Neural Network Connectivity Following Opioid Dependence is Altered by a Common Genetic Variant in the µ-Opioid Receptor, OPRM1 A118G.

Opioid use disorder is a chronic, relapsing disease associated with persistent changes in brain plasticity. A common single nucleotide polymorphism (SNP) in the µ-opioid receptor gene, OPRM1 A118G, is associated with altered vulnerability to opioid addiction. Reconfiguration of neuronal connectivity may explain dependence risk in individuals with this SNP. Mice with the equivalent Oprm1 variant, A112G, demonstrate sex-specific alterations in the rewarding properties of morphine and heroin. To determine whether this SNP influences network-level changes in neuronal activity, we compared FOS expression in male and female mice that were opioid-naive or opioid-dependent. Network analyses identified significant differences between the AA and GG Oprm1 genotypes. Based on several graph theory metrics, including small-world analysis and degree centrality, we show that GG females in the opioid-dependent state exhibit distinct patterns of connectivity compared to other groups of the same genotype. Using a network control theory approach, we identified key cortical brain regions that drive the transition between opioid-naive and opioid-dependent brain states; however, these regions are less influential in GG females leading to sixfold higher average minimum energy needed to transition from the acute to the dependent state. In addition, we found that the opioid-dependent brain state is significantly less stable in GG females compared to other groups. Collectively, our findings demonstrate sex- and genotype-specific modifications in local, mesoscale, and global properties of functional brain networks following opioid exposure and provide a framework for identifying genotype differences in specific brain regions that play a role in opioid dependence.

Sex differences in the rodent hippocampal opioid system following stress and oxycodone associated learning processes.

Over the past two decades, opioid abuse has risen especially among women. In both sexes hippocampal neural circuits involved in associative memory formation and encoding of motivational incentives are critically important in the transition from initial drug use to drug abuse/dependence. Opioid circuits, particularly the mossy fiber pathway, are crucial for associative memory processes important for addiction. Our anatomical studies, especially those utilizing electron microscopic immunocytochemistry, have provided unique insight into sex differences in the distribution of opioid peptides and receptors in specific hippocampal circuits and how these distributions are altered following stress and oxycodone-associative learning processes. Here we review the hippocampal opioid system in rodents with respect to ovarian hormones effects and baseline sex differences then sex differences following acute and chronic stress. Next, we review sex differences in the hippocampal opioid system in unstressed and chronically stressed rats following oxycodone conditioned place preference. We show that opioid peptides and receptors are distributed within hippocampal circuits in females with elevated estrogen states in a manner that would enhance sensitivity to endogenous and exogenous opioids. Moreover, chronic stress primes the opioid system in females in a manner that would promote opioid-associative learning processes. In contrast, chronic stress has limited effects on the opioid system in males and reduces its capacity to support opioid-mediated learning processes. Interestingly, acute stress appears to prime males for opioid associative learning. On a broader scale the findings highlighted in this review have important implications in understanding sex differences in opioid drug use and abuse.

Acute Delta 9-tetrahydrocannabinol administration differentially alters the hippocampal opioid system in adult female and male rats.

Our prior studies demonstrated that the rat hippocampal opioid system can undergo sex-specific adaptations to external stimuli that can influence opioid-associated learning processes. This opioid system extensively overlaps with the cannabinoid system. Moreover, acute administration of Δ9 Tetrahydrocannabinoid (THC), the primary psychoactive constituent of cannabis, can alter cognitive behaviors that involve the hippocampus. Here, we use light and electron microscopic immunocytochemical methods to examine the effects of acute THC (5 mg/kg, i.p., 1 h) on mossy fiber Leu-Enkephalin (LEnk) levels and the distribution and phosphorylation levels of delta and mu opioid receptors (DORs and MORs, respectively) in CA3 pyramidal cells and parvalbumin dentate hilar interneurons of adult female and male Sprague-Dawley rats. In females with elevated estrogen states (proestrus/estrus stage), acute THC altered the opioid system so that it resembled that seen in vehicle-injected females with low estrogen states (diestrus) and males: (1) mossy fiber LEnk levels in CA2/3a decreased; (2) phosphorylated-DOR levels in CA2/3a pyramidal cells increased; and (3) phosphorylated-MOR levels increased in most CA3b laminae. In males, acute THC resulted in the internalization of MORs in parvalbumin-containing interneuron dendrites which would decrease disinhibition of granule cells. In both sexes, acute THC redistributed DORs to the near plasma membrane of CA3 pyramidal cell dendrites, however, the dendritic region varied with sex. Additionally, acute THC also resulted in a sex-specific redistribution of DORs within CA3 pyramidal cell dendrites which could differentially promote synaptic plasticity and/or opioid-associated learning processes in both females and males.

Nalmefene, a mu opioid receptor antagonist/kappa opioid receptor partial agonist, potentiates cocaine motivation but not intake with extended access self-administration in adult male mice.

The mu opioid receptor antagonist/kappa opioid receptor (KOR) partial agonist nalmefene (NMF), a close structural analog of naltrexone (NTX), has been shown to reduce cocaine reward in preclinical models. Given the greater KOR potency and improved bioavailability compared to NTX, NMF may be a promising pharmacotherapeutic for cocaine use disorder (CUD). Here we examine the effects of NMF pretreatment on chronic daily extended access (4h) cocaine intravenous self-administration (IVSA) in adult male C57Bl/6J mice. METHODS: separate groups of mice had daily 4h cocaine IVSA sessions (0.25 or 0.5 mg/kg/inf, FR1) for 14 days. Starting on day 8, mice were pretreated with NMF (0, 1, or 10 mg/kg) 30m before each session. A separate group of mice acquired cocaine IVSA [seven days FR1 then four FR3 of 4h daily sessions (0.5 mg/kg/inf)] prior to a single progressive ratio 3 session to examine the effect of 1 mg/kg NMF on cocaine motivation. RESULTS: No significant effect of NMF pretreatment on cocaine intake was observed. Acute pretreatment of 1 mg/kg NMF significantly potentiated cocaine motivation as measured by progressive ratio breakpoint. CONCLUSIONS: NMF did not significantly attenuate cocaine intake and increased motivation for cocaine suggesting that NMF may not be suitable for non-abstinent CUD patients. Further research is needed with KOR selective partial or full agonists to determine their effect on cocaine reinforcement.

Review of addiction risk potential associated with adolescent opioid use.

Adolescence is a critical period of development with robust behavioral, morphological, hormonal, and neurochemical changes including changes in brain regions implicated in the reinforcing effects of drugs such as opioids. Here we examine the preclinical and, where appropriate complementary clinical literature, for the behavioral and neurological changes induced by adolescent opioid exposure/use and their long-term consequences during adulthood. Adolescent opioid exposure results in a widened biphasic shift in reinforcement with increased impact of positive rewarding aspects during initial use and profound negative reinforcement during adulthood. Females may have enhanced vulnerability due to fast onset of antinociceptive tolerance and reduced severity of somatic withdrawal symptoms during adolescence. Overall, adolescent opioid exposure, be it legally prescribed protracted intake or illicit consumption, results in significant and prolonged consequences of increased opioid reward concomitant with reduced analgesic efficacy and exacerbated somatic withdrawal severity during opioid use/exposure in adulthood. These findings are highly relevant to physicians, parents, law makers, and the general public as adolescent opioid exposure/misuse results in heightened risk for substance use disorders.

Modulation of cocaine-related behaviors by low doses of the potent KOR agonist nalfurafine in male C57BL6 mice.

RATIONALE: Agonists of the kappa opioid receptor (KOR) have been shown to block the rewarding effects of drugs of abuse, but with negative side effects. The antipruritic drug nalfurafine, approved in Japan in 2009, is a potent, selective KOR agonist that does not cause significant side effects in humans. Nalfurafine has not been extensively tested for its effect on drug reward and reinforcement in preclinical models. OBJECTIVES: The goal of this study was to compare the effects of nalfurafine and a reference KOR agonist for a variety of KOR-mediated endpoints in male C57BL6 mice. Specifically, we aimed to evaluate the "therapeutic window"-doses of agonists lower than those eliciting negative side effects, while still effective for desired therapeutic effects. METHODS: In this study, several low doses of nalfurafine and U50,488 were tested for serum prolactin release, rotarod-mediated sedation, and place-conditioning in male C57BL6 mice. These agonists were also tested for effects on intravenous cocaine self-administration, both on an FR1 schedule and on a progressive ratio schedule for 0.5 mg/kg/infusion cocaine. RESULTS: Serum prolactin levels increased following doses of both nalfurafine (3 µg/kg and 10 µg/kg) and U50,488 (3 mg/kg). These doses did not cause sedation in the rotarod assay or aversion in a place-conditioning assay, but blocked conditioned place preference for cocaine. Immediate pretreatment of mice with 10 µg/kg nalfurafine and 3 mg/kg U50,488, however, potentiated cocaine self-administration. Further 10 µg/kg nalfurafine was also observed to potentiate cocaine-seeking behavior as demonstrated by increased progressive ratio break point. CONCLUSIONS: Both nalfurafine and U50,488 showed a separation of negative side effects and the modulation of cocaine reward, suggesting this effect of KOR agonists at low doses may be characteristic of the KOR system in general. At higher doses, nalfurafine had similar effects to traditional KOR agonists like U50,488, indicating that its relative potency, rather than differences in KOR signaling, may be responsible for its unique effects in humans.

Effects of Kappa opioid receptor blockade by LY2444296 HCl, a selective short-acting antagonist, during chronic extended access cocaine self-administration and re-exposure in rat.

RATIONALE: Cocaine addiction is a chronic brain disease characterized by compulsive drug intake and dysregulation of brain reward systems. Few preclinical studies have modeled the natural longitudinal course of cocaine addiction. Extended access self-administration protocols are powerful tools for modeling the advanced stages of addiction; however, few studies have duration of drug access longer than 12 h/session, potentially limiting their construct validity. Identification of changes in cocaine intake patterns during the development of addictive-like states may allow better treatments for vulnerable subjects. The kappa opioid receptor (KOPr) system has been implicated in the neurobiological regulation of addictive states as well as mood and stress disorders, with selective KOPr antagonists proposed as possible pharmacotherapeutic agents. Chronic cocaine exposure increases the expression of KOPr and its endogenous agonists, the dynorphins, in several brain areas in rodents. OBJECTIVES: To examine the behavioral pattern of intake during chronic (14 days) 18 h intravenous cocaine self-administration (0.5 mg/kg/infusion) and the effect of a novel short-acting KOPr antagonist LY2444296 HCl (3 mg/kg) administered during sessions 8 to 14 of chronic 18 h/day cocaine self-administration and prior to a single re-exposure session after 2 cocaine-free withdrawal days. RESULTS: Both daily and hourly cocaine intake patterns changed over 14 days of 18 h self-administration. LY pretreatment affected the pattern of self-administration across the second week of extended access cocaine self-administration and prevented the increase in cocaine intake during re-exposure. CONCLUSIONS: Overall, the KOPr antagonist attenuated escalated cocaine consumption in a rat model of extended access cocaine self-administration.

Naltrexone and nalmefene attenuate cocaine place preference in male mice.

Cocaine addiction treatment is difficult due to the current lack of approved pharmacotherapuetics. Several preclinical and clinical studies have demonstrated that the mu opioid receptor (MOPr) antagonist/kappa opioid receptor (KOPr) partial agonist naltrexone (NTX) reduces the subjective effects and self-administration of cocaine. However, very limited research has examined the ability of the structurally similar MOPr antagonist/KOPr partial agonist nalmefene (NMF) to reduce cocaine reward. Here we examine the effect of low (1 mg/kg) and high (10 mg/kg) doses of NTX or NMF on cocaine place preference. In vivo characterization of these NTX and NMF doses were performed to examine their effectiveness at MOPr and KOPr. RESULTS: Both NTX doses and high dose NMF significantly reduced cocaine place preference. Conversely, a significant place avoidance was observed for high dose NTX and both NMF doses. Interestingly, neither NTX nor NMF blocked cocaine-induced hyperlocomotion. High dose NTX and both NMF doses fully blocked MOPr agonist morphine-induced thermal analgesia as well as KOPr agonist U50,488H-induced locomotor discoordination. However, low dose NTX fully blocked morphine analgesia but not U50,488H locomotor discoordination suggesting that low dose NTX is effective at MOPr but not KOPr. CONCLUSION: Both NTX and NMF block the place preference, but not locomotor activating, effects of cocaine. These results suggest that both NTX and NMF may be viable pharmacotheraputics for some aspects of cocaine addiction. This is an important step to understanding the potential mechanism(s) of action of NTX and NMF for the development of more efficacious pharmacological treatments for substance use disorders.

Alcohol affects the P3 component of an adaptive stop signal task ERP.

BACKGROUND: The P3 component of the event-related potential (ERP) has been particularly useful in alcohol research for identifying endophenotypes of alcohol-use disorder (AUD) risk in sober subjects. However, practice and/or fatigue reduce P3 amplitude, limiting the ability to ascertain acute and adaptive effects of alcohol exposure. Here, we report acute alcohol effects on P3 amplitude and latency using an adaptive stop signal task (aSST). METHODS: One hundred forty-eight non-dependent moderate to heavy social drinkers, ages 21 to 27, participated in two single-blind, alcohol or placebo, counterbalanced sessions approximately 1 week apart. During each session, subjects performed an adaptive stop signal task (aSST) at 1) baseline, 2) upon reaching the target 60 mg/dL breath alcohol concentration or at the equivalent time during the placebo session, and 3) approximately 135 min later while the breath alcohol concentration was clamped. Here, we report on differences between baseline and first subsequent measurements across the experimental sessions. During each aSST run, the stop signal delay (SSD, the time between stop and go signals) adjusted trial-by-trial, based on the subject's performance. RESULTS: The aSST reliably generated a STOP P3 component that did not change significantly with repeated task performance. The pre-infusion SSD distribution was bimodal, with mean values several hundred msec apart (FAST: 153 msec and SLOW: 390 msec). This suggested different response strategies: FAST SSD favoring "going" over "stopping", and SLOW SSD favoring "stopping" over "going". Exposure to alcohol at 60 mg/dL differentially affected the amplitude and latency of the STOP P3 according to SSD group. Alcohol significantly reduced P3 amplitude in the SLOW SSD compared to the FAST SSD group, but significantly increased P3 latency in the FAST SSD compared to the SLOW SSD group. CONCLUSIONS: The aSST is a robust and sensitive task for detecting alcohol-induced changes in inhibition behavior as measured by the P3 component in a within-subject design. Alcohol was associated with P3 component changes, which varied by SSD group, suggesting a differential effect as a function of task strategy. Overall, the data support the potential utility of the aSST in the detection of alcohol response-related AUD risk.

Effects of group II metabotropic glutamate receptor modulation on ethanol- and sucrose-seeking and consumption in the rat.

Previous studies suggest that group II metabotropic glutamate receptors (mGluR2/3) are involved in regulating ethanol-seeking and consumption. The mGluR2/3 agonist LY379268 (LY37) and selective mGluR2 positive allosteric modulator biphenyl­indanone A (BINA) were used to investigate the relative contribution of mGlu2 and mGlu3 receptors on ethanol- and sucrose-seeking and consumption. A microinjection study was then performed to examine the role of nucleus accumbens (NAc) core mGluR2/3 on ethanol-seeking. For the systemic experiments, separate groups of male Wistar rats [LY37 (0-2.0 mg/kg); BINA (0-20 mg/kg)] were trained to complete a response requirement (RR) resulting in access to 10% ethanol or 2% sucrose (in separate groups) for a 20­min drinking period. Animals then underwent consummatory testing (weekly drug injections with RR1) followed by appetitive testing (weekly drug injections followed by extinction session). A separate group of male Wistar rats was surgically implanted with bilateral guide cannulae directed toward the NAc core and had weekly microinjections followed by an extinction session. Systemic administration of the mGluR2/3 agonist LY37 significantly reduced ethanol- and sucrose-seeking. The same treatment also reduced sucrose consumption and body weight (24­h post injection). Systemic administration of the selective mGluR2 PAM BINA, however, had no effect on either seeking or consumption of ethanol or sucrose. Intra-accumbens core LY37 significantly reduced ethanol-seeking. These findings suggest that systemic mGluR2/3 agonism, but not allosteric modulation of mGluR2, reduces reinforcer-seeking. In particular, NAc core group II mGluR may be involved in regulating ethanol-seeking.

Adolescent oxycodone self administration alters subsequent oxycodone-induced conditioned place preference and anti-nociceptive effect in C57BL/6J mice in adulthood.

Adolescent and young adult abuse of short-acting MOP-r agonists such as oxycodone is a pressing public health issue. Few preclinical studies have examined how adolescent exposure to oxycodone impacts its effects in the transition to adulthood. OBJECTIVE: To determine in mice how chronic adolescent oxycodone self-administration (SA) affects subsequent oxycodone-induced conditioned place preference (CPP), locomotor activity, and anti-nociception once mice reach early adulthood. METHODS: Adolescent C57BL/6J male mice (4 weeks old, n = 6-11) and adult mice (10 weeks old, n = 6-10) were surgically implanted with indwelling jugular catheters. Mice then acquired oxycodone self-administration (14 consecutive 2-hr daily sessions; 0.25 mg/kg/infusion) followed by a 14-day drug-free (withdrawal) period in home cage. After the 14-day drug-free period, mice underwent a 10-day oxycodone CPP procedure (0, 1, 3, 10 mg/kg i.p.) or were tested for acute oxycodone-induced antinociception in the hot plate assay (3.35, 5, 7.5 mg/kg i.p.). RESULTS: Mice that self-administered oxycodone during adolescence exhibited greater oxycodone-induced CPP (at the 3 mg/kg dose) than their yoked saline controls and mice that self-administered oxycodone during adulthood. Oxycodone dose-dependently increased locomotor activity, but sensitization developed only to the 3 mg/kg in the mice that underwent oxycodone self-administration as adolescents. Mice that self-administered oxycodone as adolescents decreased in the anti-nociceptive effects of oxycodone in one dose (5 mg/kg), whereas animals that self-administered oxycodone as adults did not show this effect. CONCLUSION: Chronic adolescent oxycodone self-administration led to increased oxycodone-induced CPP (primarily 1 and 3 mg/kg, i.p.) and reduced antinociceptive effect of oxycodone (5 mg/kg, i.p.) in adulthood.

Adaptation of Subjective Responses to Alcohol is Affected by an Interaction of GABRA2 Genotype and Recent Drinking.

BACKGROUND: Subjective perceptions of alcohol intoxication are associated with altered risk for alcohol abuse and dependence. Acute adaptation of these perceptions may influence such risk and may involve genes associated with pleasant perceptions or the relief of anxiety. This study assessed the effect of variation in the GABAA receptor genes GABRG1 and GABRA2 and recent drinking history on the acute adaptation of subjective responses to alcohol. METHODS: One hundred and thirty-two nondependent moderate to heavy drinkers, aged 21 to 27, participated in 2 single-blind, counterbalanced sessions, approximately 1 week apart. One session was an intravenous alcohol "clamp," during which breath alcohol concentration was held steady at 60 mg/dl (60 mg%) for 3 hours, and the other an identical session using saline infusion. Subjective perceptions of Intoxication, Enjoyment, Stimulation, Relaxation, Anxiety, Tiredness, and Estimated Number of Drinks were acquired before (baseline), and during the first and final 45 minutes of the clamp. A placebo-adjusted index of the subject's acute adaptation to alcohol was calculated for each of the 7 subjective measures and used in a principal component analysis to create a single aggregate estimate for each subject's adaptive response to alcohol. Analysis of covariance tested whether GABRA2 and GABRG1 single nucleotide polymorphism (SNP) genotypes, gender, placebo session, family history of alcoholism, recent drinking history, and the genotype × recent drinking history interaction significantly predicted the adaptive response. RESULTS: Recent drinking history (p = 0.01), and recent drinking history × genotype interaction (p = 0.01) were significantly associated with acute adaptation of the subjective responses to alcohol for the GABRA2 SNP rs279858. CONCLUSIONS: Higher recent drinking was found to be associated with reduced acute tolerance to positive, stimulating effects of alcohol in carriers of the rs279858 risk allele. We postulate that the GABRA2 effect on alcohol dependence may, in part, be due to its effect on subjective responses to alcohol.

Intravenous alcohol self-administration in the P rat.

Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be temporally separate from time of intake) and the more temporally "relevant" effects (primarily olfactory and taste) that bridge the time from intake to onset of the pharmacological effects. Intravenous (IV) self-administration of ethanol limits the confounding "non-pharmacological" effects associated with oral consumption, allows for controlled and precise dosing, and bypasses first order absorption kinetics, allowing for more direct and better-controlled assessment of alcohol's effect on the brain. IV ethanol self-administration has been reliably demonstrated in mouse and human experimental models; however, models of IV self-administration have been historically problematic in the rat. An operant multiple-schedule study design was used to elucidate the role of each component of a compound IV-ethanol plus oral-sucrose reinforcer. Male alcohol-preferring P rats had free access to both food and water during all IV self-administration sessions. Animals were trained to press a lever for orally delivered 1% sucrose (1S) on a fixed ratio 4 schedule, and then surgically implanted with an indwelling jugular catheter. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5-min components across 30-min sessions. For the multiple schedule, two components were used: an oral 1S only and an oral 1S plus IV 20% ethanol (25 mg/kg/injection). Average total ethanol intake was 0.47 ± 0.04 g/kg. We found significantly higher earning of sucrose-only reinforcers and greater sucrose-lever error responding relative to the compound oral-sucrose plus IV-ethanol reinforcer. These response patterns suggest that sucrose, not ethanol, was responsible for driving overall responding. The work with a compound IV ethanol-oral sucrose reinforcer presented here suggests that the existing intravenous ethanol self-administration methodology cannot overcome the aversive properties of ethanol via this route in the rat.

Subjective perceptions associated with the ascending and descending slopes of breath alcohol exposure vary with recent drinking history.

BACKGROUND: The differentiator model predicts that individuals with a positive family history of alcoholism (FHA) or heavy alcohol consumers will feel more sensitive to the effects of alcohol on the ascending phase of the blood alcohol content while feeling less sedated on the descending phase. This study tested whether subjective perceptions are sensitive to the slope of breath alcohol concentration (BrAC) and whether that sensitivity is associated with an FHA and/or recent drinking history (RDH). METHODS: Family-history-positive (FHP, n = 27) and family-history-negative (FHN, n = 27) young adult nondependent drinkers were infused intravenously with alcohol in 2 sessions separated by 1 week. After 20 minutes, one session had an ascending BrAC (+3.0 mg%/min), while the other session had a descending BrAC (-1 mg%/min). The BrAC for both sessions at this point was approximately 60 mg%, referred to as the crossover point. Subjective perceptions of intoxication, high, stimulated, and sedation were sampled frequently and then interpolated to the crossover point. Within-subject differences between ascending and descending responses were examined for associations with FHA and/or RDH. RESULTS: Recent moderate drinkers reported increased perceptions of feeling intoxicated (p < 0.023) and high (p < 0.023) on the ascending slope compared with the descending slope. In contrast, recent light drinkers felt more intoxicated and high on the descending slope. CONCLUSIONS: Subjective perceptions in young adult social drinkers depend on the slope of the BrAC when examined in association with RDH. These results support the differentiator model hypothesis concerning the ascending slope and suggest that moderate alcohol consumers could be at risk for increased alcohol consumption because they feel more intoxicated and high on the ascending slope. Subjects did not feel less sedated on the descending slope, contrary to the differentiator model but replicating several previous studies.