Estrogen receptor beta signaling enhances extinction memory recall for heroin-conditioned cues in a sex- and region-specific manner.

Return to use, or relapse, is a major challenge in the treatment of opioid use disorder (OUD). Relapse can be precipitated by several factors, including exposure to drug-conditioned cues. Identifying successful treatments to mitigate cue-induced relapse has been challenging, perhaps due to extinction memory recall (EMR) deficits. Previously, inhibition of estradiol (E2) signaling in the basolateral amygdala (BLA) impaired heroin-cue EMR. This effect was recapitulated by antagonism of BLA estrogen receptors (ER) in a sex-specific manner such that blocking ERα in males, but ERß in females, impaired EMR. However, it is unclear whether increased E2 signaling, in the BLA or systemically, enhances heroin-cue EMR. We hypothesized that ERß agonism would enhance heroin-cue EMR in a sex- and region-specific manner. To determine the capacity of E2 signaling to improve EMR, we pharmacologically manipulated ERß across several translationally designed experiments. First, male and female rats acquired heroin or sucrose self-administration. Next, during a cued extinction session, we administered diarylpropionitrile (DPN, an ERß agonist) and tested anxiety-like behavior on an open field. Subsequently, we assessed EMR in a cue-induced reinstatement test and, finally, measured ERß expression in several brain regions. Across all experiments, females took more heroin and sucrose than males and had greater responses during heroin-cued extinction. Administration of DPN in the BLA enhanced EMR in females only, driven by ERß's impacts on memory consolidation. Interestingly, however, systemic DPN administration improved EMR for heroin cues in both sexes across several different tests, but did not impact sucrose-cue EMR. Immunohistochemical analysis of ERß expression across several different brain regions showed that females only had greater expression of ERß in the basal nucleus of the BLA. Here, in several preclinical experiments, we demonstrated that ERß agonism enhances heroin-cue EMR and has potential utility in combatting cue-induced relapse.

Novel psychoplastogen DM506 reduces cue-induced heroin-seeking and inhibits tonic GABA currents in the Prelimbic Cortex.

Opioid use disorder is a major public health crisis that is manifested by persistent drug-seeking behavior and high relapse frequency. Most of the available treatments rely on targeting opioid receptors using small molecules that do not provide sustained symptom alleviation. Psychoplastogens are a novel class of non-opioid drugs that produce rapid and sustained effects on neuronal plasticity, intended to produce therapeutic benefits. Ibogalogs are synthetic derivatives of iboga alkaloids that lack hallucinogenic or adverse side effects. In the current study, we examine the therapeutic potential of DM506, a novel ibogalog lacking any cardiotoxic or hallucinogenic effects, in cue-induced seeking behavior following heroin self-administration. At a single systemic dose of 40 mg/kg, DM506 significantly decreased cue-induced seeking in both male and female rats at abstinence day 1 (AD1) following heroin self-administration. Upon re-testing for cue-induced seeking at AD14, we found that males receiving DM506 continued to show decreased cue-induced seeking, an effect not observed in females. Since there is evidence of psychedelics influencing tonic GABA currents, and opioid and psychoplastogen-mediated neuroadaptations in the medial prefrontal cortex (PrL) underlying its functional effects, we performed patch-clamp recordings on PrL slices of drug-naïve rats with an acute application or chronic incubation with DM506. Tonic GABA current was decreased in slices incubated with DM506 for 2 h. qPCR analysis did not reveal any differences in the mRNA levels of GABAA receptor α and δ subunits at AD14 in heroin and saline self-administered animals that received vehicle or DM506 at AD1. Overall, our data indicate that DM506 attenuates cue-induced heroin seeking and inhibits tonic GABA current in the prelimbic cortex.

A novel tramadol-polycaprolactone implant could palliate heroin conditioned place preference and withdrawal in rats: behavioral and neurochemical study.

Drug dependence is a chronic brain disease characterized by craving and recurrent episodes of relapse. Tramadol HCl is a promising agent for withdrawal symptoms management, considering its relatively low abuse potential and safety. Oral administration, however, is not preferred in abstinence maintenance programs. Introducing an implantable, long-lasting formula is suggested to help outpatient abstinence programs achieve higher rates of treatment continuation. Tramadol implants (T350 and T650) were prepared on polycaprolactone polymer ribbons by the wet method. Male Wistar rats were adapted to heroin-conditioned place preference (CPP) at escalating doses (3-30 mg/kg, intraperitoneally, for 14 days). Implants were surgically implanted in the back skin of rats. After 14 days, the CPP score was recorded. Naloxone (1 mg/kg, intraperitoneally) was used to induce withdrawal on day 15, and symptoms were scored. Elevated plus maze and open field tests were performed for anxiety-related symptoms. Striata were analyzed for neurochemical changes reflected in dopamine, 3,4-dihydroxyphenyl acetic acid, gamma-aminobutyric acid, and serotonin levels. Brain oxidative changes including glutathione and lipid peroxides were assessed. The tramadol implants (T350 and T650) reduced heroin CPP and limited naloxone-induced withdrawal symptoms. The striata showed increased levels of 3,4-dihydroxyphenyl acetic acid, and serotonin and decreased levels of gamma-aminobutyric acid and dopamine after heroin withdrawal induction, which were reversed after implanting T350 and T650. Implants restore the brain oxidative state. Nonsignificant low naloxone-induced withdrawal score after the implant was used in naive subjects indicating low abuse potential of the implants. The presented tramadol implants were effective at diminishing heroin CPP and withdrawal in rats, suggesting further investigations for application in the management of opioid withdrawal.

Pan-striatal reduction in the expression of the astrocytic dopamine transporter precedes the development of dorsolateral striatum dopamine-dependent incentive heroin seeking habits.

The emergence of compulsive drug-seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatal control over behaviour. This process involves the dopamine-dependent functional coupling of the anterior dorsolateral striatum (aDLS) with the nucleus accumbens core, but the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), the level of which is altered in individuals with heroin use disorder. Astrocytes are therefore uniquely placed functionally to bridge dopamine-dependent mechanisms across the striatum. Here we tested the hypothesis that exposure to heroin influences the expression of DAT in striatal astrocytes across the striatum before the development of DLS-dependent incentive heroin seeking habits. Using Western-blot, qPCR, and RNAscope™, we measured DAT protein and mRNA levels in whole tissue, culture and in situ astrocytes from striatal territories of rats with a well-established cue-controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior aDLS that was preceded by a heroin-induced reduction in DAT mRNA and protein in astrocytes across the striatum. Striatal astrocytes were also shown to be susceptible to direct dopamine- and opioid-induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits.

Insular cortex subregions have distinct roles in cued heroin seeking after extinction learning and prolonged withdrawal in rats.

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

Regulation of histone acetylation by garcinol blocks the reconsolidation of heroin-associated memory.

Drug-associated long-term memories underlie substance use disorders, including heroin use disorder (HUD), which are difficult to eliminate through existing therapies. Addictive memories may become unstable when reexposed to drug-related cues and need to be stabilized again through protein resynthesis. Studies have shown the involvement of histone acetylation in the formation and reconsolidation of long-term drug-associated memory. However, it remains unknown whether and how histone acetyltransferases (HAT), the essential regulators of histone acetylation, contribute to the reconsolidation of heroin-associated memories. Herein, we investigated the function of HAT in the reconsolidation concerning heroin-conditioned memory by using a rat self-administration model. Systemic administration of the HAT inhibitor garcinol inhibited cue and heroin-priming induced reinstatement of heroin seeking, indicating the treatment potential of garcinol for relapse prevention.

Ventilatory Effects of Fentanyl, Heroin, and d-Methamphetamine, Alone and in Mixtures in Male Rats Breathing Normal Air .

The number of drug overdoses and deaths has increased significantly over the past decade and co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures, particularly methamphetamine, are not well characterized. Two structurally different drugs with agonist properties at µ-opioid receptors (MOR), fentanyl and heroin, and d-methamphetamine, alone and in mixtures, were assessed for their effects on ventilation in rats breathing normal air. Whole-body phethysmography chambers were equipped with a tower and swivel allowing infusions to indwelling intravenous catheters. After a 45-minute habituation period, saline, fentanyl, heroin, or d-methamphetamine, alone and in mixtures, was administered. Five minutes later, the opioid receptor antagonist naloxone or vehicle was injected. Fentanyl (0.0032-0.1 mg/kg) and heroin (0.32-3.2 mg/kg) decreased ventilation [frequency (f) and tidal volume (VT)] in a dose-related manner whereas d-methamphetamine (0.1-3.2 mg/kg) increased f to >400% of control and decreased VT to <60% of control, overall increasing minute volume (product of f and VT) to >240% of control. When combined, d-methamphetamine (0.1-3.2 mg/kg) attenuated the ventilatory depressant effects of fentanyl (0.1 mg/kg) and heroin (3.2 mg/kg). d-Methamphetamine did not alter the potency of naloxone to reverse the ventilatory depressant effects of fentanyl or heroin. These studies demonstrate that d-methamphetamine can attenuate the ventilatory depressant effects of moderate doses of opioid receptor agonists while not altering the potency of naloxone to reverse opioid hypoventilation. SIGNIFICANCE STATEMENT: Co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures are not well characterized. This study reports that 1) d-methamphetamine attenuates the ventilatory depressant effects of moderate doses of two structurally different opioid receptor agonists, fentanyl and heroin, and 2) d-methamphetamine does not alter potency or effectiveness of naloxone to reverse the ventilatory depressant effects of these opioid receptor agonists.

Inhibition of Estradiol Signaling in the Basolateral Amygdala Impairs Extinction Memory Recall for Heroin-Conditioned Cues in a Sex-Specific Manner.

INTRODUCTION: Relapse is a major treatment barrier for opioid use disorder. Environmental cues become associated with the rewarding effects of opioids and can precipitate relapse, even after numerous unreinforced cue presentations, due to deficits in extinction memory recall (EMR). Estradiol (E2) modulates EMR of fear-related cues, but it is unknown whether E2 impacts EMR of reward cues and what brain region(s) are responsible for E2s effects. Here, we hypothesize that inhibition of E2 signaling in the basolateral amygdala (BLA) will impair EMR of a heroin-associated cue in both male and female rats. METHODS: We pharmacologically manipulated E2 signaling to characterize the role of E2 in the BLA on heroin-cue EMR. Following heroin self-administration, during which a light/tone cue was co-presented with each heroin infusion, rats underwent cued extinction to extinguish the conditioned association between the light/tone and heroin. During extinction, E2 signaling in the BLA was blocked by an aromatase inhibitor or specific estrogen receptor (ER) antagonists. The next day, subjects underwent a cued test to assess heroin-cue EMR. RESULTS: In both experiments, females took more heroin than males (mg/kg) and had higher operant responding during cued extinction. Inhibition of E2 synthesis in the BLA impaired heroin-cue EMR in both sexes. Notably, E2s actions are mediated by different ER mechanisms, ERα in males but ERß in females. CONCLUSIONS: This study is the first to demonstrate a behavioral role for centrally-produced E2 in the BLA and that E2 also impacts EMR of reward-associated stimuli in both sexes.

The effects of gonadal hormones on heroin Self-Administration in male gonadectomized rats.

RATIONALE: Previous studies have shown that gonadal hormones influence opioid self-administration in female rodents, but very few studies have examined these effects in male rodents. OBJECTIVES: The purpose of this study was to examine the effects of chronic hormone treatment on intravenous heroin self-administration in gonadectomized male rats using both physiological and supraphysiological doses of testosterone, estradiol, or progesterone. METHODS: Gonadectomized male rats were surgically implanted with intravenous catheters and trained to self-administer heroin on a fixed ratio (FR1) schedule of reinforcement. Using a between-subjects design, rats were treated daily with testosterone (0.175 or 1.75 mg, sc), estradiol (0.0005 or 0.005 mg, sc), progesterone, (0.0125 or 0.125 mg, sc), or their vehicles. After 14 days of chronic treatment, a dose-effect curve was determined for heroin (0.0003-0.03 mg/kg/infusion) over the course of one week. RESULTS: Neither testosterone nor estradiol altered responding maintained by heroin. In contrast, the high dose of progesterone (0.125 mg) reduced responding maintained by all doses of heroin to saline-control levels. This dose of progesterone did not reduce responding maintained by food on a progressive ratio schedule in either food-restricted or food-sated rats. CONCLUSIONS: These data indicate that exogenous progesterone or a pharmacologically active metabolite selectively decreases heroin intake in male rodents, which may have therapeutic implications for men with opioid use disorder.

Rats chasing the dragon: A new heroin inhalation method.

BACKGROUND: Despite extensive human use of inhalation for ingesting opioids, models in rodents have mostly been limited to parenteral injection and oral dosing. Methods using electronic drug delivery systems (EDDS; "e-cigarettes") have shown efficacy in rodent models but these do not faithfully mimic the most popular human inhalation method of heating heroin to the point of vaporization. NEW METHOD: Middle aged rats were exposed to vapor created by direct heating of heroin HCl powder in a ceramic e-cigarette type atomizer. Efficacy was determined with a warm water tail withdrawal nociception assay, rectal temperature and self-administration. RESULTS: Ten minutes of inhalation of vaporized heroin slowed response latency in a warm water tail withdrawal assay and increased rectal temperature in male rats, in a dose-dependent manner. Similar antinociceptive effects in female rats were attenuated by the opioid antagonist naloxone (1.0 mg/kg, s.c.). Female rats made operant responses for heroin vapor in 15-minute sessions, increased their response rate when the reinforcement ratio increased from FR1 to FR5, and further increased their responding when vapor delivery was omitted. Anti-nociceptive effects of self-administered volatilized heroin were of a similar magnitude as those produced by the 10-minute non-contingent exposure. COMPARISON WITH EXISTING METHODS: Inhalation of directly volatilized heroin successfully produces heroin-typical effects, comparable to EDDS inhalation delivery. CONCLUSIONS: This study shows that "chasing the dragon" methods of inhalation of heroin can be modeled successfully in the rat. Inhalation techniques may be particularly useful for longer term studies deep into the middle age of rats.

Cocaine and heroin interact differently with nondrug reinforcers in a choice situation.

The present study used a rat choice model to test how cocaine or heroin economically interacted with two different nondrug reinforcers along the substitute-to-complement continuum. In Experiment 1, the nondrug alternative was the negative reinforcer timeout-from-avoidance (TOA)-that is, rats could press a lever to obtain a period of safety from footshock. One group of rats chose between cocaine and TOA and another group chose between heroin and TOA. The relative prices of the reinforcers were manipulated across phases while controlling for potential income effects. When cocaine was the reinforcer, rats reacted to price changes by increasing their allocation of behavior to the more expensive option, thereby maintaining relatively proportional intake of cocaine and TOA reinforcers across prices, suggesting these reinforcers were complements here. In contrast, when heroin became relatively cheap, rats increased allocation of income to heroin and decreased allocation of income to TOA, suggesting that heroin substituted for safety. Additionally, rats were willing to accept more footshocks when heroin was easily available. In Experiment 2, the nondrug alternative was saccharin, a positive reinforcer. Heroin and saccharin were complements, but there was no consistent effect of price changes on the allocation of behavior between cocaine and saccharin. As a model of the processes that could be involved in human drug use, these results show that drug-taking behavior depends on the type of drug, the type of nondrug alternative available, and the prices of both. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Enhancing Protective Antibodies against Opioids through Antigen Display on Virus-like Particles.

Opioid use disorder (OUD) has become a public health crisis, with recent significant increases in the number of deaths due to overdose. Vaccination can provide an attractive complementary strategy to combat OUD. A key for high vaccine efficacy is the induction of high levels of antibodies specific to the drug of abuse. Herein, a powerful immunogenic carrier, virus-like particle mutant bacteriophage Qß (mQß), has been investigated as a carrier of a small molecule hapten 6-AmHap mimicking heroin. The mQß-6-AmHap conjugate was able to induce significantly higher levels of IgG antibodies against 6-AmHap than mice immunized with the corresponding tetanus toxoid-6-AmHap conjugate in head-to-head comparison studies in multiple strains of mice. The IgG antibody responses were persistent with high anti-6-AmHap titers 600 days after being immunized with mQß-6-AmHap. The antibodies induced exhibited strong binding toward multiple heroin/morphine derivatives that have the potential to be abused, while binding weakly to medications used for OUD treatment and pain relief. Furthermore, vaccination effectively reduced the impacts of morphine on mice in both ambulation and antinociception assays, highlighting the translational potential of the mQß-6-AmHap conjugate to mitigate the harmful effects of drugs of abuse.

Restoration of a paraventricular thalamo-accumbal behavioral suppression circuit prevents reinstatement of heroin seeking.

Lack of behavioral suppression typifies substance use disorders, yet the neural circuit underpinnings of drug-induced behavioral disinhibition remain unclear. Here, we employ deep-brain two-photon calcium imaging in heroin self-administering mice, longitudinally tracking adaptations within a paraventricular thalamus to nucleus accumbens behavioral inhibition circuit from the onset of heroin use to reinstatement. We find that select thalamo-accumbal neuronal ensembles become profoundly hypoactive across the development of heroin seeking and use. Electrophysiological experiments further reveal persistent adaptations at thalamo-accumbal parvalbumin interneuronal synapses, whereas functional rescue of these synapses prevents multiple triggers from initiating reinstatement of heroin seeking. Finally, we find an enrichment of µ-opioid receptors in output- and cell-type-specific paraventricular thalamic neurons, which provide a mechanism for heroin-induced synaptic plasticity and behavioral disinhibition. These findings reveal key circuit adaptations that underlie behavioral disinhibition in opioid dependence and further suggest that recovery of this system would reduce relapse susceptibility.

The Impact of Heroin Self-Administration and Environmental Enrichment on Ventral Tegmental CRF1 Receptor Expression.

BACKGROUND: There is a strong link between chronic stress and vulnerability to drug abuse and addiction. Corticotropin releasing factor (CRF) is central to the stress response that contributes to continuation and relapse to heroin abuse. Chronic heroin exposure can exacerbate CRF production, leading to dysregulation of the midbrain CRF-dopamine-glutamate interaction. METHODS: Here we investigated the role of midbrain CRF1 receptors in heroin self-administration and assessed neuroplasticity in CRF1 receptor expression in key opioid addiction brain regions. RESULTS: Infusions of antalarmin (a CRF1 receptor antagonist) into the ventral tegmental area (VTA) dose dependently reduced heroin self-administration in rats but had no impact on food reinforcement or locomotor activity in rats. Using RNAscope in situ hybridization, we found that heroin, but not saline, self-administration upregulated CRF1 receptor mRNA in the VTA, particularly on dopamine neurons. AMPA GluR1 and dopamine reuptake transporter mRNA in VTA neurons were not affected by heroin. The western-blot assay showed that CRF1 receptors were upregulated in the VTA and nucleus accumbens. No significant changes in CRF1 protein expression were detected in the prefrontal cortex, insula, dorsal hippocampus, and substantia nigra. In addition, we found that 15 days of environmental enrichment implemented after heroin self-administration does not reverse upregulation of VTA CRF1 receptor mRNA but it downregulates dopamine transporter mRNA. CONCLUSIONS: Overall, these data suggest that heroin self-administration requires stimulation of VTA CRF1 receptors and upregulates their expression in brain regions involved in reinforcement. Such long-lasting neuroadaptations may contribute to continuation of drug use and relapse due to stress exposure and are not easily reversed by EE exposure.

Role of dopamine D1 receptor in the modulation of memory consolidation by passive and self-administered heroin and associated conditioned stimuli.

It has been proposed that opiates modulate memory consolidation, but recent work has indicated that this effect may be mediated by how the drug is experienced (i.e., passive injections vs. self-administration). Because the dopamine (DA) D1 receptor is involved in processing of learning signals and attribution of salience to events experienced by an organism, two studies in male Sprague-Dawley rats tested the effect of blocking this receptor on modulation of memory consolidation by passive and self-administered heroin, in addition to conditioned memory modulation by heroin-paired cues. Using the object location memory task, Study 1 employed SCH23390 (0, 0.05, 0.10 mg/kg, SC) to modulate enhancement of memory consolidation induced by post-training injections of heroin (1 mg/kg, SC) as well as by exposure to the environment paired with heroin injections (6 pairings, 1 h each, 1 mg/kg). Study 2 was conducted in rats that could self-administer heroin (0.05 mg/kg/infusion, IV) and tested whether SCH23390 (0 and 0.1 mg/kg, SC) could prevent memory modulation induced by a change in schedule of self-administration (from fixed to variable ratio). It was found that while repeated passive injections of heroin retained their enhancing effect on memory, when self-administered, heroin enhanced consolidation of object location memory only at the beginning of self-administration and after a change in schedule. Importantly, SCH23390 blocked memory modulation by heroin when passively administered and when the drug was self-administered on a novel schedule. SCH23390 also blocked conditioned memory modulation induced by post-training exposure to heroin-paired cues. Taken together, these results suggest that modulation of memory consolidation by unconditioned and conditioned opiate reinforcers involve a D1-dependent mechanism of salience attribution linked to the anticipation of drug effects.

The Cdk5 inhibitor ß-butyrolactone impairs reconsolidation of heroin-associated memory in the rat basolateral amygdala.

The persistence of maladaptive heroin-associated memory, which is triggered by drug-related stimuli that remind the individual of the drug's pleasurable and rewarding effects, can impede abstinence efforts. Cyclin-dependent kinase 5 (Cdk5), a neuronal serine/threonine protein kinase that plays a role in multiple neuronal functions, has been demonstrated to be involved in drug addiction and learning and memory. Here, we aimed to investigate the role of cdk5 activity in the basolateral amygdala (BLA) in relapse to heroin seeking, using a self-administration rat model. Male rats underwent 10 days of heroin self-administration training, during which an active nose poke resulted in an intravenous infusion of heroin that was accompanied by a cue. The rats then underwent nose poke extinction for 10 days, followed by subsequent tests of heroin-seeking behaviour. We found that intra-BLA infusion of ß-butyrolactone (100 ng/side), a Cdk5 inhibitor, administered 5 min after reactivation, led to a subsequent decrease in heroin-seeking behaviour. Further experiments demonstrated that the effects of ß-butyrolactone are dependent on reactivated memories, temporal-specific and long-lasting on relapse of heroin-associated memory. Results provide suggestive evidence that the activity of Cdk5 in BLA is critical for heroin-associated memory and that the specific inhibitor, ß-butyrolactone, may hold potential as a substance for the treatment of heroin abuse.

Heroin Self-Administration and Extinction Increase Prelimbic Cortical Astrocyte-Synapse Proximity and Alter Dendritic Spine Morphometrics That Are Reversed by N-Acetylcysteine.

Clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission significantly contribute to heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce cellular adaptations in both neurons and astrocytes within the nucleus accumbens (NA) core that are required for cue-induced heroin seeking. Specifically, decreased glutamate clearance and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate release from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Normalizing astrocyte glutamate homeostasis with drugs like the antioxidant N-acetylcysteine (NAC) prevents cue-induced heroin seeking. Surprisingly, little is known about heroin-induced alterations in astrocytes or pyramidal neurons projecting to the NAcore in the PrL cortex (PrL-NAcore). Here, we observe functional adaptations in the PrL cortical astrocyte following heroin self-administration (SA) and extinction as measured by the electrophysiologically evoked plasmalemmal glutamate transporter 1 (GLT-1)-dependent current. We likewise observed the increased complexity of the glial fibrillary acidic protein (GFAP) cytoskeletal arbor and increased association of the astrocytic plasma membrane with synaptic markers following heroin SA and extinction training in the PrL cortex. Repeated treatment with NAC during extinction reversed both the enhanced astrocytic complexity and synaptic association. In PrL-NAcore neurons, heroin SA and extinction decreased the apical tuft dendritic spine density and enlarged dendritic spine head diameter in male Sprague-Dawley rats. Repeated NAC treatment during extinction prevented decreases in spine density but not dendritic spine head expansion. Moreover, heroin SA and extinction increased the co-registry of the GluA1 subunit of AMPA receptors in both the dendrite shaft and spine heads of PrL-NAcore neurons. Interestingly, the accumulation of GluA1 immunoreactivity in spine heads was further potentiated by NAC treatment during extinction. Finally, we show that the NAC treatment and elimination of thrombospondin 2 (TSP-2) block cue-induced heroin relapse. Taken together, our data reveal circuit-level adaptations in cortical dendritic spine morphology potentially linked to heroin-induced alterations in astrocyte complexity and association at the synapses. Additionally, these data demonstrate that NAC reverses PrL cortical heroin SA-and-extinction-induced adaptations in both astrocytes and corticostriatal neurons.

P-glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Main Active Heroin Metabolites 6-monoacetylmorphine (6-MAM) and Morphine in Mice.

BACKGROUND & PURPOSE: Heroin (diacetylmorphine; diamorphine) is a highly addictive opioid prodrug. Heroin prescription is possible in some countries for chronic, treatment-refractory opioid-dependent patients and as a potent analgesic for specific indications. We aimed to study the pharmacokinetic interactions of heroin and its main pharmacodynamically active metabolites, 6-monoacetylmorphine (6-MAM) and morphine, with the multidrug efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2 using wild-type, Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice. METHODS & RESULTS: Upon subcutaneous (s.c.) heroin administration, its blood levels decreased quickly, making it challenging to detect heroin even shortly after dosing. 6-MAM was the predominant active metabolite present in blood and most tissues. At 10 and 30 min after heroin administration, 6-MAM and morphine brain accumulation were increased about 2-fold when mouse (m)Abcb1a/1b and mAbcg2 were ablated. Fifteen minutes after direct s.c. administration of an equimolar dose of 6-MAM, we observed good intrinsic brain penetration of 6-MAM in wild-type mice. Still, mAbcb1 limited brain accumulation of 6-MAM and morphine without affecting their blood exposure, and possibly mediated their direct intestinal excretion. A minor contribution of mAbcg2 to these effects could not be excluded. CONCLUSIONS: We show that mAbcb1a/1b can limit 6-MAM and morphine brain exposure. Pharmacodynamic behavioral/postural observations, while non-quantitative, supported moderately increased brain levels of 6-MAM and morphine in the knockout mouse strains. Variation in ABCB1 activity due to genetic polymorphisms or environmental factors (e.g., drug interactions) might affect 6-MAM/morphine exposure in individuals, but only to a limited extent.

Cognitive Enhancer Donepezil Attenuates Heroin-Seeking Behavior Induced by Cues in Rats.

PURPOSE: Opioid use disorder is a significant global problem. Chronic heroin use is associated with impairment of cognitive function and conscious control ability. The cholinergic system can be disrupted following heroin administration, indicating that activation of the cholinergic system may prevent chronic heroin misuse. Donepezil as an inhibitor of cholinesterase has been reported to clinically improve cognition and attention. In this study, the inhibition of heroin self-administration and heroin-seeking behaviours by donepezil were evaluated in rats. METHODS: Rats were trained to self-administer heroin every four hours for 14 consecutive days under a fixed ratio 1 (FR1) reinforcement schedule, then underwent withdrawal for two weeks. A progressive ratio schedule was then used to evaluate the relative motivational value of heroin reinforcement. After withdrawal, a conditioned cue was introduced for the reinstatement of heroin-seeking behaviour. Donepezil (0.3-3 mg/kg, i.p.) was used during both the FR1 heroin self-administration and progressive ratio schedules. Immunohistochemistry was used to investigate the mechanism of action of donepezil in the rat brain. RESULTS: Pre-treatment with high dose donepezil (3 mg/kg) but not low doses (0.3-1 mg/kg) significantly inhibited heroin self-administration under the FR1 schedule. Donepezil decreased motivation values under the progressive ratio schedule in a dose-dependent manner. All doses of donepezil (1-3 mg/kg) decreased the reinstatement of heroin seeking induced by cues. Correlation analysis indicated that the inhibition of donepezil on heroin-seeking behaviour was positively correlated with an increased expression of dopamine receptor 1 (D1R) and dopamine receptor 2 (D2R) in the nucleus accumbens (NAc) and increased expression of choline acetyltransferase (ChAT) in the ventral tegmental area (VTA). CONCLUSIONS: The present study demonstrated that donepezil could inhibit heroin intake and heroin-seeking behaviour. Further, donepezil could regulate dopamine receptors in the NAc via an increase of acetylcholine. These results suggested that donepezil could be developed as a potential approach for the treatment of heroin misuse.

Blockade of the Dopamine D3 Receptor Attenuates Opioids-Induced Addictive Behaviours Associated with Inhibiting the Mesolimbic Dopamine System.

Opioid use disorder (OUD) has become a considerable global public health challenge; however, potential medications for the management of OUD that are effective, safe, and nonaddictive are not available. Accumulating preclinical evidence indicates that antagonists of the dopamine D3 receptor (D3R) have effects on addiction in different animal models. We have previously reported that YQA14, a D3R antagonist, exhibits very high affinity and selectivity for D3Rs over D2Rs, and is able to inhibit cocaine- or methamphetamine-induced reinforcement and reinstatement in self-administration tests. In the present study, our results illustrated that YQA14 dose-dependently reduced infusions under the fixed-ratio 2 procedure and lowered the breakpoint under the progressive-ratio procedure in heroin self-administered rats, also attenuated heroin-induced reinstatement of drug-seeking behavior. On the other hand, YQA14 not only reduced morphine-induced expression of conditioned place preference but also facilitated the extinguishing process in mice. Moreover, we elucidated that YQA14 attenuated opioid-induced reward or reinforcement mainly by inhibiting morphine-induced up-regulation of dopaminergic neuron activity in the ventral tegmental area and decreasing dopamine release in the nucleus accumbens with a fiber photometry recording system. These findings suggest that D3R might play a very important role in opioid addiction, and YQA14 may have pharmacotherapeutic potential in attenuating opioid-induced addictive behaviors dependent on the dopamine system.