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Heroin-induced suppression of saccharin intake in OPRM1 A118G mice
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Heroin-induced suppression of saccharin intake in OPRM1 A118G mice ⁎
Christopher S. Freeta, , Danielle N. Alexandera, Caesar G. Imperioa, Victor Ruiz-Velascob, Patricia S. Grigsona a
Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, United States Department of Anesthesiology and Perioperative Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, United States b
A R T I C L E I N F O
A B S T R A C T
Keywords: Addiction Natural rewards Opiates Reward comparison
The single nucleotide polymorphism of the μ-opioid receptor, OPRM1 A118G, has been associated with greater drug and alcohol use, increased sensitivity to pain, and reduced sensitivity to the antinociceptive effects of opiates. In the present studies, we employed a ‘humanized’ mouse model containing the wild-type (118AA) or variant (118GG) allele to examine behavior in a model of heroin-induced devaluation of an otherwise palatable saccharin cue when repeated saccharin-heroin pairings occurred every 24 h (Experiment 1) or every 48 h (Experiment 2). The results showed that, while both the 118AA and 118GG mice demonstrated robust avoidance of the heroin-paired saccharin cue following daily taste-drug pairings, only the 118AA mice suppressed intake of the heroin-paired saccharin cue when 48 h elapsed between each taste-drug pairing. Humanized 118GG mice, then, defend their intake of the sweet cue despite saccharin-heroin pairings and this effect is illuminated by the use of spaced, rather than massed, trials. Given that this pattern of strain difference is not evident with saccharincocaine pairings (Freet et al., 2015), reduced avoidance of the heroin-paired saccharin cue by the 118GG mice may be due to an interaction between the opiate and the subjects’ drive for the sweet or, alternatively, to differential downstream sensitivity to the aversive kappa mediated properties of the drug. These alternative hypotheses are addressed.
1. Introduction According to the National Institute on Drug Abuse (NIDA), abuse of illicit drugs, tobacco, and alcohol costs up to $700 billion annually due to drug-related crime, lost work productivity, and health care costs in the United States (https://www.drugabuse.gov). Substance use disorder (SUD) is, in part, such a costly condition because it is a brain disease characterized by compulsive consumption of drug and chronic relapse, even following extended periods of abstinence. Thus, while initial use of the drug is voluntary, or even prescribed by a physician, drugs of abuse alter gene expression and brain circuitry resulting in behavioral changes that manifest as compulsive drug craving, seeking, and use (Kalivas and O’Brien, 2008; Koob et al., 1998; Nestler, 2004; Robinson and Berridge, 2003). It is now well understood that the development and progression of substance use disorder is determined by both environmental and hereditary factors. One such genetic factor associated with an increased risk for the development of addiction is the single nucleotide polymorphism (SNP) of the human mu (μ) opioid receptor (OPRM1) gene,
A118G (Bond et al., 1998; Miller et al., 2004; Ray and Hutchison, 2007; van den Wildenberg et al., 2007). The OPRM1 A118G SNP is the substitution of the adenine (A) in the wildtype for a guanine (G) in the mutant; this modification results in the replacement of the asparagine for an aspartate residue in the OPRM1 N-terminus and decreases the number of putative glycosylation sites from five to four (Shabalina et al., 2009). Functionally, the A118G variant creates a new CpG-methylation site in the OPRM1 DNA that prevents the upregulation of OPRM1 in response to chronic opioid use (Oertel et al., 2012). In addition, the A118G variant also results in reduced expression of OPRM1 at the cell surface (Kroslak et al., 2007; Zhang et al., 2005) and reduces the accumulation of cAMP in response to morphine, methadone, and DAMGO (Kroslak et al., 2007). In contrast, the endogenous OPRM1 ligand β-endorphin is approximately 3 times more potent and binds approximately 3 times more tightly to OPRM1 with the variant allele compared with the common allele (Bond et al., 1998). Behaviorally, the polymorphism is associated with greater intake of, and responsiveness to, a range of abused drugs including opiates, alcohol, cocaine, cannabis, and nicotine in humans and in non-humans (Haerian and
⁎ Corresponding author at: Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, 500 University Drive, H181, Hershey, PA 17033, United States. E-mail address: [email protected] (C.S. Freet).
http://dx.doi.org/10.1016/j.brainresbull.2017.09.008 Received 31 May 2017; Received in revised form 12 September 2017; Accepted 15 September 2017 0361-9230/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Freet, C.S., Brain Research Bulletin (2017), http://dx.doi.org/10.1016/j.brainresbull.2017.09.008
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to provide purified water and saccharin access. Pipettes were converted to plastic cylinders by removing the tapered ends. A stainless steel spout (Girton Manufacturing Co., Inc.; ST300C) was placed in the bottom of the cylinder, while a silicon stopper (Fisher Scientific; 09-704-1D) sealed the top of the cylinder. Intake of water and saccharin was recorded in 1/10 ml.
Haerian, 2013; Ray and Hutchison, 2007; Schwantes-An et al., 2016; van den Wildenberg et al., 2007; Zhang et al., 2015). In an effort to more closely examine the consequence of this A118G variant in SUD and addiction, a number of studies have employed a ‘humanized’ OPRM1 A118G mouse model in which the native exon 1 of the μ-opioid receptor gene has been replaced with the human exon 1. These mice have been reported to exhibit increased responsiveness to alcohol (Bilbao et al., 2015; Ramchandani et al., 2011), increased nicotine self-administration (Bernardi et al., 2016), and decreased morphine-induced antinociception (Henderson-Redmond et al., 2016; Mahmoud et al., 2011). Likewise, we previously have demonstrated that humanized OPRM1 A118G mice exhibit greater avoidance of a cocaine-paired saccharin cue in an animal model of the devaluation of natural rewards by drugs of abuse (Freet et al., 2015). It is well known that rodents will avoid a lesser reward if it predicts the future availability of a more preferred, intense reward (for review, see Flaherty, 1996) and it has been hypothesized that, with taste-drug pairings (e.g., saccharin and cocaine), avoidance of intake of the lesser saccharin reward cue is, at least in part, due to a decrease in the relative value of the taste cue when compared with the potent drug of abuse that is expected in the near future (Grigson, 2002, 1997; Grigson et al., 2009). Stimulants and depressants, however, are not one in the same and avoidance of a drug-paired cue has been found to differ when paired with cocaine or morphine. For example, reward-preferring Lewis rats demonstrate greater suppression of intake of a cocaine-paired saccharin cue compared with relatively reward-insensitive Fischer rats (Grigson and Freet, 2000); however, the inverse is true (i.e., Lewis rats exhibit lesser suppression) when saccharin is paired with morphine. This strain difference has been linked to greater sensitivity to aversive kappa receptor activation in the Fischer rats (Freet et al., 2013b). The present study will test whether the same is true for the A118G mice vs. the A118A mice. Specifically, we employed humanized mice containing either the wild-type (118AA) or variant (118GG) allele to examine behavior in heroin-induced suppression of intake of a saccharin reward cue when the saccharin-heroin pairings occurred every 24 h (Experiment 1) or every 48 h (Experiment 2). In our experience, massed trials (every 24 h) can lead to more robust suppression of intake of the taste cue than spaced trials.
2.1.3. Procedure: Saccharin-heroin pairings All subjects were weighed once a day throughout the study. For baseline measurements, all mice were placed on a water restriction schedule that consisted of access to purified water for 1 h in the morning (testing period) and unpurified tap water for 2 h in the afternoon (rehydration period). Baseline intake and body weight were recorded for 7 days and experimental groups were determined by matching terminal baseline morning water intake and age. All mice remained on the water restriction schedule as described above for the duration of the experiment. For each taste-drug pairing (which occurred every 24 h), all mice received 0.15% saccharin for the morning access period followed immediately by an i.p. injection of either saline (118AA: n = 14, 118GG: n = 15) or heroin (118AA: n = 15, 118GG: n = 14). The dose of heroin increased over trials (trials 1–7: 30 mg/kg, trials 8–10: 40 mg/kg). After the 10th trial, ad lib water was returned to all mice for four days. Mice were then water deprived overnight and an eleventh conditioning trial was conducted with saccharin access only (no injection). All mice received 2 h access to tap water each afternoon to maintain hydration throughout conditioning. The saccharin solution was presented at room temperature and the heroin was generously provided by the National Institute on Drug Abuse (NIDA). 2.2. Results 2.2.1. Saccharin Intake Saccharin intake was analyzed using a 2 × 2 x 11 repeated measures analyses of variance (ANOVA) varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–11). The main effects of drug, F(1.54) = 73.16, p < 0.0001, and trials, F(10,540) = 4.66, p < 0.0001, were significant. However, the main effect of genotype, F < 1, and the 3-way interaction, F(10,540) = 1.19, p = 0.29, did not reach statistical significance. Separate 2 × 11 ANOVAs were conducted varying drug and trials for each genotype. Although this approach did not allow direct statistical comparison between the genotypes, it allowed us to look more closely at each genotype individually. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(10,270) = 5.79, p < 0.0001, and the 118GG genotype, F(10,270) = 4.60, p < 0.0001. Observation of Fig. 1 shows that, compared to their saccharin-saline controls, both 118AA and 118GG mice in the saccharinheroin condition demonstrated significant suppression of intake of the saccharin cue on trials 2–9, ps < 0.05 when pairings occurred every 24 h.
2. Experiment 1: 24 h saccharin-heroin pairings in OPRM1 A118G mice 2.1. Methods 2.1.1. Subjects The subjects were 58 adult (60–200 days) naïve male 118AA mice (n = 29, mean weight: 35.6 g) and 118GG mice (n = 29, mean weight: 34.1 g). The original breeding pairs were obtained from Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD. The remaining mice were bred in the barrier facility at the Pennsylvania State University College of Medicine, Hershey, PA using a low-intensity monogamous stud mating system. A heterozygous mating scheme was used (AG x AG); back- and intercross lines were maintained and bred into the population. Pups were weaned at 21 days. Tail cuts were performed, and the DNA isolated (DNeasy Blood & Tissue Kit, Qiagen), amplified (GoTaq DNA Poly- merase Kit, Promega), run in gels, extracted (DNA Gel Purification Kit, Qiagen), and sequenced to determine genotype. All subjects were housed individually in standard, clear plastic pan cages in a temperature-controlled (21 °C) animal care facility with a 12:12 h light:dark cycle and mice were maintained with free access to dry Harlan Teklad rodent diet (W) 8604 and water, except where otherwise noted.
2.2.2. Body weight Body weight was analyzed using a 2 × 2 x 10 repeated measures ANOVA varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–10). Although there was a non-significant main effect of genotype, F(1,54) = 1.06, p = 0.31, and a non-significant 3-way interaction, F < 1, confirming that 118AA mice were not significantly heavier than 118GG mice overall, the main effect of drug, F(1,54) = 6.32, p = 0.01, and main effect of trials, F(9486) = 51.42, p < 0.0001, were significant indicating that body weight increased over successive trials for all mice and that heroin exposure decreased body weight independent of genotype (data not shown).
2.1.2. Apparatus All behavioral manipulations were conducted in the home cages. Modified serological pipettes (Fisher Scientific; 07-200-574) were used
2.2.3. PM water intake Intake of water for the 2 h access period in the afternoon for all trials is presented in Fig. 2. Although a 2 × 2 × 9 repeated measures 2
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Fig. 1. Heroin-induced suppression of saccharin intake in humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 24 h (Experiment 1). Mean ( ± SEM) intake (ml/hr) of 0.15% saccharin following pairings with either saline (118AA: open squares; 118GG: open circles) or 30–40 mg/kg heroin (118AA: closed squares; 118GG: closed circles).
3. Experiment 2: 48 h saccharin-heroin pairings in OPRM1 A118G mice
ANOVA did not reveal a significant three-way interaction, F(8432) = 1.78, p = 0.08, or main effect of genotype, F < 1, the main effects of drug, F(1,54) = 40.43, p < 0.0001, and trials, F(8432) = 19.38, p < 0.0001, did attain statistical significance. Separate 2 × 9 ANOVAs were conducted varying drug and trials for each genotype. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(8216) = 2.82, p < 0.01, and 118GG genotype, F(8216) = 8.08, p < 0.0001, showing that heroin treated 118AA and 118GG mice made up for low morning intake by consuming more afternoon water than their saline treated counterparts on trials 2–9 and 4–9, respectively, ps < 0.05.
3.1. Methods 3.1.1. Subjects The subjects were 58 adult (60–200 days) naïve male 118AA mice (n = 28, mean weight: 32.3 g) and 118GG mice (n = 30, mean weight: 32.1 g). The original breeding pairs were obtained from Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD. The remaining mice were bred in the barrier Fig. 2. Afternoon water intake for humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 24 h in Experiment 1. Mean ( ± SEM) intake (ml/2hr) of PM water following saccharinheroin pairings every 24 h. Saccharin-saline groups (118AA: open squares; 118GG: open circles); Saccharin-heroin groups (118AA: closed squares; 118GG: closed circles).
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Fig. 3. Heroin-induced suppression of saccharin intake in humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 48 h (Experiment 2). Mean ( ± SEM) intake (ml/hr) of 0.15% saccharin following pairings with either saline (118AA: open squares; 118GG: open circles) or 30–40 mg/kg heroin (118AA: closed squares; 118GG: closed circles).
effect of genotype, separate 2 × 9 ANOVAs were conducted varying drug and trials for each genotype. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(8208) = 7.05, p < 0.001, showing that the 118AA mice in the saccharin-heroin group demonstrated significant suppression of saccharin intake on trials 2–9 compared to their saccharin-saline treated controls, ps < 0.05 (left panel). For the 118GG mice, on the other hand, neither the main effect of drug, F (1.27) = 1.45, p = 0.24, nor the drug x trials interaction attained statistical significance, though the drug x trials interaction was close, F (8216) = 1.95, p = 0.053. Unlike the 118AA mice, then, the 118GG mice failed to demonstrate significant suppression of saccharin intake when saccharin-heroin pairings occurred at 48 h intervals.
facility at the Pennsylvania State University College of Medicine, Hershey, PA as described in Experiment 1. All subjects were housed and maintained as described above. 3.1.2. Apparatus Behavioral manipulations and apparatuses were identical to that described in Experiment 1. 3.1.3. Procedure All subjects were weighed once a day throughout the study and adapted to the water restriction schedule as described in Experiment 1. For each taste-drug pairing (which occurred every 48 h), all mice received 0.15% saccharin for the morning access period followed immediately by an i.p. injection of either saline (118AA: n = 14, 118GG: n = 15) or heroin (118AA: n = 14, 118GG: n = 15). The dose of heroin increased over trials (trials 1–3: 30 mg/kg, trials 4–6: 35 mg/kg, trials 7–9: 40 mg/kg). Thus, taste-drug pairings occurred every other morning (0.5 h into the light phase) for a total of 9 trials. All mice received 1 h access to purified water in the mornings between conditioning trials and 2 h access to purified water each afternoon to maintain hydration for all conditioning and non-conditioning trials. The saccharin solution was presented at room temperature and NIDA generously provided the heroin.
3.2.2. Body weight Body weight was analyzed using a 2 × 2 x 19 repeated measures ANOVA varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–10, W1-W9). Drug exposure did not significantly alter body weight (data not shown). This conclusion was supported by a nonsignificant main effect of drug, F < 1, and a non-significant 3-way interaction, F(18,972) = 1.27, p = 0.20. The main effect of trials was significant, F(18,972) = 20.93, p < 0.0001, indicating that body weight increased over successive trials for all mice. The main effect of genotype also did not attain statistical significance, F < 1, confirming no differences in body weight between the 118AA and the 118GG mice overall (data not shown).
3.1.3.1. Two-bottle intake test. A 10th trial occurred 48 h after the last taste-drug pairing where all mice were tested for saccharin preference using a two-bottle intake test. All mice simultaneously received a bottle of 0.15% saccharin and a bottle of purified water for 1 h. The left/right position of bottles was counter-balanced across groups.
3.2.3. AM water intake Morning intake of water (ml/h) on the days between conditioning trials (trials W1-W9) was evaluated. A 2 × 2 × 9 repeated measures ANOVA revealed that drug exposure did not significantly alter morning water intake as a function of genotype, as indicated by a non-significant main effect of genotype, F < 1, genotype x drug interaction, F < 1, and genotype x drug x trials interaction, F < 1. The main effect of drug did not reach statistical significance, F < 1. The main effect of trials was significant, F(8432) = 10.53, p < 0.0001, but follow-up post hoc tests revealed no meaningful differences, ps > 0.05 (data not shown).
3.2. Results 3.2.1. Saccharin intake Saccharin intake was analyzed using a 2 × 2 x 9 repeated measures analyses of variance (ANOVA) varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–9). The main effects of genotype, F (1,53) = 4.77, p = 0.03, drug, F(1,53) = 8.03, p < 0.01, and trials, F (8424) = 6.68, p < 0.01, were significant. However, the three-way interaction did not reach significance, F < 1. Given the robust main 4
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Fig. 4. Afternoon water intake for humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 48 h in Experiment 2. Mean ( ± SEM) intake (ml/2hr) of PM water following 48 h saccharinheroin pairings. Saccharin-saline groups (118AA: open squares; 118GG: open circles); Saccharin-heroin groups (118AA: closed squares; 118GG: closed circles).
heroin-induced suppression of saccharin intake, though avoidance of the heroin-paired cue was less marked in the 118AA mice when pairings occurred at 48 h vs. 24 h intervals. For the 118GG mice, on the other hand, no significant suppression was observed (Fig. 3). Despite the differential suppression of saccharin intake, however, both genotypes compensated by increasing intake of afternoon water on heroin injections days and the two-bottle saccharin preference, evidenced by saline-treated 118AA and 118GG mice, was eliminated in both genotypes following a history of saccharin-heroin pairings (Fig. 5). This finding suggests that, while heroin-induced suppression of saccharin intake may have been reduced in the 118GG vs. the 118AA mice, it was not eliminated, as evidenced by the two-bottle challenge. This pattern, i.e., less drug-induced avoidance by the more drugpreferring strain, is not unprecedented. For example, drug-preferring C57BL/6J mice exhibit less cocaine-induced suppression of saccharin intake than less drug-preferring DBA/2J mice (Freet et al., 2013a). Likewise, drug-preferring transgenic NSE-tTA x TetOp-ΔFosB mice demonstrate less cocaine-induced suppression of saccharin intake than their non ΔFosB over-expressing controls (Freet et al., 2009). In the case of the mice, both drug-preferring strains (C57Bl/6J and transgenic NSEtTA x TetOp-ΔFosB mice) also exhibit a greater preference for saccharin over water in a two-bottle test. This finding led us to conclude that these subjects were not merely drug-preferring, but actually were reward preferring (‘reward generalists’), allowing their drive for the sweet to override the suppressive effects of the drug. Here, however, while the saline-treated 118GG mice tend to exhibit a stronger preference for saccharin vs. water compared to the saline-treated 118AA mice, this effect was not statistically significant. Indeed, we might argue that the pattern of data in the 118AA vs. 118GG mice most closely parallels that obtained in the Fischer vs. Lewis rats. Lewis rats, like the 118GG mice, exhibit less avoidance of a saccharin cue when paired with an opiate (i.e., morphine) than Fischer rats (Freet et al., 2013b), but greater avoidance of a saccharin cue when paired with cocaine (Glowa et al., 1994; Grigson and Freet, 2000). Likewise, the humanized 118GG mice also demonstrate lesser avoidance of the opiate-paired saccharin solution (show above), but greater avoidance of a saccharin cue when paired with cocaine than their 118AA counterparts (Freet et al., 2015). But, why this dissociation
3.2.4. PM water intake Intake of water for the 2 h access period in the afternoon for all trials is presented in Fig. 4. Although a 2 × 2 x 18 repeated measures ANOVA did not reveal a statistically significant three-way interaction, F < 1, or main effect of genotype, F(1,54) = 1.54, p = 0.22, the main effects of drug, F(1,54) = 33.25, p < 0.0001, and trials, F(24, 1296) = 16.61, p < 0.0001, did attain statistical significance, as did the drug x trials interaction, F(24, 1296) = 7.45, p < 0.001. Separate 2 × 18 ANOVAs were conducted varying drug and trials for each genotype. A post hoc Newman-Keuls test (alpha = 0.05) was conducted on the significant drug x trials interaction for the 118AA genotype, F(17,442) = 3.78, p < 0.0001, and 118GG genotype, F(17,476) = 3.54, p < 0.0001, confirming, once again, that lower morning intake by mice in the saccharin-heroin condition is followed by greater afternoon water intake on trials 5–9 in the 118AA mice, and on trials 3, 4, 6–9 in the 118GG mice, ps < 0.05, compared to their saccharin-saline controls. 3.2.5. Two-bottle intake test The results of the two-bottle intake test for saccharin against water are presented in Fig. 5. Preference for saccharin was analyzed using t tests for dependent samples with alpha = 0.05 for each cell. The results confirmed that both 118AA (t = 2.70, p = 0.02) and 118GG (t = 4.60, p < 0.001) mice preferred saccharin over water under the saline condition, while neither genotype preferred saccharin over water when tested following saccharin-heroin pairings (118AA: t = −1.69, p = 0.11; 118GG: t = −0.19, p = 0.85). 4. Discussion The data from Experiment 1 demonstrate that both 118AA and 118GG mice exhibit robust heroin-induced suppression of saccharin intake when the saccharin-heroin pairings occurred every 24 h (Fig. 1). As there is a fair amount of literature demonstrating differential responding between these genotypes to drugs of abuse, Experiment 2 was conducted to unmask any potential differences by spacing out the tastedrug pairings (48 h). The results of Experiment 2 demonstrated that, under the 48 h condition, 118AA mice continued to exhibit robust 5
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Fig. 5. Two-bottle intake test for 0.15% saccharin and water following 48 h saccharin-saline or saccharin-heroin pairings in humanized 118AA and 118GG mice. Saccharin-saline groups (saccharin: solid; water: diagonal lines); Saccharin-heroin groups (saccharin: dense crossed lines; water: horizontal lines).
Conflict of interest
between an opiate and cocaine? A follow up study showed greater avoidance of a saccharin cue that had been paired with spiradoline (a kappa agonist) in the Fischer vs. Lewis rats (Freet et al., 2013b). Fischer rats, then, may exhibit greater avoidance of a morphine-paired saccharin cue because they are more sensitive than the Lewis rats to the aversive kappa agonist properties of the opiate. While heroin does not directly activate opioid receptors (Inturrisi et al., 1983), it acts as a lipid soluble prodrug for the central distribution of morphine (Hubner and Kornetsky, 1992) and rapidly metabolizes to 6-monoacetylmorphine and morphine; both metabolites strongly bind to μ-opioid receptors (Inturrisi et al., 1983; Selley et al., 2001), but also bind with δ-receptors and with κ-opioid receptors (Kristensen et al., 1995; Rady et al., 1994). We might speculate that a similar kappa sensitivity may be evidenced in 118AA vs. 118GG mice, but this would seem a stretch. Presumably, the humanized A118G mice differ only in their μ-opioid receptor; κ- and δreceptors should be the same (Browne et al., 2017). Even so, there is evidence that carrying the A118G allele in humans whom have overdosed on heroin is associated with downstream 2-fold higher levels of dynorphin A in the caudate nucleus and reduced preprodynorphin mRNA in the nucleus accumbens core (Drakenberg et al., 2006). As such, an assessment of the impact of carrying the A118G allele on kappa sensitivity should be examined in this paradigm. In sum, both the 118AA and the 118GG mice exhibit robust avoidance of an otherwise palatable saccharin solution when it predicts the administration of heroin via once daily pairings. The magnitude of this effect is reduced in the 118AA mice, and eliminated in the 118GG mice, when the saccharin-heroin trials are spaced at 48 h, rather than 24 h, intervals. That being said, when assessed using the two-bottle test (where a water alternative is provided), conditioned avoidance of the heroin-paired saccharin cue is revealed in both the 118AA and the 118GG strain of mice. Attenuated avoidance of the heroin-paired saccharin cue (at least in the one-bottle test across trials) by the 118GG mice may be due to an interaction between the opiate and the individuals’ drive for the sweet (it has long been known that opiates can increase responding for sweets (Zhang and Kelley, 1997), or due to differential downstream sensitivity to the aversive kappa mediated properties of the opiate. Further studies will need to be designed to test the merits of these two hypotheses.
None. Competing interests None. Acknowledgements This research was supported by PHS grants DA024519 and DA009815 to PSG and DA025574 to VR-V. We thank Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD, for the original 118AA and 118GG breeding pairs. References Bernardi, R.E., Zohsel, K., Hirth, N., Treutlein, J., Heilig, M., Laucht, M., Spanagel, R., Sommer, W.H., 2016. A gene-by-sex interaction for nicotine reward: evidence from humanized mice and epidemiology. Transl. Psychiatry 6, e861. http://dx.doi.org/10. 1038/tp.2016.132. Bilbao, A., Robinson, J.E., Heilig, M., Malanga, C.J., Spanagel, R., Sommer, W.H., Thorsell, A., 2015. A Pharmacogenetic Determinant of Mu-Opioid Receptor Antagonist Effects on Alcohol Reward and Consumption: Evidence from Humanized Mice. Biol. Psychiatry 77, 850–858. http://dx.doi.org/10.1016/j.biopsych.2014.08. 021. 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. U. S. A. 95, 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. http://dx.doi.org/10.1016/j. neuropharm.2017.02.005. Drakenberg, K., Nikoshkov, A., Horvath, M.C., Fagergren, P., Gharibyan, A., Saarelainen, K., Rahman, S., Nylander, I., Bakalkin, G., Rajs, J., Keller, E., Hurd, Y.L., 2006. Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers. Proc. Natl. Acad. Sci. U. S. A. 103, 7883–7888. http://dx.doi.org/10.1073/pnas.0600871103. Flaherty, C.F., 1996. Incentive relativity. Problems in the Behavioural Sciences. Cambridge University Press, New York. Freet, C.S., Steffen, C., Nestler, E.J., Grigson, P.S., 2009. Overexpression of ⌬FosB is associated with attenuated cocaine-Induced suppression of saccharin intake in mice. Behav. Neurosci. 123, 397–407. http://dx.doi.org/10.1037/a0015033. Freet, C.S., Arndt, A., Grigson, P.S., 2013a. Compared with DBA/2J mice, C57BL/6J mice demonstrate greater preference for saccharin and less avoidance of a cocaine-paired saccharin cue. Behav. Neurosci. 127, 474–484.
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P.H., Lötsch, J., 2012. Genetic-epigenetic interaction modulates μ-opioid receptor regulation. Hum. Mol. Genet. 21, 4751–4760. http://dx.doi.org/10.1093/hmg/ dds314. Rady, J.J., Aksu, F., Fujimoto, J.M., 1994. The heroin metabolite, 6-monoacetylmorphine, activates delta opioid receptors to produce antinociception in Swiss-Webster mice. J. Pharmacol. Exp. Ther. 268, 1222–1231. 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. http://dx.doi.org/10.1038/mp.2010.56. Ray, L.A., Hutchison, K.E., 2007. Effects of naltrexone on alcohol sensitivity and genetic moderators of medication response. Arch. Gen. Psychiatry 64, 1069. http://dx.doi. org/10.1001/archpsyc.64.9.1069. Robinson, T.E., Berridge, K.C., 2003. Addiction. Annu. Rev. Psychol. 54, 25–53. http:// dx.doi.org/10.1146/annurev.psych.54.101601.145237. 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., Andrews, J.A., Aveyard, P., Beltcheva, O., Brown, S.A., Cannon, D.S., Cichon, S., Corley, R.P., Dahmen, N., Degenhardt, L., Foroud, T., Gaebel, W., Giegling, I., Glatt, S.J., Grucza, R.A., Hardin, J., Hartmann, A.M., Heath, A.C., Herms, S., Hodgkinson, C.A., Hoffmann, P., Hops, H., Huizinga, D., Ising, M., Johnson, E.O., Johnstone, E., Kaneva, R.P., Kendler, K.S., Kiefer, F., Kranzler, H.R., Krauter, K.S., Levran, O., Lucae, S., Lynskey, M.T., Maier, W., Mann, K., Martin, N.G., Mattheisen, M., Montgomery, G.W., Müller-Myhsok, B., Murphy, M.F., Neale, M.C., Nikolov, M.A., Nishita, D., Nöthen, M.M., Nurnberger, J., Partonen, T., Pergadia, M.L., Reynolds, M., Ridinger, M., Rose, R.J., Rouvinen-Lagerström, N., Scherbaum, N., Schmäl, C., Soyka, M., Stallings, M.C., Steffens, M., Treutlein, J., Tsuang, M., Wall, T.L., Wodarz, N., Yuferov, V., Zill, P., Bergen, A.W., Chen, J., Cinciripini, P.M., Edenberg, H.J., Ehringer, M.A., Ferrell, R.E., Gelernter, J., Goldman, D., Hewitt, J.K., Hopfer, C.J., Iacono, W.G., Kaprio, J., Kreek, M.J., Kremensky, I.M., Madden, P.A.F., McGue, M., Munafò, M.R., Philibert, R.A., et al., 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. http://dx.doi.org/10.1007/s10519-015-9737-3. Selley, D.E., Cao, C.C., Sexton, T., Schwegel, J.A., Martin, T.J., Childers, S.R., 2001. mu opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine. Biochem. Pharmacol. 62, 447–455. Shabalina, S.A., Zaykin, D.V., Gris, P., Ogurtsov, A.Y., Gauthier, J., Shibata, K., Tchivileva, I.E., Belfer, I., Mishra, B., Kiselycznyk, C., Wallace, M.R., Staud, R., Spiridonov, N.A., Max, M.B., Goldman, D., Fillingim, R.B., Maixner, W., Diatchenko, L., 2009. Expansion of the human mu-opioid receptor gene architecture: novel functional variants. Hum. Mol. Genet. 18, 1037–1051. http://dx.doi.org/10.1093/ hmg/ddn439. Zhang, M., Kelley, A.E., 1997. Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats. Psychopharmacol. (Berl). 132, 350–360. Zhang, Y., Wang, D., Johnson, A.D., Papp, A.C., Sadee, W., 2005. Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G. J. Biol. Chem. 280, 32618–32624. http://dx.doi.org/10.1074/jbc.M504942200. 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 behavior compared with wild-type mice. Neuropsychopharmacology 40, 1091–1100. http://dx.doi.org/10.1038/npp.2014.286. van den Wildenberg, E., Wiers, R.W., Dessers, J., Janssen, R.G.J.H., Lambrichs, E.H., Smeets, H.J.M., van Breukelen, G.J.P., 2007. A functional polymorphism of the muopioid receptor gene (OPRM1) influences cue-induced craving for alcohol in male heavy drinkers. Alcohol. Clin. Exp. Res. 31, 1–10. http://dx.doi.org/10.1111/j.15300277.2006.00258.x.
Freet, C.S., Wheeler, R.A., Leuenberger, E., Mosblech, N.A.S., Grigson, P.S., 2013b. Fischer rats are more sensitive than Lewis rats to the suppressive effects of morphine and the aversive kappa-opioid agonist spiradoline. Behav. Neurosci. 127, 763–770. Freet, C.S., Ballard, S.M., Alexander, D.N., Cox, T.A., Imperio, C.G., Anosike, N., Carter, A.B., Mahmoud, S., Ruiz-Velasco, V., Grigson, P.S., 2015. Cocaine-induced suppression of saccharin intake and morphine modulation of Ca2+ channel currents in sensory neurons of OPRM1 A118G mice. Physiol. Behav. 139, 216–223. http://dx. doi.org/10.1016/j.physbeh.2014.11.040. Glowa, J.R., Shaw, A.E., Riley, A.L., 1994. Psychopharmacology Cocaine-induced conditioned taste aversions: comparisons between effects in LEW/N and F344/N rat strains. Psychopharmacol 114, 229–232. Grigson, P.S., Freet, C.S., 2000. The suppressive effects of sucrose and cocaine, but not lithium chloride, are greater in lewis than in fischer rats: evidence for the reward comparison hypothesis. Behav. Neurosci. 114, 353–363. http://dx.doi.org/10. 1037//0735-7044.114.2.353. Grigson, P.S., Twining, R.C., Freet, C.S., Wheeler, R.A., Geddes, R.I., 2009. Drug-induced suppression of conditioned stimulus intake: reward, aversion, and addiction. In: Reilly, S., Schachtman, T.R. (Eds.), Conditioned Taste Aversion: Behavioral and Neural Processes. Oxford University Press, New York, pp. 74–91. Grigson, P.S., 1997. Conditioned taste aversions and drugs of abuse: a reinterpretation. Behav. Neurosci. 111, 129–136. http://dx.doi.org/10.1037/0735-7044.111.1.129. Grigson, P.S., 2002. Like drugs for chocolate: separate rewards modulated by common mechanisms? Physiol. Behav. 76, 389–395. Haerian, B.S., Haerian, M.S., 2013. OPRM1 rs1799971 polymorphism and opioid dependence: evidence from a meta-analysis. Pharmacogenomics 14, 813–824. http:// dx.doi.org/10.2217/pgs.13.57. Henderson-Redmond, A.N., Yuill, M.B., Lowe, T.E., Kline, A.M., Zee, M.L., Guindon, J., Morgan, D.J., 2016. Morphine-induced antinociception and reward in humanized mice expressing the mu opioid receptor A118G polymorphism. Brain Res. Bull. 123, 5–12. http://dx.doi.org/10.1016/j.brainresbull.2015.10.007. Hubner, C.B., Kornetsky, C., 1992. Heroin, 6-acetylmorphine and morphine effects on threshold for rewarding and aversive brain stimulation. J. Pharmacol. Exp. Ther. 260, 562–567. Inturrisi, C.E., Schultz, M., Shin, S., Umans, J.G., Angel, L., Simon, E.J., 1983. Evidence from opiate binding studies that heroin acts through its metabolites. Life Sci. 33 (Suppl 1), 773–776. Kalivas, P.W., O’Brien, C., 2008. Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacol. Rev. 33, 166–180. http://dx.doi.org/10.1038/sj.npp. 1301564. Koob, G.F., Sanna, P.P., Bloom, F.E., 1998. Neuroscience of addiction. Neuron 21, 467–476. http://dx.doi.org/10.1016/S0896-6273(00)80557-7. Kristensen, K., Christensen, C.B., Christrup, L.L., 1995. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci. 56, PL45–PL50. 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. http://dx.doi.org/10.1111/j.1471-4159. 2007.04738.x. Mahmoud, S., Thorsell, A., Sommer, W.H., Heilig, M., Holgate, J.K., Bartlett, S.E., RuizVelasco, V., 2011. Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine- and fentanyl-mediated modulation of Ca2+ channels in humanized mouse sensory Neurons. Anesthesiology 115, 1054–1062. http://dx.doi. org/10.1097/ALN.0b013e318231fc11. Miller, G.M., Bendor, J., Tiefenbacher, S., Yang, H., Novak, M.A., Madras, B.K., 2004. A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression. Mol. Psychiatry 9, 99–108. http://dx.doi.org/10. 1038/sj.mp.4001378. Nestler, E.J., 2004. Molecular mechanisms of drug addiction. Neuropharmacology 47, 24–32. http://dx.doi.org/10.1016/j.neuropharm.2004.06.031. Oertel, B.G., Doehring, A., Roskam, B., Kettner, M., Hackmann, N., Ferreirós, N., Schmidt,
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Research report
Heroin-induced suppression of saccharin intake in OPRM1 A118G mice ⁎
Christopher S. Freeta, , Danielle N. Alexandera, Caesar G. Imperioa, Victor Ruiz-Velascob, Patricia S. Grigsona a
Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, United States Department of Anesthesiology and Perioperative Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, United States b
A R T I C L E I N F O
A B S T R A C T
Keywords: Addiction Natural rewards Opiates Reward comparison
The single nucleotide polymorphism of the μ-opioid receptor, OPRM1 A118G, has been associated with greater drug and alcohol use, increased sensitivity to pain, and reduced sensitivity to the antinociceptive effects of opiates. In the present studies, we employed a ‘humanized’ mouse model containing the wild-type (118AA) or variant (118GG) allele to examine behavior in a model of heroin-induced devaluation of an otherwise palatable saccharin cue when repeated saccharin-heroin pairings occurred every 24 h (Experiment 1) or every 48 h (Experiment 2). The results showed that, while both the 118AA and 118GG mice demonstrated robust avoidance of the heroin-paired saccharin cue following daily taste-drug pairings, only the 118AA mice suppressed intake of the heroin-paired saccharin cue when 48 h elapsed between each taste-drug pairing. Humanized 118GG mice, then, defend their intake of the sweet cue despite saccharin-heroin pairings and this effect is illuminated by the use of spaced, rather than massed, trials. Given that this pattern of strain difference is not evident with saccharincocaine pairings (Freet et al., 2015), reduced avoidance of the heroin-paired saccharin cue by the 118GG mice may be due to an interaction between the opiate and the subjects’ drive for the sweet or, alternatively, to differential downstream sensitivity to the aversive kappa mediated properties of the drug. These alternative hypotheses are addressed.
1. Introduction According to the National Institute on Drug Abuse (NIDA), abuse of illicit drugs, tobacco, and alcohol costs up to $700 billion annually due to drug-related crime, lost work productivity, and health care costs in the United States (https://www.drugabuse.gov). Substance use disorder (SUD) is, in part, such a costly condition because it is a brain disease characterized by compulsive consumption of drug and chronic relapse, even following extended periods of abstinence. Thus, while initial use of the drug is voluntary, or even prescribed by a physician, drugs of abuse alter gene expression and brain circuitry resulting in behavioral changes that manifest as compulsive drug craving, seeking, and use (Kalivas and O’Brien, 2008; Koob et al., 1998; Nestler, 2004; Robinson and Berridge, 2003). It is now well understood that the development and progression of substance use disorder is determined by both environmental and hereditary factors. One such genetic factor associated with an increased risk for the development of addiction is the single nucleotide polymorphism (SNP) of the human mu (μ) opioid receptor (OPRM1) gene,
A118G (Bond et al., 1998; Miller et al., 2004; Ray and Hutchison, 2007; van den Wildenberg et al., 2007). The OPRM1 A118G SNP is the substitution of the adenine (A) in the wildtype for a guanine (G) in the mutant; this modification results in the replacement of the asparagine for an aspartate residue in the OPRM1 N-terminus and decreases the number of putative glycosylation sites from five to four (Shabalina et al., 2009). Functionally, the A118G variant creates a new CpG-methylation site in the OPRM1 DNA that prevents the upregulation of OPRM1 in response to chronic opioid use (Oertel et al., 2012). In addition, the A118G variant also results in reduced expression of OPRM1 at the cell surface (Kroslak et al., 2007; Zhang et al., 2005) and reduces the accumulation of cAMP in response to morphine, methadone, and DAMGO (Kroslak et al., 2007). In contrast, the endogenous OPRM1 ligand β-endorphin is approximately 3 times more potent and binds approximately 3 times more tightly to OPRM1 with the variant allele compared with the common allele (Bond et al., 1998). Behaviorally, the polymorphism is associated with greater intake of, and responsiveness to, a range of abused drugs including opiates, alcohol, cocaine, cannabis, and nicotine in humans and in non-humans (Haerian and
⁎ Corresponding author at: Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, 500 University Drive, H181, Hershey, PA 17033, United States. E-mail address: [email protected] (C.S. Freet).
http://dx.doi.org/10.1016/j.brainresbull.2017.09.008 Received 31 May 2017; Received in revised form 12 September 2017; Accepted 15 September 2017 0361-9230/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Freet, C.S., Brain Research Bulletin (2017), http://dx.doi.org/10.1016/j.brainresbull.2017.09.008
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to provide purified water and saccharin access. Pipettes were converted to plastic cylinders by removing the tapered ends. A stainless steel spout (Girton Manufacturing Co., Inc.; ST300C) was placed in the bottom of the cylinder, while a silicon stopper (Fisher Scientific; 09-704-1D) sealed the top of the cylinder. Intake of water and saccharin was recorded in 1/10 ml.
Haerian, 2013; Ray and Hutchison, 2007; Schwantes-An et al., 2016; van den Wildenberg et al., 2007; Zhang et al., 2015). In an effort to more closely examine the consequence of this A118G variant in SUD and addiction, a number of studies have employed a ‘humanized’ OPRM1 A118G mouse model in which the native exon 1 of the μ-opioid receptor gene has been replaced with the human exon 1. These mice have been reported to exhibit increased responsiveness to alcohol (Bilbao et al., 2015; Ramchandani et al., 2011), increased nicotine self-administration (Bernardi et al., 2016), and decreased morphine-induced antinociception (Henderson-Redmond et al., 2016; Mahmoud et al., 2011). Likewise, we previously have demonstrated that humanized OPRM1 A118G mice exhibit greater avoidance of a cocaine-paired saccharin cue in an animal model of the devaluation of natural rewards by drugs of abuse (Freet et al., 2015). It is well known that rodents will avoid a lesser reward if it predicts the future availability of a more preferred, intense reward (for review, see Flaherty, 1996) and it has been hypothesized that, with taste-drug pairings (e.g., saccharin and cocaine), avoidance of intake of the lesser saccharin reward cue is, at least in part, due to a decrease in the relative value of the taste cue when compared with the potent drug of abuse that is expected in the near future (Grigson, 2002, 1997; Grigson et al., 2009). Stimulants and depressants, however, are not one in the same and avoidance of a drug-paired cue has been found to differ when paired with cocaine or morphine. For example, reward-preferring Lewis rats demonstrate greater suppression of intake of a cocaine-paired saccharin cue compared with relatively reward-insensitive Fischer rats (Grigson and Freet, 2000); however, the inverse is true (i.e., Lewis rats exhibit lesser suppression) when saccharin is paired with morphine. This strain difference has been linked to greater sensitivity to aversive kappa receptor activation in the Fischer rats (Freet et al., 2013b). The present study will test whether the same is true for the A118G mice vs. the A118A mice. Specifically, we employed humanized mice containing either the wild-type (118AA) or variant (118GG) allele to examine behavior in heroin-induced suppression of intake of a saccharin reward cue when the saccharin-heroin pairings occurred every 24 h (Experiment 1) or every 48 h (Experiment 2). In our experience, massed trials (every 24 h) can lead to more robust suppression of intake of the taste cue than spaced trials.
2.1.3. Procedure: Saccharin-heroin pairings All subjects were weighed once a day throughout the study. For baseline measurements, all mice were placed on a water restriction schedule that consisted of access to purified water for 1 h in the morning (testing period) and unpurified tap water for 2 h in the afternoon (rehydration period). Baseline intake and body weight were recorded for 7 days and experimental groups were determined by matching terminal baseline morning water intake and age. All mice remained on the water restriction schedule as described above for the duration of the experiment. For each taste-drug pairing (which occurred every 24 h), all mice received 0.15% saccharin for the morning access period followed immediately by an i.p. injection of either saline (118AA: n = 14, 118GG: n = 15) or heroin (118AA: n = 15, 118GG: n = 14). The dose of heroin increased over trials (trials 1–7: 30 mg/kg, trials 8–10: 40 mg/kg). After the 10th trial, ad lib water was returned to all mice for four days. Mice were then water deprived overnight and an eleventh conditioning trial was conducted with saccharin access only (no injection). All mice received 2 h access to tap water each afternoon to maintain hydration throughout conditioning. The saccharin solution was presented at room temperature and the heroin was generously provided by the National Institute on Drug Abuse (NIDA). 2.2. Results 2.2.1. Saccharin Intake Saccharin intake was analyzed using a 2 × 2 x 11 repeated measures analyses of variance (ANOVA) varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–11). The main effects of drug, F(1.54) = 73.16, p < 0.0001, and trials, F(10,540) = 4.66, p < 0.0001, were significant. However, the main effect of genotype, F < 1, and the 3-way interaction, F(10,540) = 1.19, p = 0.29, did not reach statistical significance. Separate 2 × 11 ANOVAs were conducted varying drug and trials for each genotype. Although this approach did not allow direct statistical comparison between the genotypes, it allowed us to look more closely at each genotype individually. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(10,270) = 5.79, p < 0.0001, and the 118GG genotype, F(10,270) = 4.60, p < 0.0001. Observation of Fig. 1 shows that, compared to their saccharin-saline controls, both 118AA and 118GG mice in the saccharinheroin condition demonstrated significant suppression of intake of the saccharin cue on trials 2–9, ps < 0.05 when pairings occurred every 24 h.
2. Experiment 1: 24 h saccharin-heroin pairings in OPRM1 A118G mice 2.1. Methods 2.1.1. Subjects The subjects were 58 adult (60–200 days) naïve male 118AA mice (n = 29, mean weight: 35.6 g) and 118GG mice (n = 29, mean weight: 34.1 g). The original breeding pairs were obtained from Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD. The remaining mice were bred in the barrier facility at the Pennsylvania State University College of Medicine, Hershey, PA using a low-intensity monogamous stud mating system. A heterozygous mating scheme was used (AG x AG); back- and intercross lines were maintained and bred into the population. Pups were weaned at 21 days. Tail cuts were performed, and the DNA isolated (DNeasy Blood & Tissue Kit, Qiagen), amplified (GoTaq DNA Poly- merase Kit, Promega), run in gels, extracted (DNA Gel Purification Kit, Qiagen), and sequenced to determine genotype. All subjects were housed individually in standard, clear plastic pan cages in a temperature-controlled (21 °C) animal care facility with a 12:12 h light:dark cycle and mice were maintained with free access to dry Harlan Teklad rodent diet (W) 8604 and water, except where otherwise noted.
2.2.2. Body weight Body weight was analyzed using a 2 × 2 x 10 repeated measures ANOVA varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–10). Although there was a non-significant main effect of genotype, F(1,54) = 1.06, p = 0.31, and a non-significant 3-way interaction, F < 1, confirming that 118AA mice were not significantly heavier than 118GG mice overall, the main effect of drug, F(1,54) = 6.32, p = 0.01, and main effect of trials, F(9486) = 51.42, p < 0.0001, were significant indicating that body weight increased over successive trials for all mice and that heroin exposure decreased body weight independent of genotype (data not shown).
2.1.2. Apparatus All behavioral manipulations were conducted in the home cages. Modified serological pipettes (Fisher Scientific; 07-200-574) were used
2.2.3. PM water intake Intake of water for the 2 h access period in the afternoon for all trials is presented in Fig. 2. Although a 2 × 2 × 9 repeated measures 2
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Fig. 1. Heroin-induced suppression of saccharin intake in humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 24 h (Experiment 1). Mean ( ± SEM) intake (ml/hr) of 0.15% saccharin following pairings with either saline (118AA: open squares; 118GG: open circles) or 30–40 mg/kg heroin (118AA: closed squares; 118GG: closed circles).
3. Experiment 2: 48 h saccharin-heroin pairings in OPRM1 A118G mice
ANOVA did not reveal a significant three-way interaction, F(8432) = 1.78, p = 0.08, or main effect of genotype, F < 1, the main effects of drug, F(1,54) = 40.43, p < 0.0001, and trials, F(8432) = 19.38, p < 0.0001, did attain statistical significance. Separate 2 × 9 ANOVAs were conducted varying drug and trials for each genotype. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(8216) = 2.82, p < 0.01, and 118GG genotype, F(8216) = 8.08, p < 0.0001, showing that heroin treated 118AA and 118GG mice made up for low morning intake by consuming more afternoon water than their saline treated counterparts on trials 2–9 and 4–9, respectively, ps < 0.05.
3.1. Methods 3.1.1. Subjects The subjects were 58 adult (60–200 days) naïve male 118AA mice (n = 28, mean weight: 32.3 g) and 118GG mice (n = 30, mean weight: 32.1 g). The original breeding pairs were obtained from Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD. The remaining mice were bred in the barrier Fig. 2. Afternoon water intake for humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 24 h in Experiment 1. Mean ( ± SEM) intake (ml/2hr) of PM water following saccharinheroin pairings every 24 h. Saccharin-saline groups (118AA: open squares; 118GG: open circles); Saccharin-heroin groups (118AA: closed squares; 118GG: closed circles).
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Fig. 3. Heroin-induced suppression of saccharin intake in humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 48 h (Experiment 2). Mean ( ± SEM) intake (ml/hr) of 0.15% saccharin following pairings with either saline (118AA: open squares; 118GG: open circles) or 30–40 mg/kg heroin (118AA: closed squares; 118GG: closed circles).
effect of genotype, separate 2 × 9 ANOVAs were conducted varying drug and trials for each genotype. Post hoc Newman-Keuls tests (alpha = 0.05) were conducted on the significant drug x trials interaction for the 118AA genotype, F(8208) = 7.05, p < 0.001, showing that the 118AA mice in the saccharin-heroin group demonstrated significant suppression of saccharin intake on trials 2–9 compared to their saccharin-saline treated controls, ps < 0.05 (left panel). For the 118GG mice, on the other hand, neither the main effect of drug, F (1.27) = 1.45, p = 0.24, nor the drug x trials interaction attained statistical significance, though the drug x trials interaction was close, F (8216) = 1.95, p = 0.053. Unlike the 118AA mice, then, the 118GG mice failed to demonstrate significant suppression of saccharin intake when saccharin-heroin pairings occurred at 48 h intervals.
facility at the Pennsylvania State University College of Medicine, Hershey, PA as described in Experiment 1. All subjects were housed and maintained as described above. 3.1.2. Apparatus Behavioral manipulations and apparatuses were identical to that described in Experiment 1. 3.1.3. Procedure All subjects were weighed once a day throughout the study and adapted to the water restriction schedule as described in Experiment 1. For each taste-drug pairing (which occurred every 48 h), all mice received 0.15% saccharin for the morning access period followed immediately by an i.p. injection of either saline (118AA: n = 14, 118GG: n = 15) or heroin (118AA: n = 14, 118GG: n = 15). The dose of heroin increased over trials (trials 1–3: 30 mg/kg, trials 4–6: 35 mg/kg, trials 7–9: 40 mg/kg). Thus, taste-drug pairings occurred every other morning (0.5 h into the light phase) for a total of 9 trials. All mice received 1 h access to purified water in the mornings between conditioning trials and 2 h access to purified water each afternoon to maintain hydration for all conditioning and non-conditioning trials. The saccharin solution was presented at room temperature and NIDA generously provided the heroin.
3.2.2. Body weight Body weight was analyzed using a 2 × 2 x 19 repeated measures ANOVA varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–10, W1-W9). Drug exposure did not significantly alter body weight (data not shown). This conclusion was supported by a nonsignificant main effect of drug, F < 1, and a non-significant 3-way interaction, F(18,972) = 1.27, p = 0.20. The main effect of trials was significant, F(18,972) = 20.93, p < 0.0001, indicating that body weight increased over successive trials for all mice. The main effect of genotype also did not attain statistical significance, F < 1, confirming no differences in body weight between the 118AA and the 118GG mice overall (data not shown).
3.1.3.1. Two-bottle intake test. A 10th trial occurred 48 h after the last taste-drug pairing where all mice were tested for saccharin preference using a two-bottle intake test. All mice simultaneously received a bottle of 0.15% saccharin and a bottle of purified water for 1 h. The left/right position of bottles was counter-balanced across groups.
3.2.3. AM water intake Morning intake of water (ml/h) on the days between conditioning trials (trials W1-W9) was evaluated. A 2 × 2 × 9 repeated measures ANOVA revealed that drug exposure did not significantly alter morning water intake as a function of genotype, as indicated by a non-significant main effect of genotype, F < 1, genotype x drug interaction, F < 1, and genotype x drug x trials interaction, F < 1. The main effect of drug did not reach statistical significance, F < 1. The main effect of trials was significant, F(8432) = 10.53, p < 0.0001, but follow-up post hoc tests revealed no meaningful differences, ps > 0.05 (data not shown).
3.2. Results 3.2.1. Saccharin intake Saccharin intake was analyzed using a 2 × 2 x 9 repeated measures analyses of variance (ANOVA) varying genotype (118AA or 118GG), drug (saline or heroin), and trials (1–9). The main effects of genotype, F (1,53) = 4.77, p = 0.03, drug, F(1,53) = 8.03, p < 0.01, and trials, F (8424) = 6.68, p < 0.01, were significant. However, the three-way interaction did not reach significance, F < 1. Given the robust main 4
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Fig. 4. Afternoon water intake for humanized 118AA and 118GG mice when saccharin-heroin pairings occurred every 48 h in Experiment 2. Mean ( ± SEM) intake (ml/2hr) of PM water following 48 h saccharinheroin pairings. Saccharin-saline groups (118AA: open squares; 118GG: open circles); Saccharin-heroin groups (118AA: closed squares; 118GG: closed circles).
heroin-induced suppression of saccharin intake, though avoidance of the heroin-paired cue was less marked in the 118AA mice when pairings occurred at 48 h vs. 24 h intervals. For the 118GG mice, on the other hand, no significant suppression was observed (Fig. 3). Despite the differential suppression of saccharin intake, however, both genotypes compensated by increasing intake of afternoon water on heroin injections days and the two-bottle saccharin preference, evidenced by saline-treated 118AA and 118GG mice, was eliminated in both genotypes following a history of saccharin-heroin pairings (Fig. 5). This finding suggests that, while heroin-induced suppression of saccharin intake may have been reduced in the 118GG vs. the 118AA mice, it was not eliminated, as evidenced by the two-bottle challenge. This pattern, i.e., less drug-induced avoidance by the more drugpreferring strain, is not unprecedented. For example, drug-preferring C57BL/6J mice exhibit less cocaine-induced suppression of saccharin intake than less drug-preferring DBA/2J mice (Freet et al., 2013a). Likewise, drug-preferring transgenic NSE-tTA x TetOp-ΔFosB mice demonstrate less cocaine-induced suppression of saccharin intake than their non ΔFosB over-expressing controls (Freet et al., 2009). In the case of the mice, both drug-preferring strains (C57Bl/6J and transgenic NSEtTA x TetOp-ΔFosB mice) also exhibit a greater preference for saccharin over water in a two-bottle test. This finding led us to conclude that these subjects were not merely drug-preferring, but actually were reward preferring (‘reward generalists’), allowing their drive for the sweet to override the suppressive effects of the drug. Here, however, while the saline-treated 118GG mice tend to exhibit a stronger preference for saccharin vs. water compared to the saline-treated 118AA mice, this effect was not statistically significant. Indeed, we might argue that the pattern of data in the 118AA vs. 118GG mice most closely parallels that obtained in the Fischer vs. Lewis rats. Lewis rats, like the 118GG mice, exhibit less avoidance of a saccharin cue when paired with an opiate (i.e., morphine) than Fischer rats (Freet et al., 2013b), but greater avoidance of a saccharin cue when paired with cocaine (Glowa et al., 1994; Grigson and Freet, 2000). Likewise, the humanized 118GG mice also demonstrate lesser avoidance of the opiate-paired saccharin solution (show above), but greater avoidance of a saccharin cue when paired with cocaine than their 118AA counterparts (Freet et al., 2015). But, why this dissociation
3.2.4. PM water intake Intake of water for the 2 h access period in the afternoon for all trials is presented in Fig. 4. Although a 2 × 2 x 18 repeated measures ANOVA did not reveal a statistically significant three-way interaction, F < 1, or main effect of genotype, F(1,54) = 1.54, p = 0.22, the main effects of drug, F(1,54) = 33.25, p < 0.0001, and trials, F(24, 1296) = 16.61, p < 0.0001, did attain statistical significance, as did the drug x trials interaction, F(24, 1296) = 7.45, p < 0.001. Separate 2 × 18 ANOVAs were conducted varying drug and trials for each genotype. A post hoc Newman-Keuls test (alpha = 0.05) was conducted on the significant drug x trials interaction for the 118AA genotype, F(17,442) = 3.78, p < 0.0001, and 118GG genotype, F(17,476) = 3.54, p < 0.0001, confirming, once again, that lower morning intake by mice in the saccharin-heroin condition is followed by greater afternoon water intake on trials 5–9 in the 118AA mice, and on trials 3, 4, 6–9 in the 118GG mice, ps < 0.05, compared to their saccharin-saline controls. 3.2.5. Two-bottle intake test The results of the two-bottle intake test for saccharin against water are presented in Fig. 5. Preference for saccharin was analyzed using t tests for dependent samples with alpha = 0.05 for each cell. The results confirmed that both 118AA (t = 2.70, p = 0.02) and 118GG (t = 4.60, p < 0.001) mice preferred saccharin over water under the saline condition, while neither genotype preferred saccharin over water when tested following saccharin-heroin pairings (118AA: t = −1.69, p = 0.11; 118GG: t = −0.19, p = 0.85). 4. Discussion The data from Experiment 1 demonstrate that both 118AA and 118GG mice exhibit robust heroin-induced suppression of saccharin intake when the saccharin-heroin pairings occurred every 24 h (Fig. 1). As there is a fair amount of literature demonstrating differential responding between these genotypes to drugs of abuse, Experiment 2 was conducted to unmask any potential differences by spacing out the tastedrug pairings (48 h). The results of Experiment 2 demonstrated that, under the 48 h condition, 118AA mice continued to exhibit robust 5
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Fig. 5. Two-bottle intake test for 0.15% saccharin and water following 48 h saccharin-saline or saccharin-heroin pairings in humanized 118AA and 118GG mice. Saccharin-saline groups (saccharin: solid; water: diagonal lines); Saccharin-heroin groups (saccharin: dense crossed lines; water: horizontal lines).
Conflict of interest
between an opiate and cocaine? A follow up study showed greater avoidance of a saccharin cue that had been paired with spiradoline (a kappa agonist) in the Fischer vs. Lewis rats (Freet et al., 2013b). Fischer rats, then, may exhibit greater avoidance of a morphine-paired saccharin cue because they are more sensitive than the Lewis rats to the aversive kappa agonist properties of the opiate. While heroin does not directly activate opioid receptors (Inturrisi et al., 1983), it acts as a lipid soluble prodrug for the central distribution of morphine (Hubner and Kornetsky, 1992) and rapidly metabolizes to 6-monoacetylmorphine and morphine; both metabolites strongly bind to μ-opioid receptors (Inturrisi et al., 1983; Selley et al., 2001), but also bind with δ-receptors and with κ-opioid receptors (Kristensen et al., 1995; Rady et al., 1994). We might speculate that a similar kappa sensitivity may be evidenced in 118AA vs. 118GG mice, but this would seem a stretch. Presumably, the humanized A118G mice differ only in their μ-opioid receptor; κ- and δreceptors should be the same (Browne et al., 2017). Even so, there is evidence that carrying the A118G allele in humans whom have overdosed on heroin is associated with downstream 2-fold higher levels of dynorphin A in the caudate nucleus and reduced preprodynorphin mRNA in the nucleus accumbens core (Drakenberg et al., 2006). As such, an assessment of the impact of carrying the A118G allele on kappa sensitivity should be examined in this paradigm. In sum, both the 118AA and the 118GG mice exhibit robust avoidance of an otherwise palatable saccharin solution when it predicts the administration of heroin via once daily pairings. The magnitude of this effect is reduced in the 118AA mice, and eliminated in the 118GG mice, when the saccharin-heroin trials are spaced at 48 h, rather than 24 h, intervals. That being said, when assessed using the two-bottle test (where a water alternative is provided), conditioned avoidance of the heroin-paired saccharin cue is revealed in both the 118AA and the 118GG strain of mice. Attenuated avoidance of the heroin-paired saccharin cue (at least in the one-bottle test across trials) by the 118GG mice may be due to an interaction between the opiate and the individuals’ drive for the sweet (it has long been known that opiates can increase responding for sweets (Zhang and Kelley, 1997), or due to differential downstream sensitivity to the aversive kappa mediated properties of the opiate. Further studies will need to be designed to test the merits of these two hypotheses.
None. Competing interests None. Acknowledgements This research was supported by PHS grants DA024519 and DA009815 to PSG and DA025574 to VR-V. We thank Dr. Markus Heilig at the National Institutes on Alcoholism and Alcohol Abuse (NIAAA), Bethesda, MD, for the original 118AA and 118GG breeding pairs. References Bernardi, R.E., Zohsel, K., Hirth, N., Treutlein, J., Heilig, M., Laucht, M., Spanagel, R., Sommer, W.H., 2016. A gene-by-sex interaction for nicotine reward: evidence from humanized mice and epidemiology. Transl. Psychiatry 6, e861. http://dx.doi.org/10. 1038/tp.2016.132. Bilbao, A., Robinson, J.E., Heilig, M., Malanga, C.J., Spanagel, R., Sommer, W.H., Thorsell, A., 2015. A Pharmacogenetic Determinant of Mu-Opioid Receptor Antagonist Effects on Alcohol Reward and Consumption: Evidence from Humanized Mice. Biol. Psychiatry 77, 850–858. http://dx.doi.org/10.1016/j.biopsych.2014.08. 021. 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. U. S. A. 95, 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. http://dx.doi.org/10.1016/j. neuropharm.2017.02.005. Drakenberg, K., Nikoshkov, A., Horvath, M.C., Fagergren, P., Gharibyan, A., Saarelainen, K., Rahman, S., Nylander, I., Bakalkin, G., Rajs, J., Keller, E., Hurd, Y.L., 2006. Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers. Proc. Natl. Acad. Sci. U. S. A. 103, 7883–7888. http://dx.doi.org/10.1073/pnas.0600871103. Flaherty, C.F., 1996. Incentive relativity. Problems in the Behavioural Sciences. Cambridge University Press, New York. Freet, C.S., Steffen, C., Nestler, E.J., Grigson, P.S., 2009. Overexpression of ⌬FosB is associated with attenuated cocaine-Induced suppression of saccharin intake in mice. Behav. Neurosci. 123, 397–407. http://dx.doi.org/10.1037/a0015033. Freet, C.S., Arndt, A., Grigson, P.S., 2013a. Compared with DBA/2J mice, C57BL/6J mice demonstrate greater preference for saccharin and less avoidance of a cocaine-paired saccharin cue. Behav. Neurosci. 127, 474–484.
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C.S. Freet et al.
P.H., Lötsch, J., 2012. Genetic-epigenetic interaction modulates μ-opioid receptor regulation. Hum. Mol. Genet. 21, 4751–4760. http://dx.doi.org/10.1093/hmg/ dds314. Rady, J.J., Aksu, F., Fujimoto, J.M., 1994. The heroin metabolite, 6-monoacetylmorphine, activates delta opioid receptors to produce antinociception in Swiss-Webster mice. J. Pharmacol. Exp. Ther. 268, 1222–1231. 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. http://dx.doi.org/10.1038/mp.2010.56. Ray, L.A., Hutchison, K.E., 2007. Effects of naltrexone on alcohol sensitivity and genetic moderators of medication response. Arch. Gen. Psychiatry 64, 1069. http://dx.doi. org/10.1001/archpsyc.64.9.1069. Robinson, T.E., Berridge, K.C., 2003. Addiction. Annu. Rev. Psychol. 54, 25–53. http:// dx.doi.org/10.1146/annurev.psych.54.101601.145237. 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., Andrews, J.A., Aveyard, P., Beltcheva, O., Brown, S.A., Cannon, D.S., Cichon, S., Corley, R.P., Dahmen, N., Degenhardt, L., Foroud, T., Gaebel, W., Giegling, I., Glatt, S.J., Grucza, R.A., Hardin, J., Hartmann, A.M., Heath, A.C., Herms, S., Hodgkinson, C.A., Hoffmann, P., Hops, H., Huizinga, D., Ising, M., Johnson, E.O., Johnstone, E., Kaneva, R.P., Kendler, K.S., Kiefer, F., Kranzler, H.R., Krauter, K.S., Levran, O., Lucae, S., Lynskey, M.T., Maier, W., Mann, K., Martin, N.G., Mattheisen, M., Montgomery, G.W., Müller-Myhsok, B., Murphy, M.F., Neale, M.C., Nikolov, M.A., Nishita, D., Nöthen, M.M., Nurnberger, J., Partonen, T., Pergadia, M.L., Reynolds, M., Ridinger, M., Rose, R.J., Rouvinen-Lagerström, N., Scherbaum, N., Schmäl, C., Soyka, M., Stallings, M.C., Steffens, M., Treutlein, J., Tsuang, M., Wall, T.L., Wodarz, N., Yuferov, V., Zill, P., Bergen, A.W., Chen, J., Cinciripini, P.M., Edenberg, H.J., Ehringer, M.A., Ferrell, R.E., Gelernter, J., Goldman, D., Hewitt, J.K., Hopfer, C.J., Iacono, W.G., Kaprio, J., Kreek, M.J., Kremensky, I.M., Madden, P.A.F., McGue, M., Munafò, M.R., Philibert, R.A., et al., 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. http://dx.doi.org/10.1007/s10519-015-9737-3. Selley, D.E., Cao, C.C., Sexton, T., Schwegel, J.A., Martin, T.J., Childers, S.R., 2001. mu opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine. Biochem. Pharmacol. 62, 447–455. Shabalina, S.A., Zaykin, D.V., Gris, P., Ogurtsov, A.Y., Gauthier, J., Shibata, K., Tchivileva, I.E., Belfer, I., Mishra, B., Kiselycznyk, C., Wallace, M.R., Staud, R., Spiridonov, N.A., Max, M.B., Goldman, D., Fillingim, R.B., Maixner, W., Diatchenko, L., 2009. Expansion of the human mu-opioid receptor gene architecture: novel functional variants. Hum. Mol. Genet. 18, 1037–1051. http://dx.doi.org/10.1093/ hmg/ddn439. Zhang, M., Kelley, A.E., 1997. Opiate agonists microinjected into the nucleus accumbens enhance sucrose drinking in rats. Psychopharmacol. (Berl). 132, 350–360. Zhang, Y., Wang, D., Johnson, A.D., Papp, A.C., Sadee, W., 2005. Allelic expression imbalance of human mu opioid receptor (OPRM1) caused by variant A118G. J. Biol. Chem. 280, 32618–32624. http://dx.doi.org/10.1074/jbc.M504942200. 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 behavior compared with wild-type mice. Neuropsychopharmacology 40, 1091–1100. http://dx.doi.org/10.1038/npp.2014.286. van den Wildenberg, E., Wiers, R.W., Dessers, J., Janssen, R.G.J.H., Lambrichs, E.H., Smeets, H.J.M., van Breukelen, G.J.P., 2007. A functional polymorphism of the muopioid receptor gene (OPRM1) influences cue-induced craving for alcohol in male heavy drinkers. Alcohol. Clin. Exp. Res. 31, 1–10. http://dx.doi.org/10.1111/j.15300277.2006.00258.x.
Freet, C.S., Wheeler, R.A., Leuenberger, E., Mosblech, N.A.S., Grigson, P.S., 2013b. Fischer rats are more sensitive than Lewis rats to the suppressive effects of morphine and the aversive kappa-opioid agonist spiradoline. Behav. Neurosci. 127, 763–770. Freet, C.S., Ballard, S.M., Alexander, D.N., Cox, T.A., Imperio, C.G., Anosike, N., Carter, A.B., Mahmoud, S., Ruiz-Velasco, V., Grigson, P.S., 2015. Cocaine-induced suppression of saccharin intake and morphine modulation of Ca2+ channel currents in sensory neurons of OPRM1 A118G mice. Physiol. Behav. 139, 216–223. http://dx. doi.org/10.1016/j.physbeh.2014.11.040. Glowa, J.R., Shaw, A.E., Riley, A.L., 1994. Psychopharmacology Cocaine-induced conditioned taste aversions: comparisons between effects in LEW/N and F344/N rat strains. Psychopharmacol 114, 229–232. Grigson, P.S., Freet, C.S., 2000. The suppressive effects of sucrose and cocaine, but not lithium chloride, are greater in lewis than in fischer rats: evidence for the reward comparison hypothesis. Behav. Neurosci. 114, 353–363. http://dx.doi.org/10. 1037//0735-7044.114.2.353. Grigson, P.S., Twining, R.C., Freet, C.S., Wheeler, R.A., Geddes, R.I., 2009. Drug-induced suppression of conditioned stimulus intake: reward, aversion, and addiction. In: Reilly, S., Schachtman, T.R. (Eds.), Conditioned Taste Aversion: Behavioral and Neural Processes. Oxford University Press, New York, pp. 74–91. Grigson, P.S., 1997. Conditioned taste aversions and drugs of abuse: a reinterpretation. Behav. Neurosci. 111, 129–136. http://dx.doi.org/10.1037/0735-7044.111.1.129. Grigson, P.S., 2002. Like drugs for chocolate: separate rewards modulated by common mechanisms? Physiol. Behav. 76, 389–395. Haerian, B.S., Haerian, M.S., 2013. OPRM1 rs1799971 polymorphism and opioid dependence: evidence from a meta-analysis. Pharmacogenomics 14, 813–824. http:// dx.doi.org/10.2217/pgs.13.57. Henderson-Redmond, A.N., Yuill, M.B., Lowe, T.E., Kline, A.M., Zee, M.L., Guindon, J., Morgan, D.J., 2016. Morphine-induced antinociception and reward in humanized mice expressing the mu opioid receptor A118G polymorphism. Brain Res. Bull. 123, 5–12. http://dx.doi.org/10.1016/j.brainresbull.2015.10.007. Hubner, C.B., Kornetsky, C., 1992. Heroin, 6-acetylmorphine and morphine effects on threshold for rewarding and aversive brain stimulation. J. Pharmacol. Exp. Ther. 260, 562–567. Inturrisi, C.E., Schultz, M., Shin, S., Umans, J.G., Angel, L., Simon, E.J., 1983. Evidence from opiate binding studies that heroin acts through its metabolites. Life Sci. 33 (Suppl 1), 773–776. Kalivas, P.W., O’Brien, C., 2008. Drug addiction as a pathology of staged neuroplasticity. Neuropsychopharmacol. Rev. 33, 166–180. http://dx.doi.org/10.1038/sj.npp. 1301564. Koob, G.F., Sanna, P.P., Bloom, F.E., 1998. Neuroscience of addiction. Neuron 21, 467–476. http://dx.doi.org/10.1016/S0896-6273(00)80557-7. Kristensen, K., Christensen, C.B., Christrup, L.L., 1995. The mu1, mu2, delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci. 56, PL45–PL50. 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. http://dx.doi.org/10.1111/j.1471-4159. 2007.04738.x. Mahmoud, S., Thorsell, A., Sommer, W.H., Heilig, M., Holgate, J.K., Bartlett, S.E., RuizVelasco, V., 2011. Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine- and fentanyl-mediated modulation of Ca2+ channels in humanized mouse sensory Neurons. Anesthesiology 115, 1054–1062. http://dx.doi. org/10.1097/ALN.0b013e318231fc11. Miller, G.M., Bendor, J., Tiefenbacher, S., Yang, H., Novak, M.A., Madras, B.K., 2004. A mu-opioid receptor single nucleotide polymorphism in rhesus monkey: association with stress response and aggression. Mol. Psychiatry 9, 99–108. http://dx.doi.org/10. 1038/sj.mp.4001378. Nestler, E.J., 2004. Molecular mechanisms of drug addiction. Neuropharmacology 47, 24–32. http://dx.doi.org/10.1016/j.neuropharm.2004.06.031. Oertel, B.G., Doehring, A., Roskam, B., Kettner, M., Hackmann, N., Ferreirós, N., Schmidt,
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