Epigenetic function during heroin self-administration controls future relapse-associated behavior in a cell type-specific manner.

Opioid use produces enduring associations between drug reinforcement/euphoria and discreet or diffuse cues in the drug-taking environment. These powerful associations can trigger relapse in individuals recovering from opioid use disorder (OUD). Here, we sought to determine whether the epigenetic enzyme, histone deacetylase 5 (HDAC5), regulates relapse-associated behavior in an animal model of OUD. We examined the effects of nucleus accumbens (NAc) HDAC5 on both heroin- and sucrose-seeking behaviors using operant self-administration paradigms. We utilized cre-dependent viral-mediated approaches to investigate the cell-type-specific effects of HDAC5 on heroin-seeking behavior, gene expression, and medium spiny neuron (MSN) cell and synaptic physiology. We found that NAc HDAC5 functions during the acquisition phase of heroin self-administration to limit future relapse-associated behavior. Moreover, overexpressing HDAC5 in the NAc suppressed context-associated and reinstated heroin-seeking behaviors, but it did not alter sucrose seeking. We also found that HDAC5 functions within dopamine D1 receptor-expressing MSNs to suppress cue-induced heroin seeking, and within dopamine D2 receptor-expressing MSNs to suppress drug-primed heroin seeking. Assessing cell-type-specific transcriptomics, we found that HDAC5 reduced expression of multiple ion transport genes in both D1- and D2-MSNs. Consistent with this observation, HDAC5 also produced firing rate depression in both MSN classes. These findings revealed roles for HDAC5 during active heroin use in both D1- and D2-MSNs to limit distinct triggers of drug-seeking behavior. Together, our results suggest that HDAC5 might limit relapse vulnerability through regulation of ion channel gene expression and suppression of MSN firing rates during active heroin use.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

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.

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.

Gabapentinoids Increase the Potency of Fentanyl and Heroin and Decrease the Potency of Naloxone to Antagonize Fentanyl and Heroin in Rats Discriminating Fentanyl.

Despite a significant decrease in the number of prescriptions for opioids, the opioid crisis continues, fueled in large part by the availability of the phenylpiperidine mu opioid receptor (MOR) agonist fentanyl. In contrast, the number of prescriptions for and the off-label use of gabapentinoids (gabapentin and pregabalin) has increased dramatically, with gabapentinoids commonly detected in opioid overdose victims. Although gabapentinoids can decrease the potency of the opioid receptor antagonist naloxone to reverse heroin-induced hypoventilation in male rats, the specificity and nature of interaction between gabapentinoids and MOR agonists and any potential sex difference in those interactions are not well characterized. Gabapentinoids were studied in female and male rats discriminating fentanyl (0.0032 mg/kg, i.p.) or cocaine (3.2 mg/kg, i.p.). Alone, neither gabapentin nor pregabalin significantly increased fentanyl- or cocaine-appropriate responding. In rats discriminating fentanyl, each gabapentinoid dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the left, whereas naloxone dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the right. Each gabapentinoid (100 mg/kg) significantly decreased the potency of naloxone to antagonize the discriminative stimulus effect of fentanyl or heroin. In contrast, each gabapentinoid dose-dependently shifted the cocaine and d-methamphetamine discrimination dose-effect functions to the right. There were no significant sex differences in this study. These results suggest that gabapentinoids impact the misuse of opioids, the co-use of opioids and stimulant drugs, and the increasing number of overdose deaths in individuals using opioids, stimulant drugs, and gabapentinoids in mixtures. SIGNIFICANCE STATEMENT: The number of prescriptions for and the off-label use of gabapentinoids (gabapentin and pregabalin) has increased dramatically, with gabapentinoids commonly detected in opioid overdose victims. This study reports that in rats gabapentinoids increase the potency of fentanyl and heroin to produce discriminative stimulus effects while decreasing the potency of naloxone to antagonize those effects of fentanyl and heroin. These results can help guide policies for regulating gabapentinoids and treating opioid misuse and overdose.

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.

Whole-brain white matter abnormalities in human cocaine and heroin use disorders: association with craving, recency, and cumulative use.

Neuroimaging studies in substance use disorder have shown widespread impairments in white matter (WM) microstructure suggesting demyelination and axonal damage. However, substantially fewer studies explored the generalized vs. the acute and/or specific drug effects on WM. Our study assessed whole-brain WM integrity in three subgroups of individuals addicted to drugs, encompassing those with cocaine (CUD) or heroin (HUD) use disorder, compared to healthy controls (CTL). Diffusion MRI was acquired in 58 CTL, 28 current cocaine users/CUD+, 32 abstinent cocaine users/CUD-, and 30 individuals with HUD (urine was positive for cocaine in CUD+ and opiates used for treatment in HUD). Tract-Based Spatial Statistics allowed voxelwise analyses of diffusion metrics [fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD)]. Permutation statistics (p-corrected < 0.05) were used for between-group t-tests. Compared to CTL, all individuals with addiction showed widespread decreases in FA, and increases in MD, RD, and AD (19-57% of WM skeleton, p < 0.05). The HUD group showed the most impairments, followed by the CUD+, with only minor FA reductions in CUD- (<0.2% of WM skeleton, p = 0.05). Longer periods of regular use were associated with decreased FA and AD, and higher subjective craving was associated with increased MD, RD, and AD, across all individuals with drug addiction (p < 0.05). These findings demonstrate extensive WM impairments in individuals with drug addiction characterized by decreased anisotropy and increased diffusivity, thought to reflect demyelination and lower axonal packing. Extensive abnormalities in both groups with positive urine status (CUD+ and HUD), and correlations with craving, suggest greater WM impairments with more recent use. Results in CUD-, and correlations with regular use, further imply cumulative and/or persistent WM damage.

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.

An investigation of economic interactions between social reinforcement and heroin or cocaine in rats.

The primary goal of the present study was to determine the economic relationship between heroin and social reinforcement in rats: are they substitutes, independents, or complements? In Experiment 1, one group of rats was given a budget of responses that they could allocate between heroin and social reinforcement offered at various combinations of prices. A second group chose between two levers that each resulted in social reinforcement at varying prices when pressed. There was no relationship between the relative allocation of responses between heroin and social reinforcement and changes in their relative prices, indicating that these reinforcers are best viewed as independents. In contrast, when choosing between two sources of social reinforcement, rats increased the allocation of behavior to the cheaper option, confirming that the method used here was sensitive to detecting substitution effects. In Experiment 2, the same method was used to compare one group that chose between heroin and social reinforcement with a second group that chose between cocaine and social reinforcement. The finding that heroin and social reinforcement were independents was replicated. Additionally, there was some evidence that cocaine and social reinforcement were substitutes, at least when the first few minutes of the session were excluded. These results add to our knowledge of how drug and nondrug reinforcers interact in choice situations in rats and may model factors that influence drug use in humans.

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.

A Conditioned Place Preference for Heroin Is Signaled by Increased Dopamine and Direct Pathway Activity and Decreased Indirect Pathway Activity in the Nucleus Accumbens.

Repeated pairing of a drug with a neutral stimulus, such as a cue or context, leads to the attribution of the drug's reinforcing properties to that stimulus, and exposure to that stimulus in the absence of the drug can elicit drug-seeking. A principal role for the NAc in the response to drug-associated stimuli has been well documented. Direct and indirect pathway medium spiny neurons (dMSNs and iMSNs) have been shown to bidirectionally regulate cue-induced heroin-seeking in rats expressing addiction-like phenotypes, and a shift in NAc activity toward the direct pathway has been shown in mice following cocaine conditioned place preference (CPP). However, how NAc signaling guides heroin CPP, and whether heroin alters the balance of signaling between dMSNs and iMSNs, remains unknown. Moreover, the role of NAc dopamine signaling in heroin reinforcement is unclear. Here, we integrate fiber photometry for in vivo monitoring of dopamine and dMSN/iMSN calcium activity with a heroin CPP procedure in rats to begin to address these questions. We identify a sensitization-like response to heroin in the NAc, with prominent iMSN activity during initial heroin exposure and prominent dMSN activity following repeated heroin exposure. We demonstrate a ramp in dopamine activity, dMSN activation, and iMSN inactivation preceding entry into a heroin-paired context, and a decrease in dopamine activity, dMSN inactivation, and iMSN activation preceding exit from a heroin-paired context. Finally, we show that buprenorphine is sufficient to prevent the development of heroin CPP and reduce Fos activation in the NAc after conditioning. Together, these data support the hypothesis that an imbalance in NAc activity contributes to the development of drug-cue associations that can drive addiction processes.SIGNIFICANCE STATEMENT The attribution of the reinforcing effects of drugs to neutral stimuli (e.g., cues and contexts) contributes to the long-standing nature of addiction, as re-exposure to drug-associated stimuli can reinstate drug-seeking and -taking even after long periods of abstinence. The NAc has an established role in encoding the value of drug-associated stimuli, and dopamine release into the NAc is known to modulate the reinforcing effects of drugs, including heroin. Using fiber photometry, we show that entering a heroin-paired context is driven by dopamine signaling and NAc direct pathway activation, whereas exiting a heroin-paired context is driven by NAc indirect pathway activation. This study provides further insight into the role of NAc microcircuitry in encoding the reinforcing properties of heroin.

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.

Paraventricular Nucleus of the Hypothalamus Oxytocin and Incubation of Heroin Seeking.

INTRODUCTION: There are numerous pharmacologic treatments for opioid use disorder (OUD), but none that directly target the underlying addictive effects of opioids. Oxytocin, a peptide hormone produced in the paraventricular nucleus (PVN) of the hypothalamus, has been investigated as a potential therapeutic for OUD. Promising preclinical and clinical results have been reported, but the brain region(s) and mechanism(s) by which oxytocin impacts reward processes remain undetermined. METHODS: Here, we assess peripherally administered oxytocin's impacts on cued reinstatement of heroin seeking following forced abstinence and its effects on neuronal activation in the PVN and key projection regions. We also examine how designer receptors exclusively activated by designer drug (DREADD)-mediated activation or inhibition of oxytocinergic PVN neurons alters cued heroin seeking and social interaction. RESULTS: As predicted, peripheral oxytocin administration successfully decreased cued heroin seeking on days 1 and 30 of abstinence. Oxytocin administration also led to increased neuronal activity within the PVN and the central amygdala (CeA). Activation of oxytocinergic PVN neurons with an excitatory (Gq) DREADD did not impact cued reinstatement or social interaction. In contrast, suppression with an inhibitory (Gi) DREADD reduced heroin seeking on abstinence day 30 and decreased time spent interacting with a novel conspecific. DISCUSSION: These findings reinforce oxytocin's therapeutic potential for OUD, the basis for which may be driven in part by increased PVN-CeA circuit activity. Our results also suggest that oxytocin has distinct signaling and/or other mechanisms of action to produce these effects, as inhibition, but not activation, of oxytocinergic PVN neurons did not recapitulate the suppression in heroin seeking.

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.

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.

Assessment of abuse potential of carfentanil.

Carfentanil, as a fentanyl analogue, is a potent synthetic opioid. It has been controlled in many countries, and its emergence has been highlighted by many recent reports. However, although discriminative stimulus effects of carfentanil in rats had been reported, its abuse potential has not been fully evaluated. In this study, we evaluated the abuse potential of carfentanil via the tests of conditioned place preference (CPP), drug self-administration and naloxone-precipitated opioid withdrawal assay, compared with fentanyl and heroin. Carfentanil exhibited significant place preference at a minimum dose of 1 µg/kg in mice, whereas fentanyl and heroin induced significant place preference at the minimum doses of 100 µg/kg and 1000 µg/kg, respectively. In the drug-substitution test in heroin self-administered rats (50 µg/kg/infusion), carfentanil and fentanyl acquired significant self-administrations above saline levels from 0.05-0.1 and 0.1-10.0 µg/kg/infusion, respectively. Carfentanil induced the maximum number of infusions at 0.1 µg/kg, whereas fentanyl and heroin at 1 and 25 µg/kg, respectively. In short, carfentanil showed the highest potency to induce CPP and self-administration. Furthermore, repeated treatment with escalating doses of carfentanil, fentanyl or heroin induced typical withdrawal symptoms in mice, including a greater number of jumping and weight loss than saline group. This indicated that carfentanil could produce physical dependence similar to fentanyl and heroin. Taken together, the present study demonstrated the higher abuse potential of carfentanil compared with fentanyl and heroin. The rank order of abuse potential for these compounds is carfentanil > fentanyl > heroin.

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.

Assessing the Role of Corticothalamic and Thalamo-Accumbens Projections in the Augmentation of Heroin Seeking in Chronically Food-Restricted Rats.

Drug addiction is a chronic disorder characterized by compulsive drug seeking, and involves repetitive cycles of compulsive drug use, abstinence, and relapse. In both human and animal models of addiction, chronic food restriction increases rates of relapse. Our laboratory has reported a robust increase in drug seeking following a period of withdrawal in chronically food-restricted rats compared with sated controls. Recently, we reported that activation of the paraventricular nucleus of the thalamus (PVT) abolished heroin seeking in chronically food-restricted rats. However, the precise inputs and outputs of the PVT that mediate this effect remain elusive. The goal of the current study was to determine the role of corticothalamic and thalamo-accumbens projections in the augmentation of heroin seeking induced by chronic food restriction. Male Long-Evans rats were trained to self-administer heroin for 10 d. Next, rats were removed from the self-administration chambers and were subjected to a 14 d withdrawal period while sated (unlimited access to food) or mildly food-restricted (FDR). On day 14, rats were returned to the self-administration context for a 3 h heroin-seeking test under extinction conditions during which corticothalamic and thalamo-accumbens neural activity was altered using chemogenetics. Surprisingly, chemogenetic activation or inhibition of corticothalamic projections did not alter heroin-seeking behavior. Chemogenetic activation of thalamo-accumbens shell, but not core, projectors attenuated heroin seeking in FDR rats. The results indicate an important role for the PVT to nucleus accumbens shell projections in the augmentation of heroin seeking induced by chronic food restriction.SIGNIFICANCE STATEMENT Relapse to heroin use is one of the major obstacles in the treatment of opiate addiction. Triggers for relapse are modulated by environmental challenges such as caloric restriction. Elucidating the brain mechanisms that underlie relapse is critical for evidence-based treatment development. Here we demonstrate a critical role for the input from the paraventricular thalamus (PVT), a hub for cortical, sensory, and limbic information, to the nucleus accumbens shell (an area known to be important for reward and motivation) in the augmentation of heroin seeking in food-restricted rats. Our findings highlight a previously unknown role for the PVT in heroin seeking following a period of abstinence.

Acute glucagon-like peptide-1 receptor agonist liraglutide prevents cue-, stress-, and drug-induced heroin-seeking in rats.

Substance use disorder is challenging to treat due to its relapsing nature. In the last decade, opioid use disorder has been a threat to public health, being declared an epidemic by the Centers for Disease Control and Prevention. This is a tragic situation, considering there currently are only three effective, yet not ideal, treatments to prevent relapse to opioids. Recent research has shown that hormones that modulate hunger and satiety also can modulate motivated behavior for drugs of abuse. For example, the short-acting analog of glucagon-like peptide-1 (GLP-1), an incretin hormone that regulates homeostatic feeding, has been shown to reduce responding for rewarding stimuli such as food, cocaine, heroin, and nicotine when administered over several days or weeks. This may serve as an effective adjuvant during treatment; however, whether it would be effective when used acutely to bridge a patient between cessation of use and onset of medication for the treatment of an opioid addiction is unknown. Here, we tested the acute effects of the longer acting GLP-1 analog, liraglutide, on heroin-seeking. In rats with heroin self-administration experience, we found that subcutaneous administration of an acute dose of 0.3-mg/kg liraglutide was effective in preventing drug-seeking after exposure to three major precipitators: drug-associated cues, stress (yohimbine-induced), and the drug itself. Finally, we confirmed that the reduction in drug-seeking is not due to a locomotor impairment, as liraglutide did not significantly alter performance in a rotarod test. As such, acute use of GLP-1 analogs may serve as a new and effective nonopioid bridge to treatment.

CBD-mediated regulation of heroin withdrawal-induced behavioural and molecular changes in mice.

Cannabidiol (CBD) may represent a promising therapeutic tool for treating opioid use disorder (OUD). This study was aimed to evaluate the effects of CBD on the behavioural and gene expression alterations induced by spontaneous heroin withdrawal. Thirty hours after cessation of 8-day heroin treatment (5, 10, 20 and 40 mg·kg-1 /12 h; s.c.), spontaneous heroin withdrawal was evaluated in CD1 male mice. The effects of CBD (5, 10 and 20 mg·kg-1 ; i.p.) on withdrawal-related behaviour were evaluated by measuring anxiety-like behaviour, motor activity and somatic signs. Furthermore, gene expression changes of mu-opioid receptor (Oprm1), proopiomelanocortin (Pomc), cannabinoid CB1 (Cnr1) and CB2 (Cnr2) receptors in the nucleus accumbens (NAcc) and tyrosine hydroxylase (TH) and Pomc in the ventral tegmental area (VTA) were also evaluated by real-time PCR. Anxiety-like behaviour, motor activity and withdrawal-related somatic signs were significantly increased in heroin-treated mice compared to the control group. Interestingly, CBD treatment significantly reduced these behavioural impairments and normalized gene expression of Cnr1 and Pomc in the NAcc and TH in the VTA of mice exposed to spontaneous heroin withdrawal. Also, CBD induced an up-regulation of Cnr2, whereas it did not change the increased gene expression of Oprm1 in the NAcc of abstinent animals. The results suggest that CBD alleviates spontaneous heroin withdrawal and normalizes the associated gene expression changes. Future studies are needed to determine the relevance of CBD as a potential therapeutic tool for the treatment of heroin withdrawal.