Cues predicting drug or food reward rescue morphine place conditioning in mice lacking delta opioid receptors
Julie Le Merrer, Lauren Faget, Audrey Matifas, Brigitte L. Kieffer
Supplementary methods
Somatic signs of morphine withdrawal
In this experiment, we evaluated whether impaired place preference conditioning to morphine in mice lacking the delta opioid receptors (Oprd1-/-) under certain conditions could result from altered sensitivity to morphine-induced physical dependence. Mice were injected i.p. twice daily with saline or morphine at 30 mg/kg over 5 days. On day 6 in the morning, a last single administration of morphine (30 mg/kg) was performed. Two hours after this last injection, mice were placed in rectangular clear plastic observation boxes (16x16x30 cm) under a 50 lux indirect lighting and observed for 5 min. Withdrawal was then precipitated by an injection of the opioid antagonist naloxone (1 mg/kg, s.c.). Somatic signs of withdrawal were evaluated immediately after antagonist injection during a period of 20 min. The number of head shakes and wet dog shakes, front paw tremors, scratches, jumps and sniffing episodes was counted. Body tremor, ptosis, mastication, and piloerection were scored 1 for appearance or 0 for nonappearance within 5 min bins. Locomotor activity over 5 min periods was rated from 0 for inactivity to 2 for increased activity. A quantitative value was calculated in each animal for the different checked signs by adding the scores obtained in each 5 min time period. A global withdrawal score was calculated for each animal by giving to each individual sign a relative weight (Matthes et al. 1996): 1 for the appearance of each checked sign in each 5 min time period and 0.5 for each bout of counted sign.
Drugs
Morphine hydrochloride (Francopia, Sanofi Synthelabo Laboratories, Paris, France) and naloxone hydrochloride (Natick, MA, USA) were dissolved in sterile isotonic saline (NaCl 0.9%). Both drugs were administered in a volume of 10 ml/kg. Doses refer to salt weight.
Data analyses and statistics
Somatic signs of withdrawal were analyzed using a three-way ANOVA with gender, genotype and treatment (chronic morphine versus chronic saline) as between-group factors. Following significant interactions, post-hoc analyses were performed using a Newman-Keules test where appropriate. Statistical significance was set at p < 0.05 for all tests.
Supplementary results
Preserved sensitivity to morphine-induced physical dependence in Oprd1-/- mice
Somatic signs of withdrawal to morphine were assessed in mice lacking the delta opioid receptors and their wild-type (WT) counterparts. As shown in Supplementary Figure 1, horizontal activity was higher in Oprd1-/- mice as compared to WT controls, as described previously (Filliol et al. 2000); however administering the antagonist of opioid receptors naloxone (1mg/kg) had no effect on this parameter (gender effect: F1,31=0.10, NS; genotype effect: F1,31=4.94; p<0.005; treatment effect F1,31=0.66, NS). In contrast, naloxone induced a significant expression of somatic signs of withdrawal in WT and mutant mice chronically treated with morphine (30 mg/kg), but not in vehicle control groups (see Figure 2 and Supplementary Table 1). Notably, major signs such as jumps (gender effect: F1,31=1.09, NS; genotype effect: F1,31=0.28; NS; treatment effect F1,31=14.33, p<0.001), front paw tremors (gender effect: F1,31=0.65, NS; genotype effect: F1,31=8.18; p<0.01; treatment effect F1,31=9.67, p<0.01), wet dog shakes (gender effect: F1,31=2.94, NS; genotype effect: F1,31=0.26; NS; treatment effect F1,31=4.95, p<0.05) and episodes of sniffing (gender effect: F1,31=4.87, p<0.05; genotype effect: F1,31=0.50; NS; treatment effect F1,31=9.40, p<0.01) were significantly more frequent in morphine treated mice as compared to controls. The expression of ptosis (gender effect: F1,31=0.06, NS; genotype effect: F1,31=10.78; p<0.01; treatment effect F1,31=15.82, p<0.001), piloerection (gender effect: F1,31=3.02, NS; genotype effect: F1,31=0.36; NS; treatment effect F1,31=65.28, p<0.0001) and mastication (gender effect: F1,31=1.96, NS; genotype effect: F1,31=3.02; NS; treatment effect F1,31=47.36, p<0.0001) was also increased in both lines after withdrawal from chronic morphine. Mutant mice displayed more paw tremors and less ptosis than WT animals, consistent with higher levels of anxiety in Oprd1-/- mice (Filliol et al. 2000); however naloxone-precipitated withdrawal modified these signs similarly in both mouse lines (genotype x treatment interaction; paw tremors: F1,31=1.52, NS; ptosis: F1,31=0.96, NS). Finally, a global withdrawal score was increased in mutant and WT animals chronically treated with morphine, with slightly higher basal levels in Oprd1-/- mice (gender effect: F1,31=0.49, NS; genotype effect: F1,31=5.43; p<0.05; treatment effect F1,31=29.02, p<0.0001; genotype x treatment interaction: F1,31=0.34, NS). These results indicate that sensitivity to morphine-induced physical dependence is not diminished in Oprd1-/- mice.
Supplementary Figure 1. Sensitivity to morphine-induced physical dependence is preserved in mice lacking delta opioid receptors. The incidence of selected somatic signs of withdrawal and activity and global withdrawal scores (see Supplementary Methods for calculation) are presented as mean (± SEM) (n=3-5 per gender, genotype and dose). Solid star: comparison to vehicle group (one-way analysis of variance); open star: comparison between genotypes (four-way analysis of variance). One symbol: p<0.05; two symbols: p<0.01; three symbols: p<0.001.
References
Filliol D, Ghozland S, Chluba J, Martin M, Matthes HW, Simonin F, Befort K, Gaveriaux-Ruff C, Dierich A, LeMeur M, Valverde O, Maldonado R, Kieffer BL (2000) Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat Genet 25: 195-200
Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383: 819-23