Stress-induced Relapse Research Articles

A Bicyclic Analog of the Linear Peptide Arodyn Is a Potent and Selective Kappa Opioid Receptor Antagonist.

Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

Neurobiology of Stress-Induced Nicotine Relapse.

Tobacco smoking is the leading cause of preventable death and disease. Although there are some FAD-approved medicines for controlling smoking, the relapse rate remains very high. Among the factors that could induce nicotine relapse, stress might be the most important one. In the last decades, preclinical studies have generated many new findings that lead to a better understanding of stress-induced relapse of nicotine-seeking. Several molecules such as α3β4 nicotinic acetylcholine receptor, α2-adrenergic receptors, cannabinoid receptor 1, trace amine-associated receptor 1, and neuropeptide systems (corticotropin-releasing factor and its receptors, dynorphine and kappa opioid receptor) have been linked to stress-induced nicotine relapse. In this review, we discuss recent advances in the neurobiology, treatment targets, and potential therapeutics of stress-induced nicotine relapse. We also discuss some factors that may influence stress-induced nicotine relapse and that should be considered in future studies. In the final section, a perspective on some research directions is provided. Further investigation on the neurobiology of stress-induced nicotine relapse will shed light on the development of new medicines for controlling smoking and will help us understand the interactions between the stress and reward systems in the brain.

Acute Stress Enhances Memory and Preference for Smoking-Related Associations in Smokers.

Nicotine dependence follows a chronic course that is characterized by repeated relapse, often driven by acute stress and rewarding memories of smoking retrieved from related contexts. These two triggers can also interact, with stress influencing retrieval of contextual memories. However, the roles of these processes in nicotine dependence remain unknown. We investigated how acute stress biases memory for smoking-associated contexts among smokers (N = 65) using a novel laboratory paradigm. On day 1, participants formed associations between visual stimuli of items (either neutral or related to smoking) and places (background scenes). On day 2 (24 hours later), participants were exposed to an acute laboratory-based stressor (socially evaluated cold pressor test; N = 32) or a matched control condition (N = 33) prior to being tested on their memory recognition and preferences for each item and place. We distinguished the accuracy of memory into specific (ie, precisely correct) or gist (ie, lure items with similar content) categories. Results demonstrated that the stressor significantly induced physiological and subjective perceived stress responses, and that stressed smokers exhibited a memory bias in favor of smoking-related items. In addition, the stressed group displayed greater preference for both smoking-related items and places that had been paired with the smoking-related items. We also found suggestive evidence that stronger smoking-related memory biases were associated with more severe nicotine dependence (ie, years of smoking). These results highlight the role of stress in biasing smokers toward remembering contexts associated with smoking, and amplifying their preference for these contexts. The current study elucidates the role of acute stress in promoting memory biases favoring smoking-related associations among smokers. The results suggest that the retrieval of smoking-biased associative memory could be a crucial factor in stress-related nicotine seeking. This may lead to a potential intervention targeting the extinction of smoking-related context memories as a preventive strategy for stress-induced relapse.

Chronic administration of a norepinephrine antagonist prevents and partially reverses escalation of cocaine self-administration.

Anxiety is a critical component of the development and maintenance of drug addiction; however, anti-anxiety medications such as benzodiazepines and beta-blockers (β-adrenergic receptor antagonists) are not used for the treatment of substance use disorder, except for the management of acute withdrawal syndrome. Preclinical studies have shown that beta-blockers may reduce stress-induced relapse; however, the effect of beta blockers on the escalation and maintenance of drug intake has not been tested. To address this issue, we chronically administered the β-adrenergic receptor antagonist propranolol during the escalation or maintenance of cocaine intake in a model of extended access (6h) to cocaine self-administration (0.5mg/kg). The behavioural specificity of propranolol was tested using a non-drug reward (saccharin). Daily administration of propranolol (15 mg/kg) prevented the development of escalation of cocaine self-administration and partially reversed self-administration after the establishment of escalation of intake. Moreover, propranolol dose-dependently decreased the motivation for cocaine tested under a progressive ratio schedule of reinforcement during the development of escalation and after maintenance. Finally, propranolol administration had no effect on the escalation and maintenance of saccharin self-administration. These results demonstrate that chronic treatment with propranolol provides therapeutic efficacy in reducing cocaine self-administration during the development and after the establishment of escalation of cocaine self-administration in an animal model relevant to cocaine use disorder. These results suggest that beta blockers should be further investigated as a target for medication development for the treatment of cocaine use disorder.

Dopamine D3 Receptor Modulates Akt/mTOR and ERK1/2 Pathways Differently during the Reinstatement of Cocaine-Seeking Behavior Induced by Psychological versus Physiological Stress.

Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As D3R regulates the activity of the Akt/mTOR and MEK/ERK1/2 pathways, we assessed the effects of SB-277011-A, a D3R antagonist, on the activity of these kinases during the reinstatement of cocaine-induced conditioned place preference (CPP) induced by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated an extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Cocaine-seeking behavior reactivation was correlated with decreased p-Akt, p-mTOR, and p-ERK1/2 activation in both nuclei of restrained animals. While a D3R blockade prevented stress-induced CPP reinstatement and plasma corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR, and ERK1/2 activation depending on the stressor and the dose used. Our data support the involvement of corticosterone in the SB-277011-A effects in restrained animals. Additionally, the ratios p-mTOR/mTOR and/or p-ERK1/2 /ERK1/2 in the BLA during stress-induced relapse seem to be related to the locomotor activity of animals receiving 48 mg/kg of the antagonist. Hence, our study indicates the D3R antagonist's efficacy to prevent stress-induced relapses in drug use through distinct modulation of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing nuclei.

Oxytocin Attenuates Yohimbine-Induced Reinstatement of Alcohol-Seeking in Female Rats via the Central Amygdala.

Alcohol use disorder is a significant public health concern, further exacerbated by an increased risk of relapse due to stress. In addition, factors such as biological sex may contribute to the progression of addiction, as females are especially susceptible to stress-induced relapse. While there have been many studies surrounding potential pharmacological interventions for male stress-induced ethanol reinstatement, research regarding females is scarce. Recently, the neuropeptide oxytocin has gained interest as a possible pharmacological intervention for relapse. The present study examines how oxytocin affects yohimbine-induced reinstatement of ethanol-seeking in female rats using a self-administration paradigm. Adult female rats were trained to press a lever to access ethanol in daily self-administration sessions. Rats then underwent extinction training before a yohimbine-induced reinstatement test. Rats administered with yohimbine demonstrated significantly higher lever response indicating a reinstatement of ethanol-seeking behavior. Oxytocin administration, both systemically and directly into the central amygdala, attenuated the effect of yohimbine-induced reinstatement of ethanol-seeking behavior. The findings from this study establish that oxytocin is effective at attenuating alcohol-relapse behavior mediated by the pharmacological stressor yohimbine and that this effect is modulated by the central amygdala in females. This provides valuable insight regarding oxytocin's potential therapeutic effect in female stress-induced alcohol relapse.

Estrous cycle and hormone regulation of stress-induced reinstatement of reward seeking in female mice

Women are more vulnerable to stress-induced craving, which may be associated with increased vulnerability to relapse. Susceptibility to stress-induced craving also appears to be modulated by the menstrual cycle and is negatively correlated with circulating progesterone levels in women. However, the factors that contribute to relapse vulnerability are poorly characterized in female animals. In this study, we assessed whether chronic ethanol exposure, estrous cycle, or exogenous progesterone administration modulated vulnerability to stress-induced reinstatement. To model ethanol dependence, adult female C57Bl/6J mice underwent chronic intermittent ethanol (CIE) exposure via vapor inhalation. Seventy-two hours after the final ethanol exposure, food-restricted mice began training in a conditioned place preference paradigm (CPP) for a food reward, followed by extinction training. Mice were then subjected to forced swim stress and assessed for reinstatement of their preference for the reward-paired chamber. CIE did not affect stress-induced reinstatement. However, stress-induced reinstatement was attenuated during the diestrus phase, when endogenous levels of progesterone peak in female mice. Further, administration of exogenous progesterone mimicked the attenuated reinstatement observed in diestrus. These findings indicate that circulating hormone levels modulate susceptibility to relapse-like behaviors and implicate progesterone as a potential target for treating stress-induced relapse in women.

Acute food deprivation-induced relapse to heroin seeking after short and long punishment-imposed abstinence in male rats.

Stress is a major trigger for drug relapse in humans and animal models, even after prolonged abstinence. However, animal models for stress-induced relapse were criticized for the lack of predictive and face validity. Here we investigated the effect of acute food deprivation stress in a novel stress-induced relapse model using voluntary, punishment-imposed abstinence from heroin. We also performed a detailed characterization of the development of punishment-imposed abstinence. Male rats were trained to self-administered heroin (0.1mg/kg/infusion) for 2weeks, using the seeking-taking chained schedule. Pressing the 'seeking' lever led to the insertion of the 'taking' lever and pressing the take lever resulted in heroin infusion. Following self-administration training, rats were exposed to 8 or 21days of heroin-seeking punishment. During punishment, 30% of the completed seek links resulted in a mild escalating footshock instead of take lever presentation. Next, rats were tested for heroin seeking under extinction conditions after 24h of food deprivation and sated conditions. Probabilistic punishment of seeking lever responses resulted in gradual suppression of heroin seeking and taking. Exposure to food-deprivation stress induced a robust relapse to heroin seeking after short and long punishment-imposed abstinence periods, without significant effects of time, i.e., no incubation of heroin seeking. Individual differences were observed in the development of punishment-induced abstinence and stress-induced relapse. These results suggest that stress is a reliable trigger to relapse even after a prolonged period of punishment-induced, voluntary abstinence.

Corticotropin releasing factor (CRF) systems: Promoting cocaine pursuit without distress via incentive motivation.

Corticotropin releasing factor (CRF) systems in limbic structures are posited to mediate stress-induced relapse in addiction, traditionally by generating distress states that spur drug consumption as attempts at hedonic self-medication. Yet evidence suggests that activating CRF-expressing neurons in the central amygdala (CeA) or nucleus accumbens (NAc) can magnify incentive motivation in absence of distress, at least for sucrose rewards. However, traditional CRF hypotheses in addiction neuroscience are primarily directed toward drug rewards. The question remains open whether CRF systems can similarly act via incentive motivation mechanisms to promote pursuit of drug rewards, such as cocaine. Here we tested whether optogenetic excitation of CRF-containing neurons in either NAc medial shell, lateral CeA, or dorsolateral BNST of transgenic Crh-Cre+ rats would spur preference and pursuit of a particular laser-paired cocaine reward over an alternative cocaine reward, and whether excitation served as a positively-valenced incentive itself, through laser self-stimulation tests. We report that excitation of CRF-containing neurons in either NAc or CeA recruited mesocorticolimbic circuitry to amplify incentive motivation to pursue the laser-paired cocaine: focusing preference on the laser-paired cocaine reward in a two-choice task, and spurred pursuit as doubled breakpoint in a progressive ratio task. Crucially indicating positive-valence, excitation of CRF neurons in NAc and CeA also was actively sought after by most rats in self-stimulation tasks. Conversely, CRF neuronal activation in BNST was never self-stimulated, but failed to enhance cocaine consumption. Collectively, we find that NAc and CeA CRF-containing neurons can amplify pursuit and consumption of cocaine by positively-valenced incentive mechanisms, without any aversive distress.

The Impact of HIV Infection in Cocaine Seeking Behaviors

BackgroundSubstance use disorders (SUDs) are characterized by high propensity to relapse and are highly comorbid with HIV infection. The underlying neurobiology of this relationship is poorly understood. Preclinical research on the neurobiobehavioral outcomes of progressive HIV infections may yield crucial information to improve SUD prognosis and reduce risk of relapse in people living with HIV.MethodsTo model progressive HIV, adult male and female C57BL6J mice were inoculated with EcoHIV, a chimeric HIV‐1 which infects murine cells by replacing envelope glycoprotein gp120 with gp80. Five weeks after inoculation mice were trained in a cocaine conditioned place preference (CPP) paradigm (10mg/kg, i.p.) followed by extinction training. Stress‐induced reinstatement was assessed following administration of yohimbine, an α2 adrenergic antagonist, to determine EcoHIV effects. Neuronal activation in the prefrontal cortex (PFC) and nucleus accumbens (NAc) by cocaine exposure was assessed by quantitative analysis the expression of the immediate early gene, cFos.ResultsEcoHIV and sham‐inoculated mice exhibited similar cocaine CPP and extinction. However, EcoHIV mice showed significantly increased yohimbine‐induced reinstatement rate (p<0.05). cFos analysis revealed an elevated neural activation in PFC and NAc in response to cocaine. However, only neurons within infralimbic PFC (IL) and NAc shell subregions showed further increases in activity in cocaine exposed mice infected with EcoHIV vs shams.ConclusionEcoHIV infected mice escalated stress‐induced reinstatement of cocaine seeking compared to controls, suggesting that people living with HIV may be at elevated risk for stress‐induced relapse. Progressive EcoHIV infection may dysregulate IL and NAcShell activity to facilitate the cocaine seeking and reinstatement. Ongoing research will characterize how EcoHIV infection dysregulate IL‐NAcShell pathway specific activity during cocaine reward learning and stress‐induced relapse.

A Procedure to Study Stress-induced Relapse of Heroin Seeking after Punishment-imposed Abstinence.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

Oxytocin Attenuates the Stress-Induced Reinstatement of Alcohol-Seeking in Male Rats: Role of the Central Amygdala.

Factors such as stress and anxiety often contribute to alcohol-dependent behavior and can trigger a relapse of alcohol addiction and use. Therefore, it is important to investigate potential pharmacological interventions that may alleviate the influence of stress on addiction-related behaviors. Previous studies have demonstrated that the neuropeptide oxytocin has promising anxiolytic potential in mammals and may offer a pharmacological target to diminish the emotional impact on reinstatement of alcohol-seeking. The purpose of the present study was to investigate the effect of oxytocin on stress-induced alcohol relapse and identify a neural structure mediating this effect through the use of an ethanol self-administration and yohimbine-induced reinstatement paradigm. While yohimbine administration resulted in the reinstatement of ethanol-seeking behavior, the concurrent administration of yohimbine and oxytocin attenuated this effect, suggesting that oxytocin may disrupt stress-induced ethanol-seeking behavior. The central amygdala (CeA) is a structure that drives emotional responses and robustly expresses oxytocin receptors. Intra-CeA oxytocin similarly attenuated the yohimbine-induced reinstatement of ethanol-seeking behavior. These results demonstrate that oxytocin has the potential to attenuate stress-induced relapse into ethanol-seeking behavior, and that this mechanism occurs specifically within the central amygdala.

Repeated cocaine or methamphetamine treatment alters astrocytic CRF2 and GLAST expression in the ventral midbrain.

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stress-induced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100β, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100β positive cells, and an increase of co-staining of CRF2 with both S100β and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.

Many Drugs of Abuse May Be Acutely Transformed to Dopamine, Norepinephrine and Epinephrine In Vivo.

It is well established that a wide range of drugs of abuse acutely boost the signaling of the sympathetic nervous system and the hypothalamic–pituitary–adrenal (HPA) axis, where norepinephrine and epinephrine are major output molecules. This stimulatory effect is accompanied by such symptoms as elevated heart rate and blood pressure, more rapid breathing, increased body temperature and sweating, and pupillary dilation, as well as the intoxicating or euphoric subjective properties of the drug. While many drugs of abuse are thought to achieve their intoxicating effects by modulating the monoaminergic neurotransmitter systems (i.e., serotonin, norepinephrine, dopamine) by binding to these receptors or otherwise affecting their synaptic signaling, this paper puts forth the hypothesis that many of these drugs are actually acutely converted to catecholamines (dopamine, norepinephrine, epinephrine) in vivo, in addition to transformation to their known metabolites. In this manner, a range of stimulants, opioids, and psychedelics (as well as alcohol) may partially achieve their intoxicating properties, as well as side effects, due to this putative transformation to catecholamines. If this hypothesis is correct, it would alter our understanding of the basic biosynthetic pathways for generating these important signaling molecules, while also modifying our view of the neural substrates underlying substance abuse and dependence, including psychological stress-induced relapse. Importantly, there is a direct way to test the overarching hypothesis: administer (either centrally or peripherally) stable isotope versions of these drugs to model organisms such as rodents (or even to humans) and then use liquid chromatography-mass spectrometry to determine if the labeled drug is converted to labeled catecholamines in brain, blood plasma, or urine samples.

CB1R activation in nucleus accumbens core promotes stress-induced reinstatement of cocaine seeking by elevating extracellular glutamate in a drug-paired context

Preclinical models of stress-induced relapse to drug use have shown that the dysregulation of glutamatergic transmission within the nucleus accumbens (NA) contributes notably to the reinstatement of cocaine-seeking behavior in rodents. In this sense, there has been increasing interest in the cannabinoid type-1 receptor (CB1R), due to its crucial role in modulating glutamatergic neurotransmission within brain areas involved in drug-related behaviors. This study explored the involvement of CB1R within the NA subregions in the restraint stress-induced reinstatement of cocaine-conditioned place preference (CPP), as well as in the regulation of glutamatergic transmission, by using a pharmacological approach and the in vivo microdialysis sampling technique in freely moving rats. CB1R blockade by the antagonist/inverse agonist AM251 (5 nmol/0.5 μl/side) or CB1R activation by the agonist ACEA (0.01 fmol/0.5 μl/side), prevented or potentiated restraint stress-induced reinstatement of cocaine-CPP, respectively, after local administration into NAcore, but not NAshell. In addition, microdialysis experiments demonstrated that restraint stress elicited a significant increase in extracellular glutamate in NAcore under reinstatement conditions, with the local administration of AM251 or ACEA inhibiting or potentiating this, respectively. Interestingly, this rise specifically corresponded to the cocaine-associated CPP compartment. We also showed that this context-dependent change in glutamate paralleled the expression of cocaine-CPP, and disappeared after the extinction of this response. Taken together, these findings demonstrated the key role played by CB1R in mediating reinstatement of cocaine-CPP after restraint stress, through modulation of the context-specific glutamate release within NAcore. Additionally, CB1R regulation of basal extracellular glutamate was demonstrated and proposed as the underlying mechanism.

Effects of the glucocorticoid receptor antagonist PT150 on stress-induced fentanyl seeking in male and female rats.

Opioid use disorder (OUD) is highly comorbid with stress-related disorders, and stress can serve as a trigger for reinstatement of drug seeking. Glucocorticoid receptor (GR) antagonists such as mifepristone (RU-486) may be effective against stress-induced drug seeking. In the current study, PT150 (formerly ORG-34517), a more selective GR antagonist, was tested using two models of stress-induced drug seeking, namely footshock and yohimbine. Adult male and female Sprague-Dawley rats were trained to self-administer fentanyl (2.5 μg/kg/infusion, i.v.) in a model of escalation. Rats then received 7 days of abstinence, followed by extinction; PT150 (0, 50 or 100 mg/kg in Nutella®; p.o.) treatment started on the first day of extinction training and continued daily until the end of the study. Following 14 days of extinction, rats were tested for reinstatement following footshock and yohimbine (0, 1, or 2 mg/kg; i.p.), tested in counterbalanced order; PT150 or placebo treatment occurred prior to each extinction and reinstatement session. Prior to initiation of PT150 treatment, females self-administered greater levels of fentanyl during 1-h sessions compared to males; however, when switched to 6-h sessions, males and females self-administered similar levels of fentanyl and showed a similar escalation of intake over time. PT150 had no effect on extinction of self-administration. While both footshock and yohimbine reinstated fentanyl seeking, only footshock-induced reinstatement was decreased by PT150 (50 and 100 mg/kg). The effect of PT150 on footshock-induced reinstatement was driven primarily by males. The glucocorticoid antagonist PT150 reduces shock-induced fentanyl seeking, suggesting it may be effective against stress-induced relapse, although the sex difference in response may need further exploration.

Social Rank, Behavioral Phenotypes and Kappa Opioid Receptor: PET Imaging Studies of Socially Housed Female and Male Monkey Models of Cocaine Use Disorder

In the US, cocaine use is increasing, alongside cocaine overdose deaths, which doubled from 2014 to 2016 (Nolan et al., J Urban Health 2019; John and Wu, Drug Alcohol Depend 2017). While dopamine (DA) has been implicated in the positive reinforcing effects of drugs, the kappa opioid receptor (KOR) system and its endogenous ligand, dynorphin, are implicated in the neurobiological regulation of aversive states, including stress associated with substance abuse. Both chronic stress and cocaine exposure increase dynorphin/KOR system function and sensitivity to stress-induced relapse (Tejeda and Bonci, Brain Research 2019). A recent positron emission tomography (PET) study with KOR agonist tracer [11C]EKAP, in humans, reported an inverse correlation between social status and KOR availability in “anti-reward”/stress brain regions (Matuskey et al. Neuropsychopharmacology 2019). Differences in KOR availability have also been shown to correlate with cocaine choice in subjects with CUD (Martinez et al. Neuropsychopharmacology 2019). The aim of this study is to extend the investigation of the dynorphin/KOR system using a homologous nonhuman primate model of CUD involving cocaine self-administration, primate social behavior, and PET imaging. The ongoing study utilizes cocaine-naïve male and female monkeys (N=8/sex) living in same-sex social groups of 4/pen. The first aim was to investigate the KOR system, combining [11C]EKAP PET imaging and primate social behavior to assess the relationship between social rank and KOR availability. The second aim was to extend our research to include measures of impulsivity using a delayed discounting procedure. Monkeys were trained under a 1- vs. 3-food pellets delay discounting choice procedure. The primary dependent variable was indifference point (IP), or the delay that results in 50% choice for both reinforcers. In PET studies, the lowest binding potentials (BPs) across all regions of interest were observed in dominant (Dom) females and subordinate (Sub) males; the two most vulnerable phenotypes to cocaine reinforcement (Morgan et al., Nature Neurosci 2002; Nader et al., Biol Psych 2012). We hypothesize this was due to increased basal dynorphin levels, indicative of chronic social stress. Studies are currently underway to assess cocaine acquisition. We hypothesis Dom females and Sub males, those with lower KOR BPs will acquire cocaine reinforcement at lower doses compared to Dom males and Sub females. In delay discounting studies, the IP values ranged from 23-107 seconds (Mdn = 38). Preliminary data indicates no sex or social rank differences in IP values. However, redetermining the delay discounting curve resulted in significantly higher IP values, which were correlated with BP in several brain regions in males but not females. The redetermined IP values may provide an index of adaptability; we speculate that this may be a behavioral phenotype that supports epidemiological data that women are more vulnerable than men in developing CUDs.

Allosteric Modulation of GABAA Receptors in Rat Basolateral Amygdala Blocks Stress-Enhanced Reacquisition of Nicotine Self-Administration.

Stress is a major determinant of relapse to smoked tobacco. In a rat model, repeated stress during abstinence from nicotine self-administration (SA) results in enhanced reacquisition of nicotine SA, which is dependent on the basolateral amygdala (BLA). We postulate that repeated stress during abstinence causes hyperexcitability of the BLA principal output neurons (PNs) due to disinhibition of the PNs from reduced inhibitory regulation by local GABAergic interneurons. To determine if enhanced GABAergic regulation of the BLA PNs can lessen the effects of stress on nicotine intake, positive allosteric modulators (PAMs) of GABAA receptors were infused into the BLA immediately prior to reacquisition of nicotine SA. Three selective PAMs [NS 16085 (binds the benzodiazepine site on α2/α3 GABAA); DCUK-OEt (binds a novel, benzodiazepine site on α1 or α5, β2 or β3, γ2 or δ GABAA); DS2 (binds exclusively to δ GABAA] with varied GABAA subunit specificities abolished the stress-induced amplification of nicotine taking during reacquisition. These studies indicate that highly selective PAMS targeting α3 or δ subunit-containing GABAA in the BLA may be effective in ameliorating the stress-induced relapse to smoked tobacco during abstinence from cigarettes.

Role of BNST CRFR1 Receptors in Incubation of Fentanyl Seeking

The time-dependent increase in cue-triggered opioid seeking, termed “incubation of opioid craving,” is modeled in rodents by examining responding for opioid-associated cues after a period of forced abstinence. With opioid drugs, withdrawal symptoms may heighten cue reactivity by recruiting brain systems involved in both reward seeking and stress responses. Corticotropin releasing factor (CRF) in the bed nucleus of the stria terminalis (BNST) is a critical driver of stress-induced relapse to drug seeking. Here, we sought to determine whether BNST CRF receptor 1 (CRFR1) signaling drives incubation of opioid craving in opioid dependent and non-dependent rats. First, we tested whether BNST CRFR1 signaling drives incubation of opioid craving in rats with short-access fentanyl self-administration experience (2.5 μg/kg/infusion, 3 h/day for 10 days). On Day 1 of forced abstinence, we gave bilateral intra-BNST vehicle injections to all rats and measured lever responding for opioid cues in the absence of fentanyl infusions. On Day 30 of forced abstinence, we gave an identical test after bilateral intra-BNST injections of vehicle or CRFR1 receptor antagonist, R121919 (1 μg/0.3 μL/hemisphere). Vehicle treated rats showed greater responding for opioid associated cues on Day 30 relative to Day 1, and this incubation effect was prevented by intra-BNST R121919 on Day 30. Next, we incorporated an opioid-dependence procedure to investigate whether BNST CRFR1 signaling drives opioid cue-reactivity to a greater extent in opioid-dependent relative to non-dependent rats. We trained rats to self-administer fentanyl for 5 days before initiating the dependence phase and resuming daily fentanyl self-administration sessions for 10 days. We gave intra-BNST R121919 or vehicle injections before testing during acute (Day 5) or protracted (Day 30) withdrawal. During acute withdrawal, antagonizing BNST CRFR1 decreased the number of press bouts without affecting bout size or duration. These patterns of responding with R121919 treatment resulted in less fentanyl-associated conditioned reinforcement during test. Together, these findings suggest a role for BNST CRFR1 signaling in driving cue-reinforced opioid seeking after periods of forced abstinence.