Spironolactone as a potential new pharmacotherapy for alcohol use disorder: convergent evidence from rodent and human studies.

Evidence suggests that spironolactone, a nonselective mineralocorticoid receptor (MR) antagonist, modulates alcohol seeking and consumption. Therefore, spironolactone may represent a novel pharmacotherapy for alcohol use disorder (AUD). In this study, we tested the effects of spironolactone in a mouse model of alcohol drinking (drinking-in-the-dark) and in a rat model of alcohol dependence (vapor exposure). We also investigated the association between spironolactone receipt for at least 60 continuous days and change in self-reported alcohol consumption, using the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), in a pharmacoepidemiologic cohort study in the largest integrated healthcare system in the US. Spironolactone dose-dependently reduced the intake of sweetened or unsweetened alcohol solutions in male and female mice. No effects of spironolactone were observed on drinking of a sweet solution without alcohol, food or water intake, motor coordination, alcohol-induced ataxia, or blood alcohol levels. Spironolactone dose-dependently reduced operant alcohol self-administration in dependent and nondependent male and female rats. In humans, a greater reduction in alcohol consumption was observed among those who received spironolactone, compared to propensity score-matched individuals who did not receive spironolactone. The largest effects were among those who reported hazardous/heavy episodic alcohol consumption at baseline (AUDIT-C ≥ 8) and those exposed to ≥ 50 mg/day of spironolactone. These convergent findings across rodent and human studies demonstrate that spironolactone reduces alcohol use and support the hypothesis that this medication may be further studied as a novel pharmacotherapy for AUD.

Oxytocin blocks enhanced motivation for alcohol in alcohol dependence and blocks alcohol effects on GABAergic transmission in the central amygdala.

Oxytocin administration has been reported to decrease consumption, withdrawal, and drug-seeking associated with several drugs of abuse and thus represents a promising pharmacological approach to treat drug addiction. We used an established rat model of alcohol dependence to investigate oxytocin's effects on dependence-induced alcohol drinking, enhanced motivation for alcohol, and altered GABAergic transmission in the central nucleus of the amygdala (CeA). Intraperitoneal oxytocin administration blocked escalated alcohol drinking and the enhanced motivation for alcohol in alcohol-dependent but not nondependent rats. Intranasal oxytocin delivery fully replicated these effects. Intraperitoneal administration had minor but significant effects of reducing locomotion and intake of non-alcoholic palatable solutions, whereas intranasal oxytocin administration did not. In dependent rats, intracerebroventricular administration of oxytocin or the oxytocin receptor agonist PF-06655075, which does not cross the blood-brain barrier (i.e., it would not diffuse to the periphery), but not systemic administration of PF-06655075 (i.e., it would not reach the brain), decreased alcohol drinking. Administration of a peripherally restricted oxytocin receptor antagonist did not reverse the effect of intranasal oxytocin on alcohol drinking. Ex vivo electrophysiological recordings from CeA neurons indicated that oxytocin decreases evoked GABA transmission in nondependent but not in dependent rats, whereas oxytocin decreased the amplitude of spontaneous GABAergic responses in both groups. Oxytocin blocked the facilitatory effects of acute alcohol on GABA release in the CeA of dependent but not nondependent rats. Together, these results provide converging evidence that oxytocin specifically and selectively blocks the enhanced motivation for alcohol drinking that develops in alcohol dependence likely via a central mechanism that may result from altered oxytocin effects on CeA GABA transmission in alcohol dependence. Neuroadaptations in endogenous oxytocin signaling may provide a mechanism to further our understanding of alcohol use disorder.

A closer look at alcohol-induced changes in the ghrelin system: novel insights from preclinical and clinical data.

Ghrelin is a gastric-derived peptide hormone with demonstrated impact on alcohol intake and craving, but the reverse side of this bidirectional link, that is, the effects of alcohol on the ghrelin system, remains to be fully established. To further characterize this relationship, we examined (1) ghrelin levels via secondary analysis of human laboratory alcohol administration experiments with heavy-drinking participants; (2) expression of ghrelin, ghrelin receptor, and ghrelin-O-acyltransferase (GOAT) genes (GHRL, GHSR, and MBOAT4, respectively) in post-mortem brain tissue from individuals with alcohol use disorder (AUD) versus controls; (3) ghrelin levels in Ghsr knockout and wild-type rats following intraperitoneal (i.p.) alcohol administration; (4) effect of alcohol on ghrelin secretion from gastric mucosa cells ex vivo and GOAT enzymatic activity in vitro; and (5) ghrelin levels in rats following i.p. alcohol administration versus a calorically equivalent non-alcoholic sucrose solution. Acyl- and total-ghrelin levels decreased following acute alcohol administration in humans, but AUD was not associated with changes in central expression of ghrelin system genes in post-mortem tissue. In rats, alcohol decreased acyl-ghrelin, but not des-acyl-ghrelin, in both Ghsr knockout and wild-type rats. No dose-dependent effects of alcohol were observed on acyl-ghrelin secretion from gastric mucosa cells or on GOAT acylation activity. Lastly, alcohol and sucrose produced distinct effects on ghrelin in rats despite equivalent caloric value. Our findings suggest that alcohol acutely decreases peripheral ghrelin concentrations in vivo, but not in proportion to alcohol's caloric value or through direct interaction with ghrelin-secreting gastric mucosal cells, the ghrelin receptor, or the GOAT enzyme.

Intermittent access training produces greater motivation for a non-drug reinforcer than long access training.

It has recently been proposed that the intermittent access (IntA) drug self-administration procedure better produces behavioral changes relevant to addiction than the long access (LgA) procedure. In this version of the IntA procedure, the drug is made available for a 5-min period during each half hour of a 6-h session. In contrast, on the LgA procedure, the drug is available continuously for 6 h. Previous studies have found that IntA drug self-administration produces greater drug motivation, measured by increased progressive ratio breakpoints, than LgA self-administration. It has been hypothesized that this effect is due to the rapid, "spiking" brain levels of the drug, and consequent neuroadaptations, experienced by rats during IntA sessions. However, no study has compared the effects of IntA versus LgA training on reinforcer motivation when using a non-drug reinforcer. The present study compared motivation for a saccharin reinforcer after IntA or LgA training. In Experiment 1, separate groups of rats lever-pressed for saccharin on the IntA or LgA procedures. In Experiment 2, a within-subjects design was used where rats pressed one lever on the IntA procedure and another lever on the LgA procedure for saccharin. In both experiments, IntA training produced greater breakpoints than LgA training. As no drug was used here, spiking drug levels could not have been responsible for the increased saccharin motivation observed after IntA training. Instead, it is proposed that differences in stimulus-reinforcer associations learned during IntA versus LgA training may be responsible for the effect. Future research is needed to determine the extent to which such learning factors may contribute to the increased motivation observed after IntA training with drug reinforcers.

Discriminative stimulus properties of the 5-HT1A receptor biased agonists NLX-101 and F13714, in rats trained to discriminate 8-OH-DPAT from saline.

NLX-101 and F13714 are selective, full efficacy, biased agonists of the serotonin (5-HT1A) receptor. NLX-101 preferentially activates cortical postsynaptic 5-HT1A receptors, whereas F13714 preferentially activates raphe nuclei presynaptic 5-HT1A receptors. We compared NLX-101 and F13714 for their efficacy and potency to substitute for the discriminative cue produced by the prototypical, nonbiased 5-HT1A receptor agonist 8-OH-DPAT (racemate). Male and female Sprague-Dawley rats were trained to discriminate 8-OH-DPAT (0.1 mg/kg i.p., 20 min pretreatment) from saline using a classical two-lever drug-discrimination procedure. 8-OH-DPAT (0.01 and 0.05 mg/kg i.p.) dose-dependently substituted for the training dose, with about 50% responding on the 8-OH-DPAT-associated lever at 0.05 mg/kg. F13714 fully and very potently substituted for the training dose of 8-OH-DPAT from 0.018 mg/kg i.p., whereas NLX-101 only achieved full substitution at 0.5 mg/kg i.p., a dose which is known to also activate presynaptic 5-HT1A receptors. The 5-HT1A receptor partial agonist, buspirone, partially substituted (~80%) at 1 and 2 mg/kg i.p., doses which also decreased response rates. F13714 decreased response rates at 0.05 mg/kg. The selective 5-HT1A receptor antagonist WAY-100 635 (1 mg/kg s.c., 40 min pretreatment) elicited almost no responding on the 8-OH-DPAT-associated lever by itself, but blocked the discriminative stimulus effects produced by administration (20 min pretreatment) of 8-OH-DPAT (0.1 mg/kg), F13714 (0.025 mg/kg), NLX-101 (0.5 mg/kg) or buspirone (1 mg/kg). These data suggest that the discriminative cue produced by 0.1 mg/kg i.p. 8-OH-DPAT results from activation of presynaptic 5-HT1A receptors. They also further demonstrate the distinct profiles in behavioral models of 5-HT1A receptor-biased agonists.

Probenecid Reduces Alcohol Drinking in Rodents. Is Pannexin1 a Novel Therapeutic Target for Alcohol Use Disorder?

AIMS: The development of novel and more effective medications for alcohol use disorder (AUD) is an important unmet medical need. Drug repositioning or repurposing is an appealing strategy to bring new therapies to the clinic because it greatly reduces the overall costs of drug development and expedites the availability of treatments to those who need them. Probenecid, p-(di-n-propylsulfamyl)-benzoic acid, is a drug used clinically to treat hyperuricemia and gout due to its activity as an inhibitor of the kidneys' organic anion transporter that reclaims uric acid from urine. Probenecid also inhibits pannexin1 channels that are involved in purinergic neurotransmission and inflammation, which have been implicated in alcohol's effects and motivation for alcohol. Therefore, we tested the effects of probenecid on alcohol intake in rodents. METHODS: We tested the effects of probenecid on operant oral alcohol self-administration in alcohol-dependent rats during acute withdrawal as well as in nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice. RESULTS: Probenecid reduced alcohol intake in both dependent and nondependent rats and in the DID paradigm in mice without affecting water or saccharin intake, indicating that probenecid's effect was selective for alcohol and not the result of a general reduction in reward. CONCLUSIONS: These results raise the possibility that pannexin1 is a novel therapeutic target for the treatment of AUD. The clinical use of probenecid has been found to be generally safe, suggesting that it can be a candidate for drug repositioning for the treatment of AUD.

Essential values of cocaine and non-drug alternatives predict the choice between them.

This study investigated the relationship between reinforcer value and choice between cocaine and two non-drug alternative reinforcers in rats. The essential value (EV, a behavioral economic measure based on elasticity of demand) of intravenous cocaine and food (Experiment 1) or saccharin (Experiment 2) was determined in the first phase of each experiment. Food had higher EV than cocaine, whereas the EVs of cocaine and saccharin did not differ. In the second phase of each experiment, rats were allowed to make mutually exclusive choices between cocaine and the non-drug alternative reinforcer. The main findings were that the EV of cocaine was a positive predictor of cocaine preference and the EV of food or saccharin was a negative predictor of cocaine preference. An analysis of within-session patterns of choice behavior revealed sequential dependencies, whereby rats were more likely to choose cocaine on a particular trial after having chosen the non-drug alternative on previous trials. When the time between choices was increased, these sequential dependencies disappeared. The results of these experiments are consistent with the suggestion that addiction-like behavior involves both overvaluation of drug reinforcers and undervaluation of non-drug reinforcers.

Cocaine can generate a stronger conditioned reinforcer than food despite being a weaker primary reinforcer.

The present study aimed to test the hypothesis that cues associated with drug-taking behavior become extra strong motivators of behavior compared with cues paired with non-drug reinforcers. In experiment 1, rats were trained to lever press for intravenous cocaine infusions and grain pellets. Each reinforcer was paired with a distinct audiovisual cue. When allowed to choose between these alternatives, rats chose grain on ~70-80 percent of trials. However, after extinguishing lever pressing, reintroduction of press-contingent cues during a test for cue-induced reinstatement generated more cocaine seeking than grain seeking (also observed on 3- and 8-week follow-up tests). To examine whether the same pattern of results would occur with two non-drug reinforcers, experiment 2 replicated experiment 1 using grain and sucrose as reinforcement alternatives. Rats chose sucrose over grain on ~70-80 percent of choice trials and also responded more for the sucrose cue than for the grain cue on the reinstatement test. The disconnect between primary and conditioned reinforcements in experiment 1 but not in experiment 2 suggests that drug cues may become exceptionally strong motivators of drug seeking. These results are consistent with cue-focused theories of addiction and may offer insight into the persistent cue-driven drug-seeking behavior observed in addiction.

Reinstatement in a cocaine versus food choice situation: reversal of preference between drug and non-drug rewards.

Recent studies show that when given a mutually exclusive choice between cocaine and food, rats almost exclusively choose food. The present experiment investigated potential shifts in preference between levers associated with either food or cocaine that might occur during extinction (food and cocaine no longer available) and during footshock-induced, cocaine-primed and food-primed reinstatement. During self-administration sessions where food and cocaine were simultaneously available, rats demonstrated a stable food preference, choosing food over cocaine on 83% of trials. During extinction when neither reinforcer was available, no preference between levers was evident and responding decreased until rats responded on the previously food- and cocaine-associated levers at equally low rates. Footshock resulted in a non-specific reinstatement of responding upon both levers, while cocaine priming resulted in a significant preference for cocaine seeking over food seeking. This suggests that the mechanism underlying footshock-induced reinstatement is distinct from that of cocaine-primed reinstatement. Food priming engendered a mild, non-specific increase in responding on both levers. Although rats generally prefer food over cocaine when presented with a choice between these primary reinforcers, the present results suggest that in certain situations, cocaine-seeking behavior prevails over food-seeking behavior.

Intermittent access cocaine self-administration produces context-specific escalation and increased motivation.

The intermittent-access (IntA) self-administration procedure has been reported to produce intensified addiction-like behavior compared to continuous-access (ContA) procedures. In a common variation of the IntA procedure, cocaine is available for 5 min at the beginning of each half hour of a 6-h session. In contrast, during ContA procedures, cocaine is available continuously throughout a session, typically lasting one or more hours. Previous studies comparing procedures have used between-subjects designs, where separate groups of rats self-administer cocaine on either IntA or ContA procedures. The present study used a within-subjects design where subjects self-administered cocaine on the IntA procedure in one context and self-administered cocaine on the continuous short-access (ShA) procedure in another context during separate sessions. Across sessions, rats escalated cocaine intake in the IntA, but not ShA, context. Following sessions eight and 11, rats were administered a progressive ratio test in each context to monitor the change in cocaine motivation. Rats obtained more cocaine infusions on the progressive ratio test in the IntA context than in the ShA context following 11 sessions. These results suggest that addiction-like behaviors following IntA self-administration may be influenced by context-specific learning factors.

Intermittent-access operant alcohol self-administration promotes binge-like drinking and drinking despite negative consequences in male and female heterogeneous stock rats.

The study of Alcohol Use Disorders (AUD) in preclinical models is hampered by difficulty in training rodents to voluntarily consume high levels of alcohol. The intermittency of alcohol access/exposure is well known to modulate alcohol consumption (e.g., alcohol deprivation effect, intermittent-access two-bottle-choice) and recently, intermittent access operant self-administration procedures have been used to produce more intense and binge-like self-administration of intravenous psychostimulant and opioid drugs. In the present study, we sought to systematically manipulate the intermittency of operant self-administered alcohol access to determine the feasibility of promoting more intensified, binge-like alcohol consumption. To this end, 24 male and 23 female NIH Heterogeneous Stock rats were trained to self-administer 10% w/v ethanol, before being split into three different-access groups. Short Access (ShA) rats continued receiving 30-min training sessions, Long Access (LgA) rats received 16-h sessions, and Intermittent Access (IntA) rats received 16-h sessions, wherein the hourly alcohol-access periods were shortened over sessions, down to 2 min. IntA rats demonstrated an increasingly binge-like pattern of alcohol drinking in response to restriction of alcohol access, while ShA and LgA rats maintained stable intake. All groups were tested on orthogonal measures of alcohol-seeking and quinine-punished alcohol drinking. The IntA rats displayed the most punishment-resistant drinking. In a separate experiment, we replicated our main finding, that intermittent access promotes a more binge-like pattern of alcohol self-administration using 8 male and 8 female Wistar rats. In conclusion, intermittent access to self-administered alcohol promotes more intensified self-administration. This approach may be useful in developing preclinical models of binge-like alcohol consumption in AUD.

Operant Vapor Self-administration in Mice.

Models of drug addiction in rodents are instrumental in understanding the underlying neurobiology. Intravenous self-administration of drugs in mice is currently the most commonly used model; however, several challenges exist due to complications related to catheter patency. To take full advantage of the genetic tools available to study opioid addiction in mice, we developed a non-invasive mouse model of opioid self-administration using vaporized fentanyl. This model can be used to study various aspects of opioid addiction including self-administration, escalation of drug intake, extinction, reinstatement, and drug seeking despite adversity. Further, this model bypasses the limitations of intravenous self-administration and allows the investigation of drug taking over extended periods of time and in conjunction with cutting-edge techniques such as calcium imaging and in vivo electrophysiology.

Cues conditioned to withdrawal and negative reinforcement: Neglected but key motivational elements driving opioid addiction.

Opioid use disorder (OUD) is a debilitating disorder that affects millions of people. Neutral cues can acquire motivational properties when paired with the positive emotional effects of drug intoxication to stimulate relapse. However, much less research has been devoted to cues that become conditioned to the aversive effects of opioid withdrawal. We argue that environmental stimuli promote motivation for opioids when cues are paired with withdrawal (conditioned withdrawal) and generate opioid consumption to terminate conditioned withdrawal (conditioned negative reinforcement). We review evidence that cues associated with pain drive opioid consumption, as patients with chronic pain may misuse opioids to escape physical and emotional pain. We highlight sex differences in withdrawal-induced stress reactivity and withdrawal cue processing and discuss neurocircuitry that may underlie withdrawal cue processing in dependent individuals. These studies highlight the importance of studying cues associated with withdrawal in dependent individuals and point to areas for exploration in OUD research.

Demand for fentanyl becomes inelastic following extended access to fentanyl vapor self-administration.

Opioid use disorder imposes great societal harm in the United States and in countries worldwide. Animal models that accurately capture motivational changes that occur in opioid dependence are critical to studying this disorder. The present study used a model of opioid vapor self-administration combined with a behavioral economics approach to determine whether rats would be more motivated to "work" to defend their baseline intake of fentanyl (i.e., more inelastic demand) following sufficiently frequent, intense, and chronic exposure to self-administered vaporized fentanyl. Male rats were allowed to respond for deliveries of 1.5-s of vaporized 10 mg/ml fentanyl solution. Following 15 sessions of short access (ShA; 1 h) vs. long access (LgA; 12 h) to self-administration, we conducted a between-sessions demand curve procedure, and observed significantly more inelastic demand for fentanyl (Essential Value; EV), and increased maximal response output (Omax) in LgA compared with ShA rats. In a subsequent phase, the unit-dose was doubled to 3 s of fentanyl vaporization. After seven ShA vs. LgA sessions, we assessed demand again and found that LgA rats, contrasted to ShA rats, demonstrated significantly higher baseline intake or "hedonic setpoint" (Q0), in addition to significantly increased EV and Omax. These results demonstrate that extended access to self-administration of a vaporized opioid causes changes in behavioral economic metrics consistent with development of an addiction-like state in rats. The combination of the vapor model with a translationally relevant behavioral economics framework opens new avenues to study dysregulated motivational processes in substance use disorders.

Nasal oxytocin for the treatment of psychiatric disorders and pain: achieving meaningful brain concentrations.

There is evidence of the therapeutic potential of intranasal oxytocin for the treatment of pain and various psychiatric disorders, however, there is scant evidence that oxytocin reaches the brain. We quantified the concentration and distribution pattern of [125I]-radiolabeled oxytocin in the brains and peripheral tissues of rats after intranasal delivery using gamma counting and autoradiography, respectively. Radiolabel was detected in high concentrations in the trigeminal and olfactory nerves as well as in brain regions along their trajectories. Considerable concentrations were observed in the blood, however, relatively low levels of radiolabel were measured in peripheral tissues. The addition of a mucoadhesive did not enhance brain concentrations. These results provide support for intranasal OT reaching the brain via the olfactory and trigeminal neural pathways. These findings will inform the design and interpretation of clinical studies with intranasal oxytocin.

Inactivation of the infralimbic cortex decreases discriminative stimulus-controlled relapse to cocaine seeking in rats.

Persistent susceptibility to cue-induced relapse is a cardinal feature of addiction. Discriminative stimuli (DSs) are one type of drug-associated cue that signal drug availability (DS+) or unavailability (DS-) and control drug seeking prior to relapse. We previously established a trial-based procedure in rats to isolate DSs from context, conditioned stimuli, and other drug-associated cues during cocaine self-administration and demonstrated DS-controlled cocaine seeking up to 300 abstinence days. The behavioral and neural mechanisms underlying trial-based DS-control of drug seeking have rarely been investigated. Here we show that following discrimination training in our trial-based procedure, the DS+ and DS- independently control the expression and suppression of cocaine seeking during abstinence. Using microinjections of GABAA + GABAB receptor agonists (muscimol + baclofen) in medial prefrontal cortex, we report that infralimbic, but not prelimbic, subregion of medial prefrontal cortex is critical to persistent DS-controlled relapse to cocaine seeking after prolonged abstinence, but not DS-guided discriminated cocaine seeking or DS-controlled cocaine self-admininstration. Finally, using ex vivo whole-cell recordings from pyramidal neurons in the medial prefrontal cortex, we demonstrate that the disruption of DS-controlled cocaine seeking following infralimbic cortex microinjections of muscimol+baclofen is likely a result of suppression of synaptic transmission in the region via a presynaptic mechanism of action.

Glucocorticoid receptor modulators decrease alcohol self-administration in male rats.

Alcohol use disorder (AUD) is associated with the dysregulation of brain stress and reward systems, including glucocorticoid receptors (GRs). The mixed glucocorticoid/progesterone receptor antagonist mifepristone and selective GR antagonist CORT113176 have been shown to selectively reduce alcohol consumption in alcohol-dependent rats. Mifepristone has also been shown to decrease alcohol consumption and craving for alcohol in humans with AUD. The present study tested the effects of the GR modulators CORT118335, CORT122928, CORT108297, and CORT125134 on alcohol self-administration in nondependent (air-exposed) and alcohol-dependent (alcohol vapor-exposed) adult male rats. Different GR modulators recruit different GR-associated transcriptional cofactors. Thus, we hypothesized that these GR modulators would vary in their effects on alcohol drinking. CORT118335, CORT122928, and CORT125134 significantly reduced alcohol self-administration in both alcohol-dependent and nondependent rats. CORT108297 had no effect on alcohol self-administration in either group. The present results support the potential of GR modulators for the development of treatments for AUD. Future studies that characterize genomic and nongenomic effects of these GR modulators will elucidate potential molecular mechanisms that underlie alcohol drinking in alcohol-dependent and nondependent states.

Drug addiction co-morbidity with alcohol: Neurobiological insights.

Addiction is a chronic disorder that consists of a three-stage cycle of binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. These stages involve, respectively, neuroadaptations in brain circuits involved in incentive salience and habit formation, stress surfeit and reward deficit, and executive function. Much research on addiction focuses on the neurobiology underlying single drug use. However, alcohol use disorder (AUD) can be co-morbid with substance use disorder (SUD), called dual dependence. The limited epidemiological data on dual dependence indicates that there is a large population of individuals suffering from addiction who are dependent on more than one drug and/or alcohol, yet dual dependence remains understudied in addiction research. Here, we review neurobiological data on neurotransmitter and neuropeptide systems that are known to contribute to addiction pathology and how the involvement of these systems is consistent or divergent across drug classes. In particular, we highlight the dopamine, opioid, corticotropin-releasing factor, norepinephrine, hypocretin/orexin, glucocorticoid, neuroimmune signaling, endocannabinoid, glutamate, and GABA systems. We also discuss the limited research on these systems in dual dependence. Collectively, these studies demonstrate that the use of multiple drugs can produce neuroadaptations that are distinct from single drug use. Further investigation into the neurobiology of dual dependence is necessary to develop effective treatments for addiction to multiple drugs.

κ-Opioid receptor antagonism reverses heroin withdrawal-induced hyperalgesia in male and female rats.

Although opioids are potent analgesics, a consequence of chronic opioid use is hyperalgesia during withdrawal, which may contribute to opioid misuse. Dynorphin, the endogenous ligand of κ-opioid receptors (KORs), is upregulated in opioid-dependent rats and in animal models of chronic pain. However, the role of KORs in opioid withdrawal-induced hyperalgesia remains to be determined. We hypothesized that KOR antagonism would reverse opioid withdrawal-induced hyperalgesia in opioid-dependent rats. Male and female Wistar rats received daily injections of heroin (2-6 mg/kg, SC) and were tested for mechanical sensitivity in the electronic von Frey test 4-6 h into withdrawal. Female rats required significantly more heroin than male rats to reach comparable levels of both heroin-induced analgesia and hyperalgesia (6 mg/kg vs. 2 mg/kg). Once hyperalgesia was established, we tested the effects of the KOR antagonists nor-binaltorphimine (norBNI; 30 mg/kg, SC) and 5'-guanidinonaltrindole (5'GNTI; 30 mg/kg, SC). When the animals continued to receive their daily heroin treatment (or saline treatment in the repeated saline group) five times per week throughout the experiment, both KOR antagonists reversed heroin withdrawal-induced hyperalgesia. The anti-hyperalgesia effect of norBNI was more prolonged in males than in females (14 days vs. 7 days), whereas 5'GNTI had more prolonged effects in females than in males (14 days vs. 4 days). The behavioral effects of 5'GNTI coincided with higher 5'GNTI levels in the brain than in plasma when measured at 24 h, whereas 5'GNTI did not reverse hyperalgesia at 30 min posttreatment when 5'GNTI levels were higher in plasma than in the brain. Finally, we tested the effects of 5'GNTI on naloxone-induced and spontaneous signs of opioid withdrawal and found no effect in either male or female rats. These findings indicate a functional role for KORs in heroin withdrawal-induced hyperalgesia that is observed in rats of both sexes.