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. 2011 Feb;36(3):551-8.
doi: 10.1038/npp.2010.186. Epub 2010 Oct 27.

A dopamine D1 receptor-dependent β-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice

Nikhil M Urs  1 , Tanya L Daigle, Marc G Caron
Affiliations

A dopamine D1 receptor-dependent β-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice

Nikhil M Urs et al. Neuropsychopharmacology. 2011 Feb.

Abstract

Morphine is a widely used analgesic in humans that is associated with multiple untoward effects, such as addiction and physical dependence. In rodent models, morphine also induces locomotor activity. These effects likely involve functionally selective mechanisms. Indeed, G protein-coupled receptor desensitization and adaptor protein β-arrestin 2 (βarr2) through its interaction with the μ-opioid receptor regulates the analgesic but not the rewarding properties of morphine. However, βarr2 is also required for morphine-induced locomotor activity in mice, but the exact cellular and molecular mechanisms that mediate this arrestin-dependent behavior are not understood. In this study, we show that βarr2 is required for morphine-induced locomotor activity in a dopamine D1 receptor (D1R)-dependent manner and that a βarr2/phospho-ERK (βarr2/pERK) signaling complex may mediate this behavior. Systemic administration of SL327, an MEK inhibitor, inhibits morphine-induced locomotion in wild-type mice in a dose-dependent manner. Acute morphine administration to mice promotes the formation of a βarr2/pERK signaling complex. Morphine-induced locomotor activity and formation of the βarr2/pERK signaling complex is blunted in D1R knockout (D1-KO) mice and is presumably independent of D2 dopamine receptors. However, D1Rs are not required for morphine-induced reward as D1-KO mice show the same conditioned place preference for morphine as do control mice. Taken together, these results suggest a potential role for a D1R-dependent βarr2/pERK signaling complex in selectively mediating the locomotor-stimulating but not the rewarding properties of morphine.

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Figures

Figure 1
Figure 1
β-Arrestin2 but not β-arrestin1 is required for morphine-induced locomotion. (a) Wild-type (WT) and β-arrestin2 knockout (βarr2-KO) or (b) WT and β-arrestin1 knockout (βarr1-KO) littermates were habituated in an activity monitor for 30 min and then injected with morphine (20 mg/kg, s.c.), and the total cumulative distance traveled (cm) was recorded for an additional 120 min. On morphine stimulation, βarr2-KO mice but not βarr1-KO mice show significantly decreased distance traveled as compared with WT littermates. **p<0.01, compare WT morphine with βarr2-KO morphine. n=8 for all treatment groups.
Figure 2
Figure 2
ERK is required for morphine-induced locomotion. (a) Wild-type mice were injected with either saline (Sal) or the MEK inhibitor SL327 (100 mg/kg, i.p.) and placed in an activity monitor, and the distance traveled was recorded every 5 min for 15 min and then injected with morphine (Mor) (20 mg/kg, s.c.) and the distance traveled was recorded every 5 min for 120 min. n=8 for all treatment groups. (b) Total cumulative distance recorded for 135 min for either saline or different doses of SL327 (50 and 100 mg/kg, i.p.), followed by morphine administration. *p<0.05; **p<0.01, compare Sal+Mor treatment with SL327+Mor treatments (black bars).
Figure 3
Figure 3
β-Arrestin2 and pERK form a complex on morphine stimulation. (a) Immobilized pERK antibody agarose beads were mixed with striatal lysates from saline (Sal, n=6) or morphine (Mor, n=6) (20 mg/kg, s.c.), injected into C57Bl/6J WT mice, and subjected to western blot as described in the ‘Materials and methods' section. Membranes were probed with antibodies to βarr2, DARPP-32, and pERK. The left-side panel represents the immunoprecipitated protein and right panel the total lysates. (b) βarr2 band intensity was quantified using ImageJ and normalized to pERK (loading control) band intensity. **p<0.01, compare saline with morphine.
Figure 4
Figure 4
D1 receptors regulate morphine-induced locomotion. (a) Wild-type (WT) and D1 receptor knockout (D1-KO) or (c) WT and D2 receptor knockout (D2-KO) littermates were habituated in an activity monitor, and the distance traveled was recorded every 5 min for 30 min and then injected with morphine (Mor) (20 mg/kg, s.c.), and the distance traveled was recorded every 5 min for 120 min. On morphine stimulation, D1- and D2-KO mice show significantly decreased distance traveled as compared with WT littermates. n=8 for all treatment groups. (b) Total cumulative distance for WT and D1-KO or (d) WT and D2-KO was recorded for 150 min at 5, 10, and 20 mg/kg, s.c., for D1 mice and 20 mg/kg, s.c., for D2 mice. D1-KO and D2KO mice have decreased locomotor activity at all tested doses of morphine. *p<0.05; **p<0.01, compare morphine treatment between genotypes. To test the role of post-synaptic D2 receptors in morphine-induced locomotion, DAT-KO mice were placed in activity monitors and the distance traveled was recorded every 5 min for 30 min. Mice were then injected with either (e) saline or SCH23390 (0.1mg/kg, s.c.) or (f) saline or raclopride (2mg/kg, i.p.) and activity recorded for another 30 min. All treatment groups (panels e and f) were then injected with morphine (Mor) 20 mg/kg, s.c., and activity recorded for additional 60 min. SCH but not RAC blocked the morphine-induced locomotion in DAT-KO mice. (g) Absolute change (Δ) in the total distance traveled on morphine exposure between time points 60 and 140 min for various treatments as shown in the figure. *p<0.01, compare SAL+MOR with SCH+MOR.
Figure 5
Figure 5
D1 receptors are required for β-arr2/pERK complex formation (a) Wild-type (WT) and D1-KO mice were injected with either saline (Sal, n=4) or morphine (Mor, n=5) for 60 min and striata were rapidly dissected out. Immobilized pERK antibody agarose beads were mixed with striatal lysates and subjected to western blot as described in Figure 2 and in the ‘Materials and methods' section. (b) Quantification of βarr2 band intensity normalized to pERK intensity. On morphine (Mor) stimulation, WT mice show a ∼1.5-fold increase in βarr2 interaction, which is absent in D1-KO mice. *p<0.05, compare saline with morphine.
Figure 6
Figure 6
D1 receptors are not required for morphine-induced place preference Wild-type (WT) and D1-KO mice were assessed for the time spent in either black or white compartment on the pre-conditioning day. Morphine was randomly paired with either compartment for both genotypes as described in the ‘Materials and methods' section. After the sixth day of conditioning phase (see the ‘Materials and methods'), time spent in either compartment was assessed on day 8 (post-conditioning or test phase). Data are represented as difference in time spent in the drug-paired chamber on the pre- and post-conditioning days. Morphine at 3 mg/kg, s.c. (gray bars) and 6 mg/kg s.c. (black bars) induced significant place preference (p<0.01) in both WT and D1-KO mice, without any significant difference between genotypes. n=12 for all groups.
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