rats were then trained and tested in a delayed match-to-sample (DMS) task to establish baseline working memory performance over a 5-day block of testing. Next, rats received daily systemic administration of the mGlu5 NAM 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP; 3 mg/kg), the mGlu5 PAM 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB; 30 mg/kg) or vehicle prior to DMS testing during a block of 5 days, followed by a 5-day washout DMS testing block., Results: MTEP and CDPPB decreased drug-seeking in response to cocaine-associated cues after prolonged abstinence. However, repeated treatment with MTEP impaired working memory, while CDPPB had no effects on performance., Conclusions: These results emphasize the relevance of evaluating cognitive function within the context of investigating pharmacotherapies to treat CUD. Further research is needed to determine how two mechanistically different pharmacological compounds can exert the same behavioral effects to reduce cocaine-seeking.

Introduction: Cocaine dependence affects millions of individuals worldwide; however, there are no pharmacotherapeutic and/or diagnostic solutions. Recent evidence suggests a role for lipid signaling in the development and maintenance of addiction, highlighting the need to understand how lipid remodeling mediates neuroadaptation after cocaine exposure., Methods: This study utilized shotgun lipidomics to assess cocaine-induced lipid remodeling in rats using a novel behavioral regimen that incorporated multiple sessions of extinction training and reinstatement testing., Results: Mass spectrometric imaging demonstrated widespread decreases in phospholipid (PL) abundance throughout the brain, and high-spatial resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry indicated hippocampus-specific PL alterations following cocaine exposure. We analyzed the expression of genes involved in hippocampal lipid metabolism and observed region-specific regulation. In addition, we found that cocaine exposure differentially regulates mitochondrial biogenesis in the brain., Conclusions: This work presents a comprehensive lipidomic assessment of cocaine-induced lipid remodeling in the rat brain. Further, these findings indicate a potential interplay between CNS energetics and differential lipid regulation and suggest a role for cocaine in the maintenance of energy homeostasis., (© 2019 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.)

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction., (© 2018 Society for the Study of Addiction.)

Exposure to drug-associated cues evokes drug-craving and upregulates noradrenaline (NA) and dopamine (DA) system activity. Importantly, conditional stimulus-induced drug-seeking behavior depends particularly on phasic DA signaling downstream from the ventral tegmental area (VTA), a midbrain structure key for the regulation of cocaine seeking. In particular, the activity of the alpha 1 -adrenergic receptor (α 1 -AR), which has recently been hypothesized to modulate salience encoding, is capable of bidirectional regulation of VTA dopaminergic activity. Thus, the impact of the conditional stimuli (CSs) on drug-seeking behavior might involve α 1 -AR signaling in the VTA. To date, the role of VTA α 1 -ARs in regulating CS-induced cocaine seeking has not been studied. In male Sprague-Dawley rats, we found that intra-VTA terazosin, a selective α 1 -AR antagonist, attenuated CS-induced cocaine seeking in a novel context and under extinction conditions, as well as CS-induced reinstatement of cocaine seeking. In contrast, terazosin microinfusion in a dose that attenuated CS-induced cocaine seeking had no effects on CS-induced food seeking or stress (2 mg/kg yohimbine)-evoked reinstatement of cocaine seeking. The potential nonspecific effects (sedative, anxiogenic) of α 1 -AR blockade of the VTA were also measured in the open-field test. Finally, using immunostaining, we demonstrated dopamine β-hydroxylase (DBH)-positive afferents in the VTA of cocaine-abstinent rats, providing a neuroanatomical substrate for the α 1 -AR mechanism. These results demonstrated for the first time that NAergic signaling via VTA α 1 -ARs potently and selectively regulates CS-induced cocaine seeking. Our findings provide new neuronal mechanisms that regulate cocaine craving., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.

Kappa opioid receptor (KOPr) agonists have preclinical anti-cocaine and antinociceptive effects. However, adverse effects including dysphoria, aversion, sedation, anxiety and depression limit their clinical development. MP1104, an analogue of 3-iodobenzoyl naltrexamine, is a potent dual agonist at KOPr and delta opioid receptor (DOPr), with full agonist efficacy at both these receptors. In this study, we evaluate the ability of MP1104 to modulate cocaine-induced behaviors and side-effects preclinically. In male Sprague-Dawley rats trained to self-administer cocaine, MP1104 (0.3 and 1 mg/kg) reduced cocaine-primed reinstatement of drug-seeking behavior and caused significant downward shift of the dose-response curve in cocaine self-administration tests (0.3 and 0.6 mg/kg). The anti-cocaine effects exerted by MP1104 are in part due to increased dopamine (DA) uptake by the dopamine transporter (DAT) in the dorsal striatum (dStr) and nucleus accumbens (NAc). MP1104 (0.3 and 0.6 mg/kg) showed no significant anxiogenic effects in the elevated plus maze, pro-depressive effects in the forced swim test, or conditioned place aversion. Furthermore, pre-treatment with a DOPr antagonist, led to MP1104 producing aversive effects. This data suggests that the DOPr agonist actions of MP1104 attenuate the KOPr-mediated aversive effects of MP1104. The overall results from this study show that MP1104, modulates DA uptake in the dStr and NAc, and exerts potent anti-cocaine properties in self-administration tests with reduced side-effects compared to pure KOPr agonists. This data supports the therapeutic development of dual KOPr/DOPr agonists to reduce the side-effects of selective KOPr agonists. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recent evidence indicates that activation of glucagon-like peptide-1 (GLP-1) receptors reduces cocaine-mediated behaviors and cocaine-evoked dopamine release in the nucleus accumbens (NAc). However, no studies have examined the role of NAc GLP-1 receptors in the reinstatement of cocaine-seeking behavior, an animal model of relapse. Here, we show that systemic infusion of a behaviorally relevant dose of the GLP-1 receptor agonist exendin-4 penetrated the brain and localized with neurons and astrocytes in the NAc. Administration of exendin-4 directly into the NAc core and shell subregions significantly attenuated cocaine priming-induced reinstatement of drug-seeking behavior. These effects were not due to deficits in operant responding or suppression of locomotor activity as intra-accumbal exendin-4 administration had no effect on sucrose-seeking behavior. To determine the effects of GLP-1 receptor activation on neuronal excitability, exendin-4 was bath applied to ex vivo NAc slices from cocaine-experienced and saline-experienced rats following extinction of cocaine-taking behavior. Exendin-4 increased the frequency of action potential firing of NAc core and shell medium spiny neurons in cocaine-experienced rats while no effect was observed in saline controls. In contrast, exendin-4 did not affect the frequency or amplitude of spontaneous excitatory postsynaptic currents or alter the paired-pulse ratios of evoked excitatory postsynaptic currents. These effects were not associated with altered expression of GLP-1 receptors in the NAc following cocaine self-administration. Taken together, these findings indicate that increased activation of GLP-1 receptors in the NAc during cocaine abstinence increases intrinsic, but not synaptic, excitability of medium spiny neurons and is sufficient to reduce cocaine-seeking behavior., (© 2017 Society for the Study of Addiction.)

Exposure to prolonged, uncontrollable stress reduces reward-seeking behavior, resulting in anhedonia in neuropsychiatric disorders, such as posttraumatic stress disorder. However, it is unclear to what degree stressed subjects lose interest in rewards themselves or in reward-related cues that instigate reward-seeking behavior. In the present study, we investigated the effects of single prolonged stress (SPS) on cue-directed behavior in two different procedures: Pavlovian conditioned approach (PCA) and cue-induced reinstatement of cocaine-seeking. In Experiment 1, rats were exposed to SPS and tested for the acquisition of sign-tracking (cue-directed) and goal-tracking (reward-directed) behaviors during a PCA procedure. In Experiment 2, rats were exposed to SPS and tested for the expression of sign- and goal-tracking as well as cue-induced reinstatement of cocaine-seeking. Because dopaminergic activity in the nucleus accumbens is known to play a central role in many cue-directed behaviors, including both sign-tracking and cue-induced reinstatement, Experiment 3 used in vivo microdialysis to measure the effect of SPS on baseline and evoked dopamine levels in the nucleus accumbens. SPS decreased sign-tracking and increased goal-tracking during the acquisition of PCA behavior without affecting reward consumption. In addition, SPS decreased cue-induced reinstatement without affecting cocaine self-administration. Finally, SPS decreased evoked but not baseline levels of dopamine in the nucleus accumbens. These results suggest that SPS decreases the motivational, but not consummatory, aspects of reward-seeking behavior, which may result from long-term, SPS-induced reductions in dopamine release in the nucleus accumbens., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recreational drug use peaks during adolescence. Our research with adolescent vs adult male rats, however, shows that rats taking cocaine as adolescents have lower levels of cue-induced reinstatement of drug-seeking than adults, despite similar levels of intravenous (i.v.) cocaine self-administration. Lower rates of reinstatement in younger rats could be explained by higher levels of brain plasticity. Two neuroplasticity-related genes, activity-regulated cytoskeletal-associated gene (Arc) and brain-derived neurotrophic factor (Bdnf), influence cocaine self-administration and cue-induced reinstatement. We tested whether reinstatement of cocaine seeking correlates with expression of these genes in reinforcement-related brain regions. Adolescent and adult male rats (postnatal day 35 or 83-95 at start) were allowed to acquire lever-pressing maintained by i.v. infusions of cocaine in daily 2-h sessions over 13 days. At one of three experimental time points, rats were sacrificed and tissue collected to analyze Arc and Bdnf mRNA by in situ hybridization in the entire medial prefrontal cortex and entire nucleus accumbens, as well as relevant subregions: prelimbic cortex, infralimbic cortex, and nucleus accumbens core and shell. Despite taking similar amounts of cocaine, adolescents reinstated less than adults. Gene expression was most notable in the prelimbic cortex, was generally higher in adolescent-onset groups, and was higher with longer abstinence. These data partially support the hypothesis that higher levels of Arc and/or Bdnf gene expression in reinforcement-related brain regions of younger animals contribute to lower rates of extinction responding and/or reinstatement. Future studies should include mechanistic analysis of Arc, Bdnf, and their signaling pathways in age-dependent effects of cocaine., (© 2018 Society for the Study of Addiction.)

Experience-dependent synaptic plasticity is an important component of both learning and motivational disturbances found in addicted individuals. Here, we investigated the role of cocaine experience-dependent plasticity at excitatory synapses in the nucleus accumbens shell (NAcSh) in relapse-related behavior in mice with a history of volitional cocaine self-administration. Using an extinction/reinstatement paradigm of cocaine-seeking behavior, we demonstrate that cocaine-experienced mice with extinguished cocaine-seeking behavior show potentiation of synaptic strength at excitatory inputs onto NAcSh medium spiny neurons (MSNs). Conversely, we found that exposure to various distinct types of reinstating stimuli (cocaine, cocaine-associated cues, yohimbine "stress") after extinction can produce a relative depotentiation of NAcSh synapses that is strongly associated with the magnitude of cocaine-seeking behavior exhibited in response to these challenges. Furthermore, we show that these effects are due to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-specific mechanisms that differ depending on the nature and context of the reinstatement-inducing stimuli. Together, our findings identify common themes as well as differential mechanisms that are likely important for the ability of diverse environmental stimuli to drive relapse to addictive-like cocaine-seeking behavior., (Copyright © 2018. Published by Elsevier Ltd.)

There are currently no approved medications to effectively counteract the effects of methamphetamine (METH), reduce its abuse and prolong abstinence from it. Data accumulated in recent years have shown that a range of N-substituted benztropine (BZT) analogues possesses psychopharmacological features consistent with those of a potential replacement or "substitute" treatment for stimulant addiction. On the other hand, the evidence that antidepressant therapy may effectively prevent relapse to stimulant seeking is controversial. Here, we compared in rats the ability of the BZT analogue and high affinity dopamine (DA) reuptake inhibitor, JHW-007, and the antidepressant, trazodone, administered during extinction sessions after chronic METH self-administration, to alter METH-primed reinstatement of drug seeking. The data showed that trazodone produced paradoxical effects on lever pressing during extinction of METH self-administration, decreasing active, but increasing inactive, lever pressing. JHW-007 did not have any observable effects on extinction training. Importantly, JHW-007 significantly attenuated METH-primed reinstatement, whereas trazodone enhanced it. These findings lend support to the candidacy of selective DA uptake blockers, such as JHW-007, as potential treatments for METH addiction, but not to the use of antidepressant medication as a single therapeutic approach for relapse prevention., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Exposure to drug-associated cues evokes drug-seeking behavior and is regarded as a major cause of relapse. Conditional stimulus upregulates noradrenaline (NA) system activity, but the drug-seeking behavior depends particularly on phasic dopamine signaling downstream from the ventral tegmental area (VTA). The VTA dopamine-ergic activity is regulated via the signaling of alpha 1 -adrenergic and alpha 2 -adrenergic receptors (α 1 -ARs and α 2 -ARs); thus, the impact of the conditional stimulus on drug-seeking behavior might involve NAergic signaling in the VTA. To date, the role of VTA ARs in regulating cocaine seeking was not studied. We found that cocaine seeking under extinction conditions in male Sprague-Dawley rats was attenuated by intra-VTA prazosin or terazosin-two selective α 1 -AR antagonists. In contrast, cocaine seeking was facilitated by intra-VTA administration of the selective α 1 -AR agonist phenylephrine as well as α 2 -AR antagonist RX 821002, whereas the selective β-AR antagonist propranolol had no effects. In addition, blockade of α 1 -AR in the VTA prevented α 2 -AR antagonist-induced enhancement of cocaine seeking. Importantly, the potential non-specific effects of the VTA AR blockade on cocaine seeking could be excluded, because none of the AR antagonists influenced sucrose seeking under extinction conditions or locomotor activity in the open field test. These results demonstrate that NAergic signaling potently and selectively regulates cocaine seeking during early cocaine withdrawal via VTA α 1 -AR and α 2 -AR but not β-AR. Our findings provide new insight into the NAergic mechanisms that underlie cocaine craving., (© 2017 Society for the Study of Addiction.)

Cocaine self-administration in rats results in dysfunctional neuroadaptations in the prelimbic (PrL) cortex during early abstinence. Central to these adaptations is decreased phospho-extracellular signal-regulated kinase (p-ERK), which plays a key role in cocaine seeking. Normalizing ERK phosphorylation in the PrL cortex immediately after cocaine self-administration decreases subsequent cocaine seeking. The disturbance in ERK phosphorylation is accompanied by decreased phosphorylation of striatal-enriched protein tyrosine phosphatase (STEP), indicating increased STEP activity. STEP is a well-recognized ERK phosphatase but whether STEP activation during early abstinence mediates the decrease in p-ERK and is involved in relapse is unknown. Here, we show that a single intra-PrL cortical microinfusion of the selective STEP inhibitor, TC-2153, immediately after self-administration suppressed post-abstinence context-induced relapse under extinction conditions and cue-induced reinstatement, but not cocaine prime-induced drug seeking or sucrose seeking. Moreover, an intra-PrL cortical TC-2153 microinfusion immediately after self-administration prevented the cocaine-induced decrease in p-ERK within the PrL cortex during early abstinence. Interestingly, a systemic TC-2153 injection at the same timepoint failed to suppress post-abstinence context-induced relapse or cue-induced reinstatement, but did suppress cocaine prime-induced reinstatement. These data indicate that the STEP-induced ERK dephosphorylation in the PrL cortex during early abstinence is a critical neuroadaptation that promotes relapse to cocaine seeking and that systemic versus intra-PrL cortical inhibition of STEP during early abstinence differentially suppresses cocaine seeking., (© 2017 Society for the Study of Addiction.)

Cocaine use disorder is a chronic relapsing condition characterized by compulsive drug seeking and taking even after prolonged abstinence periods. Subsequent exposure to drug-associated cues can promote intense craving and lead to relapse in abstinent humans and rodent models. The responsiveness to these cocaine-related cues, or 'cue reactivity', can trigger relapse and cocaine-seeking behaviors; cue reactivity is measurable in cocaine-dependent humans as well as rodent models. Cue reactivity is thought to be predictive of cocaine craving and relapse. Here we report that PPARγ agonism during abstinence from cocaine self-administration reduced previously active lever pressing in Sprague Dawley rats during cue-reactivity tests, while administration of the PPARγ antagonist, GW9662, reversed this effect. PPARγ agonism also normalized nuclear ERK activity in the medial prefrontal cortex and hippocampus which was reversed with GW9662. Our results support the utility of PPARγ agonism as a relapse prevention strategy to maintain abstinence in the presence of cocaine-associated cues., (© 2016 Society for the Study of Addiction.)

d-Cycloserine (DCS), a partial NMDA receptor agonist, has been proposed as a cognitive enhancer to facilitate the extinction of drug-related memories. However, it is unknown whether there are individual differences in the efficacy of DCS. Here, we set out to investigate the influence of serotonin transporter (5-HTT) genotype on DCS treatment outcome and the underlying neural mechanism. To that end, we first determined the mRNA levels of several NMDA receptor subunits and observed a reduction in NR1/NR2C receptors in the ventromedial prefrontal cortex and nucleus accumbens of 5-HTT -/- compared with 5-HTT +/+ rats. Based on this finding, we hypothesized a lower sensitivity to DCS in the 5-HTT -/- rats. To test this, rats were trained in a cocaine-induced conditioned place preference (CPP) paradigm. A significant extinction of CPP was observed in 5-HTT +/+ rats receiving 1 mg/kg i.v. DCS, while a similar effect was found in the 5-HTT -/- rats only after 5 mg/kg. Following CPP, we tested if DCS were able to reduce FosB/∆FosB protein expression, a molecular switch for cocaine-seeking behaviour. We observed an overall lower number of FosB/∆FosB positive cells in 5-HTT -/- ventromedial prefrontal cortex and amygdala and an overall effect of DCS treatment on the number of positive cells in the nucleus accumbens. In conclusion, in this study, we show that the dosing of DCS to facilitate the extinction of cocaine-seeking behaviour is, at least partially, determined by 5-HTT genotype., (© 2016 Society for the Study of Addiction.)

Evidence suggests that the infralimbic cortex (IL), a subregion of the ventromedial prefrontal cortex (vmPFC), suppresses cocaine-seeking behavior in a self-administration paradigm, whereas the more anterior vmPFC subregion, the medial orbitofrontal cortex (mOFC), has received very little attention in this regard. Despite the established dopaminergic innervation of the vmPFC, whether dopamine receptor blockade in each subregion alters the reinstatement of cocaine seeking is unclear. To address this issue, male Sprague-Dawley rats underwent 2 weeks of cocaine self-administration, followed by extinction training and reinstatement testing. Immediately prior to each reinstatement test, rats received microinjections of the D1 receptor antagonist SCH 23390, the D2 receptor antagonist sulpiride or their respective vehicles. D1 receptor blockade in the IL reduced cued reinstatement but had no effect on cocaine prime and cue + cocaine-prime reinstatement, whereas D2 receptor blockade in the IL had no effect on reinstatement. For the mOFC, however, D1 receptor blockade reduced cocaine seeking in all reinstatement types, whereas blocking D2 receptors in the mOFC had no effect on any form of cocaine seeking. These findings suggest different roles for D1 receptors in the IL versus the mOFC in regulating cocaine-seeking behavior. Moreover, even as previous work indicates that IL inactivation does not affect reinstatement but, rather, induces cocaine seeking during extinction, the present findings suggest that dopamine receptor activation in the IL is necessary for cocaine seeking under some circumstances., (© 2016 Society for the Study of Addiction.)

The prelimbic prefrontal cortex is necessary for associating actions with their consequences, enabling goal-directed decision making. We find that the strength of action-outcome conditioning correlates with dendritic spine density in prelimbic cortex, suggesting that new action-outcome learning involves dendritic spine plasticity. To test this, we inhibited the cytoskeletal regulatory factor Rho kinase. We find that the inhibitor fasudil enhances action-outcome memory, resulting in goal-directed behavior in mice that would otherwise express stimulus-response habits. Fasudil transiently reduces prelimbic cortical dendritic spine densities during a period of presumed memory consolidation, but only when paired with new learning. Fasudil also blocks habitual responding for cocaine, an effect that persists over time, across multiple contexts, and depends on actin polymerization. We suggest that Rho kinase inhibition promotes goal-oriented action selection by augmenting the plasticity of prelimbic cortical dendritic spines during the formation of new action-outcome memories.

Manipulations of memory reconsolidation can interfere with the ability of a drug-paired cue to drive drug-seeking behavior. However, the typical reconsolidation paradigm that reactivates the memory through the presentation of the cue (conditioned stimulus (CS)) only interferes with the memory of the reactivated CS while leaving other drug-paired CSs intact and able to continue driving drug-seeking behavior. Here, we used a novel unconditioned-stimulus (US) reactivation paradigm to interfere with the ability of multiple cues to drive drug-seeking behavior after just one reactivation and treatment session. Rats were trained to self-administer cocaine, during which time each active lever press resulted in an i.v. cocaine infusion paired with one of two cues that alternated within each session. The drug memory was later reactivated with either i.v. or i.p. cocaine presentation in the absence of any cue. The histone acetyltransferase (HAT) inhibitor garcinol or vehicle was injected following US reactivation to impair reconsolidation. Rats were later tested on cue-induced reinstatement to both cues. Garcinol administered after either i.v. or i.p. cocaine reactivation significantly decreased cue-induced reinstatement to both cues, indicative of reconsolidation impairment. In addition, garcinol administered in the absence of reconsolidation or at a 6 h delay when the memory should be restabilized had no effect on reinstatement, further suggesting that garcinol's effects on reinstatement are through reconsolidation-based mechanisms. Our results demonstrate that a US-reactivation paradigm may be preferable to traditional CS-reactivation paradigms for treating disorders that involve multiple CS-US associations and support investigations of garcinol as a therapeutic pharmacological agent.

In humans, reward cues, including drug cues in individuals experiencing addiction, are especially effective in biasing attention towards them, so much so they can disrupt ongoing task performance. It is not known, however, whether this happens in rats. To address this question, we developed a behavioral paradigm to assess the capacity of an auditory drug (cocaine) cue to evoke cocaine-seeking behavior, thus distracting thirsty rats from performing a well-learned sustained attention task (SAT) to obtain a water reward. First, it was determined that an auditory cocaine cue (tone-CS) reinstated drug-seeking equally in sign-trackers (STs) and goal-trackers (GTs), which otherwise vary in the propensity to attribute incentive salience to a localizable drug cue. Next, we tested the ability of an auditory cocaine cue to disrupt performance on the SAT in STs and GTs. Rats were trained to self-administer cocaine intravenously using an Intermittent Access self-administration procedure known to produce a progressive increase in motivation for cocaine, escalation of intake, and strong discriminative stimulus control over drug-seeking behavior. When presented alone, the auditory discriminative stimulus elicited cocaine-seeking behavior while rats were performing the SAT, but it was not sufficiently disruptive to impair SAT performance. In contrast, if cocaine was available in the presence of the cue, or when administered non-contingently, SAT performance was severely disrupted. We suggest that performance on a relatively automatic, stimulus-driven task, such as the basic version of the SAT used here, may be difficult to disrupt with a drug cue alone. A task that requires more top-down cognitive control may be needed., Competing Interests: Conflicts of Interest: None declared., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Rationale: Cocaine produces significant aversive/anxiogenic actions whose underlying neurobiology remains unclear. A possible substrate contributing to these actions is the serotonergic (5-HT) pathway projecting from the dorsal raphé (DRN) to regions of the extended amygdala, including the bed nucleus of the stria terminalis (BNST) which have been implicated in the production of anxiogenic states., Objectives: The present study examined the contribution of 5-HT signaling within the BNST to the anxiogenic effects of cocaine as measured in a runway model of drug self-administration., Methods: Male Sprague-Dawley rats were fitted with bilateral infusion cannula aimed at the BNST and then trained to traverse a straight alley once a day for a single 1 mg/kg i.v. cocaine infusion delivered upon goal-box entry on each of 16 consecutive days/trials. Intracranial infusions of CP 94,253 (0, 0.25, 0.5, or 1.0 μg/side) were administered to inhibit local 5-HT release via activation of 5-HT 1B autoreceptors. To confirm receptor specificity, the effects of this treatment were then challenged by co-administration of the selective 5-HT 1B antagonist NAS-181., Results: Intra-BNST infusions of the 5-HT 1B autoreceptor agonist attenuated the anxiogenic effects of cocaine as reflected by a decrease in runway approach-avoidance conflict behavior. This effect was reversed by the 5-HT 1B antagonist. Neither start latencies (a measure of the subject's motivation to seek cocaine) nor spontaneous locomotor activity (an index of motoric capacity) were altered by either treatment., Conclusions: Inhibition of 5-HT 1B signaling within the BNST selectively attenuated the anxiogenic effects of cocaine, while leaving unaffected the positive incentive properties of the drug.

High rates of relapse after prolonged abstinence are often triggered by exposure to drug-associated cues that induce drug craving. Incubation of drug craving is a phenomenon that consists of time-dependent increases in cue-induced drug craving during withdrawal. Plasticity mechanisms in the nucleus accumbens (NAc) underlie drug-seeking responses and involve changes in excitatory synaptic transmission's efficacy. In particular, the prefrontal cortex (PFC) glutamatergic input to the NAc core has been well characterized regarding cocaine-evoked plasticity following non-contingent versus contingent exposure to cocaine or alternatively after protracted abstinence. Still, the synaptic strength during the course of withdrawal compared to drug-naïve condition is unknown, since electrophysiological characterizations are mainly performed in brain slices or focus on distinct time points during cocaine-evoked plasticity in vivo. Here we used an incubation paradigm, in which rats had extended accessed to cocaine self-administration, and underwent cue-induced reinstatement at withdrawal day 1 and 30. Longitudinal in vivo field potential recordings in awake rats showed that chronic contingent exposure to cocaine strengthened the prelimbic PFC to NAc core pathway when compared to pre-cocaine condition. This strengthening was associated with decreased paired-pulse ratios (PPR), indicative of presynaptic enhancement of glutamate release, which persisted throughout withdrawal. Moreover, both field potential increase and PPR reduction after chronic cocaine exposure correlated with the number of cocaine infusions received during training. The present results together with previous findings of withdrawal-dependent postsynaptic enhancement of the PFC-NAc core pathway, suggest an additional presynaptic strengthening that is initiated during self-administration and maintained throughout abstinence in drug-seeking rats. These cocaine-driven neuroadaptations may provide a neural substrate for maladaptive processing of cues that can ultimately trigger craving and relapse., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rationale: Repeated intermittent exposure to ketamine has rapid and long-lasting antidepressant effects, but the abuse potential has only been assessed at high doses. Furthermore, while females are more susceptible to depression and more sensitive to ketamine's antidepressant-like effects, the abuse potential for ketamine in females is unknown., Objectives: The objectives of this study are to determine the reinforcing properties of low-dose intermittent ketamine in adult rats of both sexes and determine whether cycling gonadal hormones influence females' response to ketamine. In male rats, we also aimed to determine whether reinstatement to intermittent ketamine is comparable to intermittent cocaine., Methods: Male rats intravenously self-administered cocaine (0.75 mg/kg/infusion) or ketamine (0.1 mg/kg/infusion) once every fourth day, while intact cycling female rats self-administered ketamine only during preidentified stages of their 4-day estrus cycle, when gonadal hormones are either high (proestrus) or low (diestrus). After acquiring self-administration, rats underwent daily extinction training followed by cue-primed and drug-primed reinstatement to assess drug-seeking behavior., Results: Diestrus-trained females fail to maintain ketamine self-administration and did not display reinstatement to ketamine-paired cues. Males and proestrus-trained females reinstated to ketamine-paired cues. Ketamine-primed reinstatement was dependent on simultaneous cue presentation. Male rats reinstated to cocaine priming independent of cue presentation., Conclusion: These findings indicate that females's responsivity to this dose of ketamine depends on stage of cycle, as only proestrus-trained females and males respond to ketamine's reinforcing effects under this treatment paradigm.

Rationale: Contemporary animal models of cocaine addiction focus on increasing the amount of drug consumption to produce addiction-like behavior. However, another critical factor is the temporal pattern of consumption, which in humans is characterized by intermittency, both within and between bouts of use., Objective: To model this, we combined prolonged access to cocaine (∼70 days in total) with an intermittent access (IntA) self-administration procedure and used behavioral economic indicators to quantify changes in motivation for cocaine., Results: IntA produced escalation of intake, a progressive increase in cocaine demand (incentive-sensitization), and robust drug- and cue-induced reinstatement of drug-seeking behavior. We also asked whether rats that vary in their propensity to attribute incentive salience to reward cues (sign-trackers [STs] vs. goal-trackers [GTs]) vary in the development of addiction-like behavior. Although STs were more motivated to take cocaine after limited drug experience, after IntA, STs and GTs no longer differed on any measure of addiction-like behavior., Conclusions: Exposure to large quantities of cocaine is not necessary for escalation of intake, incentive-sensitization, or other addiction-like behaviors (IntA results in far less total cocaine consumption than 'long access' procedures). Also, the ST phenotype may increase susceptibility to addiction, not because STs are inherently susceptible to incentive-sensitization (perhaps all individuals are at risk), but because this phenotype promotes continued drug use, subjecting them to incentive-sensitization. Thus, the pharmacokinetics associated with the IntA procedure are especially effective in producing a number of addiction-like behaviors and may be valuable for studying associated neuroadaptations and for assessing individual variation in vulnerability.

Rationale: Chronic exposure to drugs of abuse changes glutamatergic transmission in human addicts and animal models. N-acetylcysteine (NAC) is a cysteine prodrug that indirectly activates cysteine-glutamate antiporters. In the extrasynaptic space, NAC restores basal glutamate levels during drug abstinence and normalizes increased glutamatergic tone in rats during reinstatement to drugs of abuse. In initial clinical trials, repeated NAC administration seems to be promising for reduced craving in cocaine addicts., Objective: In this study, NAC-amide, called AD4 or NACA, was examined in intravenous cocaine self-administration and extinction/reinstatement procedures in rats. We investigated the behavioral effects of AD4 in the olfactory bulbectomized (OBX) rats, considered an animal model of depression. Finally, we tested rats injected with AD4 or NAC during 10-daily extinction training sessions to examine subsequent cocaine seeking., Results: AD4 (25-75 mg kg(-1)) given acutely did not alter the rewarding effects of cocaine in OBX rats and sham-operated controls. However, at 6.25-50 mg kg(-1), AD4 decreased dose-dependently cocaine seeking and relapse triggered by cocaine priming or drug-associated conditioned cues in both phenotypes. Furthermore, repeated treatment with AD4 (25 mg kg(-1)) or NAC (100 mg kg(-1)) during daily extinction trials reduced reinstatement of drug-seeking behavior in sham-operated controls. In the OBX rats only, AD4 effectively blocked cocaine-seeking behavior., Conclusions: Our results demonstrate that AD4 is effective at blocking cocaine-seeking behavior, highlighting its potential clinical use toward cocaine use disorder.

Rationale: The glutamatergic system plays a key role in the maintenance of drug use and development of drug-related conditioned behaviours. In particular, hyper-glutamatergic activity and N-methyl-D-aspartate receptor (NMDAR) activation may drive drug craving and relapse. Inhibition of kynurenine-3-monooxygenase (KMO) shifts the metabolic kynurenine pathway towards production of kynurenic acid, which leads to a reduction of glutamatergic/NMDAR activity via different mechanisms., Objectives: In this study, we investigated whether drug-seeking and relapse behaviour could be modified by the metabolic shift of endogenous kynurenine pathway., Methods: An inhibitor of kynurenine-3-monooxygenase (KMO) Ro61-8048 (4 and 40 mg/kg) and its prodrug JM6 (100 and 200 mg/kg) were tested in two behavioural rat models for drug seeking and relapse-the alcohol deprivation effect (ADE) model in long-term alcohol-drinking rats and the model of cue-induced reinstatement of alcohol- and cocaine-seeking behaviour., Results: Our results show that relapse-like alcohol drinking during the ADE was abolished by repeated intraperitoneal administration of Ro61-8048 and significantly reduced by its oral prodrug JM6. Cue-induced reinstatement of both alcohol- and cocaine-seeking behaviour was also abolished by administration of Ro61-8048., Conclusions: Pharmacological enhancement of endogenous kynurenic acid levels provides a novel treatment strategy to interfere with glutamatergic/NMDAR activity as well as with craving and relapse in alcohol-dependent patients and drug addicts.

Rationale: Caffeine is one of the psychoactive substances most widely used as an adulterant in illicit drugs, such as cocaine. Animal studies have demonstrated that caffeine is able to potentiate several cocaine actions, although the enhancement of the cocaine reinforcing property by caffeine is less reported, and the results depend on the paradigms and experimental protocols used., Objectives: We examined the ability of caffeine to enhance the motivational and rewarding properties of cocaine using an intravenous self-administration paradigm in rats. Additionally, the role of caffeine as a primer cue during extinction was evaluated., Methods: In naïve rats, we assessed (1) the ability of the cocaine (0.250-0.125 mg/kg/infusion) and caffeine (0.125-0.0625 mg/kg/infusion) combination to maintain self-administration in fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement compared with cocaine or caffeine alone and (2) the effect of caffeine (0.0625 mg/kg/infusion) in the maintenance of responding in the animals exposed to the combination of the drugs during cocaine extinction., Results: Cocaine combined with caffeine and cocaine alone was self-administered on FR and PR schedules of reinforcement. Interestingly, the breaking point determined for the cocaine + caffeine group was significantly higher than the cocaine group. Moreover, caffeine, that by itself did not maintain self-administration behavior in naïve rats, maintained drug-seeking behavior of rats previously exposed to combinations of cocaine + caffeine., Conclusions: Caffeine enhances the reinforcing effects of cocaine and its motivational value. Our results highlight the role of active adulterants commonly used in cocaine-based illicit street drugs.

Rationale: Impulsive choice, or an inability to delay immediate gratification, has been strongly linked to the development and persistence of drug abuse. Indeed, delaying drug use itself may underlie drug addiction and relapse. Thus, employing treatments that are efficacious in reducing impulsive choice (atomoxetine; ATO) or drug-seeking behavior (progesterone; PRO) may be an effective means of treating drug addiction., Objective: The current study assessed sex differences in the effects of PRO, ATO, and their combination in a delay discounting paradigm for cocaine and for sucrose pellets., Method: Male and female rats chose between a small-immediate or a large-delayed (0, 7.5, 15, 30, 60 s) outcome in an impulsive choice procedure for sucrose pellets (1 vs. 3 pellets) or for iv cocaine infusions (0.3 vs. 0.9 mg/kg). Following baseline assessment of impulsive choice, rats received daily treatment of vehicle (VEH), PRO (0.5 mg/kg), ATO (1.5 mg/kg), or a combination (PRO + ATO) until a second assessment of impulsive choice was determined., Results: Compared to the VEH group, females were less impulsive for cocaine following PRO or the PRO + ATO combined treatment, whereas males were less impulsive for cocaine following ATO. No treatment effects were observed on impulsive choice for sucrose pellets., Conclusions: The present results indicate that impulsive choice for cocaine is reduced by PRO in females and by ATO in males. These findings suggest both treatments may be an effective intervention in treating cocaine abuse, but that their effectiveness differs by sex.

Background: Neuroimaging studies in psychostimulant addicts have reported functional neural activity changes in brain regions involved in relapse. However, the difference between the effects of the psychostimulants methamphetamine and cocaine on neuronal activity in a similar setting not been clarified. Since studies in humans are limited by the inability to study the initial impact of psychostimulant drugs, we addressed this issue in a rat model., Objective: Here, we report methamphetamine and cocaine-induced blood-oxygen-level dependent (BOLD) signal change using functional magnetic resonance imaging (fMRI) in rats receiving drug for the first time during the imaging session., Methods: Twenty-three male Long Evans rats underwent fMRI imaging and received an intravenous infusion of methamphetamine, cocaine, or saline. Anatomical and pharmacological fMRI (pfMRI) were performed on a 7T BioSpec dedicated research MR scanner under isoflurane gas (1.5-2%). After collecting baseline data for 10min, rats received drug over the next 10min for a total 40min scan time. Data were then preprocessed and statistically analyzed in anatomically defined regions of interest (ROIs) that have been implicated in persistent drug seeking and relapse., Results: Methamphetamine during the imaging session resulted in a sustained negative BOLD signal change in key regions of the relapse circuit, except for the prefrontal cortex. In contrast, cocaine evoked a positive or unchanged BOLD signal in these same regions. In all of the investigated ROIs, there were no changes in BOLD signal following saline., Conclusion: Acute methamphetamine and cocaine have distinct patterns of functional activity as measured by pfMRI., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Locus coeruleus norepinephrine (LC-NE) and corticotropin releasing factor (CRF) neurons are involved in stress responses, including stress's ability to drive drug relapse. Previous animal studies indicate that female rats exhibit greater drug seeking than male rats during initial drug abstinence. Moreover, females are more sensitive to the effect of stress to drive drug seeking than males. Finally, LC-NE neurons are more sensitive to CRF in females compared to males. We hypothesized that increased drug seeking in females on extinction day one (ED1) is due to increased response to the stress of early withdrawal and is dependent upon the increased response of LC in females to CRF. We predicted that LC-NE neurons would exhibit Fos activation on ED1, and that blocking CRF1 signaling would decrease drug seeking on ED1 measured by responding on an active lever previously associated with cocaine self- administration. After chronic cocaine self-administration, female and male rats underwent a test for initial extinction responding by measuring lever pressing in the absence of cocaine. Prior to this Extinction Day 1 (ED1) session, rats were injected with vehicle or the selective CRF1 antagonist (CP) to measure effects of CRF antagonism on drug seeking during early abstinence. ED1 increased corticosterone in female rats, in proportion to lever responding in male and female, indicating that ED1 was stressful. Pretreatment with CP decreased cocaine seeking on ED1 more effectively in female compared to male rats. This increase in responding was associated with an increase in activation of LC NE neurons. Together, these findings indicate that stress, and signaling at CRF receptors in LC, may be involved in the increased drug seeking during initial abstinence.

Intensification of craving elicited by drug-associated cues during abstinence occurs over time in human cocaine users while elevation of cue reactivity ("incubation") is observed in rats exposed to extended forced abstinence from cocaine self-administration. Incubation in rodents has been linked to time-dependent neuronal plasticity in the medial prefrontal cortex (mPFC). We tested the hypothesis that incubation of cue reactivity during abstinence from cocaine self-administration is accompanied by lower potency and/or efficacy of the selective serotonin (5-HT) 5-HT2C​ receptor (5-HT2CR) agonist WAY163909 to suppress cue reactivity and a shift in the subcellular localization profile of the mPFC 5-HT2CR protein. We observed incubation of cue reactivity (measured as lever presses reinforced by the discrete cue complex) between Day 1 and Day 30 of forced abstinence from cocaine relative to sucrose self-administration. Pharmacological and biochemical analyses revealed that the potency of the selective 5-HT2CR agonist WAY163909 to suppress cue reactivity, the expression of synaptosomal 5-HT2CR protein in the mPFC, and the membrane to cytoplasmic expression of the 5-HT2CR in mPFC were lower on Day 30 vs. Day 1 of forced abstinence from cocaine self-administration. Incubation of cue reactivity assessed during forced abstinence from sucrose self-administration did not associate with 5-HT2CR protein expression in the mPFC. Collectively, these outcomes are the first indication that neuroadaptations in the 5-HT2CR system may contribute to incubation of cocaine cue reactivity., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Relapse following removal of an alternative source of reinforcement introduced during extinction of a target behavior is called resurgence. This form of relapse may be related to relapse of drug taking following loss of alternative non-drug reinforcement in human populations. Laboratory investigations of factors mediating resurgence with food-maintained behavior suggest higher rates of alternative reinforcement produce faster suppression of target behavior but paradoxically generate more relapse when alternative reinforcement is discontinued. At present, it is unknown if a similar effect occurs when target behavior is maintained by drug reinforcement and the alternative is a non-drug reinforcer. In the present experiment three groups of rats were trained to lever press for infusions of cocaine during baseline. Next, during treatment, cocaine reinforcement was suspended and an alternative response was reinforced with either high-rate, low-rate, or no alternative food reinforcement. Finally, all reinforcement was suspended to test for relapse of cocaine seeking. Higher rate alternative reinforcement produced faster elimination of cocaine seeking than lower rates or extinction alone, but when treatment was suspended resurgence of cocaine seeking occurred following only high-rate alternative reinforcement. Thus, although higher rate alternative reinforcement appears to more effectively suppress drug seeking, should it become unavailable, it can have the unfortunate effect of increasing relapse., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Adolescent rats take cocaine more readily than adults, are more sensitive to lower doses of the drug and work harder for it. It remains unknown if adolescent-onset of cocaine use has long-term consequences on adult relapse liability. Therefore, we tested if self-administering cocaine during adolescence impacts subsequent stress-induced reinstatement to cocaine seeking and taking, after a prolonged drug-free period. Adolescent (~P42) or adult (P88) rats self-administered cocaine (0.6 or 1.2 mg/kg/infusion) for 7 or 10 days. Then, they underwent a prolonged drug-free period (21-40 days), after which they were tested for reinstatement of cocaine-seeking (i.e. responding in the absence of cocaine) induced by the stress hormone corticosterone, the pharmacological stressor yohimbine or electric footshock. Studies employed either single extinction session (within-session extinction/reinstatement) or repeated extinction prior to reinstatement (between-session extinction/reinstatement). Finally, in a separate set of experiments, rats underwent a prolonged drug-free period (~40 days) and were then allowed to self-administer cocaine again, using progressive-ratio procedures that appraise the reinforcing efficacy of cocaine. Rats with adolescent-onset of cocaine use showed greater stress-induced reinstatement of cocaine seeking than rats with adult-onset of cocaine use. This was observed across conditions, providing external validity to these results. Groups did not differ on drug taking in progressive-ratio tests. Our studies indicate that experiencing cocaine during adolescence renders subjects particularly responsive to the subsequent effects of stress on drug seeking. This heightened propensity for reinstatement puts adolescent-onset drug users at heightened risk for relapse., (© 2015 Society for the Study of Addiction.)

Repeated drug exposure induces short- and long-term neuroadaptations in brain reward circuitries that are normally involved in the regulation of motivation. Hence, repeated drug exposure has been suggested to also affect the drive to acquire natural reinforcers. Here, we tested how chronic exposure of rats to cocaine, as well as a subsequent withdrawal period, affects acquisition of natural reinforcers in high- and low-demanding tasks (HD and LD tasks, respectively). We chronically administered cocaine (i.p., 15 mg/kg once daily, or saline in control) for 30 days, followed by a 30-day withdrawal period. We tested the effect of this treatment on the acquisition of two natural appetitive reinforcers, namely self-administering a 10% sucrose solution and mounting a receptive female, under LD and HD conditions. During the cocaine exposure period, behavioral testing took place 18 hours after cocaine injection, namely after the acute pharmacologic effect of the drug dissipated. We show that chronic i.p. cocaine exposure decreased procurement of both reinforcers in HD but not in LD tasks. The effect was observed throughout the administration period with partial recovery after withdrawal. Taken together, we present empirical evidence that chronic exposure to a constant dose of cocaine is sufficient to reduce natural reinforcement, and that this decrease can outlast drug exposure. Importantly, such effects are observed only when high demands are opposing the consumption of the natural reinforcer., (© 2014 Society for the Study of Addiction.)

Environmentally induced relapse to cocaine seeking requires the retrieval of context-response-cocaine associative memories. These memories become labile when retrieved and must undergo reconsolidation into long-term memory storage to be maintained. Identification of the molecular underpinnings of cocaine-memory reconsolidation will likely facilitate the development of treatments that mitigate the impact of cocaine memories on relapse vulnerability. Here, we used the rat extinction-reinstatement procedure to test the hypothesis that the Src family of tyrosine kinases (SFK) in the dorsal hippocampus (DH) critically controls contextual cocaine-memory reconsolidation. To this end, we evaluated the effects of bilateral intra-DH microinfusions of the SFK inhibitor, PP2 (62.5 ng per 0.5 μl per hemisphere), following re-exposure to a cocaine-associated (cocaine-memory reactivation) or an unpaired context (no memory reactivation) on subsequent drug context-induced instrumental cocaine-seeking behavior. We also assessed alterations in the phosphorylation state of SFK targets, including GluN2A and GluN2B N-methyl-D-aspartate (NMDA) and GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits at the putative time of memory restabilization and following PP2 treatment. Finally, we evaluated the effects of intra-DH PEAQX (2.5 μg per 0.5 μl per hemisphere), a GluN2A-subunit-selective NMDAR antagonist, following, or in the absence of, cocaine-memory reactivation on subsequent drug context-induced cocaine-seeking behavior. GluN2A phosphorylation increased in the DH during putative memory restabilization, and intra-DH PP2 treatment inhibited this effect. Furthermore, PP2-as well as PEAQX-attenuated subsequent drug context-induced cocaine-seeking behavior, in a memory reactivation-dependent manner, relative to VEH. These findings suggest that hippocampal SFKs contribute to the long-term stability of cocaine-related memories that underlie contextual stimulus control over cocaine-seeking behavior.

Lorcaserin (Lorqess, Belviq(®)) is a selective 5-HT(2C) receptor agonist that has received FDA approval for the treatment of obesity. 5-HT(2C) receptor agonists are also efficacious in decreasing multiple aspects of cocaine motivation and reward in preclinical models. This would suggest that lorcaserin is a clinically available therapeutic with the potential to treat cocaine addiction. Here we report the effects of lorcaserin (0.1 mg/kg-1.0 mg/kg) on multiple aspects of cocaine-related behaviours in rats. We find that lorcaserin dose-dependently decreases cocaine self-administration on progressive and fixed ratio schedules of reinforcement. Lorcaserin also reduces reinstatement of cocaine-seeking behaviour in response to priming injections of cocaine and/or reintroduction of cocaine-associated cues. Finally, lorcaserin dose-dependently decreases cocaine-induced hyperlocomotion. Our results, when considered in concert with similar emergent findings in non-human primates, strongly support continued research into the potential of lorcaserin as a clinical treatment for cocaine addiction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A growing body of evidence indicates that the transport of GluA1 subunit-containing calcium-permeable AMPA receptors (CP-AMPARs) to synapses in subregions of the nucleus accumbens promotes cocaine seeking. Consistent with these findings, the present results show that administration of the CP-AMPAR antagonist, Naspm, into the caudal lateral core or caudal medial shell of the nucleus accumbens attenuated cocaine priming-induced reinstatement of drug seeking. Moreover, viral-mediated overexpression of 'pore dead' GluA1 subunits (via herpes simplex virus (HSV) GluA1-Q582E) in the lateral core or medial shell attenuated the reinstatement of cocaine seeking. The overexpression of wild-type GluA1 subunits (via HSV GluA1-WT) in the medial shell, but not the lateral core, enhanced the reinstatement of cocaine seeking. These results indicate that activation of GluA1-containing AMPARs in subregions of the nucleus accumbens reinstates cocaine seeking. SAP97 and 4.1N are proteins involved in GluA1 trafficking to and stabilization in synapses; SAP97-GluA1 interactions also influence dendritic growth. We next examined potential roles of SAP97 and 4.1N in cocaine seeking. Viral-mediated expression of a microRNA that reduces SAP97 protein expression (HSV miSAP97) in the medial accumbens shell attenuated cocaine seeking. In contrast, a virus that overexpressed a dominant-negative form of a 4.1N C-terminal domain (HSV 4.1N-CTD), which prevents endogenous 4.1N binding to GluA1 subunits, had no effect on cocaine seeking. These results indicate that the GluA1 subunit accessory protein SAP97 may represent a novel target for pharmacotherapeutic intervention in the treatment of cocaine craving.

Previous investigations indicate that the dopamine-β-hydroxylase (DBH) inhibitors disulfiram and nepicastat suppress cocaine-primed reinstatement of cocaine self-administration behaviour. Moreover, both inhibitors increase dopamine release in the rat medial prefrontal cortex (mPFC) and markedly potentiate cocaine-induced dopamine release in this region. This study was aimed to clarify if the suppressant effect of DBH inhibitors on cocaine reinstatement was mediated by the high extracellular dopamine in the rat mPFC leading to a supra-maximal stimulation of D1 receptors in the dorsal division of mPFC, an area critical for reinstatement of cocaine-seeking behaviour. In line with previous microdialysis studies in drug-naïve animals, both DBH inhibitors potentiated cocaine-induced dopamine release in the mPFC, in the same animals in which they also suppressed reinstatement of cocaine seeking. Similar to the DBH inhibitors, L-DOPA potentiated cocaine-induced dopamine release in the mPFC and suppressed cocaine-induced reinstatement of cocaine-seeking behaviour. The bilateral microinfusion of the D1 receptor antagonist SCH 23390 into the dorsal mPFC not only prevented cocaine-induced reinstatement of cocaine seeking but also reverted both disulfiram- and L-DOPA-induced suppression of reinstatement. Moreover, the bilateral microinfusion of the D1 receptor agonist chloro-APB (SKF 82958) into the dorsal mPFC markedly attenuated cocaine-induced reinstatement of cocaine seeking. These results suggest that stimulation of D1 receptors in the dorsal mPFC plays a crucial role in cocaine-induced reinstatement of cocaine seeking, whereas the suppressant effect of DBH inhibitors and L-DOPA on drug-induced reinstatement is mediated by a supra-maximal stimulation of D1 receptors leading to their inactivation., (© 2014 Society for the Study of Addiction.)

Drug seeking is maintained by encounters with drug-associated cues, and disrupting retrieval of these drug-cue associations would reduce the risk of relapse. Retrieval of cocaine-associated memories is dependent on β-adrenergic receptor (β-AR) activation, and blockade of these receptors induces a persistent retrieval deficit. Whether retrieval of cocaine-associated memory is mediated by a specific β-AR subtype, however, remains unclear. Using a cocaine conditioned place preference (CPP) procedure, we examined whether retrieval of a cocaine CPP memory is mediated collectively by β1- and β2-ARs, or by one of these β-AR subtypes alone. We show that co-blockade of β1- and β2-ARs abolished CPP expression on that and subsequent drug-free CPP tests, resulting in a long-lasting retrieval deficit that prevented subsequent cocaine-induced reinstatement. To dissociate the necessity of either β1- or β2-ARs alone, we administered subtype-specific antagonists prior to retrieval. Administration of a β1-AR antagonist before the initial CPP trial dose-dependently reduced expression of a CPP on that and subsequent drug-free trials as compared to vehicle administration. In contrast, administration of a β2-AR antagonist had no effect on initial CPP expression, although the highest dose reduced subsequent CPP expression. Importantly, either β1- or β2-AR blockade prior to an initial retrieval trial prevented subsequent cocaine-induced reinstatement. Our findings indicate that the β1-AR subtype mediates retrieval of a cocaine CPP, and that acutely blocking either β1- or β2-ARs can prevent subsequent cocaine-induced reinstatement. Thus, β-AR antagonists, particularly β1-ARs antagonists, could serve as adjuncts for addiction therapies to prevent retrieval of drug-associated memories and provide protection against relapse., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Activation of AMPA receptors (AMPARs) in the nucleus accumbens is necessary for the reinstatement of cocaine-seeking behavior, an animal model of drug craving and relapse. AMPARs are tetrameric protein complexes that consist of GluA1-4 subunits, of which GluA2 imparts calcium permeability. Adenosine deaminase acting on RNA 2 (ADAR2) is a nuclear enzyme that is essential for editing GluA2 pre-mRNA at Q/R site 607. Unedited GluA2(Q) subunits form calcium-permeable AMPARs (CP-AMPARs), whereas edited GluA2(R) subunits form calcium-impermeable channels (CI-AMPARs). Emerging evidence suggests that the reinstatement of cocaine seeking is associated with increased synaptic expression of CP-AMPARs in the nucleus accumbens. However, the role of GluA2 Q/R site editing and ADAR2 in cocaine seeking is unclear. In the present study, we investigated the effects of forced cocaine abstinence on GluA2 Q/R site editing and ADAR2 expression in the nucleus accumbens. Our results demonstrate that 7 days of cocaine abstinence is associated with decreased GluA2 Q/R site editing and reduced ADAR2 expression in the accumbens shell, but not core, of cocaine-experienced rats compared with yoked saline controls. To examine the functional significance of ADAR2 and GluA2 Q/R site editing in cocaine seeking, we used viral-mediated gene delivery to overexpress ADAR2b in the accumbens shell. Increased ADAR2b expression in the shell attenuated cocaine priming-induced reinstatement of drug seeking and was associated with increased GluA2 Q/R site editing and surface expression of GluA2-containing AMPARs. Taken together, these findings support the novel hypothesis that an increased contribution of accumbens shell CP-AMPARs containing unedited GluA2(Q) promotes cocaine seeking. Therefore, CP-AMPARs containing unedited GluA2(Q) represent a novel target for cocaine addiction pharmacotherapies.

Prior studies suggest that the insular cortex (IC), and particularly its posterior region (the PIc), is involved in nicotine craving and relapse in humans and rodents. The present experiments were conducted to determine whether the IC and its different subregions regulate relapse to cocaine-seeking behavior in rats. To address this issue, male Sprague-Dawley rats underwent cocaine self-administration followed by extinction training and reinstatement tests. Before each reinstatement, the PIc or the more anterior dorsal agranular IC (AId) was inactivated to determine their roles in the reinstatement to cocaine seeking. In contrast to the nicotine findings, PIc inactivation had no effect on cue-induced reinstatement for cocaine seeking. However, AId inactivation reduced cued reinstatement while having no effect on cocaine-prime reinstatement. AId inactivation had no effect on reinstatement of food-seeking behavior induced by cues, a food-prime, or cues+food-prime. Based on previous work hypothesizing a role for corticotropin-releasing factor (CRF) in the IC during craving and relapse, a subsequent experiment found that CRF receptor-1 (CRF1) blockade in the AId similarly reduced cued reinstatement. Our results suggest that the AId, along with CRF1 receptors in this region, regulates reinstatement to cocaine seeking, but not food seeking, depending on the type of reinstatement, whereas PIc activity does not influence cue-induced reinstatement.

People with cocaine addiction retain some degree of prefrontal cortex (PFC) inhibitory control of cocaine craving, a brain capacity that may underlie the efficacy of cognitive behavioral therapy for addiction. Similar findings were recently found in rats after extended access to and escalation of cocaine self-administration. Rats' inhibitory control of cocaine seeking was flexible, sufficiently strong to suppress cocaine-primed reinstatement and depended, at least in part, on neuronal activity within the prelimbic (PL) PFC. Here, we used a large-scale and high-resolution Fos mapping approach to identify, beyond the PL PFC, how top-down and/or bottom-up PFC-subcortical circuits are recruited during inhibition of cocaine seeking. Overall, we found that effective inhibitory control of cocaine seeking is associated with the coordinated recruitment of different top-down cortical-striatal circuits originating from different PFC territories, and of different bottom-up dopamine (DA) and serotonin (5-HT) midbrain subsystems that normally modulate activity in these circuits. This integrated brain response suggests that rats concomitantly engage and experience intricate cognitive and affective processes when they have to inhibit intense cocaine seeking. Thus, even after extended drug use, rats can be successfully trained to engage whole-brain inhibitory control mechanisms to suppress cocaine seeking., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Relapse represents one of the most significant problems in the long-term treatment of drug addiction. Cocaine blocks plasma membrane monoamine transporters and increases dopamine (DA) overflow in the brain, and DA is critical for the motivational and primary reinforcing effects of the drug as well as cocaine-primed reinstatement of cocaine seeking in rats, a model of relapse. Thus, modulators of the DA system may be effective for the treatment of cocaine dependence. The endogenous neuropeptide galanin inhibits DA transmission, and both galanin and the synthetic galanin receptor agonist, galnon, interfere with some rewarding properties of cocaine. The purpose of this study was to further assess the effects of galnon on cocaine-induced behaviors and neurochemistry in rats. We found that galnon attenuated cocaine-induced motor activity, reinstatement and DA overflow in the frontal cortex at a dose that did not reduce baseline motor activity, stable self-administration of cocaine, baseline extracellular DA levels or cocaine-induced DA overflow in the nucleus accumbens (NAc). Similar to cocaine, galnon had no effect on stable food self-administration but reduced food-primed reinstatement. These results indicate that galnon can diminish cocaine-induced hyperactivity and relapse-like behavior, possibly in part by modulating DA transmission in the frontal cortex., (© 2014 Society for the Study of Addiction.)