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Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocaine-related contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocaine-memory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. Next, they were briefly re-exposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 µL/hemisphere) immediately after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed the glutamatergic neuronal marker, vesicular glutamate transporter 3. Together, these findings suggest that the DR CRF  → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF and/or glutamate release in the BLA., (© 2024. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Converging evidence indicates that both dopamine and glutamate neurotransmission within the nucleus accumbens (NAc) play a role in psychostimulant self-administration and relapse in rodent models. Increased NAc dopamine release from ventral tegmental area (VTA) inputs is critical to psychostimulant self-administration and NAc glutamate release from prelimbic prefrontal cortex (PFC) inputs synapsing on medium spiny neurons (MSNs) is critical to reinstatement of psychostimulant-seeking after extinction. The regulation of the activity of MSNs by VTA dopamine inputs has been extensively studied, and recent findings have demonstrated that VTA glutamate neurons target the NAc medial shell. Here, we determined whether the mesoaccumbal glutamatergic pathway plays a role in psychostimulant conditioned place preference and self-administration in mice. We used optogenetics to induce NAc release of glutamate from VTA inputs during the acquisition, expression, and reinstatement phases of cocaine- or methamphetamine-induced conditioned place preference (CPP), and during priming-induced reinstatement of cocaine-seeking behavior. We found that NAc medial shell release of glutamate resulting from the activation of VTA glutamatergic fibers did not affect the acquisition of cocaine-induced CPP, but it blocked the expression, stress- and priming-induced reinstatement of cocaine- and methamphetamine CPP, as well as it blocked the priming-induced reinstatement of cocaine-seeking behavior after extinction. These findings indicate that in contrast to the well-recognized mesoaccumbal dopamine system that is critical to psychostimulant reward and relapse, there is a parallel mesoaccumbal glutamatergic system that suppresses reward and psychostimulant-seeking behavior., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Preclinical research has demonstrated the efficacy of CB1 receptor (CB1R) antagonists in reducing drug-taking behavior. However, clinical trials with rimonabant, a CB1R antagonist with inverse agonist profile, failed due to severe adverse effects, such as depression and suicidality. As a result, efforts have shifted towards developing novel neutral CB1R antagonists without an inverse agonist profile for treating substance use disorders. Here, we assessed AM6527, a CB1R neutral antagonist, in addiction animal models. Our findings revealed that AM6527 did not affect cocaine self-administration under fixed-ratio reinforcement schedules but dose-dependently inhibited it under progressive-ratio reinforcement schedules. Additionally, AM6527 dose-dependently inhibited heroin self-administration under both fixed-ratio and progressive-ratio reinforcement schedules and oral sucrose self-administration under a fixed-ratio reinforcement schedule, as well as cocaine- or heroin-triggered reinstatement of drug-seeking behavior in rats. However, chronic AM6527 administration for five consecutive days significantly inhibited heroin self-administration only during the initial two days, indicating tolerance development. Notably, AM6527 did not produce rewarding or aversive effects by itself in classical electrical intracranial self-stimulation and conditioned place preference tests. However, in optical intracranial self-stimulation (oICSS) maintained by optogenetic stimulation of midbrain dopamine neurons in DAT-cre mice, both AM6527 and rimonabant dose-dependently inhibited dopamine-dependent oICSS behavior. Together, these findings suggest that AM6527 effectively reduces drug-taking and seeking behaviors without rimonabant-like adverse effects. Thus, AM6527 warrants further investigation as a potential pharmacotherapy for opioid and cocaine use disorders., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Evidence indicates that the anterior (aIC), but not posterior (pIC), insular cortex promotes cued reinstatement of cocaine seeking after extinction in rats. It is unknown whether these subregions also regulate heroin seeking and whether such involvement depends on prior extinction learning. To address these questions, we used baclofen and muscimol (BM) to inactivate the aIC or pIC bilaterally during a seeking test after extinction or prolonged withdrawal from heroin. Male Sprague-Dawley rats in the extinction groups underwent 10+ days of heroin self-administration, followed by 6+ days of extinction sessions, and subsequent cued or heroin-primed reinstatement. Results indicate that aIC inactivation increased cued reinstatement of heroin seeking after extinction, whereas pIC inactivation prevented cued reinstatement. To determine whether these effects were extinction-dependent, we conducted a subsequent study using both sexes with prolonged withdrawal. Male and female rats in the withdrawal groups underwent 10+ days of heroin self-administration, followed by cued seeking tests after 1 and 14 days of homecage withdrawal to measure incubation of heroin craving. In this case, the findings indicate that aIC inactivation had no effect on incubation of heroin craving after withdrawal in either sex, whereas pIC inactivation decreased heroin craving only in males. These findings suggest that the aIC and pIC have opposing roles in suppressing vs promoting cued heroin seeking after extinction and that these roles are distinct from those in cocaine seeking. Moreover, the incubation of craving results suggest that new contingency learning is necessary to recruit the aIC in cued heroin seeking., (© 2024. The Author(s).)

DNA cytosine methylation has been documented as a potential epigenetic mechanism of transcriptional regulation underlying opioid use disorder. However, methylation of RNA cytosine residues, which would drive another level of biological influence as an epitranscriptomic mechanism of gene and protein regulation has not been studied in the context of addiction. Here, we probed whether chronic morphine exposure could alter tRNA cytosine methylation (m 5 C) and resulting expression levels in the medial prefrontal cortex (mPFC), a brain region crucial for reward processing and executive function that exhibits opioid-induced molecular restructuring. We identified dynamic changes in glycine tRNA (tRNA Gly GCC ) cytosine methylation, corresponding to altered expression levels of this tRNA at multiple timepoints following 15 days of daily morphine. Additionally, a robust increase in methylation, coupled with decreased expression, was present after 30 days of withdrawal, suggesting that repeated opioid administration produces changes to the tRNA regulome long after discontinuation. Furthermore, forebrain-wide knockout of neuronal Nsun2, a tRNA methyltransferase, was associated with disruption of opioid conditioned place preference, and this effect was recapitulated by regional mPFC Nsun2 knockout. Taken together, these studies provide a foundational link between the regulation of tRNA cytosine methylation and opioid reward and highlight the tRNA machinery as a potential therapeutic target in addiction., (© 2024. The Author(s).)

Methylphenidate (MP) is commonly prescribed to treat attention-deficit hyperactivity disorder (ADHD). MP is also taken for non-medical purposes as a recreational drug or "cognitive enhancer". Combined exposure to MP and selective serotonin reuptake inhibitors such as fluoxetine (FLX) can also occur, such as in the treatment of ADHD with depression comorbidity or when patients taking FLX use MP for non-medical purposes. It is unclear if such exposure could subsequently increase the risk for relapse in former cocaine users. We investigated if an acute challenge with MP, FLX, or the combination of MP + FLX could trigger reinstatement of cocaine seeking behavior in a model for relapse in rats. Juvenile rats self-administered cocaine (600 µg/kg/infusion, 1-2 h/day, 7-8 days) and then underwent extinction and withdrawal during late adolescence-early adulthood. Reinstatement was tested at a low dose of MP (2 mg/kg, I.P., comparable to doses used therapeutically) or a high dose of MP (5 mg/kg, comparable to doses used recreationally or as a cognitive enhancer), with or without FLX (2.5-5 mg/kg, I.P.). An acute challenge with the high dose of MP (5 mg/kg), with or without FLX, reinstated cocaine seeking behavior to levels comparable to those seen after an acute challenge with cocaine (15 mg/kg, I.P.). The low dose of MP (2 mg/kg) with or without FLX did not reinstate cocaine seeking behavior. Our results suggest that acute exposure to a high dose of MP, with or without FLX, may increase the risk for relapse in individuals who used cocaine during the juvenile period., (© 2023. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Opioid use disorder is a chronic relapsing disorder encompassing misuse, dependence, and addiction to opioid drugs. Long term maintenance of associations between the reinforcing effects of the drug and the cues associated with its intake are a leading cause of relapse. Indeed, exposure to the salient drug-associated cues can lead to drug cravings and drug seeking behavior. The dorsal hippocampus (dHPC) and locus coeruleus (LC) have emerged as important structures for linking the subjective rewarding effects of opioids with environmental cues. However, their role in cue-induced reinstatement of opioid use remains to be further elucidated. In this study, we showed that chemogenetic inhibition of excitatory dHPC neurons during re-exposure to drug-associated cues significantly attenuates cue-induced reinstatement of morphine-seeking behavior. In addition, the same manipulation reduced reinstatement of sucrose-seeking behavior but failed to alter memory recall in the object location task. Finally, intact activity of tyrosine hydroxylase (TH) LC-dHPC Th afferents is necessary to drive cue induced reinstatement of morphine-seeking as inhibition of this pathway blunts cue-induced drug-seeking behavior. Altogether, these studies show an important role of the dHPC and LC-dHPC Th pathway in mediating cue-induced reinstatement of opioid seeking., (© 2024. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

In the classical incubation of drug craving rat model, drug seeking is assessed after homecage forced abstinence. However, human abstinence is often voluntary because negative consequences of drug seeking outweigh the desire for the drug. Here, we developed a rat model of incubation of opioid craving after electric barrier-induced voluntary abstinence and determined whether the dopamine stabilizer (-)-OSU6162 would decrease this new form of incubation. We trained male and female rats to self-administer oxycodone (0.1 mg/kg/infusion, 6 h/day) for 14 days. We then exposed them to either homecage forced abstinence or voluntary abstinence induced by an electric barrier of increasing intensity near the drug-paired lever. On abstinence days 1, 15, or 30, we tested the rats for oxycodone seeking without shock and drug. We also examined the effect of (-)-OSU6162 (7.5 and 15 mg/kg) on oxycodone seeking on abstinence day 1 or after 15 days of either voluntary or forced abstinence. Independent of sex, the time-dependent increase in oxycodone seeking after cessation of opioid self-administration (incubation of opioid craving) was stronger after voluntary abstinence than after forced abstinence. In males, (-)-OSU6162 decreased incubated (day 15) but not non-incubated (day 1) oxycodone seeking after either voluntary or forced abstinence. In females, (-)-OSU6162 modestly decreased incubated oxycodone seeking after voluntary but not forced abstinence. Results suggest that voluntary abstinence induced by negative consequences of drug seeking can paradoxically potentiate opioid craving and relapse. We propose the dopamine stabilizer (-)-OSU6162 may serve as an adjunct pharmacological treatment to prevent relapse in male opioid users.

Despite the effectiveness of current medications to treat opioid use disorder, there is still a high rate of relapse following detoxification. Thus, there is critical need for innovative studies aimed at identifying novel neurobiological mechanisms that could be targeted to treat opioid use disorder. A growing body of preclinical evidence indicates that glucagon-like peptide-1 (GLP-1) receptor agonists reduce drug reinforcement. However, the efficacy of GLP-1 receptor agonists in attenuating opioid-mediated behaviors has not been thoroughly investigated. Using recently established models of opioid-taking and -seeking behaviors, we showed that systemic administration of the GLP-1 receptor agonist exendin-4 reduced oxycodone self-administration and the reinstatement of oxycodone-seeking behavior in rats. We also identified behaviorally selective doses of exendin-4 that reduced opioid-taking and -seeking behaviors and did not produce adverse feeding effects in oxycodone-experienced rats. To identify a central site of action, we showed that systemic exendin-4 penetrated the brain and bound putative GLP-1 receptors on dopamine D1 receptor- and dopamine D2 receptor-expressing medium spiny neurons in the nucleus accumbens shell. Consistent with our systemic studies, infusions of exendin-4 directly into the accumbens shell attenuated oxycodone self-administration and the reinstatement of oxycodone-seeking behavior without affecting ad libitum food intake. Finally, exendin-4 did not alter the analgesic effects of oxycodone, suggesting that activation of GLP-1 receptors attenuated opioid reinforcement without reducing the thermal antinociceptive effects of oxycodone. Taken together, these findings suggest that GLP-1 receptors could serve as potential molecular targets for pharmacotherapies aimed at reducing opioid use disorder.

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.

Contextual cues associated with drugs of abuse, such as ethanol, can trigger craving and drug-seeking behavior. Pavlovian procedures, such as place conditioning, have been widely used to study the rewarding/aversive properties of drugs and the association between environmental cues and drug seeking. Previous research has shown that ethanol as an unconditioned stimulus can induce a strong conditioned place preference (CPP) or aversion (CPA) in rodents. However, the neural mechanisms underlying ethanol-induced reward and aversion have not been thoroughly investigated. The bed nucleus of the stria terminalis (BNST), an integral part of the extended amygdala, is engaged by both rewarding and aversive stimuli and plays a role in ethanol-seeking behavior. Here, we used ex-vivo slice physiology to probe learning-induced synaptic plasticity in the BNST following ethanol-induced CPP and CPA. Male DBA/2 J mice (2-3 months old) were conditioned using previously reported ethanol-induced CPP/CPA procedures. Ethanol-induced CPP was associated with increased neuronal excitability in the ventral BNST (vBNST). Conversely, ethanol-induced CPA resulted in a significant decrease in spontaneous glutamatergic transmission without alterations in GABAergic signaling. Ethanol-CPA also led to a significant increase in the paired-pulse ratio at excitatory synapses, suggestive of a decrease in presynaptic glutamate release. Collectively, these data demonstrate that the vBNST is involved in the modulation of contextual learning associated with both the rewarding and the aversive properties of ethanol in mice.

The current opioid crisis remains a significant public health issue and there is a critical need for biomedical research to develop effective and easily deployable candidate treatments. One emerging treatment strategy for opioid use disorder includes immunopharmacotherapies or opioid-targeted vaccines. The present study determined the effectiveness of a fentanyl-tetanus toxoid conjugate vaccine to alter fentanyl self-administration using a fentanyl-vs.-food choice procedure in male and female rats under three experimental conditions. For comparison, continuous 7-day naltrexone (0.01-0.1 mg/kg/h) and 7-day clonidine (3.2-10 μg/kg/h) treatment effects were also determined on fentanyl-vs.-food choice. Male and female rats responded for concurrently available 18% diluted Ensure® (liquid food) and fentanyl (0-10 μg/kg/infusion) infusions during daily sessions. Under baseline and saline treatment conditions, fentanyl maintained a dose-dependent increase in fentanyl-vs.-food choice. First, fentanyl vaccine administration significantly blunted fentanyl reinforcement and increased food reinforcement for 15 weeks in non-opioid dependent rats. Second, surmountability experiments by increasing the unit fentanyl dose available during the self-administration session 10-fold empirically determined that the fentanyl vaccine produced an approximate 22-fold potency shift in fentanyl-vs.-food choice that was as effective as the clinically approved treatment naltrexone. Clonidine treatment significantly increased fentanyl-vs.-food choice. Lastly, fentanyl vaccine administration prevented the expression of withdrawal-associated increases in fentanyl-vs.-food choice following introduction of extended 12 h fentanyl access sessions. Overall, these results support the potential and further consideration of immunopharmacotherapies as candidate treatments to address the current opioid crisis.

The orexin system is a potential treatment target for drug addiction. Orexin-1 receptor (OxR1) antagonism reduces demand for cocaine and remifentanil, indicating that orexin-based therapies may reduce demand for many classes of abused drugs. However, pharmacokinetics vary greatly among opioids and it is unclear if OxR1 antagonism would reduce demand for all opioids, particularly ones with high abuse liability. Here, we established a behavioral economics (BE) procedure to assess the effects of OxR1 antagonism on demand for the highly abused opioid fentanyl. We also investigated the utility of our procedure to predict OxR1 antagonism efficacy and relapse propensity. Demand parameters α (demand elasticity or price sensitivity of consumption, an inverse measure of drug motivation) and Q o (drug consumption at null cost) were assessed. The OxR1 antagonist SB-334867 (SB) decreased motivation (increased α) for fentanyl without affecting Q o . Baseline α values predicted SB efficacy, such that SB was most effective at reducing motivation (increasing α) in highly motivated rats. Baseline α values predicted the amount of cued reinstatement of fentanyl seeking; this reinstatement behavior was attenuated by SB administration. These results highlight the promise of the orexin system as a treatment target for opioid addiction and emphasize the usefulness of BE procedures in the study of opioid abuse.

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.

Vulnerability for cocaine abuse in humans is associated with low dopamine D2 receptor (D2R) availability in the striatum. The mechanisms driving this vulnerability are poorly understood. In this study, we found that downregulating D2R expression selectively in striatal indirect-pathway neurons triggers a multitude of changes in D1 receptor (D1R)-expressing direct-pathway neurons, which comprise the other main subpopulation of striatal projection neurons. These changes include a leftward shift in the dose-response to a D1-like agonist that indicates a behavioral D1R hypersensitivity, a shift from PKA to ERK intracellular signaling cascades upon D1R activation, and a reduction in the density of bridging collaterals from D1R-expressing neurons to pallidal areas. We hypothesize that the D1R hypersensitivity underlies abuse vulnerability by facilitating the behavioral responses to repeated cocaine, such as locomotor sensitization and drug self-administration. We found evidence that littermate control mice develop D1R hypersensitivity after they are sensitized to cocaine. Indeed, D1-like agonist and cocaine cross-sensitize in control littermates and this effect was potentiated in mice lacking striatal D2Rs from indirect-pathway neurons. To our surprise, mice with low striatal D2Rs acquired cocaine self-administration similarly to littermate controls and showed no significant change in motivation to take cocaine but lower seeking. These findings indicate that downregulation of striatal D2Rs triggers D1R hypersensitivity to facilitate cocaine locomotor sensitization, which by itself was not associated with greater cocaine taking or seeking under the conditions tested.

Previous preclinical and clinical investigations have focused on the L-type calcium channel (LTCC) as a potential therapeutic target for substance abuse. While some clinical studies have examined the ability of LTCC blockers to alter cocaine's subjective effects, very few LTCC studies have examined cocaine relapse. Here, we examined whether ventral tegmental area (VTA)-specific or systemic administration of the LTCC inhibitor, isradipine, altered cocaine-seeking behavior in a rat model. Male Sprague-Dawley rats first received 10 days of cocaine self-administration training (2 h sessions), where active lever depression resulted in delivery of a ∼0.5 mg/kg cocaine infusion paired with a tone + light cue. Rats then underwent 10 days of forced abstinence, without access to cocaine or cocaine cues. Rats were then returned to the opertant chamber for the cue-induced cocaine-seeking test, where active lever depression in the original training context resulted in tone + light cue presentation. We found VTA specific or systemic isradipine administration robustly attenuated cocaine-seeking, without altering cocaine-taking nor natural reward seeking. Dopamine (DA) signaling in the nucleus accumbens (NAc) core is necessary and sufficient for cue-induced drug-seeking. Surprisingly in our study, isradipine enhanced tonic and phasic DA signaling in cocaine abstinent rats, with no change in sucrose abstinent nor naïve rats. Strikingly, isradipine's behavioral effects were dependent upon NAc core DA receptor activation. Together, our findings reveal a novel mechanism by which the FDA-approved drug, isradipine, could act to decrease cocaine relapse.

The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling.

Cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, has received attention for therapeutic potential in treating neurologic and psychiatric disorders. Recently, CBD has also been explored for potential in treating drug addiction. Substance use disorders are chronically relapsing conditions and relapse risk persists for multiple reasons including craving induced by drug contexts, susceptibility to stress, elevated anxiety, and impaired impulse control. Here, we evaluated the "anti-relapse" potential of a transdermal CBD preparation in animal models of drug seeking, anxiety and impulsivity. Rats with alcohol or cocaine self-administration histories received transdermal CBD at 24 h intervals for 7 days and were tested for context and stress-induced reinstatement, as well as experimental anxiety on the elevated plus maze. Effects on impulsive behavior were established using a delay-discounting task following recovery from a 7-day dependence-inducing alcohol intoxication regimen. CBD attenuated context-induced and stress-induced drug seeking without tolerance, sedative effects, or interference with normal motivated behavior. Following treatment termination, reinstatement remained attenuated up to ≈5 months although plasma and brain CBD levels remained detectable only for 3 days. CBD also reduced experimental anxiety and prevented the development of high impulsivity in rats with an alcohol dependence history. The results provide proof of principle supporting potential of CBD in relapse prevention along two dimensions: beneficial actions across several vulnerability states and long-lasting effects with only brief treatment. The findings also inform the ongoing medical marijuana debate concerning medical benefits of non-psychoactive cannabinoids and their promise for development and use as therapeutics.

Novel molecular targets are needed to develop new medications for the treatment of cocaine addiction. Here we investigated a role for glucagon-like peptide-1 (GLP-1) receptors in the reinstatement of cocaine-seeking behavior, an animal model of relapse. We showed that peripheral administration of the GLP-1 receptor agonist exendin-4 dose dependently reduced cocaine seeking in rats at doses that did not affect ad libitum food intake, meal patterns or body weight. We also demonstrated that systemic exendin-4 penetrated the brain where it putatively bound receptors on both neurons and astrocytes in the ventral tegmental area (VTA). The effects of systemic exendin-4 on cocaine reinstatement were attenuated in rats pretreated with intra-VTA infusions of the GLP-1 receptor antagonist exendin-(9-39), indicating that the suppressive effects of systemic exendin-4 on cocaine seeking were due, in part, to activation of GLP-1 receptors in the VTA. Consistent with these effects, infusions of exendin-4 directly into the VTA reduced cocaine seeking. Finally, extinction following cocaine self-administration was associated with decreased preproglucagon mRNA expression in the caudal brainstem. Thus, our study demonstrated a novel role for GLP-1 receptors in the reinstatement of cocaine-seeking behavior and identified behaviorally relevant doses of a GLP-1 receptor agonist that selectively reduced cocaine seeking and did not produce adverse effects.

Prior work suggests a role of kappa-opioid signaling in the control of alcohol drinking, in particular when drinking is escalated due to alcohol-induced long-term neuroadaptations. Here, we examined the small molecule selective kappa antagonist CERC-501 in rat models of alcohol-related behaviors, with the objective to evaluate its potential as a candidate therapeutic for alcohol use disorders. We first tested the effect of CERC-501 on acute alcohol withdrawal-induced anxiety-like behavior. CERC-501 was then tested on basal as well as escalated alcohol self-administration induced by 20% alcohol intermittent access. Finally, we determined the effects of CERC-501 on relapse to alcohol seeking triggered by both stress and alcohol-associated cues. Control experiments were performed to confirm the specificity of CERC-501 effects on alcohol-related behaviors. CERC-501 reversed anxiety-like behavior induced by alcohol withdrawal. It did not affect basal alcohol self-administration but did dose-dependently suppress self-administration that had escalated following long-term intermittent access to alcohol. CERC-501 blocked relapse to alcohol seeking induced by stress, but not when relapse-like behavior was triggered by alcohol-associated cues. The effects of CERC-501 were observed in the absence of sedative side effects and were not due to effects on alcohol metabolism. Thus, in a broad battery of preclinical alcohol models, CERC-501 has an activity profile characteristic of anti-stress compounds. Combined with its demonstrated preclinical and clinical safety profile, these data support clinical development of CERC-501 for alcohol use disorders, in particular for patients with negatively reinforced, stress-driven alcohol seeking and use.

Kappa opioid receptor (KOR) agonists have known anti-addiction properties and can reduce drug seeking. Their potential for clinical use has largely been daunted by their aversive properties mediated through p38 MAPK signaling. Here we examined the therapeutic potential of the KOR agonist U50,488 (U50) to reduce cocaine seeking in a self-administration model. Following cocaine self-administration and 7 days of forced home-cage abstinence, rats were administered a single dose of U50 (5 mg/kg, i.p.) 30 min prior to the first extinction training session, wherein cocaine and the discrete cocaine-paired cues were no longer available. U50 reduced cocaine seeking on this first extinction session, but did not alter extinction training over subsequent days. 2 weeks after U50 treatment, rats underwent a test of cue-induced reinstatement, and rats that had received U50 reinstated less than controls. Central inhibition of p38 MAPK at the time of U50 administration prevented its long-term therapeutic effect on reinstatement, but not its acute reduction in drug seeking on extinction day 1. The long-term therapeutic effect of U50 required operant extinction during U50 exposure, extended to cocaine-primed reinstatement, and was not mimicked by another aversive drug, lithium chloride (LiCl). These data suggest U50 elicits its long-term anti-relapse effects through a KOR-p38 MAPK-specific aversive counterconditioning of the operant cocaine-seeking response. A single, albeit aversive treatment that is able to reduce relapse long-term warrants further consideration of the therapeutic potential of KOR agonists in the treatment of addiction.

Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M 5 muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M 5 mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M 5 mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M 5 mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.

Opiate abuse and addiction have become a worldwide epidemic with great societal and financial burdens, highlighting a critical need to understand the neurobiology of opiate addiction. Although several studies have focused on drug-dependent changes in neurons, the role of glia in opiate addiction remains largely unstudied. RNA sequencing pathway analysis from the prefrontal cortex (PFC) of male rats revealed changes in several genes associated with oligodendrocyte differentiation and maturation following heroin self-administration. Among these genes changed was Sox10, which is regulated, in part, by the chromatin remodeler BRG1/SMARCA4. To directly test the functional role of Sox10 in mediating heroin-induced behavioral plasticity, we selectively overexpressed Sox10 and BRG1 in the PFC. Overexpression of either Sox10 or BRG1 decreased the motivation to obtain heroin infusions in a progressive ratio test without altering the acquisition or maintenance of heroin self-administration. These data demonstrate a critical, and perhaps compensatory, role of Sox10 and BRG1 in oligodendrocytes in regulating the motivation for heroin.

Previous work indicated that activation of D1-like dopamine receptors (D1DRs) in the nucleus accumbens shell promoted cocaine seeking through a process involving the activation of PKA and GluA1-containing AMPA receptors (AMPARs). A-kinase anchoring proteins (AKAPs) localize PKA to AMPARs leading to enhanced phosphorylation of GluA1. AKAP150, the most well-characterized isoform, plays an important role in several forms of neuronal plasticity. However, its involvement in drug addiction has been minimally explored. Here we examine the role of AKAP150 in cocaine reinstatement, an animal model of relapse. We show that blockade of PKA binding to AKAPs in the nucleus accumbens shell of Sprague-Dawley rats attenuates reinstatement induced by either cocaine or a D1DR agonist. Moreover, this effect is specific to AKAP150, as viral overexpression of a PKA-binding deficient mutant of AKAP150 also impairs cocaine reinstatement. This viral-mediated attenuation of cocaine reinstatement was accompanied by decreased phosphorylation of GluA1-containing AMPARs and attenuated AMPAR eEPSCs. Collectively, these results suggest that AKAP150 facilitates the reinstatement of cocaine-seeking behavior by amplifying D1DR/PKA-dependent AMPA transmission in the nucleus accumbens.

The prelimbic cortex (PL) and infralimbic cortex (IL) play a role in context-induced reinstatement of heroin seeking in an animal model of drug relapse. Both the PL and IL receive direct glutamatergic projections from the ventral CA1 (vCA1), which is also involved in context-induced reinstatement of cocaine and heroin seeking. Here we studied the role of vCA1-PL and vCA1-IL projections in context-induced reinstatement of heroin seeking by using electrophysiological, neuropharmacological, chemogenetic, and molecular methods. We showed that context-induced reinstatement of heroin seeking caused selective activation of the vCA1-IL but not vCA1-PL glutamatergic projections, decreased synaptosomal GluA2 expression in the IL, impaired basal synaptic transmission, and facilitation of long-term depression (LTD) in the vCA1-IL pathway. Additionally, chemogenetic inactivation of the vCA1-IL but not vCA1-PL pathway decreased context-induced reinstatement of heroin seeking. Inactivation of the vCA1-IL pathway also reversed synaptosomal GluA2 downregulation and basal transmission reduction, and blocked LTD induction. Taken together, our results demonstrate a critical role of the vCA1-IL glutamatergic projection in context-induced reinstatement of heroin seeking in a rat model of drug relapse.

Adaptations in glutamate signaling within the brain's reward circuitry are observed following withdrawal from several abused drugs, including cocaine. These include changes in intrinsic cellular excitability, glutamate release, and glutamate uptake. Pharmacological or optogenetic reversal of these adaptations have been shown to reduce measures of cocaine craving and seeking, raising the hypothesis that regulation of glutamatergic signaling represents a viable target for the treatment of substance use disorders. Here, we tested the hypothesis that administration of the compound riluzole, which regulates glutamate dynamics in several ways, would reduce cocaine seeking in the rat self-administration and reinstatement model of addiction. Riluzole dose-dependently inhibited cue- and cocaine-primed reinstatement to cocaine, but did not affect locomotor activity or reinstatement to sucrose seeking. Moreover, riluzole reversed bidirectional cocaine-induced adaptations in intrinsic excitability of prelimbic (PL) and infralimbic (IL) pyramidal neurons; a cocaine-induced increase in PL excitability was decreased by riluzole, and a cocaine-induced decrease in IL excitability was increased to normal levels. Riluzole also reversed the cocaine-induced suppression of the high-affinity glutamate transporter 1 (EAAT2/GLT-1) in the nucleus accumbens (NAc). GLT-1 is responsible for the majority of glutamate uptake in the brain, and has been previously reported to be downregulated by cocaine. These results demonstrate that riluzole impairs cocaine reinstatement while rectifying several cellular adaptations in glutamatergic signaling within the brain's reward circuitry, and support the hypothesis that regulators of glutamate homeostasis represent viable candidates for pharmacotherapeutic treatment of psychostimulant relapse.

Lateral septum (LS) has re-emerged as an important structure in reward and addiction; however, LS afferents that drive addiction behaviors are unknown. Here, we used a modified self-administration/reinstatement procedure combined with anatomical, pharmacological, and chemogenetic techniques to characterize LS, and hippocampal inputs to LS, in two established triggers of drug relapse-context- and cue-induced reinstatement of cocaine seeking. We found that inactivation of LS neurons attenuated both context- and cue-induced reinstatement of cocaine seeking. However, dorsal hippocampus inputs to LS showed enhanced neuronal activation (as measured by Fos expression) during context-induced, but not cue-induced reinstatement. Additionally, chemogenetic inhibition of dorsal, but not ventral, hippocampal inputs to LS specifically attenuated context-induced reinstatement. Together these findings elucidate the importance of LS in reinstatement of cocaine seeking, and indicate that dorsal hippocampal inputs to LS mediate context-, but not cue-induced, reinstatement of cocaine seeking.

Stressors often contribute to difficulties in maintaining behavior change following a period of abstinence, and may play a significant role in drug relapse. The activation of pituitary adenylate cyclase-activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Here we ask whether PACAP is also involved in producing reinstatement in a model of stress-induced relapse to drug taking. Rats self-administered cocaine for 1 h daily over 10 days that was followed by 20 days of extinction training in which lever pressing no longer produced cocaine. In experiment 1, quantitative PCR (qPCR) was performed at several stages to determine transcript levels of PACAP and corresponding receptors. Reinstatement of cocaine seeking was then tested after footshock exposure in different groups of rats that were pretreated with vehicle solution, a PAC1 receptor antagonist (experiment 2), or a PACAP agonist (experiment 3) without footshock. In experiment 1, cocaine self-administration increased BNST PACAP transcript levels similar to what we have previously reported with chronic stress. In experiment 2, intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38, prevented footshock-induced reinstatement of extinguished cocaine seeking. In experiment 3, intra-BNST PACAP infusion reinstated previously extinguished cocaine-seeking behavior in the absence of footshock. Cocaine self-administration elevated BNST PACAP, and BNST PACAP receptor activation was necessary and sufficient for stress-induced reinstatement of cocaine seeking. These data suggest that BNST PACAP systems may be viable targets for relapse prevention.

Clinical observations imply that female cocaine addicts experience enhanced relapse vulnerability compared with males, an effect tied to elevated estrogen phases of the ovarian hormone cycle. Although estrogens can enhance drug-seeking behavior, they do not directly induce reinstatement on their own. To model this phenomenon, we tested whether an estrogen could augment drug-seeking behavior in response to an ordinarily subthreshold reinstatement trigger. Following cocaine self-administration and extinction, female rats were ovariectomized to isolate estrogen effects on reinstatement. Although neither peak proestrus levels of the primary estrogen 17β-estradiol (E2; 10 μg/kg, i.p., 1-h pretreatment) nor a subthreshold cocaine dose (1.25 mg/kg, i.p.) alone were sufficient to reinstate drug-seeking behavior, pretreatment with E2 potentiated reinstatement to the ordinarily subthreshold cocaine dose. Furthermore, E2 microinfusions revealed that E2 (5 μg/0.3 μl, 15-min pretreatment) acts directly within the prelimbic prefrontal cortex (PrL-PFC) to potentiate reinstatement. As E2 has been implicated in endocannabinoid mobilization, which can disinhibit PrL-PFC projection neurons, we investigated whether cannabinoid type-1 receptor (CB1R) activation is necessary for E2 to potentiate reinstatement. The CB1R antagonist AM251 (1 or 3 mg/kg, i.p., 30-min pretreatment) administered prior to E2 and cocaine suppressed reinstatement in a dose-dependent manner. Finally, PrL-PFC AM251 microinfusions (300 ng/side, 15-min pretreatment) also suppressed E2-potentiated reinstatement. Together, these results suggest that E2 can augment reactivity to an ordinarily subthreshold relapse trigger in a PrL-PFC CB1R activation-dependent manner.

κ-Opioid receptors (KORs) and their endogenous ligand dynorphin are involved in stress-induced alcohol seeking but the mechanisms involved are largely unknown. We previously showed that systemic injections of the KOR agonist U50,488, which induce stress-like aversive states, reinstate alcohol seeking after extinction of the alcohol-reinforced responding. Here, we used the neuronal activity marker Fos and site-specific injections of the KOR antagonist nor-BNI and U50,488 to study brain mechanisms of U50,488-induced reinstatement of alcohol seeking. We trained male Long-Evans rats to self-administer alcohol (12% w/v) for 23-30 days. After extinction of the alcohol-reinforced responding, we tested the effect of U50,488 (0, 1.25, 2.5, and 5 mg/kg) on reinstatement of alcohol seeking. Next, we correlated regional Fos expression with reinstatement induced by the most effective U50,488 dose (5 mg/kg). Based on the correlational Fos results, we determined the effect of bed nucleus of the stria terminalis (BNST) injections of nor-BNI (4 μg/side) on U50,488-induced reinstatement of alcohol seeking, and reinstatement induced by injections of U50,488 (0, 0.3, 1, and 3 μg/side) into the BNST. U50,488-induced reinstatement of alcohol seeking was associated with increased Fos expression in multiple brain areas, including the BNST, where it was significantly correlated with lever pressing. U50,488-induced reinstatement was blocked by BNST nor-BNI injections, and BNST U50,488 injections partially mimicked the drug's systemic effect on reinstatement. Our data indicate that the BNST is a critical site for U50,488-induced reinstatement of alcohol seeking and suggest that KOR/dynorphin mechanisms in this brain area play a key role in stress-induced alcohol seeking.

Excessive drinking to intoxication is the major behavioral characteristic of those addicted to alcohol but it is not the only one. Indeed, individuals addicted to alcohol also crave alcoholic beverages and spend time and put much effort into compulsively seeking alcohol, before eventually drinking large amounts. Unlike this excessive drinking, for which treatments exist, compulsive alcohol seeking is therefore another key feature of the persistence of alcohol addiction since it leads to relapse and for which there are few effective treatments. Here we provide novel evidence for the existence in rats of an individual vulnerability to switch from controlled to compulsive, punishment-resistant alcohol seeking. Alcohol-preferring rats given access to alcohol under an intermittent 2-bottle choice procedure to establish their alcohol-preferring phenotype were subsequently trained instrumentally to seek and take alcohol on a chained schedule of reinforcement. When stable seeking-taking performance had been established, completion of cycles of seeking responses resulted unpredictably either in punishment (0.45 mA foot-shock) or the opportunity to make a taking response for access to alcohol. Compulsive alcohol seeking, maintained in the face of the risk of punishment, emerged in only a subset of rats with a predisposition to prefer and drink alcohol, and was maintained for almost a year. We show further that a selective and potent μ-opioid receptor antagonist (GSK1521498) reduced both alcohol seeking and alcohol intake in compulsive and non-compulsive rats, indicating its therapeutic potential to promote abstinence and prevent relapse in individuals addicted to alcohol.

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.

(±)Modafinil ((±)MOD) and its R-enantiomer (R-modafinil; R-MOD) have been investigated for their potential as treatments for psychostimulant addiction. We recently reported a series of (±)MOD analogs, of which JJC8-016 (N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)-3-phenylpropan-1-amine) was selected for further development. JJC8-016 and R-MOD were evaluated for binding across ~70 receptors, transporters, and enzymes. Although at a concentration of 10 μM, there were many hits for JJC8-016, binding affinities in the range of its DAT affinity were only observed at the serotonin transporter (SERT), dopamine D 2 -like, and sigma 1 receptors. R-MOD was more selective, but had much lower affinity at the DAT (K i =3 μM) than JJC8-016 (K i =116 nM). In rats, systemic administration of R-MOD alone (10-30 mg/kg i.p.) dose-dependently increased locomotor activity and electrical brain-stimulation reward, whereas JJC8-016 (10-30 mg/kg i.p.) did not produce these effects. Strikingly, pretreatment with JJC8-016 dose-dependently inhibited cocaine-enhanced locomotion, cocaine self-administration, and cocaine-induced reinstatement of drug-seeking behavior, whereas R-MOD inhibited cocaine-induced reinstatement only at the high dose of 100 mg/kg. Notably, JJC8-016 alone neither altered extracellular dopamine in the nucleus accumbens nor maintained self-administration. It also failed to induce reinstatement of drug-seeking behavior. These findings suggest that JJC8-016 is a unique DAT inhibitor that has no cocaine-like abuse potential by itself. Moreover, pretreatment with JJC8-016 significantly inhibits cocaine-taking and cocaine-seeking behavior likely by interfering with cocaine binding to DAT. In addition, off-target actions may also contribute to its potential therapeutic utility in the treatment of cocaine abuse.

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.

GABAergic signaling is involved in modulating the reinforcing properties of alcohol, and GABA B receptors have been proposed as a potential target for clinical treatment of alcoholism. The orthosteric GABA B receptor agonist baclofen has been shown to suppress operant self-administration of alcohol in animals and alcohol use in alcohol-dependent patients, but its utility is limited by a narrow therapeutic index. We tested the effects of ADX71441, a novel GABA B receptor positive allosteric modulator, on alcohol-related behaviors in rats. We first assessed the effects of ADX71441 (1, 3, 10 and 30 mg/kg, I.P.) on both non-dependent and dependent male Wistar rats trained to self-administer 20% alcohol. We then determined the effects of ADX71441 on stress-induced as well as cue-induced relapse-like behavior. Finally, we sought to identify the brain regions through which ADX71441 may act to prevent relapse-like behavior by mapping the neuronal activation induced by stress-induced reinstatement of alcohol-seeking using c-Fos immunohistochemistry. ADX71441 dose-dependently decreased alcohol self-administration of both dependent and non-dependent animals, but its potency was higher in alcohol-dependent rats. Furthermore, both cue- and stress-induced alcohol seeking were blocked by the GABA B receptor positive allosteric modulator. Finally, pretreatment with 3 mg/kg of ADX71441 before stress-induced reinstatement significantly decreased c-Fos expression in a network of brain regions implicated in stress-induced relapse, comprising the nucleus accumbens shell, the dorsal raphe nucleus and the medial prefrontal cortex. Our findings support a causal role of GABA B receptors in alcohol reinforcement and relapse to alcohol seeking. These effects are observed in the absence of significant sedative side effects. Jointly, these observations indicate that GABA B receptor positive allosteric modulators merit being tested clinically for the treatment of alcoholism. Our data also point to a potential biomarker of target engagement for early clinical studies.

The lateral habenula (LHb) is a brain structure receiving inputs from limbic forebrain areas and innervating major midbrain monoaminergic nuclei. Evidence indicates LHb involvement in sleep control, reward-based decision making, avoidance of punishment, and responses to stress. Additional work has established that the LHb mediates negative feedback in response to aversive events. As a hallmark of drug addiction is the inability to limit drug use despite negative consequences, we hypothesize that LHb dysfunction may have a role in the lack of control over drug seeking. Here we examine the effects of LHb inactivation in control over drug seeking in several cocaine self-administration (SA) paradigms in rats. We find that inhibition of the LHb with GABAergic agonists did not alter cocaine SA under progressive ratio or seeking/taking chained reinforcement schedules, or during punishment-induced suppression of cocaine-reinforced responding. In contrast, LHb inhibition increased cocaine seeking when the drug was not available in rats trained to discriminate its presence using an environmental cue. This effect of LHb inhibition was selective for cocaine, as it did not impair responding for sucrose reinforcement. The effect of LHb injection of GABA agonists was mimicked by intra-LHb muscarinic cholinergic (mACh) antagonist injection, and activation of mACh receptors excited a majority of LHb neurons in in vitro electrophysiology experiments. These results indicate that the LHb participates in the suppression of impulsive responding for cocaine through the activation of a cholinergic circuit, and they suggest that LHb dysfunction may contribute to impaired impulse control associated with drug addiction.

Use of marijuana (Cannabis sativa) often begins in adolescence, and heavy adolescent marijuana use is often associated with impaired cognitive function in adulthood. However, clinical reports of long-lasting cognitive deficits, particularly in subjects who discontinue use in adulthood, are mixed. Moreover, dissociating innate differences in cognitive function from cannabis-induced deficits is challenging. Therefore, the current study sought to develop a rodent model of adolescent cannabinoid self-administration (SA), using the synthetic cannabinoid receptor agonist WIN55,212-2 (WIN), in order to assess measures of relapse/reinstatement of drug seeking and long-term effects on cognitive function assessed in a delay-match-to-sample working memory task and a spatial recognition task. Adolescent male rats readily self-administered WIN in 2-h or 6-h sessions/day, but did not demonstrate an escalation of intake with 6-h access. Rats exhibited significant cue-induced reinstatement of WIN seeking that increased with 21 days of abstinence (ie, 'incubation of craving'). Cognitive testing occurred in adulthood under drug-free conditions. Both 2-h and 6-h adolescent WIN SA groups exhibited significantly better working memory performance in adulthood relative to sucrose SA controls, and performance was associated with altered expression of proteins regulating GABAergic and glutamatergic signaling in the prefrontal cortex. Self-administered WIN did not produce either acute or chronic effects on short-term memory, but experimenter administration of WIN in adolescence, at doses previously reported in the literature, produced acute deficits in short-term memory that recovered with abstinence. Thus, SA of a rewarding cannabinoid in adolescence does not produce long-term cognitive dysfunction.

The gateway hypothesis posits that initial exposure to legal drugs promotes subsequent addiction to illicit drugs. However, epidemiological studies are correlational and cannot rule out the alternative hypothesis of shared addiction vulnerability to legal and illegal drugs. We tested the gateway hypothesis using established rat alcohol exposure procedures and cocaine self-administration and reinstatement (relapse) procedures. We gave Wistar or alcohol-preferring (P) rats intermittent access to water or 20% alcohol in their homecage for 7 weeks (three 24-h sessions/week). We also exposed Wistar rats to air or intoxicating alcohol levels in vapor chambers for 14-h/day for 7 weeks. We then tested the groups of rats for acquisition of cocaine self-administration using ascending cocaine doses (0.125, 0.25, 0.5, 1.0 mg/kg/infusion) followed by a dose-response curve after acquisition of cocaine self-administration. We then extinguished lever pressing and tested the rats for reinstatement of drug seeking induced by cocaine-paired cues and cocaine priming (0, 2.5, 5, 10 mg/kg, i.p.). Wistar rats consumed moderate amounts of alcohol (4.6 g/kg/24 h), P rats consumed higher amounts of alcohol (7.6 g/kg/24 h), and Wistar rats exposed to alcohol vapor had a mean blood alcohol concentration of 176.2 mg/dl during the last week of alcohol exposure. Alcohol pre-exposure had no effect on cocaine self-administration, extinction responding, and reinstatement of drug seeking. Pre-exposure to moderate, high, or intoxicating levels of alcohol had no effect on cocaine self-administration and relapse to cocaine seeking. Our data do not support the notion that alcohol is a gateway drug to cocaine.

Varenicline, a pharmacotherapy for tobacco addiction, reduces alcohol consumption in humans and rodents. The therapeutic potential of varenicline would escalate if it also diminished conditioned responses elicited by alcohol-predictive cues, which can precipitate relapse in abstinent individuals. We investigated this application, along with the underlying neural substrates, using a robust preclinical assay in which relapse to alcohol-seeking was triggered by re-exposure to an alcohol-associated environmental context. Male, Long-Evans rats received Pavlovian conditioning sessions in which one auditory conditioned stimulus (CS+) was paired with 15% ethanol and a second conditioned stimulus (CS-) was not. Ethanol was delivered into a port for oral consumption and port entries triggered by each CS were recorded. Extinction was then conducted in a different context where the CS+ and CS- were presented without ethanol. To stimulate relapse, both cues were subsequently presented without ethanol in the prior conditioning context. Systemic varenicline (0, 0.5 or 2.5 mg/kg; intraperitoneal) blocked context-induced relapse to alcohol-seeking without affecting the ability to make a port entry. It also reduced context-induced relapse to sucrose-seeking, but only at the 2.5 mg/kg dose. Neuropharmacological studies showed that context-induced relapse to alcohol-seeking was attenuated by bilateral microinfusion of varenicline (0.3 μl/side) into the nucleus accumbens (NAc; 0 or 3.5 μg), but not the ventral tegmental area (0, 2 or 4 μg). These data show for the first time that varenicline reduces relapse triggered by contexts that predict alcohol, and suggest that nicotinic acetylcholine receptors in the NAc are critical for this effect.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3) may control relapse of alcohol seeking, but previously available Group II agonists were unable to discriminate between mGluR2 and mGluR3. Here we use AZD8529, a novel positive allosteric mGluR2 modulator, to determine the role of this receptor for alcohol-related behaviors in rats. We assessed the effects of AZD8529 (20 and 40 mg/kg s.c.) on male Wistar rats trained to self-administer 20% alcohol and determined the effects of AZD8529 on self-administration, as well as stress-induced and cue-induced reinstatement of alcohol seeking. The on-target nature of findings was evaluated in Indiana P-rats, a line recently shown to carry a mutation that disrupts the gene encoding mGluR2. The behavioral specificity of AZD8529 was assessed using self-administration of 0.2% saccharin and locomotor activity tests. AZD8529 marginally decreased alcohol self-administration at doses that neither affected 0.2% saccharin self-administration nor locomotor activity. More importantly, cue- but not stress-induced alcohol seeking was blocked by the mGluR2 positive allosteric modulator. This effect of AZD8529 was completely absent in P rats lacking functional mGluR2s, demonstrating the receptor specificity of this effect. Our findings provide evidence for a causal role of mGluR2 in cue-induced relapse to alcohol seeking. They contribute support for the notion that positive allosteric modulators of mGluR2 block relapse-like behavior across different drug categories.

Nicotine, the main psychoactive component of tobacco, and (-)-Δ(9)-tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis, play major roles in tobacco and marijuana dependence as reinforcers of drug-seeking and drug-taking behavior. Drugs that act as inverse agonists of cannabinoid CB1 receptors in the brain can attenuate the rewarding and abuse-related effects of nicotine and THC, but their clinical use is hindered by potentially serious side effects. The recently developed CB1-receptor neutral antagonists may provide an alternative therapeutic approach to nicotine and cannabinoid dependence. Here we compare attenuation of nicotine and THC reinforcement and reinstatement in squirrel monkeys by the CB1-receptor inverse agonist rimonabant and by the recently developed CB1-receptor neutral antagonist AM4113. Both rimonabant and AM4113 reduced two effects of nicotine and THC that play major roles in tobacco and marijuana dependence: (1) maintenance of high rates of drug-taking behavior, and (2) priming- or cue-induced reinstatement of drug-seeking behavior in abstinent subjects (models of relapse). In contrast, neither rimonabant nor AM4113 modified cocaine-reinforced or food-reinforced operant behavior under similar experimental conditions. However, both rimonabant and AM4113 reduced cue-induced reinstatement in monkeys trained to self-administer cocaine, suggesting the involvement of a common cannabinoid-mediated mechanism in the cue-induced reinstatement for different drugs of abuse. These findings point to CB1-receptor neutral antagonists as a new class of medications for treatment of both tobacco dependence and cannabis dependence.

Addiction is a behavioral disease, of which core components can be modeled in rodents. Much evidence implicates drug-evoked synaptic plasticity in cocaine-evoked locomotor sensitization, cue-induced cocaine seeking, and incubation of cocaine craving. However, the type of plasticity evoked by different modalities of cocaine administration (eg contingent vs non-contingent) and its role in reshaping circuit function remains largely elusive. Here we exposed mice to various regimens of cocaine and recorded excitatory transmission onto identified medium-sized spiny neurons (MSN, expressing fluorescent proteins under the control of either D1R or D2R dopamine receptor promotor) in the nucleus accumbens at time points when behavioral adaptations are observed. In D1-MSN, we found the presence of GluA2-lacking α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) after single or chronic non-contingent exposure to cocaine as well as after cocaine self-administration (SA). We also report an increase in the AMPA/NMDA ratio (A/N) in D1-MSN, which was observed only after repeated passive injections associated with locomotor sensitization as well as in a condition of SA leading to seeking behavior. Remarkably, insertion of GluA2-lacking AMPARs was also detected in D2-MSN after SA of a high dose of cocaine but not regular dose (1.5 vs 0.75 mg/kg), which was the only condition where incubation of cocaine craving was observed in this study. Moreover, synapses containing GluA2-lacking AMPARs belonged to amygdala inputs in D2-MSN and to medial prefrontal cortex inputs in D1-MSN. Taken together this study allows for a refinement of a circuit model of addiction based on specific synaptic changes induced by cocaine.

Cocaine addiction and depression are comorbid disorders. Although it is well recognized that 5-hydroxytryptamine (5-HT; serotonin) plays a central role in depression, our understanding of its role in addiction is notably lacking. The 5-HT system in the brain is carefully controlled by a combined process of regulating 5-HT neuron firing through 5-HT autoreceptors, neurotransmitter release, enzymatic degradation, and reuptake by transporters. This study tests the hypothesis that activation of 5-HT1A autoreceptors, which would lessen 5-HT neuron firing, contributes to cocaine-seeking behaviors. Using 5-HT neuron-specific reduction of 5-HT1A autoreceptor gene expression in mice, we demonstrate that 5-HT1A autoreceptors are necessary for cocaine conditioned place preference. In addition, using designer receptors exclusively activated by designer drugs (DREADDs) technology, we found that stimulation of the serotonergic dorsal raphe nucleus (DRN) afferents to the nucleus accumbens (NAc) abolishes cocaine reward and promotes antidepressive-like behaviors. Finally, using a rat model of compulsive-like cocaine self-administration, we found that inhibition of dorsal raphe 5-HT1A autoreceptors attenuates cocaine self-administration in rats with 6 h extended access, but not 1 h access to the drug. Therefore, our findings suggest an important role for 5-HT1A autoreceptors, and thus DRNNAc 5-HT neuronal activity, in the etiology and vulnerability to cocaine reward and addiction. Moreover, our findings support a strategy for antagonizing 5-HT1A autoreceptors for treating cocaine addiction.

Alcohol addiction is a problem of great societal concern, for which there is scope to improve current treatments. One potential new treatment for alcohol addiction is based on disrupting the reconsolidation of the maladaptive Pavlovian memories that can precipitate relapse to drug-seeking behavior. In alcohol self-administering rats, we investigated the effects of bidirectionally modulating adrenergic signaling on the strength of a Pavlovian cue-alcohol memory, using a behavioral procedure that isolates the specific contribution of one maladaptive Pavlovian memory to relapse, the acquisition of a new alcohol-seeking response for an alcohol-associated conditioned reinforcer. The β-adrenergic receptor antagonist propranolol, administered in conjunction with memory reactivation, persistently disrupted the memory that underlies the capacity of a previously alcohol-associated cue to act as a conditioned reinforcer. By contrast, enhancement of adrenergic signaling by administration of the adrenergic prodrug dipivefrin at reactivation increased the strength of the cue-alcohol memory and potentiated alcohol seeking. These data demonstrate the importance of adrenergic signaling in alcohol-associated memory reconsolidation, and suggest a pharmacological target for treatments aiming to prevent relapse through the disruption of maladaptive memories.

Preclinical studies revealed contribution of N-methyl-D-aspartate receptors (NMDARs) to a variety of neuropsychiatric diseases including alcoholism, but development of NMDAR antagonists for therapeutic use has been a challenge, in part due to severe side effects. One of the key intracellular events resulting from stimulation of NMDAR is activation of calpains-calcium-dependent cysteine proteases. Here we studied whether inhibition of calpains would produce therapeutic-like effects of NMDAR antagonists but without their NMDAR-mediated side-effect profile. The calpain inhibitor A-705253 (3-10 mg/kg) was tested in a model of cue-induced reinstatement of alcohol-seeking behavior in post-dependent Wistar rats and in an alcohol deprivation effect (ADE) model in long-term alcohol drinking Wistar rats, two behavioral models for alcohol-seeking and relapse, respectively. We also tested the effect of A-705253 on the saccharine deprivation effect (SDE) as a selectivity measure. Acute treatment with A-705253 dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior. Repeated administration of A-705253 caused significant reductions of relapse-like excessive alcohol intake during the post-abstinence drinking days, an effect that persisted during two more successive drug-free drinking weeks, which was selective for the ADE as the SDE was unaffected. However, A-705253 did not produce psychostimulant, cognition impairing (delayed-matching-to-position), or psychotomimetic effects (specifically, phencyclidine discriminative stimulus effects). Taken together, these results demonstrate the involvement of calpains in alcohol-seeking and relapse and present a rationale for a novel pharmacological intervention that may reduce craving and relapse with minimal side effects in alcohol-dependent patients.

Cocaine is habit-forming because of its ability to enhance dopaminergic neurotransmission in the forebrain. In addition to neuronal inputs, forebrain dopamine circuits are modulated by hormonal influences; one of these is leptin, an adipose-derived hormone that attenuates the rewarding effects of food- and hunger-associated brain stimulation reward. Here we report reciprocal inhibition between the reward-related effects of leptin and the reward-related effects of cocaine in rats. First, we report that cocaine and the expectancy of cocaine each depresses plasma leptin levels. Second, we report that exogenous leptin, given systemically or directly into the ventral tegmental area, attenuates the ability of cocaine to elevate dopamine levels in the nucleus accumbens, the ability of cocaine to establish a conditioned place preference, and the ability of cocaine-predictive stimuli to prolong responding in extinction of cocaine-seeking. Thus, whereas leptin represents an endogenous antagonist of the habit-forming and habit-sustaining effects of cocaine, this antagonism is attenuated by cocaine and comes to be attenuated by the expectancy of cocaine.

Cocaine abuse remains a public health concern for which pharmacotherapies are largely ineffective. Comorbidities between cocaine abuse, depression, and anxiety support the development of novel treatments targeting multiple symptom clusters. Selective negative allosteric modulators (NAMs) targeting the metabotropic glutamate receptor 5 (mGlu5) subtype are currently in clinical trials for the treatment of multiple neuropsychiatric disorders and have shown promise in preclinical models of substance abuse. However, complete blockade or inverse agonist activity by some full mGlu5 NAM chemotypes demonstrated adverse effects, including psychosis in humans and psychotomimetic-like effects in animals, suggesting a narrow therapeutic window. Development of partial mGlu5 NAMs, characterized by their submaximal but saturable levels of blockade, may represent a novel approach to broaden the therapeutic window. To understand potential therapeutic vs adverse effects in preclinical behavioral assays, we examined the partial mGlu5 NAMs, M-5MPEP and Br-5MPEPy, in comparison with the full mGlu5 NAM MTEP across models of addiction and psychotomimetic-like activity. M-5MPEP, Br-5MPEPy, and MTEP dose-dependently decreased cocaine self-administration and attenuated the discriminative stimulus effects of cocaine. M-5MPEP and Br-5MPEPy also demonstrated antidepressant- and anxiolytic-like activity. Dose-dependent effects of partial and full mGlu5 NAMs in these assays corresponded with increasing in vivo mGlu5 occupancy, demonstrating an orderly occupancy-to-efficacy relationship. PCP-induced hyperlocomotion was potentiated by MTEP, but not by M-5MPEP and Br-5MPEPy. Further, MTEP, but not M-5MPEP, potentiated the discriminative-stimulus effects of PCP. The present data suggest that partial mGlu5 NAM activity is sufficient to produce therapeutic effects similar to full mGlu5 NAMs, but with a broader therapeutic index.

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.

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.