Figure 3. Gpr88 null mice display modified mu-opioid mediated behavioral responses.

(A) In a locomotor sensitization paradigm (n = 5 to 11 mice per treatment and genotype) morphine induced an increase in locomotor activity that sensitized upon repeated administration in Gpr88^+/+ and Gpr88^-/- mice (panel a); morphine-induced locomotion and sensitization, however, were significantly greater in mutant mice (panel b) (Genotype effect: F_1,27=13.5, p=0.0010; Treatment: F_1,27=99.9, p=0.0000; Genotype x Treatment interaction: F_1,27=14.6, p=0.0007; Day: F_4,108=7.9, p=0.0000; Day x Treatment: F_4,108=10.2, p=0.0000; Day x Genotype x Treatment = 2.9, p=0.0253); solid stars: treatment effect (two-way ANOVA with one repeated measure – day), asterisks: genotype effect, dagger: Day x Genotype x Treatment interaction. (B) Upon exposure to escalating doses of morphine (n = 6 per treatment and genotype), Gpr88^-/- lost more body weight than controls (panel a; Treatment: F_1,20=43.7, p=0.0000; Day: F_4,80=13.6, p=0.0000; Day x Genotype: F_4,80=5.1, p=0.001; Day x Treatment: F_4,80=9.9, p=0.0000; body weight was measured daily upon morphine treatment); when withdrawal was triggered by acute naloxone administration (1 mg/kg), mutant animals displayed sniffing episodes (panel b; Genotype/Treatment: F_1,20=25.5, p=0.0000; Genotype x Treatment: F_1,20=23.1, p=0.0001; Time: F_3,60=10.5, p=0.0000; Time x Genotype/Treatment: F_3,60=9.3, p=0.0000; Time x Genotype x Treatment: F_3,60=8.3, p=0.0001) and vegetative signs of withdrawal (panel c; Genotype: F_1,20=5.3, p=0.0324; Treatment: F_1,20=5.3, p=0.0324; Time: F_3,60=14.4, p=0.0000) quicker than their Gpr88^+/+ counterparts, despite similar final withdrawal scores (panel d; Treatment: F_1,20=38.9, p=0.0000); solid stars: treatment effect (two-way ANOVA with one repeated measure – day/5 min time bin), double daggers: Time x Genotype interaction, daggers: Time x Genotype x Treatment interaction, asterisk: genotype effect. More withdrawal signs are displayed in Figure 3—figure supplement 1. (C) In a CPP paradigm (n = 8 per treatment and genotype), Gpr88^-/-mice acquired preference for a compartment associated to morphine administration (10 mg/Kg) similarly as Gpr88^+/+ animals (panel a; Treatment: F_1,28=45.8, p=0.0000; Conditioning: F_1,28=15.7, p=0.0005; Conditioning x Treatment: F_1,28=31.1, p=0.0000); they extinguished this conditioning quicker than wild-type counterparts (panel b; Genotype: F_1,28=10.3, p=0034; Treatment: F_1,28=6.5, p=0166; Genotype x Treatment: F_1,28=5.0, p=0329; Session: F_9,252=5.2, p=0.0000; Session x Treatment: F_9,252=3.7, p=0.0002) and finally reinstated morphine place preference at comparable levels as the latter (panel c; Treatment: F_1,28=9.2, p=0.0052); solid stars: Treatment effect (two-way ANOVA with one repeated measure – day/5 min time bins); asterisks: Genotype effect. (D) In the tail flick test (n = 7–9 per treatment and genotype), Gpr88^-/- mice were significantly less sensitive to morphine analgesia at 48°C and 52°C (Genotype: F_1,83=22.0, p=0.0000; Treatment: F_1,83=84.9, p=0.0000; Temperature: F_2,83=162.5, p=0.0000; Genotype x Treatment: F_1,83=15.9, p=0.0001; Treatment x Temperature: F_2,83=5.4, p=0.0065); (E) in the hot plate test (n = 15–16 per treatment and genotype), morphine-induced analgesia was detected by increased jumping latency in treated animals (right panel); this effect was increased in Gpr88 null mice versus controls (Treatment: F_1,59=79.7, p=0.0000; Genotype x Treatment: F_1,59=4.0, p=0.0490); solid stars: Treatment effect (two-way ANOVA), asterisks: Genotype x Treatment interaction (Newman Keules post-hoc test). Increased morphine-induced locomotor sensitization in Gpr88 null mice was not associated with modified pERK/tERK ratio in three brain regions (Figure 3—figure supplements 2 and 3). (F) We administered the GPR88 agonist Compound 19 (icv, 10 nmoles) to mice exposed to a morphine-induced locomotor sensitization paradigm (n = 8 to 9 mice per treatment condition). Exposure to morphine (IP, 10 mg/kg) induced an increase in locomotor activity that sensitized upon repeated administration in both vehicle and Compound 19-treated groups (panel a); pharmacological activation of GPR88 drastically reduced morphine-induced locomotor activity but left the amplitude of sensitization unchanged (Morphine effect: F_1,30=305.1, p=0.0000; Compound 19: F_1,30=55.3, p=0.0000; Day: F_2,60=33.1, p=0.0000; Day x Morphine: F_2,60=31.6, p=0.0000; Day x Morphine x Compound 19: F_2,60=2.1, NS), solid stars: treatment effect (two-way ANOVA with one repeated measure – day), asterisks: genotype effect. Data are presented as mean ± SEM. One symbol: p<0.05, two symbols: p<0.01, three symbols: p<0.001.

Figure 3—figure supplement 1. Additional morphine-induced withdrawal signs in Gpr88^-/- versus Gpr88^+/+ mice.

After an exposure to escalating doses of morphine, Gpr88^+/+ and Gpr88^-/- mice (n = 6 per treatment and genotype) received an acute injection of naloxone (1 mg/kg) and withdrawal signs were quoted for 20 min. Gpr88 null animals and wild-type controls displayed similar numbers of jumps (panel a; Genotype: F_1,20=0.02, p=0.9016; Treatment: F_1,20=5.9, p=0.0246), wet dog shakes (panel b; Genotype: F_1,20=2.4, p=0.1383; Treatment: F_1,20=2.6, p=0.1222) and paw tremors (panel c; Genotype: F_1,20=1.8, p=0.1913; Treatment: F_1,20=9.0, p=0.0070; Time: F_3,60=5.42, p=0.0023; Time x Treatment: F_3,60=4.7, p=0054). Data are presented as mean ± SEM. Solid stars: treatment effect (two-way ANOVA with one repeated measure – day/5 min time bin), one symbol: p<0.05, two symbols: p<0.01; three symbols: p<0.001.

Figure 3—figure supplement 2. Increased morphine-induced locomotor sensitization in Gpr88 null mice was not associated with modified pERK/tERK ratio in three brain regions.

(A) In a two-day locomotor sensitization paradigm (n = 8 mice per treatment and genotype), morphine (5 mg/kg) induced an increase in locomotor activity (Day 1; Genotype: F_1,28=0.83, p=0.3690; Treatment: F_1,28=63.4, p=0.0000; Time: F_17,176=11.1, p=0.0000) that sensitized after a second administration (Day 2) in Gpr88^-/- but not Gpr88^+/+ mice (right panel; Genotype: F_1,28=78.2, p=0.0000; Treatment: F_1,28=365.5, p=0.0000; Genotype x Treatment: F_1,28=58.9, p=0.0000; Time: F_8,224=76.8, p=0.0000; Time x Genotype: F_8,224=7.0, p=0.0000; Time x Treatment: F_8,224=139.9, p=0.0000; Time x Genotype x Treatment: F_8,224=13.13, p=0.0000). Data are presented as mean ± SEM. Open stars: Time x Treatment interaction, asterisks: Genotype x Treatment (three-way ANOVA with one repeated measure – time bin), three symbols: p<0.001. (B) The mice were sacrificed 60 min after the second morphine challenge for western blot analysis. Upper panels: in contrast with in vitro results, no difference was observed in the pERK/tERK ratio between Gpr88^-/- and Gpr88^+/+ animals in either striatal regions (caudate putamen: Genotype: F_1,25=0.10, p=0.7516; Treatment: F_1,25=0.28, p=0.5978; nucleus accumbens: Genotype: F_1,24=1.4, p=0.2556; Treatment: F_1,24=0.18, p=0.6719), where µOR activation induces morphine sensitization, or in a µOR-expressing region not involved in the sensitization process, the periaqueductal gray (Genotype: F_1,28=0.80, p=0.3776; Treatment: F_1,28=011, p=0.7378). Lower panels: representative western blotting images. Levels of phosphorylated-ERK (pERK) and total-ERK (tERK) were normalized to the loading control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as individual values and mean ± SEM of n = 3–4 independent experiments. Two brain punches were lost for striatal regions in the Gpr88^+/+ vehicle group.

Figure 3—figure supplement 3. Gels from western blot experiments; ERK phosphorylation assay in brain samples from Gpr88^+/+ and Gpr88^-/-mice.