Murine model of OPRM1 A118G alters oxycodone self-administration and locomotor activation, but not conditioned place preference

Journal Pre-proof Murine model of OPRM1 A118G alters oxycodone self-administration and locomotor activation, but not conditioned place preference

Devon Collins, Yong Zhang, Julie Blendy, Mary Jeanne Kreek PII:

S0028-3908(19)30430-7

DOI:

https://doi.org/10.1016/j.neuropharm.2019.107864

Reference:

NP 107864

To appear in:

Neuropharmacology

Received Date:

02 August 2019

Accepted Date:

24 November 2019

Please cite this article as: Devon Collins, Yong Zhang, Julie Blendy, Mary Jeanne Kreek, Murine model of OPRM1 A118G alters oxycodone self-administration and locomotor activation, but not conditioned place preference, Neuropharmacology (2019), https://doi.org/10.1016/j.neuropharm. 2019.107864

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof

Murine model of OPRM1 A118G alters oxycodone selfadministration and locomotor activation, but not conditioned place preference

Devon Collins, PhD1*; Yong Zhang, PhD1; Julie Blendy, PhD2; Mary Jeanne Kreek, MD1 Author affiliations

1Laboratory

of the Biology of Addictive Diseases, The Rockefeller University, New York,

NY 10065 2Department

of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104

*Corresponding

author

Journal Pre-proof

Abstract Mu-opioid receptors (MORs) mediate the rewarding properties of oxycodone and other prescription opioid medications, which have played a central role in the current opioid epidemic in the United States. The human mu-opioid receptor gene (OPRM1) contains a functional single nucleotide polymorphism (SNP), A118G, which has been associated with altered opioid addiction risk, however the mechanisms responsible for this are not well understood. To explore this, we examined oxycodone conditioned place preference (CPP) and self-administration behavior (SA) in A112G mice, which possess a functionally analogous SNP in the mouse mu-opioid receptor gene (Oprm1). For CPP, male and female A112G mice homozygous for the A112 (wild-type; AA) or G112 (GG) allele were conditioned with doses of 1 and 3 mg/kg across an 8-day period. For SA, mice were allowed to self administer oxycodone (unit dose 0.25 mg/kg/infusion, FR1) for 4h/day for 10 consecutive days. We observed no effects of genotype or sex on

1

Journal Pre-proof

conditioned place preference behavior. Oxycodone 3 mg/kg increased locomotor activity in AA mice but not GG mice, and both male and female GG mice selfadministered significantly more oxycodone compared to their wild-type AA littermates. These studies suggest that the G allele promotes greater opioid intake, which may underlie

greater

opioid

addiction

morbidity

in

G-allele

carriers.

2

Journal Pre-proof

Introduction Nonmedical use of oxycodone is a major public health concern. A semi-synthetic derivative of thebaine, oxycodone is a relatively selective, short-acting MOR agonist with clinical indications similar to morphine. While it has slightly lower MOR efficacy than morphine (Nakamura et al., 2013; Narita et al., 2008; Peckham and Traynor, 2006), oxycodone possesses similar rewarding effects when administered peripherally (Maruta et al., 1979; Olmstead and Burns, 2005; Rutten et al., 2011).

In recent

decades, clinical use of oxycodone has risen sharply, and in some countries, oxycodone may have surpassed morphine as the most commonly prescribed opioid analgesic (Söderberg Löfdal et al., 2013). The number prescriptions for oxycodone and other opioid painkillers filled by pharmacies in the United States nearly tripled over the period from 1991 to 2011 (“U.S. Prescribing Rate Maps | Drug Overdose | CDC Injury Center,” n.d.). This increase in opioid prescription rates is concomitant with a near

3

Journal Pre-proof

quadrupling of deaths related to prescription opioid overdoses (“Results from the 2016 National Survey on Drug Use and Health: Detailed Tables, SAMHSA, CBHSQ,” 2017). In humans, a common single nucleotide polymorphism (SNP), OPRM1 A118G, occurs in exon 1 of the MOR gene. Several studies have associated the A118G SNP with increased risk for mu opioid and alcohol dependence in European and East Asian populations (Bart et al., 2006, 2004; Kim et al., 2004; Nishizawa et al., 2006; Schwantes-An et al., 2016) although some studies have suggested no association (Bergen et al., 1997; Coller et al., 2009; Franke et al., 2001; Xuei et al., 2007). Clinical studies have investigated behavioral and functional effects of the G118 allele in humans. The G118 allele has been reported to be associated with greater daily drug intake in heroin users (Shi et al., 2002), greater automatic approach tendencies for alcohol and appetitive cues (Wiers et al., 2009), and increased neuronal activity in response to alcohol cues in cortical and striatal regions as shown by blood oxygenation level dependent imaging (BOLD) (Filbey et al., 2008). The G118 allele also significantly alters stress responsivity and hypothalamic-pituitary-adrenal (HPA) axis regulation in

4

Journal Pre-proof

healthy humans. G118 carriers show elevated basal cortisol levels (Bart et al., 2006) and genotype-dependent differences in response to endocrine challenge of the HPA axis by metyrapone (Ducat et al., 2013). Further, the G118 allele causes reductions in cortisol response to acute psychological stressors and greater cortisol release following MOR blockade by naltrexone or naloxone (Chong et al., 2006; Hernandez-Avila et al., 2007, 2003; Lovallo et al., 2015; Oslin et al., 2003; Wand et al., 2002).

OPRM1

A118G

has

been

demonstrated

pharmacodynamic changes (Kroslak et al., 2007).

to

produce

a

number

of

In vitro studies in AV12 cells

expressing the G118 MOR variant revealed a three-fold increase in beta-endorphin binding affinity as well as a three-fold increase in beta-endorphin potency as measured by activation of G protein-coupled inward rectifying potassium channels (Bond et al., 1998). These alterations are thought to contribute to the observed effects of the G118 allele in clinical and preclinical studies. While clinical and in vitro studies have helped elucidate some of the consequences of the A118G SNP, animal studies can provide valuable insights into the

5

Journal Pre-proof

mechanistic and behavioral effects of this polymorphism. There is high nucleotide homology between the mouse and human MOR genes, and likewise, the resultant amino acid sequences share a high degree of homology (Mura et al., 2013). Mague, Blendy, and colleagues took advantage of this to generate a knock-in mouse model bearing a point mutation equivalent to the A118G variant, A112G, in the mouse MOR gene (Oprm1) (Mague et al., 2009). Several studies have evaluated various behavioral endpoints in A112G mice. Homozygous G112 mice show reduced buprenorphinemediated antinociception, anxiolysis, and hyperlocomotion (Browne et al., 2017), as well as reduced morphine antinociception (Mague et al., 2009). Further, in a previous study of conditioned reward in A112G mice, female G112 homozygotes failed to develop morphine-induced conditioned place preference (Mague et al., 2009). A recent study by our group used a ten-day, extended-access self-administration paradigm and found that G112 homozygotes self-administered more heroin than their A112 homozygote counterparts (Zhang et al., 2015), suggesting that the G112 allele may modulate the rewarding and reinforcing effects of mu agonists. To our knowledge,

6

Journal Pre-proof

there are no published studies of oxycodone reward and reinforcement in A112G mice. As oxycodone is a major component of the current opioid epidemic, and the G allele is a common mu opioid receptor gene variant associated with greater mu opioid intake, we sought to evaluate oxycodone self-administration and conditioned place preference in male and female, homozygous A112 and G112 mice.

Materials and methods Animals Heterozygous A112G mice (Mague et al., 2009) were mated to obtain homozygous (112AA or 112GG; AA or GG) offspring. Heterozygous mice (112AG) were also produced, but only homozygous (AA or GG) male and female mice are examined in the present study. Both AA and GG mice were chosen from the litters produced by these matings, therefore siblings are included in the present study. Mice were genotyped by PCR using genomic DNA obtained by tail snip (forward primer, 5’7

Journal Pre-proof

GCTCCA

TCTTGGATCCCCTTT-3’;

reverse

primer,

5’-

GAGCTACCCAGCAATTCCAGA-3’). Mice were used for experiments between 10 and 12 weeks of age. We have observed no differences in size, health, or mortality between AA and GG mice of either sex (data not shown). GG mouse birth rates are slightly lower than the expected Mendelian rates as has been previously described (Mague et al., 2009), however mice carrying the G112 allele are fertile and produce litters of normal size. All mice were housed in groups of four to five in light- and climate-controlled (~25 °C) conditions. Mice used for oxycodone conditioned place preference/locomotor activation assays were housed in the CPP procedure room, which was maintained on a 12:12 hr light-dark cycle (lights-on at 0900 hr, lights-off at 2100 hr). Mice used for oxycodone self-administration were housed in a dedicated self-administration procedure room, which was maintained on a reversed 12:12 hr light-dark cycle (lights-off 0700 hr, lights-on 1900 hr). Food and water were available ad libitum. All experimental

8

Journal Pre-proof

procedures were approved by the Institutional Animal Care and Use Committee at The Rockefeller University.

Drugs Oxycodone hydrochloride (Sigma Aldrich, St. Louis, MO), ketamine hydrochloride, (Ketaset, Fort Dodge Animal Health, Fort Dodge, IA), and xylazine hydrochloride (AnaSed, Santa Cruz Animal Health, Dallas, TX) were dissolved in saline for injection.

Conditioned place preference Conditioning apparatus Mouse place preference apparatus were purchased from Med Associates (ENV3013; St. Albans, VT). Each chamber comprises three compartments: a central gray compartment and two, flanking conditioning compartments, one black and one white. In addition to color cues, the compartments also feature distinct tactile cues: the gray compartment has a smooth PVC floor and the conditioning compartments have either a stainless steel bar floor or a stainless steel mesh floor. The individual compartments can

9

Journal Pre-proof

be separated by removable, guillotine-style doors. Automated data collection was accomplished by individual infrared photobeams on photobeam strips, with two beams in the gray compartment and six beams in the white and black compartments. Each apparatus was contained within a dimly lit, sound-attenuating cabinet to minimize disturbances to the mice during conditioning.

Conditioning paradigm The oxycodone doses chosen were based on published data in adult mice (Narita et al., 2008) and the conditioned place preference paradigm was based on our earlier studies (Collins et al., 2016; Niikura et al., 2013; Schlussman et al., 2008; Zhang et al., 2009). Oxycodone conditioning comprised three phases: a preconditioning test, an eight-day conditioning period, and a postconditioning test. In the preconditioning test, each animal was placed in the central gray compartment with free access to the black and white compartments; the amount of time spent in each compartment was recorded for 20 minutes. During the conditioning sessions, mice were placed into and restricted to the

10

Journal Pre-proof

appropriate conditioning compartment for 20 min following oxycodone (1 or 3 mg/kg) or saline injection. The animals were injected with oxycodone and saline on alternate days, for a total of eight conditioning sessions (four oxycodone sessions and four saline sessions) for each animal. Conditioning sessions were conducted daily. The postconditioning test (without injection; i.e., a drug-free state) was performed on the day after the last conditioning session, and was identical to the preconditioning test. That is, each mouse was placed into the center compartment and had free access to both the white and black compartments for 20 minutes.

Determination of conditioned place preference and locomotor activity during place conditioning sessions

Development of conditioned place preference was determined by calculating the difference in time spent in the oxycodone-paired compartment during the pre- and postconditioning sessions (postconditioning time minus preconditioning time). Locomotor activity counts was also recorded during conditioning sessions and was calculated as number crossovers (number of times the animal traversed the chamber breaking the 11

Journal Pre-proof

beams at one end and then the other) in the conditioning compartment during conditioning sessions.

Intravenous self-administration

Catheter implantation surgery Mice were anesthetized with a ketamine-xylazine solution (80 mg/kg ketamine and 8.0 mg/kg xylazine, i.p.). Incision sites were shaved and disinfected with alcohol and iodine solutions. Two incisions were made – one dorsal incision in the midscapular region and a ventral incision anteromedial to the right foreleg. A 6-cm catheter (SILASTIC® tubing, 0.31 mm ID, 0.64 mm OD; Helix Medical, Inc., CA) was passed subcutaneously from the dorsal to the ventral incision. A 22-gauge needle was inserted into the right jugular vein to guide the catheter into the vein. The catheter was inserted to the level of a silicone ball marker placed 1.1 cm from the end, secured to the vein using surgical silk, and flushed with physiological saline (to avoid clotting). The catheter was then capped with a stopper. Surgical wounds were sutured and treated with 12

Journal Pre-proof

antibiotic ointment. Immediately following surgery, mice were allowed to recover from anesthesia in a separate cage before they were returned to their home cages, where they were group housed and allowed to rest for five days prior to the self-administration procedure. Intravenous self-administration chambers Polycarbonate

self-administration

chambers

were

purchased

from

Med

Associates (ENV-307W, 21.6 cm x 17.8 cm x 12.7 cm; St. Albans, VT). Each chamber was contained within a light- and sound-attenuating cabinet (Med Associates). In each chamber, one wall contained two nose poke ports, each comprising a photocell situated within a small hole 0.9 cm in diameter. Nose poke ports were 4.2 cm apart and 1.5 cm from the floor of the chamber. One port was defined as active and the other inactive. When the photocell in the active port was triggered by a nose poke, an infusion pump delivered an infusion (oxycodone, 0.25 mg/kg/infusion or 0.9% saline [yoked saline]) of 20 µL/3 s from a 5-mL syringe located above the chamber. The syringe was connected to the mouse’s catheter via Tygon tubing connected to a swivel. During an infusion, a

13

Journal Pre-proof

cue light above the active hole was illuminated; each infusion was followed by a 20-s timeout period during which nose pokes were recorded but would not result in additional infusions. When the photocell in the inactive port was triggered, a nose poke was recorded but did not result in an infusion.

Oxycodone self-administration Prior to the first self-administration session, mice were placed into the selfadministration chambers allowed to habituate to the chambers for 4 hours. Four-hour self-administration sessions were conducted once daily for 10 consecutive days. Sessions were conducted during the dark phase (between 0700 hr and 1100 hr). Each day, mice were removed from their home cages, weighed and their catheters were flushed with 0.01 mL heparinized saline solution (30 IU/mL) to maintain catheter patency. For each session, mice were placed in the self-administration chambers and their catheters were connected to the infusion syringe. Nose pokes at the active hole resulted in an infusion of oxycodone (0.25 mg/kg/infusion; Sigma Aldrich, St. Louis, MO) according to an FR1 reinforcement schedule. For each mouse, drug dose was 14

Journal Pre-proof

controlled by computer, which took into account the animal’s body weight. Control (saline) mice received yoked saline infusions. That is, saline was infused in the control mouse whenever the yoked oxycodone mouse self-administered an infusion of oxycodone. After the final self-administration session, mice underwent a catheter patency test: 30 µL of ketamine (5 mg/mL) was administered through the catheter and mice were observed for loss of muscle tone which was taken to indicate patency. Data from mice passing the catheter patency test were included for analysis. Statistical analysis Data were analyzed using Prism 7 (GraphPad Software, La Jolla, CA). Differences in oxycodone conditioned place preference, oxycodone-induced locomotor activity during oxycodone conditioning sessions, and oxycodone self-administration were analyzed by two-, three-, or four-way ANOVA where appropriate. If ANOVA revealed significant main or interaction effects, post-hoc tests with Sidak (two-way), Tukey HSD, or Benjamini-Hochberg (False Discovery Rate; 5%) correction for multiple

15

Journal Pre-proof

comparisons were carried out where appropriate (Benjamini and Hochberg, 1995; Šidák, 1967).

Results

Conditioned place preference in AA and GG mice We conditioned male and female AA and GG mice with doses of oxycodone (1 mg/kg and 3 mg/kg) that are known to produce a conditioned place preference in male and female C57BL6 mice (the A112G mouse background strain) (Collins et al., 2016; Niikura et al., 2013). Figure 1 shows the change in the amount of time spent in the saline-

or

drug-paired

compartment

between

the

preconditioning

test

and

postconditioning test. A three-way ANOVA, genotype x sex x dose, revealed a preference for the oxycodone-paired compartment (significant main effect of oxycodone dose, F2,124 = 41.284, p < 0.001) with no significant differences by genotype (F1,124 = 0.309, ns) or sex (F1,124 = 0.057, ns). There were no significant interaction effects. Sidak post-hoc tests revealed a significant increase in the amount of time spent in the

16

Journal Pre-proof

drug-paired chamber for both 1 mg/kg (p > 0.001) and 3 mg/kg (p > 0.001) oxycodone compared to saline, while the increase in time spent in the drug-paired chamber did not differ between 1 mg/kg and 3 mg/kg.

Figure 1. Oxycodone conditioned place preference in A112G mice. Oxycodone produced a significant place preference regardless of sex or genotype (*p < 0.05,

17

Journal Pre-proof

**p<0.01, ***p<0.001, ****p<0.0001 versus corresponding saline control animals; Sidak correction). Mean ± SEM (0 mg/kg: 12 AA males, 8 AA females, 12 GG males, 8 GG females; 1 mg/kg: 8 AA males, 8 AA females, 8 GG males, 8 GG females; 3 mg/kg: 8 AA

males,

8

AA

females,

16

GG

males,

16

GG

females)

18

Journal Pre-proof

Locomotor activity during conditioning sessions Since the G112 allele has been demonstrated to alter the motoric effects of both buprenorphine (Browne et al., 2017) and morphine (Mague et al., 2009), we examined locomotor activity in AA and GG mice during oxycodone conditioning sessions. Locomotor activity (number of infrared beam breaks) during conditioning sessions is shown in Figure 2. A four-way ANOVA, genotype x sex x dose x session (repeated measure) revealed no significant main effect of sex and no significant interactions of sex with any other factor, and so data from male and female mice were combined. A three way ANOVA revealed significant main effects of genotype (F1,360 = 43.747, p < 0.0001), a significant main effect of dose (F2,360 = 23.136, p < 0.0001), and no significant main effect of session (F3,360 = 0.245 , p =0.865). There was a significant interaction of genotype and dose (F2,360 = 23.402, p < 0.0001). 1 mg/kg oxycodone did not produce a significant elevation in locomotor activity in AA or GG mice; 3 mg/kg oxycodone

19

Journal Pre-proof

produced hyperlocomotion in AA mice, but failed to produce hyperlocomotion in GG mice.

20

Journal Pre-proof

Figure 2. Locomotor activity during conditioning sessions. 3 mg/kg oxycodone increased locomotor activity with respect to saline controls in AA mice only (*p < 0.05, ***p < 0.001, ****p < 0.0001, compared to corresponding saline animals; Sidak correction). Mean ± SEM. (0 mg/kg: 12 AA males, 8 AA females, 12 GG males, 8 GG females; 1 mg/kg: 8 AA males, 8 AA females, 8 GG males, 8 GG females; 3 mg/kg: 8 AA males, 8 AA

females,

16

GG

males,

16

GG

females)

21

Journal Pre-proof

Oxycodone self-administration in AA & GG mice (extended-access 4hr/day) The total oxycodone self-administered during sessions is shown in Figure 3. A two-way ANOVA, genotype x sex, revealed a significant main effect of genotype (F1,25 = 24.98, p < 0.0001), no significant main effect of sex (F1,25 = 0.03197, p = 0.8595), and no significant interaction of genotype and sex (F1,25 = 0.3138, p = 0.5803). GG mice of both sexes self-administered more oxycodone in total across sessions compared to AA mice (Figure 3). We also examined nose poke behavior at the active and inactive ports in mice receiving oxycodone or yoked saline. A four-way ANOVA, genotype x sex x drug condition (oxycodone vs yoked saline) x session (repeated measure) showed a significant main effect of genotype (F1,37 = 6.710, p = 0.014), no significant main effect of sex (F1,37 = 0.240, p = 0.627), a significant main effect of drug condition (F1,37 = 82.218, p < 0.001), and a significant main effect of session (F9,333 = 7.053, p < 0.001). There was a significant genotype x drug condition interaction (F1,37 = 15.110, p = 22

Journal Pre-proof

0.00041). Mice receiving oxycodone showed increased nose pokes across sessions regardless of genotype or sex. GG mice in the oxycodone group nose poked significantly more at the active hole compared to AA mice. Yoked saline controls showed no significant differences in nose pokes between genotypes or sexes, as well as no increase in number of nose pokes across sessions. Noncontingent nose pokes at the inactive hole also showed no increase across sessions (data not shown).

Figure 3. Total oxycodone administered across the 10 days of extended access selfadministration sessions. The total daily oxycodone (0.25 mg/kg/infusion) selfadministration (SA) in (a) males in each genotype, (b) females in each genotype, and (c) total oxycodone self-administration across the ten, daily extended-access sessions. GG mice of both sexes self-adminstered more oxycodone compared to AA mice (**p < 0.01 GG versus AA; Sidak correction). Mean ± SEM. (7 AA males, 8 AA females, 6 GG males, 8 GG females)

23

Journal Pre-proof

24

Journal Pre-proof

Discussion In the present study, we examined oxycodone conditioned place preference, locomotor response, and self-administration in Oprm1 A112G mice which bear nucleotide and resultant amino acid substitutions that are functionally analogous to the human SNP

OPRM1 A118G. We found that male and female, homozygous AA and GG mice developed oxycodone conditioned place preference and acquired oxycodone selfadministration behavior. Further, we found that GG mice of both sexes selfadministered more oxycodone than AA mice during the course of 10-day, extendedaccess self-administration sessions, similar to an earlier study in which male and female GG mice self-administered more heroin compared to AA mice (Zhang et al., 2015). We also observed a lack of oxycodone-induced hyperlocomotion in GG mice, consistent with previous studies in which morphine- (Mague et al., 2009) and buprenorphineinduced (Browne et al., 2017) hyperlocomotion was blunted in GG mice. Mu opioid receptors mediate the rewarding properties of mu opioid agonists, including morphine, heroin, and oxycodone (Contet et al., 2004; Kieffer and Gavériaux25

Journal Pre-proof

Ruff, 2002; Matthes et al., 1996). Global MOR knockout in mice leads to a loss of morphine conditioned place preference (Matthes et al., 1996) and MOR knockout mice do not self-administer morphine (Becker et al., 2000).

While these MOR-mediated

behaviors may be absent or attenuated in MOR knockout mice, there may be different effects in mouse strains carrying genetic variants of the mu opioid receptor. Several studies have examined the A118G SNP’s effects in human subjects and have reported that the G118 allele is associated with vulnerability to mu opioid agonist addiction (Haerian and Haerian, 2013). The underlying mechanisms remain unknown, however clinical and in vitro data suggest alterations in receptor function that may affect mu opioid agonist reward and reinforcement. It is unclear, however, whether the effects of the A118G SNP reflect a loss or a gain in overall MOR function. Cell culture and postmortem data showing reduced MOR gene expression and protein levels (Zhang et al., 2005) support a loss of function, while in vitro binding studies have shown increased beta-endorphin binding affinity and beta-endorphin-mediated signaling at MORs encoded by the G allele (Bond et al., 1998), supporting increased receptor function.

26

Journal Pre-proof

Data from A112G mice are also equivocal. Multiple studies have revealed reduced MOR mRNA expression, MOR protein levels, and mu agonist-stimulated G-protein signaling (Huang et al., 2012; Mague et al., 2009; Wang et al., 2014) in GG mice, as well as deficits in mu agonist-mediated conditioned reward, analgesia, and hyperlocomotion (Browne et al., 2017; Mague et al., 2009). In support of these findings, a recent electrophysiological study found that accumbens shell-projecting dopaminergic VTA neurons showed reduced sensitivity and lower action potential firing rate in GG mice compared to AA mice (Popova et al., 2019). Other studies have revealed potentiated MOR-mediated effects in GG mice, including increased heroin selfadministration and heroin-induced striatal dopamine release (Zhang et al., 2015). Mague, Blendy, and colleagues found that female GG mice did not develop morphine conditioned place preference and that morphine’s locomotor-activating effects were blunted in male GG mice (only male mice were studied for this behavioral endpoint) (Mague et al., 2009), suggesting deficits in MOR-mediated behavior.

In

contrast, we observed no genotype or sex differences in oxycodone conditioned place

27

Journal Pre-proof

preference in A112G mice in the present study, suggesting that oxycodone reward is intact in GG mice. Despite a lack of effect of genotype or sex on oxycodone conditioned place preference, we did find that oxycodone failed to produce hyperlocomotion in GG mice. Mu opioid agonists such as morphine and oxycodone cause dose-dependent increases in locomotor activity in C57BL6 mice (Collins et al., 2016; Crawley et al., 1997; Murphy et al., 2001; Niikura et al., 2013). Consistent with this, we observed increased locomotor activity in response to 3 mg/kg oxycodone in AA mice. In contrast, we did not observe oxycodone-induced hyperlocomotion in GG mice. This observed genotype difference does not appear to reflect a general deficit in locomotor activity in GG mice, as salinetreated AA and GG mice showed similar locomotor activity. This lack of locomotor activation is consistent with other studies in GG mice, which have found blunted locomotor responses to morphine and buprenorphine. We found a striking increase in oxycodone self-administration in GG mice compared to AA mice. A potential mechanism leading to greater oxycodone intake in

28

Journal Pre-proof

GG mice is that the G112 allele causes alterations in striatal dopamine efflux. A key mechanism by which mu agonists exert their acute rewarding effects is inhibition of GABAergic neurons in mesolimbic and striatonigral circuits, which themselves inhibit dopaminergic cells, modulating activity in multiple brain regions, including striatal areas related mu opioid reward and reinforcement (Di Chiara and Imperato, 1988; Johnson and North, 1992; Wise, 1989). While GG and AA mice show similar basal striatal dopamine levels, GG mice show greater mu agonist (heroin)-stimulated dopamine release in striatum (Zhang et al., 2015). Further, in another mouse model of the A118G SNP, in which exon 1 of the mouse MOR gene has been replaced by exon 1 of the human MOR gene with either adenine or guanine at position 118, alcohol administration results in a fourfold greater striatal dopamine release (Ramchandani et al., 2011). Contrastingly, in the same humanized mouse model, morphine-stimulated (but not fentanyl-stimulated) accumbal dopamine release was reduced in 118GG mice compared to 118AA mice (Robinson et al., 2015), suggesting that genotype differences in dopamine release are drug class- and subtype-dependent.

29

Journal Pre-proof

Because GG mice show reduced levels of MOR protein compared to AA littermates (Mague et al., 2009; Wang et al., 2014, 2012), the increased oxycodone intake in GG mice could reflect attenuated overall rewarding effect or decreased sensitivity to mu opioid agonists due to reduction of MOR protein levels. Evidence suggests that it is unlikely that increased oxycodone self-administration in GG mice is due to differences in levels of MOR protein alone. At least one previous study using heterozygous knockout mice, in which MOR density is reduced approximately two-fold, revealed reduced morphine self-administration compared to wild-type controls (Sora et al., 2001). This knockout study used a knockout line generated on a mixed C57/129 background (and subsequently backcrossed to a C57BL6 for 6 generations), while the current study used a mouse line generated on a C57BL6 background. It is possible that the difference in mu agonist self-administration between the two studies could owe various epistatic alleles that exist in the different strains. Accumbens shell-projecting VTA dopamine neurons in GG mice are hyporesponsive to DAMGO and morphine, and GG mice show reduced MOR-dependent action potential firing, as well as blunted

30

Journal Pre-proof

MOR-dependent suppression of both inhibitory and excitatory input in mesolimbic circuits. Moreover, these neuronal populations appear to be regulated by cooperative MOR and GABAB signaling (Popova et al., 2019). These deficits could lead to reduced mu-agonist sensitivity and lead GG mice to continue responding for oxycodone for a longer period than AA mice, resulting in a greater number of oxycodone infusions and thus greater oxycodone intake. This would be consistent with a loss of function in MOR signaling that could reconcile increased mu agonist self-administration (Zhang et al. 2015) and reduced mu agonist-induced hyperlocomotion (Mague et al. 2009; Browne et al. 2017). Further studies are necessary to link the genotype-dependent changes in receptor function with the ultimate behavioral consequences of the G allele. For example, the present study found an increase in oxycodone intake in GG mice despite similar conditioned place preference. Autoradiography studies have found reduced expression and reduced MOR-dependent G-protein signaling in some but not all brain regions in GG mice (Wang et al., 2014, 2012). Conditioned reward, locomotor

31

Journal Pre-proof

activation, and operant behavior are mediated by different circuitry, and so it is likely that region-specific differences in MOR levels and signaling may lead to changes in some behavioral endpoints but not others. It would be of interest to better understand how oxycodone self-administration alters MOR gene and protein expression in brain areas related mu agonist-mediated reward and reinforcement in GG mice. We have previously found that drug-naive GG mice show profound differences in expression of multiple genes known to be related to mu-agonist addiction in multiple brain regions, including reductions in striatal Oprm1 gene expression (Collins et al., 2018), however, the mechanism underlying this difference is unknown. It is also not known how chronic oxycodone exposure affects the expression of these genes. It is likely that altered MOR function owing to the G allele affects the expression of multiple proteins that interact with MORs directly or indirectly to modulate mu agonist reward and reinforcement and that mechanisms downstream of the MOR are involved in the genotype-dependent differences in oxycodone responsivity that we have observed between AA and GG mice. Finally, it is possible that greater

32

Journal Pre-proof

initial oxycodone intake in GG mice is the result of differential adaptations in MORmediated circuits rather than the initial differences in MOR protein level or MORdependent signaling that exist in mice that have not self-administered mu agonists. Additional studies will be needed to examine the impact of chronic oxycodone exposure on receptor function in circuits related to operant self-administration. Furthermore, although we observed a robust effect of the GG allele on oxycodone selfadministration,

the current self-administration study features only a single drug

concentration and a single fixed-ratio reinforcement schedule, so it is not clear whether GG mice will continue to respond for oxycodone at higher ratios or alter their response frequency under variable-ratio schedules. It is also unclear how variations in oxycodone dose would affect self-administration behavior in GG mice. Future studies would be strengthened by examining additional reinforcer-response relationships and doseresponse relationships. Overall, the present study supports a pattern of increased mu agonist selfadministration and reduced mu agonist-induced hyperlocomotion in GG mice. These

33

Journal Pre-proof

findings provide additional insights into the potential behavioral mechanism by which the carriers of the A118G SNP have increased vulnerability to mu opioid addiction.

Funding and Disclosure The work contained herein was supported by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (DC, YZ, MJK). The authors declare no financial support, compensation, nor financial holdings that could be perceived as constituting a potential conflict of interest.

Acknowledgments The authors wish to thank the laboratory of Dr. Julie Blendy at University of Pennsylvania for their donation of the A112G mouse. We would also like to thank Eduardo Butelman, Brian Reed, and Vadim Yuferov for their assistance in the preparation of this manuscript, as well as the staff of the Comparative Bioscience Center at the Rockefeller University for animal care.

34

Journal Pre-proof

Bibliography Bart, G., Heilig, M., LaForge, K.S., Pollak, L., Leal, S.M., Ott, J., Kreek, M.J., 2004. Substantial attributable risk related to a functional mu-opioid receptor gene polymorphism in association with heroin addiction in central Sweden. Mol. Psychiatry 9, 547–549. doi:10.1038/sj.mp.4001504 Bart, G., LaForge, K.S., Borg, L., Lilly, C., Ho, A., Kreek, M.J., 2006. Altered levels of basal cortisol in healthy subjects with a 118G allele in exon 1 of the Mu opioid receptor gene. Neuropsychopharmacology 31, 2313–2317. doi:10.1038/sj.npp.1301128 Becker, A., Grecksch, G., Brödemann, R., Kraus, J., Peters, B., Schroeder, H., Thiemann, W., Loh, H.H., Höllt, V., 2000. Morphine self-administration in mu-opioid receptor-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 361, 584–589. Benjamini, Y., Hochberg, Y., 1995. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289–300. Bergen, A.W., Kokoszka, J., Peterson, R., Long, J.C., Virkkunen, M., Linnoila, M., Goldman, D., 1997. Mu opioid receptor gene variants: lack of association with alcohol dependence. Mol. Psychiatry 2, 490–494. Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S., Borg, L., Gong, J., Schluger, J., Strong, J.A., Leal, S.M., Tischfield, J.A., Kreek, M.J., Yu, L., 1998. Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc Natl Acad Sci USA 95, 35

Journal Pre-proof

9608–9613. Browne, C.A., Erickson, R.L., Blendy, J.A., Lucki, I., 2017. Genetic variation in the behavioral effects of buprenorphine in female mice derived from a murine model of the OPRM1 A118G polymorphism. Neuropharmacology 117, 401–407. doi:10.1016/j.neuropharm.2017.02.005 Chong, R.Y., Oswald, L., Yang, X., Uhart, M., Lin, P.-I., Wand, G.S., 2006. The muopioid receptor polymorphism A118G predicts cortisol responses to naloxone and stress. Neuropsychopharmacology 31, 204–211. doi:10.1038/sj.npp.1300856 Coller, J.K., Beardsley, J., Bignold, J., Li, Y., Merg, F., Sullivan, T., Cox, T.C., Somogyi, A.A., 2009. Lack of association between the A118G polymorphism of the mu opioid receptor gene (OPRM1) and opioid dependence: A meta-analysis. Pharmgenomics Pers Med 2, 9–19. Collins, D., Randesi, M., da Rosa, J.C., Zhang, Y., Kreek, M.J., 2018. Oprm1 A112G, a single nucleotide polymorphism, alters expression of stress-responsive genes in multiple brain regions in male and female mice. Psychopharmacology (Berl) 235, 2703–2711. doi:10.1007/s00213-018-4965-x Collins, D., Reed, B., Zhang, Y., Kreek, M.J., 2016. Sex differences in responsiveness to the prescription opioid oxycodone in mice. Pharmacol. Biochem. Behav. 148, 99– 105. doi:10.1016/j.pbb.2016.06.006 Contet, C., Kieffer, B.L., Befort, K., 2004. Mu opioid receptor: a gateway to drug addiction. Curr. Opin. Neurobiol. 14, 370–378. doi:10.1016/j.conb.2004.05.005 Crawley, J.N., Belknap, J.K., Collins, A., Crabbe, J.C., Frankel, W., Henderson, N., Hitzemann, R.J., Maxson, S.C., Miner, L.L., Silva, A.J., Wehner, J.M., WynshawBoris, A., Paylor, R., 1997. Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology (Berl) 132, 107–124. doi:10.1007/s002130050327 36

Journal Pre-proof

Di Chiara, G., Imperato, A., 1988. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci USA 85, 5274–5278. doi:10.1073/pnas.85.14.5274 Ducat, E., Ray, B., Bart, G., Umemura, Y., Varon, J., Ho, A., Kreek, M.J., 2013. Muopioid receptor A118G polymorphism in healthy volunteers affects hypothalamicpituitary-adrenal axis adrenocorticotropic hormone stress response to metyrapone. Addict. Biol. 18, 325–331. doi:10.1111/j.1369-1600.2011.00313.x Filbey, F.M., Ray, L., Smolen, A., Claus, E.D., Audette, A., Hutchison, K.E., 2008. Differential neural response to alcohol priming and alcohol taste cues is associated with DRD4 VNTR and OPRM1 genotypes. Alcohol. Clin. Exp. Res. 32, 1113–1123. doi:10.1111/j.1530-0277.2008.00692.x Franke, P., Wang, T., Nöthen, M.M., Knapp, M., Neidt, H., Albrecht, S., Jahnes, E., Propping, P., Maier, W., 2001. Nonreplication of association between μ‐opioid‐receptor gene (OPRM1) A118G polymorphism and substance dependence. American Journal of Medical Genetics. Haerian, B.S., Haerian, M.S., 2013. OPRM1 rs1799971 polymorphism and opioid dependence: evidence from a meta-analysis. Pharmacogenomics 14, 813–824. doi:10.2217/pgs.13.57 Hernandez-Avila, C.A., Covault, J., Wand, G., Zhang, H., Gelernter, J., Kranzler, H.R., 2007. Population-specific effects of the Asn40Asp polymorphism at the mu-opioid receptor gene (OPRM1) on HPA-axis activation. Pharmacogenet. Genomics 17, 1031–1038. doi:10.1097/FPC.0b013e3282f0b99c Hernandez-Avila, C.A., Wand, G., Luo, X., Gelernter, J., Kranzler, H.R., 2003. Association between the cortisol response to opioid blockade and the Asn40Asp polymorphism at the mu-opioid receptor locus (OPRM1). Am. J. Med. Genet. B Neuropsychiatr. Genet. 118B, 60–65. doi:10.1002/ajmg.b.10054 37

Journal Pre-proof

Huang, P., Chen, C., Mague, S.D., Blendy, J.A., Liu-Chen, L.-Y., 2012. A common single nucleotide polymorphism A118G of the μ opioid receptor alters its Nglycosylation and protein stability. Biochem. J. 441, 379–386. doi:10.1042/BJ20111050 Johnson, S.W., North, R.A., 1992. Opioids excite dopamine neurons by hyperpolarization of local interneurons. J. Neurosci. 12, 483–488. Kieffer, B.L., Gavériaux-Ruff, C., 2002. Exploring the opioid system by gene knockout. Prog. Neurobiol. 66, 285–306. doi:10.1016/S0301-0082(02)00008-4 Kim, S.-G., Kim, C.-M., Kang, D.-H., Kim, Y.-J., Byun, W.-T., Kim, S.-Y., Park, J.-M., Kim, M.-J., Oslin, D.W., 2004. Association of functional opioid receptor genotypes with alcohol dependence in Koreans. Alcohol. Clin. Exp. Res. 28, 986–990. Kroslak, T., Laforge, K.S., Gianotti, R.J., Ho, A., Nielsen, D.A., Kreek, M.J., 2007. The single nucleotide polymorphism A118G alters functional properties of the human mu opioid receptor. J. Neurochem. 103, 77–87. doi:10.1111/j.1471-4159.2007.04738.x Lovallo, W.R., Enoch, M.-A., Acheson, A., Cohoon, A.J., Sorocco, K.H., Hodgkinson, C.A., Vincent, A.S., Glahn, D.C., Goldman, D., 2015. Cortisol Stress Response in Men and Women Modulated Differentially by the Mu-Opioid Receptor Gene Polymorphism OPRM1 A118G. Neuropsychopharmacology 40, 2546–2554. doi:10.1038/npp.2015.101 Mague, S.D., Isiegas, C., Huang, P., Liu-Chen, L.-Y., Lerman, C., Blendy, J.A., 2009. Mouse model of OPRM1 (A118G) polymorphism has sex-specific effects on drugmediated behavior. Proc Natl Acad Sci USA 106, 10847–10852. doi:10.1073/pnas.0901800106 Maruta, T., Swanson, D.W., Finlayson, R.E., 1979. Drug abuse and dependency in patients with chronic pain. Mayo Clin. Proc. 54, 241–244. Matthes, H.W., Maldonado, R., Simonin, F., Valverde, O., Slowe, S., Kitchen, I., Befort, 38

Journal Pre-proof

K., Dierich, A., Le Meur, M., Dollé, P., Tzavara, E., Hanoune, J., Roques, B.P., Kieffer, B.L., 1996. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383, 819–823. doi:10.1038/383819a0 Mura, E., Govoni, S., Racchi, M., Carossa, V., Ranzani, G.N., Allegri, M., van Schaik, R.H., 2013. Consequences of the 118A>G polymorphism in the OPRM1 gene: translation from bench to bedside? J. Pain Res. 6, 331–353. doi:10.2147/JPR.S42040 Murphy, N.P., Lam, H.A., Maidment, N.T., 2001. A comparison of morphine-induced locomotor activity and mesolimbic dopamine release in C57BL6, 129Sv and DBA2 mice. J. Neurochem. 79, 626–635. doi:10.1046/j.1471-4159.2001.00599.x Nakamura, A., Hasegawa, M., Minami, K., Kanbara, T., Tomii, T., Nishiyori, A., Narita, M., Suzuki, T., Kato, A., 2013. Differential activation of the μ-opioid receptor by oxycodone and morphine in pain-related brain regions in a bone cancer pain model. Br. J. Pharmacol. 168, 375–388. doi:10.1111/j.1476-5381.2012.02139.x Narita, M., Nakamura, A., Ozaki, M., Imai, S., Miyoshi, K., Suzuki, M., Suzuki, T., 2008. Comparative pharmacological profiles of morphine and oxycodone under a neuropathic pain-like state in mice: evidence for less sensitivity to morphine. Neuropsychopharmacology 33, 1097–1112. doi:10.1038/sj.npp.1301471 Niikura, K., Ho, A., Kreek, M.J., Zhang, Y., 2013. Oxycodone-induced conditioned place preference and sensitization of locomotor activity in adolescent and adult mice. Pharmacol. Biochem. Behav. 110, 112–116. doi:10.1016/j.pbb.2013.06.010 Nishizawa, D., Han, W., Hasegawa, J., Ishida, T., Numata, Y., Sato, T., Kawai, A., Ikeda, K., 2006. Association of mu-opioid receptor gene polymorphism A118G with alcohol dependence in a Japanese population. Neuropsychobiology 53, 137–141. doi:10.1159/000093099 39

Journal Pre-proof

Olmstead, M.C., Burns, L.H., 2005. Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive effects of opiate withdrawal in rats. Psychopharmacology (Berl) 181, 576–581. doi:10.1007/s00213-005-0022-7 Oslin, D.W., Berrettini, W., Kranzler, H.R., Pettinati, H., Gelernter, J., Volpicelli, J.R., O’Brien, C.P., 2003. A functional polymorphism of the mu-opioid receptor gene is associated with naltrexone response in alcohol-dependent patients. Neuropsychopharmacology 28, 1546–1552. doi:10.1038/sj.npp.1300219 Peckham, E.M., Traynor, J.R., 2006. Comparison of the antinociceptive response to morphine and morphine-like compounds in male and female Sprague-Dawley rats. J. Pharmacol. Exp. Ther. 316, 1195–1201. doi:10.1124/jpet.105.094276 Popova, D., Desai, N., Blendy, J.A., Pang, Z.P., 2019. Synaptic regulation by OPRM1 variants in reward neurocircuitry. J. Neurosci. 39, 5685–5696. doi:10.1523/JNEUROSCI.2317-18.2019 Ramchandani, V.A., Umhau, J., Pavon, F.J., Ruiz-Velasco, V., Margas, W., Sun, H., Damadzic, R., Eskay, R., Schoor, M., Thorsell, A., Schwandt, M.L., Sommer, W.H., George, D.T., Parsons, L.H., Herscovitch, P., Hommer, D., Heilig, M., 2011. A genetic determinant of the striatal dopamine response to alcohol in men. Mol. Psychiatry 16, 809–817. doi:10.1038/mp.2010.56 Results from the 2016 National Survey on Drug Use and Health: Detailed Tables, SAMHSA, CBHSQ [WWW Document], n.d. URL https://www.samhsa.gov/data/sites/default/files/NSDUH-DetTabs-2016/NSDUHDetTabs-2016.htm#lotsect1pe (accessed 2.26.18). Robinson, J.E., Vardy, E., DiBerto, J.F., Chefer, V.I., White, K.L., Fish, E.W., Chen, M., Gigante, E., Krouse, M.C., Sun, H., Thorsell, A., Roth, B.L., Heilig, M., Malanga, C.J., 2015. Receptor reserve moderates mesolimbic responses to opioids in a humanized mouse model of the OPRM1 A118G polymorphism. 40

Journal Pre-proof

Neuropsychopharmacology 40, 2614–2622. doi:10.1038/npp.2015.109 Rutten, K., van der Kam, E.L., De Vry, J., Tzschentke, T.M., 2011. Critical evaluation of the use of extinction paradigms for the assessment of opioid-induced conditioned place preference in rats. Pharmacology 87, 286–296. doi:10.1159/000327680 Schlussman, S.D., Zhang, Y., Hsu, N.M., Allen, J.M., Ho, A., Kreek, M.J., 2008. Heroininduced locomotor activity and conditioned place preference in C57BL/6J and 129P3/J mice. Neurosci. Lett. 440, 284–288. doi:10.1016/j.neulet.2008.05.103 Schwantes-An, T.-H., Zhang, J., Chen, L.-S., Hartz, S.M., Culverhouse, R.C., Chen, X., Coon, H., Frank, J., Kamens, H.M., Konte, B., Kovanen, L., Latvala, A., Legrand, L.N., Maher, B.S., Melroy, W.E., Nelson, E.C., Reid, M.W., Robinson, J.D., Shen, P.-H., Yang, B.-Z., Saccone, N.L., 2016. Association of the OPRM1 Variant rs1799971 (A118G) with Non-Specific Liability to Substance Dependence in a Collaborative de novo Meta-Analysis of European-Ancestry Cohorts. Behav. Genet. 46, 151–169. doi:10.1007/s10519-015-9737-3 Shi, J., Hui, L., Xu, Y., Wang, F., Huang, W., Hu, G., 2002. Sequence variations in the mu-opioid receptor gene (OPRM1) associated with human addiction to heroin. Hum. Mutat. 19, 459–460. doi:10.1002/humu.9026 Šidák, Z., 1967. Rectangular Confidence Regions for the Means of Multivariate Normal Distributions. J. Am. Stat. Assoc. 62, 626–633. doi:10.1080/01621459.1967.10482935 Söderberg Löfdal, K.C., Andersson, M.L., Gustafsson, L.L., 2013. Cytochrome P450mediated changes in oxycodone pharmacokinetics/pharmacodynamics and their clinical implications. Drugs 73, 533–543. doi:10.1007/s40265-013-0036-0 Sora, I., Elmer, G., Funada, M., Pieper, J., Li, X.F., Hall, F.S., Uhl, G.R., 2001. Mu opiate receptor gene dose effects on different morphine actions: evidence for differential in vivo mu receptor reserve. Neuropsychopharmacology 25, 41–54. 41

Journal Pre-proof

doi:10.1016/S0893-133X(00)00252-9 U.S. Prescribing Rate Maps | Drug Overdose | CDC Injury Center [WWW Document], n.d. URL https://www.cdc.gov/drugoverdose/maps/rxrate-maps.html (accessed 1.25.18). Wand, G.S., McCaul, M., Yang, X., Reynolds, J., Gotjen, D., Lee, S., Ali, A., 2002. The mu-opioid receptor gene polymorphism (A118G) alters HPA axis activation induced by opioid receptor blockade. Neuropsychopharmacology 26, 106–114. doi:10.1016/S0893-133X(01)00294-9 Wang, Y.-J., Huang, P., Blendy, J.A., Liu-Chen, L.-Y., 2014. Brain region- and sexspecific alterations in DAMGO-stimulated [(35) S]GTPγS binding in mice with Oprm1 A112G. Addict. Biol. 19, 354–361. doi:10.1111/j.1369-1600.2012.00484.x Wang, Y.J., Huang, P., Ung, A., Blendy, J.A., Liu-Chen, L.Y., 2012. Reduced expression of the μ opioid receptor in some, but not all, brain regions in mice with OPRM1 A112G. Neuroscience 205, 178–184. doi:10.1016/j.neuroscience.2011.12.033 Wiers, R.W., Rinck, M., Dictus, M., van den Wildenberg, E., 2009. Relatively strong automatic appetitive action-tendencies in male carriers of the OPRM1 G-allele. Genes Brain Behav. 8, 101–106. doi:10.1111/j.1601-183X.2008.00454.x Wise, R.A., 1989. Opiate reward: sites and substrates. Neurosci. Biobehav. Rev. 13, 129–133. doi:10.1016/S0149-7634(89)80021-1 Xuei, X., Flury-Wetherill, L., Bierut, L., Dick, D., Nurnberger, J., Foroud, T., Edenberg, H.J., 2007. The opioid system in alcohol and drug dependence: family-based association study. Am. J. Med. Genet. B Neuropsychiatr. Genet. 144B, 877–884. doi:10.1002/ajmg.b.30531 Zhang, Y., Picetti, R., Butelman, E.R., Ho, A., Blendy, J.A., Kreek, M.J., 2015. Mouse model of the OPRM1 (A118G) polymorphism: differential heroin self-administration 42

Journal Pre-proof

behavior compared with wild-type mice. Neuropsychopharmacology 40, 1091–1100. doi:10.1038/npp.2014.286 Zhang, Y., Picetti, R., Butelman, E.R., Schlussman, S.D., Ho, A., Kreek, M.J., 2009. Behavioral and neurochemical changes induced by oxycodone differ between adolescent and adult mice. Neuropsychopharmacology 34, 912–922. doi:10.1038/npp.2008.134 Zhang, Y., Wang, D., Johnson, A.D., Papp, A.C., Sadée, W., 2005. Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G. J. Biol. Chem. 280, 32618–32624. doi:10.1074/jbc.M504942200

43

Journal Pre-proof

Highlights ● ● ● ● ●

Oxycodone conditioned place preference, locomotor activity, and self-administration were evaluated in adult male and female Oprm1 A112G mice 112AA and 112GG mice showed equivalent conditioned place preference at the doses tested 112GG mice showed blunted locomotor responses to oxycodone 112GG mice self-administered more oxycodone than 112AA mice No significant sex differences or sex-by-genotype were observed in any behavioral endpoint