Nucleus accumbens-specific interventions in RGS9-2 activity modulate responses to morphine

Abstract

Regulator of G protein signalling 9-2 (Rgs9-2) modulates the actions of a wide range of CNS-acting drugs by controlling signal transduction of several GPCRs in the striatum. RGS9-2 acts via a complex mechanism that involves interactions with Gα subunits, the Gβ5 protein, and the adaptor protein R7BP. Our recent work identified Rgs9-2 complexes in the striatum associated with acute or chronic exposures to mu opioid receptor (MOR) agonists. In this study we use several new genetic tools that allow manipulations of Rgs9-2 activity in particular brain regions of adult mice in order to better understand the mechanism via which this protein modulates opiate addiction and analgesia. We used adeno-associated viruses (AAVs) to express forms of Rgs9-2 in the dorsal and ventral striatum (nucleus accumbens, NAc) in order to examine the influence of this protein in morphine actions. Consistent with earlier behavioural findings from constitutive Rgs9 knockout mice, we show that Rgs9-2 actions in the NAc modulate morphine reward and dependence. Notably, Rgs9-2 in the NAc affects the analgesic actions of morphine as well as the development of analgesic tolerance. Using optogenetics we demonstrate that activation of Channelrhodopsin2 in Rgs9-2-expressing neurons, or in D1 dopamine receptor (Drd1)-enriched medium spiny neurons, accelerates the development of morphine tolerance, whereas activation of D2 dopamine receptor (Drd2)-enriched neurons does not significantly affect the development of tolerance. Together, these data provide new information on the signal transduction mechanisms underlying opiate actions in the NAc.

Figure 1

Increased regulator of G protein signalling 9-2 (Rgs9-2) activity in the nucleus accumbens (NAc) blocks morphine reward. Mice infected with adeno-associated viruses (AAV)-Rgs9-2 or control AAV-GFP constructs in the NAc via stereotaxic surgery were tested in the CPP paradigm. As shown in Figure 1a, overexpression of Rgs9-2 in the NAc prevents place morphine-conditioned place preference (Mor, 5 or 10 mg/kg s.c. n=6–7 per group, *P<0.01 two-way ANOVA followed by Bonferroni post hoc test). Saline injections did not produce place preference in wild-type or mutant mice (Figure 1b, n=8 per group). Figure 1c shows western blot analysis of Rgs9-2 levels in the NAc of AAV-GFP and AAV-Rgs9-2-infected mice and the distribution of EGFP in the NAc ( × 10 ZEISS AXIO Fluorescent Microscope), 3 weeks post-viral infection. Placements were circumscribed within the dorsomedial part of the NAc, including parts of both shell and core at A/P coordinates between +1.5 and +1.7 from bregma.

Figure 2

A role of regulator of G protein signalling 9-2 (Rgs9-2) in morphine withdrawal. qPCR analysis from Fluorescence-activated cell sorting (FACS)-sorted GFP-positive D1 or D2 type striatal neurons reveals that chronic morphine (25 mg pellets, on days 1 and 3 of a 5 day paradigm) leads to a robust downregulation of Rgs9 expression in D2 type but not in D1 type neurons (Figures 2a, n=3−4 per group, *P<0.05, t-test). Mice were infected with the adeno-associated viruses (AAV)-Rgs9-2 or AAV-GFP constructs in the nucleus accumbens (NAc) and used in a morphine withdrawal paradigm 2 weeks after surgery. In Figure 2b, Animals were injected with increasing morphine doses for 4 days, and on day 5 morphine withdrawal was precipitated by naloxone injection (1 mg/kg s.c.). Increased Rgs9-2 activity in the NAc significantly reduces the number of wet dog shakes, diarhhea occurrence, and % weight loss. Interestingly, overexpression of Rgs9-2 in the dorsal striatum had a small but significant effect on weight loss (Figure 2c, n=9−12 per group). Data are expressed as mean±SEM. *P<0.05, two-way ANOVA followed by Bonferroni test.

Figure 3

Regulator of G protein signalling 9-2 (Rgs9-2) actions in the nucleus accumbens (NAc) modulate morphine analgesia and tolerance. Mice infected with adeno-associated viruses (AAV)-Rgs9-2 or AAV-GFP constructs in the NAc were evaluated in the hot plate paradigm in order to determine the role of Rgs9-2 in the NAc in analgesic responses to morphine. As shown in Figure 3a, overexpression of Rgs9-2 in the NAc reduces the analgesic actions of morphine in the 52 ^oC hot plate test (n=10 per group, *P<0.05, t-test). Overexpression of Rgs9-2 in the dorsomedial striatum does not affect hot plate responses to morphine (Figure 3b, n=10 per group). In Figure 3c, western blot analysis studies reveal that Rgs9-2 expression in the NAc increases by the fifth day of morphine (Mor) administration in a hot plate tolerance paradigm (P<0.01 t-test, n=5 per group, Sal=Saline). A separate group of animals received a high morphine dose (30 mg/kg i.p.) and were evaluated in the hot plate test for 5 consecutive days. As shown in Figure 3d, overexpression of Rgs9-2 in the NAc accelerates the development of morphine tolerance, whereas expression of the dominant-negative DEPlessRgs9-2 in the NAc delays the development of tolerance (n=7−8 per group, *P<0.05 for overexpressors vs AAV-GFP controls, ^#P<0.05 for AAV-GFP day 5 vs day 1 and for AAV-GFP day 5 vs AAV-DEPlessRgs9-2 day 5). All data are expressed as means±SEM, two-way ANOVA followed by Bonferroni post hoc test.

Figure 4

Optogenetic activation of neuronal subpopulations in the nucleus accumbens (NAc) modulates morphine tolerance. Optogenetic activation of regulator of G protein signalling 9-2 (Rgs9)-expressing neurons in the NAc accelerated the development of morphine tolerance in the hot plate paradigm (Figure 4a, n=8 per group, P<0.05, two-way ANOVA followed by Bonferroni post hoc test). In Figure 4b, Drd2-Cre mice were infected in the NAc with inverted double floxed DIO-AAV (adeno-associated viruses)-ChR2-EYFP or control DIO-AAV-EYFP vectors. Optogenetic activation of D2-type neurons in the NAc does not have a significant effect on the development of morphine tolerance (n=5−6 per group). Drd1-Cre mice were infected in the NAc with inverted double floxed DIO-AAV-ChR2-EYFP or control DIO-AAV-EYFP vectors. As shown in Figure 4c, optogenetic activation of D1-type neurons in the NAc accelerates the development of morphine tolerance n=5−7 per group, *P<0.05 two-sway ANOVA followed by Bonferroni post hoc test. A separate group of DIO-AAV-ChR2-EYFP or control DIO-AAV-EYFP injected mice were optogenetically activated until day 3 of morphine (or saline) administration in hot plate tolerance assay and the NAc was dissected 1 h after the last hot plate assay (Sal=Saline, Mor=morphine). Western blot analysis reveals that persistent activation of the NAc in morphine-treated DIO-AAV-ChR2-EYFP-expressing mice increases Rgs9-2 levels (Figures 4d, n=3−5 per group *P<0.05, one-way ANOVA, followed by Dunnett test).