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Naltrexone (NTX), a homolog of the opiate antidote naloxone, is an orally active long-acting general opioid receptor antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of few FDA-approved medications for treatment of alcohol use disorder (AUD). While it was early on established that NTX acts by blocking the binding of endogenous opioid peptide ligands released by alcohol, experimental evidence emerged that could not be fully accounted for by this explanation alone, suggesting that NTX may have additional modes of action. Mu- and kappa-opioid receptors (MOP and KOP, respectively) are structurally related G-protein-coupled receptors (GPCRs), but they are anatomically differently distributed and functionally distinct, often mediating opposite responses, with MOP typically promoting euphoria and reward, while KOP is associated with dysphoria and aversive states. While the actions of NTX on MOP are extensively characterized, the interactions with KOP are not. Here, we used sensitive fluorescence-based methods with single-molecule sensitivity to study in live cells the influence of alcohol (ethanol, EtOH) on KOP and the interaction between KOP and NTX. Our data show that alcohol, at relevant concentrations (10-40 mM), alters KOP interactions with the lipid environment in the plasma membrane. The counteracting effects of NTX are exerted by both its canonical action on KOP and its hitherto unrevealed effects on the lateral dynamics and organization of lipids in the plasma membrane. The KOP-specific antagonist LY2444296, in clinical trial for major depressive disorder (MDD), blocks KOP but does not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may in part be due to direct actions on KOP and in part due to its effect on the surrounding lipid environment., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: All experiments were done in the laboratory and did not involve humans or animals. The experiments did not involve tissues from humans or other vertebrates. All experiments were done in accordance with scientific standards., (© 2024. The Author(s).)

Background: The orexin (OX) system has received increasing interest as a potential target for treating substance use disorder. OX transmission in the posterior paraventricular nucleus of the thalamus (pPVT), an area activated by highly salient stimuli that are both reinforcing and aversive, mediates cue- and stress-induced reinstatement of reward-seeking behavior. Oral administration of suvorexant (SUV), a dual OX receptor (OXR) antagonist (DORA), selectively reduced conditioned reinstatement of oxycodone-seeking behavior and stress-induced reinstatement of alcohol-seeking behavior in dependent rats., Aims: This study tested whether OXR blockade in the pPVT with SUV reduces oxycodone or sweetened condensed milk (SCM) seeking elicited by conditioned cues or stress., Methods: Male Wistar rats were trained to self-administer oxycodone (0.15 mg/kg, i.v., 8 h/day) or SCM (0.1 ml, 2:1 dilution [v/v], 30 min/day). After extinction, we tested the ability of intra-pPVT SUV (15 µg/0.5 µl) to prevent reinstatement of oxycodone or SCM seeking elicited by conditioned cues or footshock stress., Results: The rats acquired oxycodone and SCM self-administration, and oxycodone intake correlated with signs of physical opioid withdrawal, confirming dependence. Following extinction, the presentation of conditioned cues or footshock elicited reinstatement of oxycodone- and SCM-seeking behavior. Intra-pPVT SUV blocked stress-induced reinstatement of oxycodone seeking but not conditioned reinstatement of oxycodone or SCM seeking or stress-induced reinstatement of SCM seeking., Conclusions: The results indicate that OXR signaling in the pPVT is critical for stress-induced reinstatement of oxycodone seeking, further corroborating OXRs as treatment targets for opioid use disorder., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Alcohol use disorder (AUD) remains a major public health concern. The dynorphin (DYN)/κ-opioid receptor (KOP) system is involved in actions of alcohol, particularly its withdrawal-associated negative affective states. This study tested the ability of LY2444296, a selective, short-acting, KOP antagonist, to decrease alcohol self-administration in dependent male and female Wistar rats at 8 h abstinence. Animals were trained to orally self-administer 10% alcohol (30 min/day for 21 sessions) and were made dependent via chronic intermittent alcohol vapor exposure for 6 weeks or exposed to air (nondependent). After 6 weeks, the effect of LY2444296 (0, 3, and 10 mg/kg, p.o.) was tested on alcohol self-administration at 8 h of abstinence. A separate cohort of rats was prepared in parallel, and their somatic withdrawal signs and alcohol self-administration were measured after LY2444296 administration at 8 h, 2 weeks, and 4 weeks abstinence. LY2444296 at 3 and 10 mg/kg significantly reduced physical signs of withdrawal in dependent rats at 8 h abstinence, only. Furthermore, 3 and 10 mg/kg selectively decreased alcohol self-administration in dependent rats at only 8 h abstinence. These results highlight the DYN/KOP system in actions of alcohol during acute abstinence, suggesting KOP antagonism could be beneficial for mitigating acute withdrawal signs and, in turn, significantly reduce excessive alcohol consumption associated with AUD., (© 2024. The Author(s).)

Chronic opioid use disturbs circadian rhythm and sleep, encouraging opioid use and relapse. The orexin (OX) system is recruited by opioids and regulates physiological processes including sleep. Dual OX receptor antagonists (DORAs), developed for insomnia treatment, may relieve withdrawal-associated sleep disturbances. This study investigated whether DORA-12, a recently developed DORA, reduces physiological activity disturbances during oxycodone abstinence and consequently prevents oxycodone-seeking behavior. Male and female Wistar rats were trained to intravenously self-administer oxycodone (0.15 mg/kg, 21 sessions; 8 h/session) in the presence of a contextual/discriminative stimulus (S D ). The rats were subsequently housed individually (22 h/day) to monitor activity, food and water intake. They received DORA-12 (0-30 mg/kg, p.o.) after undergoing daily 1-h extinction training (14 days). After extinction, the rats were tested for oxycodone-seeking behavior elicited by the S D . Hypothalamus sections were processed to assess oxycodone- or DORA-12-associated changes to the OX cell number. In males, oxycodone-associated increases in activity during the light-phase, reinstatement, and decreases in the number of OX cells observed in the vehicle-treated group were not observed with DORA-12-treatment. Oxycodone-associated increases in light-phase food and water intake were not observed by day 14 of 3 mg/kg DORA-12-treatment and dark-phase water intake was increased across treatment days. In females, OX cell number was unaffected by oxycodone or DORA-12. Three and 30 mg/kg DORA-12 increased females' day 7 dark-phase activity and decreased reinstatement. Thirty mg/kg DORA-12 reduced oxycodone-associated increases in light-phase food and water intake. The results suggest that DORA-12 improves oxycodone-induced disruptions to physiological activities and reduces relapse., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Opioid abuse and overdose have risen to epidemic proportions in the United States. Oxycodone is the most abused prescription opioid. Treatments for opioid use disorder (OUD) seek to reduce vulnerability to relapse by reducing sources of reinforcement to seek drug (i.e., acute drug effects or drug withdrawal/craving). Accumulating evidence that glutamate release elicits drug-seeking behaviors has generated interest in pharmacotherapies targeting the glutamate system. Agonists and positive allosteric modulators of the metabotropic glutamate 2 (mGlu2) receptor decrease glutamate activity, reducing drug taking and seeking. The present study tested whether the mGlu2 receptor positive allosteric modulator ADX106772 reduces oxycodone self-administration and the conditioned reinstatement of oxycodone seeking without affecting behaviors directed toward a highly palatable nondrug reinforcer (sweetened condensed milk). Male Wistar rats were trained to self-administer oxycodone (0.15 mg/kg/infusion, i.v., 12 h/day) or sweetened condensed milk (SCM; diluted 2:1 v/v in H 2 O, orally, 30 min/day) for 13 days in the presence of a contextual/discriminative stimulus (S D ), and the ability of ADX106772 (0, 0.3, 1, 3 and-10 mg/kg, s. c.) to decrease self-administration was tested. The rats then underwent extinction training, during which oxycodone, SCM, and the S D were withheld. After extinction, the ability of ADX106772 to prevent S D -induced conditioned reinstatement of oxycodone and SCM seeking was tested. ADX106772 reduced oxycodone self-administration and conditioned reinstatement without affecting SCM self-administration or conditioned reinstatement. ADX106772 reduced oxycodone taking and seeking and did not affect the motivation for the palatable conventional reinforcer, SCM, suggesting that activating mGlu2 receptors with a positive allosteric modulator is a potential approach for prescription OUD treatment., Competing Interests: Declaration of competing interest None, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Over the past two decades, the escalating prescription of opioid medications for pain management has culminated in a widespread opioid epidemic, significantly impacting public health, social dynamics, and economic stability. The urgent need for improved treatments for opioid addiction necessitates a deeper understanding of its biological underpinnings, with genetic variations playing a crucial role in individual susceptibility to opioid use disorder (OUD) and influencing clinical practices. In this study, we leverage the genetic diversity of four rat strains (ACI/N, BN/NHsd, WKY/N, and F344/N) to examine the contribution of genetic factors to oxycodone metabolism and addiction-like behaviors. We used the extended access to intravenous oxycodone self-administration procedure (12 h/day, 0.15 mg/kg/injection) to comprehensively characterize oxycodone-related behaviors and pharmacokinetics. We measured escalation of oxycodone self-administration, motivation for drug consumption, tolerance to the analgesic effects of oxycodone, withdrawal-induced hyperalgesia, and oxycodone-induced respiratory depression. Additionally, we examined oxycodone-seeking behavior after four weeks of withdrawal by reintroducing the animals to environmental and cue stimuli previously associated with oxycodone self-administration. The findings revealed notable strain differences in several behavioral measures, including oxycodone metabolism. Intriguingly, BN/NHsd and WKY/N strains exhibited similar drug intake and escalation patterns but displayed significant disparities in oxycodone and oxymorphone metabolism. Minimal sex differences were observed within strains, primarily relating to oxycodone metabolism. In conclusion, this study identifies strain differences in the behavioral responses and pharmacokinetics associated with oxycodone self-administration in rats, providing a robust foundation for identifying genetic and molecular variants associated with various facets of the opioid addiction process., Competing Interests: Declaration of competing interest This work was supported by the National Institute on Drug Abuse [DA051972 and DA056602 to FT, CB and GdG, DA044451 to OG and DA053443 to RM-F], by the National Institute on Alcohol Abuse and Alcoholism [T32 AA007456 to MRD, AA026999, AA028549 and AA006420 to RM-F] by the Brain & Behavior Research Foundation [2020 NARSAD Young Investigator Award to GdG] and from the Preclinical Addiction Research Consortium (PARC) at the University of California San Diego. The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human heroin addicts and mice administered morphine for a 2 week period show a greatly increased number of hypothalamic hypocretin (Hcrt or orexin) producing neurons with a concomitant reduction in Hcrt cell size. Male rats addicted to cocaine similarly show an increased number of detectable Hcrt neurons. These findings led us to hypothesize that humans with alcohol use disorder (AUD) would show similar changes. We now report that humans with AUD have a decreased number and size of detectable Hcrt neurons. In addition, the intermingled melanin concentrating hormone (MCH) neurons are reduced in size. We saw no change in the size and number of tuberomammillary histamine neurons in AUD. Within the Hcrt/MCH neuronal field we found that microglia cell size was increased in AUD brains. In contrast, male rats with 2 week alcohol exposure, sufficient to elicit withdrawal symptoms, show no change in the number or size of Hcrt, MCH and histamine neurons, and no change in the size of microglia. The present study indicates major differences between the response of Hcrt neurons to opioids and that to alcohol in human subjects with a history of substance abuse., (Published by Elsevier Ltd.)

Background and Purpose: A major problem managing alcohol use disorder is the high vulnerability to relapse, even after long periods of abstinence. Chronic alcohol use dysregulates stress responsivity, rendering this system hyporesponsive and making individuals vulnerable to relapse. Orexin (hypocretin) plays a role in diverse physiological processes, including stress. Orexin neurons in the hypothalamus, project to the infralimbic cortex. This study asked does infralimbic cortex orexin transmission play a significant role in stress-induced reinstatement of alcohol-seeking behaviour in alcohol-dependent rats., Experimental Approach: Male and female rats were trained to self-administer 10% alcohol (3 weeks) and then made dependent via chronic intermittent alcohol vapour exposure. Following extinction (5 days·week -1 at 8 h abstinence for 10 sessions), rats received an intra- infralimbic cortex microinfusion of the OX 1/2 antagonist TCS 1102 (15 μg/0.5 μl per side) and then tested for footshock stress-induced reinstatement of alcohol seeking. In a separate cohort, orexin regulation of infralimbic cortex neuronal activity at the time of reinstatement was investigated using ex vivo electrophysiology., Key Results: TCS 1102 prevented reinstatement in dependent animals only. Moreover, Hcrtr mRNA expression in the hypothalamus and Hcrtr1/2 in the infralimbic cortex increased in alcohol-dependent animals at the time of testing. Dependence dampened basal orexin/OX receptor influence over infralimbic cortex GABAergic synapses (using TCS 1102) allow for greater stimulated orexin effects., Conclusion and Implications: Infralimbic cortex transmission is implicate in stress-induced reinstatement of alcohol-seeking behaviour in subjects with a history of alcohol dependence and show maladaptive recruitment of infralimbic cortex transmission by alcohol dependence., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: The Department of Health and Human Services reports that prescription pain reliever (e.g., oxycodone) misuse was initiated by 4,400 Americans each day in 2019. Amid the opioid crisis, effective strategies to prevent and treat prescription opioid use disorder (OUD) are pressing. In preclinical models, the orexin system is recruited by drugs of abuse, and blockade of orexin receptors (OX receptors) prevents drug-seeking behavior. The present study sought to determine whether repurposing suvorexant (SUV), a dual OX receptor antagonist marketed for the treatment of insomnia, can treat two features of prescription OUD: exaggerated consumption and relapse. Methods: Male and female Wistar rats were trained to self-administer oxycodone (0.15 mg/kg, i. v., 8 h/day) in the presence of a contextual/discriminative stimulus (S D ) and the ability of SUV (0-20 mg/kg, p. o.) to decrease oxycodone self-administration was tested. After self-administration testing, the rats underwent extinction training, after which we tested the ability of SUV (0 and 20 mg/kg, p. o.) to prevent reinstatement of oxycodone seeking elicited by the S D . Results: The rats acquired oxycodone self-administration and intake was correlated with the signs of physical opioid withdrawal. Additionally, females self-administered approximately twice as much oxycodone as males. Although SUV had no overall effect on oxycodone self-administration, scrutiny of the 8-h time-course revealed that 20 mg/kg SUV decreased oxycodone self-administration during the first hour in males and females. The oxycodone S D elicited strong reinstatement of oxycodone-seeking behavior that was significantly more robust in females. Suvorexant blocked oxycodone seeking in males and reduced it in females. Conclusions: These results support the targeting of OX receptors for the treatment for prescription OUD and repurposing SUV as pharmacotherapy for OUD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Illenberger, Flores-Ramirez, Matzeu, Mason and Martin-Fardon.)

Alcohol use disorder (AUD) is one of the most treatment-resistant medical conditions globally. The orexin (Orx) system regulates diverse physiological processes, including stress, and is a system of interest for the development of pharmaceuticals to treat substance use disorders, particularly AUD. The present study tested the ability of the dual orexin receptor antagonist suvorexant (SUV), marketed by Merck as Belsomra ® , for the treatment of insomnia, to decrease alcohol self-administration and the stress-induced reinstatement of alcohol-seeking behavior in male Wistar rats with a history of alcohol dependence. Rats were trained to orally self-administer 10% alcohol (30 min/day for 3 weeks) and were either made dependent via chronic intermittent alcohol vapor exposure (14 h ON, 10 h OFF) for 6 weeks or exposed to air (non-dependent). Starting on week 7, the effect of SUV (0-20 mg/kg, p.o.) was tested on alcohol self-administration at acute abstinence (8 h after vapor was turned OFF) twice weekly. A separate cohort of rats that were prepared in parallel was removed from alcohol vapor exposure and then subjected to extinction training for 14 sessions. Once extinction was achieved, the rats received SUV (0 and 5 mg/kg, p.o.) and were tested for the footshock stress-induced reinstatement of alcohol-seeking behavior. Suvorexant at 5, 10, and 20 mg/kg selectively decreased alcohol intake in dependent rats. Furthermore, 5 mg/kg SUV prevented the stress-induced reinstatement of alcohol-seeking behavior in dependent rats only. These results underscore the significance of targeting the Orx system for the treatment of substance use disorders generally and suggest that repurposing SUV could be an alternative approach for the treatment of AUD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Flores-Ramirez, Illenberger, Pascasio, Matzeu, Mason and Martin-Fardon.)

Background: Alcohol use disorder (AUD) is a leading preventable cause of death. The central amygdala (CeA) is a hub for stress and AUD, while dysfunction of the noradrenaline stress system is implicated in AUD relapse., Methods: Here, we investigated whether alcohol (ethanol) dependence and protracted withdrawal alter noradrenergic regulation of the amygdala in rodents and humans. Male adult rats were housed under control conditions, subjected to chronic intermittent ethanol vapor exposure to induce dependence, or withdrawn from chronic intermittent ethanol vapor exposure for 2 weeks, and ex vivo electrophysiology, biochemistry (catecholamine quantification by high-performance liquid chromatography), in situ hybridization, and behavioral brain-site specific pharmacology studies were performed. We also used real-time quantitative polymerase chain reaction to assess gene expression of α 1 B , β 1 , and β 2 adrenergic receptors in human postmortem brain tissue from men diagnosed with AUD and matched control subjects., Results: We found that α 1 receptors potentiate CeA GABAergic (gamma-aminobutyric acidergic) transmission and drive moderate alcohol intake in control rats. In dependent rats, β receptors disinhibit a subpopulation of CeA neurons, contributing to their excessive drinking. Withdrawal produces CeA functional recovery with no change in local noradrenaline tissue concentrations, although there are some long-lasting differences in the cellular patterns of adrenergic receptor messenger RNA expression. In addition, postmortem brain analyses reveal increased α 1 B receptor messenger RNA in the amygdala of humans with AUD., Conclusions: CeA adrenergic receptors are key neural substrates of AUD. Identification of these novel mechanisms that drive alcohol drinking, particularly during the alcohol-dependent state, supports ongoing new medication development for AUD., (Copyright © 2022 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Alcohol use dysregulates responsivity to stress, which is mediated by corticotropin-releasing factor (CRF). With repeated cycles of alcohol use, the hypothalamic-pituitary-adrenal axis becomes hyporesponsive, rendering individuals vulnerable to the reinstatement of alcohol-seeking behavior during stressful episodes. Orexin (Orx; also called hypocretin) plays a well-established role in regulating diverse physiological processes, including stress, and interacts with CRF. The infralimbic cortex (IL) is a CRF-rich region. Anatomical evidence suggests that CRF and Orx interact in this area. To test the behavioral implication of CRF and Orx transmission in the IL during the stress-induced reinstatement of alcohol-seeking behavior, male Wistar rats were trained to self-administer 10% alcohol for 3 weeks. The rats then underwent two weeks of extinction training (identical to the alcohol self-administration sessions, but alcohol was withheld). The day after the last extinction session, the rats received a bilateral intra-IL injection of the CRF 1 receptor antagonist CP154,526 (0.6 μg/0.5 μl/side), the dual Orx receptor antagonist TCS1102 (15 μg/0.5 μl/side), or their combination and then were tested for the footshock stress-induced reinstatement of alcohol-seeking behavior. CP154,526 significantly prevented reinstatement, but TCS1102 did not produce such an effect. Interestingly, the co-administration of TCS1102 and CP154,526 reversed the effect of CP154,526 alone, and footshock stress induced a significant increase in Crhr1 and Hcrtr2 mRNA expression in the IL. These results demonstrate a functional interaction between Orx receptor and CRF 1 receptor signaling and suggest that CRF 1 receptor antagonism may ameliorate stress-induced alcohol-seeking behavior., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background and Purpose: 'Food addiction' is the subject of intense public and research interest. However, this nosology based on neurobehavioural similarities among obese individuals, patients with eating disorders and those with substance use disorders (drug addiction) remains controversial. We thus sought to determine which aspects of disordered eating are causally linked to preclinical models of drug addiction. We hypothesized that extensive drug histories, known to cause addiction-like brain changes and drug motivation in rats, would also cause addiction-like food motivation., Experimental Approach: Rats underwent extensive cocaine, alcohol, caffeine or obesogenic diet histories and were subsequently tested for punishment-resistant food self-administration or 'compulsive appetite', as a measure of addiction-like food motivation., Key Results: Extensive cocaine and alcohol (but not caffeine) histories caused compulsive appetite that persisted long after the last drug exposure. Extensive obesogenic diet histories also caused compulsive appetite, although neither cocaine nor alcohol histories caused excess calorie intake and bodyweight during abstinence. Hence, compulsive appetite and obesity appear to be dissociable, with the former sharing common mechanisms with preclinical drug addiction models., Conclusion and Implications: Compulsive appetite, as seen in subsets of obese individuals and patients with binge-eating disorder and bulimia nervosa (eating disorders that do not necessarily result in obesity), appears to epitomize 'food addiction'. Because different drug and obesogenic diet histories caused compulsive appetite, overlapping dysregulations in the reward circuits, which control drug and food motivation independently of energy homeostasis, may offer common therapeutic targets for treating addictive behaviours across drug addiction, eating disorders and obesity., (© 2022 The British Pharmacological Society.)

Orexins (also known as hypocretins) are neuropeptides that participate in the regulation of energy metabolism, homeostasis, sleep, feeding, stress responses, arousal, and reward. Particularly relevant to the scope of the present review is the involvement of the orexin system in brain mechanisms that regulate motivation, especially highly motivated behavior, arousal, and stress, making it an ideal target for studying addiction and discovering treatments. Drug abuse and misuse are thought to induce maladaptive changes in the orexin system, and these changes might promote and maintain uncontrolled drug intake and contribute to relapse. Dysfunctional changes in this neuropeptidergic system that are caused by drug use might also be responsible for alterations of feeding behavior and the sleep-wake cycle that are commonly disrupted in subjects with substance use disorder. Drug addiction has often been associated with an increase in activity of the orexin system, suggesting that orexin receptor antagonists may be a promising pharmacological treatment for substance use disorder. Substantial evidence has shown that single orexin receptor antagonists that are specific to either orexin receptor 1 or 2 can be beneficial against drug intake and relapse. Interest in the efficacy of dual orexin receptor antagonists, which were primarily developed to treat insomnia, has grown in the field of drug addiction. Treatments that target the orexin system may be a promising strategy to reduce drug intake, mitigate relapse vulnerability, and restore "normal" physiological functions, including feeding and sleep. The present review discusses preclinical and clinical evidence of the involvement of orexins in drug addiction and possible beneficial pharmacotherapeutic effects of orexin receptor antagonists to treat substance use disorder., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Matzeu and Martin-Fardon.)

Background: Orexin-A (OrxA) administration in the posterior paraventricular nucleus of the thalamus (pPVT) reinstates extinguished cocaine-seeking behaviour following extended access to the drug (a model of dependence). The pPVT receives and integrates information associated with emotionally salient events and sends excitatory inputs to brain regions involved in the expression of emotional states, such as those driving cocaine-seeking behaviour (i.e., the nucleus accumbens, the central nucleus of the amygdala [CeA], the basolateral amygdala, the bed nucleus of the stria terminalis [BNST] and the prefrontal cortex)., Methods: We monitored the activation pattern of these regions (measured by Fos) during cocaine-seeking induced by OrxA administered to the pPVT. The BNST and CeA emerged as being selectively activated. To test whether the functionality of these regions was pivotal during OrxA-induced cocaine-seeking behaviour, we transiently inactivated these regions concomitantly with OrxA administration to the pPVT. We then tested the participation of corticotropin-releasing factor receptors (CRF1) in the CeA during OrxA-induced cocaine-seeking using the CRF1 antagonist CP154526., Results: We observed selective activation of the CeA and BNST during cocaine-seeking induced by OrxA administered to the pPVT, but only transient inactivation of the CeA prevented cocaine-seeking behaviour. Administration of CP154526 to the CeA prevented OrxAinduced cocaine-seeking behaviour., Limitations: The use of only male rats could have been a limitation. Other limitations could have been the use of an indirect approach to test the hypothesis that administration of OrxA to the pPVT drives cocaine-seeking via CRF1 signalling in the CeA, and a lack of analysis of the participation of CeA subregions., Conclusion: Cocaine-seeking behaviour induced by OrxA administered to the pPVT is driven by activation of the CeA via CRF1 signalling., Competing Interests: None declared., (© 2021 CMA Joule Inc. or its licensors.)

Hypothalamic orexin (Orx) projections to the paraventricular nucleus of the thalamus (PVT) have received growing interest because of their role in drug-seeking behavior. Using an established model of cocaine dependence (i.e., long access [LgA] to cocaine), we previously showed that OrxA injections in the posterior PVT (pPVT) reinstated extinguished cocaine-seeking behavior in rats after an intermediate period of abstinence (2-3 weeks). Considering the long-lasting nature of drug-seeking behavior, the present study examined whether the priming effect of intra-pPVT OrxA administration was preserved after a period of protracted abstinence (4-5 weeks) in rats that self-administered cocaine under LgA conditions. Furthermore, to better understand whether a history of cocaine dependence affects the Orx system-particularly the hypothalamic Orx↔pPVT connection-the number of Orx-expressing cells in the lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), and perifornical area (PFA) and number of orexin receptor 1 (OrxR1)- and OrxR2-expressing cells in the pPVT were quantified. Orexin A administration in the pPVT induced cocaine-seeking behavior after intermediate abstinence, as reported previously. At protracted abstinence, however, the priming effect of OrxA was absent. A higher number of cells that expressed Orx was observed in the LH/DMH/PFA at both intermediate and protracted abstinence. In the pPVT, the number of OrxR2-expressing cells was significantly higher only at intermediate abstinence, with no changes in the number of OrxR1-expressing cells. These data build on our previous findings that the hypothalamic Orx↔pPVT connection is strongly recruited shortly after cocaine abstinence and demonstrate that the priming effect of OrxA is not long lasting. Furthermore, these findings suggest that throughout abstinence, the Orx↔pPVT connection undergoes neuroadaptive changes, reflected by alterations of the number of OrxR2-expressing cells in the pPVT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Matzeu and Martin-Fardon.)

The endocannabinoid system is involved in the regulation of the stress response, but the relative contribution of N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) and their mechanisms have to be elucidated. In this study, we compared the effects of the pharmacological inhibition of the two major endocannabinoid-degrading enzymes [fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) for AEA and 2-AG, respectively] on stress-coping [forced swim test (FST) and tail suspension test (TST)] and anxiety-like [elevated-plus maze (EPM) and light-dark test (LDT)] behaviors in wild-type and FAAH knockout mice. In vivo microdialysis estimated the effects of FAAH and MAGL inhibition on dopamine (DA) and serotonin (5-HT) levels in the medial prefrontal cortex (mPFC) during an FST. Mice were treated with PF-3845 (FAAH inhibitor), JZL184 (MAGL inhibitor), JZL195 (dual FAAH/MAGL inhibitor) or vehicle. Our data showed that PF-3845 increased latency to immobility and decreased total immobility time in FST, but no effects were observed in TST compared with vehicle-treated wild-type mice. By contrast, JZL184 decreased latency and increased immobility in TST and FST. JZL195 in wild-type mice and JZL184 in FAAH knockout mice reproduced the same passive coping behaviors as JZL184 in wild-type mice in TST and FST. In the microdialysis experiment, FST was associated with increased DA and 5-HT levels in the mPFC. However, JZL184-treated wild-type mice displayed a significant attenuation of forced swim stress-induced DA release compared with vehicle-treated wild-type mice and PF-3845-treated wild-type mice. Finally, FAAH and/or MAGL inhibitors induced robust and consistent anxiolytic-like effects in EPM and LDT. These results suggested differences between FAAH and MAGL inhibition in stress-coping behaviors. Notably, MAGL inhibition induced a consistent avoidant coping behavior and attenuated the stress-induced mPFC DA response in FST. However, more investigation is needed to elucidate the functional association between DA and 2-AG signaling pathways, and the molecular mechanism in the regulation of passive coping strategies during inescapable stress., Competing Interests: All authors declare that they have no conflicts of interest., (© 2021 The Authors.)

Neural systems involved in processing natural rewards and drugs of abuse overlap and exposure to drugs of abuse induce neuroadaptations that can cause compulsive-like behavior. For example, the recruitment of the orexin (Orx) system by drugs of abuse has been proposed to induce neuroadaptations that in turn alter its function, reflected by maladaptive, compulsive, and addictive behavior. Orexin neurons project to the paraventricular nucleus of the thalamus (PVT)-particularly the posterior part (pPVT), a structure that plays a key role in stress regulation. This study investigated whether Orx transmission in the pPVT plays a role in stress-induced reinstatement of reward-seeking behavior toward ethanol (EtOH) and a highly palatable food reward [sweetened condensed milk (SCM)] in rats and whether this role changes with EtOH dependence. After being trained to orally self-administer EtOH or SCM, the rats were made dependent (EtOH D and SCM D ) by chronic intermittent EtOH vapor exposure. The control nondependent groups (EtOH ND and SCM ND ) were exposed to air. Following extinction, the rats were tested for stress-induced reinstatement of EtOH- and SCM-seeking behavior. Stress reinstated EtOH- and SCM-seeking behavior in all groups (EtOH D/ND and SCM D/ND ). Administration of the dual Orx receptor (OrxR) antagonist TCS1102 (15 μg) in the pPVT prevented stress-induced reinstatement only in dependent rats (EtOH D and SCM D ). In parallel, the qPCR analysis showed that Orx mRNA expression in the hypothalamus and OrxR1 / R2 mRNA expression in the pPVT were increased at the time of testing in the EtOH D and SCM D groups. These results are the first to implicate Orx transmission in the pPVT in the stress-induced reinstatement of reward-seeking behavior in EtOH dependent rats and indicate the maladaptive recruitment of Orx transmission in the pPVT by EtOH dependence., (Copyright © 2020 Matzeu and Martin-Fardon.)

Background: Ethanol (EtOH) self-administration is particularly sensitive to the modulation of CB 1 signaling in the nucleus accumbens (NAc) shell, and EtOH consumption increases extracellular levels of the endogenous cannabinoid CB 1 receptor agonist 2-arachidonoyl glycerol (2-AG) in this brain region. Stimulation of CB 1 receptor with agonists increases EtOH consumption, suggesting that EtOH-induced increases in 2-AG might sustain motivation for EtOH intake., Methods: In order to further explore this hypothesis, we analyzed the alterations in operant EtOH self-administration induced by intra-NAc shell infusions of 2-AG itself, the CB 1 inverse agonist SR141716A, the 2-AG clearance inhibitor URB602, anandamide, and the cyclooxygenase-2 (COX-2) inhibitor nimesulide., Results: Surprisingly, self-administration of 10% EtOH was dose-dependently reduced by either intra-NAc shell SR141716A or 2-AG infusions. Similar effects were found by intra-NAc shell infusions of URB602, suggesting again a role for accumbal 2-AG on the modulation of EtOH intake. Intra-NAc shell anandamide did not alter EtOH self-administration, pointing to a specific role for 2-AG in the modulation of EtOH self-administration. Finally, the inhibitory effect of intra-NAc shell 2-AG on EtOH intake was significantly reversed by pretreatment with nimesulide, suggesting that oxidative metabolites of 2-AG might mediate these inhibitory effects on operant self-administration., Conclusions: We propose that 2-AG signaling in the NAc exerts an inhibitory influence on EtOH consumption through a non-CB1 receptor mechanism involving the COX-2 pathway., (© 2020 by the Research Society on Alcoholism.)

Administration of selective serotonin reuptake inhibitors (SSRIs), typically used as antidepressants, induces long-lasting behavioral changes associated with alcohol use disorder (AUD). However, the contribution of SSRI (fluoxetine)-induced alterations in neurobiological processes underlying alcohol relapse such as endocannabinoid and glutamate signaling in the central amygdala (CeA) remains largely unknown. We utilized an integrative approach to study the effects of repeated fluoxetine administration during abstinence on ethanol drinking. Gene expression and biochemical and electrophysiological studies explored the hypothesis that dysregulation in glutamatergic and endocannabinoid mechanisms in the CeA underlie the susceptibility to alcohol relapse. Cessation of daily treatment with fluoxetine (10 mg/kg) during abstinence resulted in a marked increase in ethanol seeking during re-exposure periods. The increase in ethanol self-administration was associated with (a) reductions in levels of the endocannabinoids N-arachidonoylethanolomine and 2-arachidonoylglycerol in the CeA, (b) increased amygdalar gene expression of cannabinoid type-1 receptor (CB1), N-acyl phosphatidylethanolamine phospholipase D (Nape-pld), fatty acid amid hydrolase (Faah), (c) decreased amygdalar gene expression of ionotropic AMPA (GluA2 and GluA4) and metabotropic (mGlu3) glutamate receptors, and (d) increased glutamatergic receptor function. Overall, our data suggest that the administration of the antidepressant fluoxetine during abstinence dysregulates endocannabinoid signaling and glutamatergic receptor function in the amygdala, facts that likely facilitate alcohol drinking behavior during relapse., (© 2019 Society for the Study of Addiction.)

Prescription opioids are potent analgesics that are used for clinical pain management. However, the nonmedical use of these medications has emerged as a major concern because of dramatic increases in abuse and overdose. Therefore, effective strategies to prevent prescription opioid use disorder are urgently needed. The orexin system has been implicated in the regulation of motivation, arousal, and stress, making this system a promising target for the treatment of substance use disorder. This review discusses recent preclinical studies that suggest that orexin receptor blockade could be beneficial for the treatment of prescription opioid use disorder.

Prescription opioids, such as oxycodone, are potent analgesics that are used to treat and manage pain. However, oxycodone is one of the most commonly abused prescription drugs. Finding an effective strategy to prevent prescription opioid use disorder is urgent. Orexin receptors (OrxR1 and OrxR2) have been implicated in the regulation of motivation, arousal, and stress, making them possible targets for the treatment of substance use disorder. To study the significance of environmental stimuli in maintaining the vulnerability to relapse to oxycodone use, resistance to the extinction of oxycodone-seeking behavior that was elicited by an oxycodone-related stimulus was examined. Rats were trained to self-administer oxycodone in the presence of a contextual/discriminative stimulus (S D ). Using this procedure, the rats readily acquired oxycodone self-administration and exhibited increases in physical signs of opioid withdrawal. Following extinction, response-reinstating effects of re-exposure to the S D perseverated. We then tested whether OrxR blockade prevents oxycodone intake and relapse. The effects of the OrxR1 antagonist SB334867 and OrxR2 antagonist TCSOX229 on oxycodone self-administration were tested. SB334867 significantly decreased oxycodone self-administration, whereas TCSOX229 did not produce any effect. To investigate whether OrxR1 and OrxR2 blockade prevents oxycodone seeking, the rats were tested for the ability of SB334867 and TCSOX229 to prevent the S D -induced conditioned reinstatement of oxycodone-seeking behavior. SB334867 decreased oxycodone-seeking behavior, whereas TCSOX229 was ineffective. These results suggest that OrxR1 antagonism prevents excessive prescription opioid use and relapse and might be beneficial for the treatment of prescription opioid use disorder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Alcohol use disorder (AUD) is a chronic, relapsing disorder that is characterized by the compulsive use of alcohol despite numerous health, social, and economic consequences. Initially, the use of alcohol is driven by positive reinforcement. Over time, however, alcohol use can take on a compulsive quality that is driven by the desire to avoid the negative consequences of abstinence, including negative affect and heightened stress/anxiety. This transition from positive reinforcement- to negative reinforcement-driven consumption involves the corticotropin-releasing factor (CRF) system, although mounting evidence now suggests that the CRF system interacts with other neural systems to ultimately produce behaviors that are symptomatic of compulsive alcohol use, such as the hypocretin (Hcrt) system. Hypocretins are produced exclusively in the hypothalamus, but Hcrt neurons project widely throughout the brain and reach regions that perform regulatory functions for numerous behavioral and physiological responses-including the infralimbic cortex (IL) of the medial prefrontal cortex (mPFC). Although the entire mPFC undergoes neuroadaptive changes following prolonged alcohol exposure, the IL appears to undergo more robust changes compared with other mPFC substructures. Evidence to date suggests that the IL is likely involved in EtOH-seeking behavior, but ambiguities with respect to the specific role of the IL in this regard make it difficult to draw definitive conclusions. Furthermore, the manner in which CRF interacts with Hcrt in this region as it pertains to alcohol-seeking behavior is largely unknown, although immunohistochemical and electrophysiological experiments have shown that CRF and Hcrt directly interact in the mPFC, suggesting that the interaction between CRF and Hcrt in the IL may be critically important for the development and subsequent maintenance of compulsive alcohol seeking. This review aims to consolidate recent literature regarding the role of the IL in alcohol-seeking behavior and to discuss evidence that supports a functional interaction between Hcrt and CRF in the IL., (© 2019 by the Research Society on Alcoholism.)

Deletion of fatty acid amide hydrolase (FAAH), enzyme responsible for degrading endocannabinoids, increases alcohol consumption and preference. However, there is a lack of data on neurochemical events in mice exposed to alcohol in the absence of FAAH. Extracellular levels of endocannabinoids and relevant neurotransmitters were measured by in vivo microdialysis in the nucleus accumbens (NAc) of FAAH knockout (KO) and wild-type (WT) mice during an ethanol (EtOH; 2 g/kg, ip) challenge in EtOH-naive and repeated (r) EtOH-treated mice. In both genotypes, EtOH treatment caused no changes in baseline endocannabinoid levels, although FAAH KO mice displayed higher baseline N-arachidonoylethanolamine levels than WT mice. EtOH challenge caused a sustained increase in 2-arachidonoylglycerol (2-AG) levels in EtOH-naive WT mice but not in FAAH KO mice. In contrast, 2-AG levels were decreased following EtOH challenge in (r)EtOH-treated mice in both genotypes. Whereas (r)EtOH-treated mice showed higher baseline dopamine and serotonin levels than EtOH-naive mice in WT mice, these differences were attenuated in FAAH KO mice. Significant differences in baseline γ-aminobutyric acid (GABA) and glutamate levels by EtOH history were observed in WT mice but not in FAAH KO mice. Moreover, opposed effects on glutamate response were observed after EtOH challenge in EtOH-naive and (r)EtOH-treated FAAH KO mice. Finally, FAAH deletion failed to show EtOH-induced locomotion sensitivity. These data provide evidence of a potential influence of 2-AG in the neurochemical response to EtOH exposure in the NAc., (© 2018 Society for the Study of Addiction.)

Background: Despite considerable efforts, few drugs are available for the treatment of alcohol (ethanol [EtOH]) use disorder (AUD). EtOH directly or indirectly modulates several aspects of the central nervous system, including neurotransmitter/neuromodulator systems. Relapse vulnerability is a challenge for the treatment of EtOH addiction. EtOH withdrawal symptoms create motivational states that lead to compulsive EtOH drinking and relapse even after long periods of abstinence. Among the therapeutics to treat AUD, naltrexone (NTX) is a pharmacological treatment for relapse. The present study evaluated the effect of NTX on EtOH drinking in male and female EtOH-dependent rats during abstinence., Methods: Wistar rats (males and females) were first trained to orally self-administer 10% EtOH. Half of the rats were then made dependent by chronic intermittent EtOH (CIE) vapor exposure, and the other half were exposed to air. Using this model, rats exhibit somatic and motivational signs of withdrawal. At the end of EtOH vapor (or air) exposure, the rats were tested for the effects of NTX (10 mg/kg, oral) on EtOH self-administration at 3 abstinence time points: acute abstinence (A-Abst, 8 hours), late abstinence (L-Abst, 2 weeks), and protracted abstinence (P-Abst, 6 weeks)., Results: NTX decreased EtOH intake in nondependent rats, regardless of sex and abstinence time point. In postdependent rats, NTX decreased EtOH intake only at a delayed abstinence time point (P-Abst) in males, whereas it similarly reduced EtOH drinking in females at all abstinence time points., Conclusions: The therapeutic efficacy of NTX depends on the time of intervention during abstinence and is different between males and females. The data further suggest that EtOH dependence causes different neuroadaptations in male and female rats, reflected by differential effects of NTX. The results underscore the significance of considering the duration of EtOH abstinence and sex as a biological variable as important factors when developing pharmacotherapies for AUD., (© 2018 by the Research Society on Alcoholism.)