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The amount of time-out pressing was not changed by drug dosage or drug-associated cues. (A) The number of lever presses on the active lever during the 20-s time-out period that followed each infusion was measured for each experimental protocol. (B) The amount of time-out pressing was calculated by adjusting for the total number of infusions made in each experimental protocol. Shown are active lever time-out presses divided by the number of infusions for experiments 1 and 2. Error ± SEM.
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Patients with opioid use disorder experience high rates of relapse during recovery, despite successful completion of rehabilitation programs. A key factor contributing to this problem is the long-lasting nature of drug-seeking behavior associated with opioid use. We modeled this behavior in a rat drug self-administration paradigm in which drug-seek...
Citations
... In 2021, opioid use disorder (OUD) and subsequent overdose resulted in an estimated 80,411 deaths in the US, incurring a medical cost of more than 1-1.5 trillion dollars (Serdarevic et al., 2017;Torralva and Janowsky, 2019;CDC, 2020;National Center on Health Statistics, 2021;NIDA, 2023). OUD is a chronically relapsing disorder that is characterized by compulsive drug-seeking behavior that can be enhanced by drug abstinence and is maintained by encounters with drug-associated cues which elicit strong cravings and promote drug-seeking behaviors (Mueller et al., 2002;Zanda et al., 2021;Zhang et al., 2022). A large body of literature shows that sex differences impose clinically relevant changes in the vulnerability to OUD with women being prescribed and escalating to higher doses of opioid faster with greater craving and withdrawal symptoms compared to their male counterparts (Lee and Ho, 2013;Manubay et al., 2015;Serdarevic et al., 2017;Huhn et al., 2019a;Huhn et al., 2019b;Pisanu et al., 2019;Torralva and Janowsky, 2019;CDC, 2020;National Center on Health Statistics, 2021;Wightman et al., 2021). ...
Introduction: Despite their inclination to induce tolerance, addictive states, and respiratory depression, synthetic opioids are among the most effective clinically administered drugs to treat severe acute/chronic pain and induce surgical anesthesia. Current medical interventions for opioid-induced respiratory depression (OIRD), wooden chest syndrome, and opioid use disorder (OUD) show limited efficacy and are marked by low success in the face of highly potent synthetic opioids such as fentanyl. D-Cysteine ethylester (D-CYSee) prevents OIRD and post-treatment withdrawal in male/female rats and mice with minimal effect on analgesic status. However, the potential aversive or rewarding effects of D-CYSee have yet to be fully characterized and its efficacy could be compromised by interactions with opioid-reward pathology.
Methods: Using a model of fentanyl-induced conditioned place preference (CPP), this study evaluated 1) the dose and sex dependent effects of fentanyl to induce rewarding states, and 2) the extent to which D-CYSee alters affective state and the acquisition of fentanyl-induced seeking behaviors.
Results: Fentanyl reward-related effects were found to be dose and sex dependent. Male rats exhibited a range-bound dose response centered at 5 µg/kg. Female rats exhibited a CPP only at 50 µg/kg. This dose was effective in 25% of females with the remaining 75% showing no significant CPP at any dose. Pretreatment with 100 mg/kg, but not 10 mg/kg, D-CYSee prevented acquisition of fentanyl seeking in males while both doses were effective at preventing acquisition in females.
Discussion: These findings suggest that D-CYSee is an effective co-treatment with prescribed opioids to reduce the development of OUD.
... Rodent models of drug self-administration provide an excellent tool to model drug seeking behavior and interrogate the molecular neuroadaptations that arise in discrete brain areas following chronic drug exposure. Previous studies from our labs and others have demonstrated that chronic self-administration of opioids, such as morphine or heroin, induces drug seeking behavior that is both immediate and long lasting in the absence of drug access [6][7][8][9]. Such behaviors are accompanied by regulation of a class of small noncoding RNAs called microRNAs (miRNAs) that are~18-24 nucleotides long [10]. ...
... Self-administration of 0.03 mg/kg/infusion heroin and sucrose on an FR1 schedule was performed as previously reported [6,24,33]. Self-administration data for heroin animals was previously reported [24]. Drug-naïve animals were handled daily but did not undergo self-administration of any substance. ...
... Our lab and others have demonstrated that selfadministration of 0.03 mg/kg/infusion of heroin results in perseverant heroin-seeking behavior [6,43], defined as a significant preference for the active, drug-paired lever compared to the inactive lever during cue-induced relapse sessions. Furthermore, such a protocol induces biochemical changes in the brain that recapitulate heroin-induced neuroadaptations observed in human subjects [43][44][45][46]. ...
Opioid misuse in the United States contributes to >70% of annual overdose deaths. To develop additional therapeutics that may prevent opioid misuse, further studies on the neurobiological consequences of opioid exposure are needed. Here we sought to characterize molecular neuroadaptations involving microRNA (miRNA) pathways in the brain and blood of adult male rats that self-administered the opioid heroin. miRNAs are ∼18–24 nucleotide RNAs that regulate protein expression by preventing mRNA translation into proteins. Manipulation of miRNAs and their downstream pathways can critically regulate drug seeking behavior. We performed small-RNA sequencing of miRNAs and proteomics profiling on tissue from the orbitofrontal cortex (OFC), a brain region associated with heroin seeking, following 2 days of forced abstinence from self-administration of 0.03 mg/kg/infusion heroin or sucrose. Heroin self-administration resulted in a robust shift of the OFC miRNA profile, regulating 77 miRNAs, while sucrose self-administration only regulated 9 miRNAs that did not overlap with the heroin-induced profile. Conversely, proteomics revealed dual regulation of seven proteins by both heroin and sucrose in the OFC. Pathway analysis determined that heroin-associated miRNA pathways are predicted to target genes associated with the term “prion disease,” a term that was also enriched in the heroin-induced protein expression dataset. Lastly, we confirmed that a subset of heroin-induced miRNA expression changes in the OFC are regulated in peripheral serum and correlate with heroin infusions. These findings demonstrate that peripheral blood samples may have biomarker utility for assessment of drug-induced miRNA pathway alterations that occur in the brain following chronic drug exposure.
... After a period of time, animals are re-exposed to the same context previously paired with the drug self-administration, and the drugseeking is assessed by non-reinforced lever presses [14]. Previous research from our laboratory has demonstrated that the incubation of heroin craving after 21 days of forced abstinence requires a unique combination of discrete drug cues with drug dosage [15]. Animals that self-administered heroin at a dosage of 0.075 mg/kg/ infusion paired with discrete drug cues displayed robust incubation behavior, an effect that was not observed with only discriminative drug cues or a lower dosage of 0.03 mg/kg/infusion, despite controlling for total drug intake [15]. ...
... Previous research from our laboratory has demonstrated that the incubation of heroin craving after 21 days of forced abstinence requires a unique combination of discrete drug cues with drug dosage [15]. Animals that self-administered heroin at a dosage of 0.075 mg/kg/ infusion paired with discrete drug cues displayed robust incubation behavior, an effect that was not observed with only discriminative drug cues or a lower dosage of 0.03 mg/kg/infusion, despite controlling for total drug intake [15]. The goal of the present study was to use such divergent behavioral phenotypes as a tool to: 1) to elucidate the molecular mechanisms associated with heroin incubation behavior; and 2) to identify molecular determinants that modulate long-lasting heroin seeking. ...
... Drug self-administration studies were conducted in Skinner boxes under a fixed ratio (FR) 1 schedule of reinforcement for 10 days in 6-hour daily sessions using the discrete cues protocol as previously described [15]. Following 10 days of heroin selfadministration, animals underwent 2 or 21D forced abstinence in their homecage (n = 12/group) and were then euthanized for molecular analysis. ...
Recovery from opioid use disorder (OUD) and maintenance of abstinence from opioid use is hampered by perseverant drug cravings that may persist for months after cessation of drug use. Drug cravings can intensify during the abstinence period, a phenomenon referred to as the ‘incubation of craving’ that has been well-described in preclinical studies. We previously reported that animals that self-administered heroin at a dosage of 0.075 mg/kg/infusion (HH) paired with discrete drug cues displayed robust incubation of heroin craving behavior after 21 days (D) of forced abstinence, an effect that was not observed with a lower dosage (0.03 mg/kg/infusion; HL). Here, we sought to elucidate molecular mechanisms underlying long-term heroin seeking behavior by profiling microRNA (miRNA) pathways in the orbitofrontal cortex (OFC), a brain region that modulates incubation of heroin seeking. miRNAs are small noncoding RNAs with long half-lives that have emerged as critical regulators of drug seeking behavior but their expression in the OFC has not been examined in any drug exposure paradigm. We employed next generation sequencing to detect OFC miRNAs differentially expressed after 21D of forced abstinence between HH and HL animals, and proteomics analysis to elucidate miRNA-dependent translational neuroadaptations. We identified 55 OFC miRNAs associated with incubation of heroin craving, including miR-485-5p, which was significantly downregulated following 21D forced abstinence in HH but not HL animals. We bidirectionally manipulated miR-485-5p in the OFC to demonstrate that miR-485-5p can regulate long-lasting heroin seeking behavior after extended forced abstinence. Proteomics analysis identified 45 proteins selectively regulated in the OFC of HH but not HL animals that underwent 21D forced abstinence, of which 7 were putative miR-485-5p target genes. Thus, the miR-485-5p pathway is dysregulated in animals with a phenotype of persistent heroin craving behavior and OFC miR-485-5p pathways may function to support long-lasting heroin seeking.
... The current development status of immunotherapeutics in the context of substance use disorder is reviewed here. 20 Their development reflects both the acute need for relapse protection measures (the rate of relapse is >80% after exiting rehabilitation clinics 21,22 ) and the need for an adjunct to treatment with naloxone, the opioid receptor antagonist that is the current standard of care for overdose reversal. To date, several vaccine formulations have shown efficacy against the pharmacological effects of fentanyl and its analogs. ...
Poorly immunogenic small molecules pose challenges for the production of clinically efficacious vaccines and antibodies. To address this, we generate an immunization platform derived from the immunogenic surface coat of the African trypanosome. Through sortase-based conjugation of the target molecules to the variant surface glycoprotein (VSG) of the trypanosome surface coat, we develop VSG-immunogen array by sortase tagging (VAST). VAST elicits antigen-specific memory B cells and antibodies in a murine model after deploying the poorly immunogenic molecule fentanyl as a proof of concept. We also develop a single-cell RNA sequencing (RNA-seq)-based computational method that synergizes with VAST to specifically identify memory B cell-encoded antibodies. All computationally selected antibodies bind to fentanyl with picomolar affinity. Moreover, these antibodies protect mice from fentanyl effects after passive immunization, demonstrating the ability of these two coupled technologies to elicit therapeutic antibodies to challenging immunogens.
... : bioRxiv preprint fentanyl); the current status of vaccine development in the context of SUD is reviewed here (Pravetoni and Comer, 2019). Their development reflects both the acute need for relapse protection measures (the rate of relapse is > 80% after exiting rehabilitation clinics (Smyth et al., 2010;Zanda, Floris and Sillivan, 2021)), and the need for an adjunct to treatment with naloxone, the opioid receptor antagonist which is the current standard of care for reversal of overdose. Unfortunately, the short half-life of naloxone can lead to individuals "re-overdosing" due to "re-narcotization" from the same initial dose of fentanyl (Seth et al., 2018). ...
Poorly antigenic small molecules pose challenges for the production of clinically efficacious antibodies. To address this, we have developed an immunization platform derived from the antigenic surface coat of the African trypanosome. Through sortase-based conjugation of antigens to the trypanosome surface coat protein variant surface glycoprotein (VSG), we created the VAST ( V SG-immunogen A rray by S ortase T agging). We used VAST to elicit protective immunity to the effects fentanyl. Immunizing mice with fentanyl-VAST generated serological memory and protection from fentanyl challenge. Employing single-cell RNAseq, we then developed a streamlined method that synergizes with the VAST to identify immunization-elicited memory B cells and the antibodies they encode. All antibodies selected by this method displayed picomolar affinities for fentanyl. Passive immunization protected animals from fentanyl, while crystallography revealed that the mAbs bind fentanyl in an unusually deep pocket suited to small molecules, demonstrating the ability of the VAST to elicit high-quality therapeutic antibodies.
... The relapse test contained identical contextual and visual cues used during acquisition; however, active lever presses did not result in any morphine infusions. Consistent with literature on incubation of craving for other opioids Gyawali et al., 2020;Reiner et al., 2020;Theberge et al., 2013;Zanda et al., 2021), extended forced abstinence from morphine resulted in a time-dependent increase in drugseeking behaviour, as measured by a significant increase in active lever presses at 30D (unpaired t test, 1D vs. 30D relapse test: t(14) = 4.405, p = 0.0006; Figure 1). Subsequent analyses were performed with this experimental group to provide insight into how miRNA expression is affected by cue re-exposure in early and late abstinence. ...
... Correlation analyses of calculated ΔΔCt mir-1298-5p expression in the NAc with the total number of morphine infusions over 10 days (D) of Self-administration (SA) after 30D forced abstinence with a relapse test (a) or after 1D forced abstinence with no relapse test (b two abstinence timepoints suggested that this miRNA may be regulated in a dose-dependent manner. To examine this potential miRNA-morphine relationship, we segregated animals into two groups based on their average number of infusions during SA by performing a median split as previously described (Brabant et al., 2010;Puhl et al., 2009;Zanda et al., 2021). Animals that selfadministered less than the median (35 infusions) were considered 'low morphine', while animals that selfadministered more than the median were considered 'high morphine'. ...
A powerful motivation to seek opioids remains after drug cessation and intensifies during extended periods of abstinence. Unfortunately, biomarkers associated with continued drug seeking have not been described. Moreover, previous studies have focused on the effects of early abstinence with little exploration into the long-term drug-induced mechanisms that occur after extended abstinence. Here we demonstrated that 30 days of forced abstinence results in a time-dependent increase in morphine seeking in a rat model of morphine self-administration (SA). We measured expression of known drug-responsive microRNAs (miRNAs) in the nucleus accumbens, an area critical for reward-related plasticity, during early or late abstinence in animals that either underwent a cue-induced relapse test or no relapse test. miRNAs are small noncoding RNAs that represent suitable biomarker candidates due to their long-lasting nature. mir-32-5p levels during early abstinence negatively correlated with active lever pressing in both cue-exposed and cue-naïve animals. mir-1298-5p positively correlated with drug SA history after a relapse test during late abstinence. When animals underwent acute abstinence with no relapse test, mir-1298-5p correlated with drug infusions and active lever pressing during SA. In late abstinence with no relapse test, mir-137-3p negatively correlated with drug infusions. Regulation of mir-32-5p target genes and significant correlation of target gene mRNA with mir-32-5p was observed after abstinence. These results indicate lasting regulation of miRNA expression is associated with drug intake following morphine SA. In addition, we conclude that the miRNA profile undergoes regulation from early to late abstinence and miRNA expression may indicate past drug history.
... We demonstrated that heroin self-administration of a 0.03 mg/kg dosage can induce regulation of 76 circRNAs in the OFC. This dosage of heroin has been used by many labs to model drug-seeking behavior and we have previously demonstrated that animals that have higher amounts of drug intake at this dosage typically have more drug-seeking behavior after extended forced abstinence [4,51]. However, this dosage does not result in incubation of heroin craving behavior, and we expect that a higher dosage protocol (e.g., 0.075 or 1.0 mg/kg/infusion) may result in an additional unique set of circRNAs specifically associated with long-lasting drug seeking behavior. ...
... After 5-7 days of acclimation, animals underwent intravenous catheter surgery for heroin or saline self-administration, as previously described [51]. Following surgery, all animals were singly housed for the remainder of the study. ...
... Heroin self-administration studies were conducted in operant chambers under a fixed ratio (FR) 1 schedule, as previously described [51]. For heroin self-administration, active lever pressing resulted in activation of the infusion pump for intravenous infusion of 0.03 mg/kg/infusion of heroin solution, presentation of a 65 db, 2.9 kHz acoustic cue and illumination of a stimulus light above the active lever. ...
The number of drug overdose deaths involving opioids continues to rise in the United States. Many patients with opioid use disorder (OUD) that seek treatment still experience relapse. Perseverant opioid seeking behaviors represent a major challenge to treating OUD and additional therapeutic development will require insight into opioid-induced neurobiological adaptations. In this study, we explored the regulation of a novel class of RNAs, circular RNAs (circRNAs), by the addictive opioid heroin in the rat orbitofrontal cortex (OFC), a brain region that mediates behavioral responses to rewarding stimuli. Microarray analysis identified 76 OFC circRNAs significantly regulated in male rats after heroin self-administration. We evaluated the specificity of these findings by measuring heroin-associated circRNA expression in female rats after heroin self-administration and in rats that self-administered sucrose. We identify circGrin2b, circUbe2cp, circAnks1a, circAdcy5 and circSlc24A2 as heroin-responsive circRNAs in the OFC. Linear mRNA levels of heroin-associated circRNAs were unchanged except for Grin2b and Adcy5. An integrated bioinformatics analysis of regulated circRNAs identified microRNAs predicted to bind heroin-associated circRNAs and downstream targets of circRNA: microRNA sponging. Thus, heroin regulates the expression of OFC RNA splice variants that circularize and may impact cellular processes that contribute to the neurobiological adaptations that arise from chronic heroin exposure.
Preclinical and human studies indicate psilocybin may reduce perseverant maladaptive behaviors, including nicotine and alcohol seeking. Such studies in the opioid field are lacking, though opioids are involved in more >50% of overdose deaths. Psilocybin is an agonist at the serotonin 2A receptor (5-HT2AR), a well-documented target for modulation of drug seeking, and evidence suggests 5-HT2AR agonists may dampen motivation for opioids. We sought to investigate the therapeutic efficacy of psilocybin in mediating cessation of opioid use and maintenance of long-lasting abstinence from opioid seeking behavior in a rat model of heroin self-administration (SA). Psilocybin or 5-HT2AR antagonists ketanserin and volinanserin were administered systemically to rats prior to SA of 0.075 mg/kg/infusion of heroin, or relapse following forced abstinence. Psilocybin did not alter heroin taking, but a single exposure to 3.0 mg/kg psilocybin 4-24 hours prior to a relapse test blunted cue-induced heroin seeking. Conversely, 5-HT2AR antagonists exacerbated heroin relapse. To begin to elucidate mechanisms of psilocybin, drug-naive rats received psilocybin and/or ketanserin, and tissue was collected from the prefrontal cortex (PFC), a region critical for drug seeking and responsive to psilocybin, 24 hours later for RNA-sequencing. 3.0 mg/kg psilocybin regulated ~2-fold more genes in the PFC than 1.0 mg/kg, including genes involved in the cytoskeleton and cytokine signaling. Ketanserin blocked >90% of psilocybin-regulated genes, including the IL-17a cytokine receptor, Il17ra. Psychedelic compounds have reported anti-inflammatory properties, and therefore we performed a gene expression array to measure chemokine/cytokine molecules in the PFC of animals that displayed psilocybin-mediated inhibition of heroin seeking. Psilocybin regulated 4 genes, including Il17a, and a subset of genes correlated with relapse behavior. Selective inhibition of PFC IL-17a was sufficient to reduce heroin relapse. We conclude that psilocybin reduces heroin relapse and highlight IL-17a signaling as a potential downstream pathway of psilocybin that also reduces heroin seeking.
